Ultrapotent influenza hemagglutinin fusion inhibitors developed through SuFEx-enabled high-throughput medicinal chemistry.

Seasonal and pandemic-associated influenza strains cause highly contagious viral respiratory infections that can lead to severe illness and excess mortality. Here, we report on the optimization of our small-molecule inhibitor F0045(S) targeting the influenza hemagglutinin (HA) stem with our Sulfur-Fluoride Exchange (SuFEx) click chemistry-based high-throughput medicinal chemistry (HTMC) strategy. A combination of SuFEx- and amide-based lead molecule diversification and structure-guided design led to identification and validation of ultrapotent influenza fusion inhibitors with subnanomolar EC50 cellular antiviral activity against several influenza A group 1 strains. X-ray structures of six of these compounds with HA indicate that the appended moieties occupy additional pockets on the HA surface and increase the binding interaction, where the accumulation of several polar interactions also contributes to the improved affinity. The compounds here represent the most potent HA small-molecule inhibitors to date. Our divergent HTMC platform is therefore a powerful, rapid, and cost-effective approach to develop bioactive chemical probes and drug-like candidates against viral targets.

Recombinant Klotho attenuates IFNγ receptor signaling and SAMHD1 expression through blocking NF-κB translocation in glomerular mesangial cells.

Interferon gamma (IFNγ) is a cytokine implicated in the pathogenesis of autoimmune diseases. SAM and HD domain-containing protein 1 (SAMHD1) is an IFNγ-inducible protein that modulates cellular dNTP levels. Mutations in the human SAMHD1 gene cause Aicardi-Goutières (AG) syndrome, an autoimmune disease sharing similar clinical features with systemic lupus erythematosus (SLE). Klotho is an anti-inflammatory protein which suppresses aging through multiple mechanisms. Implication of Klotho in autoimmune response is identified in rheumatologic diseases such as SLE. Little information exists regarding the effect of Klotho in lupus nephritis, one of the prevalent symptoms of SLE. The present study verified the effect of IFNγ on SAMHD1 and Klotho expression in MES-13 glomerular mesangial cells, a special cell type in glomerulus that is critically involved in lupus nephritis. IFNγ upregulated SAMHD1 expression in MES-13 cells through the Janus kinase-signal transducer and activator of transcription 1 (JAK-STAT1) and the nuclear factor kappa B (NFκB) signaling pathways. IFNγ decreased Klotho protein expression in MES-13 cells. Treatment of MES-13 cells with recombinant Klotho protein inhibited SAMHD1 expression by blocking IFNγ-induced NFκB nuclear translocation, but showed no effect on JAK-STAT1 signaling. Collectively, our findings support the protective role of Klotho in attenuating lupus nephritis through the inhibition of IFNγ-induced SAMHD1 expression and IFNγ downstream signaling in MES-13 cells.

Rare, convergent antibodies targeting the stem helix broadly neutralize diverse betacoronaviruses.

Humanity has faced three recent outbreaks of novel betacoronaviruses, emphasizing the need to develop approaches that broadly target coronaviruses. Here, we identify 55 monoclonal antibodies from COVID-19 convalescent donors that bind diverse betacoronavirus spike proteins. Most antibodies targeted an S2 epitope that included the K814 residue and were non-neutralizing. However, 11 antibodies targeting the stem helix neutralized betacoronaviruses from different lineages. Eight antibodies in this group, including the six broadest and most potent neutralizers, were encoded by IGHV1-46 and IGKV3-20. Crystal structures of three antibodies of this class at 1.5-1.75-Å resolution revealed a conserved mode of binding. COV89-22 neutralized SARS-CoV-2 variants of concern including Omicron BA.4/5 and limited disease in Syrian hamsters. Collectively, these findings identify a class of IGHV1-46/IGKV3-20 antibodies that broadly neutralize betacoronaviruses by targeting the stem helix but indicate these antibodies constitute a small fraction of the broadly reactive antibody response to betacoronaviruses after SARS-CoV-2 infection.

Broadly neutralizing antibodies target the coronavirus fusion peptide.

The potential for future coronavirus outbreaks highlights the need to broadly target this group of pathogens. We used an epitope-agnostic approach to identify six monoclonal antibodies that bind to spike proteins from all seven human-infecting coronaviruses. All six antibodies target the conserved fusion peptide region adjacent to the S2' cleavage site. COV44-62 and COV44-79 broadly neutralize alpha- and betacoronaviruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron subvariants BA.2 and BA.4/5, albeit with lower potency than receptor binding domain-specific antibodies. In crystal structures of COV44-62 and COV44-79 antigen-binding fragments with the SARS-CoV-2 fusion peptide, the fusion peptide epitope adopts a helical structure and includes the arginine residue at the S2' cleavage site. COV44-79 limited disease caused by SARS-CoV-2 in a Syrian hamster model. These findings highlight the fusion peptide as a candidate epitope for next-generation coronavirus vaccine development.

Broadly neutralizing antibodies target the coronavirus fusion peptide.

The potential for future coronavirus outbreaks highlights the need to develop strategies and tools to broadly target this group of pathogens. Here, using an epitope-agnostic approach, we identified six monoclonal antibodies that bound to spike proteins from all seven human-infecting coronaviruses. Epitope mapping revealed that all six antibodies target the conserved fusion peptide region adjacent to the S2' cleavage site. Two antibodies, COV44-62 and COV44-79, broadly neutralize a range of alpha and beta coronaviruses, including SARS-CoV-2 Omicron subvariants BA.1 and BA.2, albeit with lower potency than RBD-specific antibodies. In crystal structures of Fabs COV44-62 and COV44-79 with the SARS-CoV-2 fusion peptide, the fusion peptide epitope adopts a helical structure and includes the arginine at the S2' cleavage site. Importantly, COV44-79 limited disease caused by SARS-CoV-2 in a Syrian hamster model. These findings identify the fusion peptide as the target of the broadest neutralizing antibodies in an epitope-agnostic screen, highlighting this site as a candidate for next-generation coronavirus vaccine development. One-Sentence Summary: Rare monoclonal antibodies from COVID-19 convalescent individuals broadly neutralize coronaviruses by targeting the fusion peptide.

Filament Negative Regulator CDC4 Suppresses Glycogen Phosphorylase Encoded GPH1 That Impacts the Cell Wall-Associated Features in Candida albicans.

We have previously identified Candida albicans GPH1 (orf19.7021) whose protein product was associated with C. albicans Cdc4. The GPH1 gene is a putative glycogen phosphorylase because its Saccharomyces cerevisiae homolog participates in glycogen catabolism, which involves the synthesis of ß-glucan of the fungal cell wall. We made a strain whose CaCDC4 expression is repressed, and GPH1 is constitutively expressed. We established a GPH1 null mutant strain and used it to conduct the in vitro virulence assays that detect cell wall function. The in vitro virulence assay is centered on biofilm formation in which analytic procedures are implemented to evaluate cell surface hydrophobicity; competence, either in stress resistance, germ tube formation, or fibronection association; and the XTT-based adhesion and biofilm formation. We showed that the constitutively expressed GPH1 partially suppresses filamentation when the CaCDC4 expression is repressed. The C. albicans Gph1 protein is reduced in the presence of CaCdc4 in comparison with the absence of CaCdc4. Compared with the wild-type strain, the gph1Δ/gph1Δ mutant displayed a reduction in the capability to form germ tubes and the cell surface hydrophobicity but an increase in binding with fibronectin. Compared with the wild-type strain, the gph1Δ/gph1Δ mutant showed a rise in adhesion, the initial stage of biofilm formation, but displayed a similar capacity to form a mature biofilm. There was no major impact on the gph1Δ/gph1Δ mutant regarding the conditions of cell wall damaging and TOR pathway-associated nutrient depletion. We conclude that GPH1, adversely regulated by the filament suppressor CDC4, contributes to cell wall function in C. albicans.

IL-4 and IL-13 Promote Proliferation of Mammary Epithelial Cells through STAT6 and IRS-1.

T helper (Th)2 cytokines such as interleukin (IL)-4 and IL-13 control immune function by acting on leukocytes. They also regulate multiple responses in non-hematopoietic cells. During pregnancy, IL-4 and IL-13 facilitate alveologenesis of mammary glands. This particular morphogenesis generates alveoli from existing ducts and requires substantial cell proliferation. Using 3D cultures of primary mouse mammary epithelial cells, we demonstrate that IL-4 and IL-13 promote cell proliferation, leading to enlargement of mammary acini with partially filled lumens. The mitogenic effects of IL-4 and IL-13 are mediated by STAT6 as inhibition of STAT6 suppresses cell proliferation and improves lumen formation. In addition, IL-4 and IL-13 stimulate tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1). Prolonged treatment with these cytokines leads to increased IRS-1 abundance, which, in turn, amplifies IL-4- and IL-13-stimulated IRS-1 tyrosine phosphorylation. Through signaling crosstalk between IL-4/IL-13 and insulin, a hormone routinely included in mammary cultures, IRS-1 tyrosine phosphorylation is further enhanced. Lowering IRS-1 expression reduces cell proliferation, suggesting that IRS-1 is involved in IL-4- and IL-13-stimulated cell proliferation. Thus, a Th2-dominant cytokine milieu during pregnancy confers mammary gland development by promoting cell proliferation.

Process development for pandemic influenza VLP vaccine production using a baculovirus expression system.

BACKGROUND: Influenza viruses cause hundreds of thousands of respiratory diseases worldwide each year, and vaccination is considered the most effective approach for preventing influenza annual epidemics or pandemics. Since 1950, chicken embryonated eggs have been used as the main method for producing seasonal influenza vaccines. However, this platform has the main drawback of a lack of scale-up flexibility, and thus, egg-based vaccine manufacturers cannot supply sufficient doses within a short period for use for pandemic prevention. As a result, strategies for reducing the manufacturing time and increasing production capacity are urgently needed. Non-virion vaccine methods have been considered an alternative strategy against an influenza pandemic, and the purpose of maintaining an immunogenic capsule structure with infectious properties appears to be met by the virus-like particle (VLP) platform. RESULTS: An influenza H7N9-TW VLP production platform using insect cells, which included the expression of hemagglutinin (HA), NA, and M1 proteins, was established. To scale up H7N9-TW VLP production, several culture conditions were optimized to obtain a higher production yield. A high level of dissolved oxygen (DO) could be critical to H7N9-TW VLP production. If the DO was maintained at a high level, the HA titer obtained in the spinner flask system with ventilation was similar to that obtained in a shake flask. In this study, the HA titer in a 5-L bioreactor with a well-controlled DO level was substantially improved by 128-fold (from 4 HA units (HAU)/50 µL to 512 HAU/50 µL). CONCLUSIONS: In this study, a multigene expression platform and an effective upstream process were developed. Notably, a high H7N9-TW VLP yield was achieved using a two-step production strategy while a high DO level was maintained. The upstream process, which resulted in high VLP titers, could be further used for large-scale influenza VLP vaccine production.

THR1 mediates GCN4 and CDC4 to link morphogenesis with nutrient sensing and the stress response in Candida albicans.

Candida albicans (C. albicans) CDC4 (CaCDC4), encoding the F­box protein for the substrate specificity of the Skp1­cullin­F­box E3 ubiquitin ligase complex, suppresses the yeast­to­filament transition in C. albicans. In our previous study, Thr1 was identified as a CaCdc4­associated protein using affinity purification. THR1 encodes a homoserine kinase, which is involved in the threonine biosynthesis pathway. The present study generated a strain with repressible CaCDC4 expression and continuous THR1 expression. Colony and cell morphology analyses, as well as immunoblotting, revealed that the Thr1 protein was detectable under conditions in which the expression of CaCDC4 was repressed and that the filaments resulting from the repressed expression of CaCDC4 were suppressed by the constitutive expression of THR1 in C. albicans. Additionally, by using the CaSAT1­flipper method, the present study produced null mutants of THR1, GCN4, and CaCDC4. The phenotypic consequences were evaluated by growth curves, spotting assays, microscopic analysis, reverse transcription­polymerase chain reaction and XTT­based biofilm formation ability. The results revealed that fewer cells lacking THR1 entered the stationary phase but had no apparent morphological alteration. It was observed that the expression of THR1 was upregulated concurrently with GCN4 during nutrient depletion and that cells lacking GCN4 rescued the lethality of cells in the absence of THR1 in conditions accumulating homoserine in the threonine biosynthesis pathway. Of note, it was found that cells with either CaCDC4 or THR1 loss were sensitive to oxidative stress and osmotic stress, with those with THR1 loss being more sensitive. In addition, it was observed that cells with loss of either CaCDC4 or THR1 exhibited the ability to increase biofilm formation, with those lacking CaCDC4 exhibiting a greater extent of enhancement. It was concluded that CaCDC4 is important in the coordination of morphogenesis, nutrient sensing, and the stress response through THR1 in C. albicans.

Upregulation of PRMT6 by LPS suppresses Klotho expression through interaction with NF-κB in glomerular mesangial cells.

Lipopolysaccharide (LPS) released from gram-negative bacteria stimulates immune responses in infected cells. Epigenetic modifications such as DNA methylation and protein methylation modulate LPS-induced innate immune gene expressions. Expression of the Klotho protein decreased with LPS treatment in rats. In a cellular model, information regarding the effect of LPS on Klotho expression was meager. In the present study, we demonstrated that LPS triggered global DNA and protein methylation in glomerular mesangial MES-13 cells. LPS upregulated protein expressions of enzymes central to cellular methylation reactions, especially protein arginine methyltransferase 6 (PRMT6) in MES-13 cells. Expression of the Klotho protein was diminished by LPS and was restored by 5-Aza-2'-deoxycytidine (5-Aza-2'-dc), AMI-1, and ammonium pyrrolidinedithiocarbamate (PDTC), but not adenosine aldehyde (AdOx). NF-κB was identified as a substrate for arginine methylation and interacted with PRMT6 in MES-13 cells. Inhibition of PRMT activity by AMI-1 blocked LPS-induced NF-κB nuclear translocation in MES-13 cells. Our data indicate that NF-κB negatively regulated Klotho expression with an interaction with PRMT6, which was upregulated by LPS in MES-13 cells.

Galangin ameliorates cisplatin-induced nephrotoxicity by attenuating oxidative stress, inflammation and cell death in mice through inhibition of ERK and NF-kappaB signaling.

Cisplatin is a chemotherapeutic agent widely used in the treatment of various cancers. However, cisplatin can induce nephrotoxicity and neurotoxicity, limiting its dosage and usage. Galangin, a natural flavonol, has been found to exhibit anti-oxidant and anti-inflammatory effects in vivo. Here, we investigated the effects of galangin on cisplatin-induced acute kidney injury (AKI) and its molecular mechanisms in mice. Galangin administration reduced the cisplatin-induced oxidative stress by decreasing renal MDA and 3-NT formations. Galangin administration also increased renal anti-oxidative enzyme activities (SOD, GPx, and CAT) and GSH levels depleted by cisplatin. Furthermore, galangin administration inactivated stress-induced Nrf2 protein and its downstream products, HO-1 and GCLC. In terms of the inflammatory response, galangin administration reduced IκBα phosphorylation, NF-κB phosphorylation and nuclear translocation, and then inhibited cisplatin-induced secretions of pro-inflammatory TNF-α, IL-1ß and IL-6. In addition, cisplatin-induced ERK and p38 phosphorylations were inhibited by galangin administration. In terms of cell death, galangin administration reduced levels of p53, pro-apoptotic Bax and activated caspase-3 to inhibit the cisplatin-induced apoptosis. Galangin administration also reduced the expression levels of RIP1 and RIP3 to inhibit cisplatin-induced RIP1/RIP3-dependent necroptosis. Therefore, galangin administration significantly ameliorates cisplatin-induced nephrotoxicity by attenuating oxidative stress, inflammation, and cell death through inhibitions of ERK and NF-κB signaling pathways. Galangin might be a potential adjuvant for clinical cisplatin therapy.

Novel findings of secreted cyclophilin A in diabetic nephropathy and its association with renal protection of dipeptidyl peptidase 4 inhibitor.

BACKGROUND: Our previous clinical indicated that urinary cyclophilin A was a good marker for diabetic nephropathy. METHODS: We used animal and cell models of diabetic nephropathy to examine the role of cyclophilin A in disease progression. RESULTS: Significantly increased urinary cyclophilin A could be detected in db/db at the 8th week. Linagliptin (3mg/kg/day and 15mg/kg/day) could suppress urinary 8-hydroxy-2'-deoxyguanosine at the 8th and 16th week but only the high dose Linagliption could suppress cyclophilin A at the 8th week. Compared to 8-hydroxy-2'-deoxyguanosine, cyclophilin A was a stronger, earlier, and more sensitive marker. Immunohistochemical staining for cyclophilin A was also positive for db/db. In cell studies, oxidative stress and hyperglycemia could stimulate MES-13 and HK-2 cells to secrete cyclophilin A. Hyperglycemia stimulated HK-2 cells to secrete TGFß1, which caused secretion of cyclophilin A. The secreted cyclophilin A further stimulated CD 147 to move outward from cytosol onto cell membrane in confocal microscopy, which was associated with the p38 MAPK pathway in the downstream. CONCLUSIONS: Secreted cyclophilin A may play an important role in diabetic nephropathy in the mouse model and is associated with TGFß1, CD 147, and the p38 MAPK pathway.

Distal M domain of cobra ADAM-like metalloproteinase mediates the binding of positively charged cysteine-rich domain to αvß3 integrin in the suppression of cell migration.

We have previously identified two new P-III type ADAM-like snake venom metalloproteinases (SVMPs), i.e., atragin and kaouthiagin-like, from Taiwan cobra venom and determined their 3D structures with a distinct C- and I-shaped metalloproteinase/disintegrin-like/cysteine-rich (MDC) modular architecture. Herein, we investigated their functional targets to elucidate the role of cobra SVMPs in perturbing wound healing in snakebite victims. We showed that the non-RGD (Arg-Gly-Asp) C-shaped SVMP atragin binds about ten-fold stronger than the RGD-containing I-shaped SVMP kaouthiagin-like to αvß3 integrin in the surface-immobilized form. Atragin binds to αvß3 integrin through a novel interaction mode involving distal M and C domains via the RRN sequence motif in the hyper variable loop. In a cell adhesion assay, the adhesion of fibroblasts to atragin was mediated by αvß3 integrin. Furthermore, atragin inhibited wound healing and suppressed cell migration in a αvß3 integrin-dependent manner. These results, together with our previous demonstration of non-cytotoxic cobra CTX A5 in targeting αvß3 integrin, suggest that cobra venom consists of several non-RGD toxins with integrin-binding specificity that could perturb wound healing in snakebite victims.

Galangin Prevents Acute Hepatorenal Toxicity in Novel Propacetamol-Induced Acetaminophen-Overdosed Mice.

Acetaminophen (APAP) overdose causes severe liver and kidney damage. APAP-induced liver injury (AILI) represents the most frequent cause of drug-induced liver failure. APAP is relatively insoluble and can only be taken orally; however, its prodrug, propacetamol, is water soluble and usually injected directly. In this study, we examined the time-dependent effects of AILI after propacetamol injection in mice. After analyses of alanine aminotransferase and aspartate aminotransferase activities and liver histopathology, we demonstrated that a novel AILI mouse model can be established by single propacetamol injection. Furthermore, we compared the protective and therapeutic effects of galangin with a known liver protective extract, silymarin, and the only clinical agent for treating APAP toxicity, N-acetylcysteine (NAC), at the same dose in the model mice. We observed that galangin and silymarin were more effective than NAC for protecting against AILI. However, only NAC greatly improved both the survival time and rate consequent to a lethal dose of propacetamol. To decipher the hepatic protective mechanism(s) of galangin, galangin pretreatment significantly decreased the hepatic oxidative stress, increased hepatic glutathione level, and decreased hepatic microsomal CYP2E1 levels induced by propacetamol injection. In addition, propacetamol injection also reproduced the probability of APAP-induced kidney injury (AIKI), appearing similar to a clinical APAP overdose. Only galangin pretreatment showed the protective effect of AIKI. Thus, we have established a novel mouse model for AILI and AIKI using a single propacetamol injection. We also demonstrated that galangin provides significant protection against AILI and AIKI in this mouse model.

Candida albicans DBF4 gene inducibly duplicated by the mini-Ura-blaster is involved in hypha-suppression.

The opportunistic human fungal pathogen Candida albicans is a natural diploid that does not have a complete sexual cycle. The ability to switch between diverse cellular forms is important to its virulence. Here, we describe the characterization of the C. albicans DBF4 gene, a Saccharomyces cerevisiae homolog that encodes a regulatory subunit of Cdc7 kinase that is known to initiate DNA replication. We made a C. albicans strain, with one DBF4 allele deleted by the mini-Ura-blaster and the other controlled by a repressible promoter. We also found a third CaDBF4 copy that was later verified to be inducibly duplicated by targeted recombination with the min-Ura-blaster. Surprisingly, the strain deleted with the third CaDBF4 copy exhibited hyphal growth under repressed conditions. We conclude that the CaDBF4 gene is prone to being duplicated by the mini-Ura-blaster and that it suppresses hyphal growth in C. albicans.

Suppression of prolactin signaling by pyrrolidine dithiocarbamate is alleviated by N-acetylcysteine in mammary epithelial cells.

Prolactin is the key hormone to stimulate milk synthesis in mammary epithelial cells. It signals through the Jak2-Stat5 pathway to induce the expression of ß-casein, a milk protein which is often used as a marker for mammary differentiation. Here we examined the effect of pyrrolidine dithiocarbamate (PDTC) on prolactin signaling. Our results show that PDTC downregulates prolactin receptor levels, and inhibits prolactin-induced Stat5 tyrosine phosphorylation and ß-casein expression. This is not due to its inhibitory action on NF-κB since application of another NF-κB inhibitor, BAY 11-7082, and overexpression of I-κBα super-repressor do not lead to the same results. Instead, the pro-oxidant activity of PDTC is involved as inclusion of the antioxidant N-acetylcysteine restores prolactin signaling. PDTC triggers great extents of activation of ERK and JNK in mammary epithelial cells. These do not cause suppression of prolactin signaling but confer serine phosphorylation of insulin receptor substrate-1, thereby perturbing insulin signal propagation. As insulin facilitates optimal ß-casein expression, blocking insulin signaling by PDTC might pose additional impediment to ß-casein expression. Our results thus imply that lactation will be compromised when the cellular redox balance is dysregulated, such as during mastitis.

Downregulation of connective tissue growth factor by LPS/IFN-γ-induced nitric oxide is reversed by aristolochic acid treatment in glomerular mesangial cells via STAT-1α and NF-κB signaling.

Aristolochic acid (AA) is a common cause of Chinese herb nephropathy. The mechanisms involved in the pathogenesis of AA nephropathy (AAN) are intricate. One well-documented effect of AA in the kidney is its pro-fibrotic activity. Nitric oxide (NO), a messenger gas generated from l-arginine, is the product of nitric oxide synthase (NOS). NO is involved in renal hemodynamics and exerts cytoprotective effects against renal injury. In the present study, the role of NO in AAN was investigated in MES-13 cells, a glomerular mesangial cell line. NO endogenously generated by the induction of inducible nitric oxide synthase (iNOS) with lipopolysaccharide (LPS)/interferon-γ (IFN-γ) significantly downregulated connective tissue growth factor (CTGF) protein expression in MES-13 cells. AA significantly suppressed LPS/IFN-γ-induced NO production and reversed CTGF expression that was downregulated by LPS/IFN-γ. AA decreased iNOS gene and protein expressions in a concentration-dependent manner. AA caused declines in LPS/IFN-γ-induced signal transducer and activator of transcription-1α (STAT-1α) phosphorylation and interferon response factor-1 (IRF-1) mRNA expression. Furthermore, AA attenuated IκB phosphorylation and reduced NF-κB translocation to the nuclear fraction. Taken together, our data indicate that AA reversed the CTGF expression inhibited by LPS/IFN-γ treatment via suppression of NO and iNOS expressions in MES-13 cells through inhibition of the JAK/STAT-1α and NF-κB signaling pathways. NO potentially exerts antifibrotic activity by down regulation of CTGF in MES-13 cells and inhibition of the iNOS gene by AA might partially account for the fibrotic effects of AA in nephropathy.

Gcse, a novel germ-cell-specific gene, is differentially expressed during meiosis and gametogenesis.

Gametogenesis is a complex process wherein germ cells develop from primordial diploid cells into haploid gametes. To understand the mechanisms controlling gametogenesis, we identified a novel germ-cell-specific gene, Gcse. Gcse produces two major transcripts that are 1589 bp (Gcse-l) and 906 bp (Gcse-s) in length. Northern blotting and reverse transcription-polymerase chain reaction (RT-PCR) analyses of multiple tissues reveal that Gcse-l is expressed in both adult testes and ovaries, but Gcse-s is expressed only in adult testes. During female gonad development, Gcse-l is expressed from embryonic day 13.5 to adulthood, specifically in oocytes, and maintained in ovulated and fertilized eggs. However, Gcse-s signals were detected only in ovulated oocytes and fertilized eggs but not in adult ovary. During male gonad development, strong Gcse-l signals were detected in late pachytene spermatocytes and round spermatids. However, Gcse-s transcripts exist only in round spermatids. Furthermore, the expression of GCSE-L proteins and their subcellular localizations within cells are stage specific. GCSE-L is detected in the nucleus of late pachytene spermatocytes. During meiosis, GCSE-L is translocated to acrosome regions in spermatids and maintained in the acrosome of spermatozoa. GCSE-L colocalizes with acrosin and lectin peanut agglutinin in the Golgi apparatus. However, GCSE-S proteins are expressed only in the nucleus of spermatids. From these results, we suggest that GCSE proteins play roles in meiosis and may be involved in acrosome biogenesis during spermiogenesis.

Secreted cyclophilin A induction during embryo implantation in a model of human trophoblast-endometrial epithelium interaction.

OBJECTIVE: Embryo implantation in the human is a complex process that is crucial for a successful pregnancy. Implantation involves complex interactions between the embryo and the maternal endometrium. In order to understand the critical events involved in human embryo implantation, we established a human trophoblast-endometrial interaction model to study the putative alterations of gene expression during implantation. STUDY DESIGN: Comparative proteomic analysis was applied to the co-culture and separated culture systems of the trophoblast cell line BeWo and human endometrial epithelial cell (EEC) line RL95-2. Comparing the secreted molecules resulted in an interesting finding that secreted cyclophilin A (CypA) was increased in the co-culture system. To further verify our observation, human recombinant CypA was applied to endometrial cells to understand the downstream gene regulation. RESULTS: Cyclophilin A (CypA) was identified by MALDI-TOF Mass with higher expression levels in the co-cultured cells when compared to the endometrial cells alone. Western blot analysis further confirmed the increased expression of CypA in co-culture conditions. Immunoblotting analysis showed that both p38 MAPK and extracellular signal-regulated kinase (ERK) were activated in EEC. CONCLUSION: Our results suggest that the secreted CypA plays a specific role in the signaling pathway of embryo implantation. The identification of CypA opens a new avenue for early embryo implantation research.