mRNA Expression Profiles Of Genes Research Articles

The landscape of N1-methyladenosine (m1A) modification in mRNA of the decidua in severe preeclampsia.

Recent discoveries in mRNA modification have highlighted N1-methyladenosine (m1A), but its role in preeclampsia (PE) pathogenesis remains unclear. In this study, we utilized methylated RNA immunoprecipitation sequencing (MeRIP-seq) and RNA sequencing (RNA-seq) to identify m1A peaks and the expression profile of mRNA in the decidua of humans with early-onset PE (EPE), late-onset PE (LPE), and normal pregnancy (NP). We assessed the m1A modification patterns in preeclamptic decidua using 10 m1A modulators. Our bioinformatic analysis focused on differentially methylated mRNAs (DMGs) and differentially expressed mRNAs (DEGs) in pairwise comparisons of EPE vs. NP, LPE vs. NP, and EPE vs. LPE, as well as m1A-related DEGs. The comparisons of EPE vs. NP, LPE vs. NP, and EPE vs. LPE identified 3110, 2801, and 2818 DMGs, respectively. We discerned three different m1A modification patterns from this data. Further analysis revealed that key PE-related DMGs and m1A-related DEGs predominantly influence signaling pathways critical for decidualization, including cAMP, MAPK, PI3K-Akt, Notch, and TGF-β pathways. Additionally, these modifications impact pathways related to vascular smooth muscle contraction, estrogen signaling, and relaxin signaling, contributing to vascular dysfunction. Our findings demonstrate that preeclamptic decidua exhibits unique mRNA m1A modification patterns and gene expression profiles that significantly alter signaling pathways essential for both decidualization and vascular dysfunction. These differences in m1A modification patterns provide valuable insights into the molecular mechanisms influencing the decidualization process and vascular function in the pathogenesis of PE. These m1A modification regulators could potentially serve as potent biomarkers or therapeutic targets for PE, warranting further investigation.

Myeloid cell differentiation-related gene signature for predicting clinical outcome, immune microenvironment, and treatment response in lung adenocarcinoma

Considering the key role of myeloid cell differentiation—related genes in the tumor microenvironment (TME), we aimed to build a prognostic risk model using these genes for Lung adenocarcinoma (LUAD). The mRNA gene expression profiles of LUAD patients from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases were downloaded as the training and validation sets. Then, “edgeR” R package was applied to screen out the differentially expressed genes (DEGs) and univariate cox regression, backward stepwise selection analyses were performed to construct a prognostic model for LUAD. ESTIMATE, TIMER, XCELL, CIBERSORT abs, QUANTISEQ, MCPCOUNTER, EPIC, and CIBERSORT algorithms were conducted to access the association of risk levels with the stromal and immune cell infiltration levels in LUAD. Six genes (F2RL1, PRKDC, TNFSF11, INHA, PLA2G3 and TUBB1) were utilized to construct the prognostic model. The risk model showed excellent prognostic performance for LUAD in both TCGA and GEO datasets. Also, compared to the low-risk patients, the high-risk patients had higher expression of immune checkpoint molecules and showed a lower IC50 value to the chemotherapy agents. Our findings provided a myeloid cell differentiation—related gene signature that could effectively predict prognosis and guide treatment strategies for LUAD patients.

Growth behavior and mRNA expression profiling during growth of IPEC-J2 cells

ObjectiveThe IPEC-J2 cell line is used as an in vitro small intestine model for swine, but it is also used as a model for the human intestine, presenting a relatively unique setting. By combining electric cell-substrate impedance sensing, with next-generation-sequencing technology, we showed that mRNA gene expression profiles and related pathways can depend on the growth phase of IPEC-J2 cells. Our investigative approach welcomes scientists to reproduce or modify our protocols and endorses putting their gene expression data in the context of the respective growth phase of the cells.ResultsThree time points are presented: (TP1) 1 h after medium change (= 6 h after seeding of cells), (TP2) the time point of the first derivative maximum of the cell growth curve, and a third point at the beginning of the plateau phase (TP3). Significantly outstanding at TP1 compared to TP2 was upregulated PLEKHN1, further FOSB and DEGS2 were significantly downregulated at TP2 compared to TP3. Any provided data can be used to improve next-generation experiments with IPEC-J2 cells.

Transcriptome research of human amniocytes identifies hub genes associated with developmental dysplasia in down syndrome.

Trisomy 21, or Down syndrome (DS), is the most frequent human autosomal chromosome aneuploidy, which leads to multiple developmental disorders, especially mental retardation in individuals. The presence of an additional human chromosome 21 (HSA21) could account for the pathological manifestations in DS. In this study, we analyzed the mRNA gene expression profile of DS-derived amniocytes compared with normal amniocytes, aiming to evaluate the relationship between candidate dysregulated HSA21 genes and DS developmental phenotypes. Differentially expressed genes (DEGs) included 1794 upregulated genes and 1411 downregulated genes, which are mainly involved in cell adhesion, inflammation, cell proliferation and thus may play an important role in inducing multiple dysplasia during DS fetal development. Furthermore, STRING protein network studies demonstrated 7 candidate HSA21 genes participated Gene Ontology (GO) terms: cell adhesion and extracellular matrix remodeling (COL6A1, COL6A2, COL18A1, ADAMTS5, JAM2, and POFUT2), inflammation and virus infection response (MX1 and MX2), histone modification and chromatin remodeling (NRIP1), glycerolipid and glycerophospholipid metabolism (AGPAT3), mitochondrial function (ATP5PF and ATP5PO), synaptic vesicle endocytosis (ITSN1 and SYNJ1) and amyloid metabolism (APP). Meanwhile, GSEA enrichment identified several transcription factors and miRNAs, which may target gene expression in the DS group. Our study established connections between dysregulated genes, especially HSA21 genes, and DS-associated phenotypes. The alteration of multiple pathways and biological processes may contribute to DS developmental disorders, providing potential pathogenesis and therapeutic targets for DS.

931. Host PFKFB3-dependent glycolytic reprogramming as a broad-spectrum antiviral strategy

Abstract Background The current therapeutic arsenal against viral infections remains poor, with limited spectrum of coverage and appears inadequate to face the emergence of pandemic and antiviral resistance. This study aims to develop a broad-spectrum antiviral strategy for rapid epidemic control and early treatment with better clinical outcome. Methods To investigate the in vivo change of glucose homeostasis under infectious circumstances, we carried out a time-course monitoring of blood glucose concentration in pdmH1N1-infected BALB/c mice and revealed that impairment of glucose tolerance and insulin sensitivity were caused when compared with the mock-infection control. To explore the change of gene expression file upon virus infection, we first plotted the pdmH1N1 virus replication kinetics in human primary bronchial/tracheal epithelial cells (hBTECs), followed by mRNA gene expression profiling of the key glycolytic enzymes at 0hpi, 6hpi and 24hpi, respectively. To discover potential enzymatic inhibitors/modulators of host glycolysis for antiviral treatment, we screened a small molecular compound library with 344 glycolysis compounds on pdmH1N1 infected hTBECs, followed by secondary validation to confirm the dose-dependent virus inhibition at non-toxic concentrations. Results Significant higher blood glucose level was detected in infected-mice with either glucose or insulin supplement, which suggested that host glucose metabolism, in particular the glycolysis homeostasis, were disrupted upon influenza virus infection. We also identified Glucose transporter 4 (GLUT4), enolase 2 (ENO2), monocarboxylate transporter 2 (MCT2), MCT4 and 6-phosphofructo-2-kinase (PFK2, also named PFKFB3) the most perturbed genes at 24hpi. KAN0438757, a potent and selective inhibitor of the metabolic kinase PFKFB3, was shown to be antiviral effective against both H1N1 and EV-A71 viruses in human primary cells. Moreover, Kan exhibited broad-spectrum antiviral activity in ZIKV-infected Huh7 cells and SARS-CoV-2 infected Caco-2 cells. Metabolic profiling reveals significant perturbations of host glucose homeostasis after influenza A virus infection. Conclusion Host PFKFB3-dependent glycolysis pathway with broad-relevance to multiple virus replications, which might be a vulnerable host target for antiviral intervention. Disclosures All Authors: No reported disclosures

Feeding rumen-protected lysine prepartum alters placental metabolism at a transcriptional level

Rumen-protected Lys (RPL) fed to Holstein cows prepartum resulted in a greater intake and improved health of their calves during the first 6 wk of life. However, whether increased supply of Lys in late gestation can influence placental tissue and, if so, which pathways are affected remain to be investigated. Therefore, we hypothesize that feeding RPL during late gestation could modulate placental metabolism, allowing for improved passage of nutrients to the fetus and thus influencing the offspring development. Therefore, we aimed to determine the effects of feeding RPL (AjiPro-L Generation 3, Ajinomoto Health and Nutrition North America) prepartum (0.54% DM of TMR) on mRNA gene expression profiles of placental samples of Holstein cows. Seventy multiparous Holstein cows were randomly assigned to 1 of 2 dietary treatments, consisting of TMR top-dressed with RPL (PRE-L) or without (control, CON), fed from 27 ± 5 d prepartum until calving. After natural delivery (6.87 ± 3.32 h), placentas were rinsed with physiological saline (0.9% sodium chloride solution) to clean any dirtiness from the environment and weighed. Then, 3 placentomes were collected, one from each placental region (cranial, central, and caudal), combined and flash-frozen in liquid nitrogen to evaluate the expression of transcripts and proteins related to protein metabolism and inflammation. Placental weights did not differ from cows in PRE-L (15.5 ± 4.03 kg) and cows in CON (14.5 ± 4.03 kg). Feeding RPL prepartum downregulated the expression of NOS3 (nitric oxide synthase 3), involved in vasodilation processes, and SOD1, which encodes the enzyme superoxide dismutase, involved in oxidative stress processes. Additionally, feeding RPL prepartum upregulated the expression of transcripts involved in energy metabolism (SLC2A3, glucose transporter 3; and PCK1, phosphoenolpyruvate carboxykinase 1), placental metabolism and cell proliferation (FGF2, fibroblast growth factor 2; FGF2R, fibroblast growth factor 2 receptor; and PGF, placental growth factor), Met metabolism (MAT2A, methionine adenosyltransferase 2-α), and tended to upregulate IGF2R (insulin-like growth factor 2 receptor). Placental FGF2 and LRP1 (low-density lipoprotein receptor-related protein 1) protein abundance were greater for cows that received RPL prepartum than cows in CON. In conclusion, feeding RPL to prepartum dairy cows altered uteroplacental expression of genes and proteins involved in cell proliferation, and in metabolism and transport of glucose. Such changes are illustrated by increased expression of SLC2A3 and PCK1 and increased protein abundance of FGF2 and LRP1 in uteroplacental tissue of cows consuming RPL.

Neurotoxicity of 4-nonylphenol in adult zebrafish: Evaluation of behaviour, oxidative stress parameters and histopathology of brain

Nonylphenol and its derivatives use as plasticizer or additives in manufacturing industries. Effluents originated from industrial areas are being added to soil, ground water, river and marine water intentionally or unintentionally. Complex mixture of these contaminants enter the food chain and produce sub-lethal deleterious effects mainly on nervous and reproductive systems of aquatic animals and human beings. The information pertaining to oxidative stress-mediated alterations in brain of zebrafish would be helpful to understand the toxicity potential of such compounds in aquatic animals. The aim of the present study was to evaluate the behavioural changes, status of oxidative stress markers; sod, cat, and NF-E2-related factor 2 (nrf2) mRNA gene expression profile; and histopathological changes in the brain of adult zebrafish exposed to 4-nonylphenol (4NP) at concentration of 100 and 200 μg/L of water for 21 days. Zebrafish were divided into four groups viz; control (C1), vehicle (C2, ethanol 10 μg/L of water), treatment 1 (T1, 4-NP, 100 μg/L) and treatment 2 (T2, 4-NP, 200 μg/L). Both exposure levels of 4-NP adversely affected the exploratory behaviour of zebrafish and produced anxiety-like symptom. Concentration-dependent reduction in activity of superoxide dismutase and catalase; and glutathione level, with increased level of malondialdehyde recorded in the brain of exposed zebrafish. Gene expression analysis showed down regulation of sod, cat, nrf2 genes in brain of zebrafish from toxicity groups indicating 4-NP induced oxidative stress in brain. However, noticeable histological alterations were not observed in 4-NP exposed brain of zebrafish.

Gene expression profiles of mRNA, lncRNA, miRNA, and circRNA and their clinical implications in chronic rhinosinusitis with nasal polyps.

Chronic rhinosinusitis with nasal polyps (CRSwNP) is a chronic inflammatory disease with complex pathophysiology and therapeutic strategies. Moreover, the molecular mechanisms underlying the pathogenesis of CRSwNP are incompletely understood. This study aimed to investigate the transcriptomic characteristics, ceRNA networks, and whether these molecular markers play a role in the occurrence and development of CRSwNP. Following RNA sequencing, a ceRNA network was predicted and constructed based on the sequencing results and multiple databases. Gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and disease ontology (DO) were applied to analyze the potential mechanisms in relation to the pathogenesis of CRSwNP. CIBERSORT was used to evaluate the immune cell infiltration levels in CRSwNP. The candidate genes of differentially expressed (DE) mRNA, DE-lncRNA, DE-miRNA, and DE-circRNA were verified by RT-qPCR, and the back-splice junction of circRNA was verified using Sanger sequencing. The clinical significance of differentially expressed genes was analyzed with correlation test and receiver operating characteristic curve. We identified 716 DE-mRNA, 230 DE-lncRNA, 42 DE-miRNA, and 46 DE-circRNA, and GO and KEGG enrichment analyses indicated that they were involved in multiple biological pathways, predominantly those associated with immunity and inflammation. DO analysis revealed CRSwNP is associated with diseases such as gastroesophageal reflux and allergic reactions. High expression of circ_0021727 was significantly and positively correlated with several important clinical indicators, and the area under the curve was 0.741. This study provides transcriptomic characteristics, which are potential biomarkers or therapeutic targets for the diagnosis and treatment of CRSwNP.

Invention of 3Mint for feature grouping and scoring in multi-omics.

Advanced genomic and molecular profiling technologies accelerated the enlightenment of the regulatory mechanisms behind cancer development and progression, and the targeted therapies in patients. Along this line, intense studies with immense amounts of biological information have boosted the discovery of molecular biomarkers. Cancer is one of the leading causes of death around the world in recent years. Elucidation of genomic and epigenetic factors in Breast Cancer (BRCA) can provide a roadmap to uncover the disease mechanisms. Accordingly, unraveling the possible systematic connections between-omics data types and their contribution to BRCA tumor progression is crucial. In this study, we have developed a novel machine learning (ML) based integrative approach for multi-omics data analysis. This integrative approach combines information from gene expression (mRNA), microRNA (miRNA) and methylation data. Due to the complexity of cancer, this integrated data is expected to improve the prediction, diagnosis and treatment of disease through patterns only available from the 3-way interactions between these 3-omics datasets. In addition, the proposed method bridges the interpretation gap between the disease mechanisms that drive onset and progression. Our fundamental contribution is the 3 Multi-omics integrative tool (3Mint). This tool aims to perform grouping and scoring of groups using biological knowledge. Another major goal is improved gene selection via detection of novel groups of cross-omics biomarkers. Performance of 3Mint is assessed using different metrics. Our computational performance evaluations showed that the 3Mint classifies the BRCA molecular subtypes with lower number of genes when compared to the miRcorrNet tool which uses miRNA and mRNA gene expression profiles in terms of similar performance metrics (95% Accuracy). The incorporation of methylation data in 3Mint yields a much more focused analysis. The 3Mint tool and all other supplementary files are available at https://github.com/malikyousef/3Mint/.

A novel risk score model based on gamma-aminobutyric acid signature predicts the survival prognosis of patients with breast cancer.

Gamma-aminobutyric acid (GABA) participates in the migration, differentiation, and proliferation of tumor cells. However, the GABA-related risk signature has never been investigated. Hence, we aimed to develop a reliable gene signature based on GABA pathways-related genes (GRGs) to predict the survival prognosis of breast cancer patients. GABA-related gene sets were acquired from the MSigDB database, while mRNA gene expression profiles and corresponding clinical data of breast cancer patients were downloaded from the Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA) databases. Univariate Cox regression analysis was used to identify prognostic-associated GRGs. Subsequently, LASSO analysis was applied to establish a risk score model. We also constructed a clinical nomogram to perform the survival evaluation. Besides, ESTIMATE and ssGSEA algorithms were used to assess the immune cell infiltration among the risk score subgroups. A GRGs-related risk score model was constructed in the TCGA cohort, and validated in the GSE21653 cohort. The risk score was significantly related to the overall survival of breast cancer patients, which could predict the survival prognosis of breast cancer patients independently of other clinical features. Breast cancer patients in the low-risk score group exhibited higher immune cell infiltration levels. A novel prognostic model containing five GRGs could accurately predict the survival prognosis and immune infiltration of breast cancer patients. Our findings provided a novel insight into investigating the immunoregulation roles of GRGs.

Phenotypic and transcriptomic acclimation of the green microalga Raphidocelis subcapitata to high environmental levels of the herbicide diflufenican

Herbicide pollution poses a worldwide threat to plants and freshwater ecosystems. However, the understanding of how organisms develop tolerance to these chemicals and the associated trade-off expenses are largely unknown. This study aims to investigate the physiological and transcriptional mechanisms underlying the acclimation of the green microalgal model species Raphidocelis subcapitata (Selenastraceae) towards the herbicide diflufenican, and the fitness costs associated with tolerance development. Algae were exposed for 12 weeks (corresponding to 100 generations) to diflufenican at the two environmental concentrations 10 and 310 ng/L. The monitoring of growth, pigment composition, and photosynthetic performance throughout the experiment revealed an initial dose-dependent stress phase (week 1) with an EC50 of 397 ng/L, followed by a time-dependent recovery phase during weeks 2 to 4. After week 4, R. subcapitata was acclimated to diflufenican exposure with a similar growth rate, content of carotenoids, and photosynthetic performance as the unexposed control algae. This acclimation state of the algae was explored in terms of tolerance acquisition, changes in the fatty acids composition, diflufenican removal rate, cell size, and changes in mRNA gene expression profile, revealing potential fitness costs associated with acclimation, such as up-regulation of genes related to cell division, structure, morphology, and reduction of cell size. Overall, this study demonstrates that R. subcapitata can quickly acclimate to environmental but toxic levels of diflufenican; however, the acclimation is associated with trade-off expenses that result in smaller cell size.

Immune Microenvironment in Sporadic Early-Onset versus Average-Onset Colorectal Cancer

The incidence of left-sided colon and rectal cancer in young people are increasing worldwide, but its causes are poorly understood. It is not clear if the tumor microenvironment is dependent on age of onset, and little is known about the composition of tumor-infiltrating T cells in early-onset colorectal cancer (EOCRC). To address this, we investigated T-cell subsets and performed gene expression immune profiling in sporadic EOCRC tumors and matched average-onset colorectal cancer (AOCRC) tumors. Left-sided colon and rectal tumors from 40 cases were analyzed; 20 EOCRC (<45 years) patients were matched 1:1 to AOCRC (70-75 years) patients by gender, tumor location, and stage. Cases with germline pathogenic variants, inflammatory bowel disease or neoadjuvant-treated tumors were excluded. For T cells in tumors and stroma, a multiplex immunofluorescence assay combined with digital image analysis and machine learning algorithms was used. Immunological mediators in the tumor microenvironment were assessed by NanoString gene expression profiling of mRNA. Immunofluorescence revealed no significant difference between EOCRC and AOCRC with regard to infiltration of total T cells, conventional CD4+ and CD8+ T cells, regulatory T cells, or γδ T cells. Most T cells were located in the stroma in both EOCRC and AOCRC. Immune profiling by gene expression revealed higher expression in AOCRC of the immunoregulatory cytokine IL-10, the inhibitory NK cell receptors KIR3DL3 and KLRB1 (CD161), and IFN-a7 (IFNA7). In contrast, the interferon-induced gene IFIT2 was more highly expressed in EOCRC. However, in a global analysis of 770 tumor immunity genes, no significant differences could be detected. T-cell infiltration and expression of inflammatory mediators are similar in EOCRC and AOCRC. This may indicate that the immune response to cancer in left colon and rectum is not related to age of onset and that EOCRC is likely not driven by immune response deficiency.

Anti-inflammatory, anti-oxidant and cardio-protective properties of novel fluorophenyl benzimidazole in L-NAME-induced hypertensive rats

BackgroundChronic inflammation and oxidative stress play important role in development of hypertension. Recently, we have reported novel fluorophenyl benzimidazole (FPD) for vasorelaxation and antihypertensive activity in SHRs. The present study envisaged the anti-inflammatory, anti-oxidant and cardio-protective properties of FPD in L-NAME model of hypertension with special emphasis on reversal of vascular remodeling, gene expression and restoration of hemodynamic. MethodsAntihypertensive activity of FPD was evaluated in L-NAME treated Wistar rats, and the parameters studied were anti-inflammatory activity, histomorphological changes, gene expression profile and anti-oxidant properties. ResultsFPD at 50 and 100 mg kg−1 once daily for 15 days significantly reduced SBP, DBP and MAP in L-NAME treated rats and the values were well comparable to vehicle control group. Further, FPD treatment showed a significant increase in hepatic GSH content, SOD, catalase activity, decreased MDA level and restoration of pro and anti-inflammatory cytokine levels. The mRNA expression profile of genes associated with regulation of vascular tone, remodeling and inflammation showed a significant level of alteration by chronic L-NAME treatment and was dose-dependently restored upon treatment with FPD. Further, FPD treatment restored serum lipid profile, CK, CK-MB and LDH level and also reversed the histomorphological changes like intimal wall thickening, hyperplasia of cardiomyocytes and ventricular wall thickening. ConclusionsTaken together, FPD produced potent antihypertensive activity in L-NAME model through vasorelaxation, anti-oxidative and anti-inflammatory properties leading to restoration of serum lipid profile, cardiac biomarker, expression profile of target genes and reversal of histomorphological changes.

P1285: IN VIVO IMPACT OF ABERRANT OVEREXPRESSION OF C-REL NF-ΚB SUBUNIT IN GERMINAL CENTER B-LYMPHOCYTES: A TUMOR ONCOGENIC EVENT IN GCB-DLBCLS

Background: Diffuse large B-cell lymphomas (DLBCLs) are the most NF-κB-related aggressive non-Hodgkin’s lymphomas. DLBCLs are clinically, molecularly and genetically heterogeneous, suggesting different oncogenic mechanisms. Gene-expression profiling studies led to the proposal of two main cell-of-origin molecular subtypes known as germinal center B-cell-like (GCB) DLBCLs, and activated B-cell like (ABC) DLBCLs. With the Lymphoma Study Association LYSA, we previously published that strong c-Rel DNA-binding activity was mostly found in GCBs and was associated with increased REL mRNA expression. These cases with both overexpression of REL mRNA and c-Rel related gene expression profile were correlated with various recurrent GCB-DLBCLs genetic events, including REL gains, BCL2 translocation, MEF2B, EZH2, CREBBP, and TNFRSF14 mutations and with the EZB GCB genetic subtype. Aims: We propose that c-Rel is a major oncogene of GCB-DLBCLs. We speculate that c-Rel confers a selective advantage in the germinal center (GC)-specific environment. We wish to study the consequences of aberrant overexpression and activation of c-Rel in vivo that could promote tumorigenesis in DLBCL precursor cells by disrupting the normal dynamics of B-cell development in the GC. Methods: We have generated the fl_STOP-REL mouse model: the murine REL transgene preceded by a STOP-Neomycin cassette flanked of loxP sites was introduced in Rosa26 locus on chromosome 6. The REL transgene is followed by an IRES to allow YFP marker concomitant expression. To reach GC B-cell specific expression, we used AID_Cre-ert2 mice: the Cre-ert2 transgene was at the AICDA gene locus on chromosome 6. On the same chromosome, the tdTomato marker preceded by a STOP cassette flanked by loxP sites was introduced at the Rosa26 locus. A pool of REL overexpressing GC B-cell was generated after tamoxifen administration and T-cell-dependent antigen stimulation with sheep red blood cells (SRBC). The fate of these YFP and tdTomato positive GC B-cells was followed over-time until 8 months. The first four months, a SRBC immune booster was performed to maintain the GCs. Results: As expected, our model fl_STOP-REL | AID_Cre-ert2 (i.e. REL mice) allowed for in vivo competition between c-Rel overexpressing (tdTomato+/YFP+) and normal (tdTomato+/YFP-) GC B-cells. In blood, the percentage of YFP positive B-lymphocytes among the tdTomato positive cells was increased over-time: 30% at 14 days, 65% at 2 months and about 80% at 8 months. From blood, spleen and lymph nodes, phenotyping analysis by flow cytometry of REL/YFP positive B-cells indicated an activated profile compared to normal GC-derived B-cells: CD19+, IgM+, IgD-, CD21+, CD80+, and B220low. At 8 months after generation of GC B-cells pool, approximatively 3-5% of B-cells were tdTomato and YFP positives in spleen of REL mice, whereas tdTomato positives cells in control mice were rare hardly detectable (less than 0.8%). Altogether, our result indicated that c-Rel reprograms B-cell to be activated and to better survive in vivo for very long time. Image:Summary/Conclusion: We bring strong experimental arguments showing that increased c-Rel expression in GC B-cell favors their survival for several months, and would thus be a primary event. We suspect that additional oncogenic events are probably needed for tumor clonal expansion such as BCL2, EZH2, CREBBP, TNFRSF14 and/or c-Myc dysregulation.

Probing Notch1-Dll4 signaling in regulating osteogenic differentiation of human mesenchymal stem cells using single cell nanobiosensor

Human mesenchymal stem cells (hMSCs) have great potential in cell-based therapies for tissue engineering and regenerative medicine due to their self-renewal and multipotent properties. Recent studies indicate that Notch1-Dll4 signaling is an important pathway in regulating osteogenic differentiation of hMSCs. However, the fundamental mechanisms that govern osteogenic differentiation are poorly understood due to a lack of effective tools to detect gene expression at single cell level. Here, we established a double-stranded locked nucleic acid (LNA)/DNA (LNA/DNA) nanobiosensor for gene expression analysis in single hMSC in both 2D and 3D microenvironments. We first characterized this LNA/DNA nanobiosensor and demonstrated the Dll4 mRNA expression dynamics in hMSCs during osteogenic differentiation. By incorporating this nanobiosensor with live hMSCs imaging during osteogenic induction, we performed dynamic tracking of hMSCs differentiation and Dll4 mRNA gene expression profiles of individual hMSC during osteogenic induction. Our results showed the dynamic expression profile of Dll4 during osteogenesis, indicating the heterogeneity of hMSCs during this dynamic process. We further investigated the role of Notch1-Dll4 signaling in regulating hMSCs during osteogenic differentiation. Pharmacological perturbation is applied to disrupt Notch1-Dll4 signaling to investigate the molecular mechanisms that govern osteogenic differentiation. In addition, the effects of Notch1-Dll4 signaling on hMSCs spheroids differentiation were also investigated. Our results provide convincing evidence supporting that Notch1-Dll4 signaling is involved in regulating hMSCs osteogenic differentiation. Specifically, Notch1-Dll4 signaling is active during osteogenic differentiation. Our results also showed that Dll4 is a molecular signature of differentiated hMSCs during osteogenic induction. Notch inhibition mediated osteogenic differentiation with reduced Alkaline Phosphatase (ALP) activity. Lastly, we elucidated the role of Notch1-Dll4 signaling during osteogenic differentiation in a 3D spheroid model. Our results showed that Notch1-Dll4 signaling is required and activated during osteogenic differentiation in hMSCs spheroids. Inhibition of Notch1-Dll4 signaling mediated osteogenic differentiation and enhanced hMSCs proliferation, with increased spheroid sizes. Taken together, the capability of LNA/DNA nanobiosensor to probe gene expression dynamics during osteogenesis, combined with the engineered 2D/3D microenvironment, enables us to study in detail the role of Notch1-Dll4 signaling in regulating osteogenesis in 2D and 3D microenvironment. These findings will provide new insights to improve cell-based therapies and organ repair techniques.

A novel four-gene signature for predicting the prognosis of hepatocellular carcinoma

Objective To identify and utilize gene signatures for the prognostic evaluation of postoperative patients with hepatocellular carcinoma (HCC). Methods The gene mRNA expression profiles and corresponding clinicopathological data of postoperative patients with HCC were downloaded from The Cancer Genome Atlas (TCGA) database. Highly differentially expressed genes (DEGs) in tumor tissues compared to adjacent tissues were identified, and their associations with the overall survival (OS) of HCC patients were analyzed. The strongly associated genes were used to develop a prognostic score for the survival stratification of HCC, and the underlying mechanisms were analyzed using bioinformatics. Results A total of 376 DEGs were identified and four DEGs (ADH4, COL15A1, RET and KCNJ16) were independently associated with OS. A prognostic score derived from the four genes could effectively stratify HCC patients with different OS outcomes, independent of clinical parameters. Patients with high scores exhibited poorer OS than patients with low scores (HR 5.526, 95% CI: 2.451–12.461, p < .001). The four genes were involved in cancer-related biological processes and were independent of each other in bioinformatics analyses. Conclusion Four genes strongly associated with the prognosis of postoperative patients with HCC were identified, and the derived prognostic score was simple and valuable for overall survival prediction.

Hairy cell leukemia: a specific 17-gene expression signature points to new targets for therapy

BackgroundHairy cell leukemia (HCL) is a rare chronic B cell malignancy, characterized by infiltration of bone marrow, blood and spleen by typical “hairy cells” that bear the BRAFV600E mutation. However, in addition to the intrinsic activation of the MAP kinase pathway as a consequence of the BRAFV600E mutation, the potential participation of other signaling pathways to the pathophysiology of the disease remains unclear as the precise origin of the malignant hairy B cells.Materials and methodsUsing mRNA gene expression profiling based on the Nanostring technology and the analysis of 290 genes with crucial roles in B cell lymphomas, we defined a 17 gene expression signature specific for HCL.ResultsSeparate analysis of samples from classical and variant forms of hairy cell leukemia showed almost similar mRNA expression profiles apart from overexpression in vHCL of the immune checkpoints CD274 and PDCD1LG2 and underexpression of FAS. Our results point to a post-germinal memory B cell origin and in some samples to the activation of the non-canonical NF-κB pathway.ConclusionsThis study provides a better understanding of the pathogenesis of HCL and describes new and potential targets for treatment approaches and guidance for studies in the molecular mechanisms of HCL.

Metabolomics based mechanistic insights to vasorelaxant and cardioprotective effect of ethanolic extract of Citrullus lanatus (Thunb.) Matsum. & Nakai. seeds in isoproterenol induced myocardial infraction

BackgroundCardiovascular diseases (CVDs) are a significant cause of morbidity and death in the current world, posing a challenge to both developing and industrialized nation's health systems. Citrullus lanatus (Thunb.) Matsum. & Nakai. seeds have long been utilized to supplement and enhance health and treat cardiovascular illnesses. However, its treatments for CVDs are still unknown. More research is required to fully comprehend the impact of C. lanatus seeds on vasorelaxation and myocardial infractions. PurposeTherefore, an integrated metabolomics profiling technique was used to investigate possible pathways of C. lanatus in isoproterenol (ISO)-induced myocardial infarction (MI). Isoproterenol causes long-term cardiac hypertrophy by causing cardiomyocyte compensatory loss, eventually leading to heart failure. MethodsIn vitro models of vasoconstriction, atrium, and in vivo models of invasive blood pressure measurement and isoproterenol (ISO) induced cardiac hypertrophy in rats were used to understand underlying mechanistic by LC-MS/MS based dynamic metabolomics analysis of the serum and heart samples to be investigated the effect of ethanolic extract of C. lanatus (Cl.EtOH). ResultsCl.EtOH exhibited vasorelaxant, negative chronotropic, and inotropic effects in in-vitro models whereas, a potent hypotensive effect was observed in normotensive rats. The Cl.EtOH protected the animals from ISO-induced myocardial infarction (MI) with therapeutic interventions in left ventricular thickness, cardiomyocyte hypertrophy, mRNA gene expression, biochemical assays, and metabolomic profiling of serum and heart tissues. ConclusionsFor the first time, our study confirmed that C. lanatus seeds (Cl.EtOH) possess significant antihypertensive and prevent ISO-induced myocardial infarction. These findings comprehensively demonstrated mechanistic insights of Cl.EtOH in vasorelaxation and myocardial infarction. The current study provides evidence for further mechanistic studies and the development of C. lanatus seeds as a potential therapeutic intervention for patients with cardiovascular disorders.

Computational Identification of Guillain-Barré Syndrome-Related Genes by an mRNA Gene Expression Profile and a Protein-Protein Interaction Network.

BackgroundIn the present study, we used a computational method to identify Guillain–Barré syndrome (GBS) related genes based on (i) a gene expression profile, and (ii) the shortest path analysis in a protein–protein interaction (PPI) network.Materials and MethodsmRNA Microarray analyses were performed on the peripheral blood mononuclear cells (PBMCs) of four GBS patients and four age- and gender-matched healthy controls.ResultsTotally 30 GBS-related genes were screened out, in which 20 were retrieved from PPI analysis of upregulated expressed genes and 23 were from downregulated expressed genes (13 overlap genes). Gene ontology (GO) enrichment and KEGG enrichment analysis were performed, respectively. Results showed that there were some overlap GO terms and KEGG pathway terms in both upregulated and downregulated analysis, including positive regulation of macromolecule metabolic process, intracellular signaling cascade, cell surface receptor linked signal transduction, intracellular non-membrane-bounded organelle, non-membrane-bounded organelle, plasma membrane, ErbB signaling pathway, focal adhesion, neurotrophin signaling pathway and Wnt signaling pathway, which indicated these terms may play a critical role during GBS process.DiscussionThese results provided basic information about the genetic and molecular pathogenesis of GBS disease, which may improve the development of effective genetic strategies for GBS treatment in the future.

Peroxiredoxin 6 Applied after Exposure Attenuates Damaging Effects of X-ray Radiation in 3T3 Mouse Fibroblasts.

Although many different classes of antioxidants have been evaluated as radioprotectors, none of them are in widespread clinical use because of their low efficiency. The goal of our study was to evaluate the potential of the antioxidant protein peroxiredoxin 6 (Prdx6) to increase the radioresistance of 3T3 fibroblasts when Prdx6 was applied after exposure to 6 Gy X-ray. In the present study, we analyzed the mRNA expression profiles of genes associated with proliferation, apoptosis, cellular stress, senescence, and the production of corresponding proteins from biological samples after exposure of 3T3 cells to X-ray radiation and application of Prdx6. Our results suggested that Prdx6 treatment normalized p53 and NF-κB/p65 expression, p21 levels, DNA repair-associated genes (XRCC4, XRCC5, H2AX, Apex1), TLR expression, cytokine production (TNF-α and IL-6), and apoptosis, as evidenced by decreased caspase 3 level in irradiated 3T3 cells. In addition, Prdx6 treatment reduced senescence, as evidenced by the decreased percentage of SA-β-Gal positive cells in cultured 3T3 fibroblasts. Importantly, the activity of the NRF2 gene, an important regulator of the antioxidant cellular machinery, was completely suppressed by irradiation but was restored by post-irradiation Prdx6 treatment. These data support the radioprotective therapeutic efficacy of Prdx6.