The local fields, such as magnetic, optic and temperature, can be used to characterize the properties of physical and biological materials. These local fields could be measured with closely placed quantum emitters. The imaging process were usually accomplished by placing the probe on a scanning tip. However, the moving of tips might change the local field distribution. Another promising method is to place an array of probes close to the sample and detect the probes with super-resolution optical far-field microscopy. In this way, the relative distance between the probe and sample is fixed, and the scanning process would not affect the results of detection. The resolution of fluorescence based optical far-field microscopy has been improved to several nanometers in recent years. It is possible to detect the nanoscale physical local effects with optical far-field microscopy.

  In this work, we used the nitrogen vacancy (NV) center in diamond as probes to detect the local optical field passing through aluminum structures. By improving the spatial resolution of optical microscopy with NV center, we can detect the shape of structures with resolution below diffraction limit. This method has the potential to detect other materials, which enable it to be a universal super-resolution microscope.

  It has been proved that the charge state conversion of NV center can be pumped by lasers with different wavelengths. The green laser can initialize the center to the negative charge state with fidelity about 75%, while red laser can initialize the center to neutral charge state with fidelity about 95%. To realize high spatial resolution microscopy, we used a Gaussian shaped 637 nm laser beam to initialize the charge state, and then a doughnut shaped 532 nm laser beam to switch the charge state of NV, as shown in Fig.1 (a). Therefore, the doughnut shaped 532 nm laser beam will change the charge state of NV from NV⁰ to NV^-. Only the charge state of NV at the center of 532 nm laser beam will not be changed by doughnut laser beam (Fig. 1 (b)). The charge state was finally detected by a weak 589 nm laser. By detecting the fluorescence of NV^-, the intensity of fluorescence would show a dark spot, which indicated the position of NV with resolution below diffraction limit. This super-resolution microscopy was named as charge state depletion (CSD) microscopy.

  In order to improve the resolution of microscopy, one way was to increase the power of 532 nm laser. Instead, we applied another 780 nm Gaussian shaped laser beam, as in Fig. 1(a). The 780 nm laser can accelerate the charge state conversion pumped by 532 nm. Therefore, the resolution of microscopy was significantly improved by applying the 780 nm laser, as shown in Fig 1(c). And the power of 532 nm laser can be reduced by a factor about 10 times by applying 0.4 mW 780 nm laser. The resolution of 50 nm can be obtained with 0.14 mW 532 nm laser. As the 780 nm laser might cause less photon damage than 532 nm laser, this method could be applied to the biological imaging.

  In further step, we used the super-resolution microscopy with NV center in bulk diamond to detect the structure of nanoscale materials, as shown in Fig.2. Aluminum was deposited on the surface of diamond plate. The laser beams were used to pump NV center through the aluminum structures. The local optical field below diamond surface was affected by the shape of aluminum material. And the charge state conversion of NV center was pumped by the local optical field below diamond surface. By applying CSD microscopy, we can detect the charge state of NV center with high spatial resolution. Subsequently, the structure of aluminum was imaged with spatial resolution higher than that with confocal microscopy, as in Fig.2 (c)-(e). This method can be used to detect properties of other nanoparticles.

  In summary, we demonstrated the super-resolution microscopy with NV center based on charge state conversion. The power of laser was decreased at least one order by applying an additional 780 nm laser. A universal super-resolution microscopy was developed by using the NV center ensemble in bulk diamond as local optical field probes.

We  describe an algorithm of processing of z-stacks of images of unlabelled live cells obtained from wide-field bright-field optical transmission microscopy to achieve the super-resolved volumes of intracellular objects in the order of 20×20×100 nm³, including the classification of pixels according to an intracellular dynamics and spectral properties [1].

This super-resolved method is based on

(1) non-interpolating de-mosaicing of a 12-bit z-stack of raw images obtained from a camera chip equipped by a Bayer mask, which preserves as much information in the image as possible to obtain RGB images.

(2) searching for pixels of unchanged (or near) intensities between two consecutive images, after either the simple subtraction of two consecutive images or the calculation of a point divergence gain (PDG).

The latter step of image processing is responsible for evaluation of the intracellular dynamics. If PDG = 0 (the same intensities at the same position in two consecutive images), large intensity homogenous non-moving objects are mainly segmented. At minimal and maximal values of PDG (a pixel of the rarest intensity is replaced by a pixel of the most frequent intensity and vice versa), we track a large moving organelles. The other values of PDG correspond either to the other intracellular objects or to other intensities in the course of the sum of point spread functions of the live cell.

(3) the selection and evaluation of each colour channel of the image. In the blue channel, we observe mainly autofluorescence. In the green channel, the light diffraction is also projected. In the red channel, the light absorption in near infrared region is further observed.

We also show a method of simplified reporting of resulting data on the example of a living mammalian cell.

References:

[1] R. Rychtáriková et al., in ISCS 2014: Interdisciplinary Symposium on Complex Systems (Emergence, Complexity and Computation 14), edited by A. Sanayei, O.E. Rössler, I. Zelinka (Switzerland: Springer), 2014, pp. 261–267.

Acknowledgements:

This work was nancially supported by CENAKVA (No. CZ.1.05/2.1.00/01.0024), CENAKVA II (No.LO1205 under the NPU I program) and The CENAKVA Centre Development (No. CZ.1.05/2.1.00/19.0380).

Microbubbles (MBs) or air bubbles have been explored for various applications in many fields of science and technology, such as water treatment, biomedical engineering and nanomaterials. The so-called nanobubble was first proposed in 1994 and also confirmed their existence at the interfaces between water and hydrophobic solids [1]. In a previous study, microbubbles may evolve spontaneously on smooth modified hydrophobic Si surfaces following the decompression [2]. It was proposed that nanobubbles may be the gas micronuclei responsible for the microbubble formation. However, direct observation of microbubble nucleation sites is not yet reported. In this work, we investigated the formation process of microbubbles at a solid/water interface using pe-degassed and N₂-saturated water. Optical microscopy was first used to observe the nucleation site of microbubbles and atomic force microscopy (AFM) was utilized to obtain high-resolution images of the nucleation sites. In figures 1(a)-(c), optical images show that the formation process of microbubbles on graphene-coated mica surface in N₂-saturated water. Microbubbles tend to form on hydrophobic graphite or graphene-coated areas but not on pure mica areas. Interestingly, there were certain sites on graphene-coated areas that microbubbles prefer to form when we inject N₂-saturated water. We zoomed in an area with the preferential gas nucleation sites [figure 1(d)] and acquired high-resolution AFM images [figure 1(e)] after injecting pre-degassed water to make the microbubbles disappear. The preferential bubble nucleation sites appeared dark in optical images, but AFM images revealed that these sites were local protrusions in the height images. In addition, AFM images showed presence of nanobubbles on flat graphene-covered areas outside the protrusions [figure 1(f)]. The relation between microbubble and nanobubble will be discussed.

Ref [1]. Parker J L, Claesson P M and Attard P, J. Phys. Chem. 98 8468–80 (1994)

Ref [2]. Arieli R., Marmur A., Respir Physiol Neurobiol. 177(1):19-23 (2011)

The recent emergence of super-resolution microscopy imaging techniques has surpassed the diffraction limit to improve image resolution. Contrary to the breakthroughs of spatial resolution, high temporal resolution remains a challenge. By introducing a detour phase term, a quadratically distorted (QD) grating, which imparts an equal but opposing focal power in the positive and negative diffracted orders, can provide a simple, versatile avenue for simultaneous multi-plane microscopy imaging when combined with a lens [1]. As Fig. 1 illustrated, objects A, B and C located at different distances from QD grating are imaged simultaneously and spatially separated on a single image plane, and the biological images on the right side are, for example, the partial views of a stage 8 drosophila egg chamber (Δz~7.3µm). This narrowband multi-plane bioimaging work was in collaboration with Prof. Ilan Davis of Oxford University.

However, QD gratings are dispersive and the multi-colour images may be chromatically smeared if the dispersion is not corrected. A correction scheme based on grism, which is a combination of blazed grating and prism, has been demonstrated earlier [2]. In principle, utilizing the inherent non-periodic chirps of QD grating, identical diffraction angles with respect to different wavelengths could be obtained, thus correcting the chromatic dispersion (Fig. 2a). Therefore a collimated beam in which the spectral components are laterally displaced has been created by a pair of grisms (Fig. 2b). The lateral shear between the polychrome components in the output beam is controlled by varying the separation between the grisms. Due to the various errors in alignment of the long optical path (>1 metre in reference [2]) and the defects in the fabrication of grisms and their mounts, the grism-based approach we proposed before is also limiting, which is only applicable for optical tests rather than the real microscopy imaging [2].

We have recently customized a new pair of grisms and their mounts, and optimized the design of QD grating with practicable parameters, such that the optical system can be effectively improved and hence available for simultaneous multi-plane, multi-colour microscopy imaging. Fabricated from Schott B270 glass, a 17.5° wedge/blazed angle was designed, and a transmission grating of 300 grooves/mm was cemented to one side of the prism, thus forming a grism with an un-deviated wavelength of 527.3nm. The grism pair was mounted between two 250mm achromatic lenses for qualitatively evaluating the wide-range chromatic correction capability. The achromats were spaced 200mm apart, and the QD grating (a nominal axial period of 50μm, a curvature W₂₀ of 50 waves and radius of 10mm) was placed 208mm from the second principal plane of the compound achromats system, thus equal magnification images can be obtained in each diffraction order. Then a simulated 3D imaging of eGFP fluorophore was implemented — a high power fibre continuum source (Fianium SC450-PP-HE) filtered by a set of 20nm bandpass filters (Thorlabs) with central wavelengths from 480nm to 600nm in 20nm steps was applied, and the system was focused for the central wavelength of ~532nm in 0^th order, with and without grism correction. The combined greyscale image of all the 7 wavebands (simulated from bandpass weighting) demonstrate that the chromatic smearing of first-order images can be effectively corrected (Fig. 3). The grism and thus the optical system can also be designed for broadband simultaneous multi-plane imaging of various fluorophores/wavebands.

This technique can be realised with a simple optical attachment fully compatible with commercial microscopes and standard camera systems, and has direct applications for studies of rapidly-changing objects in cell-biology, fluid-flow and high-speed, 3D tracking. A few biological applications are still in process, and the details of which may be published in the near future.

References

[1] P. Blanchard and A. Greenaway, Applied Optics, 38(32), 6692-6699 (1999).

[2] Y. Feng, P. Dalgarno, D. Lee, Y. Yang, R. Thomson and A. Greenaway, Optics Express 20(18), 20705-20714 (2012).

We determined the crystal structure of the K_6.4Nb_28.2Ta_8.1O₉₄ pseudo-tetragonal tungsten bronze-type material using advanced transmission electron microscopy techniques with an aberration corrected microscope. [1] The crystal structure solution obtained with TEM was afterwards refined using X-ray and neutron powder diffraction data. The crystal structure consists of a framework containing trigonal, tetragonal and pentagonal tunnels, all built from (Nb,Ta)O₆ octahedra. Using ABF-STEM and atomic resolution EDX we found that the tetragonal tunnels are occupied by K and Nb atoms, not observed before in the K₂Nb₈O₂₁ counterpart, whereas part of the trigonal tunnels are occupied with K. The occupation of the trigonal tunnels was not reported before for any of the compounds with similar structure type. 

[1] Robert Paria Sena, Artem A. Babaryk, Sergiy Khainakov, Santiago Garcia-Granda, Nikolay S. Slobodyanik, Gustaaf Van Tendeloo, Artem M. Abakumov, Hadermann, Joke, Dalton Transactions 45 (2016) 973-979

Acknowledgements

We thank Dr. E. Suard and Dr. O. Fabello for assistance collecting the neutron diffraction data. R.P.S. acknowledges funding from the European Research Council under the 7th Framework Program (FP7), ERC Grant No.246791-COUNTATOMS. The titan microscope was partly funded by the Hercules fund from the Flemish Government. The authors acknowledge financial support from the European Union under the Seventh Framework Program under a contract for an Integrated Infrastructure Initiative. Reference No. 312483-ESTEEM2.

Synapses are compartmentalized organelles containing thousands of proteins. Their precise localization at both pre- and post- synaptic elements is essential for a correct processing and transfer of information between excitable cells. To decipher cellular and molecular mechanisms that regulate and maintain neurotransmitter receptors at synapses we are using C. elegans neuromuscular junction (NMJ) as a model of synapse. In particular, we are interested in a novel mechanism of clustering acetylcholine and GABA_A receptors based on extracellular scaffolding proteins in the synaptic cleft. The transparency and simple morphology of C. elegans combined with the ease of genomic engineering to tag endogenous proteins, provide a means to address the localization and organization of these extracellular scaffolding proteins.

We used 2D and 3D direct Stochastic Optical Reconstruction Microscopy (dSTORM) on immuno-stained worms and we found that acetylcholine receptor (AChR) clusters are close but not in perfect register with presynaptic boutons. We then built a knock-in strain expressing AChR tagged with the photoswitchable protein Eos and used PALM to analyze AChR content at individual synapses. We found that the number of AChRs is variable depending on synaptic size but the density or receptors is remarkably constant. We are now mapping protein distribution on synaptic ultrastructure by combining the tomo-array technique with STORM/PALM imaging.

RISE Microscopy is a novel correlative microscopy technique that combines confocal Raman Imaging and Scanning Electron (RISE) microscopy within one microscope system (Fig. 1a). This unique combination provides distinct advantages with regard to comprehensive sample characterization: electron microscopy is an excellent technique for visualizing surface structures in the nanometer range and confocal Raman imaging is an established method for detection of the chemical and molecular components of a sample with diffraction-limited resolution. In contrast to conventional systems in which individual Raman spectra are collected from areas several micrometers across, RISE can produce diffraction-limited confocal Raman images from the same sub-micron areas investigated by SEM. It can also generate 3D-images and depth profiles to visualize the distribution of the molecular compounds within a sample volume. Both analytical methods are fully integrated into the RISE microscope. Between measurements a precise translation stage automatically transfers the sample inside the microscope's vacuum chamber and re-positions it. The RISE software carries out the required parameter adjustments and instrument alignments. The acquired results can then be correlated and the Raman and SEM images overlaid.

The instrument itself as well as example measurements demonstrating the potential of its hybrid approach will be presented. An example of correlative Raman and SEM imaging is presented in Figure 1b-d, showing the distribution of TiO2 nanoparticles. Fig. 1b shows the SEM image of TiO2, revealing nanoparticles of two different sizes. From the same sample area an array of 150 x 150 complete Raman spectra was acquired with an integration time per spectrum of 37 ms. From this array two distinct spectra were evaluated as shown in Fig. 1c. They show the two polymorphic phases of TiO2: rutile and anatase. The correlative Raman-SEM image (Fig. 1d) reveals that the large particles correspond to the rutile phase of TiO2, whereas the small particles consist of anatase TiO2, enabling the fine crystallographic structure of the nanoparticles to be differentiated.

Electron microscopy (EM) and nano secondary ion mass spectrometry (NanoSIMS) aim to acquiring nanometric information, which also imply ultraresolution and therefore these techniques require the best preservation of samples. Analytical techniques such as X-ray spectroscopy and NanoSIMS are able to identify, localize and quantify chemical elements both at the whole cell and at the intracellular level. These techniques can be coupled with biological structural analysis. The goal in sample preparation is to maintain chemical elements at their original localization site as well as at their physiological active site. Therefore, sample preparation has to prevent also delocalization of the biological molecules (e.g. proteins, lipids). In EM and NanoSIMS, samples are subjected to drastic conditions such as high vacuum and beam energy. Thus, due to the characteristics of these devices, analyses are incompatible with native biological systems. Moreover, these techniques require thin sections of samples (TEM/X-EDS (70-100nm) and NanoSIMS (200-300nm)). The sample should not be destroyed too quickly under the beam or by vacuum sublimation and must be stable chemically. Diffusible elements are quickly lost during dehydration and embedding step used for the routine preparation of biological specimens and so these methods can only be used if it is already known that the elements of interest are tightly bound. Sample preparation for EM and NanoSIMS must immobilize the biological elements, must eliminate water and must allow sectioning of the sample. As generally accepted in the literature, cryofixation by high pressure freezing followed by cryosubstitution are the best methods to limit redistribution of metal ions. In general, the preparation of biological samples for TEM and NanoSIMS is rather similar. Therefore, both techniques can be easily applied together and in a complementary way for bioimaging.

In this work, different biological sample preparation techniques will be presented. The goal is to compare several cryomethods like cryosubstitution with different resins or freeze-drying. An advanced sample preparation protocol was developed basing on high pressure freezing cryofixation follow by cryosubstitution or freeze-drying, in order to limit metal redistribution, and preparation of adjacent ultramicrotome sections for parallel TEM and NanoSIMS analyses of the same cell.

Best results were obtained by correlative imaging of a single cell by TEM and NanoSIMS combining the advantages of both techniques. As an illustration, the ultrastructure of a C. reinhardtii cell (Fig. 2) could be directly related to the spatial distribution of macro and trace elements present at basal levels in the cell (Figs. 1 and 3). Thus, metals could be localized in different cell organelles such as the pyrenoid and granules. For another example, epidermal cells on adherent culture (keratinocytes), localization of elements (e.g. Ni) demonstrates the importance of sample preparation. Correlative TEM and NanoSIMS shows potential for many future applications of subcellular imaging of trace elements in medicine and biology.

Acknowledgments

Ministère de l’Enseignement Supérieur et de la Recherche : ANR-11-EQPX-0027 MARSS, PhD fellowship for  Florent Penen and ANR-10-INBS-04 FranceBioImaging.

We propose to improve atom probe tomography (APT) reconstruction methods to increase the reliability and the accuracy of the resulting 3D volumes. Currently, 3D reconstructions are done iteratively atom by atom, to convert the ion sequence (#N) and the detected position (X_D,Y_D) into atomic coordinates (x,y,z). To obtain a more realistic reconstruction, a better estimation of the main reconstruction parameters (e.g. image compression factor, detection efficiency, evolution of the tip radius) is required [1].

Some techniques have already been developed in order to estimate the reconstruction parameters. Those calibrations are based on voltage curve, tip image, field ion microscopy or desorption images. Most of these techniques have been shown to give accurate results on homogenous or metallic materials (in this case, the crystallographic pole or atomic planes can easily be observed) [2]. Unfortunately, these techniques are poorly suited for semiconductor materials, since the projection laws and the physics of field evaporation affect the reconstruction parameters, especially in the case of multiphase materials where evaporation fields are different from one phase to another.

In this study, we aim to correlate the information obtained by transmission electron microscopy (TEM) with atom probe tomography to improve the current reconstruction model. We focused on high-K multilayer materials where the evaporation field is very different between each layer. As we can see on figure 1, the original sample contains flat and chemically well-defined interfaces, whereas those interfaces are strongly distorted (figure2.a) when a classic reconstruction algorithm (with cone angle consideration) is used.  This distortion comes from the assumption that two atoms arriving one after the other on the detector were close to one another in the tip, which is not the case in wide field of view APT.

In our model, the reconstruction is performed by dealing separately with different (and previously defined) areas of the detector surface. The ions are labeled independently in every sub-detector, allowing different reconstruction parameters for each subdetector and therefore a better match of the theoretical thickness of every layer. The new reconstruction (figure 2.b) shows homogenous atom density, flat interfaces and more accurate layer thicknesses, making the chemical quantification more reliable. This study could be extended to 3D objects, where the microstructural features observed in TEM could be used to constrain the resulting APT volume.

[1] Vurpillot et al., Ultramicroscopy, 132, 19, 2013.

[2] B. Gault et al., Microscopy and Microanalysis, 14 (04), 296, 2008.

This work has been funded by the French ANR Recherche Technologique de Base (RTB) programme. The experiments were performed on the Nanocharacterisation platform (PFNC) at MINATEC.

Macrobrachium rosenbergii Nodavirus (MrNV) causes white tail disease (WTD) in the giant freshwater prawn M. rosenbergii. In our past studies, we generated Macrobrachium rosenbergii Nodavirus virus-like particles (MrNV-VLPs) that were capable of being “nanocontainers” through the uptake of foreign plasmids after viral capsid disassembling/reassembling treatment. Nevertheless, the studies into the binding and internalization of this particular virus into susceptible host cells have been lacking; therefore our team has utilized live MrNV and recombinantly synthesized MrNV-VLPs as a tool to examine viral entry mechanisms in Sf9 insect cells; whereby revealing the dependence upon the caveolin-mediated endocytotic pathway for internalization and infection. Furthermore, viral capsid protein subunit modeling and capsid enzyme digestion were performed for revealing viral protrusions/ligands.

CoCr alloys became commonly used in orthopaedic implants, especially for younger patients, owing to their superior wear and corrosion resistance. However, severe inflammation resulted in unexpectedly high failure rates, leading to the withdrawal of some CoCr devices from the market, and lawsuits were filed in the US. Simulation studies show that, despite exhibiting lower volumetric wear, CoCr implants produce more, smaller (50 nm-3 µm), particles; up to one trillion nanoparticles (NPs) can be produced in each patient annually. The observed inflammation in patients is believed to be caused by these wear-produced NPs. CoCr NPs have been observed in macrophage cells in periprosthetic tissue. While CoCr is extremely corrosion resistant in bulk form, Co²⁺ ions have been observed in blood and other organs such as the liver and spleen raising question on the mechanism of the dissolution of the CoCr NPs particles in vivo.

Ex situ studies of CoCr NPs in simulated biological environment have been performed in our lab with the use of an applied electrochemical potential to simulate the oxidising environment generated during inflammatory response. Electron microscopy showed morphological changes in the particles as they developed into a porous sponge-like structures (e.g. shown in Figure 1). This phenomena has not been observed in CoCr alloys before, revealing a new mechanism of dissolution of these alloys at the nanoscale. This research suggests that new testing criteria are required for implant materials, in particular where there is wear debris generated, where bulk form testing must be accompanied with studying reactivity of materials at the nanoscale.

The morphological changes apparent at the nanoscale, through electron microscopy, was correlated with chemical changes through the high energy resolution of Transmission X-ray Microscopy/X-ray Absorption Spectroscopy at Stanford Synchrotron Light Source. This was done at both the Co and Cr X-ray absorption edges, to reveal the points of oxidation of Co and Cr in the particles.

Correlative Light and Electron Microscopy (CLEM) combines two imaging techniques at different scales to figure out the precise localization of rare elements in a well-defined biological context. Light microscopy (LM) allows positional mapping of the sample labeled with a fluorochrome. Electron microscopy (EM) provides the nanometer resolution of the mapped area. The general principle of CLEM is to collect different informations from a single region in a sample. The data are then combined toward a global understanding of the sample ultrastructure. The precise localization of molecules of interest in their biological context serves to define their functional role. In the present study, we aimed to apply CLEM to tissue samples. We have chosen define regions of interest (ROI) by fiducial laser marks surrounding the ROI. For this purpose, we applied the “Near infrared branding” (NIRB) method (Bishop et al., 2011).

This NIRB technique consists to create easily detectable fiducial marks in three dimensions in a fixed tissue sample. A specific region of the sample is imaged with LM, the fiducial marks are performed, and the same region is analysed with EM. The fiducial marks are detectable both in optical and electron microscopy. They are made with a femtosecond pulsed titanium-sapphire laser. The laser is used to create a user defined pattern with line or points scans. The size of the marks depends on the fraction of laser power used or the number of laser swipes. The NIRB marks can be placed with a three-dimensional micrometer precision in close proximity to the ROI. Laser-made marks are visualized through tissue photo-oxidation that induces autofluorescence in LM and is a characteristic marker for EM. When using Green Fluorescent Protein (GFP) labelling, no GFP photo-oxidation is noticed when drawing NIRB.

The present study focused on adjustments that are necessary to adapt the NIRB method to nervous tissue and skin. These two types of tissue present a very different composition and are therefore well suited to compare the conditions of NIRB implementation. The NIRB technique has been set using immunostaining of easily detectable and strongly represented antigens with a specific location in well-defined regions. In nervous tissue, the constitutive extra-membranous mitochondrial protein TOM20 was used as a target antigen in spinal cord sections. For the skin, the development relied on Langerin immunostaining (a type II transmembrane, C-type lectin receptor on Langerhans cells). The goal is to locate the Langerhans cells in the epidermis. All immunostaining strategies are performed with a pre-embedding method. It consists in immunostaining on chemically fixed samples before embedding in resin and ultra-sectioning for TEM.

First, we set the method to achieve the ROI localization. Then, we defined our own parameter settings using our multiphoton system. Finally, it is essential to adapt the marks to the size of the region of interest, and the type of sample. The delimited ROI can size up to 70μm3. Future perspectives are to develop three-dimensional CLEM approach using electron tomography.

Acknowledgments

Ministère de l’Enseignement Supérieur et de la Recherche : ANR-10-INBS-04 FranceBioImaging.

In CLEM experiments biological samples are typically first investigated by light microscopy (LM) to locate a region of interest. Next, nanometer-scale imaging can be achieved by electron microscopy (EM). This results in identification of biomolecules within the ultrastructure and/or the possibility to correlate ultrastructure with preliminary live-cell dynamics on biomolecules [1].

We have developed an integrated CLEM with which widefield fluorescence microscopy (WF-FM) and EM can be performed in principle simultaneously [2]. Integration of these two modalities allows for fast ROI retrieval [3], optional fast switching between modalities increasing CLEM throughput, high (5 nm) correlation accuracy for the overlay [4], and novel imaging applications [5]. However, for WF-FM the depth correlation for thick specimen or live cells contained in a dedicated liquids sample holder [6] is relatively poor. WF-FM images through the entire sample and SEM only the upper ~100 nm (depending on e-beam energy). We therefore integrated a Confocal Laser Scanning Microscope (CLSM) into a scanning electron microscope (SEM) via a Delmic Secom stage (Fig. 1-4). This system will allow imaging with improved depth-correlation by suppression of out-of-focus light contributions and will provide 3D optical information in CLEM.

We present our integrated CLSM SEM, using a commercial confocal scanner unit. We also demonstrate the procedure for alignment of the CLSM and SEM axes, needed to image the same area with both modalities. Lastly, the results of proof-of-principle experiments are presented, showing optical sectioning in thick specimen such as resin blocks or whole cells for improved LM-SEM correlation along the axial direction.    

[1] P. de Boer, J.P. Hoogeboom, B.N.G. Giepmans, Correlated light and electron microscopy: ultrastructure lights up!, Nature methods, 12, 503-513 (2015)

[2] A.C. Zonnevylle, R.F.C. Van Tol, N. Liv, A.C. Narvaez, A.P.J. Effting, P. Kruit, J.P. Hoogenboom, Integration of a high-NA light microscope in a scanning electron microscope, Journal of Microscopy, 252-1, 58-70 (2013)

[3] N. Liv, A.C. Zonnevylle, A.C. Narvaez, A.P.J. Effting, P.W. Voorneveld, M.S. Lucas, J.C. Hardwick, R.A. Wepf, P. Kruit, J.P. Hoogenboom, Simultaneous Correlative Scanning Electron and High-NA Fluorescence Microscopy, PLOS one, 8-2, e55707 (2013)

[4] M.T. Haring, N. Liv, A.C. Zonnevylle, A.C. Narvaez, L.M. Voortman, P.Kruit, J.P.Hoogenboom (submitted)

[5] J. Sueters, N. Liv, J.P. Hoogenboom, Using Advanced Correlative Microscopy to Study Complex Biological Samples, Encyclopedia of analytical chemistry, a9473, (2016, in press).

[6] N. Liv, D.S.B. Van Oosten Slingeland, J-P. Baudoin, P. Kruit, D.W. Piston, J.P. Hoogenboom, Electron microscopy of living cells during in situ fluorescence microscopy, ACS Nano, 10-1, 265-273 (2016)   

While there is an explosion in Correlative Electron and Light Microscopy (CLEM) protocols, many have been developed on different eukaryotic models, though reports on plant cell exploration by CLEM remain scarce (Bell et al., 2013). Indeed, plant cells represent a challenge for imaging in many ways. Plant specificities have to be taken into account when optimizing CLEM protocols. To name some, strong autofluorescence of cellular components specifically present in plant cells (chlorophyll, pigments, etc.) and interfering with fluorescent signals of reporter proteins, presence of a large central vacuole complicating the fixation and the dehydration processes needed for TEM processing, cell wall and cuticles acting as physical barriers and impairing resin embedding processes (Hawes et al., 2001).

Here we report two “in hand” robust and easily reproducible protocols to make such CLEM approaches on plant material. The proposed protocols had been worked out on the delicate question of autophagosomes identity in plant cells. Autophagy is a degradation process of intracellular components mainly implicated in response to environmental stresses. It consists in the delivery of cytosolic cargos entrapped by a compartment, named autophagosome, to lytic compartments for degradation and recycling. In plant cells as in any eukaryotic cells, autophagosomes have been identified as ring-shape or punctate structures, thanks to the light imaging of reporter proteins of markers for autophagosomes (Le Bars et al., 2014).

This study is using a Arabidopsis transgenic lines stably expressing GFP-ATG8, which is a common marker for autophagosomes. Two CLEM protocols are used:  firstly the high pressure freezing followed by acrylic resin embedding, and on the other hand the Tokuyasu method.  Here we compare the performance of the two protocol to preserve GFP fluorescence and enhanced ultrastructural features in plant cells.

References:

Hawes, C. and Satiat-Jeunemaitre, B. 2001 Plant Cell Biology: A Pratical Approach, 2nd edn. Oxford: Oxford University Press, 1–324.

Bell, K., Mitchell, S., Paultre, D., Posch, M., Oparka, K., 2013. Correlative Imaging of Fluorescent Proteins in Resin-Embedded Plant Material. Plant Physiol. 161, 1595–1603.

Le Bars, R., Marion, J., Le Borgne, R., Satiat-Jeunemaitre, B., Bianchi, M.W., 2014. ATG5 defines a phagophore domain connected to the endoplasmic reticulum during autophagosome formation in plants. Nat. Commun. 5.

Simple CLEM method to asses rare pulmonary vascular remodeling

Péchoux C¹; Antigny F²; Hautefort A²; Ranchoux B² ; Gouadon E² ; Humbert M²; Perros F²; Rücker-Martin C²

1- GABI, Inra, AgroparisTech, Université Paris-Saclay, MIMA2, 78350 Jouy-en-Josas, France

2- Centre Chirurgical Marie Lannelongue, Inserm UMR-S999, Le Plessis-Robinson, France

Key Words: CLEM, PAH, FNG,

Pulmonary Arterial Hypertension (PAH) is a rare disease featured by obstructive lesions of small pulmonary arteries (Ø<500µm), such lesions representing only 1% of the pulmonary tissue. Recently, we evidenced the implication of the endothelial-mesenchymal transition (EndoMT), the increase of phosphorylated vimentin (P-Vim) and the decrease of KCNK3 channel in cellular processes at the origin of the occlusive vascular remodeling in PAH. For these purposes, we adapted the CLEM methodology usually performed on small samples to large tissue taking into account two major biological specificities. First, the lung is a soft organ, consisting of air-filled alveoli, on which CLEM classical preservation methods cannot be applied because of the formation of large crystals which tear and disrupt structures. Second, PAH impacted arteries are 200-500 µm large and represent only 1% of the total lung mass (Figure 1). Therefore, we have developed a complete fixation process based on constraints imposed by electron microscopy, suitable for immunoflurorescent labeling and an immunolabeling process responding to fluorescent confocal microscopy constraints (low autofluorescence) suitable for transmitted electron microscopy (pulmonary ultra-structure preservation and use of various antibodies).

Fresh lung tissue was gently inflated through the bronchi using an automated formalin-pump and immersed in buffered formalin for 24 hours. Then, lung samples (size 2×2.5 cm) were impregnated in increasing sucrose baths (10%/ 40%/ 40) before embedded in Tissu Tek® and frozen. Before immunolabeling, thick cryosections (10µm) were collected on gridded correlative slides, quenched with NH4Cl and nonspecific sites blocked. Sections were incubated with primary antibody before FluoroNanogold™ Conjugates. Pulmonary arteries of interest were identified under a LSM 700 confocal microscope. After confocal acquisition, sections were post-fixed with 1% glutaraldehyde, quenched with glycine, incubated with the Gold reagent and fixed in osmium tetroxide. After dehydratation in ethanol, sections were embedded in Epon. Areas of interest identified by confocal microscopy were sectioned to perform TEM. Ultrathin sections (70 - 80 nm) were collected onto 150 mesh cooper grids and counterstained with Oolong Tea extract (OTE) 0.05% and lead citrate before examination with a Hitachi HT7700 operated at 80kV.

In contrast to cryopreservation, this lung adapted methodology allows to preserve the tissue structure closed to results obtained with Epon embedding (Figure 2) and to follow PAH lesions unambiguously during all the steps. Moreover, such tissue fixation and embedding allow the antigens preservation without producing non-specific labeling. In PAH lesions, confocal microscopy analysis was not resolutive enough to specifically identify, localize and quantify P-Vim or KCNK3 labeling. CLEM approach confirmed Western-blot results with significant increase in P-Vim and decrease in KCNK3 in PAH lesions (Figure 3).

Hence, using correlative microscopy, we overcame the difficulties associated to the lung ultrastructure which is difficult to preserve during EM techniques (hydrophilic resins, Cryo-TEM) and to the location and identification of the small arteries of interest. We found out a good compromise that allowed us to evidence the presence and the exact location of proteins in rare pulmonary lesions, little or not visible in confocal fluorescence microscopy.

Understanding the biological role of molecules requires to know their precise localization and structural environment. Thanks to fluorescence microscopy and biotechnologies, the localization of proteins of interest has become relatively easy. But in more and more cases, this resolution is not enough and the lack of data on the molecule/cell environment does not allow the characterization of their biological function. Transmission Electron Microscopy (TEM) and immuno-gold labelling can be used to characterize at the same time the cellular compartments associated with the protein of interest and the ultrastructure of the cellular environment at a high resolution. However, the antigenicity preservation as well as the antibody production are sometimes difficult. Moreover, TEM observations only give access to a static “snapshot” of a fixed, dead sample. For several years, many attempts have been made to correlate Fluorescent and Electron Microscopy in order to combine the advantages of both microscopy techniques on a unique sample. In this way, we develop on the BIC new protocols of sample preparation for animal and vegetal sample (culture cells and tissues), allowing to maintain simultaneously the fluorescence and the overall ultrastructure (Figure 1 and 2), using Quick Freeze Substitution (McDonald KL and Webb RI, J. Microsc, 2011). This technique will allow the correlation of both Light and Electron Microscopy data on the same section (CLEM: Correlative Light Electron Microscopy), works with classical fluorescent tags and improve the antigenicity for gold immunolabelling.

Acknowledgement: We acknowledge France BioImaging, IDEX Bordeaux and the CNRS for the support.

Plant lines and reconstructed tissue are respectively friendly given by Claire Bréhélin (UMR5200, Laboratoire de Biogénèse membranaire, Bordeaux) and, Murielle Rémy and Olivia Kérourédan (INSERM U1026 BioTis, Université de Bordeaux)

SEM can be used to characterize the crystal structure at smooth surfaces, e.g. by mapping of electron channeling pattern (ECP). Layers of GaN grown on foreign substrates usually include a huge amount of threading dislocations (TDs). ECP are also used to align a sample in specific diffraction conditions of the crystal structure for evaluating the density of TDs and its type by electron channeling contrast imaging (ECCI)¹.

Beside columnar rods ^2,3 also elongated µm-structures like fins ⁴ with high aspect ratios are supposed to have substantial advantages over conventional planar optoelectronic and sensing devices. Thus the synthesis of such 3D-structures, in particular by bottom up growth using molecular beam epitaxy and metalorganic vapor phase epitaxy (MOVPE) on patterned substrates, is under investigation - requesting methods for characterizing local properties of the crystal material.

We present results obtained with an FE-SEM which is equipped with secondary electron (SE), In-Beam SE, low-kV backscattered electron (BSE), electron beam induced current (EBIC) and monochromatic CL detection as well as a piezo controlled manipulator setup, c.f. Figure 1. Simultaneous usage of all available detectors and the manipulator is possible, only the BSE and optical detection are physically hindered by another. A modified parabolic collection mirror enables measuring luminescence from planar samples in a tilted view up to 30°, with respect to the large chamber this enables also a nondestructive investigation of full 4’’-wafers.

The electron optics (EO) of this FE-SEM is also capable of rocking the electron beam on a small area, e.g. rocking in a cone of ±12° on an area of about 15 µm in diameter. We will present how the rocking alignment can be adjusted and evaluated by using samples with dedicated contrast structures.

The EO tilt can also be used to image the sample by the SEM from a certain direction without affecting the tip contact by stage movement. This enables a fine adjustment of the diffraction conditions and subsequent ECCI images for evaluating the type of defects. A subsequent scanning of the sample from different incident directions enables topography reconstruction and generates a three dimensional impression, e.g. by a stereographic image.

By switching from the BSE detector to the mirror for light collection also a correlative analysis of ECCI with EBIC and CL can be performed at the same diffraction condition, see Figure 2. Although having a certain topography contrast also 3D-structures with dimensions of a few µm can be analyzed regarding their crystal structure and orientation using ECP. As CL is quite sensitive also to intrinsic and extrinsic point defects, such correlative images obtained on sidewall and cleavage edges of fin-GaN structures will give valuable insights for discussion of defect mechanism and optical properties.

As the resolution of channeling contrast (e.g. ECCI) and scattering volume (e.g. CL) versus the beam energy are different to another, a correlation of images obtained on the same sample area using different energies improves the identification of individual features. This demonstrates further options for investigating the material quality of 3D structures.

1. Naresh-Kumar, G. et al. Electron channelling contrast imaging for III-nitride thin film structures. Mater. Sci. Semicond. Process. 47, 44–50 (2016). doi: 10.1016/j.mssp.2016.02.007

2. Schimpke, T. et al. Phosphor-converted white light from blue-emitting InGaN microrod LEDs. Phys. status solidi 8, n/a–n/a (2016). doi: 10.1002/pssa.201532904

3. Mandl, M. et al. Group III nitride core-shell nano- and microrods for optoelectronic applications. Phys. status solidi - Rapid Res. Lett. 7, 800–814 (2013). doi: 10.1002/pssr.201307250a

4. Hartmann, J. et al. High Aspect Ratio GaN Fin Microstructures with Nonpolar Sidewalls by Continuous Mode Metalorganic Vapor Phase Epitaxy. Cryst. Growth Des. 16, 1458–1462 (2016). doi: 10.1021/acs.cgd.5b01598

The chemistry of internal interfaces plays a key role in modern physical metallurgy, in particular for the development of the new generations of steels. Despite the tremendous instrumental developments over the last decade, the atomic scale characterization of these interfaces in steels is still a very challenging problem.

Analytical electron microscopy (EDX and EELS in STEM mode) and atom probe tomography are the two techniques presenting spatial and analytical resolutions suitable for such characterizations. Their respective advantages and complementarity will be illustrated on the basis of quantitative determination of segregated amounts of interstitial and substitutional elements at interfaces, with particular emphasis on the intensive specimen preparation efforts required to reach their ultimate capabilities. 

Lactococcus lactis, a Gram(+) lactic acid-producing bacterium used for the manufacture of several fermented dairy products, is subject to infection by diverse virulent tailed phages, leading to industrial fermentation failures. Despite the availability of several antiphage measures, new phages keep emerging in this ecosystem. This constant viral risk has led to a sustained interest in the study of their biology, diversity, and evolution. Lactococcal phages now constitute a wide ensemble of at least 10 distinct genotypes within the Caudovirales order, many of them belonging to the Siphoviridae family. Lactococcal siphophage 1358, currently the only member of its group, displays a noticeably high genomic similarity to some Listeria phages as well as a host range limited to a few L. lactis strains. These genomic and functional characteristics stimulated our interest in this phage.

Here, we report the near-atomic resolution cryo electron microscopy (cryo-EM) structure of the 1358 virion mature full capsid. Recent advances in cryo-EM have enabled structure determination of macromolecular complexes at near-atomic resolution. However, structure determination and atomic modelling still remains a challenging task susceptible to model bias and overfitting especially while using de novo methods. This study demonstrates a practical approach to obtain a rigorously validated atomic resolution cryo-EM structure allowing to complement the previously published complete structural picture of a unique lactococcal phage at lower resolution and have deeper insight into structural organization of it's capsid.

Hepatitis B surface antigen (HBsAg) is overexpressed in great excess during hepatitis B (HBV) infection in the form of noninfectious (DNA-free) subviral particles. These particles greatly outnumber mature virus particles (virions) and presumably function as decoys for the immune system to facilitate infection by the DNA-containing virions. These properties are exploited in the current HBV vaccine, in which recombinant HBsAg is used as a potent antigen capable to induce a strong immune response and long-term immunological memory. Several studies have implicated that HBsAg consist of a lipid bilayer with embedded HBs subunits [1-3]. The particles appear as pleomorphic spheres with an average diameter of ~23 nm and an empty internal cavity. Particles produced for the vaccine contain monomeric HBs subunits that subsequently cross-link via formation of disulfide bridges within the HBsAg particles.

We employed cryo-electron tomography to image individual HBsAg particles in their native environment at different times of maturation. Reconstructed 3D tomograms showed different kinds of HBsAg particles that could be classified into three groups: (i) small spherical particles of ~16 nm in diameter, (ii) spherical particles of ~25 nm in diameter with an internal cavity and (iii) elongated oval particles with an internal cavity of ~20 nm diameter and 25-30 nm length. The electron-dense shell of HBsAg particles is about 5 nm thick and likely represents the lipid bilayer. The shell contains short protrusions with higher density (Figure 1) that are spaced 5-6 nm apart and likely represent lipid-embedded HBs subunits.

Parallel measurements of proton NMR spectra indicated significant structural changes in protein backbone and aliphatic side chains of lipids during HBsAg particles maturation. Those variations support a decreased flexibility and suggested more rigid structure of the lipid bilayer during HBsAg maturation [4].

We analyzed size distribution of HBsAg particles and densities corresponding to HBs subunits within the particles to assess the molecular architecture of particles during maturation. The results provide insights into how structural changes in the lipid bilayer and cross-linking of HBs subunits affect the molecular architecture of HBsAg particles that is directly related to immunogenicity of the HBV vaccine.

This research has been funded by and performed at Sanofi Pasteur.

References

[1] Gastaminza, P. et al. (2010) Ultrastructural and biophysical characterization of hepatitis C virus particles produced in cell culture. J. Virol. 84, 10999–11009.

[2] Milhiet, P. E. et al. (2011) Nanoscale topography of hepatitis B antigen particles by atomic force microscopy. Biochimie (Paris) 93, 254–259.

[3] Grélard, A. et al. (2013) Hepatitis B subvirus particles display both a fluid bilayer membrane and a strong resistance to freeze drying: a study by solid-state NMR, light scattering, and cryo-electron microscopy/tomography. FASEB J. 27, 1–11.

[4] Zhao, Q. et al. (2006) Maturation of recombinant hepatitis B virus surface antigen particles.Hum Vaccin. 2,174-80.

Septins are a highly conserved¹ family of proteins in eukaryotes required for cell division. These proteins are recognized as the fourth component of the cytoskeleton. They promote membrane remodelling by a specific phosphoinositide binding². Consequently, septins are multi-tasking proteins and have prominent role in cytokinesis³, establishing diffusion barriers  for membrane-bound proteins⁴, neuron morphogenesis⁵ and contribute to the development of neurodegenerative diseases (Alzheimer, Parkinson⁶) as well as tumor formation. Thus, recent years have seen renewed for the study of septins.

Septins are GTPases proteins which are bound to the inner cell membrane.  As opposed to other cytoskeletal proteins, they polymerize in a non-polar fashion into paired filaments^2,7. They display a remarkable plasticity, both in term of binding partners (several proteins, membranes) and self-assembly organizations. Indeed, the filaments have a potential to self-assemble into higher-order structures including rods, filaments, rings and gauzes^2,7,8.  

We focus our interest in understanding how septins self-assembly in distinct ultra-structures. To this end, we are using cryo-electron microscopy and tomography. Thus, we are now characterizing (i) liposome remodelling by septins (ii) septins organizations in the different ultra-structures (iii) physico-chemical conditions required for the formations of the ultra-structures and their functions (type of lipid, membrane curvature…). So far, we concentrate our study on Saccaharomyces Cerevisiae septins and recently on Drosophila Melanogaster ones, which is one of the first septins ultra-structure characterization in higher eukaryotes.                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              

1-       Pan and al  “Analysis of Septins across Kingdoms Reveals Orthology and New Motifs.” BMC Evolutionary Biology 7 (2007)

2-       Bertin and al  “Phosphatidylinositol-4,5-Bisphosphate Promotes Budding Yeast Septin Filament Assembly and Organization.” Journal of Molecular Biology 404, no. 4 (December 10, 2010)

3-       McMurray and al “Septins: Molecular Partitioning and the Generation of Cellular Asymmetry.” Cell Division 4 (2009)

4-       Barral and al “Compartmentalization of the Cell Cortex by Septins Is Required for Maintenance of Cell Polarity in Yeast.” Molecular Cell 5, no. 5 (May 2000)

5-       Tsang and al “Superfluous Role of Mammalian Septins 3 and 5 in Neuronal Development and Synaptic Transmission.” Molecular and Cellular Biology 28, no. 23 (December 2008)

6-       Kinoshita, Makoto. “[Role of septin cytoskeleton in dopaminergic neurotransmission and neurodegeneration].”  Japanese Journal of Psychopharmacology 32, no. 1 (February 2012)

7-       Bertin and al “Saccharomyces Cerevisiae Septins: Supramolecular Organization of Heterooligomers and the Mechanism of Filament Assembly.” Proceedings of the National Academy of Sciences 105, no. 24 (June 17, 2008)

8-      Garcia and al “Subunit-Dependent Modulation of Septin Assembly: Budding Yeast Septin Shs1 Promotes Ring and Gauze Formation.” The Journal of Cell Biology 195, no. 6 (December 12, 2011)

The nicotinic acetylcholine receptors (nAChR) are members of the Cys-loop receptor superfamily. Playing important neurophysiological roles they are in the focus of biomedical research. The acetylcholine binding proteins (AChBP) in the hemolymph of gastropods are structural and functional homologues of the ligand binding domain of nAChR. In contrast to the membrane-bound nAChR, AChBP is water soluble. Therefore, AChBP is a very efficient surrogate for indirect investigations of nAChR functions [1]. AChBP is based on 25 kDa polypeptides. Like nAChR (Fig. 1A), AChBP is usually a pentamer (Fig. 1B,C). However, in the hemolymph of the planorbid snail Biomphalaria glabrata (vector snail of the schistosomiasis parasite) it exists as a pentagonal dodecahedron (12 pentamers), 25 nm in diameter (Fig. 1D). This large size and high symmetry makes it well accessible to 3D electron microscopy [2]. Moreover, in B. glabrata two different AChBP polypeptides (designated as Bg-ACHBP1 and Bg-AChBP2) were detected, recombinantly expressed, and reassembled to functional oligomers [2]. Recombinant Bg-AChBP1 readily formed dodecahedra, whereas recombinant Bg-AChBP2 formed only pentamers and dipentamers [2]. The existence of hetero-oligomers could be excluded, but not the possibility that in the hemolymph also Bg-AChBP2 might be present as dodecahedra. Agonist and antagonist binding properties of AChBP and nAChR are comparable [1]. Concerning biological functions of AChBP in snails, several possibilities have been discussed (for references, see [1,2]): (i) suppressor of cholinergic transmission; (ii) regulator of CaCO₃ crystallization (shell growth); (iii) binding agent of algal toxins.

Here we show by in situ hybridization that in B. glabrata, AChBP is expressed in the mantle epithelium (Fig. 2A). In this context, it should be remembered that the molluscan shell is secreted by the mantle. Transmission electron microscopy revealed ultrastructural details of this epithelium (Fig. 2B). The applied cDNA probe was inefficient to discriminate between Bg-AChBP1 and Bg-AChBP2, but immunofluorescence microscopy using isoform-specific rabbit antibodies revealed the presence of Bg-AChBP1 and the absence of Bg-AChBP2 in the mantle epithelium (not shown). Therefore, Bg-AChBP1 might be involved in regulating shell growth. Although these data are preliminary, they suggest that the two Bg-AChBP isoforms significantly differ in their biological functions within the snail. We now perform CaCO₃ crystallization experiments in the presence of Bg-AChBP1.

Acknowledgments

We thank Kristiane Rusitzka, Dr. Wolfgang Gebauer, Sandra Braukmann, Dr. Nadine Leidecker, Julia Markl and Martin Haugwitz for experimental advices, Thomas Schubert, Elisabeth Sehn and Gabriele Stern-Schneider for technical support, Mirko Montigny, Prof. Dr. Wolfgang Tremel and Prof. Dr. Dirk Schneider for cooperation and the Max Planck Graduate Center (MPGC) in Mainz for financial support and a grant to DT.

[1] Shahsavar et al. Basic Clin Pharmacol Toxicol. 2015; doi: 10.1111/bcpt.12528

[2] Saur et al. PLOS One 2012; 7:e43685

Nanosheets have in recent years received considerable attention because of their unique electronic and mechanical properties. They can be prepared by exfoliation of layered materials, but can also be formed via self-assembly which offers a much larger variability in size and chemical functionality. Self-assembly of amphiphilic molecules and polymers though usually leads to the formation of spherical, cylindrical and vesicular structures. The generation of nanosheets requires one to adjust the molecular structure such that planar bilayers sheets are formed with large bending constant to prevent closure into vesicles.

We report the formation of nanosheets or nanoplatelets from an amphiphilic block copolymer consisting of a poly(caprolactone) (PCL) and a fructose-functionalized poly(acrylate) (PFA) block (Scheme 1), prepared by RAFT-polymerization, in water. The nanoplatelets have a core/shell-structure, consisting of an inner core-sheet of the water-insoluble PCL-blocks and an outer sheet of the water-soluble PFA-blocks. The nanoplatelets are stabilized by the crystallization of the poly(caprolactone) block providing a sufficiently large bending constant. We use transmission electron microscopy (TEM) with phosphotungstic acid (PTA) negative staining to determine the thickness and lateral dimension of the nanoplatelets (Fig. 1). Using selected area electron diffraction (SAED) we prove the crystallinity of the poly(caprolactone) core-sheet (Fig. 2). The fructose-functionalized nanoplatelets have a thickness of 8 nm and lateral dimensions of 50 – 200 nm and are potential vectors for targeted drug delivery, where the planar geometry allows for large-area cooperative binding to cell surface receptors.

The papillomaviruses (PV) are small dsDNA tumour viruses of significant medical importance and the prototype of the group is bovine PV (BPV-1). PVs encode one highly conserved replication enzyme, E1, that acts as an initiator and a helicase. E1 forms hexamers and unwinds double stranded DNA (dsDNA) into single stranded DNA (ssDNA) products using the energy of ATP hydrolysis. However, how the helicase engages the replication fork at the molecular level is unclear. BPV-1 E1 is comprised of four domains: a regulatory domain (residues 1-158), a sequence specific ori DNA binding domain (OBD, residues 159-299) and the C-terminal half (residues 300-605, E1HD) that has helicase activity and is further divided into the oligomerisation domain (OD, residues 308-378) and the AAA+ ATPase domain. A C-terminal acidic tail domain (AT, residues 579-605) is required for helicase processivity (Figure A). We have obtained structures of the full length E1 helicase (E1FL) in complex with a DNA replication fork with and without DNA labelling with protein tags. The 5’ end of dsDNA and the 5’ ssDNA end of the fork were labelled and helicase structures studied using single particle electron microscopy.

Negatively stained images (with 2% uranyl acetate) of E1FL/DNAfork/Fab /Streptavidin were taken and the structure obtained at a resolution of~ 20 Å (Figure B). We are currently working on the native structure by cryoEM (Figure C). The 3D reconstruction of E1FL (Figure D) has a three-tier organisation with well-defined domains (N-terminal, DBD, OD, and AAA+). Domains were localised by docking of available atomic structures. The 3D structure of the E1/ labelled DNA fork confirmed the triple ring organisation with an internal small chamber above the helicase motor domain where DNA unwinding appears to take place. Interestingly, dsDNA enters into the chamber via a side tunnel above the helicase motor domain, with the 5´-ssDNA strand leaving through a narrow tunnel located on the opposite side, while the 3´-ssDNA is pulled through the hexamer’s central tunnel (Figure D). Our findings are confirmed by DNA footprint experiments and FRET experiments. Recently we analysed a cryoEM structure of the E1FL in complex with a DNA fork at subnanometer resolution that reveals the same structural organization and provides more detail on the interaction with DNA.

 Our structural studies demonstrate that the process of DNA separation takes place inside the E1FL complex rather than on the exterior surface of E1. In the light of our results we suggest that the current ‘steric exclusion’ model for strand separation should be revised. A molecular understanding of E1 function will be essential to shed light on the early phase of DNA replication and will assist in the understanding of E1 as a therapeutic target of viral DNA replication.

DNA replication is a key cellular process and is the basis for cell division. In order to understand replication initiation in mammalian cells, viral systems such as bovine papillomavirus (BPV) are used as simplified models of the process. BPV uses a single protein E1 to perform both DNA binding and unwinding functions essential for the initiation of replication (Figure A). Biochemical assays for a cell-free replication initiation system has been established for BPV. To have a clearer understanding of a biological process at a molecular level, it is essential to determine the three-dimensional (3D) arrangement and dynamics of molecules and how macromolecular machines are assembled. Electron microscopy (EM), single particle analysis and image processing have been used in this work to reveal the structures of the BPV E1 helicase domain (E1HD), full-length E1 (E1FL), a single-labelled E1FL-replication fork DNA complex with monovalent tetrameric streptavidin (MTS) on the dsDNA of the fork and a double-labelled E1FLwith MTS on the dsDNA and FAB on the ssDNA of the fork.

The structure of BPV E1FL in complex with DNA was initially determined by EM both by applying 6-fold symmetry and without applying any symmetry. EM demonstrated that the E1FL helicase forms a hexamer that has a diameter of 130Å and a height of 100Å, consistent with an overall mass of ~410 kDa. The oligomer has a central channel inside the molecule, with a variable diameter. E1FL is a single polypeptide chain, and the OBD domain can be fitted into the structure in two different ways. In one case the DNA binding site would be located on the inner surface and in the other case it would be on the outer surface of the complex. To determine the orientation of the OBD domain, a 12-residue epitope sequence GGYPYDVPDYAG was inserted into the OBD domain after the residue 226. EM has demonstrated that the antibody was bound to the E1FL. That proves that the binding site for AB is located on the outer surface of E1FL (Figure B). The points of the entrance of dsDNA and exit of 5′ ssDNA were also determined in the complex of E1FL and DNA. Labelling with streptavidin was performed to reveal the position of the dsDNA, whereas ssDNA was labelled with DIG-FAB to reveal the 5′ ssDNA position in an asymmetrical structure of the BPV E1 hexamer bound to a replication fork DNA substrate. Comparison of the 3D structures showed that dsDNA enters the molecule between the N-terminus and oriDNA-binding domain (OBD), and the 5′ ssDNA exits the molecule between the collar domain and OBD. The angle between the point of dsDNA entrance and 5′ ssDNA exit was ~170°.

Prior to our research, the most accepted model of “steric exclusion” for dsDNA unwinding suggested that the active 3′ ssDNA strand is pulled through the helicase motor and dsDNA is wedged apart outside the protein assembly (Figure C). Our structural observations indicated that strand separation is taking place inside E1 in a chamber above the helicase domain and that the 5′ passive ssDNA strand leaves the assembly through a channel located on the opposite side to dsDNA entry. Therefore, our data suggest an alternative model for DNA unwinding by this general class of replication enzymes.

One major challenge of nanomedicine is to design nanocarriers that deliver active compounds to a target site, at a sufficient concentration and without premature degradation, in order to maximize the efficiency of the substance while limiting secondary effects. In this context, we have developed colloidal nanovectors based on cyclodextrin (CD) amphiphilic derivatives obtained by an enzymatically-catalyzed transesterification by thermolysin. We have shown that after dissolution in acetone, depending on the length of the grafted alkyl chains, the derivatives had the ability to self-organize in water, forming nanoparticles with various shapes and ultrastructures [Gèze et al., Mater. Sci. Eng. C29 (2009), 458]. The knowledge of the morphology and ultrastructure of these nanovectors is crucial in order to optimize their formulation and lyoavailability.

The present communication focuses on the βCD-C₁₄ derivative, i.e. βCDs (made of 7 glucosyl units) acylated on their secondary face with C14 chains. The resulting nanoparticle suspensions were quench-frozen and observed by cryo-transmission electron microscopy (cryo-TEM). The βCD-C₁₄ particles exhibited tortuous multidomain shapes (Figure 1a) and the corresponding small-angle X-ray scattering (SAXS) pattern collected from a concentrated suspension contained peaks whose distribution was consistent with a columnar hexagonal structure (Figure 1c). Depending on the orientation of the particles in the embedding film of vitreous ice, the cryo-TEM images revealed that some particles consisted of misoriented domains separated by sharp interfaces (Figure 1b). A direct view of the hexagonal organization was obtained when the incident beam was parallel to the columns and grain boundaries with various tilt angles were observed. The analysis of the structure of the grain boundaries was based on the concepts of coincidence site lattice (CSL) and structural units (SUs) frequently used to describe the atomic structure of interfaces in metallic alloy and semiconductor polycrystals [Thibault et al., Mat. Sci. Eng. A 164 (1993), 93-100]. An example of stepped tilt boundary is shown in Figure 2b. Assuming that the repeating motif in each grain corresponds to the projection of hollow columns made of βCD-C₁₄ molecules (Figure 2a), the boundary was described with a series of SUs differing by the number of neighboring columns (5, 6 or 7), each of them exhibiting a distinct contrast (Figure 2c) To our knowledge, it is the first time that such grain boundaries are observed in nanoparticles of self-organized amphiphilic molecules and described at the nanometric scale.

We thank Agence Nationale de la Recherche (contract #ANR-11-BS10-0007) and Institut de Chimie Moléculaire de Grenoble for financial support, Cyrille Rochas and the French CRG (BM02 beamline) at ESRF for granting beamtime for SAXS experiments, and Delphine Levilly (DPM) for the synthesis of the βCD-C₁₄ derivative.

Spontaneous assembly of anisometric colloidal particles, such as rod-like particles, in two-dimensions (2D) can be carried out via incubation of colloid-containing suspensions on solid surfaces [1]. Rod-like particles having a high aspect ratio (e.g. very long inoviruses) show liquid crystal (LC) behavior in suspensions. An ordered medium of liquid crystals often possesses a variety of defects, at which the director n(r) of the liquid crystal undergoes an abrupt change. Experimental research on these effects has remained challenging and has not been performed, to our knowledge, on confined rod-like colloidal particles on irregularly structured substrates. Therefore, we studied semi-flexible M13 phages in contact with irregular stranded webs of thin amorphous carbon (a-C) films (Figure 1a,b) using transmission electron microscopy (TEM) and theoretical considerations. In this work, we show that on the structureless and wide amorphous carbon (a-C) surface areas, far from the surface edges, the phages exhibited random orientations (Figure 1c). However, close to the surface edges the orientation of M13 phages in two-dimensional nematic films was controlled by the orientation and curvature of the edges. When constrained to surface strands, the M13 phages adopted a configuration that matched the confining boundary conditions (Figure 2). An annulus sector was superimposed on these oriented phage bundles that allowed us to derive analytic expressions for the bending energy of such oriented bundles. Our theoretical approach provides an explanation for the different number of phages orienting close to the surface edges with different local curvatures. By comparing the self-assembly on differently shaped carbon substrates, it was demonstrated that the alignment of the phages can be controlled by choosing appropriate substrate shapes [2]. This offers a convenient means to fabricate designed structures of orientationally ordered M13 phages. The understanding of such systems opens up new possibilities for defect engineering of liquid crystals, which can be beneficial for applications of liquid crystals in the presence of microscopic surface pores and irregularities.

Acknowledgements

The financial support by the DFG is gratefully acknowledged. The research leading to these results has received funding from the European Union Seventh Framework Programme [FP7/2007-2013] under grant agreement no. 312483 (ESTEEM2). P. M. thanks Dr. M. Bier (MPI-IS, Stuttgart, Germany) for discussions.

References

[1] P. Moghimian, V. Srot, D. Rothenstein, S. J. Facey, L. Harnau, B. Hauer, J. Bill, P. A. van Aken, “Adsorption and Self-Assembly of M13 Phage into Directionally Organized Structures on C and SiO2 Films”, Langmuir, 2014;30:11428-11432.

[2] P. Moghimian, L. Harnau, V. Srot, F. de la Peña, N.F. Bafi, S. J. Facey, P. A. van Aken, “Controlled Self-Assembly of Biomolecular Rods on Structured Substrates”, Soft Matter, 2016; in press, DOI: 10.1039/C6SM00073H.3.

High-resolution fluorescence imaging, combined with the latest labeling techniques available today have yet been able to provide the necessary spatiotemporal resolution to record all cellular processes in live cells. Substituting the bulky fluorescent protein tags (such as GFP) currently used in live-cell applications with much smaller fluorescent dyes that possess superior photophysical characteristics will markedly improve these advanced imaging techniques. Genetic code expansion and bioorthogonal labeling offer, for the first time, a non-invasive way to specifically and directly attach such fluorescent dyes to proteins in live cells. Here we employ this strategy to directly tag α-tubulin in live mammalian cells. By screening different conditions we have optimized the system for quantitative high-resolution recording of microtubules in live cells. We will present data demonstrating the feasibility and efficiency of the approach and will discuss the advantages and limitations of using genetic code expansion for quantitative high-resolution microscopy.

1.     INTRODUCTION

At the center of the DNA damage Fanconi Anemia repair pathway is the FANCD2/FANCI complex, which is monoubiquitinated upon DNA damage, and this monoubiquitination is indispensable for activation of the pathway. However, neither FANCD2 nor FANCI have any recognizable domains, which results in limited understanding of the molecular functions of the complex. In this study, we have taken a structural approach to better understand the function of the human FANCD2/FANCI complex.

2.     RESULTS

Experimental methods The native full-length human FANCD2/FANCI complex and the FANCD2/FANCI complex with the C-terminus FANCD2 truncated  were purified and subjected to cryo-EM analysis. The activity of the complex was studied in vivo and in vitro using various assays.

Results

Here, we provide the first structural insight into the full-length human FANCD2/FANCI complex utilizing cryo-EM. The complex has a pocket-like main body, a thumb-like structure consisting of the C-terminus of FANCI and a distinctive fork-like tower consisting of the C-terminus of FANCD2 (figure). Deletion of the C-terminus of FANCD2 leads to defective DNA interstrand cross-link repair (ICL repair). FANCD2 lacking the C-terminus cannot be monoubiquitunated, while the bona fide monoubiquitination site (Lysine 561) is still present. Furthermore, the association with FANCI and the interaction between FANCD2 and the responsible E3 ligase complex, the FA core complex, remain unaffected in the mutant.  The structure of the FANCD2/FANCI complex with the C-terminus FANCD2 truncated  was also studied using cryo-EM, this truncated domain is located in the fork-like tower (Figure ) 

3.     Conclusion

Conclusion A new domain of FANCD2 was discovered. The fork-like tower domain in the C-terminus of FANCD2 is required for its complete functions in the ICL repair. Interestingly, several disease-causing mutations lie within this region, underscoring the importance of this domain.

 Translational applicability

Our results contribute to uncover the mechanism of how the FANCD2/FANCI complex functions. A better understanding of the molecular mechanism underlying the FA pathway will increase our chances of developing better and more targeted modes of treatment.

Reference

 1- Liang CC, Zhan B, Yoshikawa Y, Haas W, Gygi SP and Cohn MA. UHRF1 Is a Sensor for DNA Interstrand Crosslinks and Recruits FANCD2 to Initiate the Fanconi Anemia Pathway. Cell reports 10, 1947-1956, doi:10.1016/j.celrep.2015.02.053 (2015

Measles a one of the most contagious human diseases. Last year we determined the structure of the helical nucleocapsid formed by the folded domain of the measles virus nucleoprotein encapsidating an RNA at 4.3 Å resolution (Gutsche et al, Science 2015). This structure revealed the molecular determinants of the nucleoprotein packing into a helix and provided first insights into the mode of nucleoprotein-RNA interaction in the nucleocapsid. We will now present our most recent high resolution cryoEM dataobtained with the goal to gain more precise structural information about the rationale of the RNA encapsidation.

Single particle cryo-electron microscopy (cryoEM) is a powerful method for determining the structures of large macromolecules and their complexes. Recent technical advances in direct electron detectors, phase plates and computational procedures for image processing have revolutionized the field, allowing 3D reconstructions to be obtained at near-atomic resolution (<4 Å). However, most monomeric proteins of biomedical interest remain too small (<100 kDa) for cryo-EM analysis.  In this work we propose a new method to overcome this size limitation by fusing a monomeric protein (target) to a homo-oligomeric scaffold protein, whose large size and symmetry facilitates cryoEM analysis. As proof-of-principle, we fused maltose-binding protein (MBP), a 40 kDa monomer, to glutamine synthetase (GS), a dodecamer formed by two hexameric rings. 

We designed an initial MBP-GS construct with a linker sufficiently long to allow the fused moieties to fold properly without mutual steric hindrance. The junction was optimized by progressively deleting linker and/or flanking residues to reduce the spatial separation between the two fusion partners, thereby restricting the relative mobility of the target subunits (Figure A). We bacterially expressed 18 MBP-GS constructs and used biophysical analysis (native poly-acrylamide gel electrophoresis, size exclusion chromatography, dynamic light scattering and differential scanning fluorimetry) to screen for particle compactness and homogeneity. Negative stain EM analysis confirmed the trend observed by the biophysical assays, and both approaches identified the same construct as the most compact and conformationally homogeneous.

Cryo-EM analysis of the most promising construct led to a reconstruction with an overall resolution of 4.2 Å (FSC=0.143 criterion) and a local resolution that varied from ~4 Å within the GS subunit to between 6 and 10 Å within the MBP subunit.  Fitting the GS crystal structure into this map revealed an excellent fit, allowing all secondary structure elements and certain large side chains to be visualized.  A good fit was also observed for MBP: the N- and C-terminal lobes enclosing the active site were clearly defined, several helical elements were well resolved, and the bound maltose ligand was partly visible (Figure B). These findings illustrate the feasibility of using homo-oligomeric scaffolds to enable cryo-EM analysis of monomeric proteins, raising the prospect of applying this strategy to more challenging structures resistant to crystallographic and NMR analysis.

DNA origami has received great attention in recent years due to its accessible self-assembly, versatility and potential for single-molecule biophysical studies. Origami is often imaged in TEM. Monolayer-thin graphene may provide the ultimate substrate for high-resolution imaging of DNA origami. However, little is known about interaction of DNA origami and graphene. Here, we report on microscopic observations of the interaction between graphene and DNA origami using transmission electron microscopy (TEM) and atomic force microscopy (AFM). We designed a symmetric origami structure that can be well recognized in imaging, which consists of different features like cavities in the middle, DNA bundles on the side arms and a free dsDNA loop at the bottom (Fig1.a). Fig1.c shows AFM images of these origami structures on highly oriented pyrolytic graphite (HOPG) substrates. The origami structures are observed to be severely distorted upon adsorption onto graphite whereas normal, undistorted adsorption is seen on mica substrates (Fig1.b). Interesting, origami exhibits excellent spreading and no severe distortions on amorphous carbon film (Fig1.d). It thus appears from Fig1.b-d that DNA origami is particularly distorted on graphite. Next, we investigated these origami structures with TEM experiments on CVD-grown graphene. We obtained images with uranyl-stained DNA origami, but interestingly also could obtain good contrast with unstained DNA origami if we employed a focused beam in STEM mode, or with dark-field microscopy with the stop of central beam (known as DF-000 technique) – see Fig2.b-d.  The imaging of unstained DNA origami is promising for future applications where possible side effects due to staining with uranyl needs to be avoided. TEM results again showed disruption of the DNA origami structures, suggesting that this is inherent to the interaction between DNA and graphene layer. These distortions may be due to DNA dehybridization caused by favorable interactions between the hydrophobic DNA bases and graphene.

The membrane targeting and insertion of proteins is facilitated by a conserved machinery that acts concomitantly with translation. Nascent proteins are recognized by the signal recognition particle (SRP) and transferred to the SRP receptor (SR) on the membrane. A series of conformational rearrangements of SRP and SR coordinates the handover of the nascent chain to the translocon, which inserts proteins into the membrane or facilitates their secretion. Here we used single particle cryo electron microscopy to obtain snapshots of co-translational protein targeting complexes at near atomic resolution [1]. We observe conformational changes of the SRP M domain induced by binding of the nascent chain, which are coupled to rearrangements of the SRP and SR. In addition we reconstructed a near atomic resolution structure of the translocon in complex with a translating ribosome. A detailed analysis of the ribosome interaction sites of SRP and translocon gives novel insight into the handover of the nascent chain to the translocon.

[1] Jomaa A, Boehringer D, Leibundgut M, Ban N. Structures of the E. coli translating ribosome with SRP and its receptor and with the translocon. 2016. Nat Commun. 7:10471

The ribosome is a large molecular machine performing protein synthesis (translation) in all living cells. During the elongation cycle of protein synthesis, new amino acids are iteratively delivered to the ribosome by aminoacylated transfer RNAs (tRNAs) and incorporated into a growing polypeptide chain, directed by the decoding of mRNA codons. In prokaryotes, this process is catalyzed by the two universal GTPase elongation factors EF-Tu and EF-G. EF-G is a large GTPase protein made of five domains. It is structurally similar to the ternary complex made by EF-Tu in complex with the incoming tRNA and GTP, with its fourth domain mimicking the anticodon stem-loop of the tRNA recognizing the codon into the ribosomal A-site. Several structures of ribosome complexes with EF-G were recently determined by X-ray or cryo-electron microscopy (cryo-EM), allowing for an accurate understanding of EF-G interactions and conformational changes during translocation. However, all the reported structures were obtained by trapping EF-G into the ribosome, using non hydrolyzable guanosine 5’-triphosphate (GTP) analogs, specific inhibitors (eg fusidic acid, thiostrepton, viomycin or dithyromycin) or a mutated form of EF-G. How EF-G binds to the ribosome in the absence of any inhibitor is still elusive. To obtain insights into the engagement of EF-G into the ribosome in a pre-translocation state, we performed cryo-electron microscopy experiments in the presence of GTP. To avoid extensive release of EF-G from the ribosome we increased the EF-G/ribosome molecular ratio and used a non-productive translation complex (NTC) stalled on a truncated mRNA and therefore unable to trigger GTP hydrolysis. We report a new high resolution structure of the ribosome complexes wtih EF-G, providing new insights into the way EF-G interacts with the decoding center.  

This study analyses the properties of nanohydroxyapatite (nHA) and layered double hydroxide (LDH) materials that can be used as non-viral gene delivery vectors. The former is a biocompatible, non-toxic calcium phosphate mineral which makes it a suitable candidate as a delivery platform¹. On the other hand, LDHs are composed of brucite-like layers with a charge compensating anionic interlayer².
   (Scanning) Transmission electron microscopy ((S)TEM) and Energy Dispersive X-ray Spectroscopy (EDX)   methods were utilised to characterize LDH and nHA nanomaterials and their respective interactions with plasmid DNA structures (pDNA). The current work also characterizes the ultrastructure of mesenchymal stem cells (MSCs), as well as investigating the uptake and localisation of the LDH gene delivery vectors at the nanoscale.
   Fig. 1(a) and Fig. 1(b) present the TEM images of the nHA and LDH particles respectively. The nHA particles are found to have a spherical structure of diameter ~50nm, indicating that nHA is a good candidate for cellular transfection. LDHs present a well-defined hexagonal platelet-like structure of ~100nm, providing promising results towards their application as a gene delivery vector.
   The interaction of nHA and LDH particles with pDNA was also investigated using STEM. Morphological variations are clearly evident in both the pDNA-nHA and pDNA-LDH composites. An annular-like structure is presented in the case of pDNA-nHA, whereas the DNA structures absorb to the surface of the LDH particles, displayed in Fig. 2(a) and Fig. 2(b) respectively.
   The application of STEM to MSCs highlight intracellular features such as membranes and nuclei, as shown in Fig. 3. The complimentary EDX techniques portray evidence of the LDH gene delivery vectors within the cell due to a presence of an Mg, Al and P peak, hence confirming uptake and localisation at the molecular level. The enhanced resolution provided by electron microscopy techniques allows for the study and understanding of cellular biological processes, such as that of pDNA-nanoparticle delivery.

References

[1] Curtin et. al, Adv. Mater. , 2012, 24, 749-754.              
[2] Xue Bi et. al, Pharmaceutics, 2014, 6, 298-332.         

The considerable growth of nano-technologies and nano-sciences requires the implementation of very well adapted characterization tools and methods to combine both a nanometer resolution and a good preservation of the nano-objects. This point is absolutely critical when the nanostructures are made of hydrated and organic compounds. Cryo-electron microscopy (cryo-TEM) made from vitrified thin films has proved to be a method of choice for characterizing the hierarchy of supramolecular assemblies of proteins and hydroxy fatty acids with maximum resolution and minimal damage.

We aim here to present a selection of applications of cryo-TEM showing the characterization of dispersions of organic or hybrid organic/inorganic nanoparticles with a large range of sizes, shapes and composition (synthetic polymers, polysaccharides, proteins, lipids).

In particular, we will detail how cryo-TEM helped to elucidate the supramolecular self-assembling of fatty acids through a multiscale approach. The unique benefits as well as the limitations of cryo-TEM will be discussed and compared with conventional SEM and TEM techniques as well as with liquid AFM.

References

Gaillard C. and Douliez JP. Cryo-TEM and AFM for the characterization of vesicle-like nanoparticle dispersions and self-assembled supramolecular fatty-acid-based structures: a few examples,   in: A. Méndez-Vilas. (Ed.) Current microscopy contributions to advances in science and technology. 2012, v. 5, 912-922

F. Dutertre, C. Gaillard, C. Chassenieux, T. Nicolai, Branched Wormlike Micelles Formed by Self-Assembled Comblike Amphiphilic Copolyelectrolytes, Macromolecules, 2015; 48(20): DOI:10.1021/acs.macromol.5b01503

H.T.P. Nguyen, E. Allard-Vannier, C. Gaillard, I. Eddaoudi, L. Miloudi, M. Sourcé, I. Chourpa, E. Munnier "On the interaction of alginate-based core-shell nanocarriers with keratinocytes in vitro" Colloids and Surfaces B: Biointerfaces, 2016, accepté

Fameau, A.-L.; Saint-Jalmes, A.; Cousin, F.; Houinsou Houssou, B.; Novales, B.; Navailles, L.; Nallet, F.; Gaillard, C.; Boué, F.; Douliez, J.-P., 2011. Smart Foams: Switching Reversibly between Ultrastable and Unstable Foams. Angewandte Chemie International Edition, 50 (36): 8264-8269.

Douliez J.P. and Gaillard C., Self-assembly of fatty acids: from foams to protocell vesicles, New Journal of Chemistry, 2014, 38, 5142-5148

Worldwide occurrence of multidrug-resistant pathogenic bacteria has increased interest in alternative treatments including bacteriophage-based therapy. Bacteriophage phi812 belongs to genus Twort-like virus, subfamily Spounavirinae and can infect at least 75% of Methicilin-resitant S. aureus strains (MRSA) and 95% of Methicillin-sensitive S. aureus strains. We have employed cryo-electron microscopy to determine structure and  genome delivery mechanism for polyvalent staphylococcal backteriophage phi812-K1. Phi812-K1 has a 90 nm diameter isometric head and 240 nm long contractile tail ended by a double layered baseplate. The tail and baseplate of the native phage are dynamic. Therefore, a divide-and-conquer strategy was employed to separately determine the cryo-EM reconstructions of the individual phage parts. The structure of the icosahedral head could be refined to 5.0 Å resolution and additional sub-averaging within the T=16 icosahedral asymmetric unit allowed determination of the major capsid protein to 3.8 Å resolution. The structures of the native tail and baseplate were solved to 8 Å and 12 Å resolution, respectively. In order to examine the mechanism of the infection process, we determined the structure of the phage in the contracted state. The phage head is not altered after the DNA ejection. However, both the baseplate and tail undergo large reorganizations documented in their 6 Å and 8 Å resolution structures. Comparison of the tail and baseplate structures in the native and contracted conformation allowed us to determine the changes  accompanying cell wall recognition and binding which is then followed by injection of the bacteriophage genome into the host bacteria.

The application of the transmission electron microscope (TEM) in essence may be regarded as one of the first instruments used to visualize molecular structures at nano-analysis level inside biological cells, although not always recognized as such. These biological structures ranged over the decades since 1939 from membranes and many other inclusions specific of cell morphology to virus structures. Different preparation techniques on cells led to different observations and conclusions. This can vary between negative staining, e.g. extracted DNA, potassium permanganate (oxidative) or a double fixation technique (reductive and oxidative), dehydration, embedding, sectioning and a variety of heavy metal staining techniques. The identification and interpretation of cellular components thus solely depend on such specific techniques. Any technique of the aforementioned is known and daily applied in electron microscopy laboratories, therefore no specific references are presented.

            Many published TEM images pertaining to specifically yeast (Ascomycetes) research, show structures in cells that were not described in either results or discussions [1]. The latest research on fermentative yeast cells highlighted the occurrence of gas in the cell cytoplasm appearing as bubbles (Fig. 1 and Fig. 2). Using TEM only, similar structures were observed in the past in yeast cells [2] but never defined, nonetheless as gas bubbles. In almost all cases the absence of membranes around such structures led many researchers either to ignore it, probably because studies covered other cell phenomena, or confusingly and incorrectly defined as lipids. The formation of gas (carbon dioxide) in yeast cells is important in the brewing and bakers industry, e.g. for the perfect leavening of bread. Therefore the importance of studies to understand gas formation, retention and release by yeast cells may help in developing improved yeast strains.

            Scanning electron microscope (SEM) techniques, however, cannot reveal internal structures of cells sufficiently. Nano scanning Auger microscopy (NanoSAM), a nano-analysis technique not typically developed for biological material, was applied in an attempt to clarify this phenomena of prominent membrane-less structures in fermenting yeast cells [3]. The NanoSAM has the additional advantage over SEM to blast the cells with an argon-gun. This process allows controlled erosion of cells and can reveal internal components such as gas bubbles. Elemental analysis can also be done at nano-levels.

            The exact same set of samples can afterwards be transferred to a high resolution field emission gun SEM to examine cell structure of bubbles in detail (Fig. 3). NanoSAM analysis of nano-areas in the exposed areas of the cells was performed to reveal chemical composition to determine the presence of zinc (Fig. 4). Zinc was added to the growth medium, which reacts to carbonic acid in cells related to fermentation gas release. Compared to TEM examination of these bubble-like structures the distribution, size and appearance correspond well to observations with this novel application of the NanoSAM in biology. Comparatively, lipids are not expected to appear as vacant structures as gas bubbles observed after argon gun erosion in the NanoSAM.

            Conclusively, this review emphasizes the importance to search and find applications such as NanoSAM with typical applications in physics research, which can then be used to reveal missing links with current and past research in other fields such as biology. Furthermore, it can also be emphasized that many areas now uncovered, can lead to other hidden phenomena in biological cells above and beyond the yeast group. Other techniques such as time-of-flight secondary ion mass spectrometry (TOF-SIMS), is also able to provide elemental, chemical state, and molecular information not only from surfaces of solid materials but from biological organisms as well. Current research in this field has already been started by this research group.

References

[1] Wilson, C.L. et al (1970). Phytopathology 60, 216-227.

[2] Van der Walt, J.P. and von Arx (1985). South African Journal of Science 81, 156-159.

[3] Swart, C.W. et al (2012). FEMS Yeast Research 12, 867-869.

Acknowledgement

The Authors wish to thank you University of the Free State (UFS) and the National Research Foundation (NRF) of South Africa for funding the project. Ms. H. Grobler, Laboratory Manager of the Laboratory for Microscopy for SEM and Dr. E. Coetzee-Hugo, Department of Physics, UFS, for NanoSAM analysis.

Chromatin in cell nuclei is formed by DNA associated with nucleosomes which consist of the core histone proteins (H2A, H2B, H3, H4). These play a crucial role in genome organization and are well-known regulators of a variety of biological processes occurring at the DNA template such as transcriptional activity. The histone subtype H3 exists in several varieties. Whereas most varieties are synthesized during S-phase of the cell cycle, the variant H3.3 is expressed throughout interphase and has been associated with transcriptionally active chromatin [1]. These differences suggest diverging roles of these variants in chromatin organization and function.

Here, we expressed histone H3 variants in normal and malignant cells and compared the nuclear expression patterns at different resolution levels using conventional epifluorescence and confocal microscopy, structured illumination imaging (SIM) and electron microscopy (Fig. 1).

We observed differing distribution patterns of the histone H3 variants within interphase cell nuclei (Fig. 2). In addition, we found distinct banding patterns after expression of histone H3.3 in mitotic chromosomes. Together this indicates differences in functions of these chromatin constituents in the interphase nucleus [2]. Surprisingly, the highly transcribed ribosomal genes within nucleoli were not labelled with histone H3.3 suggesting a difference in nucleosomal architecture or nucleosomal dynamics in active ribosomal genes to genes transcribed into mRNA molecules. In summary, we could demonstrate that histone H3 variants show diverging expression patterns in interphase nuclei and in mitotic chromosomes.

References: 1. K. Ahmad, S. Henikoff, Mol. Cell., 9, 2002; 2. L. Snyers et al., Nucleus, 5, 2014.

Introduction: Human amniotic membrane (AM) is the innermost layer of placenta and is composed of three distinct strata, namely single epithelial layer, a thick basement membrane and avascular stroma. AM has been widely adopted as a biomaterial in tissue engineering and surgical reconstructions, since it promotes wound healing and epithelialization, has low immunogenicity, reduces inflammation and scarring, and was also shown to possess anti-cancer properties (1). We have shown that AM promotes formation of highly differentiated urothelium, in particular when urothelial cells are cultured on AM stromal side (2). Although AM stroma scaffold allows establishment of differentiated urothelium, its orientation probably does not favour AM integration into surrounding tissue, if applied in vivo. Therefore, we established an in vitro AM model which would allow incorporation of fibroblasts into the AM stroma as well as promote urothelial differentiation.

Materials and Methods: Porcine bladder fibroblasts were seeded onto porous membranes and were labelled with green lipophilic dye when subconfluent. Cryopreserved human AMs mounted into Scaffadex holders were de-epithelized and laid onto cultures of fibroblasts in such a manner that fibroblasts were in close contact with the AM stromal layer. Fibroblasts were left to grow into AM stroma in medium adapted for fibroblasts for up to a week, when porcine urothelial cells were seeded on the opposite side of the AM scaffold i.e. on the AM basement membrane. The constructs were maintained for three additional weeks in the medium adapted for urothelial cells. As a control, urothelial cells seeded on the de-epithelized AM scaffold without integrated fibroblasts were used to evaluate contribution of fibroblasts on urothelial differentiation. Growth of urothelial cells was monitored daily. Integration of fibroblasts and differentiation of urothelial cells was determined by electron microscopy, histological and immunofluorescence techniques.

Results: Fibroblasts cultured in close contact with AM stroma in the medium adapted for fibroblasts integrated into AM stroma and remained viable after three additional weeks of maintenance in the medium for urothelial cells. Proliferation and differentiation of urothelial cells depended largely on the presence of fibroblasts. In comparison to the control, urothelial cells on fibroblast enriched AM scaffolds reached confluency faster, and more importantly, reached higher differentiation stage (Figure 1).

Discussion and Conclusion: Our data demonstrate that bladder fibroblast enriched AM scaffold promotes urothelial differentiation in vitro. Besides being suitable in vitro research tool to study epithelial-mesenchymal interactions, this model also indicates that de-epithelized human AM could integrate into bladder lamina propria and could be as such potentially used as a graft for urothelial reconstruction.

References

1. A. C. Mamede et al., Amniotic membrane: from structure and functions to clinical applications. Cell Tissue Res 349, 447, 2012.

2. U.D. Jerman et al., Amniotic membrane scaffolds enable the development of tissue-engineered urothelium with molecular and ultrastructural properties comparable to that of native urothelium. Tissue Eng Part C Methods 20, 317, 2014.

Use of FIB-SEM to address the ultrastructure of the periplastidial and internal chloroplast compartments of the diatom Phaeodactylum tricornutum

 Serena Flori¹, Pierre-Henri Jouneau², Benoit Gallet³, Christine Moriscot³, Leandro Estrozi³, Dimitris Petroutsos¹, Cécile Breyton³, Guy Schoehn³, Eric Maréchal¹, Giovanni Finazzi¹ and Denis Falconet¹

¹Laboratoire de Physiologie Cellulaire et Végétale; UMR5168 CNRS / Univ. Grenoble Alpes / CEA / UMR1417 INRA; Institut de Biosciences et Biotechnologies de Grenoble (BIG), France.

²Laboratoire d'Etudes des Matériaux par Microscopie Avancée (LEMMA); Institut Nanosciences et Cryogénie; Service de Physique des matériaux et Microstructures; CEA-Grenoble, France.

³UMR5075, Institut de Biologie Structurale (IBS), Grenoble, France.

 Focused Ion Beam Scanning Electron Microscope (FIB-SEM) is a state-of-the-art advanced scanning electron microscope integrated with high-resolution focused ion beam milling that enables photographic, chemical, and structural analysis of many inorganic and organic samples. Among different features FIB-SEM allows 3D tomography with nanometer-scale resolution thus becoming an instrument of choice for obtaining 3D information at the EM level.

In this study, FIB tomography has been realized in a Zeiss NVision 40 dual-beam microscope to reveal the ultrastructure of the unique chloroplast present in the diatom Phaeodactylum tricornutum, a member of these key ecological players in oceans for CO2 sequestration via photosynthesis. A striking feature of diatoms is their sophisticated ultrastructure, inside highly packed cells, including a chloroplast bounded by four membranes known as 'secondary' plastid when 'primary' plastids as those present in plant cells have two membranes. Although their photosynthetic apparatus is similar to that of plants, photosynthesis takes place in a different plastid environment, where membranes are note differentiated in appressed (grana stacks) and non-appressed regions (stroma lamellae).

Analyze of 4 nm ultrathin sections (figure 1) of disrupted Phaeodactylum tricornutum allows the detection of membrane connectivity in the three dimensions and thus the ultrastructure scanning of single organelle (chloroplast, mitochondrion or nucleus). The inner (iEM) and outer (oEM) envelope membranes of the symbiont's chloroplast together with the two outermost membranes: the chloroplastic endoplasmic reticulum membrane (cERM), which is continuous with the outer nuclear envelope and the periplastidial membrane (PPM) are well defined. Images reveal the presence of a vesicular network (VN) between the oEM and the PPM delineating the remaining of the symbiont cytoplasm and referred as the periplastidial compartment (PPC) (figure 2) [1].

Reconstitution of the three-dimensional architecture of the chloroplast in Phaeodactylum tricornutum using focused ion beam scanning electron microscopy allows proposing a structural model for the arrangement of photosynthetic complexes in this chloroplast. This model challenges the classic view of the organization of the photosynthetic membranes in chloroplasts derived form a secondary endosymbiosis, and accounts for partitioning of absorbed light between the photosystems, without restraining electron flow capacity, as required for optimum photosynthesis (figure3) [2].

[1] Flori et al. Ultrastructure of the periplastidial compartment of the diatom Phaeodactylum tricornutum. Protist (under revision)

[2] Flori et al. In preparation

Acknowledgements SF and GF were supported by a grant from the Marie Curie ITN Accliphot (FP7-PEPOPLE-2012-ITN; 316427). GF, EM and DF were supported by a grant from ANR (ANR-12-BIME-0005 DiaDomOil). EM was supported by a grant from the Investissement d’Avenir Program (Oceanomics).

Cell complexity in prokaryotic cells, whether Bacteria or Archaea, is generally considered low, with a single compartment. A few examples, however, have been recently reported showing evidence for compartmentalization [e.g. 1,2,3]. These studies gained in particular from improved sample preparation methods (plunge freezing or high-pressure freezing), adequate follow-up methods, and 3D imaging of the samples, either by FIB-SEM or by electron tomography. Here we report on our analyses of the ultrastructure of exceptional Archaea of the genera Ignicoccus and Pyrococcus, which show remarkable physiological characteristics (e.g. optimal growth at T~85 up to 100°C, under anoxic conditions), and unusual ultrastructural features. Although prokaryotes, these cells have a diameter of > 1 µm, which does not allow for reasonable cryoTEM observation without sectioning. Thus, samples were harvested by gentle concentration on ultrafiltration membranes, cryo-immobilized by high-pressure freezing, freeze-substituted, and finally resin-embedded; this enabled us to perform 3D ultrastructural analysis by (S)TEM tomography and immuno-localization studies on sections of the same resin block.

So far, we are about to routinely investigate single or serial sections (ranging from 200 up to 800 nm in thickness), on a transmission electron microscope operated as TEM or STEM at 200 keV. For STEM, the illumination is adjusted to have an almost parallel beam, i.e. small convergence angle, resulting in an extended depth of focus [4]. Under these conditions, if the sample is imaged at high tilt angle (e.g. up to 70 deg), all visible image details are seen 'in focus', which greatly facilitates image alignment during 3D image reconstruction using IMOD (Boulder, Co, USA). In addition, the system in use allows parallel acquisition of BF and DF signal. Reconstruction and visualization is done using either WBP or SIRT in IMOD, and final manual segmentation using AMIRA®.

Our 3D data show novel and remarkable ultrastructural features: (A) Ignicoccus cells possess two membranes; in the intermembrane compartment, there is a complex endogenous membrane system. Attached to the outer cellular membrane, we have localized enzyme complexes for proton gradient generation, ATP and acetyl-CoA synthesis, and CO₂ fixation [5]. (B) Pyrococccus cells possess flagella of the archaeal type [6], i.e. extracellular filaments involved in motility and cell-cell interaction, and a cytoplasmic membrane and an S-layer, and a so far unknown layer inside the cytoplasm presumbably involved in anchoring or organizing the flagellar bundle.

[1] R Santarella-Mellwig et al 2013 PLOS Biol 11, e1001565

[2] S Schlimpert et al 2012 Cell 151, 1270

[3] B Junglas et al 2008 Arch Microbiol 190: 395

[4] AA Sousa and RD Leapman 2012 Ultramicroscopy 123, 38

[5] H Huber et al 2012 Antonie van Leeuwenhoek 102, 203

[6] D Näther-Schindler et al 2014 Frontiers Microbiol 5, 695

Mitochondria play an essential metabolic role in all eukaryotic cells because they are the organelles that provide energy to drive chemical reactions. Mitochondria are highly dynamic and change their shape between discrete structures and large interconnected networks by selective fission and fusion of their membranes. The resulting fused three-dimensional networks can permeate through an entire cell, but their advantages, relative to isolated discrete organelles, are not fully understood.  It has been suggested that networks might have the following functions: dampen biochemical fluctuations; provide a mechanism for quality control and selective mitophagy; enable diffusion of proteins; and transmit calcium signals and mitochondrial membrane depolarization throughout the cell [1, 2]. Despite their highly dynamic state, useful structural information can nevertheless be obtained from electron microscopy by studying the organization of mitochondrial networks at a given time point.

We have used a SIGMA VP (Zeiss Inc.) scanning electron microscope (SEM), equipped with a 3View (Gatan Inc.) serial block face (SBF) system [3], to quantify the connectivity of mitochondrial networks in entire insulin-secreting b-cells of mouse pancreatic islets of Langerhans. Previous work has suggested that alterations in mitochondrial fission and fusion might play a role in nutrient-induced b-cell apoptosis with possible involvement in the pathophysiology of Type 2 diabetes [4].

Previously, we found that manual segmentation of SBF-SEM mitochondrial volumes in a single b-cell took about one week for a trained operator, which precluded analysis of multiple cells within a reasonable time.  We have explored a faster and more practicable approach by making use of tools within the Amira visualization software package (FEI Inc.) to partially automate the segmentation process, and enabling an entire cell to be analyzed in approximately two hours (Figure 1). Using this approach we were able to analyze quantitatively mitochondrial networks in individual pancreatic b-cells in terms of the total mitochondrial volume, average volume per network, total network length, and average network length. We found that approximately one-third of the mitochondrial volume was contained in networks of length less than 3 µm, one-third in networks of length between 3 µm and 10 µm, and one-third in highly fused networks of length between 10 µm and 60 µm (Figure 2).

SBF-SEM thus provides quantitative data on the organization of mitochondrial networks, making it feasible to test computational models for mitochondrial fusion and fission.

The authors thank Drs. A.L. Notkins, T. Cai, and H. Xu for providing the specimens of pancreatic islets of Langerhans. This research was supported by the intramural program of the National Institute of Biomedical Imaging and Bioengineering, NIH.

[1]  H. Hoitzing et al, Bioessays 37 (2015) p. 687.

[2]  B. Clancy et al, Nature 523 (2015) p. 617.

[3]  W. Denk and H. Horstmann, PLoS Biol. 2 (2004) p. 1900.

[4]  A.J.A. Molina et al, Diabetes 58 (2009) p. 2303.

[5]  C.R. Pfeifer et al, J. Struct. Biol. 189 (2015) p. 44.

[6]  A. Shomorony et al, J. Microsc. 259 (2015) p. 155.

Cryo-preparation of biological samples for electron microscopy has many advantages against the chemical fixation. Cryo-fixation is extremely rapid which can inhibit, within milliseconds, the intracellular movements of macromolecules and other substances. Due to its rapidity, it may even allow the detection of the processes of contacting between the vesicles and the membrane in the cells. One can try to capture the processes at synapse contact, which would be impossible with much slower chemical fixation.

  The problems may arrive if the biological sample is contacting to the solids for example at the interface of biological sensor. Due to different thermal expansion coefficient of the solid and biological materials, it may lead to unwonted artefacts at the interface area.

  We will present the information about the interface between our structurally modified biosensors and biological cells of different preparation techniques. We have used cryo-fixation, standard chemical fixation with critical point drying and epoxy embedding. For characterisation of the interface, we apply SEM and FIB (FEI Helios NanoLab DualBeam) sectioning at cryo- and room temperatures.

The ways in which cell architecture is modelled to meet cell function is a poorly understood facet of cell biology.  However, the compartmentalization of mutually exclusive reactions in different regions of cells by membrane-enclosed organelles or by self-assembling macromolecular complexes is a basic mechanism of life.  Fluorescent tags can display proteins confined to compartments in living cells, but provides no glimpse of the underlying ultrastructure.  The electron microscope has the resolution to display cellular ultrastructure.  Transmission electron microscopy (TEM) tomography of thin slices (~100-300nm depth) can reveal complex subcellular cellular ultrastructure.  Serial Block Face Scanning Electron Microscopy (SBFSEM) can provide ultrastructural resolution throughout a much longer (μm to mm) sample depth, enabling quantitative analysis of ultrastructural features throughout the length of most complex cells.  

We have studied the cytoarchitecture of a cell with highly specialised organisation, the cochlear inner hair cell (IHC), using multiple hierarchies of 3D electron microscopy analyses.  We have shown that synaptic terminal distribution on the IHC surface correlates with cell shape, and the distribution of a highly organised network of membranes and mitochondria encompassing the infranuclear region of the cell.  Structural linkages between organelles that underlie this organisation were identified by high resolution imaging.  Together these techniques have the potential for clarifying functionally specialised cytoarchitecture of other cell types.  Strategies employed to improve data quality will be discussed. 

Tardigrada (also known as water bears) are microscopic, cosmopolitan invertebrates that are closely related to Arthropods. They are widespread in marine and brackish, freshwater, terrestrial habitats throughout the world, and they can be found in hot springs, Himalaya mountaintops, under the ice layer and even in ocean sediments. These animals are well known due to their unbelievable abilities that allow them to survive in extreme environments (such as temperatures ranging from −272°C to 151°C, pressure, dehydration, radiation,  toxins, outer space, etc.). Although there are many more researches that are focused on tardigrades, this Phylum is still not very well known. Since knowledge about tardigrades has contributed to the discovery of dry vaccines, it seems that the research conducted on these animals is valuable.

In our research we focused on the traditional and modern methods that are used in the analysis of tardigrades. From the traditional methods, we used light microscopy, transmission and scanning electron microscopy as well as histo- and immunohistochemical methods. We also present a new method that is used in invertebrate analysis SBEM (serial block- face scanning electron microscopy), which had not previously been used for analysis of invertebrates. SBEM is a method that generates high resolution three-dimensional (3D) images from the two-dimensional (2D) images that are obtained by scanning the block-face of an analysed sample. It was invented by Winfried Denk in 2004 to analyse the connectivity of the axons in the human brain. In this method, a microscope that connects the scanning electron microscopy, transmission electron microscopy and microtome into one tool is used.

Part of this work was supported by research grant from Polish National Science Centre. Contract grant number: UMO-2014/15/N/NZ4/04350.

The rosaceae is one of the major family of flowering plants with 3200 species in 115 genera. The genus Rosa encompasses approximately 190 species and more than 18 000 cultivar forms of the plant have been identified [1]. The species Rosa damascena is well known for its intensely fragrant flowers. In the cosmetic field, essential oils and waxes are two major components which are extracted from the rose flower. Rose essential oils are very often used in fine fragrances whereas waxes can be used as active components to play a major role in the skin physiology.

Rose petals’ surfaces have already been studied by conventional SEM technologies. The rose scent compounds are emitted by both epidermal layers [2]. Residues of secretory substances have already been observed on both surfaces [2,3]. The wax components are localized in the cuticle on the surface of the epidermal cells. The cuticle of floral organs presents a micro-relief defined as cuticular patterns [4]. Although the shape of epidermal cells is variable between both the petal rose faces, the cuticle presents some common patterns which results in parallel folds. Circular wax patterns were also observed on the abaxial surface [2,3,5].

The aim of our study was principally to characterize more precisely the wax aspect at the surface of petals of the Rosa damascena. Wax microstructures are fragile and to avoid sample preparation artifacts we decided to work on fresh hydrated specimens and innovate by mixing cryomethods with different observation modes in SEM.

In a first approach we manually plunged the petal sample into liquid nitrogen and transferred it into the SEM chamber equipped with Peltier freezing stage. The surface analysis using SE and BSE detectors in variable pressure mode allowed us to distinguish two shapes of epidermal cells on the abaxial face. Both are elliptical but one of them shows globular structures (Figure 1). This latter shape would appear to be associated with circular wax models.  In a second time, the cryofixation was carried out with a semi-automated plunge freezing device. The specimen was etched, coated and then transferred from the cryo preparation chamber onto a SEM-FEG cryo-stage. High resolution imaging with an in-lens SE detector at low voltage permitted us to analyse the extreme surface of petals and observe the structure of the rose petal waxes.

We confirmed by this study the interest of mixing cryomethods to preserve the rose petal wax patterns as close as their native state. In addition using numerous and complementary detectors in different vacuum modes allowed us to collect new information on the wax aspect at the surface of petals of the Rosa damascena. The optimizing protocols of cryomethods can also be an opportunity to have a better understanding of the scent molecules release from the epidermal cells though the cuticle. Our first observations in this direction are encouraging.

References

[1] Mohammad Hossein Boskabady, Mohammad Naser Shafei, Zahra Saberi and Somayeh Amini. 2011. Pharmacological Effects of Rosa Damascena. Iran J Basic Med Sci. Vol. 14(4): 295–307.

[2] V. Bergougnoux  J.C. Caissard,  F. Jullien,  Jean-Louis Magnard, Gabriel Scalliet,  Jeremy M. Cock, Philippe Hugueney, Sylvie Baudino. 2007. Both the adaxial and abaxial epidermal layers of the rose petal emit volatile scent compounds. Planta. Vol. 226 (4): 853-866.

[3] Aneta Sulborska, Elżbieta Weryszko-Chmielewska, Mirosława Chwil. 2012.Micromorphology of Rosa rugosa Thunb. petal epidermis secreting fragrant substances. Acta Agrobotanica. Vol 65 (4): 21-28.

[4] Rea L. Antoniou Kourounioti, Leah R. Band, John A. Fozard, Anthony Hampstead, Anna Lovrics, Edwige Moyroud, Silvia Vignolini, John R. King, Oliver E. Jensen, Beverley J. Glover. 2013. Buckling as an origin of ordered cuticular patterns in flower petals. Journal of the royal society. Vol. 10 (80).

[5] Gaurav Sharma, M. L. Sharma , S. K. Sharma, and H. P. S. Kang. 2005. Single-Step Surface Replication of Flower Petals for SEM. Microscopy and Analysis, 19 (1): 21-23.

High-resolution structural visualization of animal organs at the micro- and submicrometer scale is essential for functional, comparative and developmental studies. Histology is the gold standard to examine microstructures in stained thin sections of animal organs using optical and electron microscopies. However, due to limitations intrinsic to these imaging techniques, the sample preparation protocol is usually very time-consuming, the images obtained are two-dimensional and they only exhibit structures of small sections of the organs imaged.  

Recent works have shown that the chemical staining of animal organs is a good strategy to increase the low inherent contrast of soft tissues in X-ray absorption micro-computed tomography (microCT).[1,2] However, it is repeatedly described in several works in this area that the slow and inhomogeneous diffusion of the staining agent into the samples can limit the use of stains in microCT. To overcome this issue, long staining times consisting of one or more weeks are used,[2,3] as well as highly concentrated staining solutions, that can cause sample shrinkage. 

We have recently made some significant improvements to a well known staining protocol applied to soft-tissue samples and non-distorted three-dimensional (3D) structural information of entire animal organs were easily accessed.[4] The images obtained with a bench top microCT scanner reveal rich morphological information of the whole organs analysed. Moreover, with our protocol, the chemical agents that increase the contrast were able to reach small structures and we have demonstrated that organs structures and some cells types can be discriminated in the microCT images obtained. As an example, the digital mid-sagittal section of a mouse brain stained with our protocol (Fig. 1) reveals brain regions including the cerebellum (Cb); the thalamus (Th); the hippocampus (Hp); the striatum (Str), the corpus callosum (cc) and the neocortex (NCx). In a higher-resolution measurement of the cerebellum (Fig. 2), the stratum moleculare (a); and the stratum granulosum (b) are discriminated. Moreover, we have detected microstructures of tens of mm in diameter, which due to their location, size, shape and density are presumably the Purkinje cells (Fig. 2, arrows). In a higher-resolution measurement of the brain (Fig. 3), the thalamus; the hypothalamus; the striatum, the corpus callosum and the neocortex are seen in an even more detailed fashion, and the cells organization in the corpus callosum is much more evident.  

By selecting the appropriate agent or even by combining different staining agents, we are able to improve the contrast in selected areas and a few distinct structures could then be discriminated in one entire mouse brain using microCT. These images demonstrate that staining allied to microCT is a promising strategy to increase the contrast of features of the same order of magnitude of axons in the corpus callosum and and Purkinje cells in the cerebellum, thus allowing the reconstruction in 3D of the structural  organization of some specific cells of interest and therefore being a complementary technique to histology in functional, comparative and developmental studies.

_____________

[1] Metscher, B. D. Biological applications of X-ray microtomography: imaging micro-anatomy, molecular expression and organismal
diversity. Microsc. Anal. 27, 13–16 (2013).

[2] Aslanidi, O. V. et al. Application of micro-computed tomography with iodine staining to cardiac imaging, segmentation and computational model development. IEEE Trans. Med. Imaging 32, 8–17 (2013). 

[3] Cox, P. G. & Faulkes, C. G. Digital dissection of the masticatory muscles of the naked mole-rat. PeerJ 2, e448 (2014). 

[4] Martins de Souza e Silva, J. et. al. Three-dimensional non-destructive soft-tissue visualization with X-ray staining micro-tomography. Scientific Reports 5, Article number: 14088 (2015).

Background. The existence of nanobacteria as a separate systematic unit is still under the doubt (1, 2). Nanobacteria are characterized as mycoplasmas and as L-transformed forms, too (3).

The aim of current investigations was to evaluate the possibilities of formation of "nanostructure - nanobacteria" in overgrowth bacterial cultures (death phase of growth) and in the presence of antibiotics.

Material and methods. E. coli1257 and S. flexnerii130 were used in these investigations to follow the changes in bacterial membrane structures in death phase of growth and after the treatment of antibiotics. Bacteria were grown anaerobically in LB medium (48 hours, 37C^o), solidified with 1.8% agar when necessary. The following antibiotics were used: tetracycline (15 µg/ml), doxycycline (15 µg/ml), amoxicillin  (25 µg/ml), ampicillin (35 µg/ml), cefoxitin (50 µg/ml), kanamycin (50 µg/ml), gentamicin (50 µg/ml), chloramphenicol (30 µg/ml), and streptomycin (50µg/ml).

Transmission (TEM) electron microscopy technique was used to evaluate the changes in bacterial membrane structures.

Results. The formation of spherical granules in 40-100nm surrounding by membrane structures of S. flexnerii in growth death phase (Fig. 1) and formation of outgrowths on E. coli cell walls with different length during the ampicillin-treatment (Fig. 2) were detected in these studies. These formations could be discussed as a basis for the development of nanobacteria under the specific conditions of environment.

References.

[1]. Robert J.C, Mc Lean, Brenda L. Kirland (2014) Nanostructure and Nanobacteria.p.1-10, Nanomicrobiology: Physological and Environmental CharacteristicsL\ Eds. Lary Barton, Demis Bazilski, Huifang Xu. 125p. DOI 10.1007/978-14939-16672-1

[2]. Jan Martel; Hsin-Hsin Peng; David Young; Cheng-Yeu Wu; John D Young. Of nanobacteria, nanoparticles, biofilms and their role in health and disease: facts, fancy and future.Nanomedicine. 2014;9(4):483-499.

[3]. VD Timakov, G.YA Kagan (1973) L-form bacteria and bacteria family Mycoplas-mataceae pathology. Ed. "Medicine". Moscow, 385C.

Biofilm is a major problem in the food industry produced by pathogenic microorganisms, because adhered cells to biotic or abiotic surfaces are more resistant against antimicrobials and disinfectants than planktonic cells.The retention of bacteria on food contact surfaces is an important  in food processing, catering and the domestic environments. Salmonella species and Listeria monocytogenes are the most important food-borne pathogenic bacteria. In this study, L. monocytogenesis and Salmonella species isolated from several food sources in the region of Eskisehir were screened for their biofilm forming ability on the stainless steel material and biofilm development stages were examined with scanning electron microscopy (SEM)

In our study, L.monocytogenesisveSalmonella spp isolates were identified by standard microbiological procedures. The biofilm forming capabilities of isolates were tested by microtiter plate and tube methods. Moderate and strong degree of biofilm-forming organisms on the stainless stain surfaces were taken into electron microscopic procedures. They were examined on 2, 4, 6, 24 ve 48^thhours for their structure of the biofilm and imaged by SEM (JEOL JSM-5600LV).

Stainles steel materials are  commonly used surfaces in food production, storage and processing processes.L.monocytogenesis and Salmonella spp. have been shown to adhere to stainless steel surfaces. According to our results, biofilm formation has started from 24^th  hour.  Isolation of theese microorganisms from variety foods showed that these foods can be easily infected with bacteria at any stages of food production. It carries risks for public health.

Acknowledgment: This work was supported by a grant from Eskisehir Osmangazi University (Project Number:2015-910)

In the living cells, there are many structures and organelles enveloped with a membrane that defines their border and controls the communication between the inner and outer environment. In contrast to membrane bound compartment, cells contain numerous membrane-less structures, whose formation is based on specific interactions among their components. These types of structures include Cajal bodies (CBs), PML bodies and P-Bodies found in most cell types, stress granules that appear upon environmental stress (heat shock, oxidative stress etc.) or various aggregates that form as result of protein/RNA mutations (e.g. protein inclusions in ALS). The biological role of the membrane-less structures is a topic of intensive research and the function of several bodies have been revealed. However, little is known about principles that lead to their formation. As an archetypal compartment, we employed the CB (Fig.1), the self-organizing structure involved in metabolism of various ribonucleoprotein particles. We employed time-gated Stimulated Emission Depletion (gSTED) microscopy to acquire superresolution microscopy images of the CB and surrounding nucleoplasm. We have visualized the CB scaffolding protein coilin using indirect immunofluorescence in situ. Our preliminary data clearly showed the sub-resolution structures of the CB (subCBs). Moreover, the substructures are also visible in the surrounding nucleoplasm and indicate that the CB is an aggregate of those basic building blocks (Fig.2-B). Here, we would like to present our work on a mathematical model based on the fundamental thermodynamic rules of condensation and phase-separation to describe the formation of membrane-less bodies. We will present superresolution fluorescence microscopy, photo-kinetic experiments and fast time-lapse live cell imaging results and progress of the work on determining of basic biophysical behavior of individual CB components. The project is interdisciplinary and combines advanced fluorescence microscopy techniques and mathematical modeling and simulations.

Transmission Electron Microscopy equipped with Energy Dispersive Spectroscopy (TEM-EDS) is known to be a powerful tool to study metal speciation in biological and environmental samples. The characterisation of different metal pools present in organisms is indeed essential to understand optimal development under various environmental constraints. For instance in plant cells, iron (Fe) is known to be an essential co-factor for electron transfer in many biological reactions. Fe remobilization by specific membrane transporters in seeds is especially important for germination.  In Arabidopsis thaliana seeds, the AtVIT1 transporter is involved in Fe influx into perivascular vacuoles whereas the AtNRAMP3 and AtNRAMP4 function in Fe retrieval. The vit1-1 and nramp3nramp4vit1-1 mutants display an altered Fe pattern.

To examine the subcellular localization of Fe and Mn in cotyledons from wild-type, vit1-1 and nramp3nramp4vit1-1 triple mutant dried seeds, we used Energy Dispersive Spectroscopy (EDS) combined with Transmission Electron Microscope (TEM). This technique collects the X-rays spectrum emitted by plant sample sections bombarded with a focused beam of electrons to obtain a localized chemical analysis (Figure 1). A cartography using STEM-EDX data complete the characterization of these mutants and open new questions of the iron localization and chemistry in the triple mutant.

Results (Figure 2) show that in vit1-1 mutant embryos, Fe and Mn were both concentrated in subepidermal cell globoids, whereas Fe is localized in perivascular globoids in wild-type. In the nramp3nramp4vit1-1 triple mutant Fe was detected at low level in globoids from subepidermal and perivascular cells although vit1-1 and nramp3nramp4vit1-1 have the same pattern revealed by Perl’s/DAB staining (1).

To further localize and quantify metal elements such as Fe or Ni in cells, we also developed a CEMOVIS approach which will allow to perform elemental chemical analysis from lyophilized cryosections observed by STEM-EDX.

(1)    Mary V. et al. (2015). Bypassing Iron storage in endodermal vacuoles rescues the iron mobilization defect in the natural resistance associated- macrophage protein3natural resistance associated-macrophage protein4 double mutant. Plant Physiology, 169, 748-760

Permanent progress in electron microscopy (EM) techniques enables us to reveal the finest ultrastructural details of the cell nucleus. Our recent work is focused on the previously unrecognized nucleoplasmic structures composed of phosphatidylinositol 4,5-bisphosphate, so called PIP2 islets. The super-resolution light microscopy enabled us to localize PIP2 islets inside the cell nucleus and reveal their colocalization with several components of Pol II transcription machinery and chromatin. However, only EM provides sufficient resolution to reveal the PIP2 islets ultrastructure and composition via immunostaining.

The usage of appropriate method for EM sample preparation is a crucial step in the whole procedure as PIP2 islets are lipidic structures which are predisposed to extraction or translocations. Here we compare different EM sample preparation techniques which we have optimized for the best ultrastructural preservation and antigenicity retention of PIP2 islets. We have analyzed several chemical fixation and cryoimmobilization approaches followed by various embedding media including both acrylic and epoxy resins. Moreover, different immunolabeling techniques were used such as pre-embedding, on-section labeling, and Tokuyasu method. With all these approaches we were able to observe roundish PIP2 islets (40-100 nm) recognized by anti-PIP2 antibodies. Preliminary results demonstrate that PIP2 islets are stable nuclear structures resistant to aldehyde fixation and extraction during preparation and cutting procedures. Based on the upcoming statistical analysis, the best approach for immunological studies of PIP2 islets will be selected. 

Maytenus obtusifolia Mart. is an abundant species in restingas (sandy coastal plains), mainly in Rio de Janeiro state, Brazil. Populations in restinga of Maricá are gynodioecious formed by female individuals with pistillate flowers and hermaphrodites with perfect flowers. Pistillate flowers bear a developed gynoecium and staminodes. Perfect flowers have gynoecium with varied development and fertile stamens. Hermaphrodite plants have lower fruiting rate than female plants which was associated to the presence of sterile ovules with hypertrophied synergids and egg cell. The aim of this study was to compare fertile and sterile megagametophytes under TEM. Preanthesis buds were collected at restinga of Maricá/RJ, fixed in Karnovsky, post-fixed in osmium tetroxide, embedded in Araldite, sectioned, contrasted and observed in Tecnai Spirit TEM. In fertile ovules of pistillate and perfect flowers, the egg cell presents cytoplasm dense and polarized, with vacuole facing the micropilar pole. Synergids have thin walls with evident fibrillar apparatus; polarized cytoplasm, vacuome facing the chalazal pole; mitochondria; plastids; nucleus; linear RER concentrically arranged; well developed dictyosomes with enlarged borders forming secretory vesicles. The central cell has thin walls with slightly disorganized cellulose fibrils; cytoplasm arranged in the cell periphery; developed vacuome occupying almost the entire cell; polar nucleus; ribosomes; vesicles with associated ribosomes, derived from RER; dictyosomes; oil bodies and mitochondria. In sterile ovules of perfect flowers, synergids undergo a vacuolization process, with subsequent fusion of small vacuoles; nucleus with fragmented or degenerated nuclear envelope; mitochondria and RER with signs of degeneration; organelles debris and plastids. In these hypertrophied synergids and the egg cell, disruption of tonoplast promote the cell lyses. Vacuole membranes and organelles debris are found inside synergids and central cell. Egg cell cytoplasm has fragments of nucleus. This study reveals that female sterility in perfect flowers seems to be caused by a vacuolization process followed by tonoplast disruption suggesting the occurrence of massive autophagy in megagametophyte cells. 

Acknowledgements: We thank CAPES for the PhD Fellowship by the first author; CNPq (Proc. 401053/2016-4) for the grant to S.R. Machado, and the Electron Microscopy Centre (CME) IBB, UNESP and its technicians for lab assistance.

During the last decade importance of phosphoinositides in nucleus was uncovered. We defined previously unknown nuclear structures which occupy nearly 2% of nuclear volume and contain almost 30% of the nuclear pool of phosphatidylinositol 4,5-bisphosphate (PIP2). These non-membranous structures of 40 – 100 nm are decorated by PIP2, so we name them PIP2 islets. PIP2 islets are surrounded by phosphorus and nitrogen, hence proteins and nucleic acids are in their vicinity. We showed that PIP2 on the periphery of the islets is associated with Pol II transcription complex, nuclear myosin 1, chromatin and nascent RNA transcripts. We prove that PIP2 islets contribute to efficient DNA transcription. Further, we were interested in the fine ultrastructure and precise composition of the islets to clarify their role in the nuclear architecture.

PIP2 islets are filled by carbon-rich compounds, however we were not able to identify these molecules by immunolabeling on either resin sections or Tokuyasu sections. The possible reason for this could be the high mobility of the lipids and their extraction during aldehyde fixation. To avoid problem of lipid fixation we are optimizing the method of quick- freeze fracture replica labelling for the interior of the cell nucleus. The samples are fixed physically – by high pressure freezing, and subsequently mechanically – by coating with carbon/platinum layer. This method enables a reliable fixation and immunolabeling of lipids in cellular membranes. The applicability of the method for the study of fine ultrastructure and immunolabeling of intranuclear non-membranous lipidic structures will be discussed.

Acknowledgements: This work was supported by JSPS (15K21738), GACR (15-08738S, 16-03346S), TACR (TE01020118), the Human Frontier Science Program (RGP0017/2013), and by institutional grant (long-term conceptual development of the scientific organization; RVO: 68378050). We acknowledge the Microscopy Centre, IMG AS, Prague, Czech Republic supported by the MEYS CR (LM2015062 Czech-BioImaging).

Euglena gracilis is a unicellular fresh water photosynthetic flagellate at which a bleaching phenomenon was described. Using antibacterial compounds like quinolones and cumarins, the photosynthetic activity may be destroyed and results in irreversible elimination of chloroplasts. Beside the loss of chloroplasts in E. gracilis, damage and ultrastructural transformation of mitochondria leading to a formation of giant mitochondria has been also shown [1,2,3]. In this study, we used serial block face scanning electron microscopy (SBF-SEM) for 3D reconstruction of E.gracilis to see whether the giant mitochondria are inside the bleached non-photosynthetic mutant cells or they were just an artifact/myth caused during specimen preparation.  

For SBF-SEM, biological samples are standardly prepared according the protocol described by Deerinck et al. (2010) In this case, we needed the ultrastructure preservation of our sample as close as possible to the native state, therefore we decided to use high pressure freezing followed by the freeze substitution method. To increase the image contrast in backscatter electron imaging, we added soluble salts containing heavy metals in freeze substitution solutions. We tested four modified protocols in which we used various combinations of the following compounds: osmium tetroxide, thiocarbohydrazide, potassium ferricyanide, lead nitrate, uranyl acetate, phosphotungstic acid.   Resin embedding was done using low viscosity Spurr (EMS) at room temperature.

At first, the ultrathin sections were cut from polymerized blocks using the ultramicrotome (Leica EM UC6).  Sections were examined in transmission electron microscope (TEM, JEOL 1010) and the contrast of mitochondria was compared in recorded images of mutant cells prepared by modified and standard Deerinck protocols (Fig.1). The sample with the highest contrast was roughly trimmed by a razor blade and mounted on the stub using a superglue and conductive colloidal silver paint (EMS). Small pyramids with square block face in size appr. 100x100x100 µm were prepared using a diamond trimming tool (Diatome) or glass knife in the microtome.

The high resolution SEM (Tescan Maia3 XMU FEG) equipped with ultramicrotome (Gatan 3View2XP) in the microscope chamber was used for the collection of images recorded at the accelerating voltages 1.5 and 3.0 kV. More than 1000 slices with the thickness of either 30 or 50 nm were cut from sample pyramids with the oscillating diamond knife working at cutting speed 0.5 mm/s. To prevent the sample charging, the image acquisition was performed at the chamber pressure 50 Pa. Gaussian 3D filter in Image J software was applied to recorded images to reduce a noise. 3D model was created using automatic segmentation tool in the Amira software package.

The resulting 3D reconstructions did not prove the presence of giant mitochondria in the mutant cells. However SBF-SEM in the combination with the current methods of specimen preparation has proven to be the method of choice allowing the study of the distribution of cell organelles and their mutual position in the whole cell volume.

References:

[1] L. Ebringer, J. Polónyi, J. Krajcovic, Arzneimittel-Forschung, 43(7), (1993), 777–81.

[2] J.Polonyi  et al., Folia Microbiol. 43(6), (1998),661-66.

[3] J.Polónyi et al., Zeitschrift für mikroskopisch-anatomische Forschung, 104(1),(1990), 61-78.

[4] The study was supported by the Technology Agency of the Czech Republic (TE01020118) and from the program for large research infrastructures of the Ministry of Education, Youth and Sports within the project „National Infrastructure for Biological and Medicinal Imaging  (Czech-BioImaging LM2015062)

Scanning electron microscope (SEM) is increasingly used for morphology characterization in various research fields. The technique is appealing due to the fact that it has a fairly large field of view (µm to mm) with nm lateral resolution, which makes it suitable for characterization of micrometer scale objects, which have nm scale features, such as bacteria. The major limiting factors for SEM as a visualization tool of bacteria are the sample preparation, which is prone to artefacts, and the sample interaction volume, which limits the lateral resolution. Here, we present an approach for morphology characterization, which involves minimal sample preparation and maximized lateral resolution: the use of environmental SEM (E-SEM) with a scanning transmission electron detector (STEM); wet STEM. We use wet STEM for morphological characterization (identification of cell shape, size and appendices) of two bacteria isolated from a mussel collected in the Solomon Sea (Solomon Islands) during the Danish research expedition Galathea 3: Vibrio galatheae¹ and Photobacterium galatheae².

The bacteria were grown in marine broth in flask cultures. After 24h growth at 25˚C the marine broth was washed with miliQ water and a 0.5 µL droplet placed on a plasma treated carbon film 200 mesh copper grid. The sample was negatively stained with 2 % uranyl acetate aqueous solution and imaged in an FEI Quanta 200 FEG E-SEM with an electron beam with spot 3 accelerated to 15 keV. During electron microscopy the bacterial cells were kept fully hydrated by using water as auxiliary gas and continuously condensing water on the sample. The bacteria were imaged using the transmitted electrons and a STEM detector (a 2 quad, diode back scattered electron detector mounted below the sample).

The wet STEM micrographs revealed that the Vibrio galatheae, has Vibrio monotrichous cells, 1.482±0.365 µm long, 0.743±0.181 µm width and 3.625±0.639 µm long flagellum (Figure 1), and the Photobacterium galatheae has Bacillus monotrichous cells , 2.261±0.404 µm long, 0.985±0.128 µm wide and  4.306±1.094 µm long flagellum (Figure 2).  

References

1. Vibrio galatheae sp. nov., a member of the family Vibrionaceae isolated from a mussel, Sonia Giubergia, Henrique Machado, Ramona Valentina Mateiu and Lone Gram, International Journal of Systematic and Evolutionary Microbiology (2016), 66, 347–352.

2. Photobacterium galatheae sp. nov., a bioactive bacterium isolated from a mussel in the Solomon Sea, Henrique Machado, Sonia Giubergia, Ramona Valentina Mateiu and Lone Gram, International Journal of Systematic and Evolutionary Microbiology (2015), 65, 4503–4507.

Magnetotactic bacteria (MTB) are known to produce single-domain magnetite or greigite crystals within intracellular membrane organelles and to navigate along he Earth’s magnetic field lines. MTB have been suggested as being one of the most ancient biomineralizing metabolisms on the Earth and they represent a fundamental model of intracellular biomineralization. Moreover, the determination of their specific structure and morphology is essential for paleoenvironmental studies. Yet, the mechanisms of MTB biomineralization remain poorly understood, although this process has been extensively studied in several cultured MTB strains in the Proteobacteria phylum. Here, we present a comprehensive TEM study of magnetic and structural properties down to atomic scales on bullet-shaped magnetites produced by the uncultured strain MYR-1 belonging to the Nitrospirae phylum, a deeply branching phylogenetic MTB group. HAADF-STEM imaging and XEDS elemental mapping revealed a phenotypical heterogeneity among MYR-1 cells, Phenotype III produces numerous sulfur-rich globules (fig.1a-c). Electron tomography (ET) demonstrated that the bullet-shaped magnetosomes are organized into 3–5 bundles of chains (fig.1d,e). Off-axis Electron Holography results show that each bundle of MYR-1 chains appears to behave magnetically as a large uniaxial single domain (USD) magnet that maximizes the net magnetic moment of the cell (fig.1f).

We observed a multiple-step crystal growth of MYR-1 magnetite: initial growth forming cubo-octahedral particles (fig.2), subsequent anisotropic growth and a systematic final elongation along [001] direction (fig. 3). During the crystal growth, one major {111} face is developed and preserved at the larger basal end of the crystal (fig. 4.a). The basal {111} face appears to be terminated by a tetrahedral–octahedral-mixed iron surface, suggesting dimensional advantages for binding protein(s), which may template the crystallization of magnetite  (fig. 4b,c). This study offers new insights for understanding magnetite biomineralization within the Nitrospirae phylum.

Reference : J. R. Soc. Interface 201512 20141288; DOI: 10.1098/rsif.2014.1288

            The White-Eared-Opossum (Didelphis albiventris) is a marsupial that occupies a large variety of habitats, spreading through several Brazilian biomes and some urban areas [1], with an important role in seed dissemination. As a mammalian, its tongue evolved to specialized functions and, in the neonate, in addition to milk suckling, it also serves to  keep them attached to the female nipple, as a support for protection within the marsupium, reflecting the success of organogenesis. Furthermore, the lingual papillae provides an indicative of the animal habits, mechanical uses, diet and taxonomic relationships [2].

                        The organs were collected from individuals found dead by the roads in the state of Rio Grande do Sul, southern of Brazil. This material was previously fixed in modified Karnovsky aqueous solution and, once identified the tissue quality, it was submitted to processing for scanning (SEM) and transmission (TEM) electron microscopy.

            Through TEM the epithelial layers that forms the lingual mucosa were easily distinguished and identified, both in adults and fetuses. The stratum corneum at both stages demonstrate the epithelial renewal process through the release of the most superficial cells; the granular layer is more tenuous in fetuses than in adults, where many tonofilaments and several keratohyaline granules were observed; the spinous layer was similar in both ages and marked by the large number of desmossomes between the cells.

            Two adult tongues were used in the SEM study. One was used for the mucosa analysis, and revealed filiform papillae with different morphologies according to the region. The apex is filled with "major" papillae, like acute columns. The space among them is filled with tuft of "minor" thin and flat filiform papillae. This "minor" filiform papillae covers the entire top of the body surface but is much denser than in the apex, giving a velvet appearance to the organ. The fungiform papillae are scattered in the apex and body, and partially covered by an elevation of mucosa, looking like circumvallate papillae, since it present a moat-like trough. Circumvallate papillae are 3 in number and form a triangle on the lingual root, with the apex oriented caudally. Foliate papillae are small and thin elevations in the caudolateral border of the tongue, ranging from 12 to 16.

            The other SEM study used corrosion with Sodium Hydroxide to remove the epithelial layer, reaching the connective tissue that supports the lingual mucosa. It was observed that the filiform papillae subepithelial tissue is similar to what is seen with the conventional technique. The other types of papillae present an irregular surface with some dorsal depressions. The subepithelial tissue of the two circumvallate papillae, located more rostrally, are oval like the epithelium, while the third is rounded.

            The characteristics observed in the tongue of D. albiventris are similar to those observed in other marsupial species.

            This research is supported by the São Paulo Research Foundation (FAPESP. Proc.: 2015/05065-9).

References

[1] Faria-Corrêa M; Vilella FS; Jardim MMA (2007) Biodiversidade 23:356-366.

[2]Okada S; Schraufnagel D (2005) MicroscopyandMicroanalysis11:319-332.

We made multi-scale imager including micro 2P XYZT microscope, macro 1P imager, and on-chip fluorescence imager. We utilized multi-modalities to cover from micro to macro scale for space and time axis, and we revealed thrombotic and inflammatory processes in diseased conditions.

First one is high resolution imaging system based on non-linear optics. Real-time, multi-color XYZT multi-photon imaging enabled us to visualize single platelet behavior, morphological changes, and elucidate thrombus formation in cardiovascular events. Second, macro imaging system for awake mice was developed, and free behavior monitoring revealed the tight association between metabolism and vascular reactions for daily stress.. Fluorescent imaging from body surface using 8K CMOS camera, image intensifier, and macro-lens enabled us to visualize cellular dynamics without anesthesia. Third, wearable and implantable devices for long-time recording were developed using lens-less and on-chip technologies.

We utilized these system with light-manipulation technique, to induce thrombus or inflammation reactions. We induced thrombus formation by photo-chemical reactions in vein, and observed rapidly developing thrombi composed of discoid platelets, and elucidated the novel contributing factor; Endothelial cell disruption by laser irradiations induced inflammation and thrombus formation. Remarkable transient neutrophil accumulation was induced, which was followed by spontaneous cell death and monocyte recruitment. Artery contraction reactions were induced by ROS, and elucidated the dynamics NO/ROS balances.

In sum, we developed multi-scale imaging system which can evaluate the therapeutic strategies against thrombotic and inflammatory processes in adult-common disease.

During the synaptic vesicle fusion at the active zone a fusion pore is formed that results in a formation of an Ω-shape intermediate structure (Ω-profile) at the plasma membrane for releasing contents, followed by closure (called kiss-and-run) or merging of the Ω-profile into the plasma membrane (called full fusion). Ω-profile closure limits vesicular content release and cargo delivery, but recycles vesicles economically. In contrast, Ω-profile merging allows for rapid, complete content release and cargo delivery, but couples exocytosis to classical endocytosis, involving membrane invagination, Ω-profile formation and fission, for retrieving merged vesicles. In other words, Ω-profile merging defines the mode of fusion (full fusion instead of kiss-and-run) and the mode of endocytosis (classical endocytosis instead of kiss-and-run) [1-3]. Despite these fundamental roles, the mechanism underlying Ω-profile merging is unclear in neurons, in which vesicles are less then 50 nm in diameter and fusion takes place rapidly after calcium influx.

Giant presynaptic nerve terminals in lamprey that allow intracellular microinjections of active compounds were used in our experiments to investigate the role of actin dynamics during the synaptic vesicle fusion in synapses. Compounds perturbing the actin dynamics, such as latrunculin A and cytochalasin D, phalloidin and non-dissociable profilin-actin directly tagged with fluorescence or along with a fluorescent carrier to monitor microinjections, were introduced into giant synapses stimulated at 5 Hz and 20 Hz and studied by confocal and electron microscopy. Phalloidin labeling was observed at the synaptic active zone and a large number of omega-shaped membrane invaginations with dimensions corresponded to the size of synaptic vesicles and larger were observed at active zones in synapses microinjected with compounds disrupting actin polymerization as compared to control synapses, injected with dextran or GST and stimulated at the same rate (Fig. 1). In synapses simulated at 20 Hz a larger number of vesicles was observed at active zones at sites of microinjection supporting that SV release was inhibited.

Our results indicate that actin dynamics is involved at stages when synaptic vesicle merges with the active zone and when its membrane relocates to the periactive zone for endocytosis.

[1] A.A. Alabi and R.W.Tsien, Annu. Rev. Physiol 75, 393-422 (2013).

[2] Wu,L.G., Hamid,E., Shin,W., & Chiang,H.C. Exocytosis and endocytosis: modes, functions, and coupling mechanisms. Annu. Rev. Physiol 76, 301-331 (2014).

[3] Jahn,R. & Fasshauer,D. Molecular machines governing exocytosis of synaptic vesicles. Nature 490, 201-207 (2012).

The formation of syncytial groups of germ-line cells (cysts, clusters) is a widespread phenomenon in animal gametogenesis. There are three major types of the spatial organization of germ-line cysts – linear, branched and cysts that have a central cytoplasmic mass (cytoplasmic core). In our studies, we focused on cysts with a central cytoplasm mass because this type of cyst is still poorly understood. We chose the earthworm Dendrobaena veneta as the model organism because it is easy to cultivate and a large number of germ cells clusters can be obtained from a single specimen.

Generally, clustered germ cells are interconnected via broad cell junctions called intercellular bridges or ring canals (Fig. 1, 2 and 3). Ring canals allow the cytoplasm to flow freely from one cell to another in the entire cluster. Ring canals are nothing more than modified contractile rings that do not close during late cytokinesis. The role of ring canals in the proper functioning of germ-cell clusters is best known in such model species as Caenorhabditis elegans, Drosophila melanogaster and Mus musculus, in which any disorders in the construction and functioning of ring canals result in infertility. Not only is the functioning of ring canals well known in the model species but also their structure and molecular composition. Numerous studies have shown that a rich F-actin cytoskeleton is present in the cortical layer of ring canals (Fig. 3) and that it is required to stabilize the bridges in order to keep them unobstructed, which is necessary for correct exchange of the cytoplasm. On the other hand, it is also known that microtubules play an active role in the development and functioning of germ-cell cysts(Fig. 2). Ring canals are places in which F-actin and microtubules cooperate together with other proteins (e.g. anilin) in order to stabilize the cytoplasmic channel and to optimize its functioning.

In clusters with a central cytoplasmic mass (called a cytophore in annelids), each germ cell has only one intercellular bridge that connects it to the cytophore (Fig. 1, 2 and 3). Therefore, the cytophore is an intermediate structure that mediates cytoplasm sharing between clustering cells.

Our previous studies showed that both F-actin (Fig. 3) and microtubules (Fig. 2) are present in D. veneta germ-line clusters. The aim of the presented studies was to investigate the spatial organization of the microtubules within the ring canals of the male germ-cell clusters in D. veneta. To achieve this goal, we used immunofluorescent methods to visualize the F-actin and microtubules and confocal microscopy to collect high-resolution images. Based on the collected images, we reconstructed the cytoskeletal network within the ring canals.

The main objective of the study was modified surface thin film materials deposited on polymer substrate constituting the microenvironment for the uptake and controlled differentiation of stem cells the controlled share caused by residual stress and optimized stiffness of the surface using the plasma methods.

Plasma surface modification method generating a controlled part of residual stress in the coating affect the formation of the surface topography in the form of a nano- wrinkles similar to the niches in the tissue environment where stem cells are stored. Niches like structures can catch stem cells from blood flow and prevent their rapid differentiation. Topography and stiffness of the surface of the coating allow for targeted cellular differentiation. The properly formed full confluence of the endothelial cells effectively inhibits blood clotting processes.

The challenge of the work was to develop the surfaces which would mobilize and activate stem and progenitor cells. The idea was to produce the surface containing the niche like structures, the most appropriate structure for the stem cells. The coatings were developed with controlled residual stresses participation, proper microstructure
(Fig. 1) and mechanical properties. Controlled share of residual stress created nano-wrinkles, similar to the niches where stem cells reside in the natural conditions. The creation of stress generated wrinkles did not lead to delaminating of the coating from the substrate. The coating adhered well to the substrate, and wrinkles provided the cellular microenvironment (Fig. 2). The microstructure and mechanical properties were the key factor that determined the controlled cellular differentiation. The differentiation of stem cells was carried out for endothelial cells formation. The following scientific problems were studied: the anchoring mechanisms of the coating to the polymeric substrate, the effect of ion methods for surface modification towards the microenvironment creation for the stem cells and blood- material interaction.

ACKNOWLEDGMENT

The research was financially supported by the Project no. 2014/13/B/ST8/04287 “Bio-inspired thin film materials with the controlled contribution of the residual stress in terms of the restoration of stem cells microenvironment” of the Polish National Center of Science.

Developmental neurotoxicity (DNT) of environmental chemicals has long been identified as a threat to the health of the human population, as the developing nervous system is particularly susceptible to toxicant exposure. The resulting neurological deficits can have long-term effects on families and society both financially and emotionally. Current DNT testing guidelines involve the use of animal models; primarily rodents. The testing strategy incorporates large numbers of animals, which can be extremely time- and cost-intensive; particularly due to the backlog of chemicals needing to be tested (Lein et al., 2005). This demand, in addition to current and future proposed regulations on the use of animals for testing makes it imperative that new models be found to reduce animal experimentation while providing a suitable method to test new chemicals.

Three-dimensional cell models, which incorporate human neural stem cells (hNSCs) aggregated into neurospheres, have been proposed as a viable alternative for DNT testing. The in vitro system has the ability to recapitulate the processes of brain development, including proliferation, migration, differentiation and apoptosis (Salma et al., 2015). Inclusion of human cells, as opposed to murine, also meets recommendations to circumvent the drawback of species differences between in vivo testing and actual exposure effects.

Here we will demonstrate the use of a 3D neurosphere model, composed of hNSCs, to conduct toxicity testing of potential neurotoxicants. A spheroid microplate was used to create and maintain cells in the 3D model. 3D neurosphere proliferation, multipotency, along with the continued capacity to differentiate into neurons, astrocytes, and oligodendrocytes was initially validated. Neurotoxicity testing was then performed using neurospheres maintained in the 3D spheroid plate. Detection of induced levels of oxidative stress, apoptotic, and necrotic activity within treated neurospheres, compared to negative control spheres, was evaluated. Monitoring of cell proliferation, differentiation, multipotency and experimental testing was performed using a novel cell imaging multi-mode reader. 

Laticifers are latex-producing structures which occur in some plant families. In Moraceae (the mulberry family), laticifers are of the non-articulated and branched type and are distributed throughout the whole plant, i.e., they consist of a very long single cell that branches, forming a continuous network throughout the plant tissues. Given that the laticifer wall of Moraceae species is under continuing expansion and consists mainly of pectin and cellulose, the action of pectinases and cellulases must be of great importance in the laticifer development. Thus, this work aimed to verify the subcellular producing sites of pectinases and cellulases and their role in the laticifer development in two Moraceae species, using as a tool cytochemical analysis associated with transmission electron microscopy. Reproductive meristems of Maclura tinctoria (L.) D.Don ex Steud. and Ficus montana Burm.f. were collected, fixed in Karnovsky's solution and stored in 0.1 M phosphate buffer (pH 7.2) at 4°C. For pectinase detection the samples were incubated in 0.1 M sodium acetate buffer (pH 5.0) containing 0.5% of pectin for 20 min. at room temperature. To detect cellulase the samples were incubated in 0.05 M citrate buffer (pH 4.8) containing 0.02% of carboxymethylcellulose for 10 min. at room temperature. The materials from both treatments were transferred to Benedict's reagent heated to 80°C for 10 min., and then washed in 0.1 M phosphate buffer and postfixed in 1% osmium tetroxide for 2 h at 4°C. The samples were washed in distilled water, dehydrated, embedded in Araldite resin and sectioned at ca. 70 nm thick. The obtained sections were stained with 2% uranyl acetate for 25 min and Reynolds lead citrate for 5 min. and observed in a Jeol 100CX II transmission electron microscope. The cytolocalization controls of pectinases and cellulases followed the same methods but without the incubation of the samples in pectin and carboxymethylcellulose, respectively. Pectinases and cellulases were found in regions of the laticifer wall close to the middle lamella in both species (Figures 1 and 2). The pectinases should act on the dissolution of the middle lamella, enabling intrusive apical growth of laticifers through the plant tissues. The occurrence of pectinases is expected for the development of non-articulated laticifers but it is the first record of cellulase presence in this type of laticifer. Cellulases have been reported in laticifers of the articulated type, in which they operate in the degradation of the end cell walls. In the case of the non-articulated laticifers like those of Moraceae, it is likely that cellulases act on the partial disassembly of cellulosic components of the wall near to the middle lamella facilitating the intrusive growth. Both pectinases and cellulases are synthesized in the endoplasmic reticulum (Figure 3) and transported to the cell wall by exocytosis or stored in the vacuole (Figure 4) of Maclura tinctoria and Ficus montana. The production of these enzymes in the reticulum has been recorded for the secretory cavities of Citrus; however, this is the first study in which were identified the organelles involved in the synthesis of pectinases and cellulases in laticifers.

Acknowledgments: São Paulo Research Foundation – Fapesp (process numbers: 2013/21794-5 and 2014/07453-3).

Triple negative breast cancer (TNBC) is the most aggressive and lethal subtype of breast cancer due to a higher rate of early recurrence, distant metastases and also heterogeneity in the molecular levels. Thus, there is a urgent need for effective therapies. In the recent years, PARP inhibitors have drawn considerable attention for especially BRCA-associated sporadic TNBC patients. In the present study, we aimed to determine the cytotoxic and apoptotic effects of BMN 673, which is a novel and highly potent PARP inhibitor,  on TNBC cell line.

Triple-negative BRCA1-defective HCC1937 breast cancer cell line was treated with BMN673 and the cytoxoxicity effects was evaluated by WST-1 analysis. The apoptotic effects was evaluated using Annexin V-propidium iodide (PI) and cell cycle analysis. The double stained with acridine orange/ethidium bromide of these cells was observed under the fluorescence microscope.

Initial cytotoxicity screening showed that BMN673 displayed the anti-proliferative effect, with in time (6-12 days) and dose-dependent (0.01, 0.1, 1 and 10 nM) (p<0.05). The maximum inhibition rate was measured 19.54%, 24.21%, 49.91%  and 58.0% at 0.01, 0.1, 1 and 10 nM of BMN673 at 12 days, respectively (p<0.05). There was an increase of apoptotic cell death and cell cycle progression in all concentrations compared to the untreated control. Treatment with doses of 0.01, 0.1, 1 and 10 nM BMN673 significantly induced total apoptotic cells (6.2%, 23.45%, 47.95% and 61.28%, respectively) and a significant block at the G₂/M phase. Additionally, we observed a loss of membrane integrity, chromatin condensation and increasing vacuole formation in HCC1937 cells when treated with the maximum concentration of BMN673.

In conlusion, this study has revealed that BMN673 inhibits cell proliferation and  induces cell cycle progression and apoptosis within the minimum concentration. Thus, BMN673 could represent a potentially therapeutic strategy for especially, BRCA-defective TNBC.

Diabetes is a chronic metabolic disease which lasts for the whole life. Cancer is the second cause of death in the world, according to World Health Organization data. Association of diabetes with cancer is a major health concern. Diabetes and cancer is a serious metabolic disorder with many functional and structural complications as well as having a significant impact both directly and indirectly on all systems (1). Prostate cancer has a great importance for male morbidity and mortality observed both in our country and also in the globe. It is at the second rank among cancer-related mortality cases. Prostate cancer can be determined as the alteration of the balance between cell proliferation and cell death in the prostate which causes a malign increase of the organ volume. Dunning prostate cancer model is formed by subcutaneous injection of strongly metastatic MAT-Lylu cells in a Copenhagen rats (2). Experimental diabetes model is widely induced by streptozotocin (STZ). Metformin is a drug that used for the treatment of type 2 diabetes. Besides, the studies related to reduce the risk of cancer of the metformin have recently drawn attention (3). The aim of this study is to investigate the role of metformin on testicular damage in diabetic+prostate cancer model.

Male Copenhagen rats were divided into three groups: 1) Control group: % 0.9 physiological saline was received during 14 days, 2) Diabetic+ cancer group: 2x10⁴ Mat-LyLu cells were received after injection of 65 mg/kg STZ. 3) Diabetic+ cancer+metformin group: metformin was received 250 mg/kg during experimental period, following injection of STZ and inocculation of Mat-LyLu cells. At the end of the experimental period (day 14) testes tissues were taken. Tissues were stained with hematoxylin and eosin and periodic acid-Schiff reaction and determined the degree of histopathological damage. The degree of histopathological damage in the seminiferous tubules were evaluated as: normal, regressive, degenerative and atrophic (4). Apoptotic cells in testes tissue were detected with TUNEL reaction. Biochemically, serum glucose, glutathione, malondialdehyde, prostate specific antigen levels and testis protein carbonyl levels and myeloperoxidase, xanthine oxidase activities were determined.

Testes tissue of the control group presented a normal testicular morphology and regular seminiferous tubules. The histopathological damage score of testicular tissue was significantly increased in diabetic+cancer group compared to control group. The number of regressive and degenerative tubules in diabetic+cancer+metformin group was decreased by metformin treatment. TUNEL positive cells were observed in all groups. The total number of TUNEL positive cells throughout the testes was increased in diabetic+cancer+metformin group compared to diabetic+cancer group. According to biochemical data, serum glutathione levels were decreased in diabetic+cancer group compared to control group. Serum glucose, malondialdehyde, prostate specific antigen levels, and testis protein carbonyl levels and myeloperoxidase and xanthine oxidase activities were increased in diabetic+cancer group. Treatment with metformin reversed these effects.

It was indicated that metformin has been shown to be protective against testicular damage in diabetic male rats (5). It was has been reported that metformin was used as protective agent to prevent high-fat diet induced testicular damage. Metformin inhibits the growth of cancer cell lines which suggests that it also has an inhibitory effect on cancer progression (6). Our results suggest that administration of metformin prevents the testicular damage by ameliorating the oxidative stress parameters and tissue damage. In conclusion, we can say that metformin has a potential protective effect on the testes tissue in diabetes and Dunning prostate cancer model.

Acknowledgements

This study was supported by a Research Found from Istanbul University, Project no: 48418.

References

1. Giovannucci et al. CA Cancer J Clin. 2010, 60 (4): 207–21.

2. Michaud et al. Ther Adv Urol 2015, 7(6):378-87.

3. Tao et al.. Int J Mol Sci 2013, 14: 24603-24618.

4. Hess et al. J Androl 1988, 9: 327- 342.

5. Mahmoud et al. J Vet Sci 2014, 9 (4): 277-284.

6. Sahra et al. Cancer Res 2010, 70: 2465–2475.

Skeletal muscle is usually composed of four types of muscle cells (I, IIA, IIX, IIB) varying on their speed of contraction (slow: type I; fast: type II) and their metabolism pathway of glycogen (oxidative: type I and IIA; glycolytic: Type IIX and IIB). Depending on the anatomical position and muscle function, the proportion of these different muscle fiber types is variable. The different fibers types are identified longstanding by histoenzymology. ATPase activity in type I fibers is labile at alkali pH and resistant at acidic pH. In contrast, the ATPase activity of type II fibers is alkali resistant and acid labile (For review, see Pette & Staron, 1990, 2000, Schiaffino & Reggiani, 2011). The molecular basis of this typology resides in the polymorphism of myosin heavy chains (MyHC). The use of monoclonal antibodies against MyHC isoforms allows to identify precisely the type I IIA IIX and IIB fibers and hybrid fibers expressing simultaneously different isoforms of myosin (Schiaffino & Reggiani, 2011).

Our goal was to characterise the fiber type of porcine masseter muscle.

Materiel and methodes

Ten 6 months old pigs (105-115 Kg) were slaughtered in a commercial abattoir and masseter muscles (jaw) were extracted from the head at 30 min postmortem. Muscle sample (1X1X1.5 cm) were frozen in cooled isopentane (−160 °C). Serial cross-sections (10 µm thick, cryostat Microm, HM 560) were collected on glass. Fiber types were identified by histoenzymology both by revealing ATPase activity after acidic incubation, and SDH activity that reflect oxidative metabolism. Fiber types were also identified by immunohistofluorescence using three monoclonal antibodies specific to MyHC isoform (BAD5 specific for type I, S58 H2 all except type IIa, and BF35 all types except type IIb and IIx) (Schiaffino & Reggiani, 2011). Histological sections were observed on a photonic microscope (Olympus BX 61) coupled to a high resolution digital camera (Olympus DP 71) and the Cell F software. The percentage of each fiber type was calculated according to Meunier et al. (2010) using the image analysis Visilog 6.7 Professional Software.

Results and discussion

ATPase histoenzymology (Fig.1) revealed only type I (30.6%) and IIA (69.4%) oxidative fibers according to Ström & Holm, 1997. Immunohistochemistry (Fig.2) revealed that 17% of the masseter fibers are hybrid fibers containing two MyHC isoforms IIa and IIx (Fig.3). These hybrid fibers are generally a transitional step to move from a pure type to another (Schiaffino & Reggiani, 2011). In this study, no pure type IIX is highlighted, suggesting that the transition is not completed or that the IIA-IIX hybrids are the final stage of transition in this muscle.

For a given muscle, fiber type evolves mainly with physical activity and age of the animals. In our case, pigs were reared in a building without noticeable change in their physical activity. The pigs were slaughtered at the age of 6 months, which corresponds to a period of entry into sexual maturity. It is possible that the fibers are in transition classes, however, no study has highlighted the presence of IIX fibers in pork masseter, suggesting that the hybrid could be permanent. A large proportion of IIA-IIX fibers (74 %) were found in rat masseter but several pure glycolytic fibers were detected (Pette & Staron, 1990) and unique HCIIm isoforms were identified in primate and carnivore masseter muscles (Pette & Staron, 1990, 2000). All these data highlight the special feature of masseter compared to other skeletal muscles.  

Conclusion

The use of monoclonal antibodies revealed the presence of a high proportion of hybrid fibers IIA-IIX in pork masseter and did not allow the detection of any pure type IIX. These results suggest a specificity of porcine masseter muscle in which the hybrid type IIA-IIX would not be a transitory state.

References

Pette, D., & Staron, R. S. (2000). Microscopy Research and Technique. 50, 500–509.

Pette, D. & Staron, R. S. (1990). Reviews in Physiology, Biochemistry and Pharmacology. 116, 2-76.

Ström, D. & Holm, S. (1997). Journal of oral rehabilitation. 24, 389-400.

Schiaffino, S. & Reggiani, C. (2011). Physiol. Rev. 91, 1447–1531.

Meunier, B., Picard, B., Astruc, T., Labas, R. (2010). Histochem. Cell. Biol. 134, 307-317.

The Investigation of Effect of Carvacrol on Biofilm positive Salmonella spp and Listeria monocytogenes isolates

İlknur  Dağ*,  BükayYenice Gürsu, Gökhan Dikmen,

¹ Eskişehir Osmangazi University; Central Research Laboratory Application and Research Center (ARUM)  26480, Odunpazarı, Eskişehir-Turkey

idag280@gmail.com

Salmonella spp. and Listeria monocytogenes are important pathogenic bacteria, which are transmitted by food. It is known that both microorganisms may produce biofilm on biotic or abiotic surfaces. An adherence of pathogenic microorganisms to food contact surfaces and biofilm formation is dangerous with respect to food security. Because biofilms provide several advantages to their members including such as protection from antibiotics, disinfectans and chemicals.

Recently, alternative strategies or more effective agents exhibiting activity against biofilm-producing microorganisms are of great interest. Researchs focused on effects on plant materials and essential oils. It is reported that the Carvacrol [2-methyl-5-(1-methylethyl)phenol] exhibited strong antimicrobial activity and it is one of the phenolic components of thyme. The aims of this study were (i) to investigate the biofilm existing of Salmonella ve L. monocytogenesis obtained from variety food samples, (ii) to extend the research to evalute the antimicrobial activity of carvacrol on planctonic cells of biofilm positive Salmonella spp ve L. monocytogenesis strains by microbiological and electronmicroscopic methods.

Among the 603 food samples taken for analysis, a total of 6 samples were confirmed positive L. monocytogenes; 8 samples were confirmed positive for Salmonella spp. Isolates were identified by standard microbiological procedures. Biofilm detection was evaluated by microtiter plate assay and tube method. In addition, biofilm production and developments were also investigated on granit surfaces by Scanning electron microscope. All tested Salmonella spp and L. monocytogenes isolates produced biofilm. The minimum inhibitory concentration (MIC) of carvacrol was determined using the broth microdilution method according to M7-A8 of the Clinical Laboratory Standards Institute. Carvacrol was introduced into Mueller Hinton Broth at a concentration of 0.0015, 0.003%, 0.006%, 0.013%, 0.025%, 0.05%, 0.1%, 0.2%, 0.4% and 0.8% (vol/vol) to determine the minimum inhibitory concentration (MIC) and the maximal tolerated concentration (MTC) for each isolates evaluated. Ampicillin was used as a standard drug against E. coli ATCC 25922. MIC results showed a MIC ⩽ 0.025% (vol/vol) for all isolates tested. For Scanning and Transmission electron microscopic studies, isolates were also exposured to the carvacrol at concentrations of  2xMIC, MIC and ½ MIC and results were compared with the control. According to our results, carvacrol showed a high antibacterial potential with very low MIC values on planktonic cells of Salmonella spp and L. monocytogenes. Electronmicroscopically, no growth was observed for the MIC value and a  concentrations higher; cellular damage was also determined at sub MIC concentrations.

Key words: Carvacrol, Salmonella spp, Listeria monocytogenesis, electron microscope

Acknowledgment

This work was supported by a grant from Eskisehir Osmangazi University (Project number  2015-910).

Intracellular aggregation of abnormally phosphorylated tau in neurofibrillary tangles (NFTs) is a major neuropathological hallmark of Tauopathies such as Alzheimer’s disease. Tau phosphorylation is controlled by the homeostasis of glycogen synthase kinase-3β (GSK-3β) and protein phosphatase-2A (PP2A). Okadaic acid (OKA) is a potent inhibitor of PP2A, leading to abnormal tau phosphorylation. Brain-derived neurotrophic factor (BDNF) is a member of the neurotrophin family and is selectively downregulated in AD brain. In this study, we investigated the effects of tau phosphorylation on secreted and cellular BDNF levels in primary cortical neurons. Primary cortical neurons were prepared from embryonic day 16 (E16) Sprague-Dawley rat embryos. After 7 days in vitro, neurons were treated with 25 nM OKA for inducing tau hyperphosphorylation. Tau phosphorylation was assessed by Western blot using antibody against phospho-Thr231 and non-phosphorylated tau protein was detected with the Tau-1 antibody. Levels of BDNF secreted to the culture medium were determined by ELISA at the 4th, 8th and 24th hours of treatment. Cellular localization and the protein expression of BNDF were determined by immunofluorescent labeling and fluorescent intensity measurements.

Our results show that after 8 hours of OKA treatment, tau phosphorylation at Thr231 increased, whereas Tau-1 signal decreased (p<0.0001) Compared with the control groups, BDNF levels in the OKA treated group were significantly lower after 4 and 24 hours of treatment (p<0.0001) but were not significantly different at 8 hours of treatment (p>0.05). While prominent BDNF immunoreactivity was seen in cytoplasm and neurites of the neurons in control groups, BDNF immunoreactivity significantly decreased in the OKA treated group (p<0.0001) and this attenuation was significant especially at neurites.

Our results suggested that decreased BDNF protein levels might depend on the defects in axonal transport as a result of disrupted microtubule structure caused by tau hyperphosphorylation.

Iron (Fe) is an essential micronutrient operating in several important processes. It is found in high concentrations in mangrove sediment, and in contaminated areas, the level of this element may increase substantially. Mangrove vascular plants have strategies to deal with the high concentration of this metal, involving Fe-plaque formation on the surface of the root, metal retention in the epidermis and endoderm cells, and compartmentalization in the vacuoles. Some methods have been used for the intracellular localization of iron, such as electron spectroscopic imaging and energy-dispersive X-ray analysis. Perl’s reagent has been traditionally used to detect iron in tissue plants by light microscopy; however, its use in ultrastructural studies has been limited to animal cells. This study aimed to assess whether the Perl’s reagent can be used successfully in plant material under TEM. Samples of Rhizophora mangle L. (Rhizophoraceae) mature leaves were incubated for 24 h at 4^oC in a fixative solution containing 3% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.3). Then, materials were immersed for 30 min at 25^oC in Perl’s reagent (1% potassium ferrocyanide and 2% HCl, 1v/1v). Samples were rinsed using a sucrose-cacodylate buffer, postfixed for 1 h at 4^oC with 1% O_sO₄ (0.1 M cacodylate buffer pH 7.3), dehydrated in alcohol series and propylene oxide, and embedded in Spurr’s resin. Thin sections (80 nm) were cut with a diamond knife, and stained with uranyl acetate and lead citrate, and viewed with a Tecnai Spirit TEM. Sections without contrast were analyzed for enhanced identification of the reaction. Dense iron deposits, cuboid or irregular in shape, were detected in the peripheral cytoplasm and in cell walls (Fig. 1). The use of this method was able to show intracellular sites of iron compartmentalization in plant cells, and indicates the apoplastic and symplastic routes for the iron movement throughout the leaf tissues.

Acknowledgements: We thank FAPESP (Proc. 2014/06870-0) for financial support; CAPES for the PhD Fellowship by the first author;  CNPq (Proc. 401053/2016-4 ) for the grant to S.R. Machado, and the Electron Microscopy Centre (CME) IBB, UNESP and its technicians for  lab assistance.

In plants, ions fluxes are involved in several physiological processes such as nutrient uptake, signaling pathways, cell elongation and stomata movements. However, little information is available about the intracellular anionic concentrations and anion fluxes among plant cell compartments. In the last years some promising anion concentration sensors have been developed (ClopHensor, Arosio et al. 2010) but have never been used in plant cells. The ClopHensor sensor (Figure 1) is a genetically encoded non-FRET based sensor developed in mammals cells. It is based on a modified GFP (E²GFP) that is sensitive to chloride and nitrate concentration, and coupled to a DsRed. The DsRed fluorescent protein is used as an internal control for the expression of the construction, because its fluorescence is insensitive to anions concentration and pH, and clearly separated in term of spectral properties. The E²GFP fluorescent protein, excited at 488 nm, is sensitive to pH and anions concentration. But when it excited at 458 nm, the fluorescent signal is only sensitive to anion concentration. Thus, we are able to obtain simultaneously the intracellular pH and anion concentration maps in the cytosol by calculating ratios of fluorescence emission intensity. To do that we used a scanning confocal microscope equipped with an argon laser which provides these laser lines.

In the present collaboration work, Arabidopsis thaliana transgenic lines stably expressing ClopHensor in the cytosol were generated by Elsa Demes (phD student). We are currently performing the in vivo characterization of the ClopHensor sensor in Arabidopsis cells. Indeed, we are establishing the relationship between the fluorescent signal, and pH or anion concentration values, by generating in cellulo calibration curves. This work will lead to the creation of both pH and anions maps to study those parameters at the cell level.

Background. Beneficial effects of probiotic Nissle 1917 on the host health have been shown in a range of investigations(1). During our previous studies the positive effects of E. coli 2-1 on human and animals organism have also been described(2), and the liquid-crystal state of cell walls from the E. coli cells was shown by the x-ray diffraction method under the small and large angles (Fig. 1).

Taking into account that solid-liquid interfaces could have a considerable effect on host bacterial physiology(3) we aimed to investigate the cell surface hydrophobicities of probiotic strains E. coli M-17 and E. coli 2-1 strains.

Methods. The probiotic strains E. coli 2-1 from the probiotic formulation ASAP (Armenia) and E. coli M-17 from the probiotic formulation Colibacteron (Armenia) were used during these investigations. The twenty-four gut commensal E. coli strains from the four healthy volunteers were used as controls. The cell surface hydrophobicities of the E. coli strains were determined by a MATH test according to Koss and coauthors(4 ), and the crystallization levels of cell walls (35 % water suspensions) from these strains were assessed semi-quantitatively at 25'C with polarizing microscopy MIN-8 with Bertrán's prism and increase x 300. The determination of the sizes of structural units carried out by a standard way with the help an eyepiece - a micrometer; the +150W halogen lamp was a light source.

Results. Comparative MATH test experiments show that both investigated probiotic strains’ cells surfaces are more hydrophobic than the bacterial surfaces of strains from the normal gut microbiota. At the same time, the low crystallization levels of cell wall proteins for probiotic E. coli strains were described. Most probably, the comparative hydrophobic structure of bacterial membranes from probiotic strains regulates “probiotic-characteristics” of E. coli M-17 and E. coli 2-1 strains.

References.

[1]. Sylvia K, et al. Ed. B. A. McCormick.Infection and Immunity 82.5 (2014): 1801–1812. PMC. Web. 19 Mar. 2016.

[2]. Balayan M, et al. International Journal of Biological and Medical Sciences. 2010 (68), 670-675.

[3]. De Wouters T, et al.2015; 10(8):e0136437. Epub 2015 Aug 21.

[4]. Kos, et al. J. Appl. Microbiol. 2003;94:981-987.

Inwardly-rectifying potassium (Kir) channels regulate membrane electrical excitability and K+ transport in many cell types where they control such diverse processes as heart rate, vascular tone, insulin secretion and salt/fluid balance. The physiological importance of eukaryotic Kir channels is highlighted by the fact that genetically-inherited defects in Kir channels are responsible for a number of human diseases such as in Andersen’s syndrome (Kir2.1), Bartter syndrome (Kir1.1), and neonatal diabetes (Kir6.2). To date, the available treatment is unfortunately not rational but rather empirical and this is mostly due to the lack of knowledge about atomic structure of these channels.

To elucidate how channel function becomes defective in the disease state requires a detailed understanding of channel structure in both the open and closed states. We have reported the structure of a homologous bacterian KirBac3.1 potassium channel with an open bundle crossing indicating a mechanism of channel gating determined by X-ray crystallography at 3Å resolution. In this model, the rotational twist of the cytoplasmic domain is coupled to opening of the bundle-crossing gate via a network of inter- and intra-subunit interactions [1,2]. In addition, we have also used EM analysis of 2D crystals of the same Kirbac channel trapped in an open state and compared these results with the 3D structure [3].   

We are now focusing in characterizing the structural determinants correlated to malfunctioning behind Andersen mutants forms. We are therefore studying the structure of the human potassium channel kir2.1.The full-length human Kir2.1 (50 kDa the monomer, 200kDa the tetrameric functional form) was over expressed in yeast Pichia Pastoris and subjected to various method for its characterization.

Here we show preliminary results on the expression, the purification and the imaging of negatively stained isolated kir 2.1 in detergent. The particles have an averaged size of 12 nm consistent with a tetrameric form of the native channel. The oligomeric organisation is supported by native gel electrophoresis and Dynamic Light Scattering measurements.

Our results will hopefully contribute to uncover the mechanism of clinically-relevant disease-causing mutations on the structure, dynamics, and function of kir2.1 potassium channels and at investigating the potential of pharmaceutical correctors.

References

[1] Bavro VN, De Zorzi R, Schmidt MR, Muniz JR, Zubcevic L, Sansom MS, Vénien-Bryan C, Tucker SJ  Nature Structural & Molecular Biology Structure of a KirBac potassium channel with an open bundle crossing indicates a mechanism of channel gating 7, 158–163 (2012).

[2] Zubcevic L, Bavro VN, Muniz JR, Schmidt MR, Wang S, De Zorzi R, Venien-Bryan C, Sansom MS, Nichols CG, Tucker SJ. J Biol Chem. 2014 Jan 3;289(1):143-51. Control of KirBac3.1 Potassium Channel Gating at the Interface between Cytoplasmic Domains.

 [3] De Zorzi R., Nicholson WV, Guigner JM, Erne-Brand F, Vénien- Bryan C Growth of large and highly ordered 2D crystals of a K+ channel, structural role of lipidic environement. Biophys J. 2013;105(2):398-408

Thanks to their unique physico-chemical properties, ZnO nanoparticles are widely used in consumer and industrial products, due to their higher chemical reactivity, stronger oxidation and corrosion resistance, antimicrobial properties, as compared with larger micro-sized counterparts (Madhumitha et al., 2016). Recent studies have shown that ZnO nanoparticles can be promising candidates for biomedical applications and therapeutic interventions, and also successful as drug carrier and in targeted gene delivery (Peng et al., 2015; Velmurugan et al.,2015). In our previous in vitro study, ZnO nanoparticles showed to induce oxidative stress in human colon carcinoma cells (LoVo), resulting in significant decrease of cell viability (De Berardis et al., 2010).

In order to gain insight into the mechanism of action at subcellular level, aim of the present investigation was to carry out an ultrastructural study by transmission electron microscopy (TEM) on the subcellular localization of ZnO nanoparticles and a semi-quantitative analysis of cellular uptake at multiple time points (from a few minutes up to 24 h of exposure). Electron microscopy observations of ZnO treated cells revealed two different mechanisms of cellular uptake, passive diffusion and endocytosis. Control cells show a mitochondria and nuclear normal shape (Fig. 1A). Small particles entry by passive diffusion crossing the plasma membrane without altering its structure (30 min of treatment, Fig. 1B; arrow indicates the nanoparticle in the cell membrane area). After 1h of treatment  ZnO nanoparticles are already visible in the mitochondria cristae (Fig. 1C). The induction of the apoptosis is clearly showed in Fig. 1D, after 24 h of treatment. Quantitative analysis of cell death has been performed by flow cytometry.

We also evaluated the intracellular ions release from ZnO nanoparticles, their genotoxic potential by determining 7,8-dihydro-8-oxo-deoxyguanosine (8-oxodG) levels, and the expression of phosphorylated histone H2AX (γ-H2AX). The simultaneous presence of ZnO nanoparticles and Zn²⁺ ions in the LoVo cells determined the formation of reactive oxygen species at the mitochondrial and nuclear level, inducing severe DNA damage.

In conclusion, our observations showed that ZnO nanoparticles entered LoVo cells  by either passive diffusion or endocytosis or  both, depending on the agglomeration state of the nanomaterial. ZnO nanoparticles coming into contact with acid pH of lysosomes altered organelles structure, resulting in the release of Zn²⁺ ions. Taken together, the results of this study provide the evidence that damage induced by ZnO nanoparticles in LoVo cells derives from a combined action between intact nanoparticles and Zn²⁺ ions, leading new insights into their toxicity.

References

Madhumitha, G.,et al., 2016. Biotechnological aspects of ZnO nanoparticles: overview on synthesis and its applications. Appl. Microbiol. Biotechnol. 100, 571-581.

Peng, H., et al., 2015. ultifunctional β-CD-modified Fe3O4@ZnO:Er(3+),Yb(3+) nanocarrier for antitumor drug delivery and microwave-triggered drug release.Mater. Sci. Eng. C Mater. Biol. Appl. 46, 253-263.

Velmurugan, P., et al., 2015. Phytofabrication of bioinspired zinc oxide nanocrystals for biomedical application. Artif. Cells Nanomed. Biotechnol. 27, 1-8.

De Berardis, B., et al., 2010. Exposure to ZnO nanoparticles induces oxidative stress and cytotoxicity in human Ccolon carcinoma cells. Toxicol. Appl. Pharmacol.  246, 116-127.

Ectodermally derived gut and epidermal cells in crustaceans are covered by cuticle, a chitinous apical extracellular matrix, and at the opposite side they are underlined by a basal lamina. Thus, the same cell performs synthesis and spatiotemporal control of elaboration of two extracellular matrices that differ in ultrastructure, composition and functions. Differentiation of the extracellular matrix in epithelial tissues is an integrative part of the establishment and maintenance of the characteristic apicobasal polarized cell architecture. We present here the ultrastructural description of the hindgut cells' apical matrix differentiation during embryonic and larval development of crustacean Porcellio scaber. In addition, in the same developmental stages the ultrastructure of basal lamina was described.

The ultrastructural hallmarks of the hindgut cells and their extracellular matrices (ECM) in adults are: (i) apical ECM is a chitinous cuticle, consisting of distinct horizontal layers - electron dense epicuticle and electron lucent procuticle (Fig. 1), (ii) extensive apical and basal plasma membrane infoldings, forming prominent membrane labyrinths, (iii) basal lamina that is forming an extensive interface surface to hemolymph space (Fig. 2). The ultrastructure of cuticle differs in the two anatomic regions of the hindgut, namely the anterior chamber and the papillate region. In the anterior chamber the epicuticle is much thinner than procuticle and lamellae are discernible in the procuticle due to helicoidally arranged chitin protein fibers (Fig. 1). In papillate region epicuticle and procuticle are of approximately equal thicknesses and no lamellae could be discerned in the procuticle.

Differentiation of the hindgut cuticular matrix during embryonic and larval development involves a sequential formation and shedding of several apical matrices, which gradually display ultrastructural features characteristic for adults. The outline of the pre-cuticluar apical matrix in embryos is ruffled and consists of homogenous lucent material and a distal electron dense lamina. In late embryos of stage 18 the first deposition of the apical cuticle was evidenced. In marsupial larvae the cuticle follows the outline of apical hindgut surface and is already differentiated into thin electron dense epicuticle and thicker electron lucent procuticle. In the epicuticle a trilayered surface lamina and electron dense material beneath are discernible (Fig. 3). The procuticle is homogenous along the whole length of the hindgut and non-lamellate as is characteristic in anterior chamber of adults. The basal side of the hindgut epithelium of marsupial larvae is in contact with basal lamina (Fig. 4) exhibiting fibrillary structure in certain section profiles.

Non-alcoholic fatty liver (NAFLD) occurs as simple hepato-steatosis and its evolution into severe non-alcoholic steatohepatitis (NASH) is strictly connected to obesity, but the pathogenesis is still obscure. Recently, a multiple hit theory suggested that from gut and adipose tissue different adipokines and inflammatory markers influenced the liver where steatosis, collagen deposition and inflammation might occur. Sirtuin 1 (Sirt1) is a NAD-dependent enzyme involved in longevity and in the regulation of multiple cellular functions and metabolism. Here we hypothesized that Sirt 1 down-regulation might exacerbate liver damage and mitochondrial lesions in hepatocytes in dietary-induced obesity in mice. To best analyse these events, we studied male C57BL/6 mice (WT) and Sirt 1 deficient heterozygous mice (HET) (by an established colony in CSIC generated from founders from Dr David Sinclair laboratory, Harvard University, Boston, USA). Animals housed in calorimetric rooms on a 12h: 12h light/dark cycle were placed for 16 wks, from 12 to 28 wks of age, on a standard rodent diet (8% fat -13.0 kJ/g) or on a high fat diet (HFAT- 35% lard-18.8kJ/g Tekland Research Diet–TD03584). Body weight was monitored at the beginning, at 3wks and at 16 wks before euthanasia. Livers were fixed in 4% paraformaldehyde overnight for histopathology, immunohistochemistry or in Karnowsky mixture (4% paraformaldehyde and 1% glutaraldehyde) then post-fixed in 1% osmium tetroxide for transmission electron microscopy analysis (TEM). By H&E, Masson trichrome and Sirius red staining at bright and polarized microscopy significant increase in steatosis and fibrosis in HET mice received HFAT diet vs WT fed HFAT diet were detected (Fig.1 and Fig.2). Interstitial collagen type I, type III, and basal membrane associated collagen type IV fibres were detected in pericentral vascular wall, intermediate lobular area and in thickened sinusoidal wall at light microscopy and at TEM (Fig.3). In contrast, WT or HET animals fed standard diet (as control groups) displayed negligible liver changes. Remarkably, HET mice fed HFAT diet showed abnormal giant mitochondria and activated Kupffer cells, strongly positive to Perls Prussian Blue iron staining, that were lacking in other groups. Finally, in HET obese mice myofibroblasts immersed in an abundant extracellular matrix and fibrous debris were observed by TEM (Fig.4). To highlight the crucial role of oxidative damage and caspase 1 complex in the progression to NASH, we analysed 4HNE and pro-caspase 1 expressions. Remarkably, strong 4HNE and moderate caspase 1 signals were present in pericentral and midzonal hepatocytes in HET HFAT diet mice but weak in WT HFAT diet group and absent in controls. All these data suggest that Sirt 1 is critical to limit fibrogenesis and inflammation associated to irreversible NASH in HFAT diet induced obesity in mice.

Acknowledgements

New Pet Food Italia Srl generously supports this study. Authors thank Mr G. Bozzoni and Mrs S. Castrezzati for expert TEM sample preparation and trimming.

Articular cartilage is located at the bone ends in synovial joints and enables smooth and pain-free joint motion. Its function is in large part determined by a network of collagen fibrils organized into zones and compartments, but little is known about how this organization is achieved in growing animals [1]. The articular cartilage develops superficially to a layer of temporary growth cartilage. The growth cartilage is responsible for the expansion of the bone ends and is replaced by bone during growth. The interface between the immature articular cartilage and the growth cartilage is histologically indistinguishable [2]. On the other hand, aligned collagen fibrils encapsulate cells in the articular cartilage but not in the growth cartilage, so the immature articular cartilage and the growth cartilage can be distinguished by locating these fibrils with transmission electron microscopy (TEM) in growing animals [1]. However, TEM requires extensive sample preparation and has limited field of view.

Second harmonic generation (SHG) microscopy is a promising image modality that is highly specific to collagen fibrils without any staining [3]. SHG is a coherent nonlinear optical process that is specific for non-centrosymmetric structures such as collagen fibrils and is characterized by frequency-doubling of light. In contrast to fluorescence, the SHG intensity and radiation direction is highly sensitive to the arrangement of collagen fibrils. In addition, as the arrangement of fibrils can vary at different length scales, this sensitiveness can depend on the size of the focal volume.

The aim of this study was to examine if the aligned fibrils encapsulating cells could be visualized by SHG microscopy without any staining in order to locate the immature articular cartilage of a growing animal.

The cartilage located at the distal femur of growing piglets was imaged by detecting the forward and backward directed SHG. Focusing objectives with different numerical aperture (NA), i.e. focal volume, were used to examine the collagen fibrils at different length scales. The results demonstrated that the ratio of forward to backward SHG was dependent on the NA and that the dependence on the NA varied with distance from the articular surface (Figure 1). The high forward to backward ratio at low NA near the articular surface (Figure 2) was interpreted to represent the aligned fibrils characterizing the articular cartilage. The gradual transition between the immature articular cartilage and the growth cartilage was therefore clearly visualized by SHG, and SHG microscopy is therefore a promising technique to study the development of the collagen matrix in articular cartilage.

[1] J. M. Clark, A. Norman, and H. Nötzli, J. Anat. 191, 215–221 (1997).

[2] R. Rivas and F. Shapiro, J Bone Jt Surg. 84, 85–100 (2002).

[3] G. Cox, E. Kable, A. Jones, I. Fraser, F. Manconi, and M. D. Gorrell, J. Struct. Biol. 141, 53–62 (2003)

Origin recognition complex (ORC) architecture has only been explored in depth in the opisthokont supergroup of eukaryotes, which includes yeast and mammals, with little work in protists. The Kinetoplastida is a well-studied order of eukaryotic microbes and contains several important human parasites, such as Trypanosoma brucei. Genome sequencing of T. brucei identified only a single ORC-related protein – TbORC1/CDC6. A number of TbORC1/CDC6-interacting factors have been identified, raising the possibility that an ORC is present in T. brucei. However, many of these interactors are highly diverged in sequence from canonical ORC subunits and none has been shown to have a role in replication. To compare the localization pattern of TbORC1/CDC6 with two of these factors (TbORC1b, and TbORC4), and with a subunit (TbMCM3) of the replicative MCM helicase that ORC is through to recruit, each protein was tagged with 12 copies of the c-myc epitope (12myc). Analysis of the ratio and morphology of the nucleus (N) and kinetoplast (K) delineates the cell cycle stage of cells within the population: 1N1K for G1 phase; 1N1eK (elongated - replicating - but not yet divided kinetoplast) for S phase; 1N2K cells for S-G2 phase; and 2N2K cells for postmitosis. Using Super-resolution structured illumination microscopy (SR-SIM) we characterized the distribution pattern of each factor using anti-myc antiserum and compared this with replicating DNA, which was detected with EdU labeling, and with chromatin (DAPI). TbORC1/CDC6 and TbORC4 presented a punctate distribution in non-replicating cells, and did not localize to a specific region within the nucleus. In early S phase cells there was limited co-localization between either TbORC1/CDC6 or TbORC4 and EdU, which was also seen in puncta. An overlap in the myc and EdU signals became more pronounced in 1N2K cells, where S phase is more complete, and at that cell cycle stage the protein and replication puncta become more abundant and appeared more diffuse. In 2N2K cells, after completion of replication, localization of TbORC1/CDC6 and TbORC4 returned to a similar pattern to that of 1N1K cells. SR-SIM imaging revealed that TbORC1B is only seen in the nucleus of S phase cells (1N1eK and 1N2K), where a comparable pattern to TbORC1/CDC6 and TbORC4 was seen, with signal throughout the nucleus in a similarly large number of puncta and with some overlap with EdU. TbMCM3 signal was more abundant and more homogenous in the nucleus that any of the putative ORC-factors, and displayed little obvious variation in the different cell cycle stages, indicating that each of TbORC1/CDC6, TbORC4 and TbORC1B display distinct sub-nuclear localisation to that of TbMCM3. SR-SIM imaging has helped reveal that ORC architecture and regulation appear to be diverged features of replication initiation in T. brucei.

Acknowledgements: This work was supported by the Wellcome Trust [083485], the BBSRC [BB/K006495/1] and Fundação para a Ciência e Tecnologia (FCT, Portugal) [SFRH/BD/68784/2010]. The Wellcome Trust Centre for Molecular Parasitology is supported by core funding from the Wellcome Trust [085349].

Tuberculosis remains one of the major infectious diseases in the world. Current vaccine testing relies on in vivo models including the mouse aerosol challenge model. To determine structural changes in the 3D of granulomas caused by Mycobacterium tuberculosis previous observations have relied on serial sectioning with histopathology techniques. However, this has several disadvantages including the possibility of missing or distorting information in the 3D.

Optical clearing techniques may offer a way around this by making the whole organs transparent using chemical procedures, and then imaging by confocal laser scanning microscopy to produce optical sections. Therefore this study aimed to see whether optical clearing techniques can be used to study granulomas in 3D and the structural changes to the surrounding tissue. Results to date have shown that it is possible to optically clear lobes of the mouse lung and to resolve individual nuclei. We now hope to use the same techniques to image M. tuberculosis infected lung and to compare with other organs.

Influenza is an infectious disease caused by Orthomyxoviridae viruses that infect 3-5 million people worldwide during seasonal epidemics. Influenza vaccines must be produced and administred annually to respond to the high rate of mutations and possible exchange of genomic segments between different strains. The vaccines are produced by a well established prodedure in inoculated embryonated eggs. Influenza virions are purified by sucrose gradient and then split by detergent filtered and inactivated by formaldehyde. Samples of split virions contain mainly HA and NP viral proteins.

To monitor quality of produced vaccines, quantitative information about the oligomerization state and molecular assembly of antigens in produced lots is desirable. We have developed a new approach to determine the size distribution of particles in split-viron influenza vaccine samples by electron microscopy and could thus interpret elution profiles from the asymmetrical flow field-flow fractionation (A4F) method. Samples were imaged by negative-stain electron microscopy using organo-tungstate stain (nanoW, pH 8.0) at 19,000x magnification (5.59 Å/pix) and 4-μm defocus (Figure 1). About 40-60 micrographs were initially filtered to remove variations in background and improve signal-to-noise ratio [1]. Individual particles were then segmented by a watershed algorithm [2]. Areas of the segmented 2D projections were determined in ImageJ [3] and plotted as histograms of the size distribution. (Figure 2). This approach is able to distinguish among different oligomeric states of HA and NP subunits in the sample, however all larger assemblies and aggregates are not resolved.

In parallel, we analyzed the same samples by A4F. Elution profiles resolved three peaks of small particles (diameter 200 nm). While the first three peaks correspond to the HA and NP oligomers resolved in the size distribution resolved in the analysis by electron microscopy, the latter peaks reflect presence of large lipid vesicles with embedded HA proteins. Interpretation of the A4F elution profiles allows routine screening and monitoring of produced split-virion influenza vaccines.

This study has been funded by Sanofi Pasteur.

References

[1] Tang, G. et al. (2007) J. Struct. Biol., 157, 38-46.

[2] Heymann, J.B. (2001) J. Struct. Biol.,133, 156-169.

[3] Schneider, C.A. et al. (2012) Nature Methods, 9, 671-675.

The formation of neutrophil extracellular traps (NETs) is often associated to entrapment and growth inhibition of several pathogens. However, an excessive NET formation or impaired degradation of the DNA fibres can result in the development of autoimmune diseases, such as SLE or psoriasis. Also NET-associated molecules, like host defence peptide LL-37 and cationic histones can have harmful effects. Thus, the host organism needs to rely on precautions to circumvent such deleterious effects.

Our group recently published that p33, a multi-ligand protein present on several cells types, is able to inhibit the cytolytic activity of LL-37 on endothelial cells. Additionally, it was found that the application of p33 prevents histone-mediated platelet aggregation as well as lethal haemolysis in mice treated with histones.

In order to investigate whether p33 plays a role in the formation of NETs, first NETosis was analysed microscopically and biochemically. p33 administration abolished the PMA-mediated release of DNA fibres and additionally reduced the PMA-triggered production of ROS. Moreover, both cationic LL-37 and histones significantly triggered the formation of NETs as well as the release of MPO. However, the simultaneous addition of p33 in a 1:2 ratio decreased the amount of released MPO and the protein-triggered NETosis. Utilising DNA-intercalating dye PicoGreen, the effect of p33 on the stabilisation of DNA, backbone of the NET structures, was investigated. Due to their cationic properties, histones as well as LL-37 bind and protect DNA against bacterial nuclease MN. Interestingly, additional application of p33 diminished the protection of DNA mediated by both LL-37 and Histones against S. aureus nuclease. Taking together, we hypothesise that p33 plays a role in modulating NET induction and NET stabilisation.

Phytoplasmas, obligate parasites of plants and phloem-feeding insects, belong to Mollicutes (Lee et al., 2004) and are associated with several hundreds of diseases affecting over one thousand plant species, including many economically important crops (Marcone, 2014). There is no effective curative strategy available so far, so the sole ways to limit the infection outbreaks are the use of insecticides and the removal of symptomatic plants (Bertaccini et al., 2014).

Even if not all infections are necessarily deleterious, symptoms in infected plants suggest heavy disorders of phloem functions and growth-regulator balancing (Lee et al., 2000). Upon their discovery (Doi et al., 1967), the study of phytoplasmas has been hindered by the extreme difficulty to culture them in vitro, due to their lack of fundamental metabolic pathways (Bai et al., 2006). Moreover, the study in natural plant hosts is often limited by environmental conditions, long plant life cycle and poor knowledge of host-plant biology. Therefore, in the last decade some authors suggested to use Arabidopsis thaliana as model plant for studying phytoplasma-plant interactions. This choice was supported by the correspondence between the macroscopic symptoms developed in infected A. thaliana and those observed in natural host plants (Bressan and Purcell, 2005; Hoshi et al., 2009; Cettul and Firrao, 2011; MacLean et al., 2011). Nevertheless, morphological and ultrastructural modifications occurring in infected A. thaliana tissues have never been described in detail.

In this work, we adopted a combined microscopy approach to verify if this plant is a reliable model for the study of phytoplasma-plant interactions at microscopical level.

Using DAPI and fluorescence microscopy (FM), phytoplasma presence and localization were demonstrated in every infected plant. Transmission electron microscopy (TEM) observations confirmed phytoplasma massive presence into the sieve elements (SEs) (Figure 1). Phytoplasma appeared well preserved, with typical pleomorphic shape, free-floating and dividing in the lumen or adhered to SE membrane, probably connecting to the host (Marcone et al., 2014; Buxa et al., 2015). Phytoplasmas also established relationships with sieve element reticulum (SER). Pathogen presence, probably linked to nutrient uptake (Celli et al., 2015; Musetti et al., 2016), caused SER hyperproliferation, as observed in many other plant-phytoplasma interaction (Rudzinska-Langwald and Kaminska, 2001; Buxa et al., 2015) (Figure 1). Pathogen spread was documented by the passage through sieve pores.

As remarked above, phytoplasma presence affected host plant development (Lee et al., 2000). In infected A. thaliana plants, light microscopy (LM) evidenced a profound disturbance in phloem morphology at histological level, mainly consisting in collapse, necrosis and hyperplasia of the phloem components. The relationship between necrosis and hyperplasia could be explained as a plant response to the impaired phloem functionality (Oshima et al., 2001) or due to pathogen effectors (Bai et al., 2009; Sugio et al., 2011).

At ultrastructural level, as previously observed in other phytoplasma hosts (Musetti et al., 2000; 2013; Kaminska et al., 2001; Santi et al., 2013), phloem components showed plasmolysis or were collapsed or necrotized. Even in vital SEs, abnormalities of cell membrane profile and cell wall thickness were visible. TEM observations showed two typical plant responses to phytoplasma infection: phloem-protein agglutination and callose deposition at the sieve plates, which limited sieve-pore diameter (Figure 1). These phenomena have been interpreted as a plant reaction to physically limit pathogen spread (Lherminier et al., 2003; Gamalero et al., 2010; Luna et al., 2011; Musetti et al., 2010; 2013).

Phloem functionality experiments using CFDA and confocal laser scanner microscopy (CLSM) suggested that sieve-pore obstruction leads to phloem impairment (Figure 2 A, C). This phenomenon is also associated to the accumulation of photo-assimilates, visible as chloroplast starch deposits under LM and TEM (Figure 2 B, D), as previously reported in other host plants (Maust et al., 2003; Junqueira et al., 2004; Musetti et al., 2013).

This study proved that phloem tissue of infected A. thaliana presented the main morphological and ultrastructural response to phytoplasma infection as reported in natural hosts. Moreover, analyses carried on A. thaliana were not affected by troubles linked to low phytoplasma titre and uneven distribution, typical of woody plants. Therefore, we can state that A. thaliana revealed a reliable model plant for phytoplasma-plant interactions, concerning both macroscopic symptoms and morphological and ultrastructural changes.

Being the most numerous, diverse and ubiquitous group of animals, the arthropods have established a wide array of interactions with bacteria. Despite their considerable potential in agriculture, health-care and pest control, exploration of these associations is limited to a few well studied associations (Bourtzis & Miller 2003), while comprehensive understanding of interactions between bacteria and arthropods generally remain underexplored, due to lack of appropriate model organisms. Our work in recent years has been therefore focused on bacterial associations in terrestrial isopod crustaceans (Fig. 1), a diverse and ubiquitous group of arthropods, with considerable ecological role, well-known biology and undemanding maintenance under laboratory conditions.

In order to expand our knowledge on isopod-bacteria associations we used a wide array of microscopic approaches on crustacean tissues including FE-SEM, TEM and fluorescence microscopy with structured illumination in combination with histochemistry, fluorescent ‘in situ’ hybridization and EDXS analytical techniques.

Beside diverse transitional bacterial microbiota inhabiting the gut, our observations revealed three groups of bacteria exhibiting specific adaptations to the isopod host as their environment. The first are filamentous gut bacteria, belonging to a novel and distant phylogenetic lineage named ‘Candidatus Bacilloplasma’ (2). These commensal developed specific attachment structures, enabling their attachment to the tips of the cuticular structures covering the inner surface of the hindgut of common woodlouse, Porcellio scaber (Fig. 2). The second specific interaction comprises the intracellular pathogen Rhabdochlamydia porcellionis (Fig. 3), which represents the first description of the Chlamydiae in arthropods (3). Studies of infection in established cell culture (4) and in the digestive glands of its natural host, isopod P. scaber (5), enabled insight into chlamydial interactions with the host and mechanisms of pathogenesis of R. porcellionis, currently recognized as emerging pathogen. The third association includes bacterial community inhabiting specialized organs, known as the calcium bodies (6,7). These chevron-shaped organs in the body cavity of trichoniscid isopods serve as transitional storage of calcium during moulting of the exoskeleton and comprise a bacterial community of phosphate-accumulating bacteria arranged in densely packed layers surrounding a mineralized core (Fig. 4). Due to involvement of the polyphosphate-accumulating bacteria in phosphorous metabolism, the latter of described isopod symbioses can be, in contrast to the previous two, described as beneficial to the host and therefore mutualistic.

Our results show that terrestrial isopods can be considered important, yet overlooked evolutionary playgrounds, which enabled specific bacterial adaptations to the arthropod hosts and the development of novel bacterial lineages. By displaying the full scope of bacterial symbiosis ranging from pathogens, over commensals to mutualists, the terrestrial isopods have considerable potential as model organisms for much needed studies on host-symbiont interactions.

References:

(1) Bourtzis K., Miller T.A. (2003): Insect symbiosis. CRC Press, 347 pp.

(2) Kostanjšek R. Štrus, J., Avguštin G. (2007). Appl. Environ. Microbiol. 73(17): 5566-5573.

(3) Kostanjšek R., Štrus J., Drobne D., Avguštin G. (2004).  Int J Syst Evol Microbiol, 54: 543-549.

(4) Sixt B.S., Kostanjšek R., Mustedanagic A., Toenshoff E.R., Horn M. (2013). Environ. Microbiol. 15(1): 2980-2993.

 (5) Kostanjšek R., Pirc Marolt T. (2015). J Invertbr. Pathol. 125: 56-67.

(6) Vittori M., Kostanjšek R., Žnidaršič N., Žagar K., Čeh M., Štrus J. (2012). J. Struct. Biol 180(1): 216-225.

(7) Vittori M., Rozman A., Gradodolnik J., Novak U., Štrus J. (2013). PloS one 8(3) 1-14, e58968.

Introduction: Helicobacter pylori (H.pylori) is a Gram-negative bacterium that causes chronic gastritis, peptic ulcers and stomach cancer. The activity of immune cells, including monocytes, infiltrating gastric mucosa of H. pylori infected individuals depend on the ability to recognize and react to the bacterial antigens including lipopolysaccharide (LPS) H.pylori. The LPS of this bacterium has a unique structure of lipid A and possess Lewis (Le) determinants that allow bacteria to modulate host immune responses and promote chronic infection. However little is known about the role of Le determinants in H.pylori – host cell interactions and receptors involved in binding of H.pylori LPS. Aim: To evaluate TLR receptors involved in binding H.pylori LPS with or without Lewis antigens and to evaluate the consequences of this interaction: cell signaling, activity, morphology, ROS production and tumor necrosis factor (TNF-α) secretion. Material and Methods: THP-1XBlue™ cells were used as a model of human monocytes - a reporter cell line that when activated via TLR receptors induce NFk-B transcription factor and secrete embryonic alkaline phosphatase (SEAP). Cells were seeded in 96-well tissue culture plates (1×10⁵ cells/well) and stimulated for 24 h with LPS of: H.pylori Le^XY(+) or H.pylori Le^XY(-), or standard LPS E. coli O55:B5, in a final concentration of 100ng/ml. To evaluate the involvement of particular TLR receptors and signaling molecules in LPS binding 30 min. prior stimulation monoclonal antibodies: mAbα: TLR2, TLR4, TLR6 (Novus, CO, USA) or MyD88 inhibitor (Selleckchem) were introduced. After stimulation supernatants were used for SEAP (QuantiBlue reagent) and TNF-α quantification (ELISA, ElabSciences). ROS production was estimated with Total ROS Detection Kit (Enzo Life Sciences). Simultaneously, the cells were fixed for 10 min. with 99.8% methanol and stained with DAPI (1:2000) and DiOC (2: 1000) for 15 min. and 5 min. respectively. The microscopic evaluation was performed with Leica TCS SP8 (Leica-Microsystems, Wetzlar, Germany). Results: All LPS types have potential to activate monocytes, however each LPS with different intensity. The LPS of H.pylori Le^XY(-) (OD=0.870±0.07) as well as H.pylori Le^XY(+) (OD=0.677±0.05) exhibited significantly lower potential to activate monocytes in comparison to  the standard LPS of E. coli (OD=1.361±0,.44), p<0.001). Confocal microscopy analysis showed that the cells stimulated with LPS of E. coli lost their ability to adhere (78%) and the integrity of cellular membrane was disturbed (Figure 1), whereas the cells incubated in the presence of H.pylori LPS Le^XY(-) remained in a condition similar to untreated controls. The LPS of H.pylori Le^XY(+) did not affect the adhesive properties however we observed a significant drop of fluorescence signal from DAPI which suggest its influence on cellular nuclei. The experiments with mAbαTLR receptors revealed that the activation induced by standard LPS of E. coli solely depended on the presence of TLR4 receptor (or in complex with TLR2), whereas the H.pylori Le^XY(+)/Le^XY(-) – mediated monocyte activation was related with the presence of receptor complexes: TLR2/4 (p=0.0001) or TLR2/6 (p=0.003), whereas the blocking of TLR4 did not reduce the level of monocyte activation. The experiments with LPS evaluated by confocal microscopy were consistent with cellular culture study and showed that the lower activation of monocytes mediated by H.pylori LPS is accompanied by lower ROS production in comparison to that induced by E. coli LPS (3631±184). H. pylori LPS with Lewis antigens induced significantly higher ROS production than the H.pylori LPS without Lewis residues(1446±138 vs 853±62), which might suggest the involvement of sugar moieties in this phenomenon. Further studies showed that LPS E.coli-mediated stimulation was strongly correlated with the secretion of high doses of TNF-α by monocytes (150,5±9.4 pg/ml) whereas the level of this cytokine secreted in response to H.pylori Le^XY(+) or H.pylori Le ^XY(-) was notably lower (101.5±18.1 and 64.2±5.9, respectively, p<0.001). Additional experiments with the utility of Myd88 inhibitor used before stimulation showed that in all cases TNF-α production was Myd88 dependent and resulted with at least 70% inhibition of its release. Conclusion: Our results suggest that the LPS of H.pylori is recognized not solely by classic TLR4 receptor but rather by TLR4/2 or TLR2/6 receptor complexes and that the Lewis residues present in 80% of H.pylori isolates are involved in that process. The immunomodulatory activity of H.pylori is weaker in regard to ROS production and TNF-α which is associated with the level of monoCyte activation and might explain the chronic character of this infection.

Dengue virus (DENV) is one of the most important arthropod-borne viruses that infect humans. Approximately 400 million people are infected annually in tropical and sub-tropical regions worldwide [1]. In 2015, Sanofi Pasteur released the first commercialized and approved tetravalent vaccine that is protective against the four DENV serotypes [2].

In the context of developing a better understanding of the interaction of dengue antigen with the immune system, it is important to fully understand the structure and accessibility of specific epitopes. Particularly in the case of the dengue virus, viral capsids bud into the host endoplasmic reticulum and acquire structural proteins prM and E embedded in the membrane. The prM and E proteins are assembled as trimers as part of an immature icosahedral shell and undergo significant structural rearrangement during viral exit via the Golgi apparatus. In the Golgi, Host furin-type proteases concomitantly cleave the pr peptide triggering rearrangement of the trimers into dimers, which is necessary for infectivity of the viral particle [3]. However, maturation of DENV virions is not efficient and partly immature particles are released during DENV infection as well. It has been hypothesized that these particles function as decoys to avoid neutralization of the infectious mature particles by the immune system [4].

Recombinant antigens in the form of virus-like particles are attractive as reagents for understanding antigen-antibody interactions. Here, we investigated the molecular architecture of virus like particles (VLPs) made of dengue structural subunits, which are produced by The Native Antigen Company (Oxfordshire, UK). We found by SDS-PAGE and confirmed by Western blot that DENV-2 and DENV-3 VLPs contain a significant fraction of uncleaved prM subunits. To assess the molecular architecture and conformation of subunits in the VLP particles, we determined 3D structure of individual particles by cryo-electron tomography. The particles have a spherical (DENV-2 VLPs) or elongated (DENV-3 VLPs) outer shell with short spikes and an internal cavity (Figure 1). The spikes exhibit the same morphology as the prM/E subunits in the immature dengue viral particles. We additionally imaged and reconstructed the 3D structure of VLP particles mixed with monoclonal antibodies specific to the mature conformation of DENV. We identified bound antibodies to individual particles in subtomograms after subtraction of the VLP shell (Figure 2). Binding of the antibodies to the VLP particles was subsequently confirmed by Western blot and immunogenicity assays suggesting that these VLPs could adopt a mixed conformation with mature dimers and immature trimers on the same particles, as previously described for DEN virions or adopt a specific new conformation of mature prME specific to VLPs.

This study was funded by Sanofi Pasteur using equipment installed at Sanofi Pasteur.

References

[1] Bhatt S., Gething P. W., Brady O. J., et al. (2013) Nature, 496, 504–507.

[2] Hadinegoro, S.R. et al. (2015) N Engl J Med., 373, 1195–206.

[3] Kuhn R. J., Zhang W., Rossmann M. G., et al. (2002) Cell, 108, 717–725.

[4] Rodenhuis-Zybert IA1, Wilschut J, Smit JM. (2011) Trends Microbiol., 19, 248-254.

Staphylococcus aureus is one of the most important bacterial pathogens causing a wide spectrum of diseases varying in severity. Treatment of the infections is often complicated by S. aureus resistant to antibiotics. Current development of antibiotics is not fast enough to counterpart the emergence of antibiotic-resistance. Preclinical studies showed, that phage therapy is promising alternative for treatment of bacterial infections including those caused by resistant strains, but approval of bacteriophage-based drugs requires a detailed functional understanding of the molecular mechanisms of phage infection which we are still lacking.

Bacteriophage 812K1-420 from the family Myoviridae is potential candidate to be used in phage therapy against S. aureus. Polyvalent and strictly virulent bacteriophage 812K1-420 virion is composed of an icosahedral head containing linear 145,5 kb dsDNA, contractile tail and baseplate which specifically recognizes specific surface on the bacterial host. Replication and virion-assembly mechanisms inside host cell are not fully explored yet, although few bacteriophage 812K1-420 lytic cycle intermediates of virion assembly were reproduced in vitro.

Here we present initial structural analysis of bacteriophage 812K1-420 infection in vivo using cryo-electron tomography. We used cryo FIB milling technique to thin plunge frozen S. aureus cells (600-1000 nm in diameter) infected by phage 812K1-420 into wedge geometry . The cryoET data acquired on FEI Titan Krios microscope and tomograms were reconstructed using program IMOD.

INTRODUCTION

Toxoplasma gondii is an intracellular protozoan parasite which affects animals and humans causing encephalitis, chorioretinitis and death. The tachyzoite has structures that allow it to perform cell invasion. During the invasion, the tachyzoite adheres to the target cell membrane through proteins MIC that come from micronemes and then projects a highly dynamic structure called conoid [1,2]. Subsequently the parasite enters the cell and is housed in a parasitophorous vacuole proliferating by endodyogeny. After several cycles of replication, the parasites leave the infected cell by mechanisms such as conoid extrusion and secretion, which enables the parasite to invade neighboring cells. This replication cycle results in cell destruction and is responsible for the major clinical manifestations of toxoplasmosis. Ethanol is a well-characterized inductor calcium-dependent events in different cell models. In this work we used ethanol and we analyze the asociation between conoid extrusion and MIC secretion.

 
METHODS

Cell culture. The cell model used for our invasion, proliferation and egress assays was HEP-2 cells (human epithelial laryngeal carcinoma cells) (Hep-2,ATCCCCL-23).

Parasites and conoid extrusion. The RH strain of T. gondii was maintained in BALB/c mice, purified and incubated with 0.5M ethanol, fixed, and processed for electron microscopy and immunofluorescence.

Eletronic microscopy. The samples were fixed, dehydrated and infiltrated in resin or processed for scanning electron microscopy. Thin sections obtained were contrasted with uranyl acetate and examined in a transmission electron microscope (Jeol 2000 EX).
Immunodetection. MIC2 protein detection was performed without permeating in extracellular tachyzoites. The samples were fixed and incubated with specific primary antibodies and analyzed using confocal microscopy.

RESULTS

The exposure of tachyzoites to ethanol induces reversible conoid extrusion, clearly observed the projection of the apical part of tachyzoites (Fig. 1). We also observed the presence of vesicle components accumulating in the back of tachyzoites, associated with some changes in the plasma membrane. We could detected that these vesicular components presenting on its surface was always associated with the extruded conoid(Fig. 1). For immunofluorescence, we showed that the secreted products during extrusion conoid micronemes come from. These results suggest a similar mechanism of secretion and induction for extrusion.

REFERENCES

[1] Mondragón y Frixione . J. Euk. Microbiol. 43 (1996) 120-127.

[2] González-Del Carmen, et al. Cell Microbiol 11(2009):967-82

ACKNOWLEDGEMENTS

This work was supported by the project CONACYT # 165282 

Cauliflower mosaic virus (CaMV) is transmitted from plant to plant through an interaction with aphid vectors, according to a non-circulant transmission (1). Until recently, it was admitted that CaMV transmission was not specific. However, our colleagues have recently identified a proteic receptor at the extreme tip of the aphid maxillary stylets involved in interaction with the viral protein P2, the transmission factor of  CaMV, suggesting a specific and complex interaction process (2).

As a consequence, it is now believed that  CaMV transmission involves an aphid receptor and two viral proteins, P2 and P3. P2 binds both to the aphid receptor and to P3, which is tightly associated with the CaMV viral particle, forming the transmissible viral complex (Figure 1). We have previously resolved the P3 structure by X-ray crystallography (3) and proposed a model  for the CaMV:P3 complex based on single particle cryo-electron microscopy (cryo-EM) studies (4). We are now trying to characterize P2, the viral helper-component, which acts as a bridge between the aphid receptor and the viral complex CaMV:P3, thus playing a major role in plant-vector transmission. Structure predictions indicate that P2 is composed by a N-terminal globular domain, followed by two coiled-coil domains. Due to the presence of these coiled-coil domains which seem to confer the protein a strong tendency to aggregate, it is extremely difficult to work and manipulate the native P2 protein. Hence, we have adopted a hybrid strategy combining  X-ray crystallography and cryo-EM studies to investigate the structural organization of P2. In this context, we are trying to crystallize various domains of the P2 protein over-expressed in bacteria. Additionally, we want to obtain the tertiary structure of P2 by using electron microscopy. Indeed, as His-tagged P2 protein can form paracrystals when expressed in sf9 cells (Figure 2), we use  cryo-electron tomography  and sub-tomogram averaging methods to get first structural data on P2 and interactions between coiled-coil domains. Moreover, a P2 mutant (157m) was shown to decorate microtubules in sf9 cells (Figure 3). We defined conditions of purification and used these decorated microtubules to engage a single particle analysis in cryo-EM and image processing.

This poster presents the first structural results in the organization of the P2 protein and its multiple interactions.

(1)    Martinière et al., Plant Signaling & Behavior, 4:6, 548-550, 2009

(2)    Uzest et al., PNAS, vol. 104 no. 46, 17959-17964, 2007

(3)    Hoh et al., Journal of Virology, vol. 84 no. 9, 4706-4713, 2010

(4)    Plisson et al., J. Mol. Biol, 346:267-277, 2005

Our goal was to find an optimal tissue clearing protocol for whole mount imaging of embryonic and adult hearts and whole embryos of transgenic mice that, unlike the golden standard BABB protocol used for whole mount immunohistochemistry, would preserve GFP fluorescence and to compare different currently available 3D imaging modalities.

We tested various organic solvent- or water-based clearing protocols intended to preserve GFP fluorescence in central nervous system: tetrahydrofuran dehydration and dibenzylether protocol (DBE), SCALE, CLARITY, and CUBIC and evaluated their ability to render hearts and whole embryos transparent.

DBE clearing protocol did not preserve GFP fluorescence; in addition, it caused considerable tissue shrinking artifacts when compared to the golden standard BABB protocol. The CLARITY method considerably improved tissue transparency at later stages, but also decreased GFP fluorescence intensity. The SCALE clearing resulted in sufficient tissue transparency up to ED12.5; at later stages the useful depth of imaging was limited by tissue light scattering. The best method for the cardiac specimens proved to be the CUBIC protocol, which preserved GFP fluorescence well, and cleared the specimens sufficiently even at the adult stages. In addition, CUBIC decolorized the blood and myocardium by removing iron from the tissues.

Good 3D renderings of whole fetal hearts and embryos were obtained with OPT and SPIM, although at resolutions lower than with a confocal microscope. Comparison of five tissue clearing protocols and three imaging methods for study of GFP mouse embryos and hearts shows that the optimal method depends on stage and level of detail required.

Figure 1 demonstrates 3D visualizations of specimens cleared by various techniques, acquired and visualized by an OPT microscope and VolViewer software package, Bangham Lab, A)-C) and a SPIM and Amira 3D, FEI Software, D), respectively.

A) is an ED12.5 mouse heart with a scissor cut through the right ventricle, BABB clearing, primary antibody against GFP detected with Cy5-coupled secondary. Red-grey channel is transmission one, green channels is iso-surface rendering of the fluorescent channel acquired using excitation of 628/40 nm and emission of 692/40 nm.

B) shows an ED16.5 mouse heart with a scissor cuts through both ventricles, SCALE clearing. Maximum intensity projection (MIP) visualization of overlays of red (transmission) channel and green (fluorescent) channel using excitation of 425/40 nm and emission of 475 nm (high pass). GFP-positive network of Purkinje fibers is clearly visible.

C) depicts an ED12.5 mouse embryo cleared in CUBIC. MIP visualization of fluorescent channel using excitation of 425/40 nm and emission of 475 nm (high pass). GFP positivity is seen in the head arteries and heart; lot of autofluorescence is apparent in the liver.

And, finally, D) represents ED12.5 mouse heart with a virtual cut through the right atrium and ventricle. Strong GFP signal (shown in red with the pseudocolor display) is visible in the pectinate muscles of the atrium and ventricular trabeculae. Cleared in CUBIC.

Used abbreviations: LA – left atrium, LV – left ventricle, RA – right atrium, RV – right ventricle.

Acknowledgements. Supported by 13-12412S from the Czech Science Foundation, Ministry of Education PRVOUK P35/LF1/5, institutional support RVO:67985823, and Charles University UNCE 204013.

To understand developmental processes it is essential to consider both, molecular / genetic data and structural aspects of morphogenesis and differentiation. Integration of both approaches is beneficial to interpret the data obtained and unravel tissue and organism formation. Sequential morphological changes during animal development can be followed by different microscopic methods that enable us to combine imaging of intact embryos, elucidation of the tissue and cell architecture at different size scales, localization of tissue constituents in their original location and correlative imaging, supplemented by analytical microscopy at the specific stages of development.

Here we present a combined use of light microscopy, histology, transmission (TEM) and scanning electron microscopy (SEM), labelling with specific ligands and analytical microscopy to unravel the differentiation of epidermis and gut epithelium in a crustacean model. Our study addresses the interrelation of tissue morphogenesis, epithelial cell differentiation at the ultrastructural level and differentiation of the corresponding apical extracellular matrix, the cuticle, examined in the embryos and marsupial larvae of Porcellio scaber. Cuticle is a chitin-based matrix, secreted apically by ectodermal epithelia during development and renewal in arthropods. We integrate structural information at different scale levels with data on matrix elemental and molecular composition in the selected developmental stages. Developmental stages and gross morphological changes in epidermal and gut tissues were identified by imaging the intact embryos and larvae (Fig. 1A), fluorescence labelling of nuclei and imaging of histological sections (Fig. 1B). Architecture of tissues, cells and matrix was characterized at the levels of light and electron microscopy. To determine the organic scaffold composition of the matrix, we performed localization of macromolecules containing N-acetyl-glucosamine (chitin) by labelling with lectin wheat germ agglutinin. The epidermal cuticle in crustaceans is in addition mineralized, forming a stiff exoskeleton. Analytical microscopy was applied to examine calcification of the forming cuticular matrix in marsupial larvae, performed by energy dispersive X-ray spectroscopy (EDS) to analyse elemental composition and Raman spectroscopy to determine mineral forms in the cuticle. The results show that several apical matrices are produced sequentially during epidermis and gut morphogenesis and that matrix renewal is coupled with major morphological modifications such as growth of appendages, elongation or bending of embryo body and hatching from egg envelopes. The early stages of apical matrix formation are similar in the epidermis and gut. Later in marsupial larval stages, specialization of both cuticles is evident, implying the establishment of their specific functions. Exoskeletal cuticle with elaborate surface structures (Fig. 2A) differentiates in several layers displaying chitin-protein fibres patterns (Fig. 2B) and is already prominently calcified. Hindgut cuticle displays differentiation in a homogenous electron lucent procuticle and prominent electron dense epicuticle (Fig. 3A).In concert with the stages of apical matrix formation, a gradual formation of the subapical cell junctions, one of the key factors that determines cell polarity, was evidenced (Fig. 3B). We consider our model system as valuable system to study tissue and cell differentiation in their native environment, with applying the integrated structure-function perspective.

Developmental studies are important to understand the morphology and evolution of the flower, an exclusive unique structure of the Angiosperms. For such studies, the analysis in SEM is essential to the understanding of organogenesis and organ initiation order. The Cannabaceae family, well known to include species such Humulus lupulus (hop) and Cannabis sativa (hemp), recently had its circunscription expanded, with the addition of some genera (e.g. Celtis and Trema) before belonging to Ulmaceae family. The flowers of these four genera have some diagnostic differences (dicliny, monocliny), but share some conditions (perianth single, few stamens, pseudomonomerous gynoecium) which causes the authors to consider them reduced compared to other related families. Then, the aim of this work was to study the development pathways that result in flower of the Celtis iguanaea (Jacq.) Sarg. and Trema micrantha (L.) Blume using SEM and compare them to the data available for Cannabis sativa and Humulus lupulus. From this comparison it will be possible to infer if the development of the flower is homologous in this group, which would support the inclusion of Celtis and Trema in Cannabaceae. To this end, flowers in various stages of development were fixed in buffered formalin, dehydrated in an ethanol series, critical-point dried, mounted on aluminum stubs with carbon tape, and coated with gold of 85,5 nm thick in current of 40mA. Samples were analyzed and documented in SEM (Zeiss IVO-50) with voltage 10 kV. Interpretations of electron micrographs shows that the species analyzed have similar floral development. The floral meristem is rounded and subtended by an abaxial bract and two small lateral bracts (figure 1).  The floral meristem iniates five sepals, five opposite stamens and a carpel primordia. The order of sepal and stamen initiation is helical (figure 2) and none of the species presents petals from the inception (figure 2), resulting in flowers single perianthed. The pistil starts its development as a single primordium (figure 2), which subsequently divides into two, giving rise to two carpels (figure 3 and 4); however, only one is responsible for the formation of the ovule. This pathway gives rise to the pseudomonomerous gynoecium. In Trema micrantha, a dioecious species, the dicliny occurs by abortion of the pistil in the staminate flowers and of the stamens in the pistillate flowers (figure 3). In Celtis iguanaea, an andromonoecious species, occurs abortion of the pistil in the staminate flowers (figure 4). No pistillate flowers were found in studied plants (figure 4), but perfect (=bisexual) flowers. Our results show that Celtis iguanaea, Trema micrantha, Cannabis sativa and Humulus lupulus do not have similar floral development. Only the absence of petals is shared among the four species of the four genera. The dicliny occurs by abortion in Celtis and Trema, and by inception in Cannabis and Humulus. The beginning of pistil development is also different: a single primordium initiates in Celtis and Trema while two primordia initiate in Cannabis and Humulus. Thus, we can conclude that the floral developmental features do not support the phylogenetic positioning of Celtis iguanaea and Trema micrantha in Cannabaceae. Different developmental vias give rise to the reduced flower in this group.

Acknowledgements: We thank to CAPES and FAPESP (process number 2014/07453-3) for financial support.

Urticaceae is a botanical family well known for its urticating trichomes that cause allergic reactions when touching the human skin.  The members of the family are anemophilous, which means that pollen grain transfer from staminate flowers to the stigma of pistillate flowers of the same individual occurs through the wind, without the participation of any biotic vector. They are also characterized by monoecious or dioecious sexual expression, and staminate flowers can develop a pistil called pistillode which is aborted during development. In most cases, the term pistillode refers to an aborted nonfunctional gynoecium in the mature flower that can be developed or rudimentary; the developed pistillode can perform various functions in pollination. Morphology studies based on microscopy help us to understand the floral mechanisms acting on pollen dispersal by the wind. Thus the objective of the present study was to investigate by light and scanning microscopy the morphology and functioning of staminate flowers of four species belonging to four Urticaceae genera: Boehmeria cylindrica (L.) Sw., Laportea aestuans (L.) Chew, Myriocarpa stipitata Benth. and Urera baccifera (L.) Gaudich. ex Wedd., with emphasis on the pistillode. Flower buds in a stage preceding anthesis and developed flowers were collected, fixed in buffered formalin or in Karnovsky solution, dissected with the aid of a Leica MZ 75 stereomicroscopic magnifying glass and processed for surface (scanning electron microscopy – SEM) and anatomical observations (light microscopy - LM). For SEM examination the dissected material was dried to the critical point, mounted on metal holders, placed on a carbon adhesive tape, sputtered with gold (exposure of 340 seconds) and observed with a Jeol JSM-6610LV scanning electron miroscope at 25 kV. For the LM exam the material was embedded in historesin and sectioned crosswise and lengthwise with a rotary microtome (2 to 4 μm thick sections). Serial sections were stained with 0.05% Toluidine Blue in phosphate buffer, pH 4.4, mounted on synthetic resin and observed with a light microscope. Photomicrographs were obtained with a Leica DM 4500 B photomicroscope coupled to a Leica DFC 320 digital camera. The pistillode arises as a single central protuberance after the initiation of four or five sepal primordia together with the primordia of lateral stamens. The elongation of the pistillode is rapid, exceeding the height of the stamens during the intermediate phases of flower development (figure 1). At the end of development, stamens and pistillode exhibit the same height since the stamens take on an inflex position in the flower bud. The pistillode does not contain ovules, exhibits an epidermis with voluminous and mucilaginous cells and a mesophyll consisting of aerenchyma in Laportea aestuans and Urera baccifera (figure 2), empty in Boehmeria cylindrica (figure 3), and of parenchyma in Myriocarpa stipitata (figure 4). Our results, added to those reported in the literature, indicate that the pistillode, together with the inflexed stamens, whose anthers are embedded in the sepals, acts on the explosive mechanism of release of the pollen to be transported by the wind. The pistillode, inflated by the accumulation of air in the mesophyll during development, presses the anthers, which separate from the sepals and move in the direction opposite to the flower, releasing pollen that can be agglutinated by the mucilage produced in the epidermis of the pistillode. We believe that this floral mechanism involving pistillode, stamens and sepals, optimizes anemophily, so that the pollen can reach farther distances, avoiding self-pollination and guaranteeing a greater genetic variability for these species.

Acknowledgements: This study was supported by São Paulo Research Foundation (Fapesp - process numbers: 2013/19459-3 and 2014/07453-3).

The single female germ-line cyst constitutes the whole ovary in Tubifex tubifex. The cyst is polarized and germ cells in the consecutive stages of oogenesis are localized along their long axis. The entire cyst consists of more than 2 000 germ cells. As oogenesis progresses, clustered germ cells differentiate into two morphologically distinguishable categories – the smaller but more numerous nurse cells and only eight growing oocytes at a time on average. During oogenesis, the oocytes gather organelles and storage material, i.e. lipid droplets, glycogen granules and protein yolk and grow considerably on the one side of the ovary in a linear arrangement. Vitellogenic oocytes lose their connections to the ovary and are released into the body cavity.

The spatial organization of germ-line cysts in T. tubifex is broadly similar to the other clitellate annelids known to date, and each cyst is composed of two elements – 1) a common and anuclear cytoplasmic mass (cytophore), which in T. tubifex is long and branched and snakes back and forth within a cyst and 2) germ cells located on the cytophore periphery. Each germ cell is connected to the cytophore by one stable intercellular bridge (ring canal). A rich cytoskeleton was observed within the cyst, which was mainly an inner microfilamentous rim of ring canals, prominent strands of actin filaments within the cytoplasm of the cytophore and a dense microtubular network that filled the cytoplasm of the germ cells and cytophore. Using the techniques of life cell imaging, experiments with cytotoxic substances that inhibit cytoskeletal polymerization and SBEM methods (Serial block-face scanning electron microscopy),we attempted to study the formation and functioning of germ-line cysts and the role of the cytoskeleton. It appears that the cytoskeleton within the germ-line cysts of T. tubifex plays an active role in maintaining the integrity of the huge and multicellular syncytium and takes part in the cytoplasmic transport of organelles and macromolecules from the nurse cells via the cytophore towards the growing oocytes.

A fixation technique, which allows a “near-to-native” state tissue preservation, such as high-pressure freezing in combination with electron tomography is required for studying synapses systematically at high resolution in 3D. With these techniques we are studying the ultrastructural architecture of synapses in the zebrafish Danio rerio which can be high-pressure frozen as intact animals at early stages. Currently we are focusing at a group of cytoskeletal proteins, the septins, which play important roles in synapse formation and synaptic function. We localize them with various microscopy techniques ranging from light to 3D electron microscopy in the nervous system. A limiting factor in 3D electron microscopy is the annotation of structures. We therefore developed an automatic vesicle annotation macro with Fiji, which helps us to automatically measure the size of synaptic vesicles and their density.  

The flower has always aroused great interest of researchers because it is considered the key innovation in the evolution of plants, featuring a very special group, the Angiosperms. It is conceptualized as a specialized stem apex or as a lateral branch with shortened internodes and appendices hypothetically homologous to leaves, modified for reproductive functions in sepals, petals, stamens and carpels. The predominance of angiosperm species on Earth is due probably to the success of the appearance of the flower, which enabled more effective ways in interbreeding. This fact is closely related to pollinators, which, along with the flowers, have diversified, becoming sometimes specialized in the pollination of a single plant species. A group of well-known plants, the urticalean rosids, composed of Cannabaceae, Moraceae, Ulmaceae and Urticaceae, surprises by displaying a flower of unusual morphology: small, inconspicuous, diclinous (= single-sex), perianth single or none, androecium with few stamens (1-5) arranged in one whorl, pseudomonomerous gynoecium, a single functional ovule inserted in different ways, in addition to a stigma with various forms. Thus, this flower, if compared to that of other rosids (Rosaceae, for example), can be considered reduced. Although morphological features of the flower of urticalean rosids are recorded since a long time (about 150 years), comprehensive studies on floral development, which could elucidate how this floral reduction takes place in this group are missing. This study aims to show how the ontogeny can explain the variations in flower structure of urticalean rosids. Flower morphology and pollination syndrome are also addressed. Our interpretations are based on data from at least 20 species obtained by analyses of surface in scanning electron microscopy and of histology in conventional light microscopy. The main points we raised are summarized in: (A) The condition perianth single (Fig. 1) or none (Fig. 2) results from the absence of the corolla organs or of both calyx and corolla organs from the inception. The interspecific variation in the calyx number of organs is also due to the absence of organs from the inception and not by organ abortion throughout the development. (B) The synorganization can be congenital (rare) (Fig. 3) or postgenital in the perianth and congenital in the gynoecium and androecium (rare). (C) Male and female flowers result of stamen and carpel abortion (Fig. 4) in most cases. Thus female flowers bear a pistillode and the male flowers a staminode. (D) The ontogeny of pseudomonomerous gynoecium deserves to be highlighted: one primordium arises in the center of the floral meristem, divides into two, but only one forms an ovule, although the other also participates in the style and stigma structure. (E) The arrangement of floral organs and pollination syndrome is strongly related in some species of Urticaceae (anemophily) and Moraceae (anemophily and entomophily). The inflate pistillode together with the anthers arrested by sepals in the staminate flowers of Urticaceae, for example, compose an explosive mechanism of pollen release which is then transported by the wind to a pistillate flower. In the few genera of Moraceae with entomophilous species, the union of several stigmas of different flowers within the inflorescence (fig), forming platforms (Ficus), or the offer of pollen and various exudates to pollinator insects (Artocarpus, Castilla, Dorstenia) ensures the formation of seeds. We conclude that the floral reduction in urticalean rosids takes place by different ontogenetic pathways, even among members of the same family. This finding contradicts the current paradigm which predicts that the floral ontogeny is conserved in related groups. Floral specializations, observed late in development, appear as responses to selection pressures exerted by different pollinators observed in the urticalean rosids.

We thank to FAPESP (process number 2014/07453-3, 2013/19459-3, 2012/15644-8), CNPq (process number 303493/2015-1) and CAPES for financial support.

INTRODUCTION
Sry gene expression, starting at 10,5dpc, initiates male sex determination. Sry gene initiates testis differentiation by regulating the differentiation of precursor supporting cells not into Granulosa cells but Sertoli cells (1). Pre-Sertoli cells are the first cell type to show sex differentiation in gonads. This study aimed to observe the light and electron microscopic features of pre-Sertoli cells.
MATERIAL AND METHODS
In this study 27 embryos obtained from 6 balb/c type pregnant female mice from 11,5 to 12,5 dpc were used. Sections were observed by using light and electron microscope.
RESULTS
Pre-Sertoli cells, stained with PAS, were seen as forming a cord around germ cells starting from central parts of gonads (Fig.1). Germ cells and epithelial cells lining the gonadal ridge were PAS (-). On electron microscope pre-Sertoli cells were seen mostly disorderly but also clustered and encircled the germ cells at some parts. The nuclei of pre-Sertoli cells were pulled at one pole of the cells. The cytoplasm at the other pole consisted of granuler and smooth endoplasmic reticulum, ribosomes, Golgi apparatus, mitochondria and large amount of glycogen granules (Fig. 2). Lipid granules were also seen in cytoplasm.
DISCUSSION
Pre-Sertoli cells are found disorderly in gonadal ridges as they are first developed. Shortly after that they proliferate and cumulate to form circular cords around germ cells (2). The accumulation of glycogen starting in pre-Sertoli cells is responsible for PAS(+) staining. The in vitro study of Matoba et al. stated that, testis specific accumulation of glycogen is a tissue-otonomous event as it is possible even with no serum or in the absence of adjacent mezonephrosis(4). The same study puts forward that the activation of PI3K-AKT pathway after Sry gene expression enhances glycogen accumulation in pre-Sertoli cells. Sox9, Mis, Dhh, Fgf9 are reported as pre-Sertoli cell marker genes(3). By using the fact that pre-Sertoli cells are stained PAS(+) because of glycogen they use as energy resource, it can easily be determined if the gonad is developing as male or female, before it is seen morphologically at 12,5dpc.
REFERENCES
1. Albrecht K H, and Eicher E M. (2001) Evidence That Sry Is Expressed in Pre-Sertoli Cells and Sertoli and Granulosa Cells Have a Common Precursor. Dev Biol 240, 92–107 2. Chen S, Liu Y. (2016) Testis cord maintenance in Mouse emmryos: Genes and Signaling. Biol Reprod. 94(2): 42 3. Cory AT, Boyer A, Pilon N, Lussier JG, Silversides DW.( 2007) Presumptive pre-Sertoli cells express genes involved in cell proliferation and cell signalling during a critical window in early testis differentiation. Mol Reprod Dev74(12):1491-504 4. Matoba S, Kanai Y, Kidokoro T, Kanai-Azuma M, Kawakami H, Hayashi Y, Kurohmaru M. (2005) A novel Sry-downstream cellular event which preserves the readily available energy source of glycogen in mouse sex differentiation. J Cell Sci. 1;118(Pt 7):1449-59

INTRODUCTION

Early developmentof the gonads is monitored in the same morphological appearance in both sexes. Retinoic acid, plays a role in the formation process of mammalian germ cells and is also effective in the selection of male or female identity (2).  Oct3/4, which is a transcription factor, is expressed in blastula, morula, inner cell mass of blastocyst and then in the primordial germ cells (8). Retinoic acid has been reported to be one of the possible regulators of Oct3/4 expression (3,7). This study was planned to evaluate the effects of the exogenous retinoic acid on gonadal development in mouse embryos and Oct3/4 expression of primordial germ cells.

MATERIALS AND METHODS

In this study, we used 12 Balb/c pregnant mice. No drug was given to the control group. The experimental group of Balb/c mice were given by oral gavage a dose of 80 mg / kg of retinoic acid dissolved in a mixture of absolute ethyl alcohol-corn oil (the days of 6.5, 7.5, 8.5). In the meantime, the second control group was formed and given by oral gavage only a suspension of absolute ethyl alcohol-corn oil for 3 days. Total of 73 embryos 11.5 - 12.5dpc from the control and experimental groups were evaluated.

RESULTS

In fetal gonads of the experimental group (Fig. 1and 2), a smaller number of primordial germ cells was marked with Oct 3/4  immunohistochemically compared to the first and second control groups.

DISCUSSION

A fine set of changes in the levels of retinoic acid are reported to control gonadal development at  different stages of  embryogenesis (1). Retinoic acid has been reported to have an important role in the sex differentiation of mouse gonads and shows its effect as a meiosis inducing factor  (4,5). Suppression of Oct3/4 expression in primordial germ cells that reached and settled in the gonads, is a critical step in the process of embryogenesis. A retinoic acid sensitive element localized in the promoter-enhancer region has been reported to play a role in suppression of Oct 3/4 in a study of embryonal carcinoma cells(6). The study concluded that, retinoic acid regulates the development and differentiation of primordial germ cells during embryogenesis by suppressing Oct 3/4 expression and the opinion to control this process externally by using activators or inhibitors of retinoic acid receptors, become stronger.

REFERENCES

1.  Bowles J, Knight D, Smith C, Wilhelm D, Richman J, Mamiya S, Yashiro K, Chawengsaksophak K, Wilson   MJ, Rossant J, Hamada H, Koopman P. 2006. Retinoid signaling determines germ cell fate in mice. Science, 28;312(5773), 596-600.

2.  Bowles J, Koopman P. 2007. Retinoic acid, meiosis and germ cell fate in mammals. Development, 134(19), 3401-3411.

3. Fuhrmann G, Chung AC, Jackson KJ, Hummelke G, Baniahmad A, Sutter J, Sylvester I, Schöler HR, Cooney AJ. 2001. Mouse germline restriction of Oct4 expression by germ cell nuclear factor. Developmental Cell, 1(3), 377–387.

4.  Koubova J, Menke DB, Zhou Q, Capel B, Griswold MD, Page DC. 2006. Retinoic acid regulates sex-specific timing of  meiotic initiation  in mice. Proceedings  of The National Academy of Sciences, 103(8), 2474-2479.

5. Ohta K, Lin Y, Hogg N, Yamamoto M, Yamazaki Y. 2010. Direct effects of retinoic acid on entry of fetal male germ cells into meiosis in mice. Biology of Reproduction, 83(6), 1056-1063.

6.  Pikarsky E, Sharir H, Ben-Shushan E, Bergman Y. 1994. Retinoic acid represses Oct-3/4 gene expression through several retinoic acid-responsive elements located in the promoter-enhancer region. Molecular and Cellular Biology, 14(2), 1026-1038.

7.  Saiti D, Lacham-Kaplan O. 2007. Mouse germ cell development in-vivo and in-vitro. Biomark Insights, 2, 241-252.

8.  Spiller CM, Bowles J. 2015. Sex determination in mammalian germ cells. Asian Journal of Andrology, 17(3), 427-432.

Noni is a native fruit which grows abundantly from Southeast Asia to Australia and is cultivated in Polynesia, India, the Caribbean, Central and northern South America. Morinda citrifolia L. (Noni), family Rubiaceae, has been used in Polynesia for over 2000 years in traditional medicine. Noni, has been reported to show a wide range of therapeutic effects including anticancer, anti inflammatory and antioxidant effects. The 3-methyl-4-nitrophenol (PNMC) isolated from diesel exhaust particles and also a degradation product of the insecticide fenitrothion. In this study we aimed to evaluate the effects on TNF-α and iNOS immunohistochemically and investigate the effects of Noni on plasma cell distribution in the spleen of rats exposed to PNMC. In the study, fifty-six mature male rats were randomly divided into eight groups (n: 7). Control group received phosphate-buffered saline+0.05% Tween-80 (vehicle). First experimental group received only Noni and other experimental groups received 1, 10 and 100 mg/kg PNMC (S.C) alone or in combination with Noni (2 ml per rat by gavage) respectively for 5 days. At the sixth day animals were sacrificed and spleen tissues were removed and weighted. After routine histological process, paraffin sections were stained with methyl green-pyronin for identification of plasma cells and TNF-α and iNOS antibodies were applied immunohistochemically and evaluated under light microscope. Immunohistochemical staining of TNF-α and iNOS were eveluated with histological scoring system (H-scores). Spleen indices were close to control in Noni administered groups. Plasma cell numbers were decreased in 1, 10 and 100 mg/kg PNMC recevied groups (P<0.001) while plasma cell numbers were close to control in Noni recevied groups. According to TNF-α and iNOS H-score results, the values of Noni recevied groups were similar to control. TNF-α and iNOS H-Score levels were increased in all PNMC recevied groups. Noni administration showed no effect on TNF-α H-Score levels, however in Noni+1 mg/kg PNMC combination group iNOS H-Score levels were decreased obviously with the effect of Noni (P<0.001). According to these results, it has been shown that oral administration of Noni has positive effects on plasma cell numbers in the spleen of rats which exposed to various doses of PNMC and on iNOS levels in the spleen of 1 mg/kg PNMC administered rats.

Diabetes Mellitus is characterized by hyperglycemia resulting from abnormalities in insulin secretion, insulin action, or both. It is one of the most serious challanges of 21th century because of both increased prevalence due to industrialization, urbanization and changed lifestyle habits, and socio-economic impacts. Lifestyle reforms, treatment of oral antidiabetics for glycemic control and of statins to improve the dyslipidemia are crucial in the prevention and treatment of cardiovascular diseases caused by diabetes. Numerous studies proved that diabetes has negative effects on male reproductive system, however, data obtained about atorvastatin and gliclazide are conflicted.

In this study diabetes was induced in 250-300g young adult male Sprague-Dawley rats by streptozotocin. Diabetic rats were given atorvastatin, gliclazide and atorvastatin/gliclazide combination by oral gavage for 28 days. Rats were sacrificed on day 28 and hormone analysis in heart blood, morphological evaluations in sperms, light microscopic and ultrastructural examinations in testes were conducted. The obtained data were analyzed by using one-way ANOVA.

The following results were obtained: (i) diabetes and individually the drugs did not change the hormone levels, while atorvastatin/gliclazide combination reduced the testosterone level, (ii) diabetes decreased sperm count, altered sperm morphology, caused damage in testes and impaired spermatogenesis, (iii) in diabetic rats atorvastatin did not effect sperm count and not improve testes structure, yet ameliorated sperm morphology, (iv) in diabetic rats gliclazide increased sperm count, ameliorated sperm morphology, improved testes structure and spermatogenesis, (v) in diabetic rats atorvastatin/gliclazide combination did not effect sperm count, ameliorated sperm morphology, improved testes structure, however, the improvement has not reached to control level.

In conclusion, data obtained from the study, which the effects of atorvastatin and gliclazide combination in diabetes-induced damage on male reproductive system were evaluated, is believed to contribute to the literature.

Although the articular cartilage is complex and thin structure it is crucial in the structure and function of the joint. Treatment of the articular cartilage injuries has an significant role in orthopedic practice. Nevertheless, to heal the damaged articular cartilage in hyaline cartilage has not yet been achieved. In this study it was aimed to evaluate not only the effects of vitamin D to the cellular and extracellular matrix components of the articular cartilage but also the effects of vitamin D in the healing of the damaged articular cartilage by the microfracture technique which is frequently used in the treatment of the articular cartilage injuries. 

In this study, twenty-four 18-months-old New Zealand rabbits were divided into four groups based on the treatment modality. Standart full thickness articular cartilage defect was applied in the medial femoral condile of the right knee of the all rabbits in the experimental groups. In group 1, only cartilage defect was established. After cartilage defect, in group 2, microfracture technique was applied. In Group 3, vitamin D was supplemented. And in group 4 both microfracture technique and vitamin D supplementation was applied as a treatment method. All animals were sacrified after 16 weeks and were evaluated histologically by ICRS, O’Driscoll and Pineda scoring systems.

It was not observed any improvement in the area of the tissue defect in group 1. In group 2, the recovery of the defected tissue was not pure hyaline cartilage character. It was in the fibrous cartilage structure. Moreover a few round cells resembling chondrocytes were observed especially in the deep part of the healing tissue within the fibrous matrix. In group 3, the healing tissue in the defect area was a fibrous matrix character and although most of the embedded cells in matrix were fibroblast character, the existence of cartilage-like cells was also recognized. Fibrotic tissue was observed in the superficial part of the healing tissue in the cartilage defect area but also in the deep layer close to the bone hyaline cartilage-like tissue repairing was detected. Even though all other groups were reached statistically significant better results compared with Group 1, hyalin-like cartilage repair was observed in Group 4. But this repair degree could not reach statistical significance when compared with Group 2.

As a result, in this study it was shown that the systemic vitamin D supplementation has a positive effect in the treatment of local full-thickness articular cartilage defects in rabbits. We suggest that more comprehensive and further long term studies are necessary for better understanding of the effects of vitamin D on the articular cartilage defects.

In dental implant treatment, the atrophic alveolar crest remains a challenging area for optimum rehabilitation. Growth factors have great potential for bone regeneration at the atrophied alveolar crest. Among the various growth factors, recombinant human bone morphogenic protein-2 (rhBMP-2) and recombinant human platelet-derived growth factor (rhPDGF) have received a great deal of attention. This study evaluates the efficacy of rhBMP-2 and rhPDGF-BB delivered via absorbable collagen sponge (ACS) on bone formation in guinea pig.

A total of 24 adult Dunkin-Hartley guinea pigs aged 6–7 months, weighing 700–800 g were randomly assigned initially into two groups as 15 and 45 days of healing which have 12 animals in each group. Three-millimeter-circular bone defects were created on the tibias of animals as 2 defects per each tibia and totally 4 defects per each animal. The four groups in each animal corresponded to the biomaterial used to fill the created bone defects as follows: rhBMP-2+ACS, rhPDGF-BB+ACS, ACS only (positive control), and no treatment (empty; negative control). To prevent the possibility of growth factor interactions between two biomaterials, randomization was performed so that rhBMP-2+ACS and rhPDGF-BB+ACS were not applied to the same tibia consecutively. New bone formation was evaluated histologically and histomorphometrically at 15 (early healing) and 45 days (late healing). Data were subjected to statistical analysis. Quantities of new bone were analyzed with one-way ANOVA followed by post hoc Holm-Sidak testing. Statistical calculations were performed using Sigma Stat for Windows, version 3.0; P < 0.05 was considered to be significant.

At day 15, new bone formation was seen at the peripheral portion of the defect where ACS was partly resorbed. Significant fibrous callus formation was observed in the rhBMP-2+ACS group. The amounts of fibrous callus and newly formed bone trabeculae in the rhBMP-2+ACS group were significantly higher than in the other groups (P < 0.05). Highest amount of new bone per defect area was seen in the rhBMP-2+ACS group at the end of day 15. New bone formation in the rhPDGF-BB+ACS group was lower than that observed in the rhBMP-2+ACS group on day 15. However, the amount of newly formed bone trabeculae was significantly higher than in the control groups (ACS only and empty; P < 0.05). The ratio of new bone to total defect area increased significantly over time. At the end of day 45, a large part of the ACS had been resorbed. New bone per defect area at day 45 was significantly higher than at day 15 in the rhPDGF BB+ACS group (P ≤ 0.001). In the rhPDGF-BB+ACS group, at the end of day 45, the defects were almost filled with new bone, and remodeling was determined. At the end of day 45, new bone formation was significantly higher in the rhPDGF-BB+ACS group than in the control groups (P = 0.007 and P = 0.006 for empty and ACS only groups, respectively). There was no significant difference between the rhBMP-2+ACS and rhPDGF-BB+ACS groups at day 45.

Advances in suitable carriers, coupled with the osteoinductive effects of rhBMP-2 and rhPDGF-BB, could ultimately create a future gold standard for bone grafting. Although further long-term studies and clinical trials are required, the findings presented herein suggest a promising use of rhBMP-2 and rhPDGF-BB for bone regeneration applications.

Bismuth Ferrite Harmonic Nanoparticles and Biphotonic Microscopy: Innovative Approach to Track In Vivo Muscle Stem Cells, a Promising Candidate for Cell Therapy of Muscular Dystrophy

Duchenne Muscular Dystrophy (DMD) is a fatal X-linked recessive muscle disease that represents the most common form of muscular dystrophy, affecting one in 5,000 male births. It is caused by mutations or deletions in the gene encoding dystrophin leading to the lack of dystrophin protein. This results in muscle fiber degeneration followed by severe endomysial sclerosis, leading to progressive muscle weakness and premature death at the age of 20-30 years. Currently, there is no effective treatment for DMD. In 2011, we showed that a stem cell population (named MuStem cells) isolated from healthy dog skeletal muscle induces long-term muscle repair and striking clinical efficacy after its systemic delivery in the dystrophic dog representing the clinically relevant DMD animal model. During last years, our group isolated the human counterparts (hMuStem) and demonstrated that they share the same phenotypic and in vitro behavioral features of canine cells. Currently, little is known about the homing in the muscle tissue and the whole body distribution of the MuStem cells following a systemic delivery which are nevertheless essential to determine the safety and efficacy aspect of the therapeutic strategy. To address this need, we use bismuth ferrite harmonic nanoparticles (BFO-NP, 100-120 nm range) as probes for the tracking of MuStem cells to study in vivo the hMuStem cell engraftment.

The nanoparticles are very interesting tools because capable to generate both Second Harmonic Generation (SHG) and Third Harmonic Generation (THG) signals. The origin of the signals is related to the crystalline properties of the nanoparticles, in particular its peculiar non-centrosymmetric structure. Cell tracking is therefore possible from the NPs SHG and THG signals detected with a two-photon confocal microscope A1RMP Nikon and this work provides the opportunity for long-term, three-dimensional cell tracking.

Uncoated and poly-ethylene glycol(PEG)-coated BFO-NP were investigated to label hMuStem cells in vitro. The cells were exposed to BFO-NP and BFO-PEG-NP. Localisation and cytotoxicity of the NPs were investigated in vitro during two weeks.BFO-labeled cells were tracked in the mouse Tibialis anterior muscle after their intramuscular injection by using biphotonic microscopy.

Acknowledgements

This research was supported by funds from the FEDER (Fonds Européens de Développement Régional N°37085 and 38436). It was carried out in the context of the IHU-Cesti project that received French government financial support managed by the National Research Agency via the investment for the future programme ANR-10-IBHU-005. The IHU-Cesti project is also supported by Nantes Metropole and the Pays de la Loire Region.

INTRODUCTION

There are three types of corneal dystrophies: anterior, posterior and stromal. Lattice corneal dystrophies (LCD) are a part of stromal dystrophies and characterized by an accumulation of amyloid deposition with in the stroma. LCD is diognosed with biomicroscopic examination and the diognosis confirmed by histological examination after keratoplasty. Genetic analysis may be required for specific cases.

In our study, the corneal buttons of four patients, three of them belonging to the same family, who applied to the Ophthalmology Clinic of Cerrahpasa Medical Faculty with lattice corneal dystrophy, histologically examined with light and electron microscopies.

MATERIAL-METHOD

For light microscopic examination, a portion of corneal buttons were embedded into the parafine blocks after fixation and dehidration steps. Parafine blocks were cut as 5 µ sections and stained with hematoxylen & eosin and periodic acid & Schiff (PAS). For electron microscopic examination, the other pieces of corneal buttons were fixed glutaraldehyde and osmium tetroxide (OsO₄). After the dehidration, corneal buttons embedded into araldite and the blocks were sectioned 500-700Å. The sections taken on copper grids were analyzed and photographed.   

FINDINGS

In the light microscopic examination of hematoxylin & eosin stained corneal buttons; the local deposits seen in a circular arrangment, was observed and it was suggested that these were amyloid deposits. In only one of the four patients’ corneal buttons, these deposits were seen in the stroma, but in the other three patients’, belonging to the same family, deposits increased epithelial level and led to significant damage to the epithelium. When the samples were stained with periodic acid- Schiff reagent, positive reaction was observed in these deposits. Electron microscopic examination revealed that typical amyloid fibrils also occurred in deposits.

Despite local areas were damaged due to these amyloid deposits, no inflammation was observed.

CONCLUSION

As in other corneal dystrophies, a systematic approach and histopathological evaluation, including special staining and ultrastructural studies, are critical to a correct diagnosis, also in LCD. Therefore, it is important to make a histopathologic examination for all grafts clinically diagnosed or undiagnosed.

Introduction:

Biofilm infection disease has become a serious problem as it is related to various types of refractory infections. Biofilms, with a three-dimensional (3D) structure, are formed by bacterial cells covered by a thick film of extracellular polymeric substance matrix. This 3D matrix-like structure can protect the bacterial cells within the biofilm from antibacterial drugs, rendering drug therapy against biofilm infection disease ineffective. This problem accelerates several refractory infections, such as periodontal disease and nosocomial infectious disease. Therefore, we have developed drug-encapsulated polymeric nanoparticles (NPs) for drug delivery system to treat the biofilm infection disease. If microscopic observation of efficacy of prepared NPs at a nanoscale level can be carried out, preparation of suitable NPs for drug delivery can be determined. Therefore, the goal of this study is developing a scanning transmission electron microscopy-cathodoluminescence (STEM-CL) technique for evaluation of the antibacterial activity derived from NPs on the bacterial cells within the biofilm.

Materials and method:

S. epidermidis was used as a model biofilm forming bacterial strain. An ionic liquid (IL) 1-butyl-3-methylimidazolium tetrafluoroborate was employed in sample preparation of STEM-CL observation. The STEM-CL observation was carried out by a JEM 2100M using a Gatan Vulcan TEM-CL holder with a liquid nitrogen cooling system. For the TEM observation, the sample preparation method using IL and TEM observation using the cooling holder was combined. Samples for STEM and TEM observations were cooled down to 100 K. In addition, field emission-scanning electron microscopy observation was also performed using a JXA-8530FA. Using the LIVE/DEAD BacLight bacterial viability kit, antibacterial assays of the prepared NPs on the biofilm were performed. Polymeric NPs were prepared with polymeric poly (DL-lactide-co-glycolide) (lactide:glycolide = 75:25, PLGA) NPs and Soluplus^® (Sol) micelles using the emulsion solvent diffusion (ESD) method. Clarithromycin and chitosan were used for antibacterial drug and surface modifier for NPs. The particle size and zeta potential of prepared NPs were determined using a Zetasizer Nano ZS90.

Results and discussion:

We developed the STEM-CL technique for design of polymeric NPs on the biofilm. By CL imaging of fluorescence substance derived from NPs, the antibacterial activity and ability of the biofilm removal of the prepared NPs that have never been revealed were visualized at a nanoscale level. Moreover, a combination of several types of electron microscopy observations enables to reveal different antibacterial mechanism of respectively PLGA NPs and Sol micelle NPs. CS-modified Sol micelle NPs can intrude into the bacterial cells within a short time of the treatment and exert high antibacterial activity by the induction of abnormal cell division and inhibition of cell division. The results of antibacterial assay using the LIVE/DEAD BacLight bacterial viability kit supported the electron microscopy studies. Based on the information obtained from the electron microscopy measurements, NPs can be suitably designed for the inhibition of biofilm formation and treatment against biofilm infections. The developed methodology can contribute to the nanoscale visualization of the antibacterial activity and other deformable composite materials.We expect this technique to be applicable to various fields such as pharmacy, engineering, biology, and medical science.

ACKNOWLEDGMENT

This study was partially supported by Grants-in-Aid for Scientific Research on Innovative Areas "Nano Informatics" Number 25106004, JSPS KAKENHI Grant Number 50574448. A part of this work was conducted in Nagoya University, supported by Nanotechnology Platform Program (Molecule and Material Synthesis) of the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan. The authors are grateful to Prof. M. Tanemura of Nagoya Institute of Technology, Japan for useful discussion regarding TEM measurement.

Nicotine causes the formation of free radicals and oxidative damage. Vitamin E which is required in reproduction is a potent antioxidant found in nature. In our study, due to increasing tobacco consumption in women in especially recent years it was aimed to examine the nicotine induced damage in ovary, its mechanisms and the curative effects of vitamin E on these damages.

In our study, rats (12 week old- Wistar female rats) were treated with nicotine (1 mg/kg/day, intraperitoneally (ip)) and vitamin E (α-tocopherol) (200 mg/kg/day, ip) alone or in combination in experimental groups. Levels of 17ß-estradiol, LPO (Lipid peroxidation) and MDA (Malondialdehyde) were measured in serum of the animals. Hematoxylin and eosin (H+E) staining and labeling of Ki-67 (a cellular marker for proliferation), 4-HNE (4-Hydroxynonenal; produced by lipid peroxidation in cells) and TUNEL (Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling; Apoptosis Detection)  in paraffin sections of the ovaries was performed, immunohistochemical changes were assessed by scoring system. Ultrastructural changes in araldite sections were evaluated with the electron microscopy. The data were compared statistically.

Decreased estrogen level and increased levels of LPO and MDA in serum; decreased the number of ovarian follicles and Ki-67-labeling in granulosa cells of follicles; increased 4-HNE immunoreactivity and TUNEL-labeling were observed in nicotine-treated rats. In these animals, nicotine co-treatment with vitamin E significantly reduced the increased levels of LPO and MDA in serum and 4-HNE immunoreactivity and TUNEL-labeling in ovary sections; significantly increased serum estrogen level, the number of ovarian follicles and Ki-67-labeling in granulosa cells of follicles. Vitamin E demonstrated positive and curative effect on biochemical and histological parameters compared to the control group. Ultrastructurally, frequent apoptotic profiles were observed in granulosa cells in follicles in nicotine group and vitamin E co-administration led to ultrastructural alleviation of apoptotic process.

In conclusion, our biochemical, immunohistochemical and ultrastructural level findings demonstrated that oxidative stress-induced adverse effects caused by nicotine in folliculogenesis and streoidogenesis occur via lipid peroxidation in granulosa cells. Our data suggest that vitamin E may represent an alternative of pharmacologic treatment for preserving fertility owing to its beneficial effects on the nicotine induced damages occurred in the granulosa cells.

Tick-borne encephalitis virus (TBEV) is the etiological agent of Tick-borne encephalitis, a serious neurological human infection. The endemic region is Europe and Asia with more than 10,000 clinical cases every year. We used electron tomography (ET) technique for visualization of neural cells infected with TBEV prepared by high-pressure freezing followed by freeze substitution and embedded into epoxy resin [1, 2].  Images of three dimensional volume of the section enables better to understand alterations of neural cells caused by TBEV than 2D Transmission electron microscopy (TEM) projection images, where the visibility of structures depends crucially on section thickness. Here, we showed how to 3D visualization influenced an interpretation of obtained TEM results. Next, we discuss our experience with settings of optimal imaging conditions, appropriate resolution given by Crowther criterion [3], elongation factor [4] and necessity of using of the single or dual tomography.  ET was done like a single/dual axis using JEOL 2100F equipped with high tilt stage and Gatan camera Orius SC 1000 controlled with Serial EM software. Tomograms were aligned and reconstructed using IMOD software package. 3D models were generated by manually masking of the area of interest by means of IMOD or Amira.

Our results depicted mainly changes in organisation of rough endoplasmic reticulum (RER) induced by TBEV and a formation of virus-induced sub-compartments of RER called smooth membrane structures including a presence of pores. We found that cisterns of RER are often arranged into two tightly apposed cisterns. Depending on the type of host cells, used TBEV strains and length of infection, we described proliferation of RER with harboured sites of replication (3 days post infection) which are reorganised during the extensive infection stress (12 days post infection) into whorls with central part having features of an autophagosome [2]. In primary neurons infected by TBE strain Neudoerfl, virus-induced tubule-like structures (18 – 22 nm in diameter) were frequently observed inside of reorganised RER. Most of these structures were connected to the TBEV and run either in parallel lines or in different directions. In several cases, these tubule-like structures were surrounded with other tubules of 43.8 ± 4.3 nm (N = 7) in diameter [2]. Finally, we imaged vacuoles containing individual virions with nucleocapsids associated with host microtubules [2].

The study was supported by the Technology Agency of the Czech Republic (TE01020118) and from the program for large research infrastructures of the Ministry of Education, Youth and Sports within the project „National Infrastructure for Biological and Medicinal Imaging  (Czech-BioImaging LM2015062)

1. M. Palus et al., J. Gen. Virol. (2014) Nov. 95(Pt 11):2411-26. doi: 10.1099/vir.0.068411-0
2. Bily et al., Sci Rep. (2015) Jun 15; 5:10745. doi: 10.1038/srep10745
3. R. A. Crowther et al., Proc. Roc. Soc. Lond. (1970). A 317.
4. M. Radermacher, J. of El. Micro. Tech. 9 (4) (1988). 359–394

Rotavirus (Reoviridae) viral particles consist of capsids with 3 concentric protein layers surrounding the virus genetic material. The outer layer is composed of the VP4 and VP7 proteins, which independently elicit neutralizing antibodies and induce protective immunity.

Rotarix™, GSK live attenuated Rotavirus vaccine, was characterized by EM. The observations show that the vaccine contains both triple-layered particles (TLPs) containing all 3 protein layers and double-layered particles (DLPs) which lack the outer capsid proteins VP4 and VP7.

As DLPs are non-infectious, it may be of importance to address the question of their abundance vs. TLPs in bulk preparations. Although such information can be obtained by EM, the method is relatively impractical for this purpose, particularly for routine analysis, as high number of particles should be observed and sized to get accurate relative abundances.

To determine the size and quantify the relative abundance of TLPs and DLPs on large populations of viral particles in bulk preparations, two size-based, orthogonal analytical techniques were applied to detect these particles: disc centrifuge (DC) and capillary electrophoresis (CE).
To confirm the capacity of both methods to distinguish clearly the two types of capsids, TLPs and DLPs were partially purified using CsCL gradient. They were characterized by EM negative staining (Fig. 1) and cryo-TEM (Fig. 2). Their apparent diameter difference of about 10 nm was well correlated in both techniques. The profiles obtained by DC and CE (Fig. 3) show the well-resolved identification.

In addition, further confirmation was obtained by incubation of the preparations with EDTA, to remove the calcium required for VP7 protein stabilisation and thus to convert TLPs into DLPs [¹]. The shift from TLPs to DLPs is clearly visible with CE (not shown), DC and also EM (Fig. 4).
Dynamic Light Scattering (DLS) was also evaluated and works fine with the partially purified samples of either TLPs or DLPs. The shift from TLPs to DLPs by this method is clearly observable as well (not shown). However, DLS does not allow distinguishing clearly the two populations of capsids in real samples with mixed TLPs and DLPs.

These orthogonal structural and sizing analysis methods can therefore be combined to assess and measure accurately DLPs/TLPs ratio. They thus provide a tool to understand the possible link of this ratio with the immunological potency of vaccine preparations.

1. Mathis PK, Ciarlet M, Campbell KM, Wang S, Einterz Owen K, Ranheim      TS (2010) Separation of rotavirus double-layered particles and      triple-layered particles by capillary zone electrophoresis. Journal of      Virological Methods 169: 13-21

Protection of organ function is important factor for the success of organ transplantation. Protection activity and also protection time period are dependent on a large scale of  pre-transplant preservation solutions. Therefore, donor organs are being preserved in special preservation solutions such as University of Wisconsin (UW), Histidin-Triptophan-Ketogluterate (HTK) and Celcior solution. For that aim, UW is the most preferred solution, even so, this solution could not preserve the organs over 36 hours. In this study, the ingredients of the UW solution were changed to extend the preservation time and also to provide more effective protection. Rat model is used for investigation of time related morphological changes between UW (group I) and Modified UW (Mod UW) (group II) solutions during preservation time. Rat kidneys were perfused with respective solutions. Totally perfused kidneys were removed and placed either in UW or Mod UW solutions and also kept during 2, 10, 24 and 72 h at 4⁰ C. Kidney tissue samples were taken at given time intervals and fixed with 10% formaldehyde, and then these samples were prepared for paraffin sections. These tissue samples were also taken at 72nd hour, they were fixed in 2.5% glutaraldehyde and processed for epoxy resin embedding. For light microscopical evaluation, renal injury, based on tubular and glomerular degeneration, inflammatory cell infiltration, and vasocongestion has been scored with using a scale ranging from 0 to 3 (0: none; 1: mild; 2: moderate; and 3:severe) for each criterion. Liquid samples were also taken 2, 10, 24 and 72 h at 4⁰ C from the storage media and investigated for LDH activity which was measured using standard chemistry method (Siemens ADVIA 1800 chemistry System). Microscopical examinations showed that Mod UW solution provided good preservation for all time intervals than UW solution. These results are consistent with lactate dehydrogenase (LDH) levels. Vacuolated tubular cells, as well as decreased renal tubules and glomerular degeneration described a prominent tissue injury revealed at 72nd h of UW group.  Podocyte degeneration, formation of hump with GBM thickening revealed in 72nd h of UW group at transmission electron microscopical level. These degeneration parameters were observed less than in 72nd h of Mod UW group. It was concluded that Mod UW solution at low temperature is very effective and suitable for preservation up to 72 h.

References

1. Goktas C, Coskun A, Bicik Z, Horuz R, Unsal I, Serteser M, Albayrak S, Sarica K. Evaluating ESWL-induced renal injury based on urinary TNF-α, IL-1α, and IL-6 levels. Urol Res, 40: 569–73, 2012

2. Gunal O, Coskun A, Aslaner A, Yildirim U. Does melatonin alleviate cold preservation injury of the liver Turk J Med Sci, 40: 465–70, 2010

3. S Öner, F Ercan, S Arbak: time-dependent morphological alterations of cold-stored small bowel in Euro-Collins and Ringer’s lactate solutions. Acta Histochemica, 10: 207–217,2004

This study was financed by The Scientific and Technological Research Council of Turkey (TUBITAK), project number 113S847

Organ transplantation solutions are used for protection of organ functions for a long time. In order to provide long lasting time period for organ preservation, donor organs are kept in special preservation solutions such as University of Wisconsin (UW), Histidine-Triptophane-Ketogluterate (HTK) and Celcior solutions. Although UW is the most preferred solution, preservation time even for the kidney tissue, has not exceeded over 36 hours. The aim of this study is to extend the preservation time and also provide more effective protection. In order to provide better preservation,  the ingredients of the UW solution were changed (Mod UW), as well as melatonin was included (Mod UW+M)  in the preservation medium. Time-related morphological changes of rat kidneys in UW (group I), Modified UW (group II) and Modified UW+Melatonin (Mod UW+M) (group III) were comparatively investigated within this study. Totally perfused kidneys were removed and placed in UW, Mod UW and Mod UW +M solutions and kept during 2, 10, 24 and 72 h at 4⁰ C. Kidney tissue samples were taken at all given time intervals and these samples were prepared for paraffin sections. Kidney tissue samples, taken at 72nd hour, were fixed in 2.5% glutaraldehyde and prepared for epoxy resin embedding. Histopathological scoring system were performed for light microscopical evaluation by using a scale ranging from 0 to 3 (0: none; 1: mild; 2: moderate; and 3:severe) for each criterion. Histopathological parameters were renal injury, based on tubular and glomerular degeneration, inflammatory cell infiltration and vasocongestion. Liquid samples, taken at 2, 10, 24 and 72 h at 4⁰ C from the storage media, investigated for lactate dehydrogenase (LDH) activity which was measured using standard chemistry method (Siemens ADVIA 1800 chemistry System). Comparative findings at light and transmission electron microscopical levels revealed that preservation in Mod UW+M solution was statistically much more prominent in all time intervals, significantly at 72nd hour of preservation.  In all groups, LDH levels were consistent with morphological results. We could conclude that Mod UW+M was the most effective solution among the  experimental groups, especially  suitable for preservation up to 72 h. In this study,  the important efficacies of UW solution, a better protectant and melatonin,  an effective agent to be used in such systems, were highlighted.

References

1. Goktas C, Coskun A, Bicik Z, Horuz R, Unsal I, Serteser M, Albayrak S, Sarica K. Evaluating ESWL-induced renal injury based on urinary TNF-α, IL-1α, and IL-6 levels. Urol Res, 40: 569–73, 2012

2. Gunal O, Coskun A, Aslaner A, Yildirim U. Does melatonin alleviate cold preservation injury of the liver Turk J Med Sci, 40: 465–70, 2010

3. S Öner, F Ercan, S Arbak: time-dependent morphological alterations of cold-stored small bowel in Euro-Collins and Ringer’s lactate solutions. Acta Histochemica, 10: 207–217,2004

This study was financed by The Scientific and Technological Research Council of Turkey (TUBITAK), project number 113S847

Starting in the 1960ies, with a common availability of high resolution microscopes and the introduction of negative staining and ultrathin sectioning of microwave embedded infectious tissues, pathogen diagnosis on basis of electron microscopy (EM) has become increasingly important. An EM-based lab diagnosis turned out to be useful in a wide spectrum of indications like "rapid diagnosis in emerging diseases", "search for otherwise undetectable agents" ("open view"), the need for "catch-all-methods" and/or "rapid differential diagnosis". Since then, the use of transmission electron microscopy, and to a lesser extent scanning electron microscopy provide a multiplex platform for the detection and differentiation of a wide range of pathogens (Fig. 1-3). Examples will be given. In past, EM was involved with the detection and identification of broad range of viruses some of which have impacted on human, veterinary and wildlife health. Many of the diseases caused by viruses have emerged from anthropogenic based perturbations of the environment including altered habitat e.g. changes in the number of vector breeding sites and/or host reservoirs, niche invasions, changes in biodiversity, genetic changes of disease vectors or pathogens, and environmental contamination of infectious agents.

However, the use of diagnostic EM declines radically since the late decade of the 20th century which was characterized by a continuous shifting of research interests to molecular biology. With whole genome sequencing some of the indications mentioned above like "catch-all-method" or "search for undetectable pathogens - open view" seem to be on first sight no longer existing. Is it now a change of paradigm, a still going onward decline or may diagnostic EM play still an essential role in pathogen diagnosis? The future of diagnostic EM, its constraints and its welfare for the society is discussed. Be aware, since September 11^th executive authorities have noticed that molecular biology and EM complement each other and most modern diagnostic centres of disease control still execute both techniques in their diagnostic programme; but for how long?. Additionally increased mobility, changes in life style, global trade and social unrest are supporting the spread of emerging or re-emerging infections. Anyway, we should be aware that molecular biology of today with whole genome sequencing as well as electron microscopy bear a lot of uncertainties in their technical procedure - technical sources of error which should be always be in mind during diagnostic procedure.

Spinal muscular atrophy (SMA) the second most common autosomal recessive disorder causes of childhood mortality affecting between 1/6000 to 1/10000 live births. SMA is due to deletion/mutations in the Survival Motor Neuron 1 (SMN1) gene. The disease is characterized by degeneration of spinal motor neurons (MNs), atrophy of skeletal muscles, and generalized weakness. SMA is clinically classified into four main types (I, II, III and IV) based on the age of onset and clinical severity. Currently, no curative treatment exists for SMA type II/III SMA patients representing the majority currently awaiting treatment.

Gene therapy, restoring SMN1 activity in MNs, is a promising therapeutic strategy for SMA. In the moderate forms (type II/III) of the disease, no lesion is found in peripheral organs. Thus, a targeted therapy directly into the CNS should be more appropriate. We have, recently, demonstrated that a single administration of an rAAV9 vector into the cerebrospinal fluid leads to an efficient transduction of MNs in non-human primates.

In order to treat our mouse model that can mimick the human pathology of type II/III SMA, the first main challenge was to develop an efficient and safety route of delivery that can target whole spinal cord and brain. In this context, we develop the lumbar intrathecal delivery (itlumb), by single percutaneous lumbar puncture. We demonstrate that a single lumbar intrathecal injection of rAAV9-eGFP vector using specific neuronal promoter can efficiently and specifically target MNs from proximal to distal part of the spinal cord in adult mice, wherease, using rAAV9-eGFP ubiquitous promoter, we observe both glial cells and MNs targeting (figure a, b and c). Surprisingly, by lumbar administration, we efficiently reach the brain and target neurons and glial cells using rAAV9 ubiquitous promoter (figure g).

Then, we compared the serotypes 9 and 10 of AAV vector in order to demonstrate which serotype is the most efficient to target MNs after intrathecal delivery. We used the Synapsin 1 (SYN1) promoter, that lead to specific expression in neuron cells population, in adult mice, by lumbar intrathecal administration. We observed a spectacular and specific MNs expression with the serotype 9 around 50% and 10 % in the lumbar and cervical spinal cord respectively. Wherease the serotype 10 transduce less significantly MNs cells (30% and 5 % in the lumbar and cervical spinal cord respectively).

            This results, offer new perspectives for the treatment of motor neuron diseases such as moderate forms of spinal muscular atrophy.

Acknowledgments : We thank the vector core of the Atlantic Gene Therapies Institute (AGT) in Nantes for the preparation of the rAAV vectors, Véronique Blouin and Philippe Moullier (INSERM UMR1089) for vector production and the technical staff of Oniris rodent facility for animal care. This work was supported by a  grant from “Investissement d'Avenir - ANR-11-INBS-0011”  - NeurATRIS :  A Translational Research Infrastructure for Biotherapies in Neurosciences.

Gastric digestion is the result of physical disintegration, acidic hydrolysis and enzymatic reactions leading to the release of nutrients which are absorbed in the upper intestinal tract. Protein is one of the essential macro-nutrient and can be eaten in a great variety of forms (solubilized, cross-linked, in their native or denatured states). Controlling food protein gelation conditions result in the formation of particles with specific structural features. Several in vivo and in vitro studies have shown an influence of the macro- and microstructure on the kinetics of milk protein hydrolysis. Nevertheless, the mechanisms by which the structure of dairy gels can affect the digestion kinetics remain largely unknown.

The aim of the study was to assess the part play by HCl and gastric enzyme (i.e. pepsin) during gastric digestion using a dynamic and label-free imaging technique on the DISCO beamline of Synchrotron SOLEIL to visualize in situ the milk protein gels breakdown kinetics. The DISCO beamline uses the deep ultraviolet range to probe the intrinsic UV tryptophan fluorescence without the need of specific external probes. Two milk gels with the same protein concentration but different microstructures were prepared either by rennet or acid coagulation of non-fat milk. The disintegration of the different networks was monitored under digestion at body temperature in simulated gastric fluids and the effect of the acidic environment uncoupled from the enzyme effect. The evolution of particle area and mean fluorescence intensity has been determined, and used to estimate the kinetics of food particles breakdown. 

The kinetics of acid gel in vitro digestion was significantly reduced compared to rennet gel. Our data indicate that rennet gel has a two-step behavior during the acidification phase with a swelling followed by a contraction of the particle, not observed for acid gel. In addition, these microstructural modifications of rennet gel affect negatively the enzymatic breakdown kinetics of particles compared to acid gel.

This study leads to original methodological developments both from the point of view of the acquisition of data and their joint analysis. Getting in situ information about digestion kinetics, microstructural transformation and enzymatic reaction, allow further analysis of the digestion process.

The strong relation between inhaled asbestos fibers and development of important respiratory diseases (asbestosis, pulmonary carcinoma, mesothelioma) is now demonstrated by many researches and accepted by the international community. The peculiarity of the minerals defined by the law “asbestos” is their growth as long and thin fibers that, as a consequence of the crystal habit, reach the respiratory apparatus, where may lie during long times –decades- displaying their carcinogenic effects. The longer asbestos fibers may be coated by iron-proteins, forming the well-known “ferruginous bodies”, which represent a way to isolate the fiber in the biological system [1]. It must be remarked that the observation of fibers or ferruginous bodies allow to prove exposure, environmental or working, to asbestos. Nevertheless “ferruginous bodies” may be formed also around elongated minerals/crystals, not belonging to asbestos group. In order to determine surely exposure to asbestos it appears therefore very important to recognize the chemical composition of the fiber inside the coating and identify the mineral phase. A new methodology allowing the sure identification of the mineral phase of a fiber coated in a “ferruginous body” [2], using Environmental Scanning Electron Microscopy (E-SEM) coupled with Energy Dispersive Spectroscopy (EDS), will be presented.

Our University is placed near Casale Monferrato, a town of Piedmont, Italy, sadly famous for the high incidence of asbestos related diseases, in particular mesothelioma, as consequence of the presence of an Eternit factory working large contents of asbestos until 1985. In several patients suffering of mesothelioma also serious disorders of the biliary tract and/or of the gallbladder have been often diagnosed. Nevertheless, the seriousness of the damages to the respiratory system reduce the attention to other organs. In order to determine the presence of asbestos fibers in the gallbladder or in the biliary tract of patients suffering both of mesothelioma and of disorders in this gland, histological sections of gallbladder and of bile have been observed under E-SEM/EDS. All the inorganic phases observed have been characterized under their morphological and chemical aspects [3]. By this work it can be proved the presence of asbestos fibers or bundles of asbestos fibers, as shown in the figure, in particular of crocidolite and chrysotile, in almost all the examined sections. The results obtained during this work will be presented and discussed.

References

1. Rinaudo C, Croce A, Musa M, Fornero E, Allegrina M, Trivero P, Bellis D, Sferch D, Toffalorio F, Veronesi G, Pelosi G. Study of inorganic particles, fibers, and asbestos bodies by Variable Pressure Scanning Electron Microscopy with annexed Energy Dispersive Spectroscopy and micro-Raman spectroscopy in thin sections of lung and pleural plaque. Applied Spectroscopy, 2010, 64:571-577.

2. Croce A, Musa M, Allegrina M, Trivero P, Rinaudo C. Environmental scanning electron microscopy technique to identify asbestos phases inside ferruginous bodies. Microscopy and Microanalysis, 2013, 19:420–424.

3. Grosso F, Randi L, Croce A, Mirabelli D, Libener R, Magnani C, Bellis D, Allegrina M, Bertolotti M, Degiovanni D, Rinaudo C. Asbestos fibers in the gallbladder of patients affected by benign biliary tract diseases. European Journal of Gastroenterology and Hepatology, 2015, 27:860-864.

Introduction

Nanomedical approaches in cancer treatment have the potential to enhance the effectiveness of localized therapy while minimizing side effects on the surrounding healthy tissue. The aim of our study was the development of a novel hybrid nanoparticles (hNPs) based on magnetic core (FePt) and photothermaly active shell (SiO₂/Au) for targeting of cancer tissue. hNPs should enable controlled triggering of photothermic drug release, followed by extraction of hNPs from the body using an external magnetic field. We established biomimetic in vitro models of normal and cancer urothelial cells for testing proof-of-concept, biocompatibility, targeting, release, photothermic effects and extraction capabilities.

Materials, methods

Superparamagnetic SiO₂-coated FePt nanoparticles have been used as seeds for the growth of the Au shells [1]. Au nanoshells were coated with polyethylene glycol (PEG), because of its known biocompatibility, and improved in vivo stability. Finally, for the sterilization of the final suspension the sterile filters were used (sterile Millex-GP syringe filter unit). Normal porcine urothelial cells – (NPU), low-grade (RT4) –  and high-grade (T24) cancer urothelial cells - were grown on plastic petri dishes in growth media with different basic ingredients (UroM or A-DMEM/F12) at 37 °C in CO₂-incubator for up to 3 weeks [2]. To determine cytotoxic effects, hNPs were added to the culture media (100 µg/mL) for 2h in the presence and absence of the magnetic field (a Nd-Fe-B permanent magnet m₀H=0.3 T measured right at the top of the magnet)). The viability was measured by counting trypan-blue stained cells. The cellular distribution of hNPs was determined by TEM. Cells were fixed with 4% FA + 2% GA in 0.1M cacodilate buffer for 3h at 4°C, post-fixed with 1% OsO₄ for 1h at 4°C, dehydrated in ethanol and embedded in Epon. Ultrathin sections were observed under transmission electron microscope (TEM, Philips CM100) running at 80kV.

Results

Schematic representation and a TEM image of hNPs are shown in Figure 1. The survival of urothelial cells, which were exposed to hNPs, has been >85 % in the absence as well as presence of the magnetic field. TEM showed hNP on the apical plasma membrane and in endocytotic structures of urothelial cells. While hNPs were primarily distributed individually or in small clusters (>20 particles) on the apical surface of the urothelial cells, they were also found individually or accumulated in the endosomal compartments of the urothelial cells (Figure 2). High-grade cancer urothelial cells showed higher intensity of endocytosis compared to low-grade cancer and normal urothelial cells.

Discussion and Conclusion

We have (i) optimized the culture conditions for long-term growth and differentiation of normal and cancer urothelial cells and therefore established relevant biomimetic in vitro models of normal and cancer urothelial cells, (ii) shown that used hNPs are biocompatible in the presence and absence of magnetic field, and (iii) that hNPs could be internalized into the endosomal compartments of the cells. Our study confirmed proof-of-concept and provides a foundation for the next stage studies of hybrid nanomaterials, aiming at the development of smart diagnostic, targeted drug delivery and stimuli-responsive release system.

References

[1] Kostevšek N.,  Žužek Rožman K.,  Arshad M. S.,  Spreitzer M.,  Kobe S., Šturm S. Multimodal hybrid FePt/SiO2/Au nanoparticles for nanomedical applications: combining photothermal stimulation and manipulation with an external magnetic field. J Phy Chem C. 2015;119(28):16374−16382

[2] Imani R, Veranič P, Iglič A, Kreft ME, Pazoki M, Hudoklin S. Combined cytotoxic effect of UV-irradiation and TiO2 microbeads in normal urothelial cells, low-grade and high-grade urothelial cancer cells. Photochem Photobiol Sci. 2015 Mar;14(3):583-90

Normal Betula pollen grains are triporate, i.e. having three pores, whereas genetically abnormal or deformed grains are usually not. We collected samples of pollen from 92 individual Betula trees/shrubs growing in natural birch woodlands in Iceland.  The trees were previously identified as being diploid (2n=28) dwarf birch Betula nana (31 plants) and tetraploid (2n=56) downy birch B. pubescens (39 plants), whereas 22 plants were found to be triploid (2n=42) hybrids of the two species [1]. The results [2] showed clearly that the two species mostly produced normal triporate pollen, whereas damaged and deformed grains were significantly more frequent among pollen samples from triploid hybrids. The most frequent type of deformity in pollen morphology was pollen with four pores instead of the normal three. Meiosis in the microspore mother cells was also examined and as expected the triploid plants had irregular meiotic figures and produced deformed microspores (unpublished). We therefore investigated the fertility of these triploid trees, by testing pollen viability and assessing seed germination. The results (unpublished) confirmed that the fertility of triploid hybrids was severely reduced. The good news is that triploid hybrids are not completely sterile, and a few individuals under study are even as fertile as the parental species can be. This discovery supports our botanical and molecular studies of introgressive hybridization in Betula [1, 3], whereby triploid hybrids serve as a bridge of gene flow across the two species via back-crossing.

The knowledge that triploid birch hybrids produce abnormal pollen has been utilized in our search for past hybridization events in the Holocene vegetation history of Iceland. Samples from peat were collected in three locations in Iceland: Eyjafjördur (N), Grímsnes (SW) and Thistilfjördur (E). In all three places, periods of elevated proportions of abnormal Betula pollen were detected [4 - 6]. By comparison to climate data from the Greenland Ice Core Project, the effect of climate on the birch woodlands can be seen. The hybridisation periods were found to be connected to the advance of woodland-forming downy birch over dwarf birch habitat in warming climate. Such hybridisation may have taken place in most parts of northern Europe when woodland expanded in the beginning of the Holocene. In Iceland the climate stayed near the lower limits of birch woodland tolerance for most of the Holocene, repeatedly creating conditions that facilitated hybridization. With the warming of climate in the last few decades a new wave of birch hybridisation has started.

References:

[1] Thórsson ÆTh, Pálsson S, Sigurgeirsson A, Anamthawat-Jónsson K (2007) Ann Bot 99: 1183-1193.  [2] Karlsdóttir L, Hallsdóttir M, Thórsson ÆTh, Anamthawat-Jónsson K (2008) Grana 47: 52-59.  [3] Thórsson ÆTh, Pálsson S, Lascoux M, Anamthawat-Jónsson K (2010) J Biogeogr 37: 2098-2110.  [4] Karlsdóttir L, Hallsdóttir M, Thórsson ÆTh, Anamthawat-Jónsson K (2009) Rev Palaeobot Palynol 156: 350-357.  [5] Karlsdóttir L, Hallsdóttir M, Thórsson ÆTh, Anamthawat-Jónsson K (2012) Rev Palaeobot Palynol 181: 1-10.  [6] Karlsdóttir L, Hallsdóttir M, Eggertsson Ó, Thórsson ÆTh, Anamthawat-Jónsson K (2014) Iceland Agric Sci 27: 95-109.

Natural materials can inspire the production and development of materials that can be environmentally friendly, cheap and have a variety of applications. Fungal hydrophobin proteins, in particular, have several interesting traits that make them an ideal sample to investigate. These proteins are found only in the fungal kingdom and are known to play several distinct roles in the life and development of different fungi: acting as a biosurfactants to allow formation of aerial hyphae, protecting the spores from the environment, acting as an adhesive film during germination. This is all possible due to the hydrophobin protein’s unique amphiphile structure and the ability to self-assemble into an amphiphilic film at any hydrophobic:hydrophilic interface. At such an interface, the hydrophobins undergo a conformational change to form amphiphilic monolayers. With certain hydrophobins, known as class I, these monolayer films are very robust under acidic and basic conditions, and have been shown to be able to reverse the hydrophobicity of any surface.

This body of research has been investigating the structure of the amphiphilic films formed by the hydrophobin EAS_∆15, a truncated version of the wild type hydrophobin EAS, found on the surface of spores of Neurospora crassa. Additionally, we have compared the ability of EAS_∆15 and the hydrophobin RodA, which is found on spores produced by Aspergillus fumigatus, to form hydrophobin protein films with the ability to alter surface hydrophobicity.

By transmission electron microscopy (TEM), it was possible to visualise the morphology of the hydrophobin films formed by EAS_∆15 at the hydrophobic:hydrophilic interface upon a holey carbon grid. Changes in the film morphology were observed when the film was formed from protein solutions with increasing amounts of alcohol, and hence exhibiting variation in surface tension. A TEM tomogram of the hydrophobin film was constructed from 168 TEM images tilted at ±60ᵒ. 3D topography information was collected from atomic force microscopy (AFM) to confirm the morphology visualised on TEM and compare the resulting models. Both imaging techniques revealed rodlet structures, which exhibit a “double track” structure and forms nanosized ridges.

Hydrophobin proteins can vary in sequence length and the amount of hydrophobic residues within the sequence; this provides a variety of functionalities for different fungi.  Two different class 1 hydrophobin proteins, RodA and EAS_∆15, were used to compared the difference in their ability to alter the surface hydrophobicity of a) hydrophobic surfaces (OTS silicon wafer) and b) hydrophilic surfaces (StarFrost^(R) Superclean hydrophilic glass slides). Hydrophobin proteins were solubilised in deionised water (5µg/mL) and a 50 µL droplet of the protein solution was allow dry down to form a film upon the OTS silicon and hydrophilic glass. The hydrophobin protein films were able to reduce the contact angle of water from 104±2 ᵒC on bare OTS silicon wafer to 73±4 ᵒC for EAS_∆15 coated surface and 81±7 ᵒC for RodA coated surface. For the hydrophilic glass, the contact angle for water on the bare surface was increased from 31±3 ᵒC to 59±4 ᵒC for the EAS_∆15 coated surface and the 58±8 ᵒC for RodA coated surface. AFM images indicates that even when protein films are formed from solutions with such low protein concentration, both surfaces were coated with enough hydrophobin protein to form a layer that effects the hydrophobicity of both the OTS silicon wafer and hydrophilic glass surfaces.

Nowadays pollen allergies become an increasing problem for human population. Mugwort (Artemisia vulgaris) and hazel (Corylus avellana ) are major herbaceous allergenic plants in Europe [1-3]. In this study the effect of vehicle pollutants on the structure and chemical composition of mugwort and hazel pollen were investigated. For this purpose pollen of the respective plants were collected from three sites with different vehicle pollution level. Scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR) and curve-fitting analysis of amide I profile was performed to assess the structural changes of mugwort and hazel pollen. SEM imaging did not reveal any differences in shape or any physical degradation of the hazel (Fig. 1a, b) and mugwort (Fig. 1c, d) pollen grains collected from the different sites. It was found that infrared spectra look the same for pollen collected at sites with high traffic pollution. Conversely, they differ a lot from spectra of respective pollen types collected from sites without pollution. Moreover, structural changes in proteins, observed in the second derivative of the FTIR spectra and in the curve-fitting analysis of amide I profile, are a consequence of mutations occurring in the genetic material of pollen, which can be caused inter alia by air pollutants [4]. The results suggest, that mugwort and hazel pollen chemical composition may be a good indicator of air quality and FTIR may be applied in biomonitoring.

Acknowledgements:

Grant n° UMO-2014/13/B/ST5/04497 is acknowledged for the financial support of the SEM Tescan Vega 3 instrument.

References:

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2. Sozańska B., Błaszczyk M., Pearce M., Cullinan P.  2014. Atopy and allergic respiratory disease in rural Poland before and after accession to the European Union. J. Allergy Clin. Immunol. 133(5):1347-1353.

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According to the US National Nanotechnology Initiative, nanoparticles are a set of solid particles with a diameter ranged from 1 to 100 nm. Numerous domains employ nanoparticles (such as industry, cosmetic, construction, medicine…) due to their specific properties.

The significant arise of nanomaterials application leads to a boost of environmental discharges and especially in aquatic systems which represent an important pollutants receptacles. Nevertheless, the lack of knowledge about the toxicity of nanomaterials can be detrimental for the aquatic ecosystems sustainability. Trophic transfer is often referred as an important pathway of nanoparticles contamination in aquatic systems being reportedly the main exposure route to organisms. Among their great variety of nature and characteristics, gold nanoparticles (AuNPs, PEG coating, diameter 10nm) have been chosen as model contaminant due to their high stability in solution.

This work aims to characterize: (i) AuNPs transfer within food chain and (ii) a histological study of AuNPs damages in fish. Thereby natural river biofilms contaminated in laboratory for 48h at environmental AuNPs’ concentrations were grazed by the fish Hypostomus plecostomus during a 21-days laboratory experiment. Gold analyses (by atomic absorption spectroscopy) revealed that biofilms presented a high AuNPs retention capacity. Secondly, results point out that AuNPs were effectively transferred from natural biofilms to the grazer fish showing their ability to enter the food chain. Regarding AuNP fish distribution, organs involved in metabolism and excretion (i.e. liver and kidney) presented a significant bioaccumulation. Moreover, transmission electron microscopy observations showed tissue alterations indicating inflammatory responses for all organs studied. AuNPs appeared to be: (a) distributed by the circulatory system into fish organs without entering the erythrocytes and (b) not degraded once internalized by the fish.

Nanomaterials represent today a major economic and technological issue, because of their innovating properties rising from their nanometric size (1 to 100 nm) and in particular of their chemical compositions, sizes, specific surfaces and varied surface qualities. These nanomaterials are present in sectors as varied as building, car industry, chemistry, energy or health, and therefore can be found in natural ecosystems without knowing yet their potential impacts.

In this context, trophic transfer of gold nanoparticles (AuNPs) between periphytic biofilms and a periphytophage fish Hypostomus plecostomusas final consumer has been studied in laboratory. Histological analyses have been performed at the two trophic levels with a particular focus on transmission electronic microscopy (TEM) observations in fishes.

In upstream running waters, periphytic biofilms are constituted by the association of micro-organisms secreting an organic polymermatrix. Diatoms (brown microalgae) as a major component of these biofilms ensure a great part of the primary productionin the upstream running watersand are at the basis of their food web.

A contamination of these microorganisms by AuNPs could be vector of a trophic transfer of these nanomaterials along the trophic chain. Thus we determined if a trophic transfer of AuNPs was possible between natural periphytic biofilms and the fish Hypostomus plecostomus. H. Plecostomus is a tropical benthic fish with a ventral mouth modified into suction cup and adapted to graze the periphytic biofilms.

Analyses of gold bio-accumulation in fishes were performed after 7 days of contamination, followed by a period of depuration of 14 days. These analyses were supplemented by an observation of AuNPs localization in tissues and cells by TEM and the toxic effects of AuNPs were also observed in them.

The results showed a trophic transfer of AuNPs between biofilms and fish, with a significant gold bioaccumulation in several organs (heart, spleen, liver, muscles). TEM observations revealed also a concentration of AuNPs in the spleen of the fish, localized especially in the erythrocytes and an important destructuration of the muscle fibers. After depuration, AuNPs totally disappeared from the erythrocytes, but were concentrated in the melanomacrophages, along with a recuperation of the muscles fibers, suggesting an important capacity of this fish to detoxify this type of nanoparticles.