One-step cell biomanufacturing platform:porous gelatin microcarrier beads promote human embryonic stem cell-derived midbrain dopaminergic progenitor cell differentiation in vitro and survival after transplantation in vivo

Numerous studies have shown that cell replacement therapy can replenish lost cells and rebuild neural circuitry in animal models of Parkinson’s disease.Transplantation of midbrain dopaminergic progenitor cells is a promising treatment for Parkinson’s disease.However,transplanted cells can be injured by mechanical damage during handling and by changes in the transplantation niche.Here,we developed a one-step biomanufacturing platform that uses small-aperture gelatin microcarriers to produce beads carrying midbrain dopaminergic progenitor cells.These beads allow midbrain dopaminergic progenitor cell differentiation and cryopreservation without digestion,effectively maintaining axonal integrity in vitro.Importantly,midbrain dopaminergic progenitor cell bead grafts showed increased survival and only mild immunoreactivity in vivo compared with suspended midbrain dopaminergic progenitor cell grafts.Overall,our findings show that these midbrain dopaminergic progenitor cell beads enhance the effectiveness of neuronal cell transplantation.

Genetically modified pigs:Emerging animal models for hereditary hearing loss

Hereditary hearing loss(HHL),a genetic disorder that impairs auditory function,significantly affects quality of life and incurs substantial economic losses for society.To investigate the underlying causes of HHL and evaluate therapeutic outcomes,appropriate animal models are necessary.Pigs have been extensively used as valuable large animal models in biomedical research.In this review,we highlight the advantages of pig models in terms of ear anatomy,inner ear morphology,and electrophysiological characteristics,as well as recent advancements in the development of distinct genetically modified porcine models of hearing loss.Additionally,we discuss the prospects,challenges,and recommendations regarding the use pig models in HHL research.Overall,this review provides insights and perspectives for future studies on HHL using porcine models.

Lecithin-cholesterol acyltransferase is a potential tumor suppressor and predictive marker for hepatocellular carcinoma metastasis

BACKGROUND Hepatocellular carcinoma(HCC)is a major cause of cancer mortality worldwide,and metastasis is the main cause of early recurrence and poor prognosis.However,the mechanism of metastasis remains poorly understood.AIM To determine the possible mechanism affecting HCC metastasis and provide a possible theoretical basis for HCC treatment.METHODS The candidate molecule lecithin-cholesterol acyltransferase(LCAT)was screened by gene microarray and bioinformatics analysis.The expression levels of LCAT in clinical cohort samples was detected by quantitative realtime polymerase chain reaction and western blotting.The proliferation,migration,invasion and tumor-forming ability were measured by Cell Counting Kit-8,Transwell cell migration,invasion,and clonal formation assays,respectively.Tumor formation was detected in nude mice after LCAT gene knockdown or overexpression.The immunohistochemistry for Ki67,E-cadherin,N-cadherin,matrix metalloproteinase 9 and vascular endothelial growth factor were performed in liver tissues to assess the effect of LCAT on HCC.Gene set enrichment analysis(GSEA)on various gene signatures were analyzed with GSEA version 3.0.Three machine-learning algorithms(random forest,support vector machine,and logistic regression)were applied to predict HCC metastasis in The Cancer Genome Atlas and GEO databases.RESULTS LCAT was identified as a novel gene relating to HCC metastasis by using gene microarray in HCC tissues.LCAT was significantly downregulated in HCC tissues,which is correlated with recurrence,metastasis and poor outcome of HCC patients.Functional analysis indicated that LCAT inhibited HCC cell proliferation,migration and invasion both in vitro and in vivo.Clinicopathological data showed that LCAT was negatively associated with HCC size and metastasis(HCC size≤3 cm vs 3-9 cm,P<0.001;3-9 cm vs>9 cm,P<0.01;metastatic-free HCC vs extrahepatic metastatic HCC,P<0.05).LCAT suppressed the growth,migration and invasion of HCC cell lines via PI3K/AKT/mTOR signaling.Our results indicated that the logistic regression model based on LCAT,TNM stage and the serum level of α-fetoprotein in HCC patients could effectively predict high metastatic risk HCC patients.CONCLUSION LCAT is downregulated at translational and protein levels in HCC and might inhibit tumor metastasis via attenuating PI3K/AKT/mTOR signaling.LCAT is a prognostic marker and potential therapeutic target for HCC.

Activation-induced pyroptosis contributes to the loss of MAIT cells in chronic HIV-1 infected patients

Background: Mucosal-associated invariant T(MAIT) cells are systemically depleted in human immunodeficiency virus type 1(HIV-1) infected patients and are not replenished even after successful combined antiretroviral therapy(cART).This study aimed to identify the mechanism underlying MAIT cell depletion.Methods: In the present study, we applied flow cytometry, single-cell RNA sequencing and immunohistochemical staining to evaluate the characteristics of pyroptotic MAIT cells in a total of 127 HIV-1 infected individuals, including 69 treatment-naive patients, 28 complete responders, 15 immunological non-responders, and 15 elite controllers, at the Fifth Medical Center of Chinese PLA General Hospital, Beijing, China.Results: Single-cell transcriptomic profiles revealed that circulating MAIT cells from HIV-1 infected subjects were highly activated, with upregulation of pyroptosis-related genes. Further analysis revealed that increased frequencies of pyroptotic MAIT cells correlated with markers of systemic T-cell activation, microbial translocation, and intestinal damage in cART-naive patients and poor CD4+ T-cell recovery in long-term cART patients. Immunohistochemical staining revealed that MAIT cells in the gut mucosa of HIV-1 infected patients exhibited a strong active gasdermin-D(GSDMD, marker of pyroptosis) signal near the cavity side, suggesting that these MAIT cells underwent active pyroptosis in the colorectal mucosa. Increased levels of the proinflammatory cytokines interleukin-12(IL-12) and IL-18 were observed in HIV-1 infected patients. In addition, activated MAIT cells exhibited an increased pyroptotic phenotype after being triggered by HIV-1 virions, T-cell receptor signals, IL-12 plus IL-18, and combinations of these factors, in vitro.Conclusions: Activation-induced MAIT cell pyroptosis contributes to the loss of MAIT cells in HIV-1 infected patients,which could potentiate disease progression and poor immune reconstitution.

Three New Heptelidic Acid Derivatives from the Culture of Mushroom Lentinellus ursinus

Three new heptelidic acid derivatives(1-3)including two new dimeric esters and two known heptelidic acid analogues(4 and 5)were isolated from the solid culture of mushroom Lentinellus ursinus.The structures of new compounds were confirmed by the analysis of NMR and HRESIMS spectroscopic data.The biosynthetic origin of compounds 1-5 was postulated.Compounds 1-5 exhibited no antibacterial activity against Staphylococcus aureus and Escherichia coli at the dose of 100 μM.

Basic researches on the new coronavirus and its clinical progress

The initial case of atypical pneumonia occurred in Wuhan,Hubei Province at the end of 2019 caused by a novel coronavirus,soon it broke out all over the country beyond our imagination.For the very beginning of the break-out,we were lack of acknowledge of the newly disease,but with the further researches go,we intend to know the virus and disease better in every aspect.In this review we focus on the recent paper have been published online about epidemiology,clinical characteristics and therapy,which might help doctors to have a better understanding of the very disease.

Crystal structures of RNA-dependent RNA polymerases from Jingmen tick virus and Alongshan virus

Jingmenviruses are a group of flavi-like viruses with segmented genome and have been found in various types of hosts,including humans,cattle,monkeys,bats,rodents,sheep,ticks,mosquitoes and nematodes.Jingmenviruses,including the Jingmen tick virus(JMTV)and Alongshan virus(ALSV),have been associated with febrile illness and flu-like symptoms in humans.Viral polymerase plays critical roles in genome replication and transcription and is an ideal target for antiviral drugs.Here,we determined the crystal structures of RNA-dependent RNA polymerase(RdRp)domains of JMTV and ALSV at 2.6Åand 3.2Åresolutions,respectively.The overall structures of JMTV and ALSV RdRp domains are similar to those from the typical unsegmented viruses in Flaviviridae family,especially the Flavivirus genus.JMTV and ALSV RdRps can be divided into three subdomains and the catalytical Motif A-G are conserved like the typical flaviviruses,whereas the zinc-binding pockets are absent from the JMTV and ALSV RdRps.The 50-ends of jingmenvirus genomes are varied in length and sequence,and a highly conserved 8-nucleotide element located on the tip of stem loop A was identified and shown to be required for binding with RdRp and performing de novo replication activity.These findings provide important structural insights into RdRp of segmented flavivirus and reveal the key region of virus genome responsible for replication initiation,which would promote molecular understanding of segmented flavivirus replication and the structure-based design of antiviral drugs against flaviviruses.

Enhanced potency of an IgM-like nanobody targeting conserved epitope in SARS-CoV-2 spike N-terminal domain

Almost all the neutralizing antibodies targeting the receptor-binding domain(RBD)of spike(S)protein show weakened or lost efficacy against severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)emerged or emerging variants,such as Omicron and its sub-variants.This suggests that highly conserved epitopes are crucial for the development of neutralizing antibodies.Here,we present one nanobody,N235,displaying broad neutralization against the SARS-CoV-2 prototype and multiple variants,including the newly emerged Omicron and its sub-variants.Cryo-electron microscopy demonstrates N235 binds a novel,conserved,cryptic epitope in the N-terminal domain(NTD)of the S protein,which interferes with the RBD in the neighboring S protein.The neutralization mechanism interpreted via flow cytometry and Western blot shows that N235 appears to induce the S1 subunit shedding from the trimeric S complex.Furthermore,a nano-IgM construct(MN235),engineered by fusing N235 with the human IgM Fc region,displays prevention via inducing S1 shedding and cross-linking virus particles.Compared to N235,MN235 exhibits varied enhancement in neutralization against pseudotyped and authentic viruses in vitro.The intranasal administration of MN235 in low doses can effectively prevent the infection of Omicron sub-variant BA.1 and XBB in vivo,suggesting that it can be developed as a promising prophylactic antibody to cope with the ongoing and future infection.

Regulatory functions and mechanisms of human microbiota in infectious diseases

The human microbiota,a diverse community of microorganisms living on or within their hosts,play an irreplaceable role in maintaining human health.Dysbiosis of the microbiota is associated with the pathogenesis of diverse human dis-eases.In recent years,growing evidence has been presented to support the substantial effect of human microbiota on the progression of infectious diseases.In this review,we describe the functional role of human microbiota in infec-tious diseases by highlighting their Janus-faced effects in the regulation of acute and chronic infections as well as their related co-morbidities.Thereafter,we review the latest advances elucidating the mechanisms underlying tri-directional interactions between the microbiota,hosts,and invading pathogens,with a further discussion on external environ-mental factors that shape this interconnected regulatory network.A better understanding of the regulatory functions and mechanisms of human microbiota in infectious diseases will facilitate the development of new diagnostic,preven-tive,and therapeutic approaches for infectious diseases.

Nonconserved epitopes dominate reverse preexisting T cell immunity in COVID-19 convalescents

The herd immunity against SARS-CoV-2 is continuously consolidated across the world during the ongoing pandemic.However,the potential function of the nonconserved epitopes in the reverse preexisting cross-reactivity induced by SARS-CoV-2 to other human coronaviruses is not well explored.In our research,we assessed T cell responses to both conserved and nonconserved peptides shared by SARS-CoV-2 and SARS-CoV,identifying cross-reactive CD8^(+)T cell epitopes using enzyme-linked immunospot and intracellular cytokine staining assays.Then,in vitro refolding and circular dichroism were performed to evaluate the thermal stability of the HLA/peptide complexes.Lastly,single-cell T cell receptor reservoir was analyzed based on tetramer staining.Here,we discovered that cross-reactive T cells targeting SARS-CoV were present in individuals who had recovered from COVID-19,and identified SARS-CoV-2 CD8^(+)T cell epitopes spanning the major structural antigens.T cell responses induced by the nonconserved peptides between SARS-CoV-2 and SARS-CoV were higher and played a dominant role in the cross-reactivity in COVID-19 convalescents.Cross-T cell reactivity was also observed within the identified series of CD8^(+)T cell epitopes.For representative immunodominant peptide pairs,although the HLA binding capacities for peptides from SARS-CoV-2 and SARS-CoV were similar,the TCR repertoires recognizing these peptides were distinct.Our results could provide beneficial information for the development of peptide-based universal vaccines against coronaviruses.

Transcription factor ETV4 promotes the development of hepatocellular carcinoma by driving hepatic TNF-αsignaling

Background Hepatic inflammation is the major risk factor of hepatocellular carcinoma(HCC).However,the underlying mechanism by which hepatic inflammation progresses to HCC is poorly understood.This study was designed to investigate the role of ETS translocation variant 4(ETV4)in linking hepatic inflammation to HCC.Methods Quantitative real-time PCR and immunoblotting were used to detect the expression of ETV4 in HCC tissues and cell lines.RNA sequencing and luciferase reporter assays were performed to identify the target genes of ETV4.Hepatocyte-specific ETV4-knockout(ETV4fl/fl,alb-cre)and transgenic(ETV4Hep-TG)mice and diethylnitrosamine-carbon tetrachloride(DEN-CCL4)treatment experiments were applied to investigate the function of ETV4 in vivo.The Cancer Genome Atlas(TCGA)database mining and pathological analysis were carried out to determine the correlation of ETV4 with tumor necrosis factor-alpha(TNF-α)and mitogen-activated protein kinase 11(MAPK11).Results We revealed that ETV4 was highly expressed in HCC.High levels of ETV4 predicted a poor survival rate of HCC patients.Then we identified ETV4 as a transcription activator of TNF-αand MAPK11.ETV4 was positively correlated with TNF-αand MAPK11 in HCC patients.As expected,an increase in hepatic TNF-αsecretion and macrophage accumulation were observed in the livers of ETV4Hep-TG mice.The protein levels of TNF-α,MAPK11,and CD68 were significantly higher in the livers of ETV4Hep-TG mice compared with wild type mice but lower in ETV4fl/fl,alb-cre mice compared with ETV4fl/fl mice as treated with DEN-CCL4,indicating that ETV4 functioned as a driver of TNF-α/MAPK11 expression and macrophage accumulation during hepatic inflammation.Hepatocyte-specific knockout of ETV4 significantly prevented development of DEN-CCL4-induced HCC,while transgenic expression of ETV4 promoted growth of HCC.Conclusions ETV4 promoted hepatic inflammation and HCC by activating transcription of TNF-αand MAPK11.Both the ETV4/TNF-αand ETV4/MAPK11 axes represented two potential therapeutic targets for highly associated hepatic inflammation and HCC.ETV4+TNF-αwere potential prognostic markers for HCC patients.

TRIM27 maintains gut homeostasis by promoting intestinal stem cell self-renewal

Dysregulation of gut homeostasis is associated with irritable bowel syndrome(IBS),a chronic functional gastrointestinal disorder affecting approximately 11.2%of the global population.The poorly understood pathogenesis of IBS has impeded its treatment.Here,we report that the E3 ubiquitin ligase tripartite motif-containing 27(TRIM27)is weakly expressed in IBS but highly expressed in inflammatory bowel disease(IBD),a frequent chronic organic gastrointestinal disorder.Accordingly,knockout of Trim27 in mice causes spontaneously occurring IBS-like symptoms,including increased visceral hyperalgesia and abnormal stool features,as observed in IBS patients.Mechanistically,TRIM27 stabilizesβ-catenin and thus activates Wnt/β-catenin signaling to promote intestinal stem cell(ISC)self-renewal.Consistent with these findings,Trim27 deficiency disrupts organoid formation,which is rescued by reintroducing TRIM27 orβ-catenin.Furthermore,Wnt/β-catenin signaling activator treatment ameliorates IBS symptoms by promoting ISC self-renewal.Taken together,these data indicate that TRIM27 is critical for maintaining gut homeostasis,suggesting that targeting the TRIM27/Wnt/β-catenin axis could be a potential treatment strategy for IBS.Our study also indicates that TRIM27 might serve as a potential biomarker for differentiating IBS from IBD.

Migrasomes released by HSV-2-infected cells serve as a conveyance for virus spread

Dear Editor,Migrasomes are newly discovered cellular organelles with diameters of 0.5–3μm which have been found to be produced by normal and cancer cells,and distributed in various organs of animals(Ma et al.,2015)and in human sera(Zhao et al.,2019).Migrasomes are present inside the cavities of pulmonary alveoli,blood vessels and lymph capillaries(Zhang et al.,2020),and can be captured by surrounding cells with their cargoes internalized.

OTUB1 promotes osteoblastic bone formation through stabilizing FGFR2

Bone homeostasis is maintained by the balance between osteoblastic bone formation and osteoclastic bone resorption.Dysregulation of this process leads to multiple diseases,including osteoporosis.However,the underlying molecular mechanisms are not fully understood.Here,we show that the global and conditional osteoblast knockout of a deubiquitinase Otub1 result in low bone mass and poor bone strength due to defects in osteogenic differentiation and mineralization.Mechanistically,the stability of FGFR2,a crucial regulator of osteogenesis,is maintained by OTUB1.OTUB1 attenuates the E3 ligase SMURF1-mediated FGFR2 ubiquitination by inhibiting SMURF1’s E2 binding.In the absence of OTUB1,FGFR2 is ubiquitinated excessively by SMURF1,followed by lysosomal degradation.Consistently,adeno-associated virus serotype 9(AAV9)-delivered FGFR2 in knee joints rescued the bone mass loss in osteoblast-specific Otub1-deleted mice.Moreover,Otub1 mRNA level was significantly downregulated in bones from osteoporotic mice,and restoring OTUB1 levels through an AAV9-delivered system in ovariectomy-induced osteoporotic mice attenuated osteopenia.Taken together,our results suggest that OTUB1 positively regulates osteogenic differentiation and mineralization in bone homeostasis by controlling FGFR2 stability,which provides an optical therapeutic strategy to alleviate osteoporosis.

Quantitative characterization of filamentous fungal promoters on a single-cell resolution to discover cryptic natural products

Characterization of filamentous fungal regulatory elements remains challenging because of time-consuming transformation technologies and limited quantitative methods.Here we established a method for quantitative assessment of filamentous fungal promoters based on flow cytometry detection of the superfolder green fluorescent protein at single-cell resolution.Using this quantitative method,we acquired a library of 93 native promoter elements from Aspergillus nidulans in a high-throughput format.The strengths of identified promoters covered a 37-fold range by flow cytometry.P_(zipA) and P_(sltA)were identified as the strongest promoters,which were 2.9-and 1.5-fold higher than that of the commonly used constitutive promoter P_(gpdA).Thus,we applied P_(zipA)and P_(sltA)to activate the silent nonribosomal peptide synthetase gene Afpes1 from Aspergillus fumigatus in its native host and the heterologous host A.nidulans.The metabolic products of Afpes1 were identified as new cyclic tetrapeptide derivatives,namely,fumiganins A and B.Our method provides an innovative strategy for natural product discovery in fungi.

Rapid evaluation of heterologous chimeric RBD-dimer mRNA vaccine for currently-epidemic Omicron sub-variants as booster shot after inactivated vaccine

With continuous mutations of severe acute respiratory syndrome coronavirus 2(SARS-CoV-2),the severe immune escape of Omicron sub-variants urges the development of next-generation broad-spectrum vaccines,especially as booster jabs after high-level vaccination coverage of inactivated vaccines in China and many other countries.Previously,we developed a coronavirus disease 2019(COVID-19)protein subunit vaccine ZF2001?based on the tandem homo-prototype receptor-binding domain(RBD)-dimer of the SARS-CoV-2 spike protein.We upgraded the antigen into a hetero-chimeric prototype(PT)-Beta or Delta-BA.1 RBD-dimer to broaden the cross-protection efficacy and prove its efficiency with protein subunit and mRNA vaccine platforms.Herein,we further explored the hetero-chimeric RBD-dimer mRNA vaccines and evaluated their broad-spectrum activities as booster jabs following two doses of inactivated vaccine(Ⅳ)in mice.Our data demonstrated that the chi-meric vaccines significantly boosted neutralizing antibody levels and specific T-cell responses against the vari-ants,and PT-Beta was superior to Delta-BA.1 RBD as a booster in mice,shedding light on the antigen design for the next-generation COVID-19 vaccines.

Immunogenic molecules associated with gut bacterial cell walls:chemical structures,immune-modulating functions,and mechanisms

Interactions between gut microbiome and host immune system are fundamental to maintaining the intestinal mucosal barrier and homeostasis.At the host-gut microbiome interface,cell wall-derived molecules from gut commensal bacteria have been reported to play a pivotal role in training and remodeling host immune responses.In this article,we review gut bacterial cell wall-derived molecules with characterized chemical structures,including peptidoglycan and lipid-related molecules that impact host health and disease processes via regulating innate and adaptive immunity.Also,we aim to discuss the structures,immune responses,and underlying mechanisms of these immunogenic molecules.Based on current advances,we propose cell wall-derived components as important sources of medicinal agents for the treatment of infection and immune diseases.

Deciphering the placental abnormalities associated with somatic cell nuclear transfer at single-nucleus resolution

Dear Editor,The placenta connecting the fetus to the matermal uterus provides material exchange and an immune-tolerant environment for the embryo in all eutherian mammals(Shao et al.,2022).The representative mouse placenta displays a multilayered structure with distinct characteristics and functions,including the matermal decidua,junctional zone,and labyrinth layer(Marsh and Blelloch,2020).The decidua,which is thought to be derived from the matermal endometrium(and further undergoes decidualization),provides an anchor for fetal trophoblast invasion.The junctional zone predominantly contains spongiotrophoblasts(SpT),glycogen trophoblasts(GlyT),and trophoblast giant cells(TGCs).The labyrinth is the innermost two-layer structure,which mainly consists of syncytiotrophoblast cells(SynTI and SynTII),sinusoidal TGCs(S-TGCs),and fetal endothelial cells(Simmons and Cross,2005).

Heterologous production of bioactive xenoacremone analogs in Aspergillus nidulans

Tyrosine-decahydrofluorene derivatives are a class of hybrid compounds that integrate the properties of polyketides and nonribosomal peptides.These compounds feature a[6.5.6]tricarbocyclic core and a para-cyclophane ether moiety in their structures and exhibit anti-tumor and anti-microbial activities.In this study,we constructed the biosynthetic pathway of xenoacremones from Xenoacremonium sinensis ML-31 in the Aspergillus nidulans host,resulting in the identification of four novel tyrosine-decahydrofluorene analogs,xenoacremones I–L(1-4),along with two known analogs,xenoacremones A and B.Remarkably,compounds 3 and 4 contained a 12-membered para-cyclophane ring system,which is unprecedented among tyrosine-decahydrofluorene analogs in X.sinensis.The successful reconstruction of the biosynthetic pathway and the discovery of novel analogs demonstrate the utility of heterologous expression strategy for the generation of structurally diverse natural products with potential biological activities.

Geneformer:a deep learning model for exploring gene networks

Uncovering gene regulatory networks is vital for identifying core regulators,understanding disease progression mechanisms,and identifying potential therapeutic targets,in which large amounts of transcriptome data are required.However,most research projects face limitations in obtaining only limited data,constrained by factors such as individual heterogeneity and sequencing technology.