Altered Gene Expression Profiles Research Articles

Transcriptomic analysis of glucosidase II beta subunit (GluIIß) knockout A549 cells reveals its roles in regulation of cell adhesion molecules (CAMs) and anti-tumor immunity

Glucosidase II beta subunit (GluIIß), encoded from PRKCSH, is a subunit of the glucosidase II enzyme responsible for quality control of N-linked glycoprotein folding and suppression of GluIIß led to inhibitory effect of the receptor tyrosine kinase (RTKs) activities known to be critical for survival and development of cancer. In this study, we investigated the effect of GluIIß knockout on the global gene expression of cancer cells and its impact on functions of immune cells. GluIIß knockout lung adenocarcinoma A549 cell line was generated using CRISPR/Cas9-based genome editing system and subjected to transcriptomic analysis. Among 23,502 expressed transcripts, 1068 genes were significantly up-regulated and 807 genes greatly down-regulated. The KEGG enrichment analysis showed significant down-regulation of genes related extracellular matrix (ECM), ECM-receptor interaction, cytokine-cytokine receptor interaction and cell adhesion molecules (CAMs) in GluIIß knockout cells. Of 9 CAMs encoded DEG identified by KEGG enrichment analysis, real time RT-PCR confirmed 8 genes to be significantly down-regulated in all 3 different GluIIß knockout clones, which includes cadherin 4 (CDH4), cadherin 2 (CDH2), versican (VCAN), integrin subunit alpha 4 (ITGA4), endothelial cell-selective adhesion molecule (ESAM), CD274 (program death ligand-1 (PD-L1)), Cell Adhesion Molecule 1 (CADM1), and Nectin Cell Adhesion Molecule 3 (NECTIN3). Whereas PTPRF (Protein Tyrosine Phosphatase Receptor Type F) was significantly decreased only in 1 out of 3 knockout clones. Microscopic analysis revealed distinctively different cell morphology of GluIIβ knockout cells with lesser cytoplasmic and cell surface area compared to parental A549 cells and non-targeted transfected cells.Further investigations revealed that Jurkat E6.1 T cells or human peripheral blood mononuclear cells (PBMCs) co-cultured with GluIIß knockout A549 exhibited significantly increased viability and tumor cell killing activity compared to those co-cultured with non-target transfected cells. Analysis of cytokine released from Jurkat E6.1 T cells co-cultured with GluIIß knockout A549 cells showed significant increased level of angiogenin and significant decreased level of ENA-78. In conclusion, knockout of GluIIß from cancer cells induced altered gene expression profile that improved anti-tumor activities of co-cultured T lymphocytes and PBMCs thus suppression of GluIIß may represent a novel approach of boosting anti-tumor immunity.

Unstable environmental conditions constrain the fine-tune between opsin sensitivity and underwater light in an Amazon forest stream fish.

Visual adaptations can stem from variations in amino acid composition, chromophore utilization, and differential opsin gene expression levels, enabling individuals to adjust their light sensitivity to environmental lighting conditions. In stable environments, adaptations often involve amino acid substitutions, whereas in unstable conditions, differential gene expression may be a more relevant mechanism. Amazon forest streams present diverse underwater lighting conditions and experience short-term water colour fluctuations. In these environments, it is less likely for genetic and amino acid sequences to undergo modifications that tailor opsin proteins to the prevailing lighting conditions, particularly in species having several copies of the same gene. The sailfin tetra, Crenuchus spilurus, inhabits black and clear water Amazon forest streams. The long-wavelength sensitivity (LWS) is an important component for foraging and courtship. Here, we investigated LWS opsin genes in the sailfin tetra. Three copies of LWS1 and two copies of LWS2 genes were found. The maximum absorbance wavelength (λmax) estimated from the amino acid sequences of LWS1 genes exhibited variation among the different copies. In contrast, the copies of LWS2 genes showed identical expected λmax values. Although the amino acid positions affecting λmax varied among LWS genes, they remained consistent among populations living in different water colours. The relative expression levels of LWS genes differed between gene copies. While not formally tested, our results suggest that in fluctuating environments, visual adaptations may primarily stem from alterations in gene expression profiles and/or chromophore usage rather than precise genetic tuning of protein light sensitivity to environmental lighting conditions.

DEEPSIDE A DEEP LEARNING FRAMEWORK FOR DRUG SIDE EFFECT PREDICTION

Drug failures brought on by unanticipated side effects during clinical trials put participants' health at risk and result in significant financial losses. Algorithms for predicting side effects may direct the development of new drugs. The LINCS L1000 dataset establishes a knowledge foundation for context-specific characteristics and offers a wealth of cell line gene expression data that has been altered by various medications. The state-of-the-art method, which seeks to use context-specific information, discards a significant number of the trials and only uses the high-quality experiments in LINCS L1000. Our aim in this research is to maximize the prediction performance by fully using this data. Five deep learning architectures are used in our experiments. We discover that when drug chemical structure (CS) and the whole collection of drug altered gene expression profiles (GEX) are employed as modalities, a multi-modal architecture yields the greatest prediction performance among multi-layer perceptron-based designs. We find that overall, the CS provides more information than the GEX. The best results are obtained by a convolutional neural network-based model that just employs the SMILES string representation of the medications; this model outperforms the state-of-the-art by 3:1% micro-AUC and 13:0% macro-AUC. Additionally, we demonstrate that the model can forecast drug-side effect couples that are absent from the ground truth side effect dataset but are described in the literature.

Dual PI3K/mTOR inhibitor PF-04979064 regulates tumor growth in gastric cancer and enhances drug sensitivity of gastric cancer cells to 5-FU

Gastric cancer (GC) is characterized by high tumor heterogeneity, increased surgical difficulty, and limited chemotherapy efficacy, and it is associated with a poor prognosis. The abnormal proliferation of cells involves abnormal activation of the PI3K/AKT/mTOR signaling pathway. Inhibition of this signaling pathway can inhibit tumor cell proliferation and induce cell apoptosis. This study evaluated the effect of PF-04979064, a dual inhibitor of PI3K and mTOR, on human GC cells. PF-04979064 significantly inhibited the proliferation of human gastric adenocarcinoma AGS cells and the undifferentiated GC cell line HGC-27, promoting cell apoptosis. Combination treatment with PF-04979064 and the GC first-line clinical drug 5-FU showed synergistic effects, and PF-04979064 markedly increased the sensitivity of GC cells to chemotherapy drugs. Western blot results showed that PF-04979064 significantly inhibited the PI3K/AKT/mTOR signaling pathway in GC cells, whereas RNA seq results demonstrated substantial alterations in gene expression profiles upon treatment with PF-04979064. This study provides insight into the effects of PF-04979064, thereby establishing a solid foundation for its potential clinical application in the treatment of GC.

Methylation and transcriptomic expression profiles of HUVEC in the oxygen and glucose deprivation model and its clinical implications in AMI patients.

The obstructed coronary artery undergoes a series of pathological changes due to ischemic-hypoxic shocks during acute myocardial infarction (AMI). However, the altered DNA methylation levels in endothelial cells under these conditions and their implication for the etiopathology of AMI have not been investigated in detail. This study aimed to explore the relationship between DNA methylation and pathologically altered gene expression profile in human umbilical vein endothelial cells (HUVECs) subjected to oxygen-glucose deprivation (OGD), and its clinical implications in AMI patients. The Illumina Infinium MethylationEPIC BeadChip assay was used to explore the genome-wide DNA methylation profile using the Novaseq6000 platform for mRNA sequencing in 3 pairs of HUVEC-OGD and control samples. GO and KEGG pathway enrichment analyses, as well as correlation, causal inference test (CIT), and protein-protein interaction (PPI) analyses identified 22 hub genes that were validated by MethylTarget sequencing as well as qRT-PCR. ELISA was used to detect four target molecules associated with the progression of AMI. A total of 2,524 differentially expressed genes (DEGs) and 22,148 differentially methylated positions (DMPs) corresponding to 6,642 differentially methylated genes (DMGs) were screened (|Δβ|>0.1 and detection p < 0.05). After GO, KEGG, correlation, CIT, and PPI analyses, 441 genes were filtered. qRT-PCR confirmed the overexpression of VEGFA, CCL2, TSP-1, SQSTM1, BCL2L11, and TIMP3 genes, and downregulation of MYC, CD44, BDNF, GNAQ, RUNX1, ETS1, NGFR, MME, SEMA6A, GNAI1, IFIT1, and MEIS1. DNA fragments BDNF_1_ (r = 0.931, p < 0.0001) and SQSTM1_2_NEW (r = 0.758, p = 0.0043) were positively correlated with the expressions of corresponding genes, and MYC_1_ (r = -0.8245, p = 0.001) was negatively correlated. Furthermore, ELISA confirmed TNFSF10 and BDNF were elevated in the peripheral blood of AMI patients (p = 0.0284 and p = 0.0142, respectively). Combined sequencing from in vitro cellular assays with clinical samples, aiming to establish the potential causal chain of the causal factor (DNA methylation) - mediator (mRNA)-cell outcome (endothelial cell ischemic-hypoxic injury)-clinical outcome (AMI), our study identified promising OGD-specific genes, which provided a solid basis for screening fundamental diagnostic and prognostic biomarkers of coronary endothelial cell injury of AMI. Moreover, it furnished the first evidence that during ischemia and hypoxia, the expression of BNDF was regulated by DNA methylation in endothelial cells and elevated in peripheral blood.

DNA methylation extends lifespan in the bumblebee Bombus terrestris.

Epigenetic alterations are a primary hallmark of ageing. In mammals, age-related epigenetic changes alter gene expression profiles, disrupt cellular homeostasis and physiological functions and, therefore, promote ageing. It remains unclear whether ageing is also driven by epigenetic mechanisms in invertebrates. Here, we used a pharmacological hypomethylating agent (RG108) to evaluate the effects of DNA methylation (DNAme) on lifespan in an insect-the bumblebee Bombus terrestris. RG108 extended mean lifespan by 43% and induced the differential methylation of genes involved in hallmarks of ageing, including DNA damage repair and chromatin organization. Furthermore, the longevity gene sirt1 was overexpressed following the treatment. Functional experiments demonstrated that SIRT1 protein activity was positively associated with lifespan. Overall, our study indicates that epigenetic mechanisms are conserved regulators of lifespan in both vertebrates and invertebrates and provides new insights into how DNAme is involved in the ageing process in insects.

Taurine supplementation alters gene expression profiles in white adipose tissue of obese C57BL/6J mice: Inflammation and lipid synthesis perspectives

This work aimed to identify the mechanisms by which taurine exerts its anti-obesity effects in the C57BL/6J ob/ob mice model and determine if taurine supplementation increases the amelioration of inflammation and lipogenesis linked genes in the adipose and liver tissues. Three groups of C57BL/6J mice were fed a standard chow diet for a period of 10 weeks the C57BL/6J normal group, the C57BL/6J ob/ob negative control group with no taurine intake and C57BL/6J ob/ob taurine group with taurine intake. Real time PCR was used to examine the gene expression profile in the experimental groups intrascapular brown adipose tissue (BAT), inguinal white adipose tissue (WAT) and liver. TNF-alpha, Ccl2, Adgre and illb genes that are associated with inflammation were found to have varying level of expression in the three tissues. In comparison to BAT and liver these genes were expressed at a much lower level in WAT, with enhanced serum adiponectin levels.

De Novo RNA Sequencing and Transcriptome Analysis of Sclerotium rolfsii Gene Expression during Sclerotium Development.

Sclerotium rolfsii is a destructive soil-borne fungal pathogen that causes stem rot in cultivated plants. However, little is known about the genetic basis of sclerotium development. In this study, we conducted de novo sequencing of genes from three different stages of S. rolfsii (mycelia, early sclerotium formation, and late sclerotium formation) using Illumina HiSeqTM 4000. We then determined differentially expressed genes (DEGs) across the three stages and annotated gene functions. STEM and weighted gene-co-expression network analysis were used to cluster DEGs with similar expression patterns. Our analysis yielded an average of 25,957,621 clean reads per sample (22,913,500-28,988,848). We identified 8929, 8453, and 3744 DEGs between sclerotium developmental stages 1 versus 2, 1 versus 3, and 2 versus 3, respectively. Additionally, four significantly altered gene expression profiles involved 220 genes related to sclerotium formation, and two modules were positively correlated with early and late sclerotium formation. These results were supported by the outcomes of qPCR and RNA-sequencing conducted on six genes. This is the first study to provide a gene expression map during sclerotial development in S. rolfsii, which can be used to reduce the re-infection ability of this pathogen and provide new insights into the scientific prevention and control of the disease. This study also provides a useful resource for further research on the genomics of S. rolfsii.

CX43-Mediated Mitochondria Transfer Enhances the Stemness of Leukemia Stem Cells Though Metabolic Remodeling

Background :Despite improvements in treatment options for acute myeloid leukemia (AML), chemotherapy resistance remains an important clinical concern with a high recurrence rate in patients.Leukemia stem cells (LSCs) play a decisive role in the relapse resistance of leukemia. Bone marrow mesenchymal stem cells(BMSCs) protect LSCs from being completely removed from the bone marrow, but the underlying mechanisms are poorly understood.In this study, we aimed to uncover the mechanism of BMSCs-regulated the stemness of leukemia stem cells through mitochondrial transfer mediated by Connexin43(CX43), thus providing a promising clinical therapy target. Methods: Primary BMSCs were isolated from the primary bone marrow specimens of patients with AML and cultured in MSCM culture base within 10% serum.CX43 overexpressing BMSCs (CX43-BMSCS) were constructed by lentivirus transfection.The efficiency of CX43 overexpression was verified by immunofluorescence, qPCR and Western Blot.In vitro, CX43-BMSCs and control BMSCs (EV-BMSCs) were cocultured with KG-1a cells.In some experiments,vincristine (1nM) was added to inhibit the mitochondrial transferring.Bidirectional mitochondrial transfer was detected by flow cytometry and confocal microscopy.The mechanism of mitochondria transfer was analyzed by inhibitor assays.The proliferation of KG-1a from the coculture system were assessed by Flow cytometry and EdU assay.After co-culture, the clone formation of KG-1a was detected by using semisolid cloning assay.The mitochondrial respiration of KG-1a was analyzed by using Seahorse.The transcriptomes of KG-1a after co-culture using RNA-seq.Finally,we verified our findings using LC-MS non-targeted metabolomics to detect the metabolites changes of KG-1a after co-culture. Results: Compared with Control-BMSCs and EV-BMSCs,the mRNA and protein expression levels of CX43 in CX43-MSCs were significantly increased (P&amp;lt;0.05).In the co-culture system without serum for 48h,BMSCs transfer mitochondria to KG-1a cells but receive no mitochondria from KG-1a.Moreover,CX43-BMSCs transferred more mitochondria to KG-1a than EV-BMSCs group (P&amp;lt;0.05),while there was no obvious mitochondrial transfer was observed in the transwell and 10% serum co-culture system.In addition,compared with EV-BMSCs,CX43-BMSCs significantly increased the proliferation and adhesion of KG-1a (P&amp;lt;0.05) and clone formation ability (P&amp;lt;0.05).Vincristine significantly reduced the mitochondrial transfer from CX43-BMSCs to KG-1a,and decreased the adhesion of CX43-BMSCs to KG-1a,as well as the proliferation and clone formation of KG-1a after co-culture.The metabolic capacity of KG-1a cells in CX43-BMSCs was significantly increased compared with EV-BMSCs (P&amp;lt;0.05),and this metabolic enhancement could be reversed by vincristine.Hierarchical cluster analysis (HC) of metabolites showed that there were significant differences in metabolites among treatment groups (p&amp;lt;0.05),showed unique metabolic characteristics related to CX43-induced changes.The GO analysis revealed that the enrichments occurring in KG-1a after co-cultured with CX43-BMSCs mainly included cell adhesion and migration,cell proliferation and regulation,signal transduction,PI3K/Akt pathway,transmembrane transport and intracellular protein metabolism.Metabolism-related pathways,like nucleotide metabolism,lipid metabolism and amino acid metabolism,were enriched in KG-1a after co-cultured with CX43-BMSCs.Furthermore,KG-1a after co-cultured with CX43-BMSCs showed up-regulated of promoting the proliferation and the stemness of AML-LSCs,including N-cadherin (CDH2),CD244,SLC43A2,CYBB and IGLL1. Conclusion: We show evidence that CX43 mediated mitochondria transfer from BMSCs to LSCs which promoting the adhesion of BMSCs to LSCs under nutrient deficiency condition,and then enhanced the proliferation and cloning ability of LSCs,altered gene expression profiles related to cell proliferation,adhesion,and energy metabolism,and increased the expression of genes related to maintaining stemness of LSCs, allows metabolic remodeling to regulate LSCs stemness, may be a potential therapeutic target for AML treatment. Keywords:Leukemia stem cells;Bone marrow microenvironment;Connexin;Mitochondrial transfer;Metabolic remodeling

Identification of cell-intrinsic effects of mutated circulating cells due to clonal hematopoiesis in patients with heart failure

Abstract Clonal hematopoiesis of indeterminate potential (CHIP) is caused by somatic mutations in hematopoietic stem cells and is associated with worse prognosis in patients with heart failure with reduced ejection fraction (HFrEF). Circulating immune cells derived from patients with HFrEF harboring CHIP mutations show enhanced proinflammatory signatures. However, it is unclear, whether these signatures are derived from the relatively low number of cells harboring CHIP-driver mutations, or are indicators of a systemic pro-inflammatory activation associated with CHIP. Methods and Results We assessed the cell-intrinsic effects of the most prevalent CHIP-driver gene (DNMT3A) mutant-carrying circulating immune cells in a total of 5 patients with HFrEF. Using an improved protocol to detect mutant cells on a single cell level (MutDetect-Seq), as illustrated in figure 1A, we show that CHIP mutations can be detected in all major immune cell types (e.g. T cells, monocytes, B cells, NK cells). DNMT3A mutant monocytes demonstrated the highest number of significantly upregulated genes relative to other cell types. Specifically, DNMT3A mutant monocytes exhibit altered gene expression profiles associated with inflammation and phagolysosome function (figure 1B). In addition, DNMT3A mutant carrying CD4+, but not CD8+ T cells, showed significant changes in gene expression patterns towards effector cells and away from a naïve T cell state. Finally, increased paracrine signaling pathways involved in amplifying inflammatory circuits are predicted between mutated and non-mutated monocytes. Conclusion This is the first study to decipher the genetic signature of DNMT3A-CHIP-driver mutation carrying circulating immune cells, thereby providing first evidence for the cell intrinsic effects of DNMT3A mutations on peripheral blood cell subsets in patients with HFrEF. The altered gene expression profiles associated with inflammation and phagolysosome function of mutant monocyte as well as the skewing of mutated T cells towards effector cells provide novel insights into how CHIP might promote worse prognosis in heart failure patients.MutDetect-seq

Abstract 17672: Implications of Pcsk9 on Abdominal Aortic Aneurysm

Objective: Recent genome-wide association studies revealed a potential role of proprotein convertase subtilisin/kexin type9(Pcsk9) in the human abdominal aortic aneurysm(AAA) disease.Altered Pcsk9 gene expression and smaller AAA diameter were observed in murine AAA using the porcine pancreatic elastase(PPE) model in our lab. Pcsk9 increases plasma LDL-cholesterol,enhances tissue inflammation and induces vascular smooth muscle cell (VSMC) senescence, hallmarks in AAA.However, the exact cellular mechanisms of Pcsk9 in AAA disease remain unclear.Despite the absence of a pharmacological treatment for AAA, monoclonal Pcsk9 antibody therapies have proven effective against statin-resistant hypercholesterolemia.This study aimed to explore the impacts of Pcsk9 on aortic vascular inflammation and VSMC dedifferentiation with a view to better understanding AAA pathophysiology and providing a basis for future investigations into Pcsk9 inhibitors. Hypothesis: We hypothesized that local vascular injury escalates Pcsk9 expression,amplifying vascular inflammation and driving VSMC towards dedifferentiation that further advances AAA. Conversely,Pcsk9 inhibition will mitigate these processes. Methods: AAA was induced by PPE model in wildtype C57Bl/6 mice and Pcsk9 knock-out (KO)mice.Sonography was used to evaluate the aneurysm growth. Single-cell RNA sequencing was employed to examine local changes in the aortic wall. Local vascular Pcsk9,pro-inflammatory and VSMC marker gene/protein expression were quantified using qPCR and immunohistochemistry.VSMCs were treated with exogenous Pcsk9 in vitro to assess metabolism via Seahorse. Results: Reduced AAA diameters were observed in Pcsk9 KO mice compared to wildtype controls. Significant upregulation of local Pcsk9 expression accompanied AAA induction. Single-cell RNA sequencing revealed altered gene expression profiles across vascular cell populations. In vitro,VSMCs displayed changes in marker gene/protein expression after treatment with exogenous Pcsk9, alongside metabolic changes (Mann-Whitney Test). Conclusions: The findings suggest a significant role of Pcsk9 in AAA pathogenesis. Its inhibition appears to mitigate AAA growth,indicating a promising treatment target for AAA.

Gray matter volume abnormalities in vascular cognitive impairment and their association with gene expression profiles

BackgroundIt has been revealed that brain gray matter volume (GMV) abnormalities are present in patients with vascular cognitive impairment (VCI). However, the GMV alterations that have been uncovered are highly inconsistent, and their correlation with gene expression profiles is still largely unknown. PurposeTo establish a correlation between VCI-related GMV alterations and gene expression patterns and uncover potential genetic profiles underlying GMV abnormalities in VCI. Materials and methodsHere, a quantitative meta-analysis that compared voxel-based GMV between VCI patients and healthy controls (HCs) was carried out on 11 datasets (10 from previous studies and 1 newly collected), comprising 385 VCI individuals and 334 ​HCs, to investigate GMV alterations in VCI patients. Partial least squares regression analysis was then conducted to investigate the relationship between the GMV alterations in VCI and gene expression profiles obtained from Allen Human Brain Atlas database. ResultsCompared with healthy controls, patients with VCI showed consistent decreased GMV which predominantly included the right insula, right Rolandic operculum, right putamen, right superior temporal gyrus, left medial superior frontal gyrus, and right median cingulate and paracingulate gyri. Meta-regression analysis revealed that decreased GMV in left medial superior frontal gyrus was negatively correlated with Mini-Mental State Examination score in VCI. Furthermore, 2835 genes were identified whose expression patterns were correlated with VCI-related GMV changes, and these genes were enriched in distinct biological processes, brain cell types and lifespan windows across brain regions. ConclusionTogether, these findings could provide the potential neurobiological underpinnings and the genetic substrates underlying GMV abnormalities of VCI.

Analysis of fatty acid composition and miRNA profile in adipose tissue after application of Allium mongolicum Regel extract in diet of sheep

Extensive regulation of microRNAs (miRNAs) that belong to a type of endogenous small noncoding RNAs on gene expression in sheep has been recognised. Meanwhile, utilisation of water extract of Allium mongolicum Regel (WEA) in diet of sheep has been proved to regulate fatty acid composition and flavour in mutton. However, the regulatory mechanism remains unclear. We conducted miRNA and mRNA sequencing in this study. Briefly, 30 male small-tailed Han sheep (4.5 months old) were randomly assigned to two groups. A basal diet with 3.4 g·sheep−1·d−1 WEA supplementation (WEA group) or with no WEA supplementation (CON group) was fed to sheep, respectively. Subcutaneous and visceral adipose tissue samples were collected during slaughter after 75-day feeding experiment. We obtained 30 differentially expressed miRNAs (DE-miRNAs) after application of WEA in diet of sheep in subcutaneous adipose tissue. Likewise, 15 DE-miRNAs in visceral adipose tissue were identified. A total of 746 differentially expressed mRNAs (DE-mRNAs) with 90 lipid-related DE-mRNAs in subcutaneous adipose tissue and 569 DE-mRNAs with 73 lipid-related DE-mRNAs in visceral adipose tissue were identified after WEA supplementation. The intersection genes from predicted target genes of DE-miRNAs by databases and actual DE-mRNAs by mRNA sequencing were enriched in several lipid-related pathways both in subcutaneous adipose and in visceral adipose tissue. We constructed differential miRNAs-mRNAs networks by Cytoscape software. Our results will help to explain regulatory mechanism of fatty acid composition in mutton by dietary WEA and will provide a theoretical basis for rational use of the plant extract in diet of sheep. HIGHLIGHTS Utilisation of Allium mongolicum Regel water extract led to alterations of endogenous miRNA and gene expression profiles in subcutaneous and visceral adipose tissue of sheep. Allium mongolicum Regel water extract improved nutritional value and flavour of mutton by changing fatty acid composition in adipose tissue. Establishment of miRNA-mRNA regulatory network with regard to manipulation of fatty acid composition in mutton by Allium mongolicum Regel water extract, and provided a theoretical basis for rational utilisation of this plant extract in diet of sheep.

A highly specific antibody against the core fucose of the N-glycan in IgG identifies the pulmonary diseases and its regulation by CCL2

Glycan structure is often modulated in disease or predisease states, suggesting that such changes might serve as biomarkers. Here, we generated a monoclonal antibody (mAb) against the core fucose of the N-glycan in human IgG. Notably, this mAb can be used in Western blotting and ELISA. ELISA using this mAb revealed a low level of the core fucose of the N-glycan in IgG, suggesting that the level of acore fucosylated (noncore fucosylated) IgG was increased in the sera of the patients with lung cancer, chronic obstructive pulmonary disease, and interstitial pneumonia compared to healthy subjects. In a coculture analysis using human lung adenocarcinoma A549cells and antibody-secreting B cells, the downregulation of the FUT8 (α1,6 fucosyltransferase) gene and a low level of core fucose of the N-glycan in IgG in antibody-secreting B cells were observed after coculture. A dramatic alteration in gene expression profiles for cytokines, chemokines, and their receptors were also observed after coculturing, and we found that the identified C-C motif chemokine 2 was partially involved in the downregulation of the FUT8 gene and the low level of core fucose of the N-glycan in IgG in antibody-secreting B cells. We also developed a latex turbidimetric immunoassay using this mAb. These results suggest that communication with C-C motif chemokine 2 between lung cells and antibody-secreting B cells downregulate the level of core fucose of the N-glycan in IgG, i.e., the increased level of acore fucosylated (noncore fucosylated) IgG, which would be a novel biomarker for the diagnosis of patients with pulmonary diseases.

Characterizing Early Cardiac Metabolic Programming via 30% Maternal Nutrient Reduction during Fetal Development in a Non-Human Primate Model.

Intra-uterine growth restriction (IUGR) is a common cause of fetal/neonatal morbidity and mortality and is associated with increased offspring predisposition for cardiovascular disease (CVD) development. Mitochondria are essential organelles in maintaining cardiac function, and thus, fetal cardiac mitochondria could be responsive to the IUGR environment. In this study, we investigated whether in utero fetal cardiac mitochondrial programming can be detectable in an early stage of IUGR pregnancy. Using a well-established nonhuman IUGR primate model, we induced IUGR by reducing by 30% the maternal diet (MNR), both in males (MNR-M) and in female (MNR-F) fetuses. Fetal cardiac left ventricle (LV) tissue and blood were collected at 90 days of gestation (0.5 gestation, 0.5 G). Blood biochemical parameters were determined and heart LV mitochondrial biology assessed. MNR fetus biochemical blood parameters confirm an early fetal response to MNR. In addition, we show that in utero cardiac mitochondrial MNR adaptations are already detectable at this early stage, in a sex-divergent way. MNR induced alterations in the cardiac gene expression of oxidative phosphorylation (OXPHOS) subunits (mostly for complex-I, III, and ATP synthase), along with increased protein content for complex-I, -III, and -IV subunits only for MNR-M in comparison with male controls, highlight the fetal cardiac sex-divergent response to MNR. At this fetal stage, no major alterations were detected in mitochondrial DNA copy number nor markers for oxidative stress. This study shows that in 90-day nonhuman primate fetuses, a 30% decrease in maternal nutrition generated early in utero adaptations in fetal blood biochemical parameters and sex-specific alterations in cardiac left ventricle gene and protein expression profiles, affecting predominantly OXPHOS subunits. Since the OXPHOS system is determinant for energy production in mitochondria, our findings suggest that these early IUGR-induced mitochondrial adaptations play a role in offspring's mitochondrial dysfunction and can increase predisposition to CVD in a sex-specific way.

The effects of heat stress on intrauterine development, reproductive function, and ovarian gene expression of F1 female mice as well as gene expression of F2 embryos†.

Exposure to heat stress (HS) in utero was postulated to trigger an adaptive molecular response that can be transmitted to the next generation. Hence, this study assessed the impact of HS exposure at different stages of the gestational period of mice on the female F1 population and their offspring. Heat stress exposure (41°C and 65% relative humidity-RH) occurred during the first half (FP), the second half (SP), or the entire pregnancy (TP). A control group (C) was maintained in normothermic conditions (25°C, 45% RH) throughout the experiment. Heat stress had a significant negative effect on intrauterine development, mainly when HS exposure occurred in the first half of pregnancy (FP and TP groups). Postnatal growth of FP and TP mice was hindered until 4 weeks of age. The total number of follicles per ovary did not vary (P > 0.05) between the control and HS-exposed groups. Mean numbers of primordial follicles were lower (P < 0.05) in the sexually mature FP than those in SP and TP F1 females. However, the mean number of viable embryos after superovulation was lower (P < 0.05) in TP compared with C group. The expression of genes associated with physiological and cellular response to HS, autophagy, and apoptosis was significantly affected in the ovarian tissue of F1 females and F2 in vivo-derived blastocysts in all HS-exposed groups. In conclusion, exposure to HS during pregnancy compromised somatic development and reproductive parameters as well as altered gene expression profile that was then transmitted to the next generation of mice.

SAT275 Generation And Characterization Of A Humanized CYP21A2 Mouse Model For Congenital Adrenal Hyperplasia

Abstract Disclosure: A. Huebner: None. S. Thirumalasetty: None. T. Schubert: None. R. Naumann: None. I. Reichardt: None. M. Rohm: None. D. Landgraf: None. F. Gembardt: None. M.F. Hartmann: None. S.A. Wudy: None. M. Peitzsch: None. N. Reisch: None. K. Koehler, PhD: None. 21-hydroxylase deficiency (21OHD) is the most common form of congenital adrenal hyperplasia (CAH) and is caused by mutations in the CYP21A2 gene. 21OHD causes a wide array of clinical symptoms that result from gluco- and mineralocorticoid deficiency and adrenal androgen excess. Treatment firstly aims to substitute lacking steroid hormones, and secondly to restore negative feedback towards CRH and pituitary ACTH secretion to diminish adrenal androgen overproduction. In most cases supra-physiological glucocorticoid doses are necessary which may cause short stature, obesity, hypertension, and cardiovascular and metabolic co-morbidity with reduced quality of life. Hence, current steroid substitution regimens have significant limitations, so novel therapeutic strategies are required. In recent years new therapeutic approaches have emerged including new non-glucocorticoid substances interfering with the HPA axis to minimize adrenal androgen production and to lower external glucocorticoid substitution to physiological levels. However, valuable in-vivo models for pre-clinical testing of such drugs are lacking. Here we present the first viable and humanized mouse model in which the mouse gene Cyp21a1 is replaced by the human orthologue CYP21A2 in which the human point mutation R484Q is integrated. Twenty-weeks-old homozygous mice show marked adrenal hyperplasia, enhanced expression of the CYP21A2 gene and a weak increase of Cyp11a1 and Cyp11b2 gene expression. Tandem mass spectrometry measurements of the mice plasma at 20 weeks show decreased corticosterone and 11-deoxycorticosterone levels in the presence of increased ACTH levels in both male and female homozygous animals. Progesterone levels in homozygous mice are significantly higher (p&amp;lt;0.01) than in wildtype mice. We also observed increased aldosterone levels in female mutants whereas blood pressure does not differ between wildtype and mutant mice strains. Histologically, mutants exhibit adrenocortical hyperplasia. While mutant male mice are fertile with normal appearing testes, females are infertile, remain in the diestrus phase and present with a reduced number of ovarian follicles. In parallel, a second mouse strain bearing the I173N mutation was developed. This mutation is frequent in human patients causing simple virilizing or rarely salt wasting CAH. Homozygous mice require dexamethasone treatment during pregnancy and until weaning but are viable without treatment afterwards. Preliminary results show adrenal hyperplasia and alteration in steroidogenic gene expression and steroid profiles. In conclusion, we demonstrate that the humanized mutant CYP21A2 mice may represent an excellent animal CAH model to test novel treatment strategies for CAH patients. We believe that this model(s) will facilitate the transition from basic research into clinical application. Presentation: Saturday, June 17, 2023

Adverse effect of environmental androgenic compounds Galaxolide and Irgacure 369 on the male reproductive system

Our recent study has found that two environmental chemicals, Galaxolide (HHCB, a raw material for synthesizing musk) and Irgacure 369 (IC-369, a photoinitiator used in packaging) are agonists for the androgen receptor in vitro and in vivo. This study aims to reveal the subchronic reproductive toxicity of these two compounds in mature male rats. The results showed that compared with the control group, HHCB and IC-369 reduced the sperm concentration and motility, increased the sperm deformity, and caused the atrophy of the seminiferous tubules in the testicles. Exposure to HHCB and IC-369 reduced testosterone level, and induced luteinizing hormone, and follicle-stimulating hormone levels in rat serum. Compared with the control group, the levels of oxidative stress markers in the serum and testicular tissue increased. HHCB and IC-369 also inhibited expression of the genes involved in androgen synthesis in testicle. The above results indicated that HHCB and IC-369 could affect the levels of sex hormones, alter gene expression profiles and induce histological damage in reproductive organs, resulting in decreased sperm quality. Therefore, HHCB and IC-369 have endocrine disruptors with prominent reproductive toxicity in males.

BM-MSCs display altered gene expression profiles in B-cell acute lymphoblastic leukemia niches and exert pro-proliferative effects via overexpression of IFI6

BackgroundThe tumor microenvironment (TME) is a supportive environment responsible for promoting the growth and proliferation of tumor cells. Current studies have revealed that the bone marrow mesenchymal stem cells (BM-MSCs), a type of crucial stromal cells in the TME, can promote the malignant progression of tumors. However, in the adult B-cell acute lymphoblastic leukemia (B-ALL) microenvironment, it is still uncertain what changes in BM-MSCs are induced by leukemia cells.MethodsIn this study, we mimicked the leukemia microenvironment by constructing a BM-MSC–leukemia cell co-culture system. In vitro cell experiments, in vivo mouse model experiments, lentiviral transfection and transcriptome sequencing analysis were used to investigate the possible change of BM-MSCs in the leukemia niche and the potential factors in BM-MSCs that promote the progression of leukemia.ResultsIn the leukemia niche, the leukemia cells reduced the MSCs' capacity to differentiate towards adipogenic and osteogenic subtypes, which also promoted the senescence and cell cycle arrest of the MSCs. Meanwhile, compared to the mono-cultured MSCs, the gene expression profiles of MSCs in the leukemia niche changed significantly. These differential genes were enriched for cell cycle, cell differentiation, DNA replication, as well as some tumor-promoting biofunctions including protein phosphorylation, cell migration and angiogenesis. Further, interferon alpha-inducible protein 6 (IFI6), as a gene activated by interferon, was highly expressed in leukemia niche MSCs. The leukemia cell multiplication was facilitated evidently by IFI6 both in vitro and in vivo. Mechanistically, IFI6 might promote leukemia cell proliferation by stimulating SDF-1/CXCR4 axis, which leads to the initiation of downstream ERK signaling pathway. As suggested by further RNA sequencing analysis, the high IFI6 level in MSCs somewhat influenced the gene expression profile and biological functions of leukemia cells.ConclusionsBM-MSCs in the leukemia niche have varying degrees of changes in biological characteristics and gene expression profiles. Overexpression of IFI6 in BM-MSCs could be a key factor in promoting the proliferation of B-ALL cells, and this effect might be exerted through the SDF-1/CXCR4/ERK signal stimulation. Targeting IFI6 or related signaling pathways might be an important measure to reduce the leukemia cell proliferation.

Cell-intrinsic effects of clonal hematopoiesis in heart failure.

Clonal hematopoiesis of indeterminate potential (CHIP) is caused by somatic mutations in hematopoietic stem cells and associates with worse prognosis in patients with heart failure. Patients harboring CHIP mutations show enhanced inflammation. However, whether these signatures are derived from the relatively low number of cells harboring mutations or are indicators of systemic pro-inflammatory activation that is associated with CHIP is unclear. Here we assess the cell-intrinsic effects of CHIP mutant cells in patients with heart failure. Using an improved single-cell sequencing pipeline (MutDetect-Seq), we show that DNMT3A mutant monocytes, CD4+ T cells and NK cells exhibit altered gene expression profiles. While monocytes showed increased genes associated with inflammation and phagocytosis, T cells and NK cells present increased activation signatures and effector functions. Increased paracrine signaling pathways are predicted and validated between mutant and wild-type monocytes and T cells, which amplify inflammatory circuits. Altogether, these data provide novel insights into how CHIP might promote a worse prognosis in patients with heart failure.