JEV infection leads to dysfunction of lysosome by downregulating the expression of LAMP1 and LAMP2.

Japanese Encephalitis Virus (JEV), the predominant cause of viral encephalitis in many Asian countries, affects approximately 68,000 people annually. Lysosomes are dynamic structures that regulate cellular metabolism by mediating lysosomal biogenesis and autophagy. Here, we showed that lysosome-associated membrane protein 1 (LAMP1) and LAMP2 were downregulated in cells after JEV infection, resulting in a decrease in the quantity of acidified lysosomes and impaired lysosomal catabolism. What's more, JEV nonstructural protein 4B plays key roles in the reduction of LAMP1/2 via the autophagy-lysosome pathway. JEV NS4B also promoted abnormal aggregation of SLA-DR, an important component of the swine MHC-II molecule family involved in antigen presentation and CD4+ cell activation initiation. Mechanistically, NS4B localized to the ER during JEV infection and interacted with GRP78, leading to the activation of ER stress-mediated autophagy. The 131-204 amino acid (aa) region of NS4B is essential for autophagy induction and LAMP1/2 reduction. In summary, our findings reveal a novel pathway by which JEV induces autophagy and disrupts lysosomal function.

Association Between Anti-Müllerian Hormone and Early Spontaneous Abortion in Assisted Reproduction Treatment: A Case-Control Study Integrated with Biological Evidence.

Early spontaneous abortion (ESA) is a common adverse pregnancy outcome mainly attributed to embryo chromosomal abnormalities. However, as a quantitative marker, whether the anti-Müllerian hormone (AMH) can reflect oocyte quality is still controversial. By integrating biological evidence and adjusting many cofounders, this study aimed to clarify the controversies about the association between AMH and ESA caused by embryo aneuploidy during assisted reproductive technology (ART) treatment. We strictly preselected 988 patients receiving first ART treatment for analyzing clinical data, while 55 of them acquired chorionic villi karyotype results. In addition, 373 biopsied embryos from 126 patients receiving preimplantation genetic diagnosis (PGT) were tracked to compare embryo karyotypes. Univariate and multiple factor regressions were applied to analyze the risk factors leading to ESA. As covariates unadjusted, AMH (odds ratio 0.87, 95% CI 0.82-0.93) was the significant variable contributing to ESA. However, AMH played no significant role in the following regression models after age was adjusted. Also, AMH had no significant association with ESA in most age-adjusted subgroups, except in the male factors engaged subgroup. Additionally, compared to the patients with euploid chorionic villi karyotypes, those with aneuploid karyotypes were older and acquired fewer oocytes, yet their AMH levels were not significantly different. Furthermore, the embryo aneuploidy was independent of AMH while associated with maternal age, retrieved oocyte number, and embryo quality. This study suggested that AMH was unassociated with the ESA caused by embryo aneuploidy in ART therapy. As a critical cofounder, age remains the variable closely related to ESA.

Interaction between hTIM-1 and Envelope Protein Is Important for JEV Infection.

Japanese encephalitis virus (JEV), a mosquito-borne zoonotic virus, is one of the most important causes of human viral encephalitis. JEV relies on various attachment or entry co-factors to enter host cells. Among these co-factors, hTIM-1 has been identified as an attachment factor to promote JEV infection through interacting with phosphatidylserine (PS) on the viral envelope. However, the reasons why JEV prefers to use hTIM-1 over other PS binding receptors are unknown. Here, we demonstrated that hTIM-1 can directly interact with JEV E protein. The interaction between hTIM-1 and JEV relies on specific binding sites, respectively, ND114115 in the hTIM-1 IgV domain and K38 of the E protein. Furthermore, during the early stage of infection, hTIM-1 and JEV are co-internalized into cells and transported into early and late endosomes. Additionally, we found that the hTIM-1 soluble ectodomain protein effectively inhibits JEV infection in vitro. Moreover, hTIM-1-specific antibodies have been shown to downregulate JEV infectivity in cells. Taken together, these findings suggested that hTIM-1 protein directly interacts with JEV E protein and mediates JEV infection, in addition to the PS-TIM-1 interaction.

Electron-Rich Triazine-Conjugated Microporous Polymers for the Removal of Dyes from Wastewater.

Conjugated microporous polymers (CMP) as porous functional materials have received considerable attention due to their unique structures and fascinating properties for the adsorption and degradation of dyes. Herein, a triazine-conjugated microporous polymer material with rich N-donors at the skeleton itself was successfully synthesized via the Sonogashira-Hagihara coupling by a one-pot reaction. These two polymers had Brunauer-Emmett-Teller (BET) surface areas of 322 and 435 m2g-1 for triazine-conjugated microporous polymers (T-CMP) and T-CMP-Me, respectively. Due to the porous effects and the rich N-donor at the framework, it displayed a higher removal efficiency and adsorption performance compared to cationic-type dyes and selectivity properties for (methylene blue) MB+ from a mixture solution of cationic-type dyes. Furthermore, the T-CMP-Me could quickly and drastically separate MB+ and (methyl orange) MO- from the mixed solution within a short time. Their intriguing absorption behaviors are supported by 13C NMR, UV-vis absorption spectroscopy, scanning electron microscopy, and X-ray powder diffraction studies. This work will not only improve the development of porous material varieties, but also demonstrate the adsorption or selectivity of porous materials for dyes from wastewater.

P34L Mutation of swine TIM-1 enhances its ability to mediate Japanese encephalitis virus infection.

Japanese encephalitis virus (JEV) is a major causative agent of neurological infection affecting humans and pigs. Human T Cell Immunoglobulin and Mucin Domain 1 (hTIM-1) enhances the infection of JEV through virion-associated phosphatidylserine (PS) binding. Here, five swine TIM-1 (sTIM-1) gene variants were cloned from pig lung tissues by reverse-transcriptase polymerase chain reaction (RT-PCR). Sequence alignment analysis revealed that the gene homology between the sTIM-1 and hTIM-1 was 42.3-43.8%. Furthermore, ectopic expression of all five sTIM-1 variants in 293 T cells can promote JEV entry and infection. However, sTIM-1 V3 exhibited significantly less potent at promoting virus entry compared to the other four variants. Further studies revealed that the 34th amino acid of sTIM-1is critical for the entry of JEV, which is Pro34 in sTIM-1V3 while Leu34 in other four sTIM-1 variants. Mechanically, leucine at locus 34 was associated with the membrane distribution of sTIM-1, thereby affecting viral entry and infection. In total, our findings provide evidence that the PS receptor sTIM-1 promotes the infection of JEV and that the 34th amino acid position is critical for sTIM-1 to mediate viral infection.

Disrupted expression of long non-coding RNAs in the human oocyte: the possible epigenetic culprits leading to recurrent oocyte maturation arrest.

PURPOSE: To depict the lncRNA expression during human oocyte maturation and explore the lncRNAs leading to recurrent oocyte maturation arrest. METHODS: LncRNA sequencing was performed on pooled RNA from 20 oocytes of each group (recurrent oocyte maturation arrest (ROMA), of germinal vesicle (GV), metaphase I (MI), or metaphase II (MII) stages. Bioinformatics software was deployed to compare the lncRNA differential expression between the normal and ROMA oocytes. The co-expression of lncRNA/mRNA was illustrated with the Cytoscape software. The pooled RNA from every 10 oocytes of each group (ROMA, GV, MI, MII) was extracted for further qPCR validation. RESULTS: There were 17 downregulated and 3 upregulated lncRNAs in the ROMA oocyte. Among them, co-expression analysis indicated that NEAT1 and NORAD were both downregulated. Basing on the KEGG enrichment analysis, PRCKA and JAK3 might be the target genes in the PI3K-Akt pathway and modulated by NEAT1 and NORAD. As validated by qPCR, the expressional levels of lncRNA candidates (NEAT1 and NORAD) and their target genes (PRKCA and JAK3) were confirmed to be extremely lower in the ROMA oocyte than in the normal oocyte. CONCLUSION: By targeting the PI3K-Akt pathway genes PRKCA and JAK3, the abnormal expression of NEAT1 and NORAD is suggested to impede oocyte maturation and impair oocyte genome integrity.

Japanese Encephalitis Virus (JEV) NS1' Enhances the Viral Infection of Dendritic Cells (DCs) and Macrophages in Pig Tonsils.

Pigs are the amplifying hosts of Japanese encephalitis virus (JEV). Currently, the safe and effective live attenuated vaccine made of JEV strain SA14-14-2, which does not express NS1', is widely used in humans and domestic animals to prevent JEV infection. In this study, we constructed the NS1' expression recombinant virus (rA66G) through a single nucleotide mutation in NS2A of JEV strain SA14-14-2. Animal experiments showed that NS1' significantly enhanced JEV infection in pig central nervous system (CNS) and tonsil tissues. Pigs shed virus in oronasal secretions in the JEV rA66G virus inoculation group, indicating that NS1' may facilitate the horizontal transmission of JEV. Additionally, dendritic cells (DCs) and macrophages are the main target cells of JEV infection in pig tonsils, which are an important site of persistent JEV infection. The reduction of major histocompatibility complex class II (MHC II) and activation of inducible nitric oxide synthase (iNOS) in pig tonsils caused by viral infection may create a beneficial environment for persistent JEV infection. These results are of significance for JEV infection in pigs and lay the foundation for future studies of JEV persistent infection in pig tonsils. IMPORTANCE Pigs are amplification hosts for Japanese encephalitis virus (JEV). JEV can persist in the tonsils for months despite the presence of neutralizing antibodies. The present study shows that NS1' increases JEV infection in pig tonsils. In addition, DCs and macrophages in the tonsils are the target cells for JEV infection, and JEV NS1' promotes virus infection in DCs and macrophages. This study reveals a novel function of JEV NS1' protein and lays the foundation for future studies of JEV persistent infection in pig tonsils.

Japanese Encephalitis Virus NS2B-3 Protein Complex Promotes Cell Apoptosis and Viral Particle Release by Down-Regulating the Expression of AXL.

Japanese encephalitis virus (JEV) is a flavivirus transmitted by mosquitoes that causes severe encephalitis in humans and animals. It has been suggested that AXL, a transmembrane protein, can promote the replication of various flaviviruses, such as dengue (DENV), Zika (ZIKV), and West Nile (WNV) viruses. However, the effect of AXL on JEV infection has not yet been determined. In the present study, we demonstrate that AXL is down-regulated after JEV infection in the late stage. JEV NS2B-3 protein specifically interacted with AXL, and promoted AXL degradation through the ubiquitin-proteasome pathway. AXL-degradation increased cell apoptosis by disrupting phosphatidylinositol 3-kinase (PI3K)/Akt signal transduction. In addition, the degradation of AXL promoted JEV release to supernatant, whereas the virus in the cell lysates decreased. The supplementation of AXL ligand Gas6 inhibited the JEV-mediated degradation of AXL. Altogether, we discover a new function of NS2B-3 during the process of JEV replication, and provide a new insight into the interactions between JEV and cell hosts.

C19orf66 Inhibits Japanese Encephalitis Virus Replication by Targeting -1 PRF and the NS3 Protein.

The Japanese encephalitis serogroup of the neurogenic Flavivirus has a specific feature that expresses a non-structural protein NS1' produced through a programmed -1 ribosomal frameshifting (-1 PRF). Herein, C19orf66, a novel member of interferon-stimulated gene (ISG) products, exhibited significant activity of antagonizing Japanese encephalitis virus (JEV) infection. Overexpression of C19orf66 in 293T cells significantly inhibited JEV replication, while knock-down of endogenous C19orf66 in HeLa cells and A549 cells significantly increased virus replication. Notably, C19orf66 had an inhibitory effect on frameshift production of JEV NS1'. The inhibition was more significant when C19orf66 and JEV NS1-NS2A were co-expressed in the 293T cells. Both C19orf66-209 and C19orf66-Zincmut did not significantly change the NS1' to NS1 ratio and had weaker antiviral effects than C19orf66. Similarly, C19orf66-209 and C19orf66-Zincmut had no significant effect on the expression of the JEV NS3 protein, whose expression was down-regulated by C19orf66 via the lysosome-dependent pathway. These findings suggest that C19orf66 may possess at least two different mechanisms of antagonizing JEV infection. This study identified C19orf66 as a novel interferon-stimulated gene product that can inhibit JEV replication by targeting -1 PRF and the NS3 protein. The study provides baseline information for the future development of broad-spectrum antiviral agents against JEV.

AXL, an Important Host Factor for DENV and ZIKV Replication.

Flaviviruses, as critically important pathogens, are still major public health problems all over the world. For instance, the evolution of ZIKV led to large-scale outbreaks in the Yap island in 2007. DENV was considered by the World Health Organization (WHO) as one of the 10 threats to global health in 2019. Enveloped viruses hijack a variety of host factors to complete its replication cycle. Phosphatidylserine (PS) receptor, AXL, is considered to be a candidate receptor for flavivirus invasion. In this review, we discuss the molecular structure of ZIKV and DENV, and how they interact with AXL to successfully invade host cells. A more comprehensive understanding of the molecular mechanisms of flavivirus-AXL interaction will provide crucial insights into the virus infection process and the development of anti-flavivirus therapeutics.

Abnormal PIWI-interacting RNA profile and its association with the deformed extracellular matrix of oocytes from recurrent oocyte maturation arrest patients.

OBJECTIVE: To depict the PIWI-interacting RNA (piRNA) profile in oocytes from patients with recurrent oocyte maturation arrest (ROMA) and explore the piRNA candidates associated with the disease. DESIGN: An observational study. SETTING: Academic research unit. PATIENT(S): Sixteen ROMA patients who provided 140 immature oocytes that arrested at metaphase I, and 146 control patients who provided 420 oocytes for in vitro culture that were collected at the stages of germinal vesicle (GV), metaphase I (MI), and MII. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Expression profiles of piRNA and quantitative reverse-transcription polymerase chain reaction (qRT-PCR) validating data of piR-hsa-17139 and its target genes. RESULT(S): After the piRNA profile was established using piRNA sequencing and hierarchical clustering, the target genes of the piRNA were predicted by bioinformatics databases and matched with mRNA sequencing data. The piRNA expression profiles showed a greater quantity of differentially expressed piRNAs in the older-stage oocytes compared with the early-stage oocytes. The piRNA and mRNA sequencing data indicated that the most affected genes were mainly concentrated in the extracellular matrix (ECM) pathway. Based on the comparison of the piRNA and mRNA sequencing data, four differentially expressed piRNAs were associated with modulation of those ECM pathway genes. The qRT-PCR validation confirmed that piR-hsa-17139 was the only up-regulated piRNA, and its target ECM genes were suppressed in ROMA oocytes. The expression level of piR-hsa-17139 declined slightly while the expression of its target ECM genes plunged dramatically during the development of normal oocytes. CONCLUSION(S): As the important genome monitors in gametogenesis, abnormally expressed piRNAs may affect the expression of ECM modulating genes, which subsequently contributes to ROMA.

SEPARATION AND PURIFICATION OF TWO MINOR COMPOUNDS FROM RADIX ISATIDIS BY INTEGRATIVE MPLC AND HSCCC WITH PREPARATIVE HPLC.

Radix isatidis has been widely used as a Chinese traditional medicine for its anti-virus and anticancer activities where the minor components may contribute to these beneficial pharmaceutical effects. In order to enrich the target minor compounds effectively and rapidly, extraction, medium-pressure liquid chromatography (MPLC), high-speed countercurrent chromatography (HSCCC) and preparative high-performance liquid chromatography (pre-HPLC) were integratively used for separation and purification of two target minor compounds indole-3-acetonitrile-6-O-ß-D-glucopyranoside (target 1) and clemastanin B (target 2) in the present study. Radix isatidis was dried, pulverized and extracted with 50% methanol at room temperature, then concentrated and subjected to pretreatment with D-101 macroporous resin chromatography and extraction by MPLC. The first target compound was separated by MPLC at the purity raised to 70-80%, but without the second minor compounds which were irreversibly adsorbed by C18 solid support. Therefore, the second target compound in the crude extract was directly separated by HSCCC at purity of 80-90%. Finally these refined samples were further separated by pre-HPLC to obtain a high purity at 98-99%. The chemical structure identification of each target compound was carried out by IR, ESI-MS and 1H NMR.

STUDIES ON A SIMPLE AND EFFICIENT METHOD FOR LARGE-SCALE PREPARATION OF GENKWANIN FROM DAPHNE GENKWA SIEB. ET ZUCC. USING NORMAL-PHASE FLASH CHROMATOGRAPHY.

Genkwanin, a flavonoid which has anti-oxidant and anti-tumor activities, was isolated and purified from flowers of Daphne genkwa Sieb. et Zucc. in a large-scale by normal-phase flash chromatography (NPFC). Dried flower buds were extracted with methanol at room temperature and concentrated. The residues were suspended in water and first extracted with petroleum ether, and then chloroform. Genkwanin was concentrated in the chloroform and insoluble fractions. Under the target-guidance of thin layer chromatography (TLC) as well as solubility, a solvent system composed of cyclohexane-acetone (22:3, v/v) was selected. At a flow rate of 30 mL/min,the insoluble and chloroform fractions were separated to yield 1.5 g and 1.35 g of genkwanin with high purities of 98.3% and 98.6% by HPLC analysis, respectively. The chemical structure of the compound was identified by ESI-MS and NMR. Results of the present study indicated that NPFC was a large preparative-scale, speedy and simple process separation technology and it was feasible to find the appropriate proportion of solvent system by transformation from TLC condition.

Selective recognition of Triamterene in biological samples by molecularly imprinted monolithic column with a pseudo template employed.

Melamine (MAM) was employed as a pseudo template to prepare a molecularly imprinted polymer monolithic column which presents the ability of selective recognition to Triamterene (TAT), whose structure was similar to that of MAM. Methacrylic acid and ethylene glycol dimethacrylate were applied as functional monomer and cross-linker, respectively, during the in situ polymerization process. Chromatographic behaviors were evaluated, the results indicated that the molecularly imprinted polymer monolithic column possessed excellent affinity and selectivity for TAT, and the imprinting factor was high up to 3.99 when 7:3 of ACN/water v/v was used as mobile phase. In addition, the dissociation constant and the binding sites were also determined by frontal chromatography as 134.31 µmol/L and 132.28 µmol/g, respectively, which demonstrated that the obtained molecularly imprinted polymer monolith had a high binding capacity and strong affinity ability to TAT. Furthermore, biological samples could be directly injected into the column and TAT was enriched with the optimized mobile phase. These assays gave recovery values higher than 91.60% with RSD values that were always less than 3.5%. The molecularly imprinted monolithic column greatly simplified experiment procedure and can be applied to preconcentration, purification, and analysis of TAT in biological samples.