A developmental landscape of 3D-cultured human pre-gastrulation embryos.

Our understanding of how human embryos develop before gastrulation, including spatial self-organization and cell type ontogeny, remains limited by available two-dimensional technological platforms1,2 that do not recapitulate the in vivo conditions3-5. Here we report a three-dimensional (3D) blastocyst-culture system that enables human blastocyst development up to the primitive streak anlage stage. These 3D embryos mimic developmental landmarks and 3D architectures in vivo, including the embryonic disc, amnion, basement membrane, primary and primate unique secondary yolk sac, formation of anterior-posterior polarity and primitive streak anlage. Using single-cell transcriptome profiling, we delineate ontology and regulatory networks that underlie the segregation of epiblast, primitive endoderm and trophoblast. Compared with epiblasts, the amniotic epithelium shows unique and characteristic phenotypes. After implantation, specific pathways and transcription factors trigger the differentiation of cytotrophoblasts, extravillous cytotrophoblasts and syncytiotrophoblasts. Epiblasts undergo a transition to pluripotency upon implantation, and the transcriptome of these cells is maintained until the generation of the primitive streak anlage. These developmental processes are driven by different pluripotency factors. Together, findings from our 3D-culture approach help to determine the molecular and morphogenetic developmental landscape that occurs during human embryogenesis.

Human cytomegalovirus infection impairs neural differentiation via repressing sterol regulatory element binding protein 2-mediated cholesterol biosynthesis.

Congenital human cytomegalovirus (HCMV) infection is a major cause of abnormalities and disorders in the central nervous system (CNS) and/or the peripheral nervous system (PNS). However, the complete pathogenesis of neural differentiation disorders caused by HCMV infection remains to be fully elucidated. Stem cells from human exfoliated deciduous teeth (SHEDs) are mesenchymal stem cells (MSCs) with a high proliferation and neurogenic differentiation capacity. Since SHEDs originate from the neural crest of the early embryonic ectoderm, SHEDs were hypothesized to serve as a promising cell line for investigating the pathogenesis of neural differentiation disorders in the PNS caused by congenital HCMV infection. In this work, SHEDs were demonstrated to be fully permissive to HCMV infection and the virus was able to complete its life cycle in SHEDs. Under neurogenic inductive conditions, HCMV infection of SHEDs caused an abnormal neural morphology. The expression of stem/neural cell markers was also disturbed by HCMV infection. The impairment of neural differentiation was mainly due to a reduction of intracellular cholesterol levels caused by HCMV infection. Sterol regulatory element binding protein-2 (SREBP2) is a critical transcription regulator that guides cholesterol synthesis. HCMV infection was shown to hinder the migration of SREBP2 into nucleus and resulted in perinuclear aggregations of SREBP2 during neural differentiation. Our findings provide new insights into the prevention and treatment of nervous system diseases caused by congenital HCMV infection.

Development of a clinical prognostic model for primary plasma cell leukemia patients treated with novel agents: a multicenter retrospective cohort study.

The prognosis of primary plasma cell leukemia (pPCL) is poor, and the relevant prognostic factors are incompletely understood. We aimed to explore the prognostic factors and develop a validated prognostic prediction model for pPCL patients in the new era. This multicenter retrospective study was conducted across 16 hospitals in China. Cox proportional hazards regression analysis was used to develop a prediction model. The predictive performance of the model was assessed using multiple metrics. Internal validation was conducted using bootstrap resampling. A total of 102 pPCL patients were included in this study, and 57 (55.9%) were male. The 12-month, 24-month, and 36-month OS rates for pPCL patients were 75.4%, 58.3%, and 47.6%, respectively. An overall survival prognostic nomogram for pPCL patients was established by integrating independent prognostic factors, including age, B2MG, and del17p. The nomogram exhibited good performance, with a C-index of 0.720 (95% CI 0.642-0.797) and an AUC of 0.653. Bootstrap validation yielded a C-index of 0.721 (95% CI 0.629-0.787) and an AUC of 0.653 (95% CI 0.546-0.759), indicating a relatively good fit of the calibration curve. A nomogram incorporating age, B2MG grade, and del17p were developed and validated to accurately and consistently predict the prognosis of pPCL patients.

Efficacy and safety of generic pomalidomide plus low-dose dexamethasone in relapsed or refractory multiple myeloma: a multicenter, open-label, single-arm trial.

This multicenter, open-label, single-arm trial (ClinicalTrials.gov, NCT05236621) was conducted to confirm the efficacy and safety of generic pomalidomide plus dexamethasone in Chinese patients with relapsed or refractory multiple myeloma (RRMM). Total 79 eligible RRMM patients were planned to be included. Patients were treated with generic pomalidomide (4 mg daily on days 1-21, orally) and low-dose dexamethasone (40 mg/day on days 1, 8, 15, and 22, orally; 20 mg for patients aged > 75 years) in 28-day cycles until disease progression with a maximum treatment duration of 2 years. The primary endpoint is the overall response rate (ORR) assessed by the independent review committee per the 2016 International Myeloma Working Group guidelines. A total of 85 eligible patients were included in this study from 32 centers in China, with a median age of 62.0 (range, 39-76) years, a median prior line of therapy of 4 (range, 1-16), and 41.2% patients with high-risk cytogenetics. The ORR was 38.8% (95% confidence interval (CI), 28.44-50.01). The disease control rate was 67.1% (95% CI, 56.02-76.87), meanwhile, the median progression-free survival was 5.55 months (95% CI, 3.68-7.52). Among the treatment-related adverse events (TRAEs), infective pneumonia (17.6%) was the most frequent non-hematologic adverse event, while a decrease in neutrophil count (52.9%) was the most common grade ≥ 3 TRAE. The study results indicated that the generic pomalidomide demonstrated consistent efficacy and a safety profile similar to the branded pomalidomide when combined with low-dose dexamethasone in Chinese RRMM patients.Registration number ClinicalTrials.gov NCT05236621, retrospectively registered on February 11, 2022.

Mandarin Fish Ranavirus (MRV) Infection Induced Inflammation and Histologic Lesions in the Gut of Mandarin Fish.

Mandarin fish ranavirus (MRV) is widely spread in China and causes huge economic losses to the mandarin fish (Siniperca chuatsi) aquaculture. However, the pathogenesis of MRV is still unclear. In the present study, mandarin fish were artificially infected with MRV, and then different gut compartments from diseased fish were subjected to histologic analysis by H&E staining, quantification of proinflammatory genes and MRV copies by qPCR. MRV-MCP protein expression was assessed using indirect fluorescence assay (IFA) and immunohistochemistry. Proliferation of IgM+ B cells was evaluated by indirect fluorescence assay (IFA). Then, we found that MRV infection caused serious histologic lesions along with inflammatory cell infiltration, especially in the foregut. A significant accumulation of IgM+ B cells was detected in the foregut (~6.5-fold) and hindgut (~3.3-fold), respectively. The expression of inflammation-related genes such as IL-1ß, IL-6, IL-8, TNF-α, CSF1r and NCF1 was significantly upregulated in the foregut, varying from ~2.8-fold to ~11.9-fold. In addition, MRV exhibited foregut tropism, according to the investigation of viral loads and MCP protein expression. Overall, our findings indicated that MRV-induced hyperinflammation in the gut eventually led to enteritis. This study provided new insights into uncovering the pathogenesis of MRV infection.

Intensive Cycloalkyl-Fused Pyridines for Aminopyridyl-Zinc-Heteroimidazoles Achieving High Efficiency toward the Ring-Opening Polymerization of Lactides.

The model precatalyst sp3- and sp2-N dinitrogen-coordinated zinc-heteroimidazole has been used as an efficient catalyst for the ring-opening polymerization of cyclic esters. Subsequent to our exceptional active 5,6,7-trihydroquinolin-8-amine-zinc catalysts for the ring-opening polymerization (ROP) of ε-caprolactone, various pyridine-fused cycloalkanones (ring size from five to eight) are developed for the correspondent fused amine-pyridine derivatives and their zinc-heteroimidazole chloride complexes Zn1-Zn8 (LZnCl2) bearing N-diphenylphosphinoethyl pendants. Activated with two equivalents of LiN(SiMe3)2, the title zinc complexes efficiently promote the ROP of L-lactide (L-LA) in situ; among them, Zn4/2Li(NSiMe3)2 catalyzed 500 equivalent L-LA at 80 °C with 92% conversion in 5 min (TOF: 5520 h-1). Under the same conditions, the catalytic efficiency for the ROP of rac-LA by Zn1-Zn8/2Li(NSiMe3)2 was slightly lower than that for L-LA (highest TOF: 4440 h-1). In both cases, cyclooctyl-fused pyridyl-zinc complexes exhibited higher activity than others, while the cycloheptyl-fused zinc complexes showed the lowest activity. The microstructure analysis of the polymers showed they possessed a linear structure capped with CH3O as major and cyclic structure as minor. In this work, all the ligands and zinc complexes were well characterized by 1H/13C/31P NMR, FT-IR spectroscopy as well as elemental analysis.

Characterization of a membrane Fcγ receptor in largemouth bass (Micropterus saloumoides) and its response to bacterial challenge.

Fc receptors (FcRs), specific to the Fc portion of immunoglobulin (Ig), are required to regulate immune responses against pathogenic infections. However, FcγR is a member of FcRs family, whose structure and function remains to be elucidated in teleost fish. In this study, the FcγRII, from largemouth bass (Micropterus saloumoides), named membrane MsFcγRII (mMsFcγRII), was cloned and identified. The opening reading frame (ORF) of mMsFcγRII was 750 bp, encoding 249 amino acids with a predicted molecular mass of 27 kDa. The mMsFcγRII contained a signal peptide, two Ig domains, a transmembrane domain, and an intracellular region, which was highly homology with FcγR from other teleost fish. The mRNA expression analysis showed that mMsFcγRII was widely distributed in all tested tissues and with the highest expression level in spleen. After bacterial challenge, the expression of mMsFcγRII was significantly upregulated in vivo (spleen and head kidney), as well as in vitro (leukocytes from head kidney). The subcellular localization assay revealed that mMsFcγRII was mostly observed on the membrane of HEK293T cells which were transfected with mMsFcγRII overexpression plasmid. Flow cytometric analysis showed that natural mMsFcγRII protein was highly expressed in head kidney lymphocytes. Moreover, indirect immunofluorescence assay and pull-down assay indicated that mMsFcγRII could bind to IgM purified from largemouth bass serum. These results suggested that mMsFcγRII was likely to play an influential role in the immune response against pathogens and provided valuable insights for studying the function of FcRs in teleost.

Protein-RNA Interactome Analysis Reveals Wide Association of Kaposi's Sarcoma-Associated Herpesvirus ORF57 with Host Noncoding RNAs and Polysomes.

Kaposi's sarcoma-associated herpesvirus (KSHV) ORF57 is an RNA-binding posttranscriptional regulator. We recently applied an affinity-purified anti-ORF57 antibody to conduct ORF57 cross-linking immunoprecipitation (CLIP) in combination with RNA-sequencing (CLIP-seq) and analyzed the genome-wide host RNA transcripts in association with ORF57 in BCBL-1 cells with lytic KSHV infection. Mapping of the CLIP RNA reads to the human genome (GRCh37) revealed that most of the ORF57-associated RNA reads were from rRNAs. The remaining RNA reads mapped to several classes of host noncoding and protein-coding mRNAs. We found that ORF57 binds and regulates expression of a subset of host long noncoding RNAs (lncRNAs), including LINC00324, LINC00355, and LINC00839, which are involved in cell growth. ORF57 binds small nucleolar RNAs (snoRNAs) responsible for 18S and 28S rRNA modifications but does not interact with fibrillarin or NOP58. We validated ORF57 interactions with 67 snoRNAs by ORF57 RNA immunoprecipitation (RIP)-snoRNA array assays. Most of the identified ORF57 rRNA binding sites (BS) overlap the sites binding snoRNAs. We confirmed ORF57-snoRA71B RNA interaction in BCBL-1 cells by ORF57 RIP and Northern blot analyses using a 32P-labeled oligonucleotide probe from the 18S rRNA region complementary to snoRA71B. Using RNA oligonucleotides from the rRNA regions that ORF57 binds for oligonucleotide pulldown-Western blot assays, we selectively verified ORF57 interactions with 5.8S and 18S rRNAs. Polysome profiling revealed that ORF57 associates with both monosomes and polysomes and that its association with polysomes increases PABPC1 binding to polysomes but prevents Ago2 association with polysomes. Our data indicate a functional correlation with ORF57 binding and suppression of Ago2 activities for ORF57 promotion of gene expression. IMPORTANCE As an RNA-binding protein, KSHV ORF57 regulates RNA splicing, stability, and translation and inhibits host innate immunity by blocking the formation of RNA granules in virus-infected cells. In this study, ORF57 was found to interact with many host noncoding RNAs, including lncRNAs, snoRNAs, and rRNAs, to carry out additional unknown functions. ORF57 binds a group of lncRNAs via the RNA motifs identified by ORF57 CLIP-seq to regulate their expression. ORF57 associates with snoRNAs independently of fibrillarin and NOP58 proteins and with rRNA in the regions that commonly bind snoRNAs. Knockdown of fibrillarin expression decreases the expression of snoRNAs and CDK4 but does not affect viral gene expression. More importantly, we found that ORF57 binds translationally active polysomes and enhances PABPC1 but prevents Ago2 association with polysomes. Data provide compelling evidence on how ORF57 in KSHV-infected cells might regulate protein synthesis by blocking Ago2's hostile activities on translation.

Aponermin or placebo in combination with thalidomide and dexamethasone in the treatment of relapsed or refractory multiple myeloma (CPT-MM301): a randomised, double-blinded, placebo-controlled, phase 3 trial.

BACKGROUND: Aponermin, a circularly permuted tumor necrosis factor-related apoptosis-inducing ligand, is a potential death receptor 4/5-targeted antitumour candidate. Previous phase 1/2 studies have demonstrated the efficacy of aponermin in patients with relapsed or refractory multiple myeloma (RRMM). To confirm the superiority of aponermin plus thalidomide and dexamethasone (aponermin group) over placebo plus thalidomide and dexamethasone (placebo group) in RRMM, a randomized, double-blinded, placebo controlled phase 3 trial was performed. METHODS: Four hundred seventeen patients with RRMM who had previously received at least two regimens were randomly assigned (2:1) to receive aponermin, thalidomide, and dexamethasone or placebo, thalidomide, and dexamethasone. The primary endpoint was progression-free survival (PFS). Key secondary endpoints included overall survival (OS) and overall response rate (ORR). RESULTS: A total of 415 patients received at least one dose of trial treatment (276 vs. 139). The median PFS was 5.5 months in the aponermin group and 3.1 months in the placebo group (hazard ratio, 0.62; 95% confidence interval [CI], 0.49-0.78; P < 0.001). The median OS was 22.4 months for the aponermin group and 16.4 months for the placebo group (hazard ratio, 0.70; 95% CI, 0.55-0.89; P = 0.003). Significantly higher rates of ORR (30.4% vs. 13.7%, P < 0.001) and very good partial response or better (14.1% vs. 2.2%, P < 0.0001) were achieved in the aponermin group than in the placebo group. Treatment with aponermin caused hepatotoxicity in some patients, as indicated by the elevated alanine transaminase, aspartate transaminase, or lactate dehydrogenase levels (52.2% vs. 24.5%, 51.1% vs. 19.4% and 44.9% vs. 21.6%, respectively), mostly grade 1/2, transient and reversible. The main grade 3/4 adverse events included neutropenia, pneumonia and hyperglycemia. The incidence of serious adverse events was similar between the two groups (40.6% vs. 37.4%). There was no evidence that aponermin leads to hematological toxicity, nephrotoxicity, cardiotoxicity, or secondary tumors. CONCLUSIONS: Aponermin plus thalidomide and dexamethasone significantly improved PFS, OS and ORR with manageable side effects in RRMM patients who had received at least two prior therapies. These results support the use of aponermin, thalidomide, and dexamethasone as a treatment option for RRMM patients. TRIAL REGISTRATION: The trial was registered at http://www.chictr.org.cn as ChiCTR-IPR-15006024, 17/11/2014.

Elevated Interleukin-10 Levels Are Associated with Low Platelet Count and Poor Prognosis in 90 Adult Patients with Hemophagocytic Lymphohistiocytosis.

INTRODUCTION: Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening syndrome characterized by excessive inflammatory responses. This study explored the association between laboratory characteristics and outcomes in adult patients with HLH. METHODS: The adult patients diagnosed with HLH at the Second Hospital of Shanxi Medical University from September 2016 to September 2021 were retrospectively analyzed in this study. RESULTS: A total of 90 HLH patients were included. Among them, 60% were males, with a median age at diagnosis of 54 years. More than 85% of HLH patients presented with fever, splenomegaly, and cytopenias. IL-10 and IL-6 were elevated in 93.3% and 91.1% of patients, respectively. Elevated IL-10 levels were associated with lower platelet counts (r = -0.37, p < 0.001). Infections were seen in 46.7% (42/90) of cases. 29 patients with malignancy-associated HLH had T- or NK-cell (n = 16) or B-cell (n = 12) lymphoma. Autoimmune diseases accounted for 21.1% (19/90). Treatment was variable. In total, 36 patients survived (40%). The median overall survival (OS) was 1.5 months (95% confidence intervals [CI]: 0.2-2.8 months), with a 1-year OS of 40.9%. Patients with autoimmune diseases had markedly longer survival than those triggered by infection and malignancy (p < 0.001). Multivariate Cox regression analysis indicated that treatment delays (hazard ratios 0.36, 95% CI: 0.14-0.94, p = 0.036), platelet count (2.33, 1.30-4.18, p = 0.005), and IL-10 (2.07, 1.16-3.68, p = 0.014) were independent risk factors for poor outcome. CONCLUSION: Infection and lymphoma are the leading causes of HLH in adult patients with heterogeneous clinical manifestations. Survival of adult HLH is frustrating, especially those associated with malignancies. Besides, elevated IL-10 levels were associated with lower platelet counts, and these two markers were independent risk factors for poor prognosis. Earlier treatment led to better outcomes.

Fcγ receptor-mediated phagocytosis pathway was involved in phagocytosis of mIgM+ B lymphocytes from largemouth bass (Micropterus salmoides).

The potential for phagocytosis has been proven in teleost B cells, but the research on the regulatory mechanism of phagocytosis remains lacking. In this study, three largemouth bass (Micropterus salmoides) (15 ± 5 g) were injected intraperitoneally with Nocardia seriolae (105 CFU/100 µl/fish) in vivo, and their spleen was collected at 72 h post-infection for mRNA-seq. After the de novo assembly of the paired-end reads, 73,622 unigenes were obtained. Gene expression profiling revealed that 2043 unigenes were differentially expressed after N. seriolae infection, comprising 1285 upregulated and 758 downregulated unigenes (q-value <0.05, log2FC > |2|) of which 181 genes were involved in phagocytosis. The Kyoto Encyclopaedia of Genes and Genomes (KEGG) analysis demonstrated that 12 differentially expressed genes (DEG) associated with phagocytosis were enriched in the Fcγ receptor-mediated phagocytosis signalling pathway. In vitro, the phagocytic ability of mIgM+ B lymphocytes was validated using indirect immunofluorescence assay (IIFA) and fluorescence activating cell sorter (FACS), and the phagocytosis rates of the mIgM+ B lymphocytes incubated with a Lyn inhibitor had decreased from 18.533 ± 6.00% to 11.610 ± 4.236% compared with the unblocked group. These results suggested that the Fcγ receptor-mediated phagocytosis signalling pathway had participated in the phagocytosis of B cells and provide further insight into the role of B cells in innate immunology.

N,N-Bis(2,4-Dibenzhydryl-6-cycloalkylphenyl)butane-2,3-diimine-Nickel Complexes as Tunable and Effective Catalysts for High-Molecular-Weight PE Elastomers.

Four examples of N,N-bis(aryl)butane-2,3-diimine-nickel(II) bromide complexes, [ArN=C(Me)-C(Me)=NAr]NiBr2 (where Ar = 2-(C5H9)-4,6-(CHPh2)2C6H2 (Ni1), Ar = 2-(C6H11)-4,6-(CHPh2)2C6H2 (Ni2), 2-(C8H15)-4,6-(CHPh2)2C6H2 (Ni3) and 2-(C12H23)-4,6-(CHPh2)2C6H2 (Ni4)), disparate in the ring size of the ortho-cycloalkyl substituents, were prepared using a straightforward one-pot synthetic method. The molecular structures of Ni2 and Ni4 highlight the variation in the steric hindrance of the ortho-cyclohexyl and -cyclododecyl rings exerted on the nickel center, respectively. By employing EtAlCl2, Et2AlCl or MAO as activators, Ni1-Ni4 displayed moderate to high activity as catalysts for ethylene polymerization, with levels falling in the order Ni2 (cyclohexyl) > Ni1 (cyclopentyl) > Ni4 (cyclododecyl) > Ni3 (cyclooctyl). Notably, cyclohexyl-containing Ni2/MAO reached a peak level of 13.2 × 106 g(PE) of (mol of Ni)-1 h-1 at 40 °C, yielding high-molecular-weight (ca. 1 million g mol-1) and highly branched polyethylene elastomers with generally narrow dispersity. The analysis of polyethylenes with 13C NMR spectroscopy revealed branching density between 73 and 104 per 1000 carbon atoms, with the run temperature and the nature of the aluminum activator being influential; selectivity for short-chain methyl branches (81.8% (EtAlCl2); 81.1% (Et2AlCl); 82.9% (MAO)) was a notable feature. The mechanical properties of these polyethylene samples measured at either 30 °C or 60 °C were also evaluated and confirmed that crystallinity (Xc) and molecular weight (Mw) were the main factors affecting tensile strength and strain at break (εb = 353-861%). In addition, the stress-strain recovery tests indicated that these polyethylenes possessed good elastic recovery (47.4-71.2%), properties that align with thermoplastic elastomers (TPEs).

Characterization of three novel cell lines derived from the brain of spotted sea bass: Focusing on cell markers and susceptibility toward iridoviruses.

Despite tens of cell lines originating from fish brain tissue have been constructed, little is known about the definite cell types they belong to. Whether fish cell lines derived from the brain shares similar characteristics is not well-answered yet. Here, we constructed three cell lines designated as LMB-S, LMB-M, LMB-L using brain tissue of spotted sea bass (Lateolabrax maculatus). Among them, LMB-L was identified as astroglia-like cells considering the high expression of GFAP, DCX, PTX, S100b, which are regarded as astrocyte-specific or astrocyte-associated cell markers. LMB-M exhibited smooth muscle-like features showing strong expression of LMOD1, SLAMP, M-cadherin, MGP, which are confirmed as muscle-restricted or myogenesis-involved cell markers. Although LMB-S was not definitely identified, it appeared an activation of WNT/ß-catenin pathway. Besides the distinct expression profiles of cell markers, the three cell lines also presented differences in transfection efficiency and susceptibility to iridovirus infection. Relying on the established cell lines, a novel megalocytivirus, named LMIV (Lateolabrax maculatus iridovirus), was first isolated from diseased spotted sea bass. Genetic analysis of major capsid protein (MCP) and adenosine triphosphatase (ATPase) manifested that LMIV was clearly distinguishable from other representative teleost iridoviruses. Further investigations revealed that LMIV could replicate most efficiently in LMB-L cells obtaining the highest viral load (2.16 × 1010 copy/mL). By contrast, LMB-S cells gave rise to the highest viral load up to 3.86 × 108 copy/mL, when the three cell lines were infected with MRV, a newly emerged ranavirus. Moreover, LMIV infection caused lots of cells to be detached from monolayers, generating adherent and non-adherent cells. An opposite expression profiling of type I IFN pathway-related genes (JAK1, STAT1, STAT2, IRF9, Mx1) was found between adherent and non-adherent cells. Combined with the analysis of MCP gene expression, it is speculated that inhibiting type I IFN pathway in non-adherent cells allowed the facilitation of virus duplication. Taken together, the present study broadens our understanding about the diversity of cell lines derived from fish brain tissue and screening cells more susceptible to virus is not only meaningful for the development of vaccine, but also provide clues for further clarification of cell-iridovirus interactions.

Analysis of phagocytosis by mIgM+ lymphocytes depending on monoclonal antibodies against IgM of largemouth bass (Micropterus salmoides).

The phagocytic actives of B cells in fish have been proven in recent years. In this study, five positive hybridomas secreting monoclonal antibodies (MAbs) against largemouth bass IgM were produced. Indirect immunofluorescence assay (IFA) demonstrated that five MAbs could specifically recognize membrane-bound IgM (mIgM) molecule of largemouth bass. Indirect ELISA and Western blotting analysis showed that all the five MAbs had no cross-reactions with the other two teleost IgMs. Flow cytometry analysis (FCM) revealed that the percentages of largemouth bass mIgM+ lymphocytes in head kidney, peripheral blood and spleen were 51.66 ± 0.608%, 16.5 ± 1.235% and 42.92 ± 1.091%, respectively. In addition, the phagocytosis rates of mIgM + lymphocytes ingesting Nocardia seriolae from head kidney, peripheral blood and spleen were calculated to be 5.413 ± 0.274%, 16.6 ± 0.289% and 26.3 ± 0.296%, respectively. The qPCR results of sorted cells indicated that most inflammatory cytokines (IFNγ, IL-1ß, IL-2, IL-12ß, IL-34, IL-10), chemokine (CXCL12), chemokines receptors (CXCR2, CXCR4) and genes (FcγRⅠa, NCF1, CFL, ARP2/3, CD45, Syk, MARCKS) related to FcγR-mediated phagocytic signaling pathway in phagocytic mIgM+ lymphocytes were up-regulated significantly (P < 0.05). Taken together, the results suggested that the MAb (MM06H) produced in this paper could be used as a tool to study mIgM+ lymphocytes of largemouth bass, and FcγR may participate in the phagocytosis of mIgM+ lymphocytes, which is helpful to further study the role of mIgM+ lymphocytes in innate immunity.

Functional Characterization of Largemouth Bass (Micropterus salmoides) Soluble FcγR Homolog in Response to Bacterial Infection.

Fc receptors (FcRs) are key players in antibody-dependent cellular phagocytosis (ADCP) with their specific recognition of the Fc portion of an immunoglobulin. Despite reports of FcγR-mediated phagocytosis in mammals, little is known about the effects of soluble FcγRs on the immune response. In this study, FcγRIα was cloned from the largemouth bass (Micropterus salmoides) (MsFcγRIα). Without a transmembrane segment or a cytoplasmic tail, MsFcγRIα was identified as a soluble form protein and widely distributed in the spleen, head kidney, and intestine. The native MsFcγRIα was detected in the serum of Nocardia seriolae-infected largemouth bass and the supernatants of transfected HEK293 cells. Additionally, it was verified that the transfected cells' surface secreted MsFcRIα could bind to largemouth bass IgM. Moreover, the expression changes of MsFcγRIα, Syk, and Lyn indicated that MsFcγRIα was engaged in the acute phase response to bacteria, and the FcγR-mediated phagocytosis pathway was activated by Nocardia seriolae stimulation. Furthermore, recombinant MsFcγRIα could enhance both reactive oxygen species (ROS) and phagocytosis to Nocardia seriolae of leukocytes, presumably through the interaction of MsFcγRIα with a complement receptor. In conclusion, these findings provided a better understanding of the function of soluble FcγRs in the immune response and further shed light on the mechanism of phagocytosis in teleosts.

MIF-mediated NF-κB signaling pathway regulates the pathogenesis of polycystic ovary syndrome in rats.

Polycystic ovary syndrome (PCOS) resulting from abnormal glucose metabolism is a relatively common and complex endocrine disorder among women in their reproductive years, However, the pathogenesis of PCOS is still unclear. The purpose of this study is to investigate the macrophage migration inhibitory factor (MIF) involvement of the nuclear factor (NF)-κB in rats with PCOS. Results indicated that testosterone promoted the increase in the levels of MIF and luteinizing hormone (LH) but inhibited the increase in the level of follicular stimulating hormone (FSH). The MIF antibody could alleviate the process of PCOS to a certain extent. Testosterone promoted the expression of interleukin 1-beta (IL-1ß), interleukin 6 (IL-6), Inducible nitric oxide synthase (iNOS), and tumor necrosis factor alpha (TNF-α); the MIF antibody could reverse this effect. Testosterone could inhibit the expression of NF-κB protein whereas MIF antibody could promote the expression in the ovarian cytoplasm. Testosterone promoted the expression of NF-κB protein in the nucleus, this effect also could be reversed by the MIF antibody. Hyperandrogenism activated the NF-κB pathway. After using the MIF antibody, this effect was reversed. This finding suggested that hyperandrogenism activated the NF-κB pathway through MIF. In short, increased MIF levels activated the NF-κB pathway in ovaries, leading to inflammation and the increase in the levels of relevant inflammatory indicators, which might be one of the important factors in the pathogenesis of PCOS.

Virulence of "white-gray-opaque" tri-stable transformation in clinical Candida albicans in vitro and in vivo.

The study aimed to induce the white-opaque-gray tri-stable transformation in clinical C. albicans and to explore their potential pathogenicity. Sixty-four clinical strains were used to induce the white, opaque and gray cells of C. albicans. Secreted aspartyl proteinases (Sap) activity of the three phenotypes was then measured, and a vulvovaginal candidiasis (VVC) animal model was constructed. Of the 64 clinical strains, only 3 strains successfully underwent white-gray-opaque tri-stable transformation, and the three strains all belonged to MTL homozygous strains. Pz values in white, opaque and gray phenotypes were 0.834 ± 0.012, 0.707 ± 0.036, and 0.628 ± 0.002, respectively, which indicated that the cells with gray phenotype had higher Sap activity. After inoculation of different fungal suspension, the fungal colony count in descending order was as follows: gray phenotype, opaque phenotype and white phenotype. After treated with fluconazole for 3 days or 10 days, the fungal colony counts were significantly decreased compared with that before treatment (P < 0.05). The Sap activity and pathogenicity of gray cells in C. albicans were the strongest, followed by opaque cells and white cells. Additionally, white, gray and opaque phenotypic cells were all susceptible to fluconazole.

Thyroid dysfunction following radiofrequency ablation for benign thyroid nodules: more likely to occur within one-week and in high-risk population.

BACKGROUND: Radiofrequency ablation (RFA) has been recommended as the treatment for benign thyroid nodules (BTNs) by some guidelines. However, detailed follow-up instructions for thyroid function about the timing and affected populations after RFA are lacked due to insufficient researches. This 12-month prospective study aimed to evaluate the incidence and risk factors of thyroid dysfunction at different time points after RFA, especially within 1 week that previous studies didn't concern. METHODS: Seventy-five euthyroid patients who underwent RFA for symptomatic BTNs were enrolled (ChiCTR-INR-16007884). The incidence of thyroid dysfunction within 1 week, at 1, 6, and 12 months after RFA was evaluated. The risk factors for different types of thyroid dysfunction in the different terms were further analyzed. RESULTS: Within 1 week after RFA, the incidence of thyroid dysfunction was as high as 36.00% unexpectedly, and only overt thyrotoxicosis and subclinical thyrotoxicosis occurred, which were significantly associated with the low-normal baseline thyrotropin (TSH) level (p = 0.001) and high ablation volume ratio (p = 0.008). From 1 to 12 months (the long term), the incidence dropped significantly and remained low (8.00-12.00%); and thyroid dysfunction presented as overt thyrotoxicosis, subclinical thyrotoxicosis, and subclinical hypothyroidism. The long-term thyrotoxicosis group had more cases with diabetes and lower baseline TSH levels. The long-term subclinical hypothyroidism group had more cases with positive thyroid peroxidase antibodies, higher baseline TSH levels, and higher ablation volume ratios. CONCLUSIONS: After the RFA of BTNs, thyroid dysfunction was more likely to occur within 1 week and in populations with risk factors.

Characterization of a novel brain cell line from Jian carp (Cyprinus carpio var. Jian).

Jian carp (Cyprinus carpio var. Jian) is an economically important cultured fish in China. Currently, it is facing threats from infectious diseases including koi herpesvirus (KHV). Here, we established a new cell line, designated CCB-J, derived from the brain tissue of the Jian carp. CCB-J cells grew well in Leibovitz's L-15 medium containing 20% fetal bovine serum at 25 °C and have been subcultured for more than 60 passages. At the 30th passage, analysis showed that the number of chromosomes was 100, which is identical to that of other carp variants. Sequencing of the 18S ribosomal DNA confirmed that CCB-J originated from Jian carp. After transfection with the pEGFP-N1 plasmid, green fluorescence was observed in CCB-J. The replication of KHV in CCB-J cells was confirmed by RT-PCR and transmission electron microscopy. The viral titers of KHV in CCB-J cells and CCB cells, which have been widely used in the study of KHV, reached 103.9 and 101.8 median tissue culture infectious dose (TCID50/mL), respectively, within 14 days. The result of TaqMan PCR revealed that CCB-J cells were more sensitive to KHV than CCB cells. Meanwhile, a cytopathic effect (CPE) was also observed in the CCB-J cells in a shorter time post-infection compared with CCB cells. In summary, the CCB-J cell line will be a useful tool in the study of viral pathogenesis and vaccine research.

Levels of human cytomegalovirus miR-US25-1-5p and miR-UL112-3p in serum extracellular vesicles from infants with HCMV active infection are significantly correlated with liver damage.

Human cytomegalovirus (HCMV)-encoded microRNAs (miRNAs) are involved in posttranscriptional regulation of gene expression. Extracellular vesicles (EVs) can incorporate miRNAs. Relationship between HCMV infection and miRNAs in EVs remains unknown. EVs were isolated from supernatants of human embryonic lung fibroblasts (HELF) cells. Profiles of miRNAs in EVs were analyzed by deep sequencing. Dynamics of candidate viral miRNAs transportation via EVs was investigated using TaqMan PCR. Levels of candidate viral miRNAs in serum EVs from infants with HCMV active infection were detected and analyzed with their clinical index levels. A total of 16 HCMV miRNAs were found in EVs from infected HELF. Levels of miR-US25-1-5p and miR-UL112-3p in EVs increased at 6 h post-infection and were correlated with those in cells (for miR-US25-1-5p: r2 = 0.9375, p value < 0.05; for miR-UL112-3p: r2 = 0.7557, p value < 0.05). Viral miRNAs were transported into recipient cells at 2 h post-incubation. Moreover, levels of miR-US25-1-5p in serum EVs showed positive correlations with serum levels of γ-glutamyl transpeptidase, direct bilirubin, and total bile acid. Levels of miR-UL112-3p in serum EVs showed a positive correlation with serum levels of direct bilirubin. HCMV miRNAs could be transported to uninfected cells via EVs. Levels of miR-US25-1-5p and miR-UL112-3p in serum EVs from infants with HCMV active infection were significantly correlated with liver damage.