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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.

Classical swine fever virus (CSFV) and porcine circovirus type 2 (PCV2) have caused severe diseases in swine populations worldwide. Here, a polycistronic baculovirus vector was developed to express a bivalent vaccine, consisting of the CSFV-E2 and PCV2-Cap protein, and an immunomodulator protein derived from the Flammulina velutipes, FVE-FIP, as well as the selection marker, green fluorescent protein. The simultaneous expression of the CSFV-E2 and PCV2-Cap protein was mediated by the 2A-like sequence derived from the Perina nuda virus (PnV), while the expression of the FVE-FIP was driven by the internal ribosome entry site (IRES) element derived from the Rhophalosipum padi virus (RhPV). The Western blot analysis result suggested that the CSFV-E2, PCV2-Cap, and FVE-FIP protein were successfully co-expressed by the infected Spodoptera frugiperda IPBL-Sf21 (Sf21) cell line. The extracted cell lysate containing all three recombinant proteins was administered to Balb/C mice with or without the supplementation of Freund’s adjuvant. The ELISA analysis of the serum collected from all the immunized groups showed detectable antibodies against CSFV-E2 and PCV2-Cap. Furthermore, the immunized group without the adjuvant supplementation demonstrated a similar level of antibodies to the group with adjuvant supplementation, suggesting the efficiency of the FVE-FIP in enhancing the immune response. These results demonstrated the polycistronic baculovirus vector could be employed to develop bivalent vaccines for pigs.

We have previously identified

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.

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.

Clinical Nephrology is a specialty of medicine that concerns itself with the study of normal kidney function, kidney problems, the treatment of kidney problems and renal replacement therapy including dialysis and kidney transplantation. The Journal of Clinical Nephrology is an initiative of Heighten Science Publication to publish distinctive and scholarly manuscripts that can play astounding role in enhancing the excellence and confidence of the doctors, scientists and researchers associated in the studies related to renal medicine, hypertension and other related fields of Clinical Nephrology.

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.

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-1β 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.

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.

Additional file 2: Table S2. Comparison of influenza VLPs production with shake flask and Bioreactor.

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 αvβ3 integrin in the surface-immobilized form. Atragin binds to αvβ3 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 αvβ3 integrin. Furthermore, atragin inhibited wound healing and suppressed cell migration in a αvβ3 integrin-dependent manner. These results, together with our previous demonstration of non-cytotoxic cobra CTX A5 in targeting αvβ3 integrin, suggest that cobra venom consists of several non-RGD toxins with integrin-binding specificity that could perturb wound healing in snakebite victims.

Human infections of novel avian influenza A virus (H7N9) emerged in early 2013 and caused about 40% case-fatality through 2017. Therefore, development of influenza H7N9 vaccines is critical for pandemic preparedness. Currently, there are three means of production of commercial influenza vaccines: egg-based, mammalian cell-based, and insect cell-based platforms. The insect cell-based platform has the advantage of high speed in producing recombinant protein. In this study, we evaluate the stability and immunogenicity of two different influenza H7 HA expression constructs generated using the baculovirus system, including membrane-based full-length HA (mH7) and secreted ectodomain-based H7 (sH7). The mH7 construct could form an oligomer-rosette structure and had a high hemagglutinin (HA) titer 8192. In contrast to mH7, the sH7 construct could not form an oligomer-rosette structure and did not have HA titer before cross-linking with anti-His antibody. Thermal stability tests showed that the sH7 and mH7 constructs were unstable at 43 °C and 52 °C, respectively. In a mice immunization study, the mH7 construct but not the sH7 construct could induce robust HI and neutralizing antibody titers. In conclusion, further development of the mH7 vaccine candidate is desirable.

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.

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 β-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 β-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 β-casein expression, blocking insulin signaling by PDTC might pose additional impediment to β-casein expression. Our results thus imply that lactation will be compromised when the cellular redox balance is dysregulated, such as during mastitis.

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.

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.

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 (3 mg/kg/day and 15 mg/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 TGFβ1, 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 TGFβ1, CD 147, and the p38 MAPK pathway.

The South China Sea (SCS) is always a hot issue in the Asia-Pacific region after World War II. The US rebalancing strategy to Asia and China’s rising power influence the order of this region. Taiwan is one of six claimants in the SCS and effectively occupies Itu Aba and the Ban Than Reef. There are three challenges that Taiwan faces in the recent situation. The first challenge is the legal war between the Philippines and China. The second one is taking sides. The last one is ensuring sovereignty. However, Taiwan could transfer those challenges into opportunities. International cooperation on environment and humanitarian issues among all claimants is the best way to deal with disputes. After creating initiatives, Taiwan could play a constructive role to carry out the details of those initiatives.

Mitochondrial DNA (mtDNA) mutations are associated with a large number of neuromuscular diseases. Myoclonus epilepsy with ragged-red fibers (MERRF) syndrome is a mitochondrial disease inherited through the maternal lineage. The most common mutation in MERRF syndrome, the A8344G mutation of mtDNA, is associated with severe defects in mitochondrial protein synthesis, which impair the assembly and function of the respiratory chain. We have previously shown that there is a decreased level of heat shock protein 27 (HSP27) in lymphoblastoid cells derived from a MERRF patient and in cytoplasmic hybrids (cybrids) harboring the A8344G mutation of mtDNA. In the present study, we found a dramatic decrease in the level of phosphorylated HSP27 (p-HSP27) in the mutant cybrids. Even though the steady-state level of p-HSP27 was reduced in the mutant cybrids, normal phosphorylation and dephosphorylation were observed upon exposure to stress, indicating normal kinase and phosphatase activities. To explore the roles that p-HSP27 may play, transfection experiments with HSP27 mutants, in which three specific serines were replaced with alanine or aspartic acid, showed that the phosphomimicking HSP27 desensitized mutant cybrids to apoptotic stress induced by staurosporine (STS). After heat shock stress, p-HSP27 was found to enter the nucleus immediately, and with a prolonged interval of recovery, p-HSP27 returned to the cytoplasm in wild-type cybrids but not in mutant cybrids. The translocation of p-HSP27 was correlated with cell viability, as shown by the increased number of apoptotic cells after p-HSP27 returned to the cytoplasm. In summary, our results demonstrate that p-HSP27 provides significant protection when cells are exposed to different stresses in the cell model of MERRF syndrome. Therapeutic agents targeting anomalous HSP27 phosphorylation might represent a potential treatment for mitochondrial diseases.

Nitric oxide (NO) that is produced by inducible nitric oxide synthase (iNOS) is associated with the pathophysiology of glomerulonephritis. Numerous studies have focused on the regulation of NO production by iNOS to reduce NO-mediated cytotoxicity. In the present study, we demonstrated the differential effects of two phosphatidylinositol 3-kinase (PI3K) inhibitors, LY294002 and wortmannin, on lipopolysaccharide- (LPS) and interferon (IFN)-γ-induced NO production in a glomerular mesangial cell line, MES-13 cells. At dosages without affecting cell viability of MES-13 cells, 5 μM LY294002 showed a more-significant inhibitory effect on LPS/IFN-γ-induced NO production, and iNOS protein and gene expressions than did 1 μM wortmannin. Akt phosphorylation in MES-13 cells declined upon the addition of wortmannin, but not upon treatment with LY294002. Suppression of PI3K expression by small interfering (si)RNA exhibited no effect on LPS/IFN-γ-stimulated NO production or iNOS protein expression in MES-13 cells. Neither LY294002 nor wortmannin reduced IFN-γ-induced STAT-1α phosphorylation. LY294002 exhibited a more-significant inhibitory effect on NF-κB luciferase activities than wortmannin in LPS/IFN-γ-stimulated MES-13 cells. Moreover, LY294002, but not wortmannin, accelerated iNOS protein degradation and reduced the iNOS dimer/monomer ratio in MES-13 cells. Although both LY294002 and wortmannin are known as PI3K inhibitors, their differential effects on iNOS expression in MES-13 cells indicate that the effects of LY294002 on inhibiting NF-κB activation and accelerating iNOS protein degradation are through a mechanism independent of PI3K.

In mammary epithelial cells (MECs), prolactin-induced signaling and gene expression requires integrin-mediated cell adhesion to basement membrane (BM). In the absence of proper cell–BM interactions, for example, culturing cells on collagen-coated plastic dishes, signal propagation is substantially impaired. Here we demonstrate that the RhoA-Rok-myosin II pathway accounts for the ineffectiveness of prolactin signaling in MECs cultured on collagen I. Under these culture conditions, the RhoA pathway is activated, leading to downregulation of prolactin receptor expression and reduced prolactin signaling. Enforced activation of RhoA in MECs cultured on BM suppresses prolactin receptor levels, and prevents prolactin-induced Stat5 tyrosine phosphorylation and β-casein expression. Overexpression of dominant negative RhoA in MECs cultured on collagen I, or inhibiting Rok activity, increases prolactin receptor expression, and enhances prolactin signaling. In addition, inhibition of myosin II ATPase activity by blebbistatin also exerts a beneficial effect on prolactin receptor expression and prolactin signaling, suggesting that tension exerted by the collagen substratum, in collaboration with the RhoA-Rok-myosin II pathway, contributes to the failure of prolactin signaling. Furthermore, MECs cultured on laminin-coated plastic have similar morphology and response to prolactin as those cultured on collagen I. They display high levels of RhoA activity and are inefficient in prolactin signaling, stressing the importance of matrix stiffness in signal transduction. Our results reveal that RhoA has a central role in determining the fate decisions of MECs in response to cell–matrix interactions. J. Cell. Physiol. 227: 1553–1560, 2012. © 2011 Wiley Periodicals, Inc.

Aristolochic acid I (AAI) is a phytotoxin that has been found in various herbal remedies and linked to the development of human carcinogenesis. To investigate the playing role of AAI in the function of macrophages, lipopolysaccharide (LPS)-stimulated macrophage cells RAW264.7 were employed as a model to examine the effect of AAI on the expression of the inducible nitric oxide synthase (iNOS) gene. AAI reduced the expression of iNOS mRNA and protein, as well as the production of NO in LPS-stimulated macrophages. Treatment of transfected macrophages with AAI effectively suppressed the luciferase activities of the iNOS promoter which is activated by LPS. The results of promoter deletion and electrophoretic gel mobility shift assay (EMSA) indicated that the NF-κB binding site at nucleotides -86 to -76 was the major site that was most responsible for the inhibitory effect of AAI. Moreover, the presence of AAI substantially reduced the phosphorylation of the inhibitory κBα (IκBα) protein in LPS-stimulated cultures. AAI also down-regulated the LPS-induction of TNF-α, a NF-κB regulated gene. On the other hand, AAI did not modulate the luciferase activities of reporter construct that contained iNOS mRNA 3'-UTR. Taken together, the data herein suggest that in activated macrophages, AAI effectively down-regulated the expression of iNOS gene by interfering with the activation of NF-κB at the transcription level. The stability of iNOS mRNA was not the target of AAI inhibition.

The mycotoxin, citrinin (CTN), is a secondary metabolite of the fermented products of Monascus. The mycotoxin can either suppress or stimulate immune responses. In the present study, the immunomodulatory role of CTN in nitric oxide (NO) production, a proinflammatory mediator in the process of inflammation, was investigated. NO is well known as a mediator of immune responses. Overproduction of NO catalyzed by inducible nitric oxide synthase (iNOS) protects host cells against microbial invasion, while aberrant iNOS induction is associated with the pathophysiology of inflammatory events. Herein, we report that CTN significantly suppressed lipopolysaccharide (LPS)/interferon (IFN)-γ-induced NO production in MES-13 cells, a glomerular mesangial cell line. The percentage of NO reduction caused by CTN was far greater than that of the decline in cell viability. CTN decreased iNOS gene and protein expressions in concentration-dependent manners. CTN caused declines in LPS/IFN-γ-induced signal transducer and activator of transcription-1α (STAT-1α) phosphorylation. Furthermore, LPS/IFN-γ's induction of interferon response factor-1 (IRF-1) mRNA expression was inhibited by CTN. Moreover, CTN attenuated IκB-α phosphorylation and reduced NF-κB's translocation to the nuclear fraction. Taken together, our data indicated that CTN significantly suppressed NO and iNOS expressions in MES-13 cells via inhibition of the JAK/STAT-1α and NF-κB signaling pathways.

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.

That both infiltrating macrophages and resident mesangial cells express inducible nitric oxide synthase (iNOS) and produce nitric oxide (NO) excessively is crucial to the progress of glomerulonephritis. Although several reports have mentioned the protective impacts of conjugated linoleic acid (CLA) in stimulated macrophages, the role of CLA in glomerular mesangial cells is unknown. The aim of the present study was to explore the ability of CLA to regulate iNOS expression and NO production in stimulated glomerular mesangial cells. Additionally, we evaluated the effect of CLA on activation of transcription factors which mediate iNOS expression. Exogenous CLA dose-dependently diminished iNOS mRNA and protein expression as well as NO production in lipopolysaccharide (LPS) plus interferon-gamma (IFN-gamma)-stimulated SV-40-transformed mouse mesangial cells. Electrophoretic mobility shift assay experiments demonstrated that CLA (100 microM) dramatically reduced activation of nuclear factor-kappaB (NF-kappaB), activator protein-1 (AP-1) and cAMP response element binding protein (CREB) induced by LPS/IFN-gamma. Moreover, addition of 100 microM CLA significantly diminished LPS-IFN-gamma-induced protein degradation of inhibitor kappaB-alpha (IkappaB-alpha) and the protein expression of phosphorylated IkappaB-alpha in the cytosolic fraction as well as nuclear p65 expression (P < 0.05). In summary, inhibition of NF-kappaB, AP-1 and CREB activation by CLA may be associated with the molecular basis for which CLA suppresses iNOS expression and NO production in stimulated mesangial cells.

Data from a number of researchers have shown that conjugated linoleic acid (CLA) has some beneficial health activities in animal models. Because inflammatory responses are associated with pathophysiology of many diseases, the aim of this study is to explore the effect and mechanism of CLA in the regulation of lipopolysaccharide (LPS)-induced inflammatory responses in RAW 264.7 macrophages. The addition of increasing levels of CLA proportionally augmented the incorporation of CLA in cultures. CLA diminished LPS-induced mRNA and protein expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX2) as well as subsequent production of nitric oxide and prostaglandin E(2), respectively. We further examined the effect of CLA on LPS-induced NF-kappaB activation by Western blot and the electrophoretic mobility shift assay. The addition of CLA at 200 microM significantly diminished LPS-induced protein expression of the cytoplasmic phosphorylated inhibitor kappaBalpha and nuclear p65 as well as NF-kappaB nuclear protein-DNA binding affinity. In conclusion, our data suggest that CLA may inhibit LPS-induced inflammatory events in RAW 264.7 macrophages and this inhibitory activity of CLA, at least in part, occurs through CLA modulating the NF-kappaB activation and therefore negatively regulating expression of inflammatory mediators.

We previously showed that phosphorylation of Ser(10) of the N terminus domain of the type VI adenylyl cyclase (ACVI) partly mediated protein kinase C (PKC)-induced inhibition of ACVI. We now report that phosphorylation of the other two cytosolic domains (C1 and C2), which form the catalytic core complex of ACVI, also contributes to PKC-mediated inhibition. In vitro phosphorylation by PKC of the recombinant C1a and C2 domains, and of the synthetic peptides representing potential PKC phosphorylation sites, suggests that Ser(568) and Ser(674) of the C1 domain and Thr(931) of the C2 domain might act as substrates for PKC. We next created several full-length ACVI mutants in which one or more of the four likely PKC phosphorylation sites (Ser(10), Ser(568), Ser(674), and Thr(931)) were mutated to alanine. Simultaneous mutation of at least two of the three likely residues located in the N and C1 domains (Ser(10), Ser(568), and Ser(674)) was required to render ACVI variants completely insensitive to PKC treatment. In contrast, a single mutation of Thr(931) was sufficient to create a functional ACVI mutant that exhibited no detectable PKC-mediated inhibition, demonstrating the essentiality of Thr(931) to PKC-mediated regulation. Based on these results, we propose that the three cytosolic domains of ACVI might form a regulatory complex. Phosphorylation of this regulatory complex at different sites might induce a fine-tuning of the catalytic core complex and subsequently lead to alternation in the catalytic activity of ACVI.

Among the various construction safety and health (S&H) training methods, 3D digital game-based learning is believed to have a great potential. This method can combine various educational strategies to engage learners that have grown up with computer gaming. To this end, an increasing number of 3D serious games have been developed for the learning of construction S&H. However, developing a virtual construction-site simulation for serious gaming is a tedious process. Little has been done to understand the simulation parameters that should be controlled in order to adequately represent the dynamics of a construction site and, as a result, to influence student learning. Therefore, the purpose of this study is to understand and prioritize the 3D construction-site simulation parameters for learning hazard recognition. In the paper, the researchers reported their findings on the S&H game design framework as an initial step to identify how simulation parameters such as texture, lighting, and animation could align with game contents and positively affect student learning. A future experiment for validating each parameter was also introduced. The researchers expect that the final research outcomes will enable construction education stakeholders to better understand and manage the design complexity of 3D construction-site modeling for S&H serious games.

Previous results from our laboratory have shown that phosphorylation of type VI adenylyl cyclase (ACVI) by protein kinase C (PKC) caused suppression of adenylyl cyclase activity. In the present study, we investigated the role of the N terminus cytosolic domain of ACVI in this PKC-mediated inhibition of ACVI. Removal of amino acids 1 to 86 of ACVI or mutation of Ser(10) (a potential PKC phosphorylation site) into alanine significantly relieved the PKC-mediated inhibition and markedly reduced the PKC-evoked protein phosphorylation. PKC also effectively phosphorylated a recombinant N terminus cytosolic domain (amino acids 1-160) protein of ACVI and a synthetic peptide representing Ser(10). In addition, the amino acids 1 to 86 truncated mutant exhibited kinetic properties similar to those of the wild type. Taken together, these data demonstrate that the highly variable N terminus cytoplasmic domain of ACVI is a regulatory domain with a critical role in PKC-mediated suppression, which is a hallmark of this adenylyl cyclase isozyme. In addition, Ser(10) was found to serve as an acceptor for the PKC-mediated phosphorylating transfer of ACVI.

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.

Panax notoginseng(PN) is a traditional Chinese herb experimentally proven to have anti-inflammatory effects, and it is used clinically for the treatment of atherosclerosis, cerebral infarction, and cerebral ischemia. This study aimed to determine the anti-inflammatory effects of PN against bleomycin-induced pulmonary fibrosis in mice. First, in an in vitro study, culture media containing lipopolysaccharide (LPS) was used to stimulate macrophage cells (RAW 264.7 cell line). TNF-αand IL-6 levels were then determined before and after treatment with PN extract. In an animal model (C57BL/6 mice), a single dose of PN (0.5 mg/kg) was administered orally on Day 2 or Day 7 postbleomycin treatment. The results showed that TNF-αand IL-6 levels increased in the culture media of LPS-stimulated macrophage cells, and this effect was significantly inhibited in a concentration-dependent manner by PN extract. Histopathologic examination revealed that PN administered on Day 7 postbleomycin treatment significantly decreased inflammatory cell infiltrates, fibrosis scores, and TNF-α, TGF-β, IL-1β, and IL-6 levels in bronchoalveolar lavage fluid when compared with PN given on Day 2 postbleomycin treatment. These results suggest that PN administered in the early fibrotic stage can attenuate pulmonary fibrosis in an animal model of idiopathic pulmonary fibrosis.

Excessive release of nitric oxide (NO) by mesangial cells contributes to the pathogenesis of glomerulonephritis. Prostaglandin E(2) (PGE(2)) produced at inflammatory sites regulates the release of NO through its downstream signaling. In glomerular mesangial cells (MES-13 cells), PGE(2) modulated NO production mainly through EP4 receptor in a cAMP-dependent manner. Lipopolysaccharide and interferon-gamma (LPS+IFNgamma)-induced NO production, inducible nitric oxide synthase (iNOS) gene and protein expression were greatly inhibited by AH23848, an EP4 antagonist. Further investigation indicated that AH23848 attenuated endogenous cAMP accumulation in MES-13 cells and modulated NO production through declination of iNOS gene expression and acceleration of iNOS protein degradation. AH23848 downregulated the iNOS protein in MES-13 cells through protein kinase A (PKA) since KT5720, a PKA-specific inhibitor, reduced iNOS protein stability. A short exposure of activated MES-13 cells to okadaic acid augmented iNOS activity. AH23848 and KT5720 attenuated serine/threonine phosphorylation of iNOS protein in LPS + IFNgamma-stimulated MES-13 cells. The results of this study led us to speculate that cAMP might regulate iNOS-stimulated NO synthesis through posttranslational mechanisms. Attenuation of cAMP signaling and the phosphorylation status of the iNOS protein may account for the effect of AH23848 in accelerating iNOS protein degradation in MES-13 cells.

An urban network of signalized intersections can be suitably modeled as discrete event system, in which the traffic light alternations are described by means of timed coloured Petri nets (TCPN). In this paper, a model of such a network via TCPN is used to state and solve the problem of coordinating several traffic lights with six and two phases. Moreover, the analysis of the control TCPN models is performed by occurrence graphs (OG) method. And then the relation of the liveliness and reversibility of the control TCPN will be obtained. Finally, an urban traffic lights' control structure with nine intersections will be realized. To our knowledge, this is the first work that employs TCPN to obtain the models of the urban traffic lights.

In this paper, we propose a method called Timed Colour Petri Nets (TCPN) in modeling a sophisticated urban traffic light control system for nine crossroads. The variation of a vehicle's direction in the crossroads is allowed two-phase, six-phase or eight-phase transitions for each different crossroad. To our knowledge, this is the first work that employs TCPN to obtain the model of the urban traffic signal control system. The first feature of the model concerns when one direction has a green light. In the eight-phase translation process, right turns are allowed under a red light in the perpendicular direction. Second, the indication displays at each crossroad can be explicitly observed. Third, the time colour feature of the employed Petri Nets enables the compactness and explicit interpretation of the system's structure. With these features, the liveness and reversibility of the model for the urban traffic signal control system is verified.

It has been documented that arginine vasopressin (AVP) and prostaglandin E(2) (PGE(2)) regulate water reabsorption in renal tubular cells. The present study was attempted to delineate the downstream signaling of AVP and PGE(2) in a cortical collecting duct cell line (M-1 cell). Using RT-PCR, we detected mRNA for V2 and VACM-1 but not for V1a and AII/AVP receptors of AVP. Furthermore, neither AVP nor V2 receptor agonist and antagonist alter cellular cAMP. These together with unchanged cellular Ca(2+) by AVP suggested that AVP pathway was not operating in M-1 cells. All four classical PGE(2) receptors with EP3 and EP4 as the most prominent were detected in M-1 cells. PGE(2), 11-deoxy-PGE(1) (EP2 and EP4 agonist), and 17-phenyl-trinor-PGE(2) (EP1 agonist) increased cellular concentration of cAMP. There was no effect of PGE(2) or EP1 agonist on cellular Ca(2+). These findings provide evidence of the involvement of PGE(2) cascade in M-1 cells. M-1 cells were capable of synthesizing nitric oxide (NO). Although individual cytokines did not affect NO production, a mixture of tumor necrosis factor-alpha, interleukin-1beta, and interferon-gamma elevated NO concentration to 4.5-fold of the control. Addition of PGE(2) and db-cAMP to the cytokine mixture further increased NO production to 7.0- and 9.8-fold, respectively, of that seen in non-treated cells. PGE(2) or db-cAMP alone, however, had no effect on NO production. The results of the study led us to speculate that enhanced production of cAMP via PGE(2) signaling pathway in M-1 cells could either stimulate or attenuate water reabsorption in renal tubule. While an increase in cAMP alone may enhance water reabsorption, a concomitant increase in cAMP and cytokines may inhibit water reabsorption in renal tubule.