An accurate assessment of host and pathogen gene expression during infection is critical for understanding the molecular aspects of host-pathogen interactions.Often,pathogen-derived transcripts are difficult to ascert...An accurate assessment of host and pathogen gene expression during infection is critical for understanding the molecular aspects of host-pathogen interactions.Often,pathogen-derived transcripts are difficult to ascertain at early infection stages owing to the unfavourable transcript representation compared to the host genes.In this study,we compare two sequencing techniques,RNAseq and enrichment sequencing(RenSeq and PenSeq)of cDNA,to investigate gene expression patterns in the doubled monoploid potato(DM)infected with the late blight pathogen Phytophthora infestans.Our results reveal distinct advantages of cDNA RenSeq and PenSeq over traditional RNAseq in terms of target gene representation and transcriptional quantification at early infection stages.Throughout the infection time course,cDNA enrichment sequencing enables transcriptomic analyses for more targeted host and pathogen genes.For highly expressed genes that were sampled in parallel by both cDNA enrichment and RNAseq,a high level of concordance in expression profiles is observed,indicative of at least semi-quantitative gene expression representation following enrichment.展开更多
BACKGROUND Adenomatous polyposis confers an increased risk of developing colorectal cancer.APC and MUTYH are the major genes investigated in patients suspected of having polyposis.In addition to APC and MUTYH genes,ot...BACKGROUND Adenomatous polyposis confers an increased risk of developing colorectal cancer.APC and MUTYH are the major genes investigated in patients suspected of having polyposis.In addition to APC and MUTYH genes,other genes,such as POLE,POLD1,NTHL1,MBD4,MSH3 and MLH3,have recently been associated with polyposis phenotypes,conferring heterogeneity in terms of the clinical,etiological and heritable aspects of patients with polyposis.AIM To investigate the underlying variant landscape in patients with suspected polyposis who lack variants in the APC and MUTYH genes using whole-exome sequencing.METHODS Twenty-seven participants were included in the study and subjected to germline whole-exome sequencing.In addition,their clinical-pathological,personal,and family history data were collected.RESULTS The mean age at diagnosis was 51 years,and most participants had attenuated forms of polyposis(88.9%),with 63.0%diagnosed with a primary tumor,mostly colorectal cancer(76.5%).Among the variants identified,17 were classified as pathogenic or likely pathogenic(in 12 participants),including variants in genes involved in the Wnt/β-catenin signaling pathway,such as ST7 L,A1CF,and DKK4,and variants in DNA-repair genes,such as NTHL1,PNKP,and PMS2,as well as a variant found at the FRK gene identified in a patient with classic polyposis at age 19 and with a family history of polyps.CONCLUSION This study identified novel genes potentially associated with polyposis in patients lacking germline pathogenic variants in the APC and MUTYH genes.These findings support the use of next-generation sequencing for screening,expanding the scope of polyposis-related variants beyond these two genes.展开更多
In order to save manpower and time costs,and to achieve simultaneous detection of multiple animal-derived components in meat and meat products,this study used multiple nucleotide polymorphism(MNP)marker technology bas...In order to save manpower and time costs,and to achieve simultaneous detection of multiple animal-derived components in meat and meat products,this study used multiple nucleotide polymorphism(MNP)marker technology based on the principle of high-throughput sequencing,and established a multi-locus 10 animalderived components identification method of cattle,goat,sheep,donkey,horse,chicken,duck,goose,pigeon,quail in meat and meat products.The specific loci of each species could be detected and the species could be accurately identified,including 5 loci for cattle and duck,3 loci for sheep,9 loci for chicken and horse,10 loci for goose and pigeon,6 loci for quail and 1 locus for donkey and goat,and an adulteration model was established to simulate commercially available samples.The results showed that the method established in this study had high throughput,good repeatability and accuracy,and was able to identify 10 animalderived components simultaneously with 100%repeatability accuracy.The detection limit was 0.1%(m/m)in simulated samples of chicken,duck and horse.Using the method established in this study to test commercially available samples,4 samples from 14 commercially available samples were detected to be inconsistent with the labels,of which 2 did not contain the target ingredient and 2 were adulterated with small amounts of other ingredients.展开更多
BACKGROUND Gallbladder neuroendocrine carcinoma(NEC)represents a subtype of gallbladder malignancies characterized by a low incidence,aggressive nature,and poor prognosis.Despite its clinical severity,the genetic alte...BACKGROUND Gallbladder neuroendocrine carcinoma(NEC)represents a subtype of gallbladder malignancies characterized by a low incidence,aggressive nature,and poor prognosis.Despite its clinical severity,the genetic alterations,mechanisms,and signaling pathways underlying gallbladder NEC remain unclear.CASE SUMMARY This case study presents a rare instance of primary gallbladder NEC in a 73-year-old female patient,who underwent a radical cholecystectomy with hepatic hilar lymphadenectomy and resection of liver segments IV-B and V.Targeted gene sequencing and bioinformatics analysis tools,including STRING,GeneMANIA,Metascape,TRRUST,Sangerbox,cBioPortal and GSCA,were used to analyze the biological functions and features of mutated genes in gallbladder NEC.Twelve mutations(APC,ARID2,IFNA6,KEAP1,RB1,SMAD4,TP53,BTK,GATA1,GNAS,and PRDM3)were identified,and the tumor mutation burden was determined to be 9.52 muts/Mb via targeted gene sequencing.A protein-protein interaction network showed significant interactions among the twelve mutated genes.Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses were used to assess mutation functions and pathways.The results revealed 40 tumor-related pathways.A key regulatory factor for gallbladder NEC-related genes was identified,and its biological functions and features were compared with those of gallbladder carcinoma.CONCLUSION Gallbladder NEC requires standardized treatment.Comparisons with other gallbladder carcinomas revealed clinical phenotypes,molecular alterations,functional characteristics,and enriched pathways.展开更多
Global brain ischemia and neurological deficit are consequences of cardiac arrest that lead to high mortality.Despite advancements in resuscitation science,our limited understanding of the cellular and molecular mecha...Global brain ischemia and neurological deficit are consequences of cardiac arrest that lead to high mortality.Despite advancements in resuscitation science,our limited understanding of the cellular and molecular mechanisms underlying post-cardiac arrest brain injury have hindered the development of effective neuroprotective strategies.Previous studies primarily focused on neuronal death,potentially overlooking the contributions of non-neuronal cells and intercellular communication to the pathophysiology of cardiac arrest-induced brain injury.To address these gaps,we hypothesized that single-cell transcriptomic analysis could uncover previously unidentified cellular subpopulations,altered cell communication networks,and novel molecular mechanisms involved in post-cardiac arrest brain injury.In this study,we performed a single-cell transcriptomic analysis of the hippocampus from pigs with ventricular fibrillation-induced cardiac arrest at 6 and 24 hours following the return of spontaneous circulation,and from sham control pigs.Sequencing results revealed changes in the proportions of different cell types,suggesting post-arrest disruption in the blood-brain barrier and infiltration of neutrophils.These results were validated through western blotting,quantitative reverse transcription-polymerase chain reaction,and immunofluorescence staining.We also identified and validated a unique subcluster of activated microglia with high expression of S100A8,which increased over time following cardiac arrest.This subcluster simultaneously exhibited significant M1/M2 polarization and expressed key functional genes related to chemokines and interleukins.Additionally,we revealed the post-cardiac arrest dysfunction of oligodendrocytes and the differentiation of oligodendrocyte precursor cells into oligodendrocytes.Cell communication analysis identified enhanced post-cardiac arrest communication between neutrophils and microglia that was mediated by neutrophil-derived resistin,driving pro-inflammatory microglial polarization.Our findings provide a comprehensive single-cell map of the post-cardiac arrest hippocampus,offering potential novel targets for neuroprotection and repair following cardiac arrest.展开更多
Glial cells play crucial roles in regulating physiological and pathological functions,including sensation,the response to infection and acute injury,and chronic neurodegenerative disorders.Glial cells include astrocyt...Glial cells play crucial roles in regulating physiological and pathological functions,including sensation,the response to infection and acute injury,and chronic neurodegenerative disorders.Glial cells include astrocytes,microglia,and oligodendrocytes in the central nervous system,and satellite glial cells and Schwann cells in the peripheral nervous system.Despite the greater understanding of glial cell types and functional heterogeneity achieved through single-cell and single-nucleus RNA sequencing in animal models,few studies have investigated the transcriptomic profiles of glial cells in the human spinal cord.Here,we used high-throughput single-nucleus RNA sequencing and spatial transcriptomics to map the cellular and molecular heterogeneity of astrocytes,microglia,and oligodendrocytes in the human spinal cord.To explore the conservation and divergence across species,we compared these findings with those from mice.In the human spinal cord,astrocytes,microglia,and oligodendrocytes were each divided into six distinct transcriptomic subclusters.In the mouse spinal cord,astrocytes,microglia,and oligodendrocytes were divided into five,four,and five distinct transcriptomic subclusters,respectively.The comparative results revealed substantial heterogeneity in all glial cell types between humans and mice.Additionally,we detected sex differences in gene expression in human spinal cord glial cells.Specifically,in all astrocyte subtypes,the levels of NEAT1 and CHI3L1 were higher in males than in females,whereas the levels of CST3 were lower in males than in females.In all microglial subtypes,all differentially expressed genes were located on the sex chromosomes.In addition to sex-specific gene differences,the levels of MT-ND4,MT2A,MT-ATP6,MT-CO3,MT-ND2,MT-ND3,and MT-CO_(2) in all spinal cord oligodendrocyte subtypes were higher in females than in males.Collectively,the present dataset extensively characterizes glial cell heterogeneity and offers a valuable resource for exploring the cellular basis of spinal cordrelated illnesses,including chronic pain,amyotrophic lateral sclerosis,and multiple sclerosis.展开更多
AIM:To estimate if nanopore targeted sequencing(NTS)could identify pathogens causing postoperative endophthalmitis and further determine the feasibility of clinical application of NTS.METHODS:A total of 55 patients(55...AIM:To estimate if nanopore targeted sequencing(NTS)could identify pathogens causing postoperative endophthalmitis and further determine the feasibility of clinical application of NTS.METHODS:A total of 55 patients(55 eyes)with postoperative endophthalmitis were retrospectively included in this study with their medical records.Intraocular fluid samples were examined by NTS and microbial culture.All included patients had undergone examinations including measurement of best corrected visual acuity(BCVA)and intraocular pressure(IOP),slit-lamp biomicroscopy,and indirect ophthalmoscopy;additionally,they underwent B-ultrasound,anterior segment photography,and fundus photography if necessary.RESULTS:Among 55 patients with postoperative endophthalmitis,the age was 65.25±15.04y and there were 30 female(54.54%)patients.Forty-one(74.54%)vitreous humor samples and fourteen(25.45%)aqueous humor samples were sent for both NTS and microbial culture.NTS had a notable higher detection rate than microbial culture in detecting pathogens(90.91%vs 38.18%,χ^(2)=33.409,P<0.001).NTS exhibited high sensitivity of pathogen detection in both microbial culture positive and negative samples(100%and 85.29%,respectively).In 16 of 21(76.19%)patients who showed culture-positivity,their results corresponded with those of NTS.Moreover,in two patients(9.52%),NTS showed a better species resolution than microbial culture;in three patients(14.28%),NTS identified additional pathogens.As for fungus,the positive detection rate of NTS was significantly higher than that of microbial culture(20%vs 3.64%,χ^(2)=7.066,P=0.008).Also,NTS could detect multi-infection by bacteria and fungi than microbial culture(32.73%vs 0,χ^(2)=21.522,P<0.001).NTS could detect bacteria as well as fungi simultaneously within 48h in all patients.Meanwhile,NTS had a shorter detection time than microbial culture(1.13±0.34 vs 2.67±0.55d,Z=-9.218,P<0.001).After the NTS results were obtained,15 patients received additional intravitreal/intracameral anti-infection treatment.At follow-up,there was a statistically significant improvement in the visual acuity relative to the baseline(Z=−5.222,P<0.001).CONCLUSION:NTS can provide rapid identification and highly sensitive detection of pathogens among patients with postoperative endophthalmitis,which can guide anti-infection treatment and improve visual prognosis.展开更多
BACKGROUND Hemorrhoids,a prevalent chronic condition globally,significantly impact patients'quality of life.While various surgical interventions,such as external stripping and internal ligation,procedure for prola...BACKGROUND Hemorrhoids,a prevalent chronic condition globally,significantly impact patients'quality of life.While various surgical interventions,such as external stripping and internal ligation,procedure for prolapse and hemorrhoids,and tissue selecting technique,are employed for treatment,they are often associated with postoperative complications,including unsatisfactory defecation,bleeding,and anal stenosis.In contrast,Xiaozhiling injection,a traditional Chinese medicine-based therapy,has emerged as a minimally invasive and effective alternative for internal hemorrhoids.This treatment offers distinct advantages,such as reduced dietary restrictions,broad applicability,and minimal induction of systemic inflammatory responses.Additionally,Xiaozhiling injection effectively eliminates hemorrhoid nuclei,prevents local tissue necrosis,preserves anal cushion integrity,and mitigates postoperative complications,including bleeding and prolapse.Despite its clinical efficacy,the molecular mechanisms underlying its therapeutic effects remain poorly understood,warranting further investigation.AIM To investigate the molecular mechanism underlying the therapeutic effect of Xiaozhiling injection in the treatment of internal hemorrhoids.METHODS An internal hemorrhoid model was established in rats,and the rats were randomly divided into a modeling group[control group(CK group)]and a treatment group.One week after injection,Stereo-seq and electron microscopy were used to study the changes in gene expression and subcellular structures in fibroblasts.RESULTS Single-cell sequencing revealed differences in the expression and transcript levels of the genes collagen 3 alpha 1,decorin,and actin alpha 2 in fibroblasts between the CK group and the treatment group.Spatial transcriptome analysis revealed that genes of the sphingosine kinase 1(Sphk1)/sphingosine-1-phosphate(S1P)pathway spatially overlapped with key genes of the transforming growth factor beta 1 pathway,namely,Sphk1,S1P receptor,and transforming growth factor beta 1,in the treatment group.The proportion of fibroblasts was lower in the treatment group than in the CK group,and Xiaozhiling treatment had a significant effect on the proportion of fibroblasts in hemorrhoidal tissue.Immunohistochemistry revealed a significant increase in the expression of a fibroblast marker.Electron microscopy showed that the endoplasmic reticulum of fibroblasts contained a large amount of glycogen,indicating cell activation.Fibroblast activation and the expression of key genes of the Sphk1-S1P pathway could be observed at the injection site,suggesting that after Xiaozhiling intervention,the Sphk1-S1P pathway could be activated to promote fibrosis.CONCLUSION Xiaozhiling injection exerts its therapeutic effects on internal hemorrhoids by promoting collagen synthesis and secretion in fibroblasts.After Xiaozhiling intervention,the Sphk1-S1P pathway can be activated to promote fibrosis.展开更多
N^(6)-methyladenine(6mA)is a prevalent DNA modification and is involved in a wide range of human diseases.Previous studies have indicated that 6mA is enriched in mitochondrial DNA(mtDNA)of mammals.By employing an evol...N^(6)-methyladenine(6mA)is a prevalent DNA modification and is involved in a wide range of human diseases.Previous studies have indicated that 6mA is enriched in mitochondrial DNA(mtDNA)of mammals.By employing an evolved adenine deaminase,we developed a deaminase-mediated sequencing(DM-seq)method that could achieve genome-wide mapping of 6mA in mammalian mtDNA at single-base resolution.In this study,we used an engineered adenine deaminase,known as TadA8e protein,to map 6mA in mtDNA of hepatocellular carcinoma(HCC)by DM-seq.Through high-throughput sequencing,we identified sixteen 6mA sites in both HCC and adjacent normal tissue mtDNA.The results revealed an increased overall 6mA level in mtDNA associated with HCC.Furthermore,an elevation in 6mA level was observed alongside a decrease in the m RNA levels of the corresponding genes,indicating that increased6mA level hindered transcription processes related to these genes.These findings demonstrate that 6mA in mtDNA is correlated with HCC and provide evidence supporting the inhibitory effect of elevated 6mA level on subsequent transcriptional activity.This research illuminates the intricate relationship between 6mA modification and transcriptional regulation in the context of HCC,offering valuable insights into the role of 6mA modification in HCC pathogenesis.展开更多
AIM:To explore the immune cell infiltration and molecular mechanisms of retinal ischemia-reperfusion injury(RIRI)to identify potential therapeutic targets.METHODS:In the bulk RNA-seq analysis,This study performed diff...AIM:To explore the immune cell infiltration and molecular mechanisms of retinal ischemia-reperfusion injury(RIRI)to identify potential therapeutic targets.METHODS:In the bulk RNA-seq analysis,This study performed differential gene expression analysis,weighted gene co-expression network analysis,and protein-protein interaction network analysis to identify hub genes.QuanTIseq was used to determine the composition of infiltrating immune cells.Following the identification of hub genes,single-cell RNA-seq analysis was employed to pinpoint the specific immune cell types expressing these hub genes.Cell-cell communication analysis to explore signaling pathways and interactions between immune cells was further performed.Finally,the expression of these key immune regulators in vivo using quantitative real-time polymerase chain reaction(qRT-PCR)was validated.RESULTS:Bulk RNA-seq analysis identified Stat2,Irf7,Irgm1,Igtp,Parp9,Irgm2,Nlrc5,and Tap1 as hub genes,with strong correlations to immune cell infiltration.Single-cell RNA-seq analysis further revealed six immune cell clusters,showing Irf7 predominantly in microglia and Tap1 in dendritic cells(DCs).And cell-cell communication analysis showed that microglia and DCs play central roles in coordinating immune activity.qRT-PCR validated the upregulation of these genes.CONCLUSION:In the acute phase of RIRI,Irf7 and Tap1 may be the potential therapeutic targets to reduce inflammation and promote neurological function recovery.展开更多
BACKGROUND Understanding the status and function of tumor-infiltrating immune cells is essential for improving immunotherapeutic effects and predicting the clinical response in human patients with carcinoma.However,li...BACKGROUND Understanding the status and function of tumor-infiltrating immune cells is essential for improving immunotherapeutic effects and predicting the clinical response in human patients with carcinoma.However,little is known about tumor-infiltrating immune cells,and the corresponding research results in hepatocellular carcinoma(HCC)are limited.AIM To investigate potential biomarker genes that are important for the development of HCC and to understand how immune cell subsets react throughout this process.METHODS Using single-cell RNA sequencing and T-cell receptor sequencing,the heterogeneity and potential functions of immune cell subpopulations from HCC tissue and normal tissue adjacent to carcinoma,as well as their possible interactions,were analyzed.RESULTS Eight T-cell clusters from patients were analyzed and identified using bioinformatics,including six typical major Tcell clusters and two newly identified T-cell clusters,among which Fc epsilon receptor 1G+T cells were characterized by the upregulation of Fc epsilon receptor 1G,tyrosine kinase binding protein,and T cell receptor delta constant,whereas metallothionein 1E+T cells proliferated significantly in tumors.Differentially expressed genes,such as regulator of cell cycle,cysteine and serine rich nuclear protein 1,SMAD7 and metallothionein 1E,were identified as significantly upregulated in tumors and have potential as biomarkers.In association with T-cell receptor analysis,we inferred the clonal expansion characteristics of each T-cell cluster in HCC patients.CONCLUSION We identified lymphocyte subpopulations and potential biomarker genes critical for HCC development and revealed the clonal amplification of infiltrating T cells.These data provide valuable resources for understanding the response of immune cell subsets in HCC.展开更多
Background:Medulloblastoma(MB)is one of the most common malignant brain tumors that mainly affect children.Various approaches have been used to model MB to facilitate investigating tumorigenesis.This study aims to com...Background:Medulloblastoma(MB)is one of the most common malignant brain tumors that mainly affect children.Various approaches have been used to model MB to facilitate investigating tumorigenesis.This study aims to compare the recapitulation of MB between subcutaneous patient-derived xenograft(sPDX),intracranial patient-derived xenograft(iPDX),and genetically engineered mouse models(GEMM)at the single-cell level.Methods:We obtained primary human sonic hedgehog(SHH)and group 3(G3)MB samples from six patients.For each patient specimen,we developed two sPDX and iPDX models,respectively.Three Patch+/-GEMM models were also included for sequencing.Single-cell RNA sequencing was performed to compare gene expression profiles,cellular composition,and functional pathway enrichment.Bulk RNA-seq deconvolution was performed to compare cellular composition across models and human samples.Results:Our results showed that the sPDX tumor model demonstrated the highest correlation to the overall transcriptomic profiles of primary human tumors at the single-cell level within the SHH and G3 subgroups,followed by the GEMM model and iPDX.The GEMM tumor model was able to recapitulate all subpopulations of tumor microenvironment(TME)cells that can be clustered in human SHH tumors,including a higher proportion of tumor-associated astrocytes and immune cells,and an additional cluster of vascular endothelia when compared to human SHH tumors.Conclusions:This study was the first to compare experimental models for MB at the single-cell level,providing value insights into model selection for different research purposes.sPDX and iPDX are suitable for drug testing and personalized therapy screenings,whereas GEMM models are valuable for investigating the interaction between tumor and TME cells.展开更多
In recent years,intensive human activities have increased the intensity of desertification,driving continual desertification process of peripheral meadows.To investigate the effects of restoration on soil microbial co...In recent years,intensive human activities have increased the intensity of desertification,driving continual desertification process of peripheral meadows.To investigate the effects of restoration on soil microbial communities,we analyzed vegetation-soil relationships in the Hulun Buir Sandy Land,northern China.Through the use of high-throughput sequencing,we examined the structure and diversity in the bacterial and fungal communities within the 0-20 cm soil layer after 9-15 a of restoration.Different slope positions were analyzed and spatial heterogeneity was assessed.The results showed progressive improvements in soil properties and vegetation with the increase of restoration duration,and the following order was as follows:bottom slope>middle slope>crest slope.During the restoration in the Hulun Buir Sandy Land,the bacterial communities were dominated by Proteobacteria,Actinobacteria,and Acidobacteria,whereas the fungal communities were dominated by Ascomycota and Basidiomycota.Eutrophic bacterial abundance increased with the restoration duration,whereas oligotrophic bacterial and fungal abundance levels decreased.The soil bacterial abundance significantly increased with the increasing restoration duration,whereas the fungal diversity decreased after 11 a of restoration,except that at the crest slope.Redundancy analysis showed that pH,soil moisture content,total nitrogen,and vegetation-related factors affected the bacterial community structure(45.43%of the total variance explained).Canonical correspondence analysis indicated that pH,total phosphorus,and vegetation-related factors shaped the bacterial community structure(31.82%of the total variance explained).Structural equation modeling highlighted greater bacterial responses(R^(2)=0.49-0.79)to changes in environmental factors than those of fungi(R^(2)=0.20-0.48).The soil bacterial community was driven mainly by pH,soil moisture content,electrical conductivity,plant coverage,and litter dry weight.The abundance and diversity of the soil fungal community were mainly driven by plant coverage,litter dry weight,and herbaceous aboveground biomass,while there was no significant correlation between the soil fungal community structure and environmental factors.These findings highlighted divergent microbial succession patterns and environmental sensitivities during sandy grassland restoration.展开更多
To understand the gene-based biological processes in-depth,the single-molecule real-time sequencing has drawn increasing attention with promoted by the Human Genome Project.Herein,a set of newly designed canonical flu...To understand the gene-based biological processes in-depth,the single-molecule real-time sequencing has drawn increasing attention with promoted by the Human Genome Project.Herein,a set of newly designed canonical fluorescent bases(A_(y),tC,G_(b),T_(p))are proposed for four-color DNA sequencing.These quasi-intrinsic probes are derived from the fluorophore replacement and ring expansion on natural bases,which still keep the pyrimidine or purine underlying skeleton and Watson–Crick hydrogen bonding face to allow minimal perturbation to the native DNA duplex.More importantly,these nucleobase analogues possess red-shifted absorption and efficient photoluminescence due to the enhancedπ-conjugation in character.Meanwhile,the four analogues could generate distinct emission wavelength(Δλ~50 nm)for real-time sequencing.To assess the biological employment of the proposed biosensors,the effects of base pairing and linking deoxyribose are also considered.展开更多
Meniscus injuries are widespread and the available treatments do not offer enough healing potential.Here,we provide critical support for using pigs as a biological model for meniscal degeneration and the development o...Meniscus injuries are widespread and the available treatments do not offer enough healing potential.Here,we provide critical support for using pigs as a biological model for meniscal degeneration and the development of cutting-edge therapies in orthopedics.We present a single-cell transcriptome atlas of the meniscus,consisting of cell clusters corresponding to four major cell types:chondrocytes,endothelial cells,smooth muscle cells,and immune cells.Five distinct chondrocyte subclusters(CH0–CH4)were annotated,of which only one was widespread in both the red and white zones,indicating a major difference in the cellular makeup of the zones.Subclusters distinct to the white zone appear responsible for cartilage-specific matrix deposition and protection against adverse microenvironmental factors,while those in the red zone exhibit characteristics of mesenchymal stem cells and are more likely to proliferate and migrate.Additionally,they induce remodeling actions in other chondrocyte subclusters and promote the proliferation and maturation of endothelial cells,inducing healing and vascularization processes.Considering that they have substantial remodeling capabilities,these subclusters should be of great interest for tissue engineering studies.We also show that the cellular makeup of the pig meniscus is comparable to that of humans,which supports the use of pigs as a model in orthopedic therapy development.展开更多
Genotyping by target sequencing(GBTS)integrates the advantages of silicon-based technology(high stability and reliability)and genotyping by sequencing(high flexibility and cost-effectiveness).However,GBTS panels are n...Genotyping by target sequencing(GBTS)integrates the advantages of silicon-based technology(high stability and reliability)and genotyping by sequencing(high flexibility and cost-effectiveness).However,GBTS panels are not currently available in pigs.In this study,based on GBTS technology,we first developed a 50K panel,including 52,000 single-nucleotide polymorphisms(SNPs),in pigs,designated GBTS50K.A total of 6,032 individuals of Large White,Landrace,and Duroc pigs from 10 breeding farms were used to assess the newly developed GBTS50K.Our results showed that GBTS50K obtained a high genotyping ability,the SNP and individual call rates of GBTS50K were 0.997–0.998,and the average consistency rate and genotyping correlation coefficient were 0.997 and 0.993,respectively,in replicate samples.We also evaluated the efficiencies of GBTS50K in the application of population genetic structure analysis,selection signature detection,genome-wide association studies(GWAS),genotyped imputation,genetic selection(GS),etc.The results indicate that GBTS50K is plausible and powerful in genetic analysis and molecular breeding.For example,GBTS50K could gain higher accuracies than the current popular GGP-Porcine bead chip in genomic selection on 2 important traits of backfat thickness at 100 kg and days to 100 kg in pigs.Particularly,due to the multiple SNPs(mSNPs),GBTS50K generated 100K qualified SNPs without increasing genotyping cost,and our results showed that the haplotype-based method can further improve the accuracies of genomic selection on growth and reproduction traits by 2 to 6%.Our study showed that GBTS50K could be a powerful tool for underlying genetic architecture and molecular breeding in pigs,and it is also helpful for developing SNP panels for other farm animals.展开更多
Background:Dengue fever,an acute insect-borne infectious disease caused by the dengue virus(DENV),poses a great challenge to global public health.Hepatic involve-ment is the most common complication of severe dengue a...Background:Dengue fever,an acute insect-borne infectious disease caused by the dengue virus(DENV),poses a great challenge to global public health.Hepatic involve-ment is the most common complication of severe dengue and is closely related to the occurrence and development of disease.However,the features of adaptive immune responses associated with liver injury in severe dengue are not clear.Methods:We used single-cell sequencing to examine the liver tissues of mild or se-vere dengue mice model to analyze the changes in immune response of T cells in the liver after dengue virus infection,and the immune interaction between macrophages and T cells.Flow cytometry was used to detect T cells and macrophages in mouse liver and blood to verify the single-cell sequencing results.Results:Our result showed CTLs were significantly activated in the severe liver injury group but the immune function-related signal pathway was down-regulated.The rea-son may be that the excessive immune response in the severe group at the late stage of DENV infection induces the polarization of macrophages into M2 type,and the macrophages then inhibit T cell immunity through the TGF-βsignaling pathway.In ad-dition,the increased proportion of Treg cells suggested that Th17/Treg homeostasis was disrupted in the livers of severe liver injury mice.Conclusions:In this study,single-cell sequencing and flow cytometry revealed the characteristic changes of T cell immune response and the role of macrophages in the liver of severe dengue fever mice.Our study provides a better understanding of the pathogenesis of liver injury in dengue fever patients.展开更多
BACKGROUND Tenofovir alafenamide fumarate(TAF)is one of the first-line treatments used to treat chronic hepatitis B patients;TAF has strong antiviral activity and a high barrier to resistance.Although virological brea...BACKGROUND Tenofovir alafenamide fumarate(TAF)is one of the first-line treatments used to treat chronic hepatitis B patients;TAF has strong antiviral activity and a high barrier to resistance.Although virological breakthroughs in patients during TAF treatment are rare,patients with incomplete responses to TAF are occasionally observed.AIM To investigate responsible mutations in the reverse transcriptase region of hepatitis B virus(HBV)for TAF-incomplete responses.METHODS Thirteen chronic hepatitis B patients who received TAF monotherapy were included.A TAF-incomplete responder was defined as one who was continuously positive for HBV DNA over 2 years after TAF treatment initiation.The emergences of mutations in TAF-incomplete responders were evaluated before,one year after,and two years after treatment by deep sequencing of HBV DNA and RNA.RESULTS Two patients were continuously positive for HBV DNA over two years.The rtL269I mutation,one of the CYEI mutations linked to tenofovir resistance,was detected in both patients by direct sequencing.The deep sequencing analysis revealed that a combination of rtT118A and rtL220I mutations and the rtL269I mutation were predominantly detected in HBV DNA even when these mutations were barely detected in HBV RNA.This suggests a superior replication capability of the HBV variants with these mutations under TAF treatment.CONCLUSION The deep sequencing analysis of HBV DNA and RNA and comparing the detection rates of mutations were useful for estimating responsible mutations for TAF-incomplete responses.Such analysis is needed to evaluate the association between mutations that emerge during TAF treatment and incomplete responses to TAF.展开更多
In rice fields,rice plants usually grow alongside wild weeds and are attacked by various invertebrate species.Viruses are abundant in plants and invertebrates,playing crucial ecological roles in controlling microbial ...In rice fields,rice plants usually grow alongside wild weeds and are attacked by various invertebrate species.Viruses are abundant in plants and invertebrates,playing crucial ecological roles in controlling microbial abundance and maintaining community structures.To date,only 16 rice viruses have been documented in rice-growing regions.These viruses pose serious threats to rice production and have traditionally been identified only from rice plants and insect vectors by isolation techniques.Advances in next-generation sequencing(NGS)have made it feasible to discover viruses on a global scale.Recently,numerous viruses have been identified in plants and invertebrates using NGS technologies.In this review,we discuss viral studies in rice plants,invertebrate species,and weeds in rice fields.Many novel viruses have been discovered in rice ecosystems through NGS technologies,with some also detected using metatranscriptomic and small RNA sequencing.These analyses greatly expand our understanding of viruses in rice fields and provide valuable insights for developing efficient strategies to manage insect pests and virus-mediated rice diseases.展开更多
基金supported by the Rural & Environment Science & Analytical Services (RESAS) Division of the Scottish Government through project JHI-B1-1the Biotechnology and Biological Sciences Research Council (BBSRC) through awards BB/ S015663/1+2 种基金BB/X009068/1Research Leaders 2025 fellowship funded by European Union’s Horizon 2020 research and innovation programme under Marie Sklodowska-Curie grant agreement no. 754380the Research/Scientific Computing teams at The James Hutton Institute and NIAB for providing computational resources and technical support for the “UK’s Crop Diversity Bioinformatics HPC” (BBSRC grant BB/ S019669/1)。
文摘An accurate assessment of host and pathogen gene expression during infection is critical for understanding the molecular aspects of host-pathogen interactions.Often,pathogen-derived transcripts are difficult to ascertain at early infection stages owing to the unfavourable transcript representation compared to the host genes.In this study,we compare two sequencing techniques,RNAseq and enrichment sequencing(RenSeq and PenSeq)of cDNA,to investigate gene expression patterns in the doubled monoploid potato(DM)infected with the late blight pathogen Phytophthora infestans.Our results reveal distinct advantages of cDNA RenSeq and PenSeq over traditional RNAseq in terms of target gene representation and transcriptional quantification at early infection stages.Throughout the infection time course,cDNA enrichment sequencing enables transcriptomic analyses for more targeted host and pathogen genes.For highly expressed genes that were sampled in parallel by both cDNA enrichment and RNAseq,a high level of concordance in expression profiles is observed,indicative of at least semi-quantitative gene expression representation following enrichment.
基金Supported by the National Oncology Care Support Program,No.25000.056766/2015-64.
文摘BACKGROUND Adenomatous polyposis confers an increased risk of developing colorectal cancer.APC and MUTYH are the major genes investigated in patients suspected of having polyposis.In addition to APC and MUTYH genes,other genes,such as POLE,POLD1,NTHL1,MBD4,MSH3 and MLH3,have recently been associated with polyposis phenotypes,conferring heterogeneity in terms of the clinical,etiological and heritable aspects of patients with polyposis.AIM To investigate the underlying variant landscape in patients with suspected polyposis who lack variants in the APC and MUTYH genes using whole-exome sequencing.METHODS Twenty-seven participants were included in the study and subjected to germline whole-exome sequencing.In addition,their clinical-pathological,personal,and family history data were collected.RESULTS The mean age at diagnosis was 51 years,and most participants had attenuated forms of polyposis(88.9%),with 63.0%diagnosed with a primary tumor,mostly colorectal cancer(76.5%).Among the variants identified,17 were classified as pathogenic or likely pathogenic(in 12 participants),including variants in genes involved in the Wnt/β-catenin signaling pathway,such as ST7 L,A1CF,and DKK4,and variants in DNA-repair genes,such as NTHL1,PNKP,and PMS2,as well as a variant found at the FRK gene identified in a patient with classic polyposis at age 19 and with a family history of polyps.CONCLUSION This study identified novel genes potentially associated with polyposis in patients lacking germline pathogenic variants in the APC and MUTYH genes.These findings support the use of next-generation sequencing for screening,expanding the scope of polyposis-related variants beyond these two genes.
基金financially supported by National Key R&D Program(2021YFF0701905)。
文摘In order to save manpower and time costs,and to achieve simultaneous detection of multiple animal-derived components in meat and meat products,this study used multiple nucleotide polymorphism(MNP)marker technology based on the principle of high-throughput sequencing,and established a multi-locus 10 animalderived components identification method of cattle,goat,sheep,donkey,horse,chicken,duck,goose,pigeon,quail in meat and meat products.The specific loci of each species could be detected and the species could be accurately identified,including 5 loci for cattle and duck,3 loci for sheep,9 loci for chicken and horse,10 loci for goose and pigeon,6 loci for quail and 1 locus for donkey and goat,and an adulteration model was established to simulate commercially available samples.The results showed that the method established in this study had high throughput,good repeatability and accuracy,and was able to identify 10 animalderived components simultaneously with 100%repeatability accuracy.The detection limit was 0.1%(m/m)in simulated samples of chicken,duck and horse.Using the method established in this study to test commercially available samples,4 samples from 14 commercially available samples were detected to be inconsistent with the labels,of which 2 did not contain the target ingredient and 2 were adulterated with small amounts of other ingredients.
基金Supported by School-Level Key Projects at Bengbu Medical College,No.2021byzd109.
文摘BACKGROUND Gallbladder neuroendocrine carcinoma(NEC)represents a subtype of gallbladder malignancies characterized by a low incidence,aggressive nature,and poor prognosis.Despite its clinical severity,the genetic alterations,mechanisms,and signaling pathways underlying gallbladder NEC remain unclear.CASE SUMMARY This case study presents a rare instance of primary gallbladder NEC in a 73-year-old female patient,who underwent a radical cholecystectomy with hepatic hilar lymphadenectomy and resection of liver segments IV-B and V.Targeted gene sequencing and bioinformatics analysis tools,including STRING,GeneMANIA,Metascape,TRRUST,Sangerbox,cBioPortal and GSCA,were used to analyze the biological functions and features of mutated genes in gallbladder NEC.Twelve mutations(APC,ARID2,IFNA6,KEAP1,RB1,SMAD4,TP53,BTK,GATA1,GNAS,and PRDM3)were identified,and the tumor mutation burden was determined to be 9.52 muts/Mb via targeted gene sequencing.A protein-protein interaction network showed significant interactions among the twelve mutated genes.Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses were used to assess mutation functions and pathways.The results revealed 40 tumor-related pathways.A key regulatory factor for gallbladder NEC-related genes was identified,and its biological functions and features were compared with those of gallbladder carcinoma.CONCLUSION Gallbladder NEC requires standardized treatment.Comparisons with other gallbladder carcinomas revealed clinical phenotypes,molecular alterations,functional characteristics,and enriched pathways.
基金supported by the National Science Foundation of China,Nos.82325031(to FX),82030059(to YC),82102290(to YG),U23A20485(to YC)Noncommunicable Chronic Diseases-National Science and Technology Major Project,No.2023ZD0505504(to FX),2023ZD0505500(to YC)the Key R&D Program of Shandong Province,No.2022ZLGX03(to YC).
文摘Global brain ischemia and neurological deficit are consequences of cardiac arrest that lead to high mortality.Despite advancements in resuscitation science,our limited understanding of the cellular and molecular mechanisms underlying post-cardiac arrest brain injury have hindered the development of effective neuroprotective strategies.Previous studies primarily focused on neuronal death,potentially overlooking the contributions of non-neuronal cells and intercellular communication to the pathophysiology of cardiac arrest-induced brain injury.To address these gaps,we hypothesized that single-cell transcriptomic analysis could uncover previously unidentified cellular subpopulations,altered cell communication networks,and novel molecular mechanisms involved in post-cardiac arrest brain injury.In this study,we performed a single-cell transcriptomic analysis of the hippocampus from pigs with ventricular fibrillation-induced cardiac arrest at 6 and 24 hours following the return of spontaneous circulation,and from sham control pigs.Sequencing results revealed changes in the proportions of different cell types,suggesting post-arrest disruption in the blood-brain barrier and infiltration of neutrophils.These results were validated through western blotting,quantitative reverse transcription-polymerase chain reaction,and immunofluorescence staining.We also identified and validated a unique subcluster of activated microglia with high expression of S100A8,which increased over time following cardiac arrest.This subcluster simultaneously exhibited significant M1/M2 polarization and expressed key functional genes related to chemokines and interleukins.Additionally,we revealed the post-cardiac arrest dysfunction of oligodendrocytes and the differentiation of oligodendrocyte precursor cells into oligodendrocytes.Cell communication analysis identified enhanced post-cardiac arrest communication between neutrophils and microglia that was mediated by neutrophil-derived resistin,driving pro-inflammatory microglial polarization.Our findings provide a comprehensive single-cell map of the post-cardiac arrest hippocampus,offering potential novel targets for neuroprotection and repair following cardiac arrest.
基金supported by the National Natural Science Foundation of China,No.82301403(to DZ)。
文摘Glial cells play crucial roles in regulating physiological and pathological functions,including sensation,the response to infection and acute injury,and chronic neurodegenerative disorders.Glial cells include astrocytes,microglia,and oligodendrocytes in the central nervous system,and satellite glial cells and Schwann cells in the peripheral nervous system.Despite the greater understanding of glial cell types and functional heterogeneity achieved through single-cell and single-nucleus RNA sequencing in animal models,few studies have investigated the transcriptomic profiles of glial cells in the human spinal cord.Here,we used high-throughput single-nucleus RNA sequencing and spatial transcriptomics to map the cellular and molecular heterogeneity of astrocytes,microglia,and oligodendrocytes in the human spinal cord.To explore the conservation and divergence across species,we compared these findings with those from mice.In the human spinal cord,astrocytes,microglia,and oligodendrocytes were each divided into six distinct transcriptomic subclusters.In the mouse spinal cord,astrocytes,microglia,and oligodendrocytes were divided into five,four,and five distinct transcriptomic subclusters,respectively.The comparative results revealed substantial heterogeneity in all glial cell types between humans and mice.Additionally,we detected sex differences in gene expression in human spinal cord glial cells.Specifically,in all astrocyte subtypes,the levels of NEAT1 and CHI3L1 were higher in males than in females,whereas the levels of CST3 were lower in males than in females.In all microglial subtypes,all differentially expressed genes were located on the sex chromosomes.In addition to sex-specific gene differences,the levels of MT-ND4,MT2A,MT-ATP6,MT-CO3,MT-ND2,MT-ND3,and MT-CO_(2) in all spinal cord oligodendrocyte subtypes were higher in females than in males.Collectively,the present dataset extensively characterizes glial cell heterogeneity and offers a valuable resource for exploring the cellular basis of spinal cordrelated illnesses,including chronic pain,amyotrophic lateral sclerosis,and multiple sclerosis.
基金Supported by Open Project of Key Laboratory of Hubei Province(No.2023KFZZ026).
文摘AIM:To estimate if nanopore targeted sequencing(NTS)could identify pathogens causing postoperative endophthalmitis and further determine the feasibility of clinical application of NTS.METHODS:A total of 55 patients(55 eyes)with postoperative endophthalmitis were retrospectively included in this study with their medical records.Intraocular fluid samples were examined by NTS and microbial culture.All included patients had undergone examinations including measurement of best corrected visual acuity(BCVA)and intraocular pressure(IOP),slit-lamp biomicroscopy,and indirect ophthalmoscopy;additionally,they underwent B-ultrasound,anterior segment photography,and fundus photography if necessary.RESULTS:Among 55 patients with postoperative endophthalmitis,the age was 65.25±15.04y and there were 30 female(54.54%)patients.Forty-one(74.54%)vitreous humor samples and fourteen(25.45%)aqueous humor samples were sent for both NTS and microbial culture.NTS had a notable higher detection rate than microbial culture in detecting pathogens(90.91%vs 38.18%,χ^(2)=33.409,P<0.001).NTS exhibited high sensitivity of pathogen detection in both microbial culture positive and negative samples(100%and 85.29%,respectively).In 16 of 21(76.19%)patients who showed culture-positivity,their results corresponded with those of NTS.Moreover,in two patients(9.52%),NTS showed a better species resolution than microbial culture;in three patients(14.28%),NTS identified additional pathogens.As for fungus,the positive detection rate of NTS was significantly higher than that of microbial culture(20%vs 3.64%,χ^(2)=7.066,P=0.008).Also,NTS could detect multi-infection by bacteria and fungi than microbial culture(32.73%vs 0,χ^(2)=21.522,P<0.001).NTS could detect bacteria as well as fungi simultaneously within 48h in all patients.Meanwhile,NTS had a shorter detection time than microbial culture(1.13±0.34 vs 2.67±0.55d,Z=-9.218,P<0.001).After the NTS results were obtained,15 patients received additional intravitreal/intracameral anti-infection treatment.At follow-up,there was a statistically significant improvement in the visual acuity relative to the baseline(Z=−5.222,P<0.001).CONCLUSION:NTS can provide rapid identification and highly sensitive detection of pathogens among patients with postoperative endophthalmitis,which can guide anti-infection treatment and improve visual prognosis.
基金Supported by the National Natural Science Foundation of China,No.81774118the Foreign Cooperation Project of Science and Technology Department of Fujian Province,No.2023I0021the Medical Innovation Project of Fujian Province,No.2024CXB013.
文摘BACKGROUND Hemorrhoids,a prevalent chronic condition globally,significantly impact patients'quality of life.While various surgical interventions,such as external stripping and internal ligation,procedure for prolapse and hemorrhoids,and tissue selecting technique,are employed for treatment,they are often associated with postoperative complications,including unsatisfactory defecation,bleeding,and anal stenosis.In contrast,Xiaozhiling injection,a traditional Chinese medicine-based therapy,has emerged as a minimally invasive and effective alternative for internal hemorrhoids.This treatment offers distinct advantages,such as reduced dietary restrictions,broad applicability,and minimal induction of systemic inflammatory responses.Additionally,Xiaozhiling injection effectively eliminates hemorrhoid nuclei,prevents local tissue necrosis,preserves anal cushion integrity,and mitigates postoperative complications,including bleeding and prolapse.Despite its clinical efficacy,the molecular mechanisms underlying its therapeutic effects remain poorly understood,warranting further investigation.AIM To investigate the molecular mechanism underlying the therapeutic effect of Xiaozhiling injection in the treatment of internal hemorrhoids.METHODS An internal hemorrhoid model was established in rats,and the rats were randomly divided into a modeling group[control group(CK group)]and a treatment group.One week after injection,Stereo-seq and electron microscopy were used to study the changes in gene expression and subcellular structures in fibroblasts.RESULTS Single-cell sequencing revealed differences in the expression and transcript levels of the genes collagen 3 alpha 1,decorin,and actin alpha 2 in fibroblasts between the CK group and the treatment group.Spatial transcriptome analysis revealed that genes of the sphingosine kinase 1(Sphk1)/sphingosine-1-phosphate(S1P)pathway spatially overlapped with key genes of the transforming growth factor beta 1 pathway,namely,Sphk1,S1P receptor,and transforming growth factor beta 1,in the treatment group.The proportion of fibroblasts was lower in the treatment group than in the CK group,and Xiaozhiling treatment had a significant effect on the proportion of fibroblasts in hemorrhoidal tissue.Immunohistochemistry revealed a significant increase in the expression of a fibroblast marker.Electron microscopy showed that the endoplasmic reticulum of fibroblasts contained a large amount of glycogen,indicating cell activation.Fibroblast activation and the expression of key genes of the Sphk1-S1P pathway could be observed at the injection site,suggesting that after Xiaozhiling intervention,the Sphk1-S1P pathway could be activated to promote fibrosis.CONCLUSION Xiaozhiling injection exerts its therapeutic effects on internal hemorrhoids by promoting collagen synthesis and secretion in fibroblasts.After Xiaozhiling intervention,the Sphk1-S1P pathway can be activated to promote fibrosis.
基金supported by the National Natural Science Foundation of China(No.22277093)the Key Research and Development Project of Hubei Province(No.2023BCB094)。
文摘N^(6)-methyladenine(6mA)is a prevalent DNA modification and is involved in a wide range of human diseases.Previous studies have indicated that 6mA is enriched in mitochondrial DNA(mtDNA)of mammals.By employing an evolved adenine deaminase,we developed a deaminase-mediated sequencing(DM-seq)method that could achieve genome-wide mapping of 6mA in mammalian mtDNA at single-base resolution.In this study,we used an engineered adenine deaminase,known as TadA8e protein,to map 6mA in mtDNA of hepatocellular carcinoma(HCC)by DM-seq.Through high-throughput sequencing,we identified sixteen 6mA sites in both HCC and adjacent normal tissue mtDNA.The results revealed an increased overall 6mA level in mtDNA associated with HCC.Furthermore,an elevation in 6mA level was observed alongside a decrease in the m RNA levels of the corresponding genes,indicating that increased6mA level hindered transcription processes related to these genes.These findings demonstrate that 6mA in mtDNA is correlated with HCC and provide evidence supporting the inhibitory effect of elevated 6mA level on subsequent transcriptional activity.This research illuminates the intricate relationship between 6mA modification and transcriptional regulation in the context of HCC,offering valuable insights into the role of 6mA modification in HCC pathogenesis.
基金Supported by the National Natural Science Foundation of China(No.82071312).
文摘AIM:To explore the immune cell infiltration and molecular mechanisms of retinal ischemia-reperfusion injury(RIRI)to identify potential therapeutic targets.METHODS:In the bulk RNA-seq analysis,This study performed differential gene expression analysis,weighted gene co-expression network analysis,and protein-protein interaction network analysis to identify hub genes.QuanTIseq was used to determine the composition of infiltrating immune cells.Following the identification of hub genes,single-cell RNA-seq analysis was employed to pinpoint the specific immune cell types expressing these hub genes.Cell-cell communication analysis to explore signaling pathways and interactions between immune cells was further performed.Finally,the expression of these key immune regulators in vivo using quantitative real-time polymerase chain reaction(qRT-PCR)was validated.RESULTS:Bulk RNA-seq analysis identified Stat2,Irf7,Irgm1,Igtp,Parp9,Irgm2,Nlrc5,and Tap1 as hub genes,with strong correlations to immune cell infiltration.Single-cell RNA-seq analysis further revealed six immune cell clusters,showing Irf7 predominantly in microglia and Tap1 in dendritic cells(DCs).And cell-cell communication analysis showed that microglia and DCs play central roles in coordinating immune activity.qRT-PCR validated the upregulation of these genes.CONCLUSION:In the acute phase of RIRI,Irf7 and Tap1 may be the potential therapeutic targets to reduce inflammation and promote neurological function recovery.
基金Supported by the Scientific Research Topic of Jiangsu Provincial Health Care Commission,No.M2021017the High-level Talent Research Project of the Second Hospital of Nanjing,No.0313504the Nanjing Second Hospital Academic Leader Program,No.0313506.
文摘BACKGROUND Understanding the status and function of tumor-infiltrating immune cells is essential for improving immunotherapeutic effects and predicting the clinical response in human patients with carcinoma.However,little is known about tumor-infiltrating immune cells,and the corresponding research results in hepatocellular carcinoma(HCC)are limited.AIM To investigate potential biomarker genes that are important for the development of HCC and to understand how immune cell subsets react throughout this process.METHODS Using single-cell RNA sequencing and T-cell receptor sequencing,the heterogeneity and potential functions of immune cell subpopulations from HCC tissue and normal tissue adjacent to carcinoma,as well as their possible interactions,were analyzed.RESULTS Eight T-cell clusters from patients were analyzed and identified using bioinformatics,including six typical major Tcell clusters and two newly identified T-cell clusters,among which Fc epsilon receptor 1G+T cells were characterized by the upregulation of Fc epsilon receptor 1G,tyrosine kinase binding protein,and T cell receptor delta constant,whereas metallothionein 1E+T cells proliferated significantly in tumors.Differentially expressed genes,such as regulator of cell cycle,cysteine and serine rich nuclear protein 1,SMAD7 and metallothionein 1E,were identified as significantly upregulated in tumors and have potential as biomarkers.In association with T-cell receptor analysis,we inferred the clonal expansion characteristics of each T-cell cluster in HCC patients.CONCLUSION We identified lymphocyte subpopulations and potential biomarker genes critical for HCC development and revealed the clonal amplification of infiltrating T cells.These data provide valuable resources for understanding the response of immune cell subsets in HCC.
基金National Key Research and Development Program of China,Grant/Award Number:2022ZD0210100Beijing Nova Star Program,Grant/Award Number:2022002+2 种基金Natural Science Foundation of Beijing and Haidian Collaboration Foundation,Grant/Award Number:L232079National Natural Science Foundation of China,Grant/Award Number:82172608,82273343,81902975 and 82101356Capital Medical University Fund for Excellent Young Scholars,Grant/Award Number:KCB2304。
文摘Background:Medulloblastoma(MB)is one of the most common malignant brain tumors that mainly affect children.Various approaches have been used to model MB to facilitate investigating tumorigenesis.This study aims to compare the recapitulation of MB between subcutaneous patient-derived xenograft(sPDX),intracranial patient-derived xenograft(iPDX),and genetically engineered mouse models(GEMM)at the single-cell level.Methods:We obtained primary human sonic hedgehog(SHH)and group 3(G3)MB samples from six patients.For each patient specimen,we developed two sPDX and iPDX models,respectively.Three Patch+/-GEMM models were also included for sequencing.Single-cell RNA sequencing was performed to compare gene expression profiles,cellular composition,and functional pathway enrichment.Bulk RNA-seq deconvolution was performed to compare cellular composition across models and human samples.Results:Our results showed that the sPDX tumor model demonstrated the highest correlation to the overall transcriptomic profiles of primary human tumors at the single-cell level within the SHH and G3 subgroups,followed by the GEMM model and iPDX.The GEMM tumor model was able to recapitulate all subpopulations of tumor microenvironment(TME)cells that can be clustered in human SHH tumors,including a higher proportion of tumor-associated astrocytes and immune cells,and an additional cluster of vascular endothelia when compared to human SHH tumors.Conclusions:This study was the first to compare experimental models for MB at the single-cell level,providing value insights into model selection for different research purposes.sPDX and iPDX are suitable for drug testing and personalized therapy screenings,whereas GEMM models are valuable for investigating the interaction between tumor and TME cells.
基金supported by the National Ecological Environment Survey and Assessment(2024-vertical-0107)the Fundamental Research Funds for the Central Public-interest Scientific Institution(2023YSKY-26)the Hulun Buir Grassland Ecological Restoration Comprehensive Survey Project(DD20230474).
文摘In recent years,intensive human activities have increased the intensity of desertification,driving continual desertification process of peripheral meadows.To investigate the effects of restoration on soil microbial communities,we analyzed vegetation-soil relationships in the Hulun Buir Sandy Land,northern China.Through the use of high-throughput sequencing,we examined the structure and diversity in the bacterial and fungal communities within the 0-20 cm soil layer after 9-15 a of restoration.Different slope positions were analyzed and spatial heterogeneity was assessed.The results showed progressive improvements in soil properties and vegetation with the increase of restoration duration,and the following order was as follows:bottom slope>middle slope>crest slope.During the restoration in the Hulun Buir Sandy Land,the bacterial communities were dominated by Proteobacteria,Actinobacteria,and Acidobacteria,whereas the fungal communities were dominated by Ascomycota and Basidiomycota.Eutrophic bacterial abundance increased with the restoration duration,whereas oligotrophic bacterial and fungal abundance levels decreased.The soil bacterial abundance significantly increased with the increasing restoration duration,whereas the fungal diversity decreased after 11 a of restoration,except that at the crest slope.Redundancy analysis showed that pH,soil moisture content,total nitrogen,and vegetation-related factors affected the bacterial community structure(45.43%of the total variance explained).Canonical correspondence analysis indicated that pH,total phosphorus,and vegetation-related factors shaped the bacterial community structure(31.82%of the total variance explained).Structural equation modeling highlighted greater bacterial responses(R^(2)=0.49-0.79)to changes in environmental factors than those of fungi(R^(2)=0.20-0.48).The soil bacterial community was driven mainly by pH,soil moisture content,electrical conductivity,plant coverage,and litter dry weight.The abundance and diversity of the soil fungal community were mainly driven by plant coverage,litter dry weight,and herbaceous aboveground biomass,while there was no significant correlation between the soil fungal community structure and environmental factors.These findings highlighted divergent microbial succession patterns and environmental sensitivities during sandy grassland restoration.
基金Project supported by the National Natural Science Foundation of China(Grant Nos.11804195,11847224,11674198,and 12274265)the Natural Science Foundation of Shandong Province,China(Grant Nos.ZR2018BA034 and ZR2022MA006)。
文摘To understand the gene-based biological processes in-depth,the single-molecule real-time sequencing has drawn increasing attention with promoted by the Human Genome Project.Herein,a set of newly designed canonical fluorescent bases(A_(y),tC,G_(b),T_(p))are proposed for four-color DNA sequencing.These quasi-intrinsic probes are derived from the fluorophore replacement and ring expansion on natural bases,which still keep the pyrimidine or purine underlying skeleton and Watson–Crick hydrogen bonding face to allow minimal perturbation to the native DNA duplex.More importantly,these nucleobase analogues possess red-shifted absorption and efficient photoluminescence due to the enhancedπ-conjugation in character.Meanwhile,the four analogues could generate distinct emission wavelength(Δλ~50 nm)for real-time sequencing.To assess the biological employment of the proposed biosensors,the effects of base pairing and linking deoxyribose are also considered.
基金supported by the National Centre for Research and Development TECHMATSTRATEG-Ⅲ/0027/2019,POWR.03.02.00-00-I006/17the IDUB UAM。
文摘Meniscus injuries are widespread and the available treatments do not offer enough healing potential.Here,we provide critical support for using pigs as a biological model for meniscal degeneration and the development of cutting-edge therapies in orthopedics.We present a single-cell transcriptome atlas of the meniscus,consisting of cell clusters corresponding to four major cell types:chondrocytes,endothelial cells,smooth muscle cells,and immune cells.Five distinct chondrocyte subclusters(CH0–CH4)were annotated,of which only one was widespread in both the red and white zones,indicating a major difference in the cellular makeup of the zones.Subclusters distinct to the white zone appear responsible for cartilage-specific matrix deposition and protection against adverse microenvironmental factors,while those in the red zone exhibit characteristics of mesenchymal stem cells and are more likely to proliferate and migrate.Additionally,they induce remodeling actions in other chondrocyte subclusters and promote the proliferation and maturation of endothelial cells,inducing healing and vascularization processes.Considering that they have substantial remodeling capabilities,these subclusters should be of great interest for tissue engineering studies.We also show that the cellular makeup of the pig meniscus is comparable to that of humans,which supports the use of pigs as a model in orthopedic therapy development.
基金supported by the grants from the Key R&D Program of Shandong Province,China(2022LZGC003)the China Agriculture Research System of MOF and MARA(CARS-35)+1 种基金the National Key Research and Development Project of China(2019YFE0106800)the 2115 Talent Development Program of China Agricultural University。
文摘Genotyping by target sequencing(GBTS)integrates the advantages of silicon-based technology(high stability and reliability)and genotyping by sequencing(high flexibility and cost-effectiveness).However,GBTS panels are not currently available in pigs.In this study,based on GBTS technology,we first developed a 50K panel,including 52,000 single-nucleotide polymorphisms(SNPs),in pigs,designated GBTS50K.A total of 6,032 individuals of Large White,Landrace,and Duroc pigs from 10 breeding farms were used to assess the newly developed GBTS50K.Our results showed that GBTS50K obtained a high genotyping ability,the SNP and individual call rates of GBTS50K were 0.997–0.998,and the average consistency rate and genotyping correlation coefficient were 0.997 and 0.993,respectively,in replicate samples.We also evaluated the efficiencies of GBTS50K in the application of population genetic structure analysis,selection signature detection,genome-wide association studies(GWAS),genotyped imputation,genetic selection(GS),etc.The results indicate that GBTS50K is plausible and powerful in genetic analysis and molecular breeding.For example,GBTS50K could gain higher accuracies than the current popular GGP-Porcine bead chip in genomic selection on 2 important traits of backfat thickness at 100 kg and days to 100 kg in pigs.Particularly,due to the multiple SNPs(mSNPs),GBTS50K generated 100K qualified SNPs without increasing genotyping cost,and our results showed that the haplotype-based method can further improve the accuracies of genomic selection on growth and reproduction traits by 2 to 6%.Our study showed that GBTS50K could be a powerful tool for underlying genetic architecture and molecular breeding in pigs,and it is also helpful for developing SNP panels for other farm animals.
基金Chinese Academy of Medical Sciences Initiative for Innovative Medicine,Grant/Award Number:2021-I2M-1-035 and 2022-I2M-1-011。
文摘Background:Dengue fever,an acute insect-borne infectious disease caused by the dengue virus(DENV),poses a great challenge to global public health.Hepatic involve-ment is the most common complication of severe dengue and is closely related to the occurrence and development of disease.However,the features of adaptive immune responses associated with liver injury in severe dengue are not clear.Methods:We used single-cell sequencing to examine the liver tissues of mild or se-vere dengue mice model to analyze the changes in immune response of T cells in the liver after dengue virus infection,and the immune interaction between macrophages and T cells.Flow cytometry was used to detect T cells and macrophages in mouse liver and blood to verify the single-cell sequencing results.Results:Our result showed CTLs were significantly activated in the severe liver injury group but the immune function-related signal pathway was down-regulated.The rea-son may be that the excessive immune response in the severe group at the late stage of DENV infection induces the polarization of macrophages into M2 type,and the macrophages then inhibit T cell immunity through the TGF-βsignaling pathway.In ad-dition,the increased proportion of Treg cells suggested that Th17/Treg homeostasis was disrupted in the livers of severe liver injury mice.Conclusions:In this study,single-cell sequencing and flow cytometry revealed the characteristic changes of T cell immune response and the role of macrophages in the liver of severe dengue fever mice.Our study provides a better understanding of the pathogenesis of liver injury in dengue fever patients.
基金Supported by the Japan Agency for Medical Research and Development(AMED),No.JP22fk0310503.
文摘BACKGROUND Tenofovir alafenamide fumarate(TAF)is one of the first-line treatments used to treat chronic hepatitis B patients;TAF has strong antiviral activity and a high barrier to resistance.Although virological breakthroughs in patients during TAF treatment are rare,patients with incomplete responses to TAF are occasionally observed.AIM To investigate responsible mutations in the reverse transcriptase region of hepatitis B virus(HBV)for TAF-incomplete responses.METHODS Thirteen chronic hepatitis B patients who received TAF monotherapy were included.A TAF-incomplete responder was defined as one who was continuously positive for HBV DNA over 2 years after TAF treatment initiation.The emergences of mutations in TAF-incomplete responders were evaluated before,one year after,and two years after treatment by deep sequencing of HBV DNA and RNA.RESULTS Two patients were continuously positive for HBV DNA over two years.The rtL269I mutation,one of the CYEI mutations linked to tenofovir resistance,was detected in both patients by direct sequencing.The deep sequencing analysis revealed that a combination of rtT118A and rtL220I mutations and the rtL269I mutation were predominantly detected in HBV DNA even when these mutations were barely detected in HBV RNA.This suggests a superior replication capability of the HBV variants with these mutations under TAF treatment.CONCLUSION The deep sequencing analysis of HBV DNA and RNA and comparing the detection rates of mutations were useful for estimating responsible mutations for TAF-incomplete responses.Such analysis is needed to evaluate the association between mutations that emerge during TAF treatment and incomplete responses to TAF.
基金supported by the National Natural Science Foundation of China(Grant Nos.31972983 and 32072487)the Key Technology R&D Program of Zhejiang Province,China(Grant No.2021C02006)the Zhejiang Provincial Natural Science Foundation of China(Grant No.LY23C140001).
文摘In rice fields,rice plants usually grow alongside wild weeds and are attacked by various invertebrate species.Viruses are abundant in plants and invertebrates,playing crucial ecological roles in controlling microbial abundance and maintaining community structures.To date,only 16 rice viruses have been documented in rice-growing regions.These viruses pose serious threats to rice production and have traditionally been identified only from rice plants and insect vectors by isolation techniques.Advances in next-generation sequencing(NGS)have made it feasible to discover viruses on a global scale.Recently,numerous viruses have been identified in plants and invertebrates using NGS technologies.In this review,we discuss viral studies in rice plants,invertebrate species,and weeds in rice fields.Many novel viruses have been discovered in rice ecosystems through NGS technologies,with some also detected using metatranscriptomic and small RNA sequencing.These analyses greatly expand our understanding of viruses in rice fields and provide valuable insights for developing efficient strategies to manage insect pests and virus-mediated rice diseases.
