Design, optimization, characterization, and in vitro evaluation of metformin-loaded liposomes for triple negative breast cancer treatment.

Recently, metformin (Met) has shown to have antineoplastic properties in cancer treatment by improving hypoxic tumor conditions, and causing reduction in the synthesis of biomolecules, which are vital for cancer growth. However, as an orally administered drug, Met has low bioavailability and rapid renal clearance. Thus, the goal of this study was to vectorize Met inside liposomes in the context of triple negative breast cancer (TNBC), which currently lacks treatment options when compared to other types of breast cancer. Vectorization of Met inside liposomes was done using Bangham method by implementing double design of experiment methodology to increase Met drug loading (minimum-run resolution V characterization design and Box-Behnken design), as it is generally extremely low for hydrophilic molecules. Optimization of Met-loaded liposome synthesis was successfully achieved with drug loading of 190 mg/g (19% w/w). The optimal Met-liposomes were 170 nm in diameter with low PdI (< 0.1) and negative surface charge (-20 mV), exhibiting sustained Met release at pH 7.4. The liposomal Met delivery system was stable over several months, and successfully reduced TNBC cell proliferation due to the encapsulated drug. This study is one the first reports addressing liposome formulation through thin-film hydration using two design of experiment methods aiming to increase drug loading of Met.

Conserved Viral Transcription Plays a Key Role in Virus-Like Particle Production of the Parasitoid Wasp Venturia canescens.

Nudiviruses are large double-stranded DNA viruses related to baculoviruses known to be endogenized in the genomes of certain parasitic wasp species. These wasp-virus associations allow the production of viral particles or virus-like particles that ensure wasp parasitism success within lepidopteran hosts. Venturia canescens is an ichneumonid wasp belonging to the Campopleginae subfamily that has endogenized nudivirus genes belonging to the Alphanudivirus genus to produce "virus-like particles" (Venturia canescens virus-like particles [VcVLPs]), which package proteic virulence factors. The main aim of this study was to determine whether alphanudivirus gene functions have been conserved following endogenization. The expression dynamics of alphanudivirus genes was monitored by a high throughput transcriptional approach, and the functional role of lef-4 and lef-8 genes predicted to encode viral RNA polymerase components was investigated by RNA interference. As described for baculovirus infections and for endogenized nudivirus genes in braconid wasp species producing bracoviruses, a transcriptional cascade involving early and late expressed alphanudivirus genes could be observed. The expression of lef-4 and lef-8 was also shown to be required for the expression of alphanudivirus late genes allowing correct particle formation. Together with previous literature, the results show that endogenization of nudiviruses in parasitoid wasps has repeatedly led to the conservation of the viral RNA polymerase function, allowing the production of viruses or viral-like particles that differ in composition but enable wasp parasitic success. IMPORTANCE This study shows that endogenization of a nudivirus genome in a Campopleginae parasitoid wasp has led to the conservation, as for endogenized nudiviruses in braconid parasitoid wasps, of the viral RNA polymerase function, required for the transcription of genes encoding viral particles involved in wasp parasitism success. We also showed for the first time that RNA interference (RNAi) can be successfully used to downregulate gene expression in this species, a model in behavioral ecology. This opens the opportunity to investigate the function of genes involved in other traits important for parasitism success, such as reproductive strategies and host choice. Fundamental data acquired on gene function in Venturia canescens are likely to be transferable to other parasitoid wasp species used in biological control programs. This study also renders possible the investigation of other nudivirus gene functions, for which little data are available.

Revisiting the mystery of centrioles at the beginning of mammalian embryogenesis.

The prevailing assumption has been that the human spermatozoon provides only one centriole to the zygote: the proximal centriole, with a canonical, cylinder-like shape. This overly simplistic view has come under challenge since discovering that the human spermatozoon provides a second, atypical centriole to the zygote. The study of human zygotes is challenging for ethical reasons, and bovine zygotes provide an important model due to a similarity in centrosome embryonic inherence and function. Detailed ultrastructural analyses by Uzbekov and colleagues identify the persistence of atypical centrioles in bovine early embryos, raising questions about the original single-centriole model. Whether the parental origin of nascent atypical centrioles or their wide structural diversity and deviation from the canonical centriolar form in blastomeres constitutes sufficient evidence to warrant a reconsideration of the single-centriole model is discussed herein. Because previous human studies identified only one canonical centriole in the zygote, atypical centrioles are likely present in the early human embryo; therefore, it is time to rethink the role of paternal centrioles in human development.

Silicon-Vacancy Nanodiamonds as High Performance Near-Infrared Emitters for Live-Cell Dual-Color Imaging and Thermometry.

Nanodiamonds (NDs) with color centers are excellent emitters for various bioimaging and quantum biosensing applications. In our work, we explore new applications of NDs with silicon-vacancy centers (SiV) obtained by high-pressure high-temperature (HPHT) synthesis based on metal-catalyst-free growth. They are coated with a polypeptide biopolymer, which is essential for efficient cellular uptake. The unique optical properties of NDs with SiV are their high photostability and narrow emission in the near-infrared region. Our results demonstrate for the first time that NDs with SiV allow live-cell dual-color imaging and intracellular tracking. Also, intracellular thermometry and challenges associated with SiV atomic defects in NDs are investigated and discussed for the first time. NDs with SiV nanoemitters provide new avenues for live-cell bioimaging, diagnostic (SiV as a nanosized thermometer), and theranostic (nanodiamonds as drug carrier) applications.

Annulate lamellae and intracellular pathogens.

Annulate lamellae (AL) have been observed many times over the years on electron micrographs of rapidly dividing cells, but little is known about these unusual organelles consisting of stacked sheets of endoplasmic reticulum-derived membranes with nuclear pore complexes (NPCs). Evidence is growing for a role of AL in viral infection. AL have been observed early in the life cycles of the hepatitis C virus (HCV) and, more recently, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), suggesting a specific induction of mechanisms potentially useful to these pathogens. Like other positive-strand RNA viruses, these viruses induce host cells membranes rearrangements. The NPCs of AL could potentially mediate exchanges between these partially sealed compartments and the cytoplasm. AL may also be involved in regulating Ca2+ homeostasis or cell cycle control. They were recently observed in cells infected with Theileria annulata, an intracellular protozoan parasite inducing cell proliferation. Further studies are required to clarify their role in intracellular pathogen/host-cell interactions.

Avian sperm increase in vitro the release of exosomes from SST-enriched organoids.

In birds, oviductal cells play a crucial role in the storage of sperm via cell-to-cell communication including extracellular vesicles (EV). We developed a culture of oviductal organoids enriched in sperm storage tubules (SSTorg) to demonstrate the release of EV. SSTorg were cultured for 24 h and added to live (LV), frozen (FZ) and lysed (LY) avian sperm, seminal plasma (SP), avian sperm conditioned medium (CM), or bovine sperm (BV). Western blot demonstrated that SSTorg contained EV protein markers, valosin-containing protein (VCP), heat shock proteins (HSP90AA1, HSPA8), and annexins (ANXA2, A4, A5). Co-culture with LV significantly decreased the intracellular level of all these proteins except HSPA8. Immunohistochemistry confirmed this result for VCP and ANXA4. LY, CM, SP and BV had no effect on the intracellular level of these proteins, whereas FZ induced a decrease in ANXA2, A4 and A5. In culture media, VCP and HSP90AA1 signals were detected in the presence of LV, FZ, BV, LY, CM and SP, but no ANXA4 signal was observed in the presence of FZ and SP. ANXA2 and A5 were only detected in the presence of LV. The most abundant EV were less than 150 nm in diameter. ANXA4 and A5 were more abundant in EV isolated from the SSTorg culture medium. This study provides a useful culture system for studying interactions between SST cells and sperm. We demonstrated the release of EV by SSTorg in vitro, and its regulation by sperm. This may be of crucial importance for sperm during storage in hens.

First insights on seminal extracellular vesicles in chickens of contrasted fertility.

Male subfertility causes are very varied and sometimes related to post-gonadic maturation disruption, involving seminal plasma constituents. Among them, extracellular vesicles are involved in key exchanges with sperm in mammals. However, in birds, the existence of seminal extracellular vesicles is still debated. The aim of the present work was first to clarify the putative presence of extracellular vesicles in the seminal plasma of chickens, secondly to characterize their size and protein markers in animals showing different fertility, and finally to make preliminary evaluations of their interactions with sperm. We successfully isolated extracellular vesicles from seminal plasma of males showing the highest differences in semen quality and fertility by using ultracentrifugation protocol (pool of 3 ejaculates/rooster, n =3/condition). Size characterization performed by electron microscopy revealed a high proportion of small extracellular vesicles (probably exosomes) in chicken seminal plasma. Smaller extracellular vesicles appeared more abundant in fertile than in subfertile roosters, with a mean diameter of 65.12 and 77.18 nm, respectively. Different protein markers of extracellular vesicles were found by western blotting (n = 6/condition). Among them, HSP90A was significantly more abundant in fertile than in subfertile males. In co-incubation experiments (n = 3/condition), extracellular vesicles enriched seminal fractions of fertile males showed a higher capacity to be incorporated into fertile than into subfertile sperm. Sperm viability and motility were impacted by the presence of extracellular vesicles from fertile males. In conclusion, we successfully demonstrated the presence of extracellular vesicles in chicken seminal plasma, with differential size, protein markers and putative incorporation capacity according to male fertility status.

Isolation and Characterization of Equine Uterine Extracellular Vesicles: A Comparative Methodological Study.

Extracellular vesicles (EVs) have been identified in the uterine fluid in different species and have been pointed as key players in the embryo-maternal dialogue, maternal recognition of pregnancy and establishment of pregnancy. However, little is known about the uterine EVs in the mare. Therefore, the present study aimed at characterizing EVs from uterine lavage of cyclic mares by comparing five EVs isolation methods and the combination of them: (1) ultracentrifugation (UC); (2) concentration of lavage volume by Centricon ultrafiltration (CE); (3) the use of CE with different washing steps (phosphate-buffered saline with or without trehalose); (4) size-exclusion chromatography with iZON-qEV columns, and (5) a combination of the methods with best results based on EVs yield, purity, and protein cargo profiles. Transmission electron microscopy and Western blotting confirmed the isolation of EVs by all methods but with quantitative and qualitative differences. Mass spectrometry provided differences in protein profiles between methods, number of identified proteins, and protein classes. Our results indicate that the combination of CE/trehalose/iZON/UC is an optimal method to isolate equine uterine EVs with good yield and purity that can be applied in future studies to determine the role of equine uterine EVs in embryo-maternal interactions.

Protein Palmitoylation in Bovine Ovarian Follicle.

Protein palmitoylation is a reversible post-translational modification by fatty acids (FA), mainly a palmitate (C16:0). Palmitoylation allows protein shuttling between the plasma membrane and cytosol to regulate protein stability, sorting and signaling activity and its deficiency leads to diseases. We aimed to characterize the palmitoyl-proteome of ovarian follicular cells and molecular machinery regulating protein palmitoylation within the follicle. For the first time, 84 palmitoylated proteins were identified from bovine granulosa cells (GC), cumulus cells (CC) and oocytes by acyl-biotin exchange proteomics. Of these, 32 were transmembrane proteins and 27 proteins were detected in bovine follicular fluid extracellular vesicles (ffEVs). Expression of palmitoylation and depalmitoylation enzymes as palmitoyltransferases (ZDHHCs), acylthioesterases (LYPLA1 and LYPLA2) and palmitoylthioesterases (PPT1 and PPT2) were analysed using transcriptome and proteome data in oocytes, CC and GC. By immunofluorescence, ZDHHC16, PPT1, PPT2 and LYPLA2 proteins were localized in GC, CC and oocyte. In oocyte and CC, abundance of palmitoylation-related enzymes significantly varied during oocyte maturation. These variations and the involvement of identified palmitoyl-proteins in oxidation-reduction processes, energy metabolism, protein localization, vesicle-mediated transport, response to stress, G-protein mediated and other signaling pathways suggests that protein palmitoylation may play important roles in oocyte maturation and ffEV-mediated communications within the follicle.

MALDI-TOF Mass Spectrometry Revealed Significant Lipid Variations in Follicular Fluid and Somatic Follicular Cells but Not in Enclosed Oocytes between the Large Dominant and Small Subordinate Follicles in Bovine Ovary.

Lipid metabolism in ovarian follicular cells supports the preparation of an enclosed oocyte to ovulation. We aimed to compare lipid composition of a dominant large follicle (LF) and subordinated small follicles (SFs) within the same ovaries. Mass spectrometry imaging displayed the differences in the distribution of several lipid features between the different follicles. Comparison of lipid fingerprints between LF and SF by Matrix Assisted Laser Desorption/Ionisation Time-Of-Flight (MALDI-TOF) mass spectrometry revealed that in the oocytes, only 8 out of 468 detected lipids (1.7%) significantly changed their abundance (p < 0.05, fold change > 2). In contrast, follicular fluid (FF), granulosa, theca and cumulus cells demonstrated 55.5%, 14.9%, 5.3% and 9.8% of significantly varied features between LF and SF, respectively. In total, 25.2% of differential lipids were identified and indicated potential changes in membrane and signaling lipids. Tremendous changes in FF lipid composition were likely due to the stage specific secretions from somatic follicular cells that was in line with the differences observed from FF extracellular vesicles and gene expression of candidate genes in granulosa and theca cells between LF and SF. In addition, lipid storage in granulosa and theca cells varied in relation to follicular size and atresia. Differences in follicular cells lipid profiles between LF and SF may probably reflect follicle atresia degree and/or accumulation of appropriate lipids for post-ovulation processes as formation of corpus luteum. In contrast, the enclosed oocyte seems to be protected during final follicular growth, likely due in part to significant lipid transformations in surrounding cumulus cells. Therefore, the enclosed oocyte could likely keep lipid building blocks and energy resources to support further maturation and early embryo development.

Long-term live cells observation of internalized fluorescent Fe@C nanoparticles in constant magnetic field.

BACKGROUND: Theranostics application of superparamagnetic nanoparticles based on magnetite and maghemite is impeded by their toxicity. The use of additional protective shells significantly reduced the magnetic properties of the nanoparticles. Therefore, iron carbides and pure iron nanoparticles coated with multiple layers of onion-like carbon sheath seem to be optimal for biomedicine. Fluorescent markers associated with magnetic nanoparticles provide reliable means for their multimodal visualization. Here, biocompatibility of iron nanoparticles coated with graphite-like shell and labeled with Alexa 647 fluorescent marker has been investigated. METHODS: Iron core nanoparticles with intact carbon shells were purified by magnetoseparation after hydrochloric acid treatment. The structure of the NPs (nanoparticles) was examined with a high resolution electron microscopy. The surface of the NPs was alkylcarboxylated and further aminated for covalent linking with Alexa Fluor 647 fluorochrome to produce modified fluorescent magnetic nanoparticles (MFMNPs). Live fluorescent imaging and correlative light-electron microscopy were used to study the NPs intracellular distribution and the effects of constant magnetic field on internalized NPs in the cell culture were analyzed. Cell viability was assayed by measuring a proliferative pool with Click-IT labeling. RESULTS: The microstructure and magnetic properties of superparamagnetic Fe@C core-shell NPs as well as their endocytosis by living tumor cells, and behavior inside the cells in constant magnetic field (150 mT) were studied. Correlative light-electron microscopy demonstrated that NPs retained their microstructure after internalization by the living cells. Application of constant magnetic field caused orientation of internalized NPs along power lines thus demonstrating their magnetocontrollability. Carbon onion-like shells make these NPs biocompatible and enable long-term observation with confocal microscope. It was found that iron core of NPs shows no toxic effect on the cell physiology, does not inhibit the cell proliferation and also does not induce apoptosis. CONCLUSIONS: Non-toxic, biologically compatible superparamagnetic fluorescent MFMNPs can be further used for biological application such as delivery of biologically active compounds both inside the cell and inside the whole organism, magnetic separation, and magnetic resonance imaging (MRI) diagnostics.

Metabolomic Profile of Oviductal Extracellular Vesicles across the Estrous Cycle in Cattle.

Oviductal extracellular vesicles (oEVs) have been proposed as key modulators of gamete/embryo maternal interactions. The aim of this study was to examine the metabolite content of oEVs and its regulation across the estrous cycle in cattle. Oviductal EVs were isolated from bovine oviducts ipsilateral and contralateral to ovulation at four stages of the estrous cycle (post-ovulatory stage, early and late luteal phases, and pre-ovulatory stage). The metabolomic profiling of EVs was performed by proton nuclear magnetic resonance spectroscopy (NMR). NMR identified 22 metabolites in oEVs, among which 15 were quantified. Lactate, myoinositol, and glycine were the most abundant metabolites throughout the estrous cycle. The side relative to ovulation had no effect on the oEVs' metabolite concentrations. However, levels of glucose-1-phosphate and maltose were greatly affected by the cycle stage, showing up to 100-fold higher levels at the luteal phase than at the peri-ovulatory phases. In contrast, levels of methionine were significantly higher at peri-ovulatory phases than at the late-luteal phase. Quantitative enrichment analyses of oEV-metabolites across the cycle evidenced several significantly regulated metabolic pathways related to sucrose, glucose, and lactose metabolism. This study provides the first metabolomic characterization of oEVs, increasing our understanding of the potential role of oEVs in promoting fertilization and early embryo development.

Steroid hormones regulate sperm-oviduct interactions in the bovine.

After insemination in the cow, a sperm reservoir is formed within the oviducts, allowing the storage and then progressive release of spermatozoa toward the ovulated oocyte. In order to investigate the hormonal regulation of these events in vitro, the ovarian steroids 17ß-estradiol (E2) and progesterone (P4) were added at various concentrations to monolayers of bovine oviduct epithelial cells (BOEC) before or during co-incubation with spermatozoa. Main findings demonstrate that (1) a 18-h pretreatment of BOEC with 100 pg/mL and 100 ng/mL of E2 decreased by 25% the ability of BOEC to bind spermatozoa after 10 min, and for the highest dose of E2, 60 min of co-incubation; (2) P4 at concentrations of 10, 100 and 1000 ng/mL induced the release within 60 min of 32-47% of bound spermatozoa from BOEC; this sperm-releasing effect was maintained after a 18-h pretreatment of BOEC with 100 pg/mL of E2; (3) E2 in concentrations above 100 pg/mL inhibited the releasing effect of P4 on bound sperm in a dose-dependent manner; (4) spermatozoa bound to BOEC, then released from BOEC by the action of P4-induced higher cleavage and blastocyst rates after in vitro fertilization than the control group. These results support the hypothesis that the dynamic changes in steroid hormones around the time of ovulation regulate the formation of the sperm reservoir and the timed delivery of capacitated spermatozoa to the site of fertilization.

Oviduct extracellular vesicles protein content and their role during oviduct-embryo cross-talk.

Successful pregnancy requires an appropriate communication between the mother and the embryo. Recently, exosomes and microvesicles, both membrane-bound extracellular vesicles (EVs) present in the oviduct fluid have been proposed as key modulators of this unique cross-talk. However, little is known about their content and their role during oviduct-embryo dialog. Given the known differences in secretions by in vivo and in vitro oviduct epithelial cells (OEC), we aimed at deciphering the oviduct EVs protein content from both sources. Moreover, we analyzed their functional effect on embryo development. Our study demonstrated for the first time the substantial differences between in vivo and in vitro oviduct EVs secretion/content. Mass spectrometry analysis identified 319 proteins in EVs, from which 186 were differentially expressed when in vivo and in vitro EVs were compared (P < 0.01). Interestingly, 97 were exclusively expressed in in vivo EVs, 47 were present only in in vitro and 175 were common. Functional analysis revealed key proteins involved in sperm-oocyte binding, fertilization and embryo development, some of them lacking in in vitro EVs. Moreover, we showed that in vitro-produced embryos were able to internalize in vivo EVs during culture with a functional effect in the embryo development. In vivo EVs increased blastocyst rate, extended embryo survival over time and improved embryo quality. Our study provides the first characterization of oviduct EVs, increasing our understanding of the role of oviduct EVs as modulators of gamete/embryo-oviduct interactions. Moreover, our results point them as promising tools to improve embryo development and survival under in vitro conditions.

Magnetocontrollability of Fe7C3@C superparamagnetic nanoparticles in living cells.

BACKGROUND: A new type of superparamagnetic nanoparticles with chemical formula Fe7C3@C (MNPs) showed higher value of magnetization compared to traditionally used iron oxide-based nanoparticles as was shown in our previous studies. The in vitro biocompatibility tests demonstrated that the MNPs display high efficiency of cellular uptake and do not affect cyto-physiological parameters of cultured cells. These MNPs display effective magnetocontrollability in homogeneous liquids but their behavior in cytoplasm of living cells under the effect of magnetic field was not carefully analyzed yet. RESULTS: In this work we investigated the magnetocontrollability of MNPs interacting with living cells in permanent magnetic field. It has been shown that cells were capable of capturing MNPs by upper part of the cell membrane, and from the surface of the cultivation substrate during motion process. Immunofluorescence studies using intracellular endosomal membrane marker showed that MNP agglomerates can be either located in endosomes or lying free in the cytoplasm. When attached cells were exposed to a magnetic field up to 0.15 T, the MNPs acquired magnetic moment and the displacement of incorporated MNP agglomerates in the direction of the magnet was observed. Weakly attached or non-attached cells, such as cells in mitosis or after cytoskeleton damaging treatments moved towards the magnet. During long time cultivation of cells with MNPs in a magnetic field gradual clearing of cells from MNPs was observed. It was the result of removing MNPs from the surface of the cell agglomerates discarded in the process of exocytosis. CONCLUSIONS: Our data allow us to conclude for the first time that the magnetic properties of the MNPs are sufficient for successful manipulation with MNP agglomerates both at the intracellular level, and within the whole cell. The structure of the outer shells of the MNPs allows firmly associate different types of biological molecules with them. This creates prospects for the use of such complexes for targeted delivery and selective removal of selected biological molecules from living cells.

Inhibition of Pathogenic Mutant SOD1 Aggregation in Cultured Motor Neuronal Cells by Prevention of Its SUMOylation on Lysine 75.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the selective death of motor neurons. Mutations in the SOD1 gene encoding the superoxide dismutase 1 are present in 15% of familial ALS cases and in 2% of sporadic cases. These mutations are associated with the formation of SOD1-positive aggregates. The mechanisms of aggregation remain unknown, but posttranslational modifications of SOD1 may be involved. Here, we report that NSC-34 motor neuronal cells expressing mutant SOD1 contained aggregates positive for small ubiquitin modifier-1 (SUMO-1), and in parallel a reduced level of free SUMO-1. CLEM (correlative light and electron microscopy) analysis showed nonorganized cytosolic aggregates for all mutations tested (SOD1A4V, SOD1V31A, and SOD1G93C). We next show that preventing the SUMOylation of mutant SOD1 by the substitution of lysine 75, the SUMOylation site of SOD1, significantly reduces the number of motor neuronal cells with aggregates. These results support the need for further research on the SUMOylation pathways, which may be a potential therapeutic target in ALS.

Effect of the alcohol consumption on osteocyte cell processes: a molecular imaging study.

We have previously shown microarchitectural tissue changes with cellular modifications in osteocytes following high chronic alcohol dose. The aim of this study was to assess the dose effect of alcohol consumption on the cytoskeleton activity, the cellular lipid content and modulation of differentiation and apoptosis in osteocyte. Male Wistar rats were divided into three groups: Control (C), Alcohol 25% v/v (A25) or Alcohol 35% v/v (A35) for 17 weeks. Bone mineral density (BMD) was assessed by DXA, osteocyte empty lacunae, lacunae surface, bone marrow fat with bright field microscopy. Osteocyte lipid content was analysed with transmission electron microscopy (TEM) and epifluorescence microscopy. Osteocyte apoptosis was analysed with immunolabelling and TEM. Osteocyte differentiation and cytoskeleton activity were analysed with immunolabelling and real time quantitative PCR. At the end of the protocol, BMD was lower in A25 and A35 compared with C, while the bone marrow lipid content was increased in these groups. More empty osteocyte lacunae and osteocyte containing lipid droplets in A35 were found compared with C and A25. Cleaved caspase-3 staining and chromatin condensation were increased in A25 and A35 versus C. Cleaved caspase-3 was increased in A35 versus A25. CD44 and phosphopaxillin stainings were higher in A35 compared with C and A25. Paxillin mRNA expression was higher in A35 versus A25 and C and sclerostin mRNA expression was higher in A35 versus C. We only observed a dose effect of alcohol consumption on cleaved caspase-3 osteocyte immunostaining levels and on the number of lipid droplets in the bone marrow.

Transient oxygen-glucose deprivation sensitizes brain capillary endothelial cells to rtPA at 4h of reoxygenation.

Thrombolysis treatment of acute ischemic stroke is limited by the pro-edematous and hemorrhagic effects exerted by reperfusion, which disrupts the blood-brain barrier (BBB) capillary endothelium in the infarct core. Most studies of the ischemic BBB overlook the complexity of the penumbral area, where the affected brain cells are still viable following deprivation. Our present objective was to examine in vitro the kinetic impact of reoxygenation on the integrity of ischemic BBB cells after oxygen-glucose deprivation. Through the use of a co-culture of brain capillary endothelial cells and glial cells, we first showed that the transendothelial permeability increase induced by deprivation can occur with both preserved cell viability and interendothelial tight junction network. The subtle and heterogeneous alteration of the tight junctions was observable only through electron microscopy. A complete permeability recovery was then found after reoxygenation, when Vimentin and Actin networks were reordered. However, still sparse ultrastructural alterations of tight junctions suggested an acquired vulnerability. Endothelial cells were then exposed to recombinant tissue-type plasminogen activator (rtPA) to define a temporal profile for the toxic effect of this thrombolytic on transendothelial permeability. Interestingly, the reoxygenated BBB broke down with aggravated tight junction disruption when exposed to rtPA only at 4h after reoxygenation. Moreover, this breakdown was enhanced by 50% when ischemic glial cells were present during the first hours of reoxygenation. Our results suggest that post-stroke reoxygenation enables retrieval of the barrier function of brain capillary endothelium when in a non-necrotic environment, but may sensitize it to rtPA at the 4-hour time point, when both endothelial breakdown mechanisms and glial secretions could be identified and targeted in a therapeutical perspective.

Sequential biogenesis of host cell membrane rearrangements induced by hepatitis C virus infection.

Like most positive-strand RNA viruses, hepatitis C virus (HCV) forms a membrane-associated replication complex consisting of replicating RNA, viral and host proteins anchored to altered cell membranes. We used a combination of qualitative and quantitative electron microscopy (EM), immuno-EM, and the 3D reconstruction of serial EM sections to analyze the host cell membrane alterations induced by HCV. Three different types of membrane alteration were observed: vesicles in clusters (ViCs), contiguous vesicles (CVs), and double-membrane vesicles (DMVs). The main ultrastructural change observed early in infection was the formation of a network of CVs surrounding the lipid droplets. Later stages in the infectious cycle were characterized by a large increase in the number of DMVs, which may be derived from the CVs. These DMVs are thought to constitute the membranous structures harboring the viral replication complexes in which viral replication is firmly and permanently established and to protect the virus against double-stranded RNA-triggered host antiviral responses.

Aurora A Kinase Begins to Localize to the Centrosome in the S-phase of the Cell Cycle in the XL2 Cell Line.

BACKGROUND: The centrosome is one of the principal cell hubs, where numerous proteins important for intracellular regulatory processes are concentrated. One of them, serine-threonine kinase 6, alias Aurora A, is involved in centrosome duplication and mitotic spindle formation and maintenance. METHODS: Long-term vital observations of cells, immunofluorescence analysis of protein localization, synchronization of cells at different phases of the cell cycle, Western blot analysis of protein content were used in the work. RESULTS: In this study, we investigated the dynamics of Aurora A protein accumulation and degradation in the XL2 Xenopus cell line during its 28-hour cell cycle. Using Western blot and immunofluorescence analyses, we demonstrated that Aurora A disappeared from the centrosome within one hour following mitosis and was not redistributed to other cell compartments. Using double Aurora A/Bromodeoxyuridine immunofluorescence labeling of the cells with precisely determined cell cycle stages, we observed that Aurora A reappeared in the centrosome during the S-phase, which was earlier than reported for all other known proteins with mitosis-specific centrosomal localization. Moreover, Aurora A accumulation in the centrosomal region and centrosome separation were asynchronous in the sister cells. CONCLUSIONS: The reported data allowed us to hypothesize that Aurora A is one of the primary links in coordinating centrosome separation and constructing the mitotic spindle.