Chromogranin A preferential interaction with Golgi phosphatidic acid induces membrane deformation and contributes to secretory granule biogenesis.

Chromogranin A (CgA) is a key luminal actor of secretory granule biogenesis at the trans-Golgi network (TGN) level but the molecular mechanisms involved remain obscure. Here, we investigated the possibility that CgA acts synergistically with specific membrane lipids to trigger secretory granule formation. We show that CgA preferentially interacts with the anionic glycerophospholipid phosphatidic acid (PA). In accordance, bioinformatic analysis predicted a PA-binding domain (PABD) in CgA sequence that effectively bound PA (36:1) or PA (40:6) in membrane models. We identified PA (36:1) and PA (40:6) as predominant species in Golgi and granule membranes of secretory cells, and we found that CgA interaction with these PA species promotes artificial membrane deformation and remodeling. Furthermore, we demonstrated that disruption of either CgA PABD or phospholipase D (PLD) activity significantly alters secretory granule formation in secretory cells. Our findings show for the first time the ability of CgA to interact with PLD-generated PA, which allows membrane remodeling and curvature, key processes necessary to initiate secretory granule budding.

Antibody-Conjugated Nanocarriers for Targeted Antibiotic Delivery: Application in the Treatment of Bacterial Biofilms.

Conventional antibiotic treatment is in most cases insufficient to eradicate biofilm-related infections, resulting in high risk of treatment failure and recurrent infections. Recent studies have shown that novel methods of antibiotic delivery can improve clinical outcomes and reduce the emergence of antibiotic resistance. The objectives of this work were to develop and evaluate a targeting nanocarrier system that enables effective delivery of antimicrobial drugs to Staphylococcus aureus, a commonly virulent human pathogen. For this purpose, we first prepared a formulation of polymeric nanoparticles (NPs) suitable for encapsulation and sustained release of antibiotics. A specific antibody against S. aureus was used as a targeting ligand and was covalently immobilized onto the surface of nanoparticulate materials. It was demonstrated that the targeting NPs preferentially bound S. aureus cells and presented an elevated accumulation in the S. aureus biofilm. Compared to free-form antibiotic, the antibiotic-loaded targeting NPs significantly enhanced in vitro bactericidal activity against S. aureus both in planktonic and biofilm forms. Using a mouse infection model, we observed improved therapeutic efficacy of these antibiotic-loaded NPs after a single intravenous administration. Taken together, our studies show that the targeting nanoparticulate system could be a promising strategy to enhance the biodistribution of antibiotics and thereby improve their efficacy.

Lymphatic and Immune Cell Cross-Talk Regulates Cardiac Recovery After Experimental Myocardial Infarction.

OBJECTIVE: Lymphatics play an essential pathophysiological role in promoting fluid and immune cell tissue clearance. Conversely, immune cells may influence lymphatic function and remodeling. Recently, cardiac lymphangiogenesis has been proposed as a therapeutic target to prevent heart failure after myocardial infarction (MI). We investigated the effects of gene therapy to modulate cardiac lymphangiogenesis post-MI in rodents. Second, we determined the impact of cardiac-infiltrating T cells on lymphatic remodeling in the heart. Approach and Results: Comparing adenoviral versus adeno-associated viral gene delivery in mice, we found that only sustained VEGF (vascular endothelial growth factor)-CC156S therapy, achieved by adeno-associated viral vectors, increased cardiac lymphangiogenesis, and led to reduced cardiac inflammation and dysfunction by 3 weeks post-MI. Conversely, inhibition of VEGF-C/-D signaling, through adeno-associated viral delivery of soluble VEGFR3 (vascular endothelial growth factor receptor 3), limited infarct lymphangiogenesis. Unexpectedly, this treatment improved cardiac function post-MI in both mice and rats, linked to reduced infarct thinning due to acute suppression of T-cell infiltration. Finally, using pharmacological, genetic, and antibody-mediated prevention of cardiac T-cell recruitment in mice, we discovered that both CD4+ and CD8+ T cells potently suppress, in part through interferon-γ, cardiac lymphangiogenesis post-MI. CONCLUSIONS: We show that resolution of cardiac inflammation after MI may be accelerated by therapeutic lymphangiogenesis based on adeno-associated viral gene delivery of VEGF-CC156S. Conversely, our work uncovers a major negative role of cardiac-recruited T cells on lymphatic remodeling. Our results give new insight into the interconnection between immune cells and lymphatics in orchestration of cardiac repair after injury.

Optimization of Advanced Live-Cell Imaging through Red/Near-Infrared Dye Labeling and Fluorescence Lifetime-Based Strategies.

Fluorescence microscopy is essential for a detailed understanding of cellular processes; however, live-cell preservation during imaging is a matter of debate. In this study, we proposed a guide to optimize advanced light microscopy approaches by reducing light exposure through fluorescence lifetime (τ) exploitation of red/near-infrared dyes. Firstly, we characterized key instrumental elements which revealed that red/near-infrared laser lines with an 86x (Numerical Aperture (NA) = 1.2, water immersion) objective allowed high transmission of fluorescence signals, low irradiance and super-resolution. As a combination of two technologies, i.e., vacuum tubes (e.g., photomultiplier) and semiconductor microelectronics (e.g., avalanche photodiode), type S, X and R of hybrid detectors (HyD-S, HyD-X and HyD-R) were particularly adapted for red/near-infrared photon counting and τ separation. Secondly, we tested and compared lifetime-based imaging including coarse τ separation for confocal microscopy, fitting and phasor plot analysis for fluorescence lifetime microscopy (FLIM), and lifetimes weighting for enhanced stimulated emission depletion (STED) nanoscopy, in light of red/near-infrared multiplexing. Mainly, we showed that the choice of appropriate imaging approach may depend on fluorochrome number, together with their spectral/lifetime characteristics and STED compatibility. Photon-counting mode and sensitivity of HyDs together with phasor plot analysis of fluorescence lifetimes enabled the flexible and fast imaging of multi-labeled living H28 cells. Therefore, a combination of red/near-infrared dyes labeling with lifetime-based strategies offers new perspectives for live-cell imaging by enhancing sample preservation through acquisition time and light exposure reduction.

Two-Photon Absorption and Cell Imaging of Fluorene-Functionalized Epicocconone Analogues.

Epicocconone 1 is a natural chromophore isolated from the fungus Epicoccum nigrum that has shown applications in proteomics and fluorescent microscopy thanks to its unique pro-fluorescence properties. The modification of the skeleton of the natural product by replacing the triene side chain by a fluorenyl scaffold can noticeably increase the fluorophore's absorption coefficient. The synthesis of the analogues of the natural product has been made possible by the use of a palladium-catalyzed carbonylation reaction, allowing the construction of the ß-keto-dioxinone key intermediate. Two-photon absorption cross-section measurements of the fluorenyl epicocconone analogues show a structure dependency with values ranging from 60 to 280 GM and live cell imaging show intense staining of intracellular vesicle-like structures around the nucleus.

A role for RASSF1A in tunneling nanotube formation between cells through GEFH1/Rab11 pathway control.

BACKGROUND: By allowing intercellular communication between cells, tunneling nanotubes (TNTs) could play critical role in cancer progression. If TNT formation is known to require cytoskeleton remodeling, key mechanism controlling their formation remains poorly understood. METHODS: The cells of human bronchial (HBEC-3, A549) or mesothelial (H2452, H28) lines are transfected with different siRNAs (inactive, anti-RASSF1A, anti-GEFH1 and / or anti-Rab11). At 48 h post-transfection, i) the number and length of the nanotubes per cell are quantified, ii) the organelles, previously labeled with specific tracers, exchanged via these structures are monitored in real time between cells cultured in 2D or 3D and in normoxia, hypoxia or in serum deprivation condition. RESULTS: We report that RASSF1A, a key-regulator of cytoskeleton encoded by a tumor-suppressor gene on 3p chromosome, is involved in TNTs formation in bronchial and pleural cells since controlling proper activity of RhoB guanine nucleotide exchange factor, GEF-H1. Indeed, the GEF-H1 inactivation induced by RASSF1A silencing, leads to Rab11 accumulation and subsequent exosome releasing, which in turn contribute to TNTs formation. Finally, we provide evidence involving TNT formation in bronchial carcinogenesis, by reporting that hypoxia or nutriment privation, two almost universal conditions in human cancers, fail to prevent TNTs induced by the oncogenic RASSF1A loss of expression. CONCLUSIONS: This finding suggests for the first time that loss of RASSF1A expression could be a potential biomarker for TNTs formation, such TNTs facilitating intercellular communication favoring multistep progression of bronchial epithelial cells toward overt malignancy.

Plant cell wall imaging by metabolic click-mediated labelling of rhamnogalacturonan II using azido 3-deoxy-D-manno-oct-2-ulosonic acid.

In plants, 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo) is a monosaccharide that is only found in the cell wall pectin, rhamnogalacturonan-II (RG-II). Incubation of 4-day-old light-grown Arabidopsis seedlings or tobacco BY-2 cells with 8-azido 8-deoxy Kdo (Kdo-N3 ) followed by coupling to an alkyne-containing fluorescent probe resulted in the specific in muro labelling of RG-II through a copper-catalysed azide-alkyne cycloaddition reaction. CMP-Kdo synthetase inhibition and competition assays showing that Kdo and D-Ara, a precursor of Kdo, but not L-Ara, inhibit incorporation of Kdo-N3 demonstrated that incorporation of Kdo-N3 occurs in RG-II through the endogenous biosynthetic machinery of the cell. Co-localisation of Kdo-N3 labelling with the cellulose-binding dye calcofluor white demonstrated that RG-II exists throughout the primary cell wall. Additionally, after incubating plants with Kdo-N3 and an alkynated derivative of L-fucose that incorporates into rhamnogalacturonan I, co-localised fluorescence was observed in the cell wall in the elongation zone of the root. Finally, pulse labelling experiments demonstrated that metabolic click-mediated labelling with Kdo-N3 provides an efficient method to study the synthesis and redistribution of RG-II during root growth.

Selective Stimulation of Cardiac Lymphangiogenesis Reduces Myocardial Edema and Fibrosis Leading to Improved Cardiac Function Following Myocardial Infarction.

BACKGROUND: The lymphatic system regulates interstitial tissue fluid balance, and lymphatic malfunction causes edema. The heart has an extensive lymphatic network displaying a dynamic range of lymph flow in physiology. Myocardial edema occurs in many cardiovascular diseases, eg, myocardial infarction (MI) and chronic heart failure, suggesting that cardiac lymphatic transport may be insufficient in pathology. Here, we investigate in rats the impact of MI and subsequent chronic heart failure on the cardiac lymphatic network. Further, we evaluate for the first time the functional effects of selective therapeutic stimulation of cardiac lymphangiogenesis post-MI. METHODS AND RESULTS: We investigated cardiac lymphatic structure and function in rats with MI induced by either temporary occlusion (n=160) or permanent ligation (n=100) of the left coronary artery. Although MI induced robust, intramyocardial capillary lymphangiogenesis, adverse remodeling of epicardial precollector and collector lymphatics occurred, leading to reduced cardiac lymphatic transport capacity. Consequently, myocardial edema persisted for several months post-MI, extending from the infarct to noninfarcted myocardium. Intramyocardial-targeted delivery of the vascular endothelial growth factor receptor 3-selective designer protein VEGF-CC152S, using albumin-alginate microparticles, accelerated cardiac lymphangiogenesis in a dose-dependent manner and limited precollector remodeling post-MI. As a result, myocardial fluid balance was improved, and cardiac inflammation, fibrosis, and dysfunction were attenuated. CONCLUSIONS: We show that, despite the endogenous cardiac lymphangiogenic response post-MI, the remodeling and dysfunction of collecting ducts contribute to the development of chronic myocardial edema and inflammation-aggravating cardiac fibrosis and dysfunction. Moreover, our data reveal that therapeutic lymphangiogenesis may be a promising new approach for the treatment of cardiovascular diseases.

Structural and functional analysis of tunneling nanotubes (TnTs) using gCW STED and gconfocal approaches.

BACKGROUND INFORMATION: Tunneling nanotubes (TnTs) are thin plasma membrane bridges mediating transfers of materials and signals between cells. Heterogeneity of heterocellular and homocellular TnTs is largely described but ultrafine imaging of these light-sensitive floating nanometric structures represents a real challenge in microscopy. We propose here imaging strategies designed to dissect structural and dynamic aspects of TnT formation and function in fixed or living PC12 cells. RESULTS: Through time-gated Continuous Wave STimulated Emission Depletion (gCW STED) nanoscopy associated with deconvolution, we provided nanoscale details of membrane and cytoskeleton organisations in two subtypes of TnTs, namely type 1 TnT (TnT1) and type 2 TnT (TnT2). In fixed PC12 cells, TnT1 (length, several tens of micrometres; diameter, 100-650 nm) exhibited a large trumpet-shaped origin, a clear cytosolic tunnel and different bud-shaped connections from closed-ended to open-ended tips. TnT1 contained both actin and tubulin. TnT2 (length, max 20 µm, diameter, 70-200 nm) only contained actin without clear cytosolic tunnel. In living PC12 cells, we observed through gCW STED additional details, unrevealed so far, including a filament spindle emerging from an organising centre at the origin of TnT1 and branched or bulbous attachments of TnT2. However, the power of depletion laser in STED nanoscopy was deleterious for TnTs and prolonged time-lapse experiments were almost prohibited. By circumventing the hazard of photoxicity, we were able to monitor dynamics of bud-shaped tips and intercellular transfer of wheat germ agglutinin labelled cellular elements through time-gated confocal microscopy. CONCLUSIONS: Our work identified new structural characteristics of two subtypes of TnTs in PC12 cells as well as dynamics of formation and transfer through complementary imaging methods combined with image processing. Therefore, we could achieve maximum lateral resolution and sample preservation during acquisitions to reveal new insights into TnT studies. SIGNIFICANCE: Due to large disparity of TnT-like structures in neuronal, immune, cancer or epithelial cells, high- and superresolution approaches can be utilised for full characterisation of these yet poorly understood routes of cell-to-cell communication.

Combining sophisticated fast FLIM, confocal microscopy, and STED nanoscopy for live-cell imaging of tunneling nanotubes.

Cell-to-cell communication via tunneling nanotubes (TNTs) is a challenging topic with a growing interest. In this work, we proposed several innovative tools that use red/near-infrared dye labeling and employ lifetime-based imaging strategies to investigate the dynamics of TNTs in a living mesothelial H28 cell line that exhibits spontaneously TNT1 and TNT2 subtypes. Thanks to a fluorescence lifetime imaging microscopy module being integrated into confocal microscopy and stimulated emission depletion nanoscopy, we applied lifetime imaging, lifetime dye unmixing, and lifetime denoising techniques to perform multiplexing experiments and time-lapses of tens of minutes, revealing therefore structural and functional characteristics of living TNTs that were preserved from light exposure. In these conditions, vesicle-like structures, and tubular- and round-shaped mitochondria were identified within living TNT1. In addition, mitochondrial dynamic studies revealed linear and stepwise mitochondrial migrations, bidirectional movements, transient backtracking, and fission events in TNT1. Transfer of Nile Red-positive puncta via both TNT1 and TNT2 was also detected between living H28 cells.

Interactions between free-living amoebae and Cryptosporidium parvum: an experimental study.

Free-Living Amebae (FLA) and Cryptosporidium oocysts occasionally share the same environment. From 2004 to 2016, Cryptosporidium was responsible for 60% of 905 worldwide waterborne outbreaks caused by protozoan parasites. The aim of this study was to evaluate interactions between C. parvum oocysts and two common FLAs (Acanthamoeba castellanii and Vermamoeba vermiformis) in a water environment. Encystment and survival of FLAs were evaluated by microscopy using trypan blue vital coloration. Oocysts were numerated on microscopy. Interactions were studied over time in conditions both unfavorable and favorable to phagocytosis. Potential phagocytosis was directly evaluated by several microscopic approaches and indirectly by numeration of microorganisms and oocyst infectivity evaluation. Occasional phagocytosis of C. parvum by FLAs was documented. However, oocyst concentrations did not decrease significantly, suggesting resistance of oocysts to phagocytosis. A temporary decrease of oocyst infectivity was observed in the presence of A. castellanii. The effect of these interactions on C. parvum infectivity is particularly interesting. The biofilm condition could favor the persistence or even the proliferation of oocysts over time. This study demonstrated interactions between C. parvum and FLAs. Further knowledge of the mechanisms involved in the decrease of oocyst infectivity in the presence of A. castellanii could facilitate the development of new therapeutic approaches.

Title: Interactions entre amibes libres et Cryptosporidium parvum : étude expérimentale. Abstract: Les amibes libres et les oocystes de Cryptosporidium partagent parfois le même environnement. Entre 2004 et 2016, Cryptosporidium a été responsable de 60 % des 905 épidémies d'origine hydrique dans le monde causées par des parasites protozoaires. Le but de cette étude était d'évaluer les interactions entre les oocystes de C. parvum et deux espèces d'amibes libres communes (Acanthamoeba castellanii et Vermamoeba vermiformis) en environnement aquatique. L'enkystement et la survie des amibes libres ont été évalués par microscopie en utilisant une coloration vitale au bleu trypan. Les oocystes ont été comptés au microscope. Les interactions ont été étudiées au cours du temps dans des conditions à la fois défavorables et favorables à la phagocytose. La phagocytose potentielle a été évaluée directement par plusieurs approches microscopiques et indirectement par la numération des micro-organismes et l'évaluation de l'infectiosité des oocystes. Une phagocytose occasionnelle de C. parvum par amibes libre a été documentée. Cependant, les concentrations d'oocystes n'ont pas diminué de manière significative, ce qui suggère une résistance des oocystes à la phagocytose. Une diminution temporaire de l'infectivité des oocystes a été observée en présence d'A. castellanii. L'effet de ces interactions sur l'infectiosité de C. parvum est particulièrement intéressant. La condition biofilm pourrait favoriser la persistance ou même la prolifération des oocystes de C. parvum au fil du temps. Cette étude a démontré des interactions entre C. parvum et amibes libres. Une meilleure connaissance des mécanismes impliqués dans la diminution de l'infectiosité des oocystes en présence d'A. castellanii pourrait faciliter le développement de nouvelles approches thérapeutiques.

Regulation and impact of cardiac lymphangiogenesis in pressure-overload-induced heart failure.

AIMS: Lymphatics are essential for cardiac health, and insufficient lymphatic expansion (lymphangiogenesis) contributes to development of heart failure (HF) after myocardial infarction. However, the regulation and impact of lymphangiogenesis in non-ischaemic cardiomyopathy following pressure-overload remains to be determined. Here, we investigated cardiac lymphangiogenesis following transversal aortic constriction (TAC) in C57Bl/6 and Balb/c mice, and in end-stage HF patients. METHODS AND RESULTS: Cardiac function was evaluated by echocardiography, and cardiac hypertrophy, lymphatics, inflammation, oedema, and fibrosis by immunohistochemistry, flow cytometry, microgravimetry, and gene expression analysis. Treatment with neutralizing anti-VEGFR3 antibodies was applied to inhibit cardiac lymphangiogenesis in mice. We found that VEGFR3-signalling was essential to prevent cardiac lymphatic rarefaction after TAC in C57Bl/6 mice. While anti-VEGFR3-induced lymphatic rarefaction did not significantly aggravate myocardial oedema post-TAC, cardiac immune cell levels were increased, notably myeloid cells at 3 weeks and T lymphocytes at 8 weeks. Moreover, whereas inhibition of lymphangiogenesis did not aggravate interstitial fibrosis, it increased perivascular fibrosis and accelerated development of left ventricular (LV) dilation and dysfunction. In clinical HF samples, cardiac lymphatic density tended to increase, although lymphatic sizes decreased, notably in patients with dilated cardiomyopathy. Similarly, comparing C57Bl/6 and Balb/c mice, lymphatic remodelling post-TAC was linked to LV dilation rather than to hypertrophy. The striking lymphangiogenesis in Balb/c was associated with reduced cardiac levels of macrophages, B cells, and perivascular fibrosis at 8 weeks post-TAC, as compared with C57Bl/6 mice that displayed weak lymphangiogenesis. Surprisingly, however, it did not suffice to resolve myocardial oedema, nor prevent HF development. CONCLUSIONS: We demonstrate for the first time that endogenous lymphangiogenesis limits TAC-induced cardiac inflammation and perivascular fibrosis, delaying HF development in C57Bl/6 but not in Balb/c mice. While the functional impact of lymphatic remodelling remains to be determined in HF patients, our findings suggest that under settings of pressure-overload poor cardiac lymphangiogenesis may accelerate HF development.

Arteriogenic therapy by intramyocardial sustained delivery of a novel growth factor combination prevents chronic heart failure.

BACKGROUND: Therapeutic angiogenesis is a promising approach for the treatment of cardiovascular diseases, including myocardial infarction and chronic heart failure. We aimed to improve proangiogenic therapies by identifying novel arteriogenic growth factor combinations, developing injectable delivery systems for spatiotemporally controlled growth factor release, and evaluating functional consequences of targeted intramyocardial growth factor delivery in chronic heart failure. METHODS AND RESULTS: First, we observed that fibroblast growth factor and hepatocyte growth factor synergistically stimulate vascular cell migration and proliferation in vitro. Using 2 in vivo angiogenesis assays (n=5 mice per group), we found that the growth factor combination results in a more potent and durable angiogenic response than either growth factor used alone. Furthermore, we determined that the molecular mechanisms involve potentiation of Akt and mitogen-activated protein kinase signal transduction pathways, as well as upregulation of angiogenic growth factor receptors. Next, we developed crosslinked albumin-alginate microcapsules that sequentially release fibroblast growth factor-2 and hepatocyte growth factor. Finally, in a rat model of chronic heart failure induced by coronary ligation (n=14 to 15 rats per group), we found that intramyocardial slow release of fibroblast growth factor-2 with hepatocyte growth factor potently stimulates angiogenesis and arteriogenesis and prevents cardiac hypertrophy and fibrosis, as determined by immunohistochemistry, leading to improved cardiac perfusion after 3 months, as shown by magnetic resonance imaging. These multiple beneficial effects resulted in reduced adverse cardiac remodeling and improved left ventricular function, as revealed by echocardiography. CONCLUSION: Our data showing the selective advantage of using fibroblast growth factor-2 together with hepatocyte growth factor suggest that this growth factor combination may constitute an efficient novel treatment for chronic heart failure.

The Diversity of Serum Anti-DSG3 IgG Subclasses Has a Major Impact on Pemphigus Activity and Is Predictive of Relapses After Treatment With Rituximab.

Introduction: We studied the distribution and in vitro pathogenicity of anti-DSG3 IgG subclasses during the course of pemphigus vulgaris (PV). Methods: We longitudinally studied the distribution of anti-DSG3 IgG subclasses (before versus after treatment) in sera from PV patients, using an addressable-laser bead immunoassay (ALBIA). The in vitro pathogenicity of corresponding sera was tested using keratinocyte dissociation and immunofluorescence assays. Results: Sixty-five sera were assessed at baseline (33 from patients treated with rituximab and 32 with corticosteroids). Sixty-three percent of these baseline sera contained 2 or more anti-DSG3 IgG subclasses versus 35.7% of sera from patients in complete remission (CR) and 75.0% of sera from patients with persistent disease activity after treatment. IgG4 was the most frequently detected anti-DSG3 IgG subclass, both in patients with disease activity and in those in CR. The presence of three or more anti-DSG3 IgG subclasses was predictive of relapse, in particular when it included IgG3, with a positive predictive value of 62.5% and a negative predictive value of 92%. While anti-DSG3 IgG4 Abs from sera collected before treatment were most often pathogenic, anti-DSG3 IgG4 from sera collected after treatment were pathogenic only after adjusting their titer to the one measured before treatment. The IgG3 fraction containing anti-DSG3 Abs also had an in vitro pathogenic effect. The disappearance of the pathogenic effect of some sera after removal of anti-DSG3 IgG3 suggested an additional effect of this IgG subclass. Conclusion: The serum levels and number of anti-DSG3 IgG subclasses drive the pathogenic effect of pemphigus sera and may predict the occurrence of relapses.

Quality assessment in light microscopy for routine use through simple tools and robust metrics.

Although there is a need to demonstrate reproducibility in light microscopy acquisitions, the lack of standardized guidelines monitoring microscope health status over time has so far impaired the widespread use of quality control (QC) measurements. As scientists from 10 imaging core facilities who encounter various types of projects, we provide affordable hardware and open source software tools, rigorous protocols, and define reference values to assess QC metrics for the most common fluorescence light microscopy modalities. Seven protocols specify metrics on the microscope resolution, field illumination flatness, chromatic aberrations, illumination power stability, stage drift, positioning repeatability, and spatial-temporal noise of camera sensors. We designed the MetroloJ_QC ImageJ/Fiji Java plugin to incorporate the metrics and automate analysis. Measurements allow us to propose an extensive characterization of the QC procedures that can be used by any seasoned microscope user, from research biologists with a specialized interest in fluorescence light microscopy through to core facility staff, to ensure reproducible and quantifiable microscopy results.

Comparative Structural and Functional Analyses of the Fusiform, Oval, and Triradiate Morphotypes of Phaeodactylum tricornutum Pt3 Strain.

The diatom Phaeodactylum tricornutum is a marine unicellular microalga that exists under three main morphotypes: oval, fusiform, and triradiate. Previous works have demonstrated that the oval morphotype of P. tricornutum Pt3 strain presents specific metabolic features. Here, we compared the cellular organization of the main morphotypes of the diatom P. tricornutum Pt3 strain through transmission electron and advanced light microscopies. The three morphotypes share similarities including spectral characteristics of the plastid, the location of the nucleus, the organization of mitochondria around the plastid as well as the existence of both a F-actin cortex, and an intracellular network of F-actin. In contrast, compared to fusiform and triradiate cells, oval cells spontaneously release proteins more rapidly. In addition, comparison of whole transcriptomes of oval versus fusiform or triradiate cells revealed numerous differential expression of positive and negative regulators belonging to the complex dynamic secretory machinery. This study highlights the specificities occurring within the oval morphotype underlying that the oval cells secrete proteins more rapidly.

3-Benzoylquinoxalinone as a photoaffinity labelling derivative with fluorogenic properties allowing reaction monitoring under "no-wash" conditions.

Described herein is a quinoxalinone-based photoaffinity probe with caged fluorescence properties. Upon visible blue LED irradiation (λmax 450 nm), this photo-crosslinker is able to covalently capture proteins with concomitant fluorescence labelling. This process enables monitoring applications under "no wash" conditions.

The Prostate Cancer Therapy Enzalutamide Compared with Abiraterone Acetate/Prednisone Impacts Motivation for Exploration, Spatial Learning and Alters Dopaminergic Transmission in Aged Castrated Mice.

Cognitive side effects after cancer treatment threatening quality of life (QoL) constitute a major challenge in oncology. Abiraterone acetate plus prednisone (AAP) and enzalutamide (ENZ) are examples of next-generation therapy (NGT) administered to metastatic castration-resistant prostate cancer (mCRPC) patients. NGT significantly improved mCRPC overall survival but neurological side effects such as fatigue and cognitive impairment were reported. We developed a behavioral 17 months-aged and castrated mouse model receiving per os AAP or ENZ for 5 days per week for six consecutive weeks. ENZ exposure reduced spontaneous activity and exploratory behavior associated with a decreased tyrosine hydroxylase (TH)-dopaminergic activity in the substantia nigra pars compacta and the ventral tegmental area. A decrease in TH+-DA afferent fibers and Phospho-DARPP32-related dopaminergic neuronal activities in the striatum and the ventral hippocampus highlighted ENZ-induced dopaminergic regulation within the nigrostriatal and mesolimbocortical pathways. ENZ and AAP treatments did not substantially modify spatial learning and memory performances, but ENZ led to a thygmotaxis behavior impacting the cognitive score, and reduced c-fos-related activity of NeuN+-neurons in the dorsal hippocampus. The consequences of the mCRPC treatment ENZ on aged castrated mouse motivation to exploration and cognition should make reconsider management strategy of elderly prostate cancer patients.

IHC_Tool: An open-source Fiji procedure for quantitative evaluation of cross sections of testicular explants.

Immunohistochemical analysis is a routine procedure for clinical and research studies in male fertility. However, most of the interpretations remain subjective and time-consuming, with inherent intra- and inter-observer variability. Given the prognostic and research implications of testicular assessment, a more objective and less time-consuming method is required. In the current study, we used in vitro matured pre-pubertal murine testes as a model. The main objective was to develop an affordable automated digital immunohistochemistry image analysis tool for an unbiased and quantitative assessment of testicular tissue sections. Testicular explants were fixed, cut, and stained for specific germ cell markers. The classical manual counting procedure was evaluated. Background and noise were reduced on brightfield images. Photomicrographs were stitched (Background_Elimination_Stitching) to create high-quality images. Two procedures were evaluated (IHC_Tool and Stained_Nuclear_Area); then a procedure (Necrotic_Area_Elimination) allowing withdrawal of the necrotic area observed after culture was assessed. Finally, the number of stained nuclei in the unaltered tissue area was extracted. The automated IHC_Tool procedure with images saved as TIFF at a ×200 magnification allowed the most rigorous cell quantification. IHC_Tool developed for testicular sample analysis can be used for various types of tissues. We foresee that this method will minimize inter-observer variations across laboratories and will be helpful for clinical trials and translational initiatives.

Longitudinal Pathogenic Properties and N-Glycosylation Profile of Antibodies from Patients with Pemphigus after Corticosteroid Treatment.

Pemphigus vulgaris is an autoimmune disease that occurs due to pathogenic autoantibodies that recognize the following epidermal adhesion proteins: desmogleins. Systemic corticosteroids usually decrease the titers of anti-desmoglein autoantibodies and improve patients' conditions. Since modifications of IgG N-glycosylation have been described in some autoimmune diseases, we hypothesized that changes in the pathogenic activity of pemphigus IgG could be related to changes in their N-glycosylation profile. The purpose of this study was to assess, longitudinally, the pathogenicity of pemphigus serum IgG and their N-glycosylation profile during phases of disease activity and clinical remission. The pathogenic activity of serum IgG was measured in vitro on immortalized keratinocytes, by immunofluorescence and dissociation assays, and IgG N-glycans were analyzed by mass spectrometry. We showed (i) a correlation between pemphigus clinical activity and the pathogenicity of serum IgG at baseline and at month 6, while the persistence of the in vitro pathogenic activity of IgG during its evolution, even in patients in clinical remission, seemed to be predictive of relapse; (ii) that modifications of the N-glycan structure were altered the in vitro pathogenicity of patients' autoantibodies; (iii) that the pathogenic properties of pemphigus IgG did not appear to be related to the disparity in IgG N-glycans during the course of pemphigus.