Increased Formation of Follicular Antrum in Aquaporin-8-Deficient Mice Is Due to Defective Proliferation and Migration, and Not Steroidogenesis of Granulosa Cells

Aquaporin-8 (AQP8) is a water channel protein expressed exclusively in granulosa cells (GCs) in mouse ovary. Our previous studies of AQP8-deficient (AQP8-/-) mice demonstrated that AQP8 participates in folliculogenesis, including in the formation of follicles, ovulation, and atresia. However, its physiological function in formation of the antral follicle is still largely unknown. In the present study, we observed significantly increased numbers of antral follicles in AQP8-/- ovaries as well as significantly increased follicular antrum formation in in vitro 3D culture of AQP8-/- follicles. Functional detection of AQP8-/- GCs indicated that cell proliferation is impaired with FSH treatment, and wound healing and Transwell migration are also impaired with or without FSH treatment, compared with that in WT. However, the biosynthesis of estradiol and progesterone as well as the mRNA levels of key steroidogenic enzyme genes (CYP19A1 and StAR) in AQP8-/- GCs did not change, even with addition of FSH and/or testosterone. In order to estimate the influence of the impaired proliferation and migration on the density of GC mass, preantral follicles were injected with FITC-dextran, which distributes only in the intercellular space, and analyzed by confocal microscopy. The micrographs showed significantly higher transmission of fluorescence in AQP8-/- follicles, suggesting increased intercellular space of GCs. Based on this evidence, we concluded that AQP8 deficiency leads to increased formation of follicular antra in vivo and in vitro, and the mechanism may be associated with increased intercellular space of GCs, which may be caused by defective proliferation and migration of GCs. This study may offer new insight into the molecular mechanisms of the formation of follicular antrum

The asymmetrical structure of Golgi apparatus membranes revealed by in situ atomic force microscope.

The Golgi apparatus has attracted intense attentions due to its fascinating morphology and vital role as the pivot of cellular secretory pathway since its discovery. However, its complex structure at the molecular level remains elusive due to limited approaches. In this study, the structure of Golgi apparatus, including the Golgi stack, cisternal structure, relevant tubules and vesicles, were directly visualized by high-resolution atomic force microscope. We imaged both sides of Golgi apparatus membranes and revealed that the outer leaflet of Golgi membranes is relatively smooth while the inner membrane leaflet is rough and covered by dense proteins. With the treatment of methyl-β-cyclodextrin and Triton X-100, we confirmed the existence of lipid rafts in Golgi apparatus membrane, which are mostly in the size of 20 nm -200 nm and appear irregular in shape. Our results may be of significance to reveal the structure-function relationship of the Golgi complex and pave the way for visualizing the endomembrane system in mammalian cells at the molecular level

Adsorption of Coxsackievirus in Sediments: Influencing Factors, Kinetics, and Isotherm Modeling

Drinking groundwater contamination by pathogenic viruses represents a serious risk to worldwide public health, particularly for enteric viruses, which exhibit high prevalence and occurrence during outbreaks. Understanding how enteric viruses adsorb in groundwater is essential to protecting human health and ensuring the sustainable use of water resources. The adsorption properties of Coxsackievirus A16 (CA16), a common gastrointestinal virus that spreads through groundwater, were investigated in this work. A typical batch equilibrium approach was used to investigate CA16 adsorption and factors that influence it. In a laboratory recognized nationally as a biosafety level 2 facility, stringent research protocols were followed to guarantee compliance with experimental standards. The variables that were investigated included the size of the sediment particles, the starting concentration of the virus, temperature, pH level, and humic acid content. The findings showed that the CA16 virus was more strongly attracted to finer sediment particles and that its adsorption increased as the size of the sediment particle decreased. Furthermore, it was discovered that higher temperatures improved the CA16 virus’s ability to bind to sediment particles. The pH of the aqueous environment has a significant effect on the effectiveness of virus adsorption; higher effectiveness was seen in acidic environments. Furthermore, it was found that the presence of humic acid decreased the ability of clay to adsorb CA16, suggesting that humic acid has a detrimental influence on clay’s ability to adsorb viruses. The examination of kinetic models demonstrated that, in every scenario examined, the adsorption process of CA16 adhered to the pseudo-second-order kinetics model. Additionally, the Langmuir and Freundlich isotherm models were used to assess the equilibrium data that were collected in this investigation. The outcomes amply proved that the most accurate representation of the adsorption equilibrium was given by the Langmuir isotherm model. The study offered a solid scientific foundation for treating groundwater and creating plans to stop the spread of viruses

Identification of a Common Epitope between Enterovirus 71 and Human MED25 Proteins Which May Explain Virus-Associated Neurological Disease

Enterovirus 71 (EV71) is a major causative pathogen of hand, foot and mouth disease with especially severe neurologic complications, which mainly account for fatalities from this disease. To date, the pathogenesis of EV71 in the central neurons system has remained unclear. Cytokine-mediated immunopathogenesis and nervous tissue damage by virus proliferation are two widely speculated causes of the neurological disease. To further study the pathogenesis, we identified a common epitope (co-epitope) between EV71 VP1 and human mediator complex subunit 25 (MED25) highly expressed in brain stem. A monoclonal antibody (2H2) against the co-epitope was prepared, and its interaction with MED25 was examined by ELISA, immunofluorescence assay and Western blot in vitro and by live small animal imaging in vivo. Additionally, 2H2 could bind to both VP1 and MED25 with the affinity constant (Kd) of 10−7 M as determined by the ForteBio Octet System. Intravenously injected 2H2 was distributed in brain stem of mice after seven days of EV71 infection. Interestingly, 2H2-like antibodies were detected in the serum of EV71-infected patients. These findings suggest that EV71 infection induces the production of antibodies that can bind to autoantigens expressed in nervous tissue and maybe further trigger autoimmune reactions resulting in neurological disease

High Linearity Microwave Photonic Up-Conversion System Based on Parallel Dual-Drive Mach–Zehnder Modulators

A large dynamic frequency up-conversion scheme based on parallel dual-drive Mach–Zehnder modulators (DD-MZM) and balance detection is proposed and demonstrated experimentally. By optimizing the distribution ratio of the optical carrier power and the IF signal power between the two DD-MZMs, the third-order intermodulation components in two sub-links cancel each other upon the balanced photodetector. The measured results show that the large spurious-free dynamic range of 112.3 dB·Hz4/5 is obtained for an intermediate frequency signal of 2 GHz up-converted to 18 GHz, which is a 14.8 dB enhancement compared with the traditional carrier suppression double-sideband modulation mixer. The frequency up-conversion performance of the established system for the broadband signal is measured with the results demonstrating the feasibility of the proposed optimization scheme

Interferon Inhibition Enhances the Pilot-Scale Production of Rabies Virus in Human Diploid MRC-5 Cells

Inactivated vaccines based on cell culture are very useful in the prevention and control of many diseases. The most popular strategy for the production of inactivated vaccines is based on monkey-derived Vero cells, which results in high productivity of the virus but has a certain carcinogenic risk due to non-human DNA contamination. Since human diploid cells, such as MRC-5 cells, can produce a safer vaccine, efforts to develop a strategy for inactivated vaccine production using these cells have been investigated using MRC-5 cells. However, most viruses do not replicate efficiently in MRC-5 cells. In this study, we found that rabies virus (RABV) infection activated a robust interferon (IFN)-β response in MRC-5 cells but almost none in Vero cells, suggesting that the IFN response could be a key limiting factor for virus production. Treatment of the MRC-5 cells with IFN inhibitors increased RABV titers by 10-fold. Additionally, the RABV titer yield was improved five-fold when using IFN receptor 1 (IFNAR1) antibodies. As such, we established a stable IFNAR1-deficient MRC-5 cell line (MRC-5IFNAR1−), which increased RABV production by 6.5-fold compared to normal MRC-5 cells. Furthermore, in a pilot-scale production in 1500 square centimeter spinner flasks, utilization of the MRC-5IFNAR1− cell line or the addition of IFN inhibitors to MRC cells increased RABV production by 10-fold or four-fold, respectively. Thus, we successfully established a human diploid cell-based pilot scale virus production platform via inhibition of IFN response for rabies vaccines, which could also be used for other inactivated virus vaccine production

The variable loop 3 in the envelope glycoprotein is critical for the atypical coreceptor usage of an HIV-1 strain.

The majority of HIV-1 strains enter CD4+ T cells using the CCR5 and/or CXCR4 co-receptor. However, we recently identified a transmitted/founder (T/F) virus (ZP6248) that efficiently used an alternative coreceptor GPR15, rather than commonly used CXCR4 and CCR5, to establish clinical infection. To understand which regions in the env gene were critical for the atypical coreceptor usage, we generated a set of V3 mutants and determined their infectivity in GHOST cells that expressed different coreceptors. When the variable loop 3 (V3) in YU2 was replaced with the ZP6248 V3 (YU2.6248V3), the chimera YU2.6248V3 infected GPR15+ cells but not CCR5+ cells. To determine which amino acids in V3 was responsible for this phenotype change, each of the eight amino acids that differed from the subtype B consensus V3 was substituted with alanine. The G306A and S322A mutations significantly reduced the replication capacity of YU2.6248V3 in GPR15+ cells, while all other alanine substitutions at positions 307, 314, 315, 316, 317 and 318 completely abrogated the infectivity of YU2.6248V3 in GPR15+ cells. The E314A mutation, as the E314G mutation reported before, also rendered the YU2.6248V3 infectious in CCR5+ cells, while none of other alanine mutants could infect CCR5+ cells. These results demonstrated that amino acids in ZP6248 V3 might form a unique conformation that was critical for the interaction with GPR15 while the amino acids at position 314 in the V3 crown of ZP6248 played a key role in interaction with both CCR5 and GPR15. The unique phenotypes of ZP6248 can serve as a model to understand how HIV-1 explores the diverse coreceptor reservoir through novel genetic variants to establish clinical infection