The ceRNA Network Indicates Its Immune Function in Peripheral Blood Leukocytes of Half-Smooth Tongue Sole(Cynoglossus semilaevis)After Stimulation with Chitosan Oligosaccharide

As an efficient immunostimulant,chitosan oligosaccharide(COS)can enhance the immunity of teleosts;however,the underlying molecular mechanisms still require elucidation.Competing endogenous RNAs(ceRNAs)are vital regulators in the immune response,but their roles in half-smooth tongue sole(Cynoglossus semilaevis)remain unclear.In this study,for the first time,we studied whole-transcriptome expression profiles and analyzed ceRNA networks in peripheral blood leukocytes of half-smooth tongue sole treated with COS.A total of 19 circRNAs(DE-circRNAs),18 miRNAs(DE-miRNAs)and 50 previously identified lncRNAs(DElncRNAs)were differentially expressed after COS stimulation.The DE-lncRNAs and DE-miRNAs targeted numerous immunity-related genes,and were enriched in important pathways,including MAPK and Toll-like receptor signaling pathways,suggesting the immunoregulatory roles of COS.Furthermore,we constructed circRNA-miRNA-mRNA and lncRNA-miRNA-mRNA regulatory networks using DE-circRNAs,DE-miRNAs,DE-lncRNAs,and DE-mRNAs.Additionally,a ceRNA network with immunity-related DEmRNAs was constructed,showing that 3 DE-circRNAs,12 DE-lncRNAs,and 29 DEGs exhibited crosstalk through 9 DE-miRNAs.Intriguingly,a DE-miRNA in the ceRNA network,miR-144-3p,was targeted by DE-lncRNA tnrc6a,and negatively regulated the genes of inhibitor of nuclear factor kappa B kinases(IKKs)(ikbkg,ikbkb,and ikbip)and c3ar1.Ikbkg,ikbkb,and c3ar1 were significantly up-regulated in macrophages stimulated by LPS.It could be inferred that ncRNAs participated in the immune and inflammatory response by acting as ceRNAs after COS stimulation in teleosts.These findings indicate that COS could enhance the immunity of teleosts by regulating ncRNAs,and lay the foundation for further practical application of COS in aquaculture.

MicroRNAs as potential diagnostic biomarkers for bipolar disorder

Abnormal expression of microRNAs is connected to brain development and disease and could provide novel biomarkers for the diagnosis and prognosis of bipolar disorder. We performed a PubMed search for microRNA biomarkers in bipolar disorder and found 18 original research articles on studies performed with human patients and published from January 2011 to June 2023. These studies included microRNA profiling in bloodand brain-based materials. From the studies that had validated the preliminary findings,potential candidate biomarkers for bipolar disorder in adults could be miR-140-3p,-30d-5p,-330-5p,-378a-5p,-21-3p,-330-3p,-345-5p in whole blood, miR-19b-3p,-1180-3p,-125a-5p, let-7e-5p in blood plasma, and miR-7-5p,-23b-5p,-142-3p,-221-5p,-370-3p in the blood serum. Two of the studies had investigated the changes in microRNA expression of patients with bipolar disorder receiving treatment. One showed a significant increase in plasma miR-134 compared to baseline after 4 weeks of treatment which included typical antipsychotics, atypical antipsychotics, and benzodiazepines. The other study had assessed the effects of prescribed medications which included neurotransmitter receptorsite binders(drug class B) and sedatives, hypnotics, anticonvulsants, and analgesics(drug class C) on microRNA results. The combined effects of the two drug classes increased the significance of the results for miR-219 and-29c with miR-30e-3p and-526b* acquiring significance. MicroRNAs were tested to see if they could serve as biomarkers of bipolar disorder at different clinical states of mania, depression, and euthymia. One study showed that upregulation in whole blood of miR-9-5p,-29a-3p,-106a-5p,-106b-5p,-107,-125a-3p,-125b-5p and of miR-107,-125a-3p occurred in manic and euthymic patients compared to controls, respectively, and that upregulation of miR-106a-5p,-107 was found for manic compared to euthymic patients. In two other studies using blood plasma,downregulation of miR-134 was observed in manic patients compared to controls, and dysregulation of miR-134,-152,-607,-633,-652,-155 occurred in euthymic patients compared to controls. Finally, microRNAs such as miR-34a,-34b,-34c,-137, and-140-3p,-21-3p,-30d-5p,-330-5p,-378a-5p,-134,-19b-3p were shown to have diagnostic potential in distinguishing bipolar disorder patients from schizophrenia or major depressive disorder patients, respectively. Further studies are warranted with adolescents and young adults having bipolar disorder and consideration should be given to using animal models of the disorder to investigate the effects of suppressing or overexpressing specific microRNAs.

Alterations of Blood Immune Cells in Elderly Patients with COVID-19 and with or without Pre-Existing Type 2 Diabetes

Elderly individuals, especially those with pre-existing conditions like type 2 diabetes mellitus (T2DM), have a high risk for developing severe cases of COVID-19. The aim of this work was to characterize the alterations of blood immune cells (BIC) in patients with symptomatic COVID-19 and confirmed SARS-CoV-2 infection, ≥60 years and who needed hospitalization in the Centro de Salud Hospital of Tucuman, Argentina, during the second peak of the pandemic in Argentina. Ten patients were enrolled from December 2020 to May 2021. Blood samples were taken at the time of admission (day 0) and five days after (day 5) for routine laboratory tests and the characterization of BIC by flow cytometry. Most of the patients were men (70%) aged between 60 and 78 years. The 70% of patients had T2DM while 50% had arterial hypertension. At day 0, all the patients had increased neutrophils and inflammatory markers (C reactive protein and D-dimers) and reduced numbers of lymphocytes, HLA-DRhi monocytes, CD16+CD56+ NK cells, CD3+HLA−DR+CD25+ cells, CD4+ and CD8+ T cells in blood. Patients received a standard treatment for COVID-19 care (O2, corticosteroids and antibiotics). The hospital treatment normalized the levels of BIC (day 5) in 30% of patients who were those with no comorbidities. In patients with T2DM, BIC recovery was variable. In T2DM patients who required administration of plasma (30%), prolonged O2 therapy (40%) or referral to the intensive care unit (10%) significant reductions of CD16+CD56+, CD3+HLA−DR+CD25+, CD4+ and CD8+ cells were observed between days 0 and 5. In line with previous studies, our results show that absolute counts of major lymphocyte subsets in blood are significantly and substantially decreased during the course of severe COVID-19 disease in elderly patients. These BIC alterations may persist despite clinical care in elderly patients with T2DM. Further studies are needed to investigate the utility of early lymphocyte subset measurements as prognostic biomarkers of disease severity, mortality, and response to treatment in COVID-19 elderly patients with T2DM.

Analysis of the HLA-A,-B allele polymorphism in 5844 umbilical cord blood samples taken from Han population of Shandong province

To investigate the HLA-A, -B allele polymorphism in Han population of Shandong province and to explore the possibility to find out the HLA-A,-B-matehed cord blood donors for stem cell transplantation to be used in other area in China, 5844 umbilical cord blood samples were taken from Han population donors of Shandong province, and assayed with PCR-sequence-oligonucleotide （PCR-SSO） assay. In Shandong Hart donors, 20 alleles at HLA-A locus and 46 alleles at HLA-B locus could be detected as revealed in the present study. Among the 20 alleles at HLA-A locus, the most prevalent five alleles included A ＊ 02（0.3041）, A ＊ 11（0. 1443）, A ＊ 24（0. 1434）, A ＊ 30（0.0975） and A 33（0.0859）, while, the alleles with lower gene frequencies included A ＊ 34（0.0006）, A ＊ 25 （0.0005）, A ＊ 66（0.0005）, A ＊ 74（0.0004） and A ＊ （0.0001）. Of the 46 HLA-B alleles detected, the most prevalent five alleles were B ＊ 13（0.1348）, B ＊ 51（0.0713）, B ＊ 62（0.0712）, B ＊ 61 （0.0676） and B ＊ 60（0.0642）; while alleles with lower gene frequencies included B ＊ 77（0.0001）, B ＊ 76（0.0002）, B ＊ 47（0.0003）, B ＊ 42（0.0003） and B ＊ 72（0.0004）. In comparison with those of the other Han population in China, the HLA-A, -B gene frequencies in the umbilical cord blood of Shandong province possess unique distribution features among the investigated populations from various regions of the same race origin, and the differences in various regions of the same race were less than those among the different race. It is evident that the HLA-A,-B alleles of the umbilical cord blood taken in Shangdong province show high degree of polymorphism, and it might be part of those of Northem Han population in China. So, it is reasonable for patients of Northern Chinese to receive HLA class Ⅰ -match transplant of cord blood stem ceils for tissue and organ transplantation from Shangdong umbilical cord blood bank.

Relationships between blood leukocyte mitochondrial DNA copy number and inflammatory cytokines in knee osteoarthritis

Osteoarthritis(OA)is a degenerative articular disorder manifested by cartilage destruction,subchondral sclerosis,osteophytes,and synovitis,resulting in chronic joint pain and physical disability in the elderly.The purpose of this study was to investigate mitochondrial DNA copy number(mtDNACN)and inflammatory cytokines in primary knee OA patients and healthy volunteers.A total of 204 knee OA patients and 169 age-matched healthy volunteers were recruited.Their relative blood leukocyte mtDNACN was assessed by quantitative real-time polymerase chain reaction(qRT-PCR),and ten inflammatory cytokines in their plasma were detected by multiplex immunoassay.Blood leukocyte mtDNACN in the OA group was significantly lower than that in the control group.Leukocyte mtDNACN in the control group was negatively correlated with their age(r=−0.380,P<0.0001),whereas mtDNACN in the OA group was positively correlated with their age(r=0.198,P<0.001).Plasma interleukin-4(IL-4)and IL-6 were significantly higher in the knee OA group than in the control group.The plasma IL-6 level was positively correlated with blood leukocyte mtDNACN in the OA group(r=0.547,P=0.0014).IL-5 showed as a major factor(coefficient 0.69)in the second dimension of principle components analysis(PCA)-transformed data and was significantly higher in the OA group(P<0.001)as well as negatively correlated with mtDNACN(r=−0.577,P<0.001).These findings suggest that elevation of plasma IL-4 and IL-6 and a relative reduction in mtDNACN might be effective biomarkers for knee OA.IL-5 is a plausible factor responsible for decreasing blood leukocyte mtDNACN in knee OA patients.

Identification of novel molecular markers of mastitis caused by Staphylococcus aureus using gene expression profiling in two consecutive generations of Chinese Holstein dairy cattle

Background: Mastitis in dairy cows caused by Staphylococcus aureus is a major problem hindering economic growth in dairy farms worldwide. It is difficult to prevent or eliminate due to its asymptomatic nature and long persistence of infection. Although transcriptomic responses of bovine mammary gland cells to pathogens that cause mastitis have been studied, the common responses of peripheral blood leukocytes to S. aureus infection across two consecutive generations of dairy cattle have not been investigated.Methods: In the current study, RNA-Seq was used to profile the transcriptomes of peripheral blood leukocytes sampled from S. aureus-infected mothers and their S. aureus-infected daughters, and also healthy non-infected mothers and their healthy daughters. Differential gene expression was evaluated as follows: 1) S. aureus-infected cows versus healthy non-infected cows(S vs. H, which include all the mothers and daughters), 2) S. aureus-infected mothers versus healthy non-infected mothers(SM vs. HM), and 3) S. aureus-infected daughters versus healthy noninfected daughters(SMD vs. HMD).Results: Analysis of all identified expressed genes in the four groups(SM, SMD, HM, and HMD) showed that EPOR,IL9, IFNL3, CCL26, IL26 were exclusively expressed in both the HM and HMD groups, and that they were significantly(P < 0.05) enriched for the cytokine-cytokine receptor interaction pathway. A total of 17, 13 and 10 differentially expressed genes(DEGs)(FDR Padj. < 0.1 and |FC| > 1.2) were detected in the three comparisons, respectively. DEGs with P < 0.05 and |FC| > 2 were used for functional enrichment analyses. For the S vs. H comparison, DEGs detected included CCL20, IL13 and MMP3, which are associated with the IL-17 signaling pathway. In the SM vs. HM and SMD vs. HMD comparisons, five(BLA-DQB, C1 R, C2, FCGR1 A, and KRT10) and six(BLA-DQB, C3 AR1, CFI, FCAR, FCGR3 A, and LOC10498484) genes, respectively, were involved in the S. aureus infection pathway.Conclusions: Our study provides insights into the transcriptomic responses of bovine peripheral blood leukocytes across two generations of cattle naturally infected with S. aureus. The genes highlighted in this study could serve as expression biomarkers for mastitis and may also contain sequence variation that can be used for genetic improvement of dairy cattle for resilience to mastitis.

Tumor necrosis factor-alpha(TNF-α) in spotted halibut Verasper variegatus at the embryonic and metamorphic stages

As an aquatic fish,the spotted halibut Verasper variegatus is highly susceptible to bacterial and virus infections.Tumor necrosis factor-alpha(TNF-α)as a cytokine could control the inflammatory responses.The functions of TNF-αin many species have been widely studied,particularly in mammals.However,little is known about the TNF-αfunctions in V.variegatus.We first cloned and sequenced the TNF-αgene in V.variegatus(VvTNF-α).The two conserved cysteine residues,transmembrane sequence,Thr-Leu motif,and TNF family signature,as well as the TA-rich motifs of its proteins related to inflammatory responses had high similarity to those of the other teleost and mammalian TNF-α.The phylogenetic analysis showed that VvTNF-αwas consistent with TNF-αgenes of other vertebrates.The VvTNF-αtranscripts were extensively distributed in the peripheral blood leukocytes(PBLs),spleen,and gill,indicating that the VvTNF-αhad a role in immune function.Furthermore,treatment with pathogen-associated molecular patterns(PAMPs)could induce a rapid and significant increase of VvTNF-αin the PBLs,which reveals that VvTNF-αdoes participate in the host immune responses against bacterial and viral pathogens.We found that VvTNF-αhad an interesting expression pattern during metamorphosis,showing that the flatfish TNF-αmay have some novel functions during specific developmental stages.In addition,the 3 D structure prediction of VvTNF-αprovided an indication of how it is likely to interact with other proteins.Therefore,VvTNF-αhas multiple functions,and provides valuable information to explore novel functions of TNF-α.

Novel Functions of Interleukin-1 Beta in Spotted Halibut(Verasper variegatus) at Its Embryonic and Metamorphic Stages

Interleukin-lbeta （IL-1β） is a cytokine involved in the control of inflammatory responses. The functions of IL-1β have been extensively studied in many vertebrates. However, few studies focused on the functions of IL-1β in the spotted halibut （Verasper variegatus）, an endangered aquatic fish that easily suffers from bacterial and viral infections. We first cloned and sequenced the IL-1β gene from V. variegatus （VvIL-1β）. Its conservative interleukin-1 propeptide and interleukin-1/18 domains corresponding regions, characteristics of the IL-1 protein family and TA-rich motifs relating to inflammatory responses were similar to those of the IL-1β genes from other teleost and mammals. The phylogenetic analysis showed that VvIL-1β was homologous to the IL-1β gene of other vertebrates. Treatment with pathogen-associated molecular patterns （PAMPs） induced a significant and rapid up-regulation of VvIL-1β expression in the peripheral blood leukocytes （PBLs）, revealing that VvIL-1β is actively involved in the host ilmnune responses against bacterial and viral pathogens. The expression level of VvIL-1β was relatively high in the PBLs, gill, and intestine, indicating the immunological function of VvIL-1β . VvIL-1β also had interesting expression patterns at specific developmental stages, implying that the IL-1β of flatfishes may have some novel functions during embryonic development and metamorphosis. The 3D structure prediction also provided information about how VvIL-113 interacts with its receptor protein. These results suggested that VvIL-1β may have multiple functions in V. variegatus and provided us valuable information for understanding the novel functions of IL- 1β.

Methylation status of DJ-1 in leukocyte DNA of Parkinson’s disease patients

Background:DJ-1 has been thought as a candidate biomarker for Parkinson’s disease(PD).It was found reduced in PD brains,CSF and saliva,although there were conflicting results.How DJ-1 expression may be regulated is not clear.Recently,blood-based DNA methylation represents a highly promising biomarker for PD by regulating the causative gene expression.Thus,in this study,we try to explore whether blood-based DNA methylation of DJ-1 could be used as a biomarker to differentiate PD patients from normal control(NC),and whether DNA methylation could regulate DJ-1 expression in a SH-SY5Y cell model.Methods:Forty PD patients and 40 NC were recruited in this study.DNA was extracted from peripheral blood leukocytes(PBLs).Methylation status of two CpG islands(CpG1 and CpG2)in promoter region of DJ-1 was explored by bisulfite specific PCR-based sequencing method.Methylation inhibitor 5-Aza-dC was used to treat SH-SY5Y cell line,DJ-1 level was detected in both mRNA and protein level.Results:CpG sites in these two CpG islands(CpG1 and CpG2)of DJ-1 were unmethylated in both PD and NC group.In SH-SY5Y cell model treated by methylation inhibitor,there was no significant change of DJ-1 expression in either mRNA level or protein level.Conclusions:Our results indicated that DNA methylation inhibitor didn’t alter DJ-1 gene expression in SH-SY5Y cell model,and DNA methylation of DJ-1 promoter region in PBLs level might not be an efficient biomarker for PD patients.