Weiheng Su

NPA motifs play a key role in plasma membrane targeting of aquaporin‐4

The two highly conserved NPA motifs (asparagine–proline–alanine, NPA) are the most important structural domains that play a crucial role in water‐selective permeation in aquaporin water channels. However, the functions of NPA motifs in aquaporin (AQP) biogenesis remain largely unknown. Few AQP members with variations in NPA motifs such as AQP11 and AQP12 do not express in the plasma membrane, suggesting an important role of NPA motifs in AQP plasma membrane targeting. In this study, we examined the role of the two NPA motifs in AQP4 plasma membrane targeting by mutagenesis. We constructed a series of AQP4 mutants with NPA deletions or single amino acid substitutions in AQP4‐M1 and AQP4‐M23 isoforms and analyzed their expression patterns in transiently transfected FRT and COS‐7 cells. Western blot analysis showed similar protein bands of all the AQP4 mutants and the wild‐type AQP4. AQP4 immunofluorescence indicated that deletion of one or both NPA motifs resulted in defective plasma membrane targeting, with apparent retention in endoplasmic reticulum (ER). The A99T mutant mimicking AQP12 results in ER retention, whereas the A99C mutant mimicking AQP11 expresses normally in plasma membrane. Furthermore, the AQP4‐M1 but not the M23 isoform with P98A substitution in the first NPA motif can target to the plasma membrane, indicating an interaction of N‐terminal sequence of AQP4‐M1 with the first NPA motif. These results suggest that NPA motifs play a key role in plasma membrane expression of AQP4 but are not involved in AQP4 protein synthesis and degradation. The NPA motifs may interact with other structural domains in the regulation of membrane trafficking during aquaporin biogenesis.

Identification of Luteolin as Enterovirus 71 and Coxsackievirus A16 Inhibitors through Reporter Viruses and Cell Viability-Based Screening

Hand, foot and mouth disease (HFMD) is a common pediatric illness mainly caused by infection with enterovirus 71 (EV71) and coxsackievirus A16 (CA16). The frequent HFMD outbreaks have become a serious public health problem. Currently, no vaccine or antiviral drug for EV71/CA16 infections has been approved. In this study, a two-step screening platform consisting of reporter virus-based assays and cell viability‑based assays was developed to identify potential inhibitors of EV71/CA16 infection. Two types of reporter viruses, a pseudovirus containing luciferase-encoding RNA replicons encapsidated by viral capsid proteins and a full-length reporter virus containing enhanced green fluorescent protein, were used for primary screening of 400 highly purified natural compounds. Thereafter, a cell viability-based secondary screen was performed for the identified hits to confirm their antiviral activities. Three compounds (luteolin, galangin, and quercetin) were identified, among which luteolin exhibited the most potent inhibition of viral infection. In the cell viability assay and plaque reduction assay, luteolin showed similar 50% effective concentration (EC50) values of about 10 μM. Luteolin targeted the post-attachment stage of EV71 and CA16 infection by inhibiting viral RNA replication. This study suggests that luteolin may serve as a lead compound to develop potent anti-EV71 and CA16 drugs.

Analysis of Cross-Reactive Neutralizing Antibodies in Human HFMD Serum with an EV71 Pseudovirus-Based Assay

Hand, foot and mouth disease, associated with enterovirus 71 (EV71) infections, has recently become an important public health issue throughout the world. Serum neutralizing antibodies are major indicators of EV71 infection and protective immunity. However, the potential for cross-reactivity of neutralizing antibodies for different EV71 genotypes and subgenotypes is unclear. Here we measured the cross-reactive neutralizing antibody titers against EV71 of different genotypes or subgenotypes in sera collected from EV71-infected children and vaccine-inoculated children in a phase III clinical trial (ClinicalTrials.gov Identifier: NCT01636245) using a new pseudovirus-based neutralization assay. Antibodies induced by EV71-C4a were cross-reactive for different EV71 genotypes, demonstrating that C4a is a good candidate strain for an EV71 vaccine. Our study also demonstrated that this new assay is practical for analyses of clinical samples from epidemiological and vaccine studies.

Increased Female Fertility in Aquaporin 8-Deficient Mice

Aquaporin‐8 (AQP8) is a water channel expressed extensively in male and female reproductive systems. But its physiological functions are largely unknown. In the present study, we first found significantly increased number of offspring delivered by AQP8−/− mothers compared with wild‐type mothers in cross‐mating experiments. Comparison of ovulation in the two genotypes demonstrated that AQP8−/− ovaries released more oocytes (9.5 ± 1.9 vs. 7.1 ± 2.1 in normal ovulation and 37.8 ± 6.7 vs. 27.9 ± 5.7 in superovulation). Histological analysis showed increased number of corpus luteums in mature AQP8−/− ovaries, suggesting increased maturation and ovulation of follicles. By RT‐PCR, western blot and immunohistochemistry analyses, we determined the expression of AQP8 in mouse ovarian granulosa cells. Granulosa cells isolated from AQP8−/− mice showed 45% of decreased membrane water permeability than wild‐type mice. As the atresia of ovarian follicles is primarily due to apoptosis of granulosa cells, we analyzed the apoptosis of isolated granulosa cells from wild‐type and AQP8−/− mice. The results indicated significantly lower apoptosis rate in AQP8−/− granulosa cells (21.3 ± 3.6% vs. 32.6 ± 4.3% in AQP8+/+ granulosa cells). Taken together, we conclude that AQP8 deficiency increases the number of mature follicles by reducing the apoptosis of granulosa cells, thus increasing the fertility of female mice. This discovery may offer new insight of improving female fertility by reducing granulosa cell apoptosis through AQP8 inhibition.

Establishment of cell lines with increased susceptibility to EV71/CA16 by stable overexpression of SCARB2

BackgroundHuman enterovirus type 71 (EV71) and Coxsackievirus A group type 16 (CA16) belong to human Enterovirus species A of the family Picornaviridae. These viruses are recognized as the major pathogens responsible for epidemics of hand-foot-mouth disease (HFMD), which presents with fever and vesicular eruptions of palms, soles of the feet or mouth. Human scavenger receptor class B, member 2 (SCARB2) has been identified as the receptor for both EV71 and CA16, as overexpression of SCARB2 in cells can enhance virus replication significantly.MethodsIn this study, we used a lentivirus packaging vector to transduce the SCARB2 gene into human embryonic kidney cells (293), human rhabdomyosarcoma cells (RD) and African green monkey kidney cells (Vero) to create stable expression lines. Expression of SCARB2 in the resulting three transgenic cell lines was confirmed by real-time RT-PCR, immunofluorescence and flow cytometry.ResultsLevels of SCARB2 mRNA determined by real-time RT-PCR in 293-SCARB2 (293S) or RD-SCARB2 (RDS) transgenic cell lines were approximately 2 × 102 times higher than those in 293 and RD cells, respectively, and three times higher in Vero-SCARB2 (VeroS) than in Vero cells. Furthermore, EV71 and CA16 virus titers in 293S and RDS cells were 102–103-fold higher (detected in RD cell) than those in the parental cells, and a 10-fold higher titer of EV71 was achieved in VeroS cells compared with that in Vero cells.ConclusionsWe established for the first time three cell lines stably overexpressing SCARB2, which showed drastic increases in susceptibility to EV71/CA16 infection. These optimal cell lines may be utilized to develop inactivated vaccines for EV71/CA16 and facilitate rapid detection and isolation of HFMD pathogens or other Enterovirus serotypes. Furthermore, these stable cell lines also can serve as tools to facilitate drug screenings as well as molecular studies on virus-host interactions and pathogenesis of causative agents for HFMD.

Anoctamin 1 Calcium-Activated Chloride Channel Downregulates Estrogen Production in Mouse Ovarian Granulosa Cells

Calcium-dependent chloride conductances have been described in chicken and human granulosa cells (GCs) and may be involved in steroidogenesis. However, the molecular identities of corresponding chloride channels remain unknown. The purpose of this study was to explore the expression and function of the Anoctamin 1 (ANO1) calcium-activated chloride channel (CaCC) in mouse ovary. ANO1 mRNA and protein expression was identified in mouse ovary GCs by RT-PCR, immunoblot, and immunostaining. Patch-clamp analysis on freshly isolated GCs identified an outwardly rectifying Ca(2+)-activated Cl(-) current that was completely blocked by a selective ANO1 inhibitor T16Ainh-A01. Knockdown of ANO1 mRNA or incubation with a selective inhibitor T16Ainh-A01 enhanced estradiol production, whereas a selective ANO1 activator Eact significantly inhibited estradiol production in primary cultured GCs. The ANO1 expression or activation increases the phosphorylation of ERK1/2 and decreases aromatase expression. The ANO1 expression level is remarkably higher at the proestrous and estrous stages in the estrous cycle. In vivo study indicated a profound induction of ANO1 expression in ovarian GCs by pregnant mare serum gonadotropin (PMSG) that can be further augmented by hCG treatment, suggesting that both FSH and LH may upregulate ANO1 expression at the proestrous and estrous stages. ANO1 expression was remarkably reduced in DHEA-induced PCOS ovary. These data identified for the first time the expression of ANO1 Ca(2+) activated Cl(-) channel in mouse ovarian GCs and determined its negative regulation on estrogen production possibly through MEK-ERK signaling cascade. The present study provided new insights into the molecular mechanisms for the regulation of folliculogenesis and ovulation.

A new EV71 VP3 epitope in norovirus P particle vector displays neutralizing activity and protection in vivo in mice

Enterovirus 71 (EV71) and Coxsackievirus A16 (CVA16), as the main agents causing hand, foot and mouth disease (HFMD), have become a serious public health concern in the Asia-Pacific region. Recently, various neutralizing B cell epitopes of EV71 were identified as targets for promising vaccine candidates. Structural studies of Picornaviridae indicated that potent immunodominant epitopes typically lie in the hypervariable loop of capsid surfaces. However, cross-neutralizing antibodies and cross-protection between EV71 and CVA16 have not been observed. Therefore, we speculated that divergent sequences of the two viruses are key epitopes for inducing protective neutralizing responses. In this study, we selected 10 divergent epitope candidates based on alignment of the EV71 and CVA16 P1 amino acid sequences using the Multalin interface page, and these epitopes are conserved among all subgenotypes of EV71. Simultaneously, by utilizing the norovirus P particle as a novel vaccine delivery carrier, we identified the 71-6 epitope (amino acid 176-190 of VP3) as a conformational neutralizing epitope against EV71 in an in vitro micro-neutralization assay as well as an in vivo protection assay in mice. Altogether, these results indicated that the incorporation of the 71-6 epitope into the norovirus P domain can provide a promising candidate for an effective synthetic peptide-based vaccine against EV71.

Enhanced Sensitivity for Detection of HIV-1 p24 Antigen by a Novel Nuclease-Linked Fluorescence Oligonucleotide Assay

The relatively high detection limit of the Enzyme-linked immunosorbent assay (ELISA) prevents its application for detection of low concentrations of antigens. To increase the sensitivity for detection of HIV-1 p24 antigen, we developed a highly sensitive nuclease-linked fluorescence oligonucleotide assay (NLFOA). Two major improvements were incorporated in NLFOA to amplify antibody-antigen interaction signals and reduce the signal/noise ratio; a large number of nuclease molecules coupled to the gold nanoparticle/streptavidin complex and fluorescent signals generated from fluorescent-labeled oligonucleotides by the nuclease. The detection limit of p24 by NLFOA was 1 pg/mL, which was 10-fold more sensitive than the conventional ELISA (10 pg/mL). The specificity was 100% and the coefficient of variation (CV) was 7.8% at low p24 concentration (1.5 pg/mL) with various concentrations of spiked p24 in HIV-1 negative sera. Thus, NLFOA is highly sensitive, specific, reproducible and user-friendly. The more sensitive detection of low p24 concentrations in HIV-1-infected individuals by NLFOA could allow detection of HIV-1 infections that are missed by the conventional ELISA at the window period during acute infection to further reduce the risk for HIV-1 infection due to the undetected HIV-1 in the blood products. Moreover, NLFOA can be easily applied to more sensitive detection of other antigens.

Podocyte‐specific VEGF down‐regulation and pathophysiological development

It is well‐known that vascular endothelial growth factor (VEGF) plays a key role in development and pathology, but its function in normal adult tissues is rarely understood. Increased use of anti‐angiogenic therapies targeting VEGF in human pathologies have shown more and more adverse effects. In this report, a conditional expression model (Tet‐On system) was used to down‐regulate podocyte VEGF in adult mice, which resulted in many kidney problems, characterized by glomerular morphological changes, proteinuria, reduced water consumption and urination, increased urine electro‐conductivity, as well as high susceptibility to BSA stress. Our findings indicated that podocyte‐specific VEGF down‐regulation resulted in poor kidney performance and led mice to be more susceptible to further kidney damages.

Transcriptome analysis reveals dynamic changes in coxsackievirus A16 infected HEK 293T cells

BackgroundCoxsackievirus A16 (CVA16) and enterovirus 71 (EV71) are two of the major causes of hand, foot and mouth disease (HFMD) world-wide. Although many studies have focused on infection and pathogenic mechanisms, the transcriptome profile of the host cell upon CVA16 infection is still largely unknown.ResultsIn this study, we compared the mRNA and miRNA expression profiles of human embryonic kidney 293T cells infected and non-infected with CVA16. We highlighted that the transcription of SCARB2, a cellular receptor for both CVA16 and EV71, was up-regulated by nearly 10-fold in infected cells compared to non-infected cells. The up-regulation of SCARB2 transcription induced by CVA16 may increase the possibility of subsequent infection of CVA16/EV71, resulting in the co-infection with two viruses in a single cell. This explanation would partly account for the co-circulation and genetic recombination of a great number of EV71 and CVA16 viruses. Based on correlation analysis of miRNAs and genes, we speculated that the high expression of SCARB2 is modulated by down-regulation of miRNA has-miR-3605-5p. At the same time, we found that differentially expressed miRNA target genes were mainly reflected in the extracellular membrane (ECM)-receptor interaction and circadian rhythm pathways, which may be related to clinical symptoms of patients infected with CVA16, such as aphthous ulcers, cough, myocarditis, somnolence and potentially meningoencephalitis. The miRNAs hsa-miR-149-3p and hsa-miR-5001-5p may result in up-regulation of genes in these morbigenous pathways related to CVA16 and further cause clinical symptoms.ConclusionsThe present study elucidated the changes in 293T cells upon CVA16 infection at transcriptome level, containing highly up-regulated SCARB2 and genes in ECM-receptor interaction and circadian rhythm pathways, and key miRNAs in gene expression regulation. These results provided novel insight into the pathogenesis of HFMD induced by CVA16 infection.