Identifying phenotype-associated subpopulations through LP_SGL.

Single-cell RNA sequencing (scRNA-seq) enables the resolution of cellular heterogeneity in diseases and facilitates the identification of novel cell types and subtypes. However, the grouping effects caused by cell-cell interactions are often overlooked in the development of tools for identifying subpopulations. We proposed LP_SGL which incorporates cell group structure to identify phenotype-associated subpopulations by integrating scRNA-seq, bulk expression and bulk phenotype data. Cell groups from scRNA-seq data were obtained by the Leiden algorithm, which facilitates the identification of subpopulations and improves model robustness. LP_SGL identified a higher percentage of cancer cells, T cells and tumor-associated cells than Scissor and scAB on lung adenocarcinoma diagnosis, melanoma drug response and liver cancer survival datasets, respectively. Biological analysis on three original datasets and four independent external validation sets demonstrated that the signaling genes of this cell subset can predict cancer, immunotherapy and survival.

A scalable unsupervised learning of scRNAseq data detects rare cells through integration of structure-preserving embedding, clustering and outlier detection.

Single-cell RNA-seq analysis has become a powerful tool to analyse the transcriptomes of individual cells. In turn, it has fostered the possibility of screening thousands of single cells in parallel. Thus, contrary to the traditional bulk measurements that only paint a macroscopic picture, gene measurements at the cell level aid researchers in studying different tissues and organs at various stages. However, accurate clustering methods for such high-dimensional data remain exiguous and a persistent challenge in this domain. Of late, several methods and techniques have been promulgated to address this issue. In this article, we propose a novel framework for clustering large-scale single-cell data and subsequently identifying the rare-cell sub-populations. To handle such sparse, high-dimensional data, we leverage PaCMAP (Pairwise Controlled Manifold Approximation), a feature extraction algorithm that preserves both the local and the global structures of the data and Gaussian Mixture Model to cluster single-cell data. Subsequently, we exploit Edited Nearest Neighbours sampling and Isolation Forest/One-class Support Vector Machine to identify rare-cell sub-populations. The performance of the proposed method is validated using the publicly available datasets with varying degrees of cell types and rare-cell sub-populations. On several benchmark datasets, the proposed method outperforms the existing state-of-the-art methods. The proposed method successfully identifies cell types that constitute populations ranging from 0.1 to 8% with F1-scores of 0.91 0.09. The source code is available at https://github.com/scrab017/RarPG.

Molecular mechanism of radiation tolerance in lung adenocarcinoma cells using single-cell RNA sequencing.

The efficacy of radiotherapy, a cornerstone in the treatment of lung adenocarcinoma (LUAD), is profoundly undermined by radiotolerance. This resistance not only poses a significant clinical challenge but also compromises patient survival rates. Therefore, it is important to explore this mechanism for the treatment of LUAD. Multiple public databases were used for single-cell RNA sequencing (scRNA-seq) data. We filtered, normalized and downscaled scRNA-seq data based on the Seurat package to obtain different cell subpopulations. Subsequently, the ssGSEA algorithm was used to assess the enrichment scores of the different cell subpopulations, and thus screen the cell subpopulations that are most relevant to radiotherapy tolerance based on the Pearson method. Finally, pseudotime analysis was performed, and a preliminary exploration of gene mutations in different cell subpopulations was performed. We identified HIST1H1D+ A549 and PIF1+ A549 as the cell subpopulations related to radiotolerance. The expression levels of cell cycle-related genes and pathway enrichment scores of these two cell subpopulations increased gradually with the extension of radiation treatment time. Finally, we found that the proportion of TP53 mutations in patients who had received radiotherapy was significantly higher than that in patients who had not received radiotherapy. We identified two cellular subpopulations associated with radiotherapy tolerance, which may shed light on the molecular mechanisms of radiotherapy tolerance in LUAD and provide new clinical perspectives.

Identification of different functions of CD8+ T cell subpopulations by a novel monoclonal antibody.

The explicit identification of CD8+ T cell subpopulation is important for deciphering the role of CD8+ T cells for protecting our body against invading pathogens and cancer. Our generated monoclonal antibody (mAb), named FE-1H10, recognized two novel subpopulations of peripheral blood CD8+ T cells, FE-1H10+ and FE-1H10- CD8+ T cells. The molecule recognized by mAb FE-1H10 (FE-1H10 molecules) had a higher distribution on effector memory CD8+ T cell subsets. The functions of FE-1H10- and FE-1H10+ CD8+ T cells were investigated. T cell proliferation assays revealed that FE-1H10- CD8+ T cells exhibited a higher proliferation rate than FE-1H10+ CD8+ T cells, whereas FE-1H10+ CD8+ T cells produced higher levels of IFN-γ and TNF-α than FE-1H10- CD8+ T cells. In T cell cytotoxicity assays, FE-1H10+ CD8+ T cells were able to kill target cells better than FE-1H10- CD8+ T cells. RNA-sequencing analysis confirmed that these subpopulations were distinct: FE-1H10+ CD8+ T cells have higher expression of genes involved in effector functions (IFNG, TNF, GZMB, PRF1, GNLY, FASL, CX3CR1) while FE-1H10- CD8+ T cells have greater expression of genes related to memory CD8+ T cell populations (CCR7, SELL, TCF7, CD40LG). The results suggested that mAb FE-1H10 identifies two novel distinctive CD8+ T cell subpopulations. The FE-1H10+ CD8+ T cells carried a superior functionality in response to tumour cells. The uncover of these novel CD8+ T cell subpopulations may be the basis knowledge of an optional immunotherapy for the selection of potential CD8+ T cells in cancer treatment.

Single-cell RNA sequencing combined with single-cell proteomics identifies the metabolic adaptation of islet cell subpopulations to high-fat diet in mice.

AIMS/HYPOTHESIS: Islets have complex heterogeneity and subpopulations. Cell surface markers representing alpha, beta and delta cell subpopulations are urgently needed for investigations to explore the compositional changes of each subpopulation in obesity progress and diabetes onset, and the adaptation mechanism of islet metabolism induced by a high-fat diet (HFD). METHODS: Single-cell RNA sequencing (scRNA-seq) was applied to identify alpha, beta and delta cell subpopulation markers in an HFD-induced mouse model of glucose intolerance. Flow cytometry and immunostaining were used to sort and assess the proportion of each subpopulation. Single-cell proteomics was performed on sorted cells, and the functional status of each alpha, beta and delta cell subpopulation in glucose intolerance was deeply elucidated based on protein expression. RESULTS: A total of 33,999 cells were analysed by scRNA-seq and clustered into eight populations, including alpha, beta and delta cells. For alpha cells, scRNA-seq revealed that the Ace2low subpopulation had downregulated expression of genes related to alpha cell function and upregulated expression of genes associated with beta cell characteristics in comparison with the Ace2high subpopulation. The impaired function and increased fragility of ACE2low alpha cells exposure to HFD was further suggested by single-cell proteomics. As for beta cells, the CD81high subpopulation may indicate an immature signature of beta cells compared with the CD81low subpopulation, which had robust function. We also found differential expression of Slc2a2 in delta cells and a potentially stronger cellular function and metabolism in GLUT2low delta cells than GLUT2high delta cells. Moreover, an increased proportion of ACE2low alpha cells and CD81low beta cells, with a constant proportion of GLUT2low delta cells, were observed in HFD-induced glucose intolerance. CONCLUSIONS/INTERPRETATION: We identified ACE2, CD81 and GLUT2 as surface markers to distinguish, respectively, alpha, beta and delta cell subpopulations with heterogeneous maturation and function. The changes in the proportion and functional status of islet endocrine subpopulations reflect the metabolic adaptation of islets to high-fat stress, which weakened the function of alpha cells and enhanced the function of beta and delta cells to bring about glycaemic homeostasis. Our findings provide a fundamental resource for exploring the mechanisms maintaining each islet endocrine subpopulation's fate and function in health and disease. DATA AVAILABILITY: The scRNA-seq analysis datasets from the current study are available in the Gene Expression Omnibus (GEO) repository under the accession number GSE203376.

EGFR marks a subpopulation of dermal mesenchymal cells highly expressing IGF1 which enhances hair follicle regeneration.

The skin harbours transcriptionally and functionally heterogeneous mesenchymal cells that participate in various physiological activities by secreting biochemical cues. In this study, we aimed to identify a new subpopulation of dermal mesenchymal cells that enhance hair follicle regeneration through a paracrine mechanism. Integrated single-cell RNA sequencing (scRNA-seq) data analysis revealed epidermal growth factor receptor (EGFR) as a marker of distinct fibroblast subpopulation in the neonatal murine dermis. Immunofluorescence staining and fluorescence-activated cell sorting (FACS) were used to validate the existence of the cell population in Krt14-rtTA-H2BGFP mouse. The difference of gene expression between separated cell subpopulation was examined by real-time PCR. Potential effect of the designated factor on hair follicle regeneration was observed after the application on excisional wounds in Krt14-rtTA-H2BGFP mouse. Immunofluorescence staining demonstrated the existence of dermal EGFR+ cells in neonatal and adult mouse dermis. The EGFR+ mesenchymal population, sorted by FACS, displayed a higher expression level of Igf1 (insulin-like growth factor 1). Co-localisation of IGF1 with EGFR in the mouse dermis and upregulated numbers of hair follicles in healed wounds following the application of exogenous IGF1 illustrated the contribution of EGFR+ cells in promoting wound-induced hair follicle neogenesis. Our results indicate that EGFR identifies a subpopulation of dermal fibroblasts that contribute to IGF1 promotion of hair follicle neogenesis. It broadens the understanding of heterogeneity and the mesenchymal cell function in skin and may facilitate the potential translational application of these cells.

REBET: a method to determine the number of cell clusters based on batch effect removal.

In single-cell RNA-seq (scRNA-seq) data analysis, a fundamental problem is to determine the number of cell clusters based on the gene expression profiles. However, the performance of current methods is still far from satisfactory, presumably due to their limitations in capturing the expression variability among cell clusters. Batch effects represent the undesired variability between data measured in different batches. When data are obtained from different labs or protocols batch effects occur. Motivated by the practice of batch effect removal, we considered cell clusters as batches. We hypothesized that the number of cell clusters (i.e. batches) could be correctly determined if the variances among clusters (i.e. batch effects) were removed. We developed a new method, namely, removal of batch effect and testing (REBET), for determining the number of cell clusters. In this method, cells are first partitioned into k clusters. Second, the batch effects among these k clusters are then removed. Third, the quality of batch effect removal is evaluated with the average range of normalized mutual information (ARNMI), which measures how uniformly the cells with batch-effects-removal are mixed. By testing a range of k values, the k value that corresponds to the lowest ARNMI is determined to be the optimal number of clusters. We compared REBET with state-of-the-art methods on 32 simulated datasets and 14 published scRNA-seq datasets. The results show that REBET can accurately and robustly estimate the number of cell clusters and outperform existing methods. Contact: H.D.L. (hongdong@csu.edu.cn) or Q.S.X. (qsxu@csu.edu.cn).

Radiogenomic analysis of cellular tumor-stroma heterogeneity as a prognostic predictor in breast cancer.

BACKGROUND: The tumor microenvironment and intercellular communication between solid tumors and the surrounding stroma play crucial roles in cancer initiation, progression, and prognosis. Radiomics provides clinically relevant information from radiological images; however, its biological implications in uncovering tumor pathophysiology driven by cellular heterogeneity between the tumor and stroma are largely unknown. We aimed to identify radiogenomic signatures of cellular tumor-stroma heterogeneity (TSH) to improve breast cancer management and prognosis analysis. METHODS: This retrospective multicohort study included five datasets. Cell subpopulations were estimated using bulk gene expression data, and the relative difference in cell subpopulations between the tumor and stroma was used as a biomarker to categorize patients into good- and poor-survival groups. A radiogenomic signature-based model utilizing dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) was developed to target TSH, and its clinical significance in relation to survival outcomes was independently validated. RESULTS: The final cohorts of 1330 women were included for cellular TSH biomarker identification (n = 112, mean age, 57.3 years ± 14.6) and validation (n = 886, mean age, 58.9 years ± 13.1), radiogenomic signature of TSH identification (n = 91, mean age, 55.5 years ± 11.4), and prognostic (n = 241) assessments. The cytotoxic lymphocyte biomarker differentiated patients into good- and poor-survival groups (p < 0.0001) and was independently validated (p = 0.014). The good survival group exhibited denser cell interconnections. The radiogenomic signature of TSH was identified and showed a positive association with overall survival (p = 0.038) and recurrence-free survival (p = 3 × 10-4). CONCLUSION: Radiogenomic signatures provide insights into prognostic factors that reflect the imbalanced tumor-stroma environment, thereby presenting breast cancer-specific biological implications and prognostic significance.

The third inactivated vaccine booster dramatically enhanced SARS-CoV-2 antibody responses and did not influence the profile of prothrombotic antibody.

The purpose of this study is to investigate the production of both severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2)-specific antibodies and autoantibodies in serum following the third booster vaccination of the inactivated COVID-19 vaccine, and to study the effect of B cell subsets with CD27 and CD38 phenotypes in peripheral blood on antibody production. Routine blood indexes, SARS-CoV-2 antibodies, platelet factor 4 and seven antiphospholipid antibodies were detected both before and 2 months after vaccination in the medical staff of the Zhongnan Hospital of Wuhan University. Peripheral blood B cell subtypes were detected before vaccination. Following immunization, the positive rate of anti-N-S1 immunoglobulin (IgG) had increased from 24.8% to 91.3% and the average antibody concentration had increased by 11 times. The positive rate of neutralizing antibody had increased from 24.8% to 91.3%, the average antibody concentration had increased by 12 times, and the primary increased anti-S1 IgG subtype was that of IgG1. Peripheral blood CD27 + CD38+ B cells were positively correlated with antibody levels after vaccination and were a predictor of the antibody response. In addition, although some indicators showed slight absolute changes, the blood parameters and antiphospholipid antibodies of most volunteers were normal both before and after COVID-19 inactivated vaccine inoculation, and there was no statistical difference in abnormal rates either before or after inoculation. Antibodies in vivo were increased after vaccination with the inactivated vaccine, and IgG1 was the main subtype involved in response to the vaccine. Vaccination with the inactivated COVID-19 vaccine did not appear to affect thrombus-related autoantibodies.

A multicentric and nationwide predictive study role of T cell sub-population in the prevalence and prognosis of cryoglobulinemia among genotype 4 chronic hepatitis C patients.

The infection caused by the hepatitis C virus (HCV) is a significant global health concern. The prevailing genotype of HCV in Egypt is 4a, commonly referred to as GT-4a. A significant proportion exceeding 50% of patients infected with HCV experience extrahepatic manifestations (EHMs), encompassing a diverse range of clinical presentations. These manifestations, including essential mixed cryoglobulinemia (MC), can serve as initial and solitary indicators of the disease. The complete understanding of the pathogenesis of EHM remains unclear, with autoimmune phenomena being recognized as the primary causative factor. In this study, we examined the predictive significance of T-cell subpopulations in relation to the occurrence and prognosis of cryoglobulinemia in HCV patients. A total of 450 CHC genotype four treatment naïve patients were enrolled in this analytic cross-sectional study after thorough clinical, laboratory, and radiological examinations. All patients underwent laboratory investigations, including testing for cryoglobulin antibodies and measurements of CD4 and CD8 levels; two groups were described according to their test results: Group 1 consists of patients who have tested positive for cryoglobulin antibodies and Group 2 consists of patients who have tested negative for cryoglobulin antibodies. The exclusion criteria encompassed individuals with HIV infection or chronic HBV infection. Additionally, pelvi-abdominal ultrasonography was performed. Our study included 450 treatment naïve CHC patients (59% male, mean age 50.8 years). The patients were categorized according to their cryoglobulin antibodys test results into two groups: group A, CHC patients with cryoglobulin antibodies (Abs) negative (364 patients), and group B, CHC patients with cryoglobulin Ab positive (86 patients). Group B demonstrated a higher average age, elevated international normalized ratio, more prolonged duration of HCV infection, lower albumin, higher alanine aminotransferase, higher aspartate aminotransferase, higher bilirubin, lower CD8, lower CD4, and lower CD4:CD8 ratio. In contrast, 27 out of 86 (31.40%) patients in group B had symptoms; 85.8% had purpura and arthralgia, 74.3% had paresthesias, 86.7% had weakness, and 12.2% had non-Hodgkin's lymphoma. The levels of CD4 and CD8 were found to be decreased in chronic HCV patients with MC. T-cell subpopulation serves as a reliable indicator for assessing the prevalence and prognosis of MC in individuals with genotype 4 chronic hepatitis C. However, additional research is needed to further understand the development and spread of various emerging infectious diseases. Nevertheless, it is noteworthy that a critical threshold may exist beyond which EHM reaches a point of no return.

Balance between Pro- and Antifibrotic Proteins in Mesenchymal Stromal Cell Secretome Fractions Revealed by Proteome and Cell Subpopulation Analysis.

Multipotent mesenchymal stromal cells (MSCs) regulate tissue repair through paracrine activity, with secreted proteins being significant contributors. Human tissue repair commonly results in fibrosis, where fibroblast differentiation into myofibroblasts is a major cellular mechanism. MSCs' paracrine activity can inhibit fibrosis development. We previously demonstrated that the separation of MSC secretome, represented by conditioned medium (CM), into subfractions enriched with extracellular vesicles (EV) or soluble factors (SF) boosts EV and SF antifibrotic effect. This effect is realized through the inhibition of fibroblast-to-myofibroblast differentiation in vitro. To unravel the mechanisms of MSC paracrine effects on fibroblast differentiation, we performed a comparative proteomic analysis of MSC secretome fractions. We found that CM was enriched in NF-κB activators and confirmed via qPCR that CM, but not EV or SF, upregulated NF-κB target genes (COX2, IL6, etc.) in human dermal fibroblasts. Furthermore, we revealed that EV and SF were enriched in TGF-ß, Notch, IGF, and Wnt pathway regulators. According to scRNAseq, 11 out of 13 corresponding genes were upregulated in a minor MSC subpopulation disappearing in profibrotic conditions. Thus, protein enrichment of MSC secretome fractions and cellular subpopulation patterns shift the balance in fibroblast-to-myofibroblast differentiation, which should be considered in studies of MSC paracrine effects and the therapeutic use of MSC secretome.

I-Impute: a self-consistent method to impute single cell RNA sequencing data.

BACKGROUND: Single-cell RNA-sequencing (scRNA-seq) is becoming indispensable in the study of cell-specific transcriptomes. However, in scRNA-seq techniques, only a small fraction of the genes are captured due to "dropout" events. These dropout events require intensive treatment when analyzing scRNA-seq data. For example, imputation tools have been proposed to estimate dropout events and de-noise data. The performance of these imputation tools are often evaluated, or fine-tuned, using various clustering criteria based on ground-truth cell subgroup labels. This limits their effectiveness in the cases where we lack cell subgroup knowledge. We consider an alternative strategy which requires the imputation to follow a "self-consistency" principle; that is, the imputation process is to refine its results until there is no internal inconsistency or dropouts from the data. RESULTS: We propose the use of "self-consistency" as a main criteria in performing imputation. To demonstrate this principle we devised I-Impute, a "self-consistent" method, to impute scRNA-seq data. I-Impute optimizes continuous similarities and dropout probabilities, in iterative refinements until a self-consistent imputation is reached. On the in silico data sets, I-Impute exhibited the highest Pearson correlations for different dropout rates consistently compared with the state-of-art methods SAVER and scImpute. Furthermore, we collected three wetlab datasets, mouse bladder cells dataset, embryonic stem cells dataset, and aortic leukocyte cells dataset, to evaluate the tools. I-Impute exhibited feasible cell subpopulation discovery efficacy on all the three datasets. It achieves the highest clustering accuracy compared with SAVER and scImpute. CONCLUSIONS: A strategy based on "self-consistency", captured through our method, I-Impute, gave imputation results better than the state-of-the-art tools. Source code of I-Impute can be accessed at https://github.com/xikanfeng2/I-Impute .

Anti-cyclic citrullinated peptide antibody titers decrease in rheumatoid arthritis patients treated with tocilizumab: A pilot study.

Objectives: To analyze the effects of tocilizumab on peripheral B-cell subpopulation and its ability to produce anti-cyclic citrullinated peptide (CCP) antibody in patients with rheumatoid arthritis (RA).Methods: Thirteen consecutive RA patients initiated with tocilizumab were enrolled in our prospective study. Anti-CCP antibody titers and clinical parameters were evaluated during treatment. Peripheral blood B-cell subsets were analyzed using flow cytometry according to the Human Immunology Project.Results: Disease activity was significantly improved and anti-CCP antibody titers significantly decreased at week 24 compared to baseline. The percentages of post-switch memory B cells in CD19+ cells transiently increased at week 12, but there was no significant difference in any of the investigated B-cell subpopulations at week 24 compared to baseline. The ratios of post-switch memory to naïve B cells (post-switch/naïve) correlated negatively with anti-CCP antibody titers regardless of the time-points.Conclusion: Our study indicated that tocilizumab has a potential to reduce anti-CCP antibody production presumably by affecting post-switch/naïve ratio, and that anti-CCP antibody titers reflect B-cell distribution/subpopulation. As anti-CCP antibodies are produced in lymph nodes or ectopic lymphoid structures in synovial tissues, not in circulation, transient increment of post-switch memory B cells after tocilizumab treatment may reflect the altered balance of B-cell distribution between circulation and arthritic joints, resulting in suppressed production of anti-CCP antibody in situ.

RPPAs for Cell Subpopulation Analysis.

Understanding cellular heterogeneity is a challenge for cell biology, particularly in the fields of stem cell and cancer biology. We recently established a reverse phase protein array (RPPA)-modified method, colony lysate array (CoLA), which enables proteomic profiling of individual subpopulations (i.e., colonies) derived from various cell proliferative conditions. Here we describe two independent CoLA assays for a functional subpopulation, drug-tolerant persisters (DTPs), which are considered to mimic the origin of chemotherapeutic cancer relapse. In the first study, we analyzed individual DTPs derived from different cell types grown in the presence of different drugs. A hierarchical clustering analysis of DTPs using CoLA revealed two types of clonal populations, including those that expressed stem cell-associated proteins as well as epithelium-associated proteins. Subsequent principal component analyses demonstrated that the DTPs clustered on the basis of their proteomic profiles, which could change in response to drugs and doses. Another study was designed to identify signaling pathways that may be associated with drug resistance using a pair of 5-fluorouracil-tolerant and parental gastric cancer cell lines. Although a hierarchical clustering analysis did not show any specific clusters for the tolerant line, a drug dose-response analysis revealed that phosphorylation of PI3K and AKT increased and decreased, respectively, specifically in the tolerant cell line. These frameworks that readily profile small subpopulations isolated from an external stresses may be applicable to a wide variety of heterogeneous cellular samples.

[Expression of granulocyte colony-stimulating factor receptor in colitis-associated colonic carcinogenesis].

Objective: To investigate the expression of granulocyte-colony stimulating factor receptor (G-CSFR) in a mouse model of colitis-associated cancer (CAC), and the roles of G-CSFR positive immune cells in the development of CAC. Methods: The C57BL/6 mouse model of CAC was established by azoxymethane and dextran sulphate sodium. Three different stages in the development of CAC, including inflammation (AD1), mild dysplasia (AD2) and adenocarcinoma (AD3) were simulated. Colon tissue was digested into single cell suspension and the expressions of G-CSF and G-CSFR were analyzed by real-time PCR and fluorescence activated cell sorter (FACS). The expressions of G-CSFR on T cell, macrophage and neutrophil were analyzed by FACS. Results: The establishment of mouse model can effectively simulate the disease progression of CAC. The results of real-time PCR detection showed that the expression level of G-CSF mRNA in AD1, AD2 and AD3 groups were 1.2, 7.3 and 18.0-fold changes of the control group, respectively. The differences between AD2, AD3 and control groups were statistically significant (P<0.05). G-CSFR mRNA levels in AD1, AD2 and AD3 groups were 1.5, 2.2 and 4.5-fold changes of the control group, respectively. The difference between AD3 and control groups was statistically significant (P<0.05). FACS showed that the percentages of CD45(+) G-CSFR(+) cells in colorectal tissues of the control group, AD1, AD2 and AD3 groups were (21.84±1.77)%, (41.48±4.15)%, (44.84±8.54)% and (57.76±1.95)%, respectively.The percentages of CD45(+) G-CSFR(+) cells in AD2 and AD3 groups were significantly higher than that of control group (P<0.05). The percentages of CD45(+) G-CSFR(+) macrophage in the colorectal tissues of the control group, AD1, AD2 and AD3 groups were (21.54±5.88)%, (47.14±5.25)%, (42.49±7.80)% and (29.25±8.24)%, respectively. The percentages of CD45(+) G-CSFR(+) T cells in these groups were (30.04±6.87)%, (29.65±8.08)%, (33.75±7.37)% and (33.32±9.85)%, respectively. The percentages of CD45(+) G-CSFR(+) granulocyte were (2.39±2.10)%, (4.05±1.56)%, (3.62±2.67)% and (2.26±0.85)%, respectively (P<0.05). The percentages of G-CSFR(+) macrophage and G-CSFR(+) T cells were significantly higher than that of G-CSFR(+) granulocyte (P<0.05). The differences between AD1 and control group, AD2 and control group, AD1 and AD2 group, AD2 and AD3 group were statistically significant (P<0.05). Conclusions: The expression of G-CSFR is significantly up-regulated in the development of CAC. The enrichment of G-CSFR(+) macrophages in the colon tissue suggests G-CSFR(+) macrophages participate in the development of CAC.

Metabolic differentiation of surface and invasive cells of yeast colony biofilms revealed by gene expression profiling.

BACKGROUND: Yeast infections are often connected with formation of biofilms that are extremely difficult to eradicate. An excellent model system for deciphering multifactorial determinants of yeast biofilm development is the colony biofilm, composed of surface ("aerial") and invasive ("root") cells. While surface cells have been partially analyzed before, we know little about invasive root cells. In particular, information on the metabolic, chemical and morphogenetic properties of invasive versus surface cells is lacking. In this study, we used a new strategy to isolate invasive cells from agar and extracellular matrix, and employed it to perform genome wide expression profiling and biochemical analyses of surface and invasive cells. RESULTS: RNA sequencing revealed expression differences in 1245 genes with high statistical significance, indicating large genetically regulated metabolic differences between surface and invasive cells. Functional annotation analyses implicated genes involved in stress defense, peroxisomal fatty acid ß-oxidation, autophagy, protein degradation, storage compound metabolism and meiosis as being important in surface cells. In contrast, numerous genes with functions in nutrient transport and diverse synthetic metabolic reactions, including genes involved in ribosome biogenesis, biosynthesis and translation, were found to be important in invasive cells. Variation in gene expression correlated significantly with cell-type specific processes such as autophagy and storage compound accumulation as identified by microscopic and biochemical analyses. Expression profiling also provided indications of cell-specific regulations. Subsequent knockout strain analyses identified Gip2p, a regulatory subunit of type 1 protein phosphatase Glc7p, to be essential for glycogen accumulation in surface cells. CONCLUSIONS: This is the first study reporting genome wide differences between surface and invasive cells of yeast colony biofilms. New findings show that surface and invasive cells display very different physiology, adapting to different conditions in different colony areas and contributing to development and survival of the colony biofilm as a whole. Notably, surface and invasive cells of colony biofilms differ significantly from upper and lower cells of smooth colonies adapted to plentiful laboratory conditions.

Dietary L-arginine modulates immunosuppression in broilers inoculated with an intermediate strain of infectious bursa disease virus.

BACKGROUND: The effects of dietary l-arginine (Arg) on immunosuppression following infectious bursal disease virus (IBDV) inoculation in broiler chickens were evaluated. The design of this study was a 5 × 2 factorial arrangement (n = 5) with five Arg concentrations (starter: 9.9, 13.9, 17.6, 21.3 and 25.3 g kg(-1) ; grower-finisher: 9.5, 13.5, 17.1, 20.1 and 23.6 g kg(-1) ) with or without IBDV inoculation (IBDV or saline inoculation at 14 days). Chickens were sampled at 2, 4 and 6 days post-inoculation (DPI) and 42 days of age. RESULTS: The IBDV inoculation decreased (P = 0.05) CD3(+) , CD4(+) , and CD8(+) T cell counts at 2 days post-inoculation (DPI) and monocyte counts at 6 DPI; and reduced (P < 0.05) bursal interleukin-1ß (IL-1ß) mRNA expression at 2 DPI and serum IL-6 concentration at 4 DPI. Increasing Arg concentration increased (P < 0.05) CD4(+) and CD8(+) T cell counts at 2 DPI, linearly increased (P = 0.05) CD3(+) T cell counts in IBDV-inoculated groups and monocyte counts in control groups at 4 DPI; increased (P < 0.05) serum IL-6 concentration in IBDV-inoculated groups at 2 DPI; and increased (P < 0.05) serum anti-IBDV antibody titres at 42 days of age. CONCLUSION: Varying concentrations of Arg supplementation attenuated IBDV inoculation induced immunosuppression via modulating circulating T cell sub-populations.

Allergenicity and Linear Epitope Analysis of Scy p 8, an Allergen from Mud Crab.

Scy p 8 (triosephosphate isomerase) as a crab allergen in inducing distinct T-helper (Th) cell differentiation and a linear epitope associated with allergenicity remain elusive. In this study, mice sensitized with Scy p 8 exhibited significantly upregulated levels of IgE, IgG1, and IL-4 release, inducing a Th2 immune response. Moreover, the release of IFN-γ (Th1) and the levels of Treg cells were downregulated, while IL-17A (Th17) was upregulated, indicating that Scy p 8 disrupted the Th1/Th2 balance and Th17/Treg balance in mice. Furthermore, bioinformatics prediction and serum samples from crab-allergic patients and mice enabled the discovery of 8 linear epitopes of Scy p 8. Meanwhile, the analysis of peptide similarity and tertiary superposition revealed that 8 epitopes of Scy p 8 exhibited conservation across various species, potentially resulting in cross-reactivity. These findings possess the potential to enhance the comprehension of crab allergens, thereby establishing a foundation for investigating cross-reactivity.

Peripheral T Cell Subpopulations as a Potential Surrogate Biomarker during Atezolizumab plus Bevacizumab Treatment for Hepatocellular Carcinoma.

The therapeutic benefits of the immunotherapeutic combination of atezolizumab and bevacizumab (Atez/Bev) in hepatocellular carcinoma (HCC) vary. Therapeutic biomarkers might help improve outcomes for HCC patients receiving Atez/Bev therapy. The role of systemic immune profiles in HCC progression also remains unclear. This study aimed to evaluate the status and dynamics of peripheral T cell subpopulations in HCC patients receiving Atez/Bev treatment and to explore biomarkers predictive of a therapeutic response. We enrolled 83 unresectable advanced HCC patients who commenced Atez/Bev treatment at our hospital between October 2020 and June 2022. Peripheral T cell subpopulations in peripheral blood mononuclear cells at baseline and 3 weeks post-treatment were investigated using flow cytometry and compared with those in control samples from 18 healthy individuals. We retrospectively analyzed the association between peripheral T cell subpopulation profiles and clinical outcomes. Baseline peripheral T cell subpopulations could be profiled in 70 patients with sufficient cell counts, among whom 3-week subpopulations could be evaluated in 51 patients. Multivariate analysis showed that a high baseline proportion of CD8+ central memory T (TCM) cells was independently associated with longer progression-free survival (PFS). Further, overall survival (OS) was significantly prolonged in patients with increased CD8+ effector memory T (TEM) cell proportions. In conclusion, TCM proportion at baseline might be a good indicator of the efficacy of Atez/Bev therapy. Furthermore, observation of increasing TEM proportions might be an early predictor of the potential clinical benefits of treatment.

Recent Achievements in the Heterogeneity of Mammalian and Human Retinal Pigment Epithelium: In Search of a Stem Cell.

Retinal pigment epithelium (RPE) cells are important fundamentally for the development and function of the retina. In this regard, the study of the morphological and molecular properties of RPE cells, as well as their regenerative capabilities, is of particular importance for biomedicine. However, these studies are complicated by the fact that, despite the external morphological similarity of RPE cells, the RPE is a population of heterogeneous cells, the molecular genetic properties of which have begun to be revealed by sequencing methods only in recent years. This review carries out an analysis of the data from morphological and molecular genetic studies of the heterogeneity of RPE cells in mammals and humans, which reveals the individual differences in the subpopulations of RPE cells and the possible specificity of their functions. Particular attention is paid to discussing the properties of "stemness," proliferation, and plasticity in the RPE, which may be useful for uncovering the mechanisms of retinal diseases associated with pathologies of the RPE and finding new ways of treating them.