Both intrinsic and microenvironmental factors contribute to the regulation of stem cell quiescence.

Precise regulation of stem cell quiescence is essential for tissue development and homeostasis. Therefore, its aberrant regulation is intimately correlated with various human diseases. However, the detailed mechanisms of stem cell quiescence and its specific role in the pathogenesis of various diseases remain to be determined. Recent studies have revealed that the intrinsic and microenvironmental factors are the potential candidates responsible for the orderly switch between the dormant and activated states of stem cells. In addition, defects in signaling pathways related to internal and external factors of stem cells might contribute to the initiation and development of diseases by altering the dormancy of stem cells. In this review, we focus on the mechanisms underlying stem cell quiescence, especially the involvement of intrinsic and microenvironmental factors. In addition, we discuss the relationship between the anomalies of stem cell quiescence and related diseases, hopefully providing therapeutic insights for developing novel treatments.

RING finger protein 122-like (RNF122L) negatively regulates antiviral immune response by targeting STING in common carp (Cyprinus carpio L.).

Stimulator of interferon genes (STING), as an imperative adaptor protein in innate immune, responds to nucleic acid from invading pathogens to build antiviral responses in host cells. Aberrant activation of STING may trigger tissue damage and autoimmune diseases. Given the decisive role in initiating innate immune response, the activity of STING is intricately governed by several posttranslational modifications, including phosphorylation and ubiquitination. Here, we cloned and characterized a novel RNF122 homolog from common carp (named CcRNF122L). Expression analysis disclosed that the expression of CcRNF122L is up-regulated under spring viremia of carp virus (SVCV) stimulation in vivo and in vitro. Overexpression of CcRNF122L hampers SVCV- or poly(I:C)-mediated the expression of IFN-1 and ISGs in a dose-dependent way. Mechanistically, CcRNF122L interacts with STING and promotes the polyubiquitylation of STING. This polyubiquitylation event inhibits the aggregation of STING and the subsequent recruitment of TBK1 and IRF3 to the signaling complex. Additionally, the deletion of the TM domain abolishes the negative regulatory function of CcRNF122L. Collectively, our discoveries unveil a mechanism that governs the STING function and the precise adjustment of the innate immune response in teleost.

Unveiling the airborne microbial menace: Novel insights into pathogenic bacteria and fungi in bioaerosols from nursery schools to universities.

Bacterial and fungal aerosol pollution is widespread in indoor school environments, and poses potential health risks to students and staff. Understanding the distribution and diversity of microbial communities within aerosols is crucial to mitigate their adverse effects. Existing knowledge regarding the composition of bacterial and fungal aerosols, particularly the presence of potential pathogenic microorganisms in fine particulate matter (PM2.5) from nursery schools to universities, is limited. To bridge this knowledge gap, in the present study, we collected PM2.5 samples from five types of schools (i.e., nursery schools, primary schools, junior schools, and high schools and universities) in China. We used advanced single-molecule real-time sequencing to analyze the species-level diversity of bacterial and fungal components in PM2.5 samples based on 16S and ITS ribosomal genes, respectively. We found significant differences in microbial diversity and community composition among the samples obtained from different educational institutions and pollution levels. In particularly, junior schools exhibited higher PM2.5 concentrations (62.2-86.6 µg/m3) than other schools (14.4-48.4 µg/m3). Moreover, microbial variations in PM2.5 samples were associated with institution type. Notably, the prevailing pathogenic microorganisms included Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus haemolyticus, Streptococcus pneumoniae, and Schizophyllum commune, all of which were identified as Class II Pathogenic Microorganisms in school settings. Four potentially novel strains of S. commune were identified in PM2.5 samples collected from the university; the four strains showed 92.4 %-94.1 % ITS sequence similarity to known Schizophyllum isolates. To the best of our knowledge, this is the first study to explore bacterial and fungal diversity within PM2.5 samples from nursery schools to universities. Overall, these findings contribute to the existing knowledge of school environmental microbiology to ensure the health and safety of students and staff and impacting public health.

The function of NLRP3 in anti-infection immunity and inflammasome assembly of common carp (Cyprinus carpio L.).

NLRP3 inflammasome can be activated by a variety of stimuli and plays an important role in protecting host from pathogen invasion and maintaining homeostasis. However, the activation mechanism of NLRP3 inflammasome in fish is still unclear. In the present study, the NLRP3 gene (CcNLRP3) was identified from common carp, which was 3069 bp in length and encoded a protein with five domains. Sequence analysis showed that NLRP3 was evolutionarily conserved, and CcNLRP3 was closely related to that in grass carp and zebrafish. Real-time PCR showed that CcNLRP3 was widely expressed in various immune-related tissues of healthy common carp, and significantly increased after stimulation with E. tarda, A. hydrophila and Cyprinus spring viremia virus (SVCV), suggesting that CcNLRP3 might be involved in the immune defense of common carp. The results of co-IP, spot formation, oligomerization and fluorescence localization showed that CcNLRP3 could interact with CcASC and assemble into inflammasome. The cytotoxicity assays showed that CcNLRP3 inflammasome was involved in the pyroptosis induced by CcGSDME. At the same time, CcNLRP3 could directly interact with CcCaspase-A/B and result in increased Caspase-B enzyme activity and LDH release, indicating that CcNLRP3 could also form inflammasome through ASC-independent pathway. Taken together, the results provide targets and theoretical basis for the prevention and control of infectious diseases in aquaculture.

Comparative analysis of the immune responses of CcIgZ3 in mucosal tissues and the co-expression of CcIgZ3 and PCNA in the gills of common carp (Cyprinus carpio L.) in response to TNP-LPS.

Fish live in an aquatic environment rich in various microorganisms and pathogens. Fish mucosal-associated lymphoid tissue (MALT) plays a very important role in immune defence. This study was conducted to characterize the immune response mediated by CcIgZ3 in common carp (Cyprinus carpio.) and investigate the proliferating CcIgZ3+ B lymphocytes in gill. We determined the expression of CcIgZ3 in many different tissues of common carp following stimulation by intraperitoneal injection of TNP-LPS (2,4,6-Trinitrophenyl hapten conjugated to lipopolysaccharide) or TNP-KLH (2,4,6-Trinitrophenyl hapten conjugated to Keyhole Limpet Hemocyanin). Compared with TNP-KLH, TNP-LPS can induce greater CcIgZ3 expression in the head kidney, gill and hindgut, especially in the gill. The results indicate that the gill is one of the main sites involved in the immune response mediated by CcIgZ3. To examine the distribution of CcIgZ3+ B lymphocytes, immunohistochemistry (IHC) experiments were performed using a polyclonal antibody against CcIgZ3. The results indicated that CcIgZ3 was detected in the head kidney, hindgut and gill. To further examine whether CcIgZ3+ B lymphocytes proliferate in the gills, proliferating CcIgZ3+ B cells were analysed by immunofluorescence staining using an anti-CcIgZ3 polyclonal antibody and an anti-PCNA monoclonal antibody. CcIgZ3 and PCNA (Proliferating Cell Nuclear Antigen) double-labelled cells in the gills were located within the epithelial cells of the gill filaments of common carp stimulated with TNP-LPS at 3 dps and 7 dps, and relatively more proliferating CcIgZ3+ B cells appeared in the gills of common carp at 7 dps. These data imply that CcIgZ3+ B cells in the gills might be produced by local proliferation following TNP-LPS stimulation. In summary, compared with those in TNP-KLH, CcIgZ3 preferentially affects the gills of common carp following challenge with TNP-LPS. CcIgZ3+ B cells proliferate in the gills to quickly produce the CcIgZ3 antibody. In addition, CcIgZ3+ B cells can be activated to induce a strong immune response very early locally in the gill and produce the antibody CcIgZ3, which helps exert an immune-protective effect. These results suggest that an effective vaccine can be designed to promote production of the mucosal antibody CcIgZ3.

Pathogenic bacteria and fungi in bioaerosols from specialized hospitals in Shandong province, East China.

Bacteria and fungi are abundant and ubiquitous in bioaerosols in hospital environments. Understanding the distribution and diversity of microbial communities within bioaerosols is critical for mitigating their detrimental effects. Our knowledge on the composition of bacteria or fungi in bioaerosols is limited, especially the potential pathogens present in fine particulate matter (PM2.5) from specialized hospitals. Thirty p.m.2.5 filter samples were collected from five hospitals (i.e., oral, dermatology, chest, eye, and general hospitals) in Shandong Province, East China. The diversity of bacteria and fungi was analyzed at the species level using single-molecule real-time sequencing of the 16 S and internal transcribed spacer 1 (ITS) ribosomal genes, respectively. Significant differences were detected across sampling sites in terms of microbial diversity and community composition in PM2.5 as well as pollution concentrations. The range of PM2.5 concentrations observed in hospital halls was higher, ranging from 39.0 to 46.2 µg/m3, compared to the wards where the concentrations ranged from 10.7 to 25.2 µg/m3. Furthermore, microbial variations in PM2.5 bioaerosols were associated with hospital type. The most dominant pathogens identified were Vibrio metschnikovii, Staphylococcus epidermidis, Staphylococcus haemolyticus, Fusarium pseudensiforme, and Aspergillus ruber. Among these, A. ruber was identified as an opportunistic fungus in a hospital setting for the first time. Nine potentially novel strains of F. pseudensiforme, showing 84.5%-92.0% ITS sequence similarity to known Fusarium isolates, were identified in PM2.5 samples from all hospitals (excluding an eye hospital). This study highlights the importance of hospital environments in shaping microbial aerosol communities. To the best of our knowledge, this is the first study to provide insights into the bacterial and fungal biodiversity of PM2.5 in specialized hospitals, enriching research in healthcare environmental microbiology and carrying significant public health implications.

Molecular characterization of the B lymphocyte-induced maturation protein-1 (blimp1) gene of common carp (Cyprinus carpio) and its transcription repression involves recruitment of histone deacetylase HDAC3.

Blimp1 is the master regulator of B cell terminal differentiation in mammals, it inhibits expression of many transcription factors including bcl6, which provides the basis for promoting further development of activated B lymphocytes into plasma cells. Blimp-1 is thought to act as a sequence-specific recruitment factor for chromatin-modifying enzymes including histone deacetylases (HDAC) and methyltransferases to repress target genes. The cDNA of Ccblimp1a (Cyprinus carpio) open reading frame is 2337 bp encoding a protein of 777 amino acids. CcBlimp1a contains a SET domain, two Proline Rich domains, and five ZnF_C2H2 domains. Blimp1 are conserved in vertebrate species. Ccblimp1a transcripts were detected in common carp larvae from 1 dpf (day post fertilization)to 31 dpf. Ccblimp1a expression was up-regulated in peripheral blood leukocytes (PBL) and spleen leukocytes (SPL) of common carp stimulated by intraperitoneal lipopolysaccharide (LPS) injection. Ccblimp1a expression in PBL and SPL of common carp was induced by TNP-LPS and TNP-KLH. The results indicated TNP-LPS induced a rapid response in PBL and TNP-KLH induced much stronger response in SPL and PBL. IHC results showed that CcBlimp1 positive cells were distributed in the head kidney, trunk kidney, liver, and gut. Immunofluorescence stain results showed that CcBlimp1 was expressed in IgM + lymphocytes. The subcellular localization of CcBlimp1 in the nuclei indicated CcBlimp1 may be involved in the differentiation of IgM + lymphocytes. Further study focusing on the function of CcBlimp1 transcriptional repression was performed using dual luciferase assay. The results showed that the transcription repression of CcBlimp1 on bcl6aa promoter was affected by the histone deacetylation inhibitor and was synergized with histone deacetylase 3 (HDAC3). The results of Co-IP in HEK293T and immunoprecipitation in SPL indicated that CcBlimp1 recruited HDAC3 and might be involved in the formation of complexes. These results suggest that CcBlimp1 is an important transcription factor in common carp lymphocytes. Histone deacetylation modification mediated by HDAC3 may have important roles in CcBlimp1 transcriptional repression during the differentiation of lymphocytes.

Intimal predominant calcification is associated with plaque instability in the vertebrobasilar artery by vessel wall magnetic resonance imaging and computed tomography.

BACKGROUND AND AIMS: It remains unknown about the relationship between vertebrobasilar artery (VBA) calcification and plaque instability. We aimed to investigate the characteristics of VBA calcification using vessel wall magnetic resonance imaging (MRI) and computed tomography (CT) and its association with acute cerebral infarction (ACI). METHODS: Nine hundred and thirty patients with VBA stenosis who underwent vessel wall MRI and CT examinations were evaluated retrospectively. Calcification morphology was classified as either intimal or non-intimal predominant using a CT-pathology-validated grading method. Qualitative and quantitative plaque MRI variables and calcification characteristics were compared between culprit and non-culprit lesions. The association between VBA calcification and the occurrence of culprit lesions was investigated using multivariate logistic regression. RESULTS: A total of 150 patients with ACI and 142 patients without ACI were eligible for subsequent analyses, respectively. In the qualitative analysis, T1 hyperintensity (p < 0.001) and intimal predominant calcification (p = 0.021) were more frequently observed in the culprit than non-culprit lesions. In the quantitative analyses, culprit lesions had a larger stenosis degree, plaque length, normal wall index, contrast enhancement ratio, lower calcification density and smaller calcification volume than non-culprit lesions (p all < 0.05). Intimal predominant calcification (odds ratio [OR], 2.51; 95 % confident interval [CI], 1.31-4.82, p = 0.006) and calcification density (OR, 0.53; 95 % CI, 0.35-0.78, p = 0.001) were independently associated with the presence of ACI after adjusting for clinical risk factors and plaque variables. CONCLUSIONS: Intimal predominant calcification in vertebrobasilar atherosclerosis is associated with the likelihood of having caused acute cerebral infarction. The morphology and density of VBA calcification may provide insight into stroke risk stratification in the posterior circulation.

Characterization of STING from common carp (Cyprinus carpio L.) involved in spring viremia of carp virus infection.

Stimulator of interferon genes (STING) serve as an endoplasmic reticulum (ER) protein and modulates innate immune responses to viral contagion. Most investigations involving teleost STING antiviral immunity have examined DNA viruses. Therefore, fish STING signaling events against RNA viruses require additional exploration. Here, common carp STING (named CcSTING) was cloned and characterized. The bioinformatics analyses of CcSTING showed evolutionary conservations and were most closely related to other cyprinid STINGs. Immunofluorescence staining discovered that the CcSTING was chiefly placed in the cytoplasm, specifically within the ER. CcSTING was ubiquitously generated in all analyzed organs, with especially strong expression in the gills and head kidney. Spring viremia of carp virus (SVCV) stimulation and poly(I:C) infection induced the generation of CcSTING in immune-associated organs, as well as in peripheral blood leukocytes. Additional investigations revealed that CcSTING overexpression strongly suppressed SVCV replication in EPC cells. Mechanistically, CcSTING enhanced IFN-1 and ISGs expression following SVCV infection. CcSTING also substantially increased both IFN and NF-κB promoter luciferase activity via a dosage-dependent fashion. Lastly, CcSTING significantly up-regulated both TBK1 and p65 phosphorylation. Collectively, these findings demonstrated the critical role and underlying mechanism of fish STING in response to RNA virus.

Common carp intelectin 3 (cITLN3) plays a role in the innate immune response.

Intelectin is a lectin with the capacity to recognize and bind to carbohydrates. In this study, we successfully cloned cITLN3 from common carp, which consists of a signal peptide domain, a FReD domain, and an intelectin domain. The expression levels of cITLN3 were detected in various organs of common carp, including the liver, head kidney, spleen, foregut, midgut, and hindgut, with the highest expression observed in the liver. Following infection with Staphylococcus aureus (S. aureus) or Aeromonas hydrophila (A. hydrophila), the expression level of cITLN3 was significantly upregulated in all organs of common carp. Subsequently, we expressed and purified the recombinant cITLN3 protein using an E. coli expression system. The cITLN3 could aggregate both gram-positive and gram-negative bacteria in the presence of Ca2+, with a stronger affinity for gram-positive bacteria. Moreover, our study demonstrated that cITLN3 displayed a higher binding affinity towards PGN compared to LPS. Furthermore, we observed that cITLN3 had the potential to inhibit bacterial proliferation in common carp and safeguard intestinal integrity during bacterial stimulation. And the results also indicated that cITLN3 might played a role in the Toll-like receptors (TLRs) signaling pathway activation.

One-tube RPA-CRISPR Cas12a/Cas13a rapid detection of methicillin-resistant Staphylococcus aureus.

At present, methicillin-resistant Staphylococcus aureus (MRSA) has caused a serious impact on a global scale. The infection and carrier rate of MRSA in the community is increasing year by year, but there is still no convenient detection system for on-site rapid detection. It is very important to select a rapid detection system to accurately and quickly detect patients infected with MRSA. We have developed a high-efficient single-tube detection platform based on RPA and CRISPR reaction system to detect the genes of mecA and clfA of MRSA. Using this detection platform, visual MRSA detection could be achieved in 30 min. It was observed that this detection platform was capable to successfully detect the target genomic as low as 5 copies µL-1, and the reaction was completed in one step without opening the lid. This detection platform could only detect MRSA, but not other common clinical pathogenic bacteria, such as Salmonella, Pseudomonas aeruginosa, Staphylococcus xylosus, Aeromonas hydrophila, Escherichia coli and Staphylococcus warneri, indicated its satisfactory selectivity for MRSA without interference from other bacteria. The results of clinical samples show that the platform has outstanding advantages in sensitivity, specificity and identification of methicillin resistance. The entire reaction can be completed in one step in the handheld instrument without opening the cover, avoiding aerosol pollution during the reaction. The detection platform combined with handheld instruments will have great application potential in point-of-care testing.

The functions of two GSDMEs in pyroptosis of common carp (Cyprinus carpio L.) in canonical and non-canonical inflammasome pathways.

Gasdermin family proteins are important effector proteins mediating pyroptosis and play an important role in innate immune response. GSDME can be cleaved by inflammatory Caspases at specific sites, releasing an active form of N-terminal fragment that binds to the plasma membrane to form pores and release cellular contents. Here, two GSDME genes, CcGSDME-like (CcGSDME-L) and CcGSDMEa, were cloned from common carp. The sequence similarity of the two genes were very high and more similar to DrGSDMEa of zebrafish in evolution. The expression levels of CcGSDME-L and CcGSDMEa can respond to the stimulation of Edwardsiella tarda. The results of cytotoxicity assay showed that CcGSDMEs were cleaved by the activation of canonical CcNLRP1 inflammasome, leading to obvious pyroptosis characteristics and increased cytotoxicity. In EPC cells, three CcCaspases responded to intracellular LPS stimulation and induced significantly cytotoxicity. In order to clarify the molecular mechanism of CcGSDME-induced pyroptosis, the N-terminal of CcGSDME-L (CcGSDME-L-NT) was expressed in 293T cells, which showed strong cytotoxicity and obvious pyroptosis characteristics. Fluorescence localization assay showed that the CcGSDME-L-NT was expressed on cell membrane, and CcGSDMEa-NT was located on the cell membrane or some organelle membranes. These findings can enrich the knowledge of CcNLRP1 inflammasome and GSDMEs mediated pyroptosis in common carp, and provide basic data for the prevention and treatment of fish infectious diseases.

Stimulator of interferon genes from Asian swamp eel (MaSTING) is involved in host defense against bacterial infection.

Stimulator of interferon genes (STING) is an endoplasmic reticulum (ER)-associated protein that plays critical roles in innate immunity and pathogenesis of various diseases. To date, teleost STING against viral stimulation has been identified, whereas STING signaling events in fish against bacteria are not well understood. In the present study, the open reading frame (ORF) of STING from Asian swamp eel (Monopterus albus) was cloned (named MaSTING) and its roles in bacterial infection were investigated. Amino acid sequence alignment and phylogenetic analysis revealed that MaSTING had conserved structures with mammalian STING and shared the closest relationship with mandarin fish STING. Subcellular localization analysis showed that MaSTING distributed in the whole cytoplasm and mainly co-localized with ER. Expression pattern analysis found that MaSTING was constitutively expressed in all the examined tissues with the highest expression in the liver and spleen. Post stimulation with bacteria and various PAMPs, the expression of MaSTING was induced at indicated time points in the immune-related organs and isolated peripheral blood leucocytes. Furthermore, the mechanism underlying MaSTING against bacterial infection was further studied. The qPCR analysis showed that MaSTING overexpression promoted 2'3'-cGAMP induced the expression of IFN-1, ISG15, Viperin, Mx, IL-1ß and TNF-α. Western blotting assay suggested that MaSTING significantly enhanced the phosphorylation of TANK-binding kinase 1 (TBK1) and p65. MaSTING also significantly increased the luciferase activity of IFN-1 and NF-κB promoters. Taken together, MaSTING is involved in host defense against bacterial infection by inducing the inflammatory response.

Comparison of the cross-protection of PPRSV sublineage 8.7 MLV vaccines against the recombinant NADC30-like strain.

The emergence of recombinant porcine reproductive and respiratory syndrome virus (PRRSV) has caused a substantial threat to the swine industry in recent years. However, the protective efficacy of different sublineage 8.7 PRRSV modified-live virus (MLV) vaccines against emerging strains were still obscure. In this study, a broad epidemiological investigation of PRRSV showed the prevalence of NADC30-like strain increased in Shandong Province, China from 2018 to 2020. Through piglet trial for vaccination and challenge with recombinant NADC30-like SDlz1601 strain, CH-1R MLV vaccine showed better protective effect than JXA1-R and TJM-F92 MLV vaccines in terms of clinical score and pathological observation. Moreover, all three MLV vaccines could reduce virus loads in the serum of piglets. This study provides valuable insights into the prevalence of the NADC30-like strain and the protective effect of PRRS MLV vaccines against recombinant NADC30-like strains, which could help to improve the prevention and control of PRRSV infections.

Genomic Analysis, Evolution and Characterization of E3 Ubiquitin Protein Ligase (TRIM) Gene Family in Common Carp (Cyprinus carpio).

Tripartite motifs (TRIM) is a large family of E3 ubiquitin ligases that play an important role in ubiquitylation. TRIM proteins regulate a wide range of biological processes from cellular response to viral infection and are implicated in various pathologies, from Mendelian disease to cancer. Although the TRIM family has been identified and characterized in tetrapods, but the knowledge about common carp and other teleost species is limited. The genes and proteins in the TRIM family of common carp were analyzed for evolutionary relationships, characterization, and functional annotation. Phylogenetic analysis was used to elucidate the evolutionary relationship of TRIM protein among teleost and higher vertebrate species. The results show that the TRIM orthologs of highly distant vertebrates have conserved sequences and domain architectures. The pairwise distance was calculated among teleost species of TRIMs, and the result exhibits very few mismatches at aligned position thus, indicating that the members are not distant from each other. Furthermore, TRIM family of common carp clustered into six groups on the basis of phylogenetic analysis. Additionally, the analysis revealed conserved motifs and functional domains in the subfamily members. The difference in functional domains and motifs is attributed to the evolution of these groups from different ancestors, thus validating the accuracy of clusters in the phylogenetic tree. However, the intron-exon organization is not precisely similar, which suggests duplication of genes and complex alternative splicing. The percentage of secondary structural elements is comparable for members of the same group, but the tertiary conformation is varied and dominated by coiled-coil segments required for catalytic activity. Gene ontology analysis revealed that these proteins are mainly associated with the catalytic activity of ubiquitination, immune system, zinc ion binding, positive regulation of transcription, ligase activity, and cell cycle regulation. Moreover, the biological pathway analyses identified four KEGG and 22 Reactome pathways. The predicted pathways correspond to functional domains, and gene ontology which proposes that proteins with similar structures might perform the same functions.

Organization of cortical microtubules in differentiated cells.

The microtubule cytoskeleton plays a critical role in a variety of cellular activities, and its structures and functions have been extensively studied. However, little is known about cell differentiation-related microtubule remodeling, its regulatory mechanisms, and its physiological functions. Recent studies have shown that microtubule-binding proteins as well as cell junctions, such as desmosomes and adherens junctions, are involved in the remodeling of microtubules in response to cell differentiation. In addition, the microtubule-organizing activity and structural integrity of centrosomes undergo dramatic changes during cell differentiation to promote microtubule remodeling. Here we summarize recent advances revealing the dynamic changes in microtubule organization and functions during cell differentiation. We also highlight the molecular mechanisms underlying microtubule modeling in differentiated cells, focusing on the key roles played by microtubule-binding proteins, cell junctions, and centrosomes.

Upregulation of TLR4-Dependent ATP Production Is Critical for Glaesserella parasuis LPS-Mediated Inflammation.

Glaesserella parasuis (G. parasuis), an important pathogenic bacterium, cause Glässer's disease, and has resulted in tremendous economic losses to the global swine industry. G. parasuis infection causes typical acute systemic inflammation. However, the molecular details of how the host modulates the acute inflammatory response induced by G. parasuis are largely unknown. In this study, we found that G. parasuis LZ and LPS both enhanced the mortality of PAM cells, and at the same time, the level of ATP was enhanced. LPS treatment significantly increased the expressions of IL-1ß, P2X7R, NLRP3, NF-κB, p-NF-κB, and GSDMD, leading to pyroptosis. Furthermore, these proteins' expression was enhanced following extracellular ATP further stimulation. When reduced the production of P2X7R, NF-κB-NLRP3-GSDMS inflammasome signaling pathway was inhibited, and the mortality of cells was reduced. MCC950 treatment repressed the formation of inflammasome and reduced mortality. Further exploration found that the knockdown of TLR4 significantly reduced ATP content and cell mortality, and inhibited the expression of p-NF-κB and NLRP3. These findings suggested upregulation of TLR4-dependent ATP production is critical for G. parasuis LPS-mediated inflammation, provided new insights into the molecular pathways underlying the inflammatory response induced by G. parasuis, and offered a fresh perspective on therapeutic strategies.

A novel l-rhamnose-binding lectin participates in defending against bacterial infection in zebrafish.

l-rhamnose-binding lectin (RBL), which is a class of animal lectins independent of Ca2+, can specifically bind l-rhamnose or d-galactose. Although several lectins in zebrafish have been reported, their functional mechanisms have not been fully uncovered. In this study, we discovered a novel l-rhamnose binding lectin (DrRBL) and studied its innate immune function. The DrRBL protein contains only one carbohydrate-recognition domain (CRD), which includes two strictly conserved motifs, "YGR" and "DPC". DrRBL was detected in all tested tissues and was present at high levels in the spleen, hepatopancreas and skin. After Aeromonas hydrophila challenge, the DrRBL mRNA level was significantly upregulated. Additionally, DrRBL was secreted into the extracellular matrix. Recombinant DrRBL (rDrRBL) could significantly inhibit the growth of gram-positive/negative bacteria, bind to several bacteria and cause obvious agglutination. The rDrRBL protein could combine with polysaccharides, such as PGN and LPS, rather than LTA. A more detailed study showed that rDrRBL could combine with monosaccharides, such as mannose, rhamnose and glucose, which are important components of PGN and LPS. However, rDrRBL could not bind to ribitol, which is an important component of LTA. The DrRBL deletion mutants, DrRBLΔ144-150 and DrRBLΔ198-200, were also constructed. DrRBLΔ144-150 ("ANYGRTD" deficient) showed weak bacterial inhibiting ability. However, DrRBLΔ198-200 ("DPC" deficient) showed weak agglutination ability. These results suggest that the "DPC" domain is important for agglutination. The conserved domain "ANYGRTD" is essential for inhibiting bacterial growth.

Common carp Peptidoglycan Recognition Protein 2 (CcPGRP2) plays a role in innate immunity for defense against bacterial infections.

PGRP is a family of pattern recognition molecules of the innate immune system. PGRPs are conserved from insects to mammals and have diverse functions in antimicrobial defense. Here we cloned a common carp PGRP ortholog, CcPGRP2 containing a conserved C-terminal PGRP domain. We tested the expression levels of CcPGRP2 in the liver, spleen, kidney, foregut, midgut, and hindgut of the highest level in the liver. The expression of CcPGRP2 upregulated in common carp infected with Aeromonas hydrophila (A. hydrophila) or Staphylococcus aureus (S. aureus). Recombinant CcPGRP2 protein expressed in Escherichia coli (E. coli) system and the purified CcPGRP2 could maintain the integrity of intestinal mucosa of common carp infected with A. hydrophila. In addition, CcPGRP2 could agglutinate or bind both gram-positive and gram-negative bacteria in a Zn2+-dependent manner. CcPGRP2 has a stronger agglutination and bacterial binding ability in gram-positive bacteria than in gram-negative bacteria. It is perhaps because CcPGRP2 could bind peptidoglycan (PGN) with a higher degree to lipopolysaccharide (LPS). And CcPGRP2 shows antimicrobial activities in the presence of Zn2+. Our results of CcPGRP2 provided new insight into the function of PGRP in the innate immunity of the common carp.

28-day repeated dose toxicity and toxicokinetics study on new melatonergic antidepressant GW117 in beagle dogs.

GW117 is new melatonergic antidepressant being developed to show better antidepressant action than agomelatine. The purpose of this study was to evaluate the toxicity and to determine potential target organs after oral (gavage) administration of the test article GW117 for 28 days and to assess the reversibility after a 4-week recovery phase in beagle dogs. Toxicokinetics was also evaluated. Four groups were designed in this study, including the vehicle control group and the GW117 50, 150 and 500 mg/kg/day groups, with 5 dogs/sex/group. Body weight, hematology, clinical chemistry, gross necropsy, organ weight, histopathology, and other indicators were examined. Results showed that animals dosed at ≥150 mg/kg/day showed gastrointestinal reactions (watery feces and dark green/red brown feces), with a dose-response relationship in the incidence and severity grade. Female dogs at 500 mg/kg/day had an increase in organ weight and ratios of the liver at the end of the dosing phase. Histopathology examination showed that some animals at 500 mg/kg/day, especially female animals, had minimal centrilobular hepatocyte hypertrophy in the liver, which reversed after 28-day recovery. With the exception of the above, no GW117-related abnormality was noted. Meanwhile, there were no sexual differences in drug exposure and accumulation after the first and last dosing. The no observed adverse effect dose level (NOAEL) was 150 mg/kg/day, under which mean Cmax and AUC0 → t were 583.5 and 2767.0 ng/ml*h for females and 663.2 and 4046.3 ng/ml*h for males on Day 28.