Expression Levels Of Isoforms Research Articles

Cannabidiol’s cytotoxicity in pancreatic cancer is induced via an upregulation of ceramide synthase 1 and ER stress

Pancreatic ductal adenocarcinoma (PDAC) remains one of the most aggressive malignancies with a median 5 year-survival rate of 12%. Cannabidiol (CBD) has been found to exhibit antineoplastic potential and may potentiate the anticancer effects of cytotoxic’s such as gemcitabine. CBD therapy has been linked to de novo synthesis of ceramide. The sphingolipid ceramide is a potent tumour suppressor lipid with roles in apoptosis and autophagy. One of the key players involved is ceramide synthase, an enzyme with six isoforms (CerS1-CerS6), reported to have disease prognostic value. Quantitative real time PCR was used to determine mRNA expression levels of ceramide synthase isoforms, GRP78, ATF4 and CHOP. Western blotting was used to analyze protein expression of these markers and knockdown of CerS1 and GRP78 were applied via an siRNA and confirmed by the two mentioned methods. Mice with PDAC xenografts were injected via intraperitoneal method with drugs and tumours were analysed with flow cytometry and processed using H&E and IHC staining. siRNA knockdown of ceramide synthase 1 (CerS1) and analysis point to evidence of a putative CerS1 dependent pathway driven by CBD in activating endoplasmic reticulum (ER) stress target; GRP78. Upon CBD treatment, CerS1 was upregulated and downstream this led to the GRP78/ATF4/CHOP arm of the unfolded protein response (UPR) pathway being activated. In an in vivo model of PDAC in which CerS1 was not upregulated on IHC, there was no observed improvement in survival of animals, however a reduction in tumour growth was observed in combination chemotherapy and CBD group, indicating further investigations in vivo. These findings provide evidence of a potential ceramide induced cytotoxic mechanism of action of CBD in pancreatic ductal adenocarcinoma.

Highly conserved and extremely variable: The paradoxical pattern of toxin expression revealed by comparative venom-gland transcriptomics of Phalotris (Serpentes: Dipsadidae)

Although non-front fanged snakes account for almost two-thirds of snake diversity, most studies on venom composition and evolution focus exclusively on front-fanged species, which comprise most of the clinically relevant accidents. Comprehensive reports on venom composition of non-front fanged snakes are still scarce for several groups. In this study, we address such shortage of knowledge by providing new insights about the venom composition among species of Phalotris, a poorly studied Neotropical dipsadid genus. Phalotris are known for their specialized venom delivery system and toxic venoms, which can cause life-threatening accidents in humans. We evaluate the venom-gland transcriptome of Phalotris, comparing the following three South American species: P. reticulatus for the Araucaria Pine forests, P. lemniscatus for the Pampa grasslands, and P. mertensi for the Brazilian Cerrado. Our results indicate similar venom profiles, in which they share a high expression level of Kunitz-type inhibitors (KUNZ). On the other hand, comparative analyses revealed substantial differences in the expression levels of C-type lectins (CTL) and snake venom metalloproteinases (SVMP). The diverse set of SVMP and CTL isoforms shows signals of positive selection, and we also identified truncated forms of type III SVMPs, which resemble type II and type I SVMPs of viperids. Additionally, we identified a CNP precursor hosting a proline-rich region containing a BPP motif resembling those commonly detected in viperid venoms with hypotensive activity. Altogether, our results suggest an evolutionary history favoring high expression levels of few KUNZ isoforms in Phalotris venoms, contrasting with a highly diverse set of SVMP and CTL isoforms. Such diversity can be comparable with the venom variability observed in some viperids. Our findings highlight the extreme phenotypic diversity of non-front fanged snakes and the importance to allocate greater effort to study neglected groups of Colubroidea.

Pregnancy influences expression of interferon-stimulated genes, progesterone receptor and progesterone-induced blocking factor in ovine thyroid.

Embryonic interferon-tau (IFNT) and progesterone affect expression of interferon-stimulated genes (ISGs), progesterone receptor (PGR) and progesterone-induced blocking factor (PIBF) in the ovine thyroid. Thyroids of ewes were sampled at day 16 of nonpregnancy, days 13, 16 and 25 of pregnancy, and RT-qPCR assay, western blot and immunohistochemistry were used to detect expression of ISGs, PGR and PIBF. Free ISG15 protein was undetected, but ISG15 conjugated proteins upregulated at day 16 of pregnancy, and expression levels of ISG15 conjugated proteins, PGR isoform (70 kDa), PIBF, interferon-gamma-inducible protein 10 and myxovirusresistance protein 1 peaked, but expression level of signal transducer and activator of transcription 1 was the lowest at day 16 of pregnancy. In addition, the expression levels of PGR isoform (70 kDa) and STAT1 decreased, but levels of PGR isoform (43 kDa), 2',5'-oligoadenylate synthetase, IP-10 and MX1 increased at day 25 of pregnancy comparing with day 16 of the estrous cycle. Early pregnancy affects expression of ISGs, PGR and PIBF in maternal thyroid through IFNT and progesterone, which may regulate thyroid autoimmunity and thyroid hormone secretion in ewes.

Overexpression of WT1 in all molecular subtypes of breast cancer and its impact on survival: exploring oncogenic and tumor suppressor roles of distinct WT1 isoforms.

Breast cancer is a highly heterogeneous solid tumor, posing challenges in developing targeted therapies effective for all mammary carcinoma subtypes. WT1 emerges as a promising target for breast cancer therapy due to its potential oncogenic role in various cancer types. Previous works have yielded inconsistent results. Therefore, further studies are needed to clarify the behavior of this complex gene in breast cancer. In this study, we examined WT1 expression in both Formalin Fixed Paraffin Embedded breast tumors (n = 41) and healthy adjacent tissues (n = 41) samples from newly diagnosed cases of ductal invasive breast cancer. The fold change in gene expression between the tumor and healthy tissue was determined by calculating 2-∆∆Ct. Disease-free survival analysis was computed using the Kaplan-Meier method. To identify the expression levels of different WT1 isoforms, we explored the ISOexpresso database. Relative quantification of the WT1 gene revealed an overexpression of WT1 in most cases. The percentage of patients surviving free of disease at 8years of follow-up was lower in the group overexpressing WT1 compared to the group with down-regulated WT1. Interestingly, this overexpression was observed in all molecular subtypes of invasive breast cancer, underscoring the significance of WT1 as a potential target in all these subtypes. The observed WT1 down-expression in a few cases of invasive breast cancer, associated with better survival outcomes, may correspond to the down-regulation of a particular WT1-KTS (-) isoform: the WT1 A isoform (EX5-/KTS-). The co-expression of this WT1 oncogenic isoform with a regulated WT1- tumor suppressor isoform, such as the major WT1 F isoform (EX5-/KTS +), could also explain such survival outcomes. Due to its capacity to adopt dual roles, it becomes imperative to conduct individual molecular expression profiling of the WT1 gene. Such an approach holds great promise in the development of personalized treatment strategies for breast cancer.

Peritumor Mucosa in Advanced Laryngeal Carcinoma Exhibits an Aberrant Proangiogenic Signature Distinctive from the Expression Pattern in Adjacent Tumor Tissue.

The field cancerization theory is an important paradigm in head and neck carcinoma as its oncological repercussions affect treatment outcomes in diverse ways. The aim of this study is to assess the possible interconnection between peritumor mucosa and the process of tumor neoangiogenesis. Sixty patients with advanced laryngeal carcinoma were enrolled in this study. The majority of patients express a canonical HIF-upregulated proangiogenic signature with almost complete predominancy of HIF-1α overexpression and normal expression levels of the HIF-2α isoform. Remarkably, more than 60% of the whole cohort also exhibited an HIF-upregulated proangiogenic signature in the peritumoral benign mucosa. Additionally, the latter subgroup had a distinctly shifted phenotype towards HIF-2α upregulation compared to the one in tumor tissue, i.e., a tendency towards an HIF switch is observed in contrast to the dominated by HIF-1α tumor phenotype. ETS-1 displays stable and identical significant overexpression in both the proangiogenic phenotypes present in tumor and peritumoral mucosa. In the current study, we report for the first time the existence of an abnormal proangiogenic expression profile present in the peritumoral mucosa in advanced laryngeal carcinoma when compared to paired distant laryngeal mucosa. Moreover, we describe a specific phenotype of this proangiogenic signature that is significantly different from the one present in tumor tissue as we delineate both phenotypes, quantitively and qualitatively. This finding is cancer heterogeneity, per se, which extends beyond the "classical" borders of the malignancy, and it is proof of a strong interconnection between field cancerization and one of the classical hallmarks of cancer-the process of tumor neoangiogenesis.

Expression of versican isoforms V0/V1 by pancreatic cancer associated fibroblasts increases fibroblast proliferation

BackgroundVersican is a large extracellular matrix (ECM) proteoglycan with four isoforms V0-3. Elevated V0/V1 levels in breast cancer and glioma regulate cell migration and proliferation, but the role of versican in pancreatic ductal adenocarcinoma (PDAC) remains unclear. MethodsIn this study, we evaluated the expression levels of versican isoforms, as well as their cellular source and interacting partners, in vivo, in human and mouse primary and metastatic PDAC tumours and in vitro, in pancreatic tumour cells and fibroblasts using immunostaining, confocal microscopy and qPCR techniques. We also investigated the effect of versican expression on fibroblast proliferation and migration using genetic and pharmacological approaches. ResultsWe found that versican V0/V1 is highly expressed by cancer-associated fibroblasts (CAFs) in mouse and human primary and metastatic PDAC tumours. Our data also show that exposing fibroblasts to tumour-conditioned media upregulates V0 and V1 expressions, while Verbascoside (a CD44 inhibitor) downregulates V0/V1 expression. Importantly, V0/V1 knockdown significantly inhibits fibroblast proliferation. Mechanistically, we found that inhibiting hyaluronan synthesis does not affect versican co-localisation with CD44 in fibroblasts. ConclusionCAFs express high levels of versican V0/V1 in primary and liver metastatic PDAC tumours and versican V0/V1 supports fibroblast proliferation.

Abstract 550: Identification of isoform switching events linked with esophageal adenocarcinoma patient survival informs novel prognostic and therapeutic targets

Abstract Esophageal adenocarcinoma (EAC) represents a growing health problem characterized by rising incidence and poor prognosis. Although it is a cancer with a high mutational burden, it lacks highly prevalent oncogenic drivers that can be therapeutically targeted. Recently, our group characterized isoform switching events, defined by the changing of dominant gene isoforms between conditions, associated with EAC progression and identified 75 switching events comparing Barrett’s low-grade dysplasia (BE.LGD) and Barrett’s high-grade dysplasia (BE.HGD) patient samples. Herein, we further investigated whether isoform-switching events may offer prognostic or therapeutic value in EAC. Isoform switching analysis was performed using RNA-seq data derived from patient samples between BE.LGD and BE.HGD combined with EAC (BE.HGD + EAC) alone or in combination with TP53 mutations. Patients were further stratified into tertiles based on isoform expression level followed by survival analysis. Thirty isoforms were significantly (P≤0.05) linked with all-cause mortality, comparing BE.LGD with BE.HGD + EAC, whereas 20 isoforms were identified when TP53 mutation status was included. In terms of cancer-specific mortality, 10 isoforms were significantly linked with survival comparing BE.LGD with BE.HGD+EAC. With the inclusion of TP53 mutation, 16 isoforms were also significantly linked with cancer-specific mortality. Isoform-specific genetic knockdown experiments were designed targeting the top isoforms identified in the analysis, including TTLL12 and HM13 isoforms. Knockdown of TTLL12 and HM13 isoforms led to significant inhibition of cell viability and migration in two EAC cell lines (OE19 and OE33). Chemotherapeutic agents (Paclitaxel + Carboplatin) further decreased cell viability with synergistic effects observed in both siRNA-treated cell lines suggesting a role for specific isoform switches in therapeutic sensitization. Protein-interaction prediction using STRING and immunoblot analysis suggest different mechanisms leading to the inhibition of cell viability and migration. TTLL12 is linked with the activation of chaperon-medicated autophagy (LAMP2A and HSC70) and protein translation, whereas HM13 is linked with unfolded protein response (IRE1α, PERK, and ATF6) and may be linked with immunotherapy outcome. An antigen-dependent cell-mediated cytotoxicity assay is currently underway to investigate whether the knockdown of HM13 isoforms can sensitize EAC cells to PD-1/PD-L1 immunotherapy agents. Future direction includes using FDA-approved agents to target the two major pathways identified above. In conclusion, isoform switching may provide fresh insight for the identification of prognostic markers and inform new potential therapeutic targets for EAC treatment or prevention. Citation Format: Yun Zhang, Rachel Israel, Bryce Vandenburg, Shari Barnett, Jean-Jack Riethoven, Jennifer L. Clarke, Michelle P. Lee, Kiran Lagisetty, Jules Lin, Rishindra Reddy, Andrew Chang, Laura A. Kresty. Identification of isoform switching events linked with esophageal adenocarcinoma patient survival informs novel prognostic and therapeutic targets [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 550.

Abstract 5448: Malignant RNA editing by inflammation-responsive RNA editase ADAR1 drives T-cell leukemia relapse via attenuating dsRNA sensing

Abstract Leukemia-initiating cells (LICs) play a crucial role in leukemia research and treatment, being considered the source of relapse and resistance, posing a substantial challenge for successful management of the disease. It's crucial to identify the key factors that drive LIC self-renewal to develop targeted methods for eliminating LICs and preventing relapse. In this study, we demonstrate that the RNA editing enzyme ADAR1 plays a vital role as a determinant of stemness, promoting LIC self-renewal by reducing the detection of abnormal double-stranded RNA (dsRNA). As a result, ADAR1-knockdown significantly impairs the ability of LICs to self-renew and extends survival in T-ALL PDX models. We performed RNA-seq studies on enriched T-ALL LICs (CD34+Lin-). The lentivirus-to-cell ratio was optimized to achieve a 50% reduction in ADAR1, resulting in differential expression of 661 genes, with downregulation of critical self-renewal genes in ADAR1-deficient leukemia-initiating cells. Importantly, additional data indicated that ADAR1 likely influences self-renewal gene expression independently of A-to-I RNA editing, revealing novel regulatory mechanisms in leukemia. Knockdown of MDA5 and ADAR1 in patient-derived xenograft (PDX) T-ALL leukemia-initiating cells (LICs) revealed diverse rescue effects on self-renewal in different PDX models. The differential response was associated with variations in gene expression patterns in Rig-I-Like signaling and cytosolic sensing pathways, suggesting intrinsic differences in IFN signaling properties among patients. Moreover, the study proposes a patient-specific reliance on the ADAR1 p150-MDA5 axis for promoting self-renewal, as evidenced by differential rescue effects and varying expression levels of ADAR1 isoforms and MDA5 in T-ALL patient samples and cell lines. ADAR1, beyond its role in RNA editing, acts as a dsRNA binding protein, exerting RNA editing-independent functions to suppress aberrant IFN signaling and promote cancer progression. Using Jurkat cells with high intrinsic MDA5 levels, an ADAR1 knockout (KO) was generated, revealing that ADAR1 is upregulated by IFN treatment, predominantly the p150 isoform. We explored the dual roles of ADAR1—RNA editing-dependent and -independent—in suppressing IFN-induced apoptosis, demonstrating the necessity of both for optimal function. Additionally, ADAR1 was found to suppress cytoplasmic dsRNA sensing pathways, such as RIG-I and PKR, and its RNA editing-independent activity contributes significantly to the suppression of interferon-stimulated gene (ISG) expression in T-ALL. In summary, our findings reveal that ADAR1 acts as a factor that promotes self-renewal while limiting the recognition of naturally occurring dsRNA. Therefore, targeting ADAR1 emerges as a safe and effective therapeutic approach for eradicating LICs in T-ALL. Citation Format: Maria Dolores Rivera, Haoran Zhang, Qingchen Zhou, Jessica Pham, Jane Isquith, Roman Sasik, Adam Mark, Sabina Enlund, Frida Holm, Ma Wenxue, Kathleen Fisch, Dennis Kuo, Catriona Jamieson, Qingfei Jiang. Malignant RNA editing by inflammation-responsive RNA editase ADAR1 drives T-cell leukemia relapse via attenuating dsRNA sensing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5448.

Abstract 862: Multi-omic analysis of allelic expression patterns of imprinted and non-imprinted genes in cancer and their association with drug response

Abstract Malignant cells undergo a broad array of epigenetic changes, including changes in imprinting patterns. In normal cells, imprinted genes have parent of origin-specific monoallelic expression, which is controlled via epigenetic mechanisms. Imprinted gene loci are often complex and frequently include multiple isoforms, which have different individual patterns of parent-of-origin monoallelic or biallelic expression in the normal tissues. Many imprinted genes promote cell proliferation and body growth, and their expression is important during embryonic and postnatal development. Dysregulation of imprinting in tumors has been suggested to contribute to growth and proliferation of cancer cells. Previously we reported that chemotherapy responses of cancer cell lines from a variety of tumors and of patient-derived acute myeloid leukemia samples were associated with copy number, expression, or DNA methylation of selected imprinted genes. In the current project, we examined allele-specific patterns of expression (monoallelic vs. biallelic) of imprinted genes and isoforms in tumors and studied their potential effect on response of cancer cells to drug treatment. We analyzed expression patterns at the whole gene, isoform, and exon levels of 59,283 autosomal protein-coding genes and ncRNA genes, with a special focus on 94 imprinted genes and their isoforms. We developed a computational pipeline to evaluate monoallelic and biallelic expression patterns of each gene in cancer cell lines and in tumor samples based on available matched RNA-seq expression data, whole exome sequencing information, and copy number data. Our initial analysis included 108 cell lines from the Cancer Cell Line Encyclopedia (CCLE), which had been derived from 9 pediatric and adult tumor histologies. We observed variation of allelic expression patterns of imprinted genes and their isoforms in tumor cell lines, including differences across and within cancer histologies. We also observed predominantly monoallelic expression of multiple additional genes. Some of them (PRIM2, BCLAF1, MAP2K3, and SEC22B) had been previously reported as potentially imprinted. Monoallelic expression of other genes identified in our analysis (e.g., GRK2, CBX4, and additional genes) was likely caused by molecular mechanisms other than imprinting. Allelic expression patterns and overall expression levels of isoforms of multiple imprinted and non-imprinted monoallelically expressed genes (e.g., CPA4, MAP2K3, and other genes) were associated with in vitro drug response. We also report our ongoing validation of allelic expression patterns of imprinted and non-imprinted genes, isoforms, and exons in primary bladder and breast tumors and in matched solid normal tissue samples using patient data from the Cancer Genome Atlas (TCGA). These results provide new knowledge about epigenetic dysregulation in cancer. Citation Format: Julia Krushkal, Travis L. Jensen, George Wright, Yingdong Zhao. Multi-omic analysis of allelic expression patterns of imprinted and non-imprinted genes in cancer and their association with drug response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 862.

A single 24 h maternal separation at PND 9 promotes behavioral resilience of female C57BL/6J mice and the possible role of hippocampal Homer1a

Early life stress (ELS) has been thought to increase vulnerability to developing psychiatric disorders later in life, while some researchers have found that adversity early in life may promote stress resilience. Studies investigating the resilient effect of maternal separation (MS) are still relatively few, and the underlying mechanisms remain unknown. In the current study, the effect of a single 24 h MS paradigm at postnatal day 9 (PND 9) in female C57BL/6J mice was investigated by assessing behavioral performance in middle adolescence. We demonstrated that, mice in MS group displayed decreased anxiety-like behavior and increased exploratory behavior than controls in the open field test and elevated plus maze test. Furthermore, MS mice exhibited improved hippocampal-dependent spatial learning in the Morris water maze test. This performance indicated behavioral resilience to early life stress. The protein expression levels of Homer1 isoforms, which are implicated in a variety of neuropsychiatric disorders, were evaluated using Western blot analysis. A significant increase in hippocampal Homer1a protein expression was observed immediately after MS, which subsequently decreased until adolescence (PND 27–42), when a significant increase was observed again. This distinctive change of hippocampal Homer1a protein expression pattern indicated that hippocampal Homer1a might play a role in behavioral resilience to MS in female C57BL/6J mice. In conclusion, this study demonstrated that exposure to a single 24 h MS at PND 9 promoted behavioral resilience of female C57BL/6J mice in middle adolescence. This behavioral resilience might be related to increased expression of hippocampal Homer1a.

Accounting for isoform expression increases power to identify genetic regulation of gene expression.

A core problem in genetics is molecular quantitative trait locus (QTL) mapping, in which genetic variants associated with changes in the molecular phenotypes are identified. One of the most-studied molecular QTL mapping problems is expression QTL (eQTL) mapping, in which the molecular phenotype is gene expression. It is common in eQTL mapping to compute gene expression by aggregating the expression levels of individual isoforms from the same gene and then performing linear regression between SNPs and this aggregated gene expression level. However, SNPs may regulate isoforms from the same gene in different directions due to alternative splicing, or only regulate the expression level of one isoform, causing this approach to lose power. Here, we examine a broader question: which genes have at least one isoform whose expression level is regulated by genetic variants? In this study, we propose and evaluate several approaches to answering this question, demonstrating that "isoform-aware" methods-those that account for the expression levels of individual isoforms-have substantially greater power to answer this question than standard "gene-level" eQTL mapping methods. We identify settings in which different approaches yield an inflated number of false discoveries or lose power. In particular, we show that calling an eGene if there is a significant association between a SNP and any isoform fails to control False Discovery Rate, even when applying standard False Discovery Rate correction. We show that similar trends are observed in real data from the GEUVADIS and GTEx studies, suggesting the possibility that similar effects are present in these consortia.

IFLAS: positive-unlabeled learning facilitates full-length transcriptome-based identification and functional exploration of alternatively spliced isoforms in maize.

The advent of full-length transcriptome sequencing technologies has accelerated the discovery of novel splicing isoforms. However, existing alternative splicing (AS) tools are either tailored for short-read RNA-Seq data or designed for human and animal studies. The disparities in AS patterns between plants and animals still pose a challenge to the reliable identification and functional exploration of novel isoforms in plants. Here, we developed integrated full-length alternative splicing analysis (iFLAS), a plant-optimized AS toolkit that introduced a semi-supervised machine learning method known as positive-unlabeled (PU) learning to accurately identify novel isoforms. iFLAS also enables the investigation of AS functions from various perspectives, such as differential AS, poly(A) tail length, and allele-specific AS (ASAS) analyses. By applying iFLAS to three full-length transcriptome sequencing datasets, we systematically identified and functionally characterized maize (Zea mays) AS patterns. We found intron retention not only introduces premature termination codons, resulting in lower expression levels of isoforms, but may also regulate the length of 3'UTR and poly(A) tail, thereby affecting the functional differentiation of isoforms. Moreover, we observed distinct ASAS patterns in two genes within heterosis offspring, highlighting their potential value in breeding. These results underscore the broad applicability of iFLAS in plant full-length transcriptome-based AS research.

FpOGT is required for fungal growth, stress response, and virulence of Fusarium proliferatum by affecting the expression of glucokinase and other glucose metabolism-related genes

O-GlcNAcylation, an important post-translational modification catalyzed by O-GlcNAc transferase (OGT), plays critical roles in several biological processes. In this study, we present our findings on the function of FpOGT in regulating physiological processes and pathogenicity of Fusarium proliferatum (Fp), the alfalfa root rot fungus. The deletion of FpOGT impaired mycelial growth and altered macroconidia morphology in Fp. Furthermore, ΔFpOGT mutant displayed altered tolerance to various stressors, including cell wall perturbing agents, osmotic stressors, metal ionic stressors, and fungicides. Deletion of FpOGT significantly decreased Fp virulence toward alfalfa. The transcriptome analysis demonstrated that FpOGT plays a regulatory role in glucose metabolic pathways, including glycolysis, tricarboxylic acid (TCA) cycle, and hexosamine biosynthesis pathway (HBP), by influencing the expression of relevant genes. The downregulation of the glucokinase gene, FpGCK, was observed in ΔFpOGT, and the disruption of FpGCK led to a decrease in Fp virulence. Additionally, FpOGT affected the expression levels of the FpGCK-AS1 isoform, thereby impacting glucokinase function. The molecular docking analysis elucidated the plausible physical interaction between FpOGT and FpGCK, thereby offering valuable insights into their interrelationship. These findings underscore the indispensable involvement of FpOGT, the sole O-GlcNAc transferase in Fp, in various biological processes and the pathogenicity through its regulation of fundamental metabolic processes. Consequently, this study emphasizes the significance and elucidates the molecular mechanism underlying the role of O-GlcNAc transferase in diverse fundamental biological processes and the pathogenicity of phytopathogenic fungi.

Gene expression changes of isocitrate dehydrogenase 1 and isocitrate dehydrogenase 2 affect carcinogenesis and survival probability

Isocitrate dehydrogenase (IDH) is an essential metabolic enzyme in the regulation of cellular metabolism. IDH gene encodes three protein isoforms, IDH1, IDH2, and IDH3, and the expression level of isoforms is altered in human cancer types. Examining the gene expression level of IDH is a therapeutic advantage that could help find a new target to use in cancer metabolism. The present study aimed to explore the gene expression level of IDH1 and IDH2 isoforms in the ten common human cancers using bioinformatic tools. In addition, the effect of gene expression changes on IDH1 and IDH2 on carcinogenesis and survival probability was examined in publicly available data deposited in the TCGA database. The results showed that the expression of IDH isoforms showed tissue-specific differences. IDH1 expression increased in esophageal and lung squamous cell carcinoma and lung and stomach adenocarcinoma tumors. Bladder urothelial, breast urothelial, and lung squamous cell carcinoma, colon, and lung adenocarcinoma displayed a significant upregulation of IDH2 expression. There was a direct relationship between the expression of IDH isoforms and the progression of various cancer types. High IDH1 expression led to decreased survival probability in esophageal carcinoma, lung, and stomach adenocarcinoma. Elevated IDH2 expression level led to decreased survival probability in bladder urothelial, breast urothelial, and lung squamous cell carcinoma and colon adenocarcinoma. In conclusion, all data showed that IDH1 could be a biomarker for esophageal carcinoma, lung and stomach adenocarcinoma, and IDH2 for bladder urothelial, breast urothelial, and lung squamous cell carcinoma, and colon adenocarcinoma.

Genome-wide characterization of soybean malate dehydrogenase genes reveals a positive role of GmMDH2 in salt stress response

Malate dehydrogenase (MDH) is a widely expressed enzyme that plays a key role in plant growth, development, and the stress response. However, information on MDH genes in the soybean genome is limited. Seventeen members of the soybean MDH family were identified by genome-wide analysis, and the presence of conserved protein motifs was analyzed. The genes were divided into five clusters according to their phylogenetic relationships. The intracellular localizations of six GmMDHs were determined by confocal microscopy on Arabidopsis mesophyll protoplasts. Transcripts of GmMDHs were significantly increased by abiotic stress (drought, salt, and alkalinity) and hormone treatments, as shown by analysis of cis-regulatory elements and quantitative real-time polymerase chain reaction (qRT-PCR). GmMDHs displayed unique expression patterns in diverse soybean tissues. It is noteworthy that under salt stress, the expression levels of a chloroplast isoform (GmMDH2) were unusually high, presumably indicating a critical role in soybean responses to salinity. Expression of GmMDH2 in Escherichia coli showed that the recombinant enzyme had NADP-dependent MDH activity. The redox states of the nicotinamide adenine dinucleotide phosphate (NADPH) pool and antioxidant activities were shown to be modulated by GmMDH2 gene overexpression, which in turn reduced reactive oxygen species (ROS) formation in transgenic soybean, significantly enhancing the salt stress resistance. Gene-based association analysis showed that variations in GmMDH2 were strongly linked to seedling salt tolerance. A polymorphism possibly associated with salt tolerance was discovered in the promoter region of GmMDH2. These findings not only improve our understanding of the stress response mechanism by identifying and characterizing the MDH gene family throughout the soybean genome but it also identified a potential candidate gene for the future enchancement of salt tolerance in the soybean.

The relative isoform expression levels of isocitrate dehydrogenase in breast cancer: IDH2 is a potential target in MDA-MB-231 cells.

The isocitrate dehydrogenase (IDH) family plays an essential role in metabolism and energy production. The relative expression levels of IDH isoforms (IDH1, IDH2, and IDH3) have prognostic significance in several malignancies, including breast carcinoma. However, the IDH isozyme expression levels in different cancer stages and types have not been determined in breast carcinoma tissues. We analyzed the messenger RNA (mRNA) and protein levels of IDH (IDH1, IDH2, and IDH3A) and α-ketoglutarate (α-KG) in 59 breast carcinoma tissues. The mRNA level of IDH2 was significantly increased at stages 2 and 3 in triple-negative and (ER-/PR-/HER+) breast cancers. However, the elevated α-KG level was only observed in stages 2 and 3, with no differences in the various breast carcinoma types. Western blotting analysis showed that IDH2 protein expression increased in the patient tissues and cell lines. An in vitro study showed IDH2 downregulation in the triple-negative breast cancer cell line MDA-MB-231 that inhibited cell proliferation and migration and induced cell cycle arrest in the G0/G1 phase. These findings suggest that different from IDH1 and IDH3, IDH2 is more highly expressed in stages 2 and 3 breast cancer tissues, especially in triple-negative breast cancer. IDH2 potentially serves as a target to detect unknown mechanisms in breast cancer.

Comprehensive gene profiling of the metabolic landscape of humanized livers in mice

The human liver transcriptome is complex and highly dynamic, e.g. one gene may produce multiple distinct transcripts, each with distinct posttranscriptional modifications. Direct knowledge of transcriptome dynamics, however, is largely obscured by the inaccessibility of the human liver to treatments and the insufficient annotation of the human liver transcriptome at transcript and RNA modification levels. We generated mice that carry humanized livers of identical genetic background and subjected them to representative metabolic treatments. We then analyzed the humanized livers with nanopore single-molecule direct RNA sequencing to determine the expression level, m6A modification and poly(A) tail length of all RNA transcript isoforms. Our system allows for the de novo annotation of human liver transcriptomes to reflect metabolic responses and for the study of transcriptome dynamics in parallel. Our analysis uncovered a vast number of novel genes and transcripts. Our transcript-level analysis of human liver transcriptomes also identified a multitude of regulated metabolic pathways that were otherwise invisible using conventional short-read RNA sequencing. We revealed for the first time the dynamic changes in m6A and poly(A) tail length of human liver transcripts, many of which are transcribed from key metabolic genes. Furthermore, we performed comparative analyses of gene regulation between humans and mice, and between two individuals using the liver-specific humanized mice, revealing that transcriptome dynamics are highly species- and genetic background-dependent. Our work revealed a complex metabolic response landscape of the human liver transcriptome and provides a novel resource to understand transcriptome dynamics of the human liver in response to physiologically relevant metabolic stimuli (https://caolab.shinyapps.io/human_hepatocyte_landscape/). Direct knowledge of the human liver transcriptome is currently very limited, hindering the overall understanding of human liver pathophysiology. We combined a liver-specific humanized mouse model and long-read direct RNA sequencing technology to establish a de novo annotation of the human liver transcriptome and identified a multitude of regulated metabolic pathways that were otherwise invisible using conventional technologies. The extensive regulatory information on human genes we provided could enable basic scientists to infer the pathological relevance of their genes of interest and physician scientists to better pinpoint the changes in metabolic networks underlying a specific pathophysiology.

Bone marrow mesenchymal stem cells to ameliorate experimental autoimmune encephalomyelitis via modifying expression patterns of miRNAs.

Clinical and experimental studies highlighted the significant therapeutic role of Mesenchymal stem cells (MSCs) in neurodegenerative diseases. MSCs possess potent immunomodulatory properties by releasing exosomes, which generate a suitable microenvironment. microRNAs (miRNAs), as one of several effective bioactive molecules of exosomes, influence cellular communication and activities in recipient cells. Recent studies revealed that miRNAs could control the progression of multiple sclerosis (MS) via differentiation and function of T helper cells (Th). Here, we investigated the therapeutic effects of syngeneic-derived BM-MSC in experimental autoimmune encephalomyelitis (EAE) mouse model of MS by evaluating expression profile of miRNAs, pro- and anti-inflammatory in serum and brain tissues. Three-time scheme groups (6th day, 6th & 12th days, and 12th day, of post-EAE induction) were applied to determine the therapeutic effects of intraperitoneally received 1*106 of BM-MSCs. The expression levels of mature isoforms of miR-193, miR-146a, miR-155, miR-21, and miR-326 showed that BM-MSCs treatment attenuated the EAE clinical score and reduced clinical inflammation as well as demyelination. The improved neurological functional outcome associated with enhanced expression of miR-193 and miR-146a, but decreased expression levels of miR-155, miR-21, and miR-326 were followed by suppressing effects on Th1/Th17 immune responses (reduced levels of IFN-γand IL-17 cytokine expression) and induction of Treg cells, immunoregulatory responses (increase of IL-10, TGF-β, and IL-4) in treatment groups. Our findings suggest that BM-MSCs administration might change expression patterns of miRNAs and downstream interactions followed by immune system modulation. However, there is a need to carry out future human clinical trials and complementary experiments.

Glucocorticoid Receptors and Disease Activity in Systemic Lupus Erythematosus Patients

Glucocorticoids (GC) have been widely used to treat patients with systemic lupus erythematosus (SLE). However, GC-insensitivity remains a major barrier in achieving remission. Therefore, understanding its mechanism is crucial to enhance the efficacy of GC treatment. Our study aimed to explore the association of the nuclear receptor subfamily 3 group C member 1 (NR3C1) gene, which encodes GC receptors GRα and GRβ, the histone deacetylase2 (HDAC2), the histone acetyltransferase-1 (HAT1), and the interleukin- 23 receptor (IL-23R), with the response to treatment in SLE patients. Methods: Quantitative real-time PCR (qPCR) was used to determine the expression levels of NR3C1 isoforms, HDAC2, HAT1, and IL23R in peripheral blood mononuclear cells (PBMC) from 30 SLE patients and 6 healthy controls (HC). Immunohistochemical (IHC) staining was used to analyze the protein expression level of GRα in 19 SLE patients’ 8 controls renal biopsies. Results: GRα mRNA expression level was associated with SLE disease (p=0.018) and correlated with the SLEDAI score (p=0,038). HDAC2 was up-regulated in patients during remission phase compared to active phase (p=0.015). The IL23R mRNA expression was associated with anemia (p=0.037) and lymphopenia (p=0.028). It was up-regulated in treated patients with the combination of hydroxychloroquine, cyclophosphamide, and methylprednisolone (p=0,028) compared to other treatments. Tubular GRα expression showed a positive correlation with the chronicity index (rs=0.607, p=0.016). Conclusion: The GRα may be involved in the pathogenesis of SLE. It appears that HDAC2 contributes to the remission phase in SLE. IL23R expression could be affected by treatments in SLE patients.

Quantitative Analysis of mRNA and Protein Expression Levels of Aldo-Keto Reductase and Short-Chain Dehydrogenase/Reductase Isoforms in the Human Intestine.

Enzymes catalyzing the reduction reaction of xenobiotics are mainly members of the aldo-keto reductase (AKR) and short-chain dehydrogenase/reductase (SDR) superfamilies. The intestine, together with the liver, is responsible for first-pass effects and is an organ that determines the bioavailability of orally administered drugs. In this study, we evaluated the mRNA and protein expression levels of 12 AKR isoforms (AKR1A1, AKR1B1, AKR1B10, AKR1B15, AKR1C1, AKR1C2, AKR1C3, AKR1C4, AKR1D1, AKR1E2, AKR7A2, and AKR7A3) and 7 SDR isoforms (CBR1, CBR3, CBR4, DCXR, DHRS4, HSD11B1, and HSD17B12) in each region of the human intestine using next-generation sequencing and data-independent acquisition proteomics. At both the mRNA and protein levels, most AKR isoforms were highly expressed in the upper regions of the intestine, namely the duodenum and jejunum, and then declined toward the rectum. Among the members in the SDR superfamily, CBR1 and DHRS4 were highly expressed in the upper regions, whereas the expression levels of the other isoforms were almost uniform in all regions. Significant positive correlations between mRNA and protein levels were observed in AKR1A1, AKR1B1, AKR1B10, AKR1C3, AKR7A2, AKR7A3, CBR1, and CBR3. The mRNA level of AKR1B10 was highest, followed by AKR7A3 and CBR1, each accounting for more than 10% of the sum of all AKR and SDR levels in the small intestine. This expression profile in the human intestine was greatly different from that in the human liver, where AKR1C isoforms are predominantly expressed. SIGNIFICANCE STATEMENT: In this study comprehensively determined the mRNA and protein expression profiles of aldo-keto reductase (AKR) and short-chain dehydrogenase/reductase isoforms involved in xenobiotic metabolism in the human intestine and found that most of them are highly expressed in the upper region, where AKR1B10, AKR7A3, and CBR1 are predominantly expressed. Since the intestine is significantly involved in the metabolism of orally administered drugs, the information provided here is valuable for pharmacokinetic studies in drug development.