Markers For Subpopulations Research Articles

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

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). 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. 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. 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. The scRNA-seq analysis datasets from the current study are available in the Gene Expression Omnibus (GEO) repository under the accession number GSE203376.

Expression of M2-associated molecules in circulating monocyte subsets in fertile non-pregnant women and pregnant women with uncomplicated pregnancy

In humans circulating monocytes include classical (CD14++CD16- ), intermediate (CD14++CD16+) and non-classical/alternative (CD14+CD16++) monocytes, which in turn can be activated via the classical or alternative pathway. Pregnancy is accompanied by significant changes in the monocyte compartment, which is manifested by an increase in the number of circulating monocytes, including the proportion of intermediate monocytes, and a change in their function. However, the functional properties of monocyte subsets during gestation remain largely unexplored. We hypothesized that circulating monocytes may be activated in an alternative pattern and acquire features of M2 polarization (anti-inflammatory / immunosuppressive properties). The aim of the investigation was to study M2-associated markers that characterize the anti-inflammatory and immunosuppressive potential of myeloid cells in subpopulations of circulating monocytes in fertile nonpregnant women and women with uncomplicated pregnancy in the 2nd trimester. It was shown that in fertile non-pregnant women intermediate and non-classical monocytes are characterized by a higher expression of M2-associated markers (CD206, Arginase 1, MerTK) compared to classical monocytes. In the 2nd trimester of pregnancy, the expression of these molecules on monocytes increases significantly, which is manifested by 1) an increase in the proportion of CD206+ cells in subpopulations of classical and intermediate monocytes, 2) an increase in the mean fluorescence intensity of Arginase 1 in all monocyte subsets, 3) an increase in the proportion of MerTK+ cells in subpopulations of classical and intermediate monocytes and mean fluorescence intensity across all monocyte subsets. The highest content of CD206+ and MerTK+ cells in pregnant women is detected in the subpopulation of intermediate monocytes, and the highest values of the mean fluorescence intensity of Arginase 1 and MerTK – in the subpopulations of intermediate and non-classical monocytes. The data obtained demonstrate that monocytes of pregnant women in the 2nd trimester of pregnancy are characterized by signs of M2 polarization. This is confirmed not only by an increase in the expression of the M2-associated mannose receptor CD206, but also by an increase in the expression of Arginase 1 and MerTK, which mediate the immunosuppressive activity of myeloid cells and, in particular, macrophages of the M2 phenotype. Further studies of M2-associated markers in monocyte subpopulations during gestation will allow a more detailed characterization of the regulatory role of circulating myeloid cells during pregnancy.

Patients with Sickle Cell Disease Treated with Hidroxiurea Have Higher Expression of PD-L1 in Monocytes

Background: Inflammation plays a key role in pathophysiology of sickle cell disease (SCD). Monocytes are important mediator cells in the inflammation pathway. Despite some evidence that monocytes are more activated in SCD and can trigger vaso-occlusion, there is a remarkable lack of knowledge about how monocytes behave in patients with SCD and whether they are modulated by different treatments. Objective: To assess possible changes in monocyte maturation and protein expression among sickle cell disease patients in different treatment modalities and compare them to healthy subjects. Methods: 25 patients with SCD followed at the outpatient service of the Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo and 3 healthy controls were included. The inclusion criteria were: patients with sickle cell anemia (HbSS), over 18 years of age, without acute complications in the prior three months. Pregnancy was an exclusion criteria. Patients were divided in three groups: treatment with Hydroxyurea (HU) (n=10) using the same dose for at least three months; chronic exchange transfusion (n=9) (TP); and no specific treatment (NST) (n=6). We collected 3-5 ml of blood and performed peripheral blood mononuclear cells (PBMC) separation. Monocytes were analyzed by flow cytometry (Dx Flex, Beckman Coulter), using CD14 and CD16 as markers of subpopulations of monocytes as classical (CD14+/CD16-), intermediate (CD14+/CD16+) and non-classical (CD14-/CD16+). We also assessed the expression of CD 86 (a co-stimulation chemokine receptor), CD 163 (receptor for the conjugate hemoglobin-haptoglobin) and PD-L1 (an inhibitor receptor). Comparison between groups were made using ANOVA and Tukey test. Results: Gender distribution yielded no significant difference. Age varied as shown: HU 30 ± 8y, TP 37 ± 9y, NST 35 ± 11y and 42 ± 19y for the controls. The mean of total leukocytes and monocytes were, respectively: 8,816 ± 2,133/mm³ and 900 ± 563/mm³ (HU), 11,487 ± 3,813/mm³ and 1,343 ± 754/mm³ (TP), 9,081 ± 1879/mm³ and 1,373 ± 432/mm³ (NST), 7,080 ± 642/mm³ and 700 ± 0/mm³ (control).Among the total nucleated cells, the percentage of classical monocytes was lower in HU compared with. Controls (3.8 ± 2.1% vs. 7.7 ± 1.3%, p 0.008), lower in TP vs NST (2.4 ± 1.8% vs. 5.6 ± 2.8%, p 0.04) and in TP vs Controls (2.4 ± 1.8% vs. 7.7 ± 1.3%, p 0.001). No differences were shown in the intermediate monocytes population. In turn, nonclassical monocytes were higher in HU compared with TP (0.44 ± 0.3% vs. 0.22 ± 0.2%, p 0.013) and in controls vs TP ( 0.35 ± 0.07% vs 0.22 ± 0.2%, p 0.017). The expression of CD 86 and CD 163 was not significantly different between any of the subsets of monocytes. In classical monocytes, expression of PD-L1 was higher in HU compared with NST (86.1 ± 17.4% vs. 43.9 ± 31.0%, p 0.015 ) and HU vs Controls ( 86.1 ± 17.4% vs. 34.9 ± 8.9%, p 0.019). Expression of PD-L1 in intermediate monocytes and in non classical monocytes were also higher in HU compared with controls (88.9 ± 16.3% vs. 58.6 ± 8.6%, p 0.02 and 79.6 ± 30.2% vs. 37.1 ± 4.6% , p 0.02 respectively). These results are depicted in table and figure 1. Conclusion: Different treatments for sickle cell disease have affected the monocyte subsets distribution and PD-L1 expression in the population studied. HU and Transfusion Program induced a reduction in the Classical Monocyte subset, more pronounced in the latter. Classical monocytes are largely associated with a pro-inflammatory profile. The use of HU in SCD has been previously associated with less classical monocytes, and induction of monocyte maturation might help explain this finding; as for chronic transfusions, a migratory effect of monocytes to tissues has been proposed. The higher expression of PD-L1 in patients submitted to exchange transfusion and in use of HU indicate inhibition of inflammation, which is more pronounced in the HU group. Both treatments have immunomodulatory effects on SCD, however, to date, no inflammatory markers that assess response to treatment are available. Our findings might contribute to uncovering the role of PD-L1 in the chronic inflammation of SCD and to identify a potential biomarker. Nevertheless, further studies with larger cohorts are warranted in these settings. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

A multifaceted and feasible prognostic model of amino acid metabolism-related genes in the immune response and tumor microenvironment of head and neck squamous cell carcinomas.

We investigated the role of amino acid metabolism (AAM) in head and neck squamous cell carcinoma (HNSCC) tissues to explore its prognostic value and potential therapeutic strategies. A risk score based on four AAM-related genes (AMG) was constructed that could predict the prognosis of HNSCC. These four genes were up-regulated in HNSCC tissues and might act as oncogenes. Internal validation in The Cancer Genome Atlas (TCGA) by bootstrapping showed that patients with high-risk scores had a poorer prognosis than patients with low-risk scores, and this was confirmed in the Gene Expression Omnibus (GEO) cohort. There were also differences between the high-risk and low-risk groups in clinical information and different anatomical sites such as age, sex, TNM stage, grade stage, surgery or no surgery, chemotherapy, radiotherapy, no radiotherapy, neck lymph node dissection or not, and neck lymphovascular invasion, larynx, overlapping lesion of lip, and oral cavity and pharynx tonsil of overall survival (OS). Immune-related characteristics, tumor microenvironment (TME) characteristics, and immunotherapy response were significantly different between high- and low-risk groups. The four AMGs were also found to be associated with the expression of markers of various immune cell subpopulations. Therefore, our comprehensive approach revealed the characterization of AAM in HNSCC to predict prognosis and guide clinical therapy.

PATIENTS WITH SICKLE CELL DISEASE TREATED WITH HIDROXIUREA HAVE HIGHER EXPRESSION OF PD-L1 IN MONOCYTES.

Inflammation plays a key role in pathophysiology of sickle cell disease (SCD). Monocytes are important mediator cells in the inflammation pathway. There is a remarkable lack of knowledge about how monocytes behave in patients with SCD and whether they are modulated by different treatments. To assess possible changes in monocyte maturation and proteinexpression among sickle cell disease patients in different treatment modalities and compare them to healthy subjects. 25 patients with SCD followed at the outpatient service of the Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo and 3 healthy controls were included. We included patients with sickle cell anemia (HbSS), over 18 years of age, without acute complications. Pregnancy was an exclusion criteria. Patients were divided in three groups: treatment with Hydroxyurea (HU) (n = 10) using the same dose for at least three months; chronic exchange transfusion (n = 9) (TP); and no specific treatment (NST) (n = 6). We performed peripheral blood mononuclear cells (PBMC) separation. Monocytes were analyzed by flow cytometry (Dx Flex, Beckman Coulter), using CD14 and CD16 as markers of subpopulations of monocytes as classical (CD14+/CD16-), intermediate (CD14+/CD16+) and non-classical (CD14-/CD16+). We also assessed the expression of CD 86 (a co-stimulation chemokine receptor), CD 163 (receptor for the conjugate hemoglobin-haptoglobin) and PD-L1 (an inhibitor receptor). Comparison between groups were made using ANOVA and Tukey test. Gender and age distribution yielded no significant difference. The mean of total leukocytes and monocytes were, respectively: 8,816 ± 2,133/mm 3 and 900 ± 563/mm 3 (HU), 11,487 ± 3,813/mm 3 and 1,343 ± 754/mm 3 (TP), 9,081 ± 1879/mm 3 and 1,373 ± 432/mm 3 (NST), 7,080 ± 642/mm 3 and 700 ± 0/mm 3 (control).Among the total nucleated cells, the percentage of classical monocytes was lower in HU compared with controls (3.8 ± 2.1%vs.7.7 ± 1.3%, p 0.008), lower in TP vs NST (2.4 ± 1.8%vs. 5.6 ± 2.8%,p 0.04) and in TP vs Controls (2.4 ± 1.8% vs. 7.7 ± 1.3%,p 0.001). No significant differences were shown in the intermediate monocytes. However, nonclassical monocytes were higher in HU compared with TP (0.44 ± 0.3% vs. 0.22 ± 0.2%, p 0.013) and in controls vs TP (0.35 ± 0.07% vs 0.22 ± 0.2%, p 0.017). In classical monocytes, expression of PD-L1 was higher in HU compared with NST (86.1 ± 17.4% vs. 43.9 ± 31.0%) and HU vs Controls (86.1 ± 17.4% vs. 34.9 ± 8.9%, p 0.019). Expression of PD-L1 in intermediate monocytes and in non classical monocytes were also higher in HU compared with controls (88.9 ± 16.3% vs. 58.6 ± 8.6%, p0.02 and 79.6 ± 30.2%vs.37.1 ± 4.6%, p 0.02). Different treatments for sickle cell disease have affected the monocyte subsets distribution and PD-L1 expression in the population studied. HU and Transfusion Program induced a reduction in the Classical Monocyte subset, more pronounced in the latter. The higher expression of PD-L1 in patients submitted to exchange transfusion and in use of HU indicate inhibition of inflammation, which is more pronounced in the HU group. Both treatments have immunomodulatory effects on SCD, however, to date, no inflammatory markers that assess response to treatment are available. Our findings might contribute to uncovering the role of PD-L1 in the chronic inflammation of SCD and to identify a potential biomarker.

Three-dimensional spheroids: a new approach for the identification and characterization of novel markers for tumor-initiating cells subpopulations in thyroid cancer

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

195P Breast cancer molecular subtypes markers in subpopulations of circulating tumor cells

Molecular subtype of breast cancer is a criterion that determines the treatment tactic and the prognosis of the clinical course of the disease. At the same time, CTCs can arise from the minor subpopulation of primary tumor cells, that do not express target molecules for prescribed therapy. Here we assessed the presence of ER/PR/Her2+ and Ki-67 in subpopulations of CTCs with different manifestations of EMT and stemness, which could dramatically effect on their capacity to migration, proliferation and finally forming a clinically detectable macrometastsis.

Differential Markers of Subpopulations of Epithelial Cells of the Larynx in Squamous Cell Carcinoma.

In squamous cell carcinoma of the larynx, the population of epithelial cells in the tumor tissue is initially heterogeneous and, in addition to tumor cells invading the organ mucosa, includes normal epithelial cells of protein-mucous glands and cells of the stratified epithelium covering the mucous membrane. A search for differential markers to separate these subpopulations was carried out. The surface marker CD44 and cytokeratins 5 and 17 that are often used to verify carcinoma cells, are common markers for all epithelial cells of the larynx. In highly differentiated carcinoma, subpopulations of normal and tumor epithelial cells can be separated by the level of expression of cytokeratins 10 and 18 and nuclear markers Ki-67 and p63. However, in moderately differentiated carcinoma, tumor cells and normal cells of the basal layer of the stratified epithelium covering the mucous membrane of the larynx have similar phenotypes, which should be taken into account when conducting experimental studies.

Effects of Coenzyme Q10 Supplementation on Oxidative Stress Markers, Inflammatory Markers, Lymphocyte Subpopulations, and Clinical Status in Dogs with Myxomatous Mitral Valve Disease.

Scarce data exist on the effects of coenzyme Q10 (CoQ10) supplementation in dogs with myxomatous mitral valve disease (MMVD). The purpose of this study was to investigate the effect of CoQ10 supplementation on oxidative stress markers (glutathione peroxidase, F2-isoprostanes), markers of inflammation (tumor necrosis factor-α, TNF soluble receptor II, leucocytes, and their subtypes), lymphocyte subpopulations (T helper and cytotoxic T lymphocytes, including activated T lymphocytes, and B lymphocytes), and echocardiographic and clinical parameters in dogs with MMVD. In this randomized, controlled, double-blind, longitudinal study, 43 MMVD dogs in stages ACVIM (American College of Veterinary Internal Medicine classification) B2 and ACVIM C and D (congestive heart failure (CHF)) received water-soluble coenzyme Q10 (100 mg twice daily) or placebo for 3 months, and 12 non-supplemented healthy dogs served as controls. All parameters were measured before and after supplementation in MMVD dogs and once in healthy dogs. CoQ10 supplementation had a positive impact on neutrophil percentage, lymphocyte percentage, and lymphocyte concentration in our cohort of dogs with CHF (ACVIM C and D). Conclusion: CoQ10 as an oral supplement may have benefits in terms of decreasing inflammation in dogs with MMVD and CHF.

Molecular markers for hemocyte subpopulations in crayfish Cherax quadricarinatus

Semigranular cells (SGCs) and granular cells (GCs) are two dominant groups of circulating hemocytes in crayfish Cherax quadricarinatus. Molecular markers are required for the clear classification of the hemocytes and the research of their function and differentiation. In this study, we compared the protein content of GCs and SGCs by using two workflows: one-dimensional gel electrophoresis followed by LC-MS/MS and in-solution digestion of cell lysate followed by LC-MS/MS. Cell type-specific proteins were identified, and their expression in SGCs and GCs was further investigated by RT-PCR, Western blotting, and immunofluorescence analysis. Three molecular markers for GCs (peroxinectin, a mannose-binding protein, and prophenoloxidase-activating enzyme 2a) and three molecular markers for SGCs (a vitelline membrane outer layer protein I-like protein, a C-type lectin, and a peptidase) were identified. The application of some of the markers in Eriocheir sinensis was also analyzed. These molecular markers are useful tools for the research of crustaceans hemocytes.

Lower Levels of GABAergic Function Markers in Corticotropin-Releasing Hormone-Expressing Neurons in the sgACC of Human Subjects With Depression

RationaleA previous transcriptome meta-analysis revealed significantly lower levels of corticotropin-releasing hormone (CRH) mRNA in corticolimbic brain regions in major depressive disorder (MDD) subjects, suggesting that cortical CRH-expressing (CRH+) cells are affected in MDD. Rodent studies show that cortical CRH is mostly expressed in GABAergic interneurons; however, the characteristic features of CRH+ cells in human brain cortex and their association with MDD are largely unknown.MethodsSubgenual anterior cingulate cortex (sgACC) of human subjects without brain disorders were labeled using fluorescent in situ hybridization (FISH) for CRH and markers of excitatory (SLC17A7), inhibitory (GAD1) neurons, as well as markers of other interneuron subpopulations (PVALB, SST, VIP). MDD-associated changes in CRH+ cell density and cellular CRH expression (n = 6/group) were analyzed. RNA-sequencing was performed on sgACC CRH+ interneurons from comparison and MDD subjects (n = 6/group), and analyzed for group differences. The effect of reduced BDNF on CRH expression was tested in mice with blocked TrkB function.ResultsAbout 80% of CRH+ cells were GABAergic and 17.5% were glutamatergic. CRH+ GABAergic interneurons co-expressed VIP (52%), SST (7%), or PVALB (7%). MDD subjects displayed lower CRH mRNA levels in GABAergic interneurons relative to comparison subjects without changes in cell density. CRH+ interneurons show transcriptomic profile suggesting lower excitability and less GABA release and reuptake. Further analyses suggested that these molecular changes are not mediated by altered glucocorticoid feedback and potentially occur downstream for a common modulator of neurotrophic function.SummaryCRH+ cells in human sgACC are a heterogeneous population of GABAergic interneurons, although largely co-expressing VIP. Our data suggest that MDD is associated with reduced markers of inhibitory function in sgACC CRH+ interneurons, and provide further evidence for impaired GABAergic function in the cortex in MDD.

Natural killer cell detection, quantification, and subpopulation identification on paper microfluidic cell chromatography using smartphone-based machine learning classification

Natural killer (NK) cells are immune cells that defend against viral infections and cancer and are used in cancer immunotherapies. Subpopulations of NK cells include CD56dim and CD56bright which either produce cytokines or cytotoxically kill cells directly. The absolute number and proportion of these cells in peripheral blood are tied to proper immune function. Current methods of cytokine detection and proportion of NK cell subpopulations require fluorescent dyes and highly specialized equipment, e.g., flow cytometry, thus rapid cell quantification and subpopulation analysis are needed in the clinical setting. Here, a smartphone-based device and a two-component paper microfluidic chip were used towards identifying NK cell subpopulation and inflammatory markers. One unit measured flow velocity via smartphone-captured video, determining cytokine (IL-2) and total NK cell concentrations in undiluted buffy coat blood samples. The other, single flow lane unit performs spatial separation of CD56dim and CD56bright and cells over its length using differential binding of anti-CD56 nanoparticles. A smartphone microscope combined with cloud-based machine learning predictive modeling (utilizing a random forest classification algorithm) analyzed both flow data and NK cell subpopulation differentiation. Limits of detection for cytokine and cell concentrations were 98 IU/mL and 68 cells/mL, respectively, and cell subpopulation analysis showed 89% accuracy.

Converging Effects of Three Different Endocrine Disrupters on Sox and Pou Gene Expression in Developing Rat Hippocampus: Possible Role of microRNA in Sex Differences.

Endocrine disrupting chemicals (EDCs) can impair hippocampus-dependent behaviors in rat offspring and in children. In search for key processes underlying this effect, we compared the transcriptomes of rat hippocampus on postnatal day 6 after gestational and lactational exposure to three different EDCs at doses known to impair development of learning and memory. Aroclor 1254, a commercial PCB mixture (5 mg/kg or 0.5 mg/kg), or bisphenol A (5 mg/kg or 0.5 mg/kg) were administered in chow, chlorpyrifos (3 mg/kg or 1 mg/kg) was injected subcutaneously. Male hippocampus exhibited a common effect of all three chemicals on genes involved in cell-autonomous processes, Sox6, Sox11, Pou2f2/Oct2, and Pou3f2/Brn2, all upregulated at the high dose. Additional genes of the Sox and Pou families were affected by only one or two of the chemicals. Real time RT PCR showed a comparable expression change for bisphenol A also at the lower dose. Female hippocampus exhibited much fewer genes with expression changes (almost none with false discovery rate <0.05), and none of the genes of the Sox and Pou families was affected. Since gene network analyses in male hippocampus suggested a link between Sox6 and miR-24, known to be repressed by activation of ER-alpha and to repress Sox6 in other tissues, this microRNA was measured. miR-24 was downregulated by all chemicals at the high dose in males. Values of Sox6 mRNA and miR-24 were inversely correlated in individual male hippocampus samples, supporting the hypothesis that the change in Sox6 expression resulted from an action of miR-24. In contrast, miR-24 levels remained unchanged in hippocampus of females. A sexually dimorphic response of miR-24 may thus be at the basis of the sex difference in Sox6 expression changes following exposure to the three chemicals. ER-alpha expression was also sex-dependent, but the expression changes did not parallel those of potential downstream genes such as Sox6. Sox6 is known to suppress differentiation of Parvalbumin (Pvalb)-expressing interneurons. Individual Sox6 levels (FPKM) were inversely correlated with levels of Pvalb, but not with markers of Sox6-independent interneuron subpopulations, Nos1 and 5HT3aR. Effects on interneuron development are further suggested, in males, by expression changes of Nrg1 and its receptor Erbb4, controlling interneuron migration. Our study disclosed new types of EDC-responsive morphogenetic genes, and illustrated the potential relevance of microRNAs in sexually dimorphic EDC actions.

Cancer Stem Cell Traits in Tumor Spheres Derived from Primary Laryngeal Carcinoma Cell Lines.

Objective:Cancer stem cells (CSCs) belong to a subpopulation of undifferentiated cells present within tumors that have the potential to regenerate, differentiate, maintenance of pluripotency, drug resistance, and tumorigenicity when transplanted into an innate host. These can influence the growth and behavior of these tumors and are used to investigate the initiation, progression, and treatment strategies of laryngeal cancer. Research on CSC science and targeted therapies were hinge on their isolation and/or enrichment procedures. The object of the study is to isolate cancer stem cells from primary laryngeal carcinoma (CSCPLC) by tumor spheres enrichment. We checked the properties of self-renewal, stemness, clonogenicity, and chemotherapeutic resistance.Materials and Methods:We performed tumor sphere formation assay (primary, secondary, and tertiary) chemotherapy resistance by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay were performed to evaluate the CSC cells. Immunofluorescence for stem cell markers (CD133+, CD44+) and gene expression of stem cell markers for CD133+, CD44+, OCT4, SOX2, and NANOG was done using the real-time polymerase chain reaction technique.Results:We were able to isolated CSC subpopulations from PLC cell lines by the tumor sphere method. These cells exhibited good primary, secondary, and tertiary tumor sphere formation efficiency and also disclosed a resistant index of more than 2. Immunofluorescence for stem cell markers (CD133+ and CD44+) confirms the presence of CSC. There was significantly higher mRNA expression of stem cell markers in CSC enriched subpopulations compared to the parental cell lines.Conclusion:We conclude that tumor spheres enrichment is an efficient, economical, and reliable approach for the isolation and characterization of CSC from PLC cell lines. These cells demonstrated the properties of self-renewal, stemness, clonogenicity, and chemotherapeutic resistance.

REACTION OF CD68-POSITIVE RAT LIVER AND SPLEEN CELLS ON SILICON INTAKE WITH DRINKING WATER

The article presents data on the long-term effect (nine months) of a silicon compound supplied with drinking water – nonahydrate sodium metasilicate (10 mg/l in terms of silicon), on CD68-positive macrophages in the liver and spleen of laboratory rats. Changes in the morphological characteristics of this cell population were found. There was a decrease in the average cell area (in the liver of the control group of rats, the average macrophage area was 179.23±5.94 microns2, and in the group receiving silicon with drinking water – 117.04±3.35 microns2; in the spleen-136.02±3.93 microns2 and 103.44±2.8 microns2, respectively). Macrophages in the liver preparations of the experimental group of rats had a fewer processes and a darker cytoplasmic membrane. The number of macrophages in the liver per unit area was comparable, for the control group of rats it was 18.78±1.24, and for the rats that received with water with the addition of silicon – 19.41±0.75 cells. CD68+ macrophages of the red splenic pulp in laboratory rats that received silicon also underwent the following morphological changes: they were located in a denser way and had fewer processes, while the number of macrophages per unit area was 73.7±2.3 for the control group, 91.6±5.0-for the experimental group, respectively. The distance between them did not change. There was a change in the intensity of CD68 expression on the surface of the cytoplasmic membrane and in the cytoplasm of liver and spleen macrophages. These changes can be interpreted as the adaptive ability of liver and spleen macrophages to silicon introduced with drinking water. Given the heterogeneity of the macrophage population in the liver and spleen, further studies using markers for different subpopulations of macrophages are needed to clarify their role in the response of tissues to silicon supplied with drinking water.

The forensic landscape and the population genetic analyses of Hainan Li based on massively parallel sequencing DNA profiling.

Due to the formation of the Qiongzhou Strait by climate change and marine transition, Hainan island was isolated from the mainland southern China during the Last Glacial Maximum. Hainan island, located at the southernmost part of China and separated from the Leizhou Peninsula by the Qiongzhou Strait, laid on one of the modern human northward migration routes from Southeast Asia to East Asia. The Hlai language-speaking Li minority, the second largest population after Han Chinese in Hainan island, is the direct descendants of the initial migrants in Hainan island and has unique ethnic properties and derived characteristics; however, the forensic-associated studies on Hainan Li population are still insufficient. Hence, 136 Hainan Li individuals were genotyped in this study using the MPS-based ForenSeq™ DNA Signature Prep Kit (DNA Primer Set A, DPMA) to characterize the forensic genetic polymorphism landscape, and DNA profiles were obtained from 152 different molecular genetic markers (27 autosomal STRs, 24 Y-STRs, 7 X-STRs, and 94 iiSNPs). A total of 419 distinct length variants and 586 repeat sequence sub-variants, with 31 novel alleles (at 17 loci), were identified across the 58 STR loci from the DNA profiles of Hainan Li population. We evaluated the forensic characteristics and efficiencies of DPMA, demonstrating that the STRs and iiSNPs in DPMA were highly polymorphic in Hainan Li population and could be employed in forensic applications. In addition, we set up three datasets, which included the genetic data of (i) iiSNPs (27 populations, 2640 individuals), (ii) Y-STRs (42 populations, 8281 individuals), and (iii) Y haplogroups (123 populations, 4837 individuals) along with the population ancestries and language families, to perform population genetic analyses separately from different perspectives. In conclusion, the phylogenetic analyses indicated that Hainan Li, with a southern East Asia origin and Tai-Kadai language-speaking language, is an isolated population relatively. But the genetic pool of Hainan Li influenced by the limited gene flows from other Tai-Kadai populations and Hainan populations. Furthermore, the establishment of isolated population models will be beneficial to clarify the exquisite population structures and develop specific genetic markers for subpopulations in forensic genetic fields.

Single-cell analysis reveals metastatic cell heterogeneity in clear cell renal cell carcinoma.

Renal cell carcinoma (RCC) is one of the leading causes of cancer‐related death worldwide. Tumour metastasis and heterogeneity lead to poor survival outcomes and drug resistance in patients with metastatic RCC (mRCC). In this study, we aimed to assess intratumoural heterogeneity (ITH) in mRCC cells by performing a combined analysis of bulk data and single‐cell RNA‐sequencing data, and develop novel biomarkers for prognosis prediction on the basis of the potential molecular mechanisms underlying tumorigenesis. Eligible single‐cell cohorts related to mRCC were acquired using the Gene Expression Omnibus (GEO) dataset to identify potential mRCC subpopulations. We then performed gene set variation analysis to understand the differential function in primary RCC and mRCC samples. Subsequently, we applied weighted correlation network analysis to identify coexpressing gene modules that were related to the external trait of metastasis. Protein‐protein interactions were used to screen hub subpopulation‐difference (sub‐dif) markers (ACTG1, IL6, CASP3, ACTB and RAP1B) that might be involved in the regulation of RCC metastasis and progression. Cox regression analysis revealed that ACTG1 was a protective factor (HR < 1), whereas the other four genes (IL6, CASP3, ACTB and RAP1B) were risk factors (HR > 1). Kaplan‐Meier survival analysis suggested the potential prognostic value of these sub‐dif markers. The expression of sub‐dif markers in mRCC was further evaluated in clinical samples by immunohistochemistry (IHC). Additionally, the genetic features of sub‐dif marker expression patterns, such as genetic variation profiles, correlations with tumour‐infiltrating lymphocytes (TILs), and targeted signalling pathway activities, were assessed in bulk RNA‐seq datasets. In conclusion, we established novel subpopulation markers as key prognostic factors affecting EMT‐related signalling pathway activation in mRCC, which could facilitate the implementation of a treatment for mRCC patients.

Thyrotropin Receptor-Specific Lymphocytes in Adenovirus-TSHR-Immunized Native and Human Leukocyte Antigen-DR3-Transgenic Mice and in Graves' Disease Patient Blood.

Background: Antigen-specific lymphocytes are increasingly investigated in autoimmune diseases and immune therapies. We sought to identify thyrotropin receptor (TSHR)-specific lymphocytes in mouse models of Graves' disease, including Graves' patient-specific immunotype human leukocyte antigen (HLA)-DR3, and in frozen and thawed Graves' patient blood samples. Methods and Results: Splenic lymphocytes of adenovirus (Ad)-TSHR-immunized BALB/c mice were stimulated with TSHR-specific peptides C, D, or J. Furthermore, CD154-expressing cells were enriched, expanded in vitro, and analyzed for binding of peptide-major histocompatibility complex (MHC) II multimers ("tetramers," immunotype H2-IAd). Only peptides C and J were able to elicit increased expression/secretion of CD154 and interferon-γ, and tetramers which were loaded with peptide C resulted in antigen-specific signals in splenic lymphocytes from Ad-TSHR-immunized mice. Accordingly, TSHR-specific HLA-DR3-MHC class II tetramers loaded with peptide p10 specifically bound to human HLA-DR3-(allele B1*03:01)-transgenic Bl/6 mouse splenic T lymphocytes. In addition, we fine-tuned a protocol to reliably measure thawed human peripheral blood mononuclear cells (PBMCs), which resulted in reliable recovery after freezing and thawing with regard to vitality and B and T cell subpopulation markers including regulatory T cells (CD3, CD4, CD25, FoxP3, CD25high, CD127low). TSHR-specific HLA-DR3-MHC class II tetramers loaded with peptide p10 identified antigen-specific T cells in HLA-DR3-positive Graves' patients' thawed PBMCs. Moreover, stimulation-dependent release of interleukin (IL)-1beta, IL-6, tumor necrosis factor-alpha from thawed PBMCs occurred at the expected levels. Conclusions: Novel MHC II tetramers identified TSHR-specific T lymphocytes in Ad-TSHR-immunized hyperthyroid BALB/c or HLA-DR3-transgenic mice and in thawed human PBMCs from patients with Graves' disease. These assays may contribute to measure both disease severity and effects of novel immune therapies in future animal studies and clinical investigations of Graves' disease.

Intrinsic Neuronal Activity during Migration Controls the Recruitment of Specific Interneuron Subtypes in the Postnatal Mouse Olfactory Bulb.

Neuronal activity has been identified as a key regulator of neuronal network development, but the impact of activity on migration and terminal positioning of interneuron subtypes is poorly understood. The absence of early subpopulation markers and the presence of intermingled migratory and postmigratory neurons make the developing cerebral cortex a difficult model to answer these questions. Postnatal neurogenesis in the subventricular zone (SVZ) offers a more accessible and compartmentalized model. Neural stem cells regionalized along the border of the lateral ventricle produce two main subtypes of neural progenitors, granule cells and periglomerular neurons that migrate tangentially in the rostral migratory stream (RMS) before migrating radially in the olfactory bulb (OB) layers. Here, we used targeted postnatal electroporation to compare the migration of these two populations in male and female mice. We do not observe any obvious differences regarding the mode of tangential or radial migration between these two subtypes. However, we find a striking increase of intrinsic calcium activity in granule cell precursors (GC-Ps) when they switch from tangential to radial migration. By decreasing neuronal excitability in GC-Ps, we find that neuronal activity has little effect on migration but is required for normal positioning and survival of GC-Ps in the OB layers. Strikingly, decreasing activity of periglomerular neuron precursors (PGN-Ps) did not impact their positioning or survival. Altogether these findings suggest that neuronal excitability plays a subtype specific role during the late stage of migration of postnatally born OB interneurons.SIGNIFICANCE STATEMENT While neuronal activity is a critical factor regulating different aspects of neurogenesis, it has been challenging to study its role during the migration of different neuronal subpopulations. Here, we use postnatal targeted electroporation to label and manipulate the two main olfactory bulb (OB) interneuron subpopulations during their migration: granule cell and periglomerular neuron precursors (PGN-Ps). We find a very striking increase of calcium activity only in granule cell precursors (GC-Ps) when they switch from tangential to radial migration. Interestingly, blocking activity in GC-Ps affected mainly their positioning and survival while PGN-Ps were not affected. These results suggest that neuronal activity is required specifically for the recruitment of GC-Ps in the OB layers.

Risk markers by sex for in-hospital mortality in patients with acute coronary syndrome: A machine learning approach

Background:Several studies have highlighted the importance of considering sex differences in the diagnosis and treatment of Acute Coronary Syndrome (ACS). However, the identification of sex-specific risk markers in ACS sub-populations has been scarcely studied. The present study aims to explore machine learning (ML) models to identify in-hospital mortality markers for women and men in ACS sub-populations collected from a public database of electronic health records (EHR). Methods:We extracted 1,299 patients with ST-elevation myocardial infarction (STEMI) and 2,820 patients with non-ST-elevation myocardial infarction (NSTEMI) from the Medical Information Mart for Intensive Care (MIMIC)-III database. We trained and validated mortality prediction models and used an interpretability technique to identify sex-specific markers for each sub-population. Results:The models based on eXtreme Gradient Boosting (XGBoost) achieved the highest performance: area under the curve (AUC) = 0.94 (95% CI:0.84–0.96) for STEMI and AUC = 0.94 (95% CI:0.80–0.90) for NSTEMI. For STEMI, the top markers in women are chronic kidney failure, high heart rate, and age over 70 years. For men, the top markers are acute kidney failure, high troponin T levels, and age over 75 years. However, for NSTEMI, the top markers in women are low troponin levels, high urea levels, and age over 80 years. For men, the top markers are high heart rate, creatinine levels, and age over 70 years. Conclusions:Our results show possible significant and coherent sex-specific risk markers of different ACS sub-populations by interpreting ML mortality models trained on EHRs. Differences are observed in the identified risk markers between women and men, highlighting the importance of considering sex-specific markers in implementing more appropriate treatment strategies and better clinical outcomes.