Spatial transcriptomics reveals that metabolic characteristics define the tumor immunosuppression microenvironment via iCAF transformation in oral squamous cell carcinoma / 国际口腔科学杂志·英文版

Tumor progression is closely related to tumor tissue metabolism and reshaping of the microenvironment. Oral squamous cell carcinoma (OSCC), a representative hypoxic tumor, has a heterogeneous internal metabolic environment. To clarify the relationship between different metabolic regions and the tumor immune microenvironment (TME) in OSCC, Single cell (SC) and spatial transcriptomics (ST) sequencing of OSCC tissues were performed. The proportion of TME in the ST data was obtained through SPOTlight deconvolution using SC and GSE103322 data. The metabolic activity of each spot was calculated using scMetabolism, and k-means clustering was used to classify all spots into hyper-, normal-, or hypometabolic regions. CD4T cell infiltration and TGF-β expression is higher in the hypermetabolic regions than in the others. Through CellPhoneDB and NicheNet cell-cell communication analysis, it was found that in the hypermetabolic region, fibroblasts can utilize the lactate produced by glycolysis of epithelial cells to transform into inflammatory cancer-associated fibroblasts (iCAFs), and the increased expression of HIF1A in iCAFs promotes the transcriptional expression of CXCL12. The secretion of CXCL12 recruits regulatory T cells (Tregs), leading to Treg infiltration and increased TGF-β secretion in the microenvironment and promotes the formation of a tumor immunosuppressive microenvironment. This study delineates the coordinate work axis of epithelial cells-iCAFs-Tregs in OSCC using SC, ST and TCGA bulk data, and highlights potential targets for therapy.

Single-Cell Mapping of Brain Myeloid Cell Subsets Reveals Key Transcriptomic Changes Favoring Neuroplasticity after Ischemic Stroke / 神经科学通报·英文版

Interactions between brain-resident and peripheral infiltrated immune cells are thought to contribute to neuroplasticity after cerebral ischemia. However, conventional bulk sequencing makes it challenging to depict this complex immune network. Using single-cell RNA sequencing, we mapped compositional and transcriptional features of peri-infarct immune cells. Microglia were the predominant cell type in the peri-infarct region, displaying a more diverse activation pattern than the typical pro- and anti-inflammatory state, with axon tract-associated microglia (ATMs) being associated with neuronal regeneration. Trajectory inference suggested that infiltrated monocyte-derived macrophages (MDMs) exhibited a gradual fate trajectory transition to activated MDMs. Inter-cellular crosstalk between MDMs and microglia orchestrated anti-inflammatory and repair-promoting microglia phenotypes and promoted post-stroke neurogenesis, with SOX2 and related Akt/CREB signaling as the underlying mechanisms. This description of the brain's immune landscape and its relationship with neurogenesis provides new insight into promoting neural repair by regulating neuroinflammatory responses.

Identification of overlay differentially expressed genes in both rats and goats with blast lung injury through comparative transcriptomics / 中华创伤杂志(英文版)

PURPOSE@#To identify the potential target genes of blast lung injury (BLI) for the diagnosis and treatment.@*METHODS@#This is an experimental study. The BLI models in rats and goats were established by conducting a fuel-air explosive power test in an unobstructed environment, which was subsequently validated through hematoxylin-eosin staining. Transcriptome sequencing was performed on lung tissues from both goats and rats. Differentially expressed genes were identified using the criteria of q ≤ 0.05 and |log2 fold change| ≥ 1. Following that, enrichment analyses were conducted for gene ontology and the Kyoto Encyclopedia of Genes and Genomes pathways. The potential target genes were further confirmed through quantitative real-time polymerase chain reaction and enzyme linked immunosorbent assay.@*RESULTS@#Observations through microscopy unveiled the presence of reddish edema fluid, erythrocytes, and instances of focal or patchy bleeding within the alveolar cavity. Transcriptome sequencing analysis identified a total of 83 differentially expressed genes in both rats and goats. Notably, 49 genes exhibited a consistent expression pattern, with 38 genes displaying up-regulation and 11 genes demonstrating down-regulation. Enrichment analysis highlighted the potential involvement of the interleukin-17 signaling pathway and vascular smooth muscle contraction pathway in the underlying mechanism of BLI. Furthermore, the experimental findings in both goats and rats demonstrated a strong association between BLI and several key genes, including anterior gradient 2, ankyrin repeat domain 65, bactericidal/permeability-increasing fold containing family A member 1, bactericidal/permeability-increasing fold containing family B member 1, and keratin 4, which exhibited up-regulation.@*CONCLUSIONS@#Anterior gradient 2, ankyrin repeat domain 65, bactericidal/permeability-increasing fold containing family A member 1, bactericidal/permeability-increasing fold containing family B member 1, and keratin 4 hold potential as target genes for the prognosis, diagnosis, and treatment of BLI.

The pattern of alternative splicing and DNA methylation alteration and their interaction in linseed (Linum usitatissimum L) response to repeated drought stresses

BACKGROUND: Drought stress has significantly hampered agricultural productivity worldwide and can also result in modifications to DNA methylation levels. However, the dynamics of DNA methylation and its association with the changes in gene transcription and alternative splicing (AS) under drought stress are unknown in linseed, which is frequently cultivated in arid and semiarid regions. RESULTS: We analysed AS events and DNA methylation patterns in drought-tolerant (Z141) and drought-sensitive (NY-17) linseed under drought stress (DS) and repeated drought stress (RD) treatments. We found that the number of intron-retention (IR) and alternative 3' splice site (Alt3'SS) events were significantly higher in Z141 and NY-17 under drought stress. We found that the linseed response to the DS treatment was mainly regulated by transcription, while the response to the RD treatment was coregulated by transcription and AS. Whole genome-wide DNA methylation analysis revealed that drought stress caused an increase in the overall methylation level of linseed. Although we did not observe any correlation between differentially methylated genes (DMGs) and differentially spliced genes (DSGs) in this study, we found that the DSGs whose gene body region was hypermethylated in Z141 and hypomethylated in NY-17 were enriched in abiotic stress response Gene Ontology (GO) terms. This finding implies that gene body methylation plays an important role in AS regulation in some specific genes. CONCLUSION: Our study is the first comprehensive genome-wide analysis of the relationship between linseed methylation changes and AS under drought and repeated drought stress. Our study revealed different interaction patterns between differentially expressed genes (DEGs) and DSGs under DS and RD treatments and differences between methylation and AS regulation in drought-tolerant and drought-sensitive linseed varieties. The findings will probably be of interest in the future. Our results provide interesting insights into the association between gene expression, AS, and DNA methylation in linseed under drought stress. Differences in these associations may account for the differences in linseed drought tolerance.

The effects of repeated doses of xylazine-ketamine and medetomidineketamine anesthesia on DNA damage in the liver and kidney

Purpose: This study evaluated the DNA damage caused by repeated doses of xylazine-ketamine and medetomidine-ketamine anesthesia in the liver and kidneys. Methods: In this study, 60 rats were used. The rats were divided into group 1 (xylazine-ketamine), and group 2 (medetomidine-ketamine), and these anesthetic combinations were administered to the rats at repeated doses with 30-min intervals. The effects of these anesthetic agents on the tumor necrosis factor-alpha gene for DNA damage were investigated. Results: According to the gene expression results, it was observed that a single dose of xylazine-ketamine was 2.9-fold expressed, while first and second repeat doses did not show significant changes in expression levels. However, in the case of the third repetition, it was observed to be 3.8-fold overexpressed. In the case of medetomidine-ketamine administration, it was observed that a single-dose application resulted in a 1.04-fold expression, while the first and the third repeat doses showed a significant down expression. The samples from the second repeat dose administration group were found to have insignificant levels of expression. Conclusions: This study can contribute to understanding the safe anesthetic combination in research and operations in which xylazine-ketamine and medetomidine-ketamine combinations are used.

O impacto da exposição química sobre o transcriptoma de trabalhadores formais e informais / The impact of chemical exposure on the transcriptome of formal and informal workers

Introdução: Exposições químicas podem variar em populações geograficamente distintas, com diferentes hábitos, estilos de vida e características individuais. Alguns elementos químicos encontrados no ambiente são capazes de alterar a expressão gênica humana. Objetivos: a) quantificar as concentrações de elementos potencialmente tóxicos (EPTs: As, Cd, Cr, Cu, Hg, Mn, Ni, Pb, Sb, Sn, e Zn) na urina da população de Limeira, e nas peças de joias e pó de solda; b) quantificar as concentrações de EPTs no sangue dos participantes de Limeira e Volta Redonda; c) avaliar os riscos de doenças associadas à exposição ocupacional; d) avaliar o impacto da exposição ocupacional na expressão gênica de trabalhadores formais e informais. Métodos: O grupo Exposto foi composto por trabalhadores informais que realizam soldagem de joias e bijuterias em ambiente domiciliar na cidade de Limeira, SP; e por trabalhadores formais da Companhia Siderúrgica Nacional, em Volta Redonda, RJ. O grupo Controle incluiu moradores dos mesmos bairros dos trabalhadores, mas que não desenvolviam nenhuma atividade diretamente relacionada à exposição química. Amostras de sangue foram coletadas para quantificação de glicose, insulina, perfil lipídico, EPTs e para análise transcriptômica. Em Limeira, também foi quantificada a concentração de EPTs na urina. Para transcriptômica, o RNA foi extraído e hibridizado com Agilent SurePrint G3 Human Gene Expression 8x60K v2 Microarray. O pré-processamento, análise estatística e de vias de interesse foram realizados no software GeneSpring GX. Todos os participantes preencheram questionários sobre hábitos, percepção de risco, morbidade referida e exposição ocupacional. A associação entre exposição a EPTs e desfechos de saúde foi testada por modelo de regressão de Poisson multivariado. Resultados: Nos trabalhadores informais, foram detectados 16 genes superexpressos e 33 subexpressos em comparação com os controles (fold-change > 2). A análise de vias indicou genes enriquecidos em vias do processo inflamatório (quimiocinas MAPK, receptor Toll-like e NF-kappa B). Nos trabalhadores formais, foram encontrados 20 genes superexpressos e 50 subexpressos, com vias relacionadas à resposta imune e ao processo de aterosclerose. O único gene diferencialmente expresso (DEG) em comum nas duas populações foi o IFI27 relacionado na literatura a diferentes tipos de câncer. A produção informal de joias de Limeira foi associada a exposição dos trabalhadores ao Cd, com concentrações significativamente maiores na urina e no sangue dos trabalhadores comparado aos controles. Além disso, foi observada uma associação positiva entre as concentrações de Cd no sangue e a glicemia. As concentrações de As e Pb também foram maiores no sangue dos trabalhadores informais comparado aos controles, sendo que participantes com concentrações de Pb superiores a 2,6 µg dL-1 apresentaram prevalência de manifestações neurológicas 2,3 vezes maior (IC 95%: 1,17 - 4,58; p = 0,02). Não foram observadas diferenças significativas nos EPTs entre os grupos de Volta Redonda, provavelmente devido ao uso de equipamentos de proteção individual e à poluição ambiental na região. Conclusão: As diferenças na expressão gênica relacionadas à exposição ocupacional estão associadas, principalmente, à inflamação e à resposta imune. Os resultados sugerem que a exposição ocupacional prolongada a elementos tóxicos pode levar a consequências negativas para a saúde, como por exemplo, um aumento da prevalência de manifestações neurológicas. Os resultados exploratórios desta tese são um ponto de partida para estudos em populações sensíveis e pouco estudadas, especialmente, de países em desenvolvimento. Análises adicionais devem ser realizadas para investigar efeitos diretos e validar associações causais.

Introduction: Chemical exposures may vary in geographically distinct populations, with different habits, lifestyles, and individual characteristics. Objectives: a) to determine potentially toxic elements' (EPTs: As, Cd, Cr, Cu, Hg, Mn, Ni, Pb, Sb, Sn, and Zn) in the urine of the population of Limeira, and in jewelry pieces and soldering powder; b) determine the PTEs concentrations in the participants' blood from Limeira and Volta Redonda municipalities; c) investigate disease risks associated with occupational exposure; d) to evaluate the impact of occupational exposure on gene expression profile. Methods: The exposed group was composed of informal workers who perform soldering of jewelry inside their houses in the city of Limeira, SP; and formal workers from a steel company in the city of Volta Redonda, RJ. Control participants were recruited from the same neighborhoods without occupational chemical exposure. Blood samples were collected for blood glucose, insulin, lipid profile, and PTE determinations, and for transcriptomic analysis. In Limeira, PTE concentration in urine was also determined. RNA was extracted and hybridized to Agilent SurePrint G3 Human Gene Expression 8x60K v2 Microarray for transcriptomics analysis. Pre-processing, statistical, and pathway analysis were performed in GeneSpring GX software. All participants completed questionnaires about household risk, reported morbidity, and occupational exposure. The association between PTEs exposure and health outcomes was tested by a multivariable robust Poisson regression model. Results: 16 up- and 33 down-regulated genes (fold-change > 2) were observed in the informal workers. Pathway analysis revealed genes enriched in inflammatory process (MAPK, Toll-like receptor, and NF-kappa B chemokine signaling pathways). In formal workers, 20 up- and 50 down-regulated genes were found with pathways related to immune response and atherosclerosis development. The gene IFI27 which has been associated with various types of cancer was the only one commonly differentially expressed between informal and formal workers. Informal jewelry production in Limeira increased exposure to Cd, with significantly higher concentrations in the urine and blood of informal exposed workers compared to controls. Furthermore, a positive association was observed between blood Cd concentrations and glycemia. The blood concentration of As and Pb were also Participants with Pb concentrations higher than 2.6 µg dL-1 showed a prevalence of neurological manifestations 2.3 times higher (95% CI: 1.17 - 4.58; p = 0.02) than those with lower lead concentrations. No significant differences were observed between formal workers from Volta Redonda and their control group, probably, because of the use of individual protection equipment and the environmental pollution in the region. Conclusion: Differences in gene expression related to occupational exposure are mainly associated with inflammation and immune response. The results suggest that prolonged occupational exposure to toxic elements could lead to negative health outcomes, such as higher prevalence of neurological manifestations. These exploratory results are a starting point for analysis in sensitive and understudied populations, especially in developing countries. Further analysis should be carried out to investigate its direct effects and to validate causal associations.

Avaliação dos aspectos moleculares e sua relevância na resposta terapêutica e prognóstico de pacientes com câncer de bexiga submetidos a quimioterapia com cisplatina / Evaluation of molecular aspects and their relevance in therapeutic response and prognosis of patients with bladder cancer undergoing chemotherapy with cisplatin

INTRODUÇÃO: O câncer de bexiga é a 10a neoplasia maligna mais comum no mundo. O tipo músculo-invasivo tem como tratamento padrão-ouro combinações de quimioterapia e cirurgia. Muitos pacientes, no entanto, não respondem a quimioterapia e são submetidos a uma terapia ineficaz. Identificar quais pacientes não irão se beneficiar da terapia pode reduzir toxicidades e custos. A base de cisplatina, a quimioterapia no tumor de bexiga age através de dano ao DNA. Levantamos a hipótese que pacientes que respondem apresentam mutação ou baixa expressão de genes de reparo ao DNA. OBJETIVO: Comparar os aspectos moleculares entre 2 grupos de pacientes com tumor de bexiga submetidos a quimioterapia: um que respondeu adequadamente e outro que não respondeu. Identificar se os genes diferencialmente expressos ou mutados estão associados ao reparo do DNA e, consequentemente, com a resposta terapêutica. MÉTODOS: O estudo foi realizado através de análise de dados moleculares e clínicos de pacientes com tumor de bexiga músculo-invasivo submetidos a quimioterapia com cisplatina. Os pacientes foram divididos em dois grupos de acordo com a resposta a quimioterapia: Respondedores e Não respondedores. Realizamos comparação do perfil de expressão gênica, regulação epigenética e mutações entre grupos. Os genes diferencialmente expressos e/ou mutados foram avaliados se estão associados com as vias de reparo do DNA e, consequentemente, com a reposta as terapias sistêmicas. Por fim, realizamos análise de sobrevida a fim de avaliar correlação entre expressão dos genes identificados com a sobrevida global dos pacientes. RESULTADOS: A casuística foi composta por 63 pacientes. 41 apresentaram resposta a quimioterapia e 22 não responderam. Após análise de expressão gênica diferencial, identificamos expressão significativamente menor dos genes DDB1 e PRPF9, associados as vias de reparo do DNA, no grupo de pacientes respondedores. Na análise de sobrevida, identificamos que a alta expressão do gene DDB1 esteve significativamente associada a uma sobrevida global pior. Na comparação de padrão mutacional, identificamos uma taxa de mutação, significativamente maior, do gene CDK12, associado ao reparo do DNA, no grupo de pacientes respondedores. Esses padrões de expressão ou mutação identificados nestes 3 genes de reparo de dano ao DNA estiveram fortemente associados a uma resposta satisfatória a quimioterapia. Dessa forma, são potenciais biomarcadores capazes de predizer resposta a quimioterapia em câncer de bexiga. CONCLUSÕES: Identificamos potenciais biomarcadores associados com a resposta terapêutica e sobrevida após uso de cisplatina em pacientes com tumor de bexiga. Estes, após validações, potencialmente poderão ser agrupado em escores capazes de predizer resposta a quimioterapia no câncer de bexiga e auxiliar na tomada de decisão, implicando em redução de toxicidades e custos.

INTRODUCTION: Bladder cancer is the 10th most common malignancy in world. The gold standard treatment for muscle-invasive type is combinations of chemotherapy and surgery. Many patients, however, do not respond to chemotherapy and are subjected to ineffective therapy. Identifying which patients will not benefit from therapy can reduce toxicities and costs. Cisplatin-based chemotherapy in bladder tumors acts through DNA damage. We hypothesize that patients who respond have mutation or low expression of DNA repair genes. OBJECTIVE: To compare the molecular aspects between 2 groups of patients with bladder tumors undergoing chemotherapy: one that responded adequately and other that did not respond. Identify whether differentially expressed or mutated genes are associated with DNA repair and, consequently, with therapeutic response. METHODS: The study was carried out through the analysis of molecular and clinical data from patients with muscle-invasive bladder tumors undergoing chemotherapy with cisplatin. Patients were divided into two groups according to their response to chemotherapy: Responders and Non-responders. We compared gene expression profile, epigenetic regulation and mutations between groups. Differentially expressed and/or mutated genes were assessed whether they are associated with DNA repair pathways and, consequently, with response to systemic therapies. Finally, we performed a survival analysis in order to evaluate the correlation between the expression of the identified genes and the overall survival of the patients. RESULTS: The sample consisted of 63 patients. 41 responded to chemotherapy and 22 did not respond. After differential gene expression analysis, we identified significantly lower expression of the DDB1 and PRPF9 genes, associated with DNA repair pathways, in Responders group. In survival analysis, we identified that high expression of the DDB1 gene was significantly associated with worse overall survival. When comparing the mutational pattern, we identified a significantly higher mutation rate in the CDK12 gene, associated with DNA repair, in Responders group. These expression or mutation patterns identified in these 3 DNA damage repair genes were strongly associated with a satisfactory response to chemotherapy. Therefore, they are potential biomarkers capable of predicting response to chemotherapy in bladder cancer. CONCLUSIONS: We identified potential biomarkers associated with therapeutic response and survival after the use of cisplatin in patients with bladder tumors. These, after validation, could potentially be grouped into scores capable of predicting response to chemotherapy in bladder cancer and assisting in decisionmaking, resulting in a reduction in toxicities and costs.

STAT3 as a candidate transcriptomic prognosticator of sepsis severity levels

Background@#Sepsis is a life-threatening multiple-organ dysfunction caused by a dysregulated host response to infection and is the leading cause of death in non-cardiac intensive care facilities. Early reliable prediction of sepsis outcomes leads to cost-efficient resource allocation and therapeutic strategies. However, there are still no reliable markers to predict the outcome of patients at the initial stage of sepsis. Analyzing transcription profiles enables researchers to predict early outcomes using transcripts and their expression patterns. Transcriptomic profiling of septic patients has been done recently; however, analysis of prognostic outcomes is still scarce.@*Objective@#This study aimed to determine transcriptional indicators that may be useful in the prognosis of the severity of sepsis.@*Methods@#This is a prospective cohort study of Filipino patients admitted for sepsis at the national tertiary referral hospital in Manila, Philippines. We conducted differentially expressed gene analysis, network analyses, and area under the curve study of publicly available datasets of surviving vs. non-surviving sepsis patients to identify candidate prognosticator markers. Quantitative PCR was used to characterize the expression of each marker. A model using ordinal logistic regression analysis was done to determine which among the markers can best predict the outcome of sepsis severity.@*Results@#We identified ACTB, RAC1, STAT3, and UBQLN1 as candidate mRNA prognosticators. The expression of STAT3, a gene involved in immunosuppression, is inversely correlated with the severity of sepsis.@*Conclusion@#Transcriptomic markers such as STAT3 can predict the severity of patients with sepsis. Early detection of its inverse expression may prompt early and more aggressive management of patients.

New strategies for the treatment of carcinoma of unknown primary / 中华肿瘤杂志

Carcinoma of unknown primary (CUP) is a kind of metastatic tumor whose primary origin cannot be identified after adequate examination and evaluation. The main treatment modality of CUP is empiric chemotherapy, and the median overall survival time is less than 1 year. Compared with immunohistochemistry, novel method based on gene expression profiling have improved the sensitivity and specificity of CUP detection, but its guiding value for treatment is still controversial. The approval of immune checkpoint inhibitors and pan-cancer antitumor agents has improved the prognosis of patients with CUP, and targeted therapy and immunotherapy based on specific molecular characteristics are the main directions of future research. Given the high heterogeneity and unique clinicopathological characteristics of CUP, "basket trial" is more suitable for clinical trial design in CUP.

Spatial transcriptome analysis of long non-coding RNAs reveals tissue specificity and functional roles in cancer / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Long non-coding RNAs (lncRNAs) play a significant role in maintaining tissue morphology and functions, and their precise regulatory effectiveness is closely related to expression patterns. However, the spatial expression patterns of lncRNAs in humans are poorly characterized. Here, we constructed five comprehensive transcriptomic atlases of human lncRNAs covering thousands of major tissue samples in normal and disease states. The lncRNA transcriptomes exhibited high consistency within the same tissues across resources, and even higher complexity in specialized tissues. Tissue-elevated (TE) lncRNAs were identified in each resource and robust TE lncRNAs were refined by integrative analysis. We detected 1 to 4684 robust TE lncRNAs across tissues; the highest number was in testis tissue, followed by brain tissue. Functional analyses of TE lncRNAs indicated important roles in corresponding tissue-related pathways. Moreover, we found that the expression features of robust TE lncRNAs made them be effective biomarkers to distinguish tissues; TE lncRNAs also tended to be associated with cancer, and exhibited differential expression or were correlated with patient survival. In summary, spatial classification of lncRNAs is the starting point for elucidating the function of lncRNAs in both maintenance of tissue morphology and progress of tissue-constricted diseases.

Analysis of Significant Genes and Pathways in Esophageal Cancer Based on Gene Expression Omnibus Database / 中国医学科学杂志(英文版)

Objective To screen antigen targets for immunotherapy by analyzing over-expressed genes, and to identify significant pathways and molecular mechanisms in esophageal cancer by using bioinformatic methods such as enrichment analysis, protein-protein interaction (PPI) network, and survival analysis based on the Gene Expression Omnibus (GEO) database.Methods By screening with highly expressed genes, we mainly analyzed proteins MUC13 and EPCAM with transmembrane domain and antigen epitope from TMHMM and IEDB websites. Significant genes and pathways associated with the pathogenesis of esophageal cancer were identified using enrichment analysis, PPI network, and survival analysis. Several software and platforms including Prism 8, R language, Cytoscape, DAVID, STRING, and GEPIA platform were used in the search and/or figure creation.Results Genes MUC13 and EPCAM were over-expressed with several antigen epitopes in esophageal squamous cell carcinoma (ESCC) tissue. Enrichment analysis revealed that the process of keratinization was focused and a series of genes were related with the development of esophageal cancer. Four genes including ALDH3A1, C2, SLC6A1,and ZBTB7C were screened with significant P value of survival curve.Conclusions Genes MUC13 and EPCAM may be promising antigen targets or biomarkers for esophageal cancer. Keratinization may greatly impact the pathogenesis of esophageal cancer. Genes ALDH3A1, C2, SLC6A1,and ZBTB7C may play important roles in the development of esophageal cancer.

Immune regulation mechanism of Saposhnikoviae Radix polysaccharide based on zebrafish model / 中国中药杂志

The immunomodulatory effect of Saposhnikoviae Radix polysaccharide(SRP) was evaluated based on the zebrafish mo-del, and its mechanism was explored by transcriptome sequencing and real-time fluorescence-based quantitative PCR(RT-qPCR). The immune-compromised model was induced by navelbine in the immunofluorescence-labeled transgenic zebrafish Tg(lyz: DsRed), and the effect of SRP on the density and distribution of macrophages in zebrafish was evaluated. The effect of SRP on the numbers of macrophages and neutrophils in wild-type AB zebrafish was detected by neutral red and Sudan black B staining. The content of NO in zebrafish was detected by DAF-FM DA fluorescence probe. The content of IL-1β and IL-6 in zebrafish was detected by ELISA. The differentially expressed genes(DEGs) of zebrafish in the blank control group, the model group, and the SRP treatment group were analyzed by transcriptome sequencing. The immune regulation mechanism was analyzed by Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment, and the expression levels of key genes were verified by RT-qPCR. The results showed that SRP could significantly increase the density of immune cells in zebrafish, increase the number of macrophages and neutrophils, and reduce the content of NO, IL-1β, and IL-6 in immune-compromised zebrafish. The results of transcriptome sequencing analysis showed that SRP could affect the expression level of immune-related genes on Toll-like receptor pathway and herpes simplex infection pathway to affect the release of downstream cytokines and interferon, thereby completing the activation process of T cells and playing a role in regulating the immune activity of the body.

Identification and expression of uridine diphosphate glycosyltransferase(UGT) gene family from Dendrobium officinale / 中国中药杂志

Uridine diphosphate glycosyltransferase(UGT) is a highly conserved protein in plants, which usually functions in secondary metabolic pathways. This study used the Hidden Markov Model(HMM) to screen out members of UGT gene family in the whole genome of Dendrobium officinale, and 44 UGT genes were identified. Bioinformatics was used to analyze the structure, phylogeny, and promoter region components of D. officinale genes. The results showed that UGT gene family could be divided into four subfamilies, and UGT gene structure was relatively conserved in each subfamily, with nine conserved domains. The upstream promoter region of UGT gene contained a variety of cis-acting elements related to plant hormones and environmental factors, indicating that UGT gene expression may be induced by plant hormones and external environmental factors. UGT gene expression in different tissues of D. officinale was compared, and UGT gene expression was found in all parts of D. officinale. It was speculated that UGT gene played an important role in many tissues of D. officinale. Through transcriptome analysis of D. officinale mycorrhizal symbiosis environment, low temperature stress, and phosphorus deficiency stress, this study found that only one gene was up-regulated in all three conditions. The results of this study can help understand the functions of UGT gene family in Orchidaceae plants and provide a basis for further study on the molecular regulation mechanism of polysaccharide metabolism pathway in D. officinale.

Comparative analysis of differentially expressed genes for biosynthesis of active ingredients in fruits of different cultivars of Lycium barbarum L. based on transcriptome sequencing / 生物工程学报

To explore the differentially expressed genes (DEGs) related to biosynthesis of active ingredients in wolfberry fruits of different varieties of Lycium barbarum L. and reveal the molecular mechanism of the differences of active ingredients, we utilized Illumina NovaSeq 6000 high-throughput sequencing technology to conduct transcriptome sequencing on the fruits of 'Ningqi No.1' and 'Ningqi No.7' during the green fruit stage, color turning stage and maturity stage. Subsequently, we compared the profiles of related gene expression in the fruits of the two varieties at different development stages. The results showed that a total of 811 818 178 clean reads were obtained, resulting in 121.76 Gb of valid data. There were 2 827, 2 552 and 2 311 DEGs obtained during the green fruit stage, color turning stage and maturity stage of 'Ningqi No. 1' and 'Ningqi No. 7', respectively, among which 2 153, 2 050 and 1 825 genes were annotated in six databases, including gene ontology (GO), Kyoto encyclopedia of genes and genomes (KEGG) and clusters of orthologous groups of proteins (KOG). In GO database, 1 307, 865 and 624 DEGs of green fruit stage, color turning stage and maturity stage were found to be enriched in biological processes, cell components and molecular functions, respectively. In the KEGG database, the DEGs at three developmental stages were mainly concentrated in metabolic pathways, biosynthesis of secondary metabolites and plant-pathogen interaction. In KOG database, 1 775, 1 751 and 1 541 DEGs were annotated at three developmental stages, respectively. Searching the annotated genes against the PubMed database revealed 18, 26 and 24 DEGs related to the synthesis of active ingredients were mined at the green fruit stage, color turning stage and maturity stage, respectively. These genes are involved in carotenoid, flavonoid, terpenoid, alkaloid, vitamin metabolic pathways, etc. Seven DEGs were verified by RT-qPCR, which showed consistent results with transcriptome sequencing. This study provides preliminary evidences for the differences in the content of active ingredients in different Lycium barbarum L. varieties from the transcriptional level. These evidences may facilitate further exploring the key genes for active ingredients biosynthesis in Lycium barbarum L. and analyzing their expression regulation mechanism.

Identification and expression analysis of MADS-box gene family in Docynia delavayi (Franch.) Schneid / 生物工程学报

MADS-box gene family is a significant transcription factor family that plays a crucial role in regulating plant growth, development, signal transduction, and other processes. In order to study the characteristics of MADS-box gene family in Docynia delavayi (Franch.) Schneid. and its expression during different stages of seed germination, this study used seedlings at different stages of germination as materials and screened MADS-box transcription factors from the transcriptome database of D. delavayi using bioinformatics methods based on transcriptome sequencing. The physical and chemical properties, protein conservative motifs, phylogenetic evolution, and expression patterns of the MADS-box transcription factors were analyzed. Quantitative real-time PCR (qRT-PCR) was used to verify the expression of MADS-box gene family members during different stages of seed germination in D. delavayi. The results showed that 81 genes of MADS-box gene family were identified from the transcriptome data of D. delavayi, with the molecular weight distribution ranged of 6 211.34-173 512.77 Da and the theoretical isoelectric point ranged from 5.21 to 10.97. Phylogenetic analysis showed that the 81 genes could be divided into 15 subgroups, among which DdMADS27, DdMADS42, DdMADS45, DdMADS46, DdMADS53, DdMADS61, DdMADS76, DdMADS77 and DdMADS79 might be involved in the regulation of ovule development in D. delavayi. The combination of the transcriptome data and the qRT-PCR analysis results of D. delavayi seeds indicated that DdMADS25 and DdMADS42 might be involved in the regulation of seed development, and that DdMADS37 and DdMADS38 might have negative regulation effects on seed dormancy. Previous studies have reported that the MIKC* subgroup is mainly involved in regulating flower organ development. For the first time, we found that the transcription factors of the MIKC* subgroup exhibited a high expression level at the early stage of seed germination, so we speculated that the MIKC* subgroup played a regulatory role in the process of seed germination. To verify the accuracy of this speculation, we selected DdMADS60 and DdMADS75 from the MIKC* subgroup for qRT-PCR experiments, and the experimental results were consistent with the expression trend of transcriptome sequencing. This study provides a reference for further research on the biological function of D. delavayi MADS-box gene family from the perspective of molecular evolution.

Genome-wide identification of the banana GLR gene family and its expression analysis in response to low temperature and abscisic acid/methyl jasmonate / 生物工程学报

Glutamate receptor-like (GLR) is an important class of Ca2+ channel proteins, playing important roles in plant growth and development as well as in response to biotic and abiotic stresses. In this paper, we performed genome-wide identification of banana GLR gene family based on banana genomic data. Moreover, we analyzed the basic physicochemical properties, gene structure, conserved motifs, promoter cis-acting elements, evolutionary relationships, and used real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) to verify the expression patterns of some GLR family members under low temperature of 4 ℃ and different hormone treatments. The results showed that there were 19 MaGLR family members in Musa acuminata, 16 MbGLR family members in Musa balbisiana and 14 MiGLR family members in Musa itinerans. Most of the members were stable proteins and had signal peptides, all of them had 3-6 transmembrane structures. Prediction of subcellular localization indicated that all of them were localized on the plasma membrane and irregularly distributed on the chromosome. Phylogenetic analysis revealed that banana GLRs could be divided into 3 subclades. The results of promoter cis-acting elements and transcription factor binding site prediction showed that there were multiple hormone- and stress-related response elements and 18 TFBS in banana GLR. RT-qPCR analysis showed that MaGLR1.1 and MaGLR3.5 responded positively to low temperature stress and were significantly expressed in abscisic acid/methyl jasmonate treatments. In conclusion, the results of this study suggest that GLR, a highly conserved family of ion channels, may play an important role in the growth and development process and stress resistance of banana.

LPS-induced endothelial cytoskeleton remodeling in human lung vessels and related miRNAs-profiling / 细胞与分子免疫学杂志

Objective To investigate the effects of lipopolysaccharide (LPS) on human pulmonary vascular endothelial cells (HPVECs) cytoskeleton and perform biological analysis of the microRNA (miRNA) spectrum. Methods The morphology of HPVECs was observed by microscope, the cytoskeleton by FITC-phalloidin staining, and the expression of VE-cadherin was detected by immunofluorescence cytochemical staining; the tube formation assay was conducted to examine the angiogenesis, along with cell migration test to detect the migration, and JC-1 mitochondrial membrane potential to detect the apoptosis. Illumina small-RNA sequencing was used to identify differentially expressed miRNAs in NC and LPS group. The target genes of differentially expressed miRNAs were predicted by miRanda and TargetScan, and the functional and pathway enrichment analysis was performed on Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). Further biological analysis of related miRNAs was carried out. Results After the LPS got induced, the cells became round and the integrity of cytoskeleton was destroyed. The decreased expression of VE-cadherin was also observed, along with the decreased ability of angiogenesis and migration, and increased apoptosis. Sequencing results showed a total of 229 differential miRNAs, of which 84 miRNA were up-regulated and 145 miRNA were down-regulated. The target gene prediction and functional enrichment analysis of these differential miRNA showed that they were mainly concentrated in pathways related to cell connection and cytoskeleton regulation, cell adhesion process and inflammation. Conclusion In vitro model of lung injury, multiple miRNAs are involved in the process of HPVECs cytoskeleton remodeling, the reduction of barrier function, angiogenesis, migration and apoptosis.

Identification of immune-related prognostic signature for colon adenocarcinoma based on weighted gene co-expression network analysis / 细胞与分子免疫学杂志

Objective To identify immune-related molecular markers in an attempt to predict prognosis of colon adenocarcinoma (COAD). Methods Immune related genes (IREGs) was analyzed based on the TCGA database. Weighted gene co-expression network analysis (WGCNA) and Cox regression analysis were used to establish risk models. According to the median risk score, COAD patients were divided into high risk and low risk groups. The prognostic difference were compared between the two groups. The function of the model was validated using GEO. Results A total of 1015 IREGs was obtained. The established model consisted of three genes: RAR related orphan receptor C (RORC), leucine-rich repeat Fli-I-interacting protein 2 (LRRFIP2) and lectin galactoside-binding soluble galectin 4 (LGALS4). The high-risk group had significantly poorer prognosis than low-risk group in the GEO database, and it was validated using a GEO database. Further analysis via univariate and multivariate Cox regression analyses revealed that risk model could function as independent prognostic factor for COAD patients. Conclusion The risk model based on IREGs can predict the prognosis of patients with COAD.

Screening of housekeeping genes in Gelsemium elegans and expression patterns of genes involved in its alkaloid biosynthesis / 生物工程学报

Gelsemium elegans is a traditional Chinese herb of medicinal importance, with indole terpene alkaloids as its main active components. To study the expression of the most suitable housekeeping reference genes in G. elegans, the root bark, stem segments, leaves and inflorescences of four different parts of G. elegans were used as materials in this study. The expression stability of 10 candidate housekeeping reference genes (18S, GAPDH, Actin, TUA, TUB, SAND, EF-1α, UBC, UBQ, and cdc25) was assessed through real-time fluorescence quantitative PCR, GeNorm, NormFinder, BestKeeper, ΔCT, and RefFinder. The results showed that EF-1α was stably expressed in all four parts of G. elegans and was the most suitable housekeeping gene. Based on the coexpression pattern of genome, full-length transcriptome and metabolome, the key candidate targets of 18 related genes (AS, AnPRT, PRAI, IGPS, TSA, TSB, TDC, GES, G8H, 8-HGO, IS, 7-DLS, 7-DLGT, 7-DLH, LAMT, SLS, STR, and SGD) involved in the Gelsemium alkaloid biosynthesis were obtained. The expression of 18 related enzyme genes were analyzed by qRT-PCR using the housekeeping gene EF-1α as a reference. The results showed that these genes' expression and gelsenicine content trends were correlated and were likely to be involved in the biosynthesis of the Gelsemium alkaloid, gelsenicine.

EHHADH is a key gene in fatty acid metabolism pathways in hepatocellular carcinoma: a transcriptomic analysis / 南方医科大学学报

OBJECTIVE@#To explore the driving gene of hepatocellular carcinoma (HCC) occurrence and progression and its potential as new therapeutic target of HCC.@*METHODS@#The transcriptome and genomic data of 858 HCC tissues and 493 adjacent tissues were obtained from TCGA, GEO, and ICGC databases. Gene Set Enrichment Analysis (GSEA) identified EHHADH (encoding enoyl-CoA hydratase/L-3-hydroxyacyl-CoA dehydrogenase) as the hub gene in the significantly enriched differential pathways in HCC. The downregulation of EHHADH expression at the transcriptome level was found to correlate with TP53 mutation based on analysis of the TCGA- HCC dataset, and the mechanism by which TP53 mutation caused EHHADH downregulation was explored through correlation analysis. Analysis of the data from the Metascape database suggested that EHHADH was strongly correlated with the ferroptosis signaling pathway in HCC progression, and to verify this result, immunohistochemical staining was used to examine EHHADH expression in 30 HCC tissues and paired adjacent tissues.@*RESULTS@#All the 3 HCC datasets showed signficnatly lowered EHHADH expression in HCC tissues as compared with the adjacent tissues (P < 0.05) with a close correlation with the degree of hepatocyte de-differentiation (P < 0.01). The somatic landscape of HCC cohort in TCGA dataset showed that HCC patients had the highest genomic TP53 mutation rate. The transcriptomic level of PPARGC1A, the upstream gene of EHHADH, was significantly downregulated in HCC patients with TP53 mutation as compared with those without the mutation (P < 0.05), and was significantly correlated with EHHADH expression level. GO and KEGG enrichment analyses showed that EHHADH expression was significantly correlated with abnormal fatty acid metabolism in HCC. The immunohistochemical results showd that the expression level of EHHADH in HCC tissues was down-regulated, and its expression level was related to the degree of hepatocytes de-differentiation and the process of ferroptosis.@*CONCLUSION@#TP53 mutations may induce abnormal expression of PPARGC1A to cause downregulation of EHHADH expression in HCC. The low expression of EHHADH is closely associated with aggravation of de-differentiation and ferroptosis escape in HCC tissues, suggesting the potential of EHHADH as a therapeutic target for HCC.