Gene Isoforms Research Articles

Revealing novel cytb and nad5 genes-based population diversity and benzimidazole resistance in Echinococcus granulosus of bovine origin.

Cystic echinococcosis (CE) is a neglected zoonotic disease caused by Echinococcus granulosus (sensu stricto). The parasite affects a wide range of livestock and wild animals. In this study, the population diversity of the Echinococcus species was investigated based on mitochondrial cytochrome b (cytb) and NADH dehydrogenase subunit 5 (nad5) genes. In addition to this, β-tubulin gene isoforms of Echinococcus granulosus were amplified to determine the resistance against benzimidazoles. For this purpose, 40 cyst samples from cattle (n = 20) and buffaloes (n = 20) were collected from the main abattoir of Sialkot. DNA extraction was performed using Qiagen Blood and Tissue Kits. Amplification was performed through PCR. Each amplicon was confirmed by GelRed™ stained agarose gel (2%). Samples were sequenced in a DNA analyzer and viewed for any misread nucleotide by using MEGA (v.11). Corrections in nucleotide sequence and multiple sequence alignment were made through the same software. NCBI-BLAST was used for sample specific sequences to identify them as belonging to a particular species. Diversity indices were estimated using DnaSP (v.6) while phylogenetic analysis was inferred using the Bayesian method using MrBayes (v.1.1). β-tubulin gene isoforms sequence analysis was performed to find out the candidate gene causing benzimidazole resistance. All 40 isolates were found positive for E. granulosus. BLAST-based searches of sequences of each isolate for each gene (nad5 and cytb) confirmed their maximum similarity with the G1 genotype. Overall, high haplotype diversity (Hd nad5 = 1.00; Hd cytb = 0.833) and low nucleotide diversity (π nad5 = 0.00560; π = cytb = 0.00763) was identified based on diversity indices. For both the genes, non-significant values of Tajima's D (nad5 = -0.81734; cytb = -0.80861) and Fu's Fs (nad5 = -1.012; cytb = 0.731) indicate recent population expansion. Bayesian phylogeny-based results of nad5 and cytb sequences confirmed their genotypic status as distinct from other Echinococcus species. This study shed light on the status of benzimidazole resistance in Echinococcus granulosus for the very first time from Pakistan. The findings of this study will significantly add in the information available on genetic diversity of Echinoccous granulosus based on cytb and nad5 genes sequences.

Abstract A006: Loss of PMEPA1 gene isoform facilitates the development of hormone and radiation therapies resistance in prostate cancer cells

Abstract Introduction: Nearly all the patients receiving hormone treatment eventually progress into castration resistance stage. In addition, approximately 30% to 40% of patients receiving radiation treatment will develop resistance. The aberrant alterations of androgen receptor (AR) and transforming growth factor-β (TGF-β) signaling play critical roles in such processes. Our studies identified five PMEPA1 (Prostate Transmembrane Protein Androgen Induced 1) gene isoforms in prostate cancer cells. PMEPA1-b isoform was androgen responsive and inhibited androgen signaling. In contrast, PMEPA1 isoforms a and d were TGF-β inducible and suppressed TGF-β signaling. Loss or reduced expressions of PMEPA1 was detected in over 60% of prostate cancer samples. In this study, we investigated the impacts of PMEPA1 isoforms (a, b and d) on the development of hormone and radiation therapies resistance in prostate cancer. Methods: RNA-seq data of unmatched 50 benign and 499 malignant prostate samples from The Cancer Genome Atlas (TCGA) dataset, as well as 101 metastatic castration resistant prostate cancer (mCRPC) biopsy samples from Quigley lab at UCSF (WCDT dataset) were used to study the transcript levels of PMEPA1 isoforms (a, b and d) and their correlations to disease progression. Hormone dependent LNCaP cells and androgen independent PC-3, DU-145 cells were utilized to investigate the impacts of PMEPA1 isoforms (a, b and d) on the responses to of androgen inhibitors (enzalutamide and bicalutamide, 0, 5.0 and 25.0 nM) and radiation (0, 1.0 and 5.0 Gy). The vectors of pcDNA3.1-PMEPA1-a, -b and -d were transfected into prostate cancer cells via Lipofectamine, and endogenous PMEPA1 isoforms were knocked down by isoforms specific siRNAs. The cell growth was assessed by cell counting, BrdU incorporation and cell plating efficiency assays. The protein levels of AR and PSA were evaluated by immunoblotting. The transcript levels of PSA, NKX3.1, NEDD9 and THBS1 were assessed by RT-PCR technology. Results: The reduced transcript levels of PMEPA1 isoforms (a, b and d) were detected in mCRPC group. Lower expression of PMEPA1-b was significantly associated with earlier biochemical recurrence. The growth inhibition and androgen signaling blockage mediated by androgen inhibitors were explicitly reversed by depletion of PMEPA1-b in LNCaP cells. Ectopic PMEPA1-b in isoform b depleted LNCaP cells reinstituted the growth inhibition induced by androgen inhibitors. Knockdown of isoforms a and d conspicuously rescued the decreased cell proliferation and suppressed TGF-β signaling resulted from radiation treatment in PC-3 and DU-145 cells. Similarly, over-expression of PMEPA1-a and -d in siRNA treated cells recapitulated the growth inhibitory effects of radiation treatments. Conclusion: Loss of PMEPA1 gene isoforms (a, b and d) induced the development of resistance to hormone ablation and radiation treatments in prostate cancer cells in vitro through activating androgen and TGF-β signaling. Our research provided a new insight into treatment resistance mechanism of prostate cancer cells. Citation Format: Shashwat Sharad, Hua Li. Loss of PMEPA1 gene isoform facilitates the development of hormone and radiation therapies resistance in prostate cancer cells [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr A006.

Clinical and genetic variants of hypertriglyceridemia in the practice of a lipidologist

Background. An important role in identifying the causes and determining the prognostic significance of dyslipidemia belongs to the level of triglycerides. High triglycerides are a risk factor for the development or early onset of cardiovascular disease.
 Aim. To assess the frequency of detection and causes of hypertriglyceridemia among patients receiving outpatient appointments with a lipidologist.
 Material and methods. An analysis of lipid metabolism disorders in patients of the Adult Lipidology Center was carried out: 1233 people aged 1884 years, including 777 (63%) women and 456 (37%) men. Examination of patients with dyslipidemia included an examination by a cardiologist-lipidologist (with the calculation of the risk of cardiovascular complications), an assessment of the probability of familial hypercholesterolemia according to the British scale and the criteria of the Dutch lipid clinics, a biochemical blood test, an analysis of the thyroid status, the content of glycated hemoglobin, extracranial duplex scanning, according to indications echocardiography. Biomaterial samples from 421 patients with the phenotype of inherited dyslipidemia were examined by next generation sequencing to identify the carriage of APOE gene isoforms, as well as genes associated with familial hypercholesterolemia (LDLR, LDLRAP1, APOB, PCSK9). For statistical processing of research data, descriptive statistics methods were used. In a non-parametric distribution, data were expressed as Me (Q1; Q3). When performing statistical processing of the obtained data, nonparametric tests (MannWhitney test, when comparing qualitative data 2 and Fisher's exact test, odds ratio and relative risk) were used. The value of p 0.05 was taken as a criterion of significance. The nature of the data distribution was assessed using the KolmogorovSmirnov test.
 Results. Elevated triglyceride levels were detected in 341 (27.66%) patients: 220 (64.5%) women and 121 (35.5%) men. Mild degree of hypertriglyceridemia occured in 42.5% of cases, moderate in 42.5%, severe in 7.6%, extremely severe in 7.4%. The genetic characteristics of patients with hypertriglyceridemia were determined, and 1 previously undescribed variant of the APOE mutation was found.
 Conclusion. The most common forms of hypertriglyceridemia were mild and moderate, the most common variants of APOE mutations were p.Cys130Arg and p.Arg176Cys.

Cyclosporine A induces cardiac remodeling through TGF-β/Smad3/miR-29 signaling pathway and alters gene expression of miR-30b-5p/CaMKIIδ isoforms pathways: alleviating effects of moderate exercise.

Cyclosporine A (CsA)-induced cardiac interstitial fibrosis and cardiac hypertrophy are highly known phenomena; however, the basic mechanisms of CsA cardiotoxicity are unclear. The present study evaluated the role of the Transforming growth factor-beta (TGF-β)/Smad3/miR-29b signaling pathway and CaMKIIδ isoforms gene expression in cardiac remodeling under CsA exposure alone or combined with moderate exercise. A total of 24 male Wistar rats were divided into control, cyclosporine (30mg/kg BW), and cyclosporine-exercise groups. After 42 days of treatment, the findings revealed a significant decline in miR-29 and miR-30b-5p gene expression and an increase in gene expression of Smad3, calcium/calmodulin-dependent protein kinaseIIδ (CaMKIIδ) isoforms, Matrix Metalloproteinases (MMPs), protein expression of TGF-β, heart tissue protein carbonyl and oxidized LDL (Ox-LDL), and plasma LDL and cholesterol levels in the CsA-treated group compared to the control group. The CsA group presented greater histological heart changes such as fibrosis, necrosis, hemorrhage, infiltrated leukocyte, and left ventricular weight/heart weight than the control group. Moreover, combined moderate exercise and CsA relatively improved gene expression changes and histological alternations compared to the CsA group. TGF-β-Smad3-miR-29 and CaMKIIδ isoforms may mainly contribute to the progression of heart fibrosis and hypertrophy due to CsA exposure, providing new insight into the pathogenesis and treatment of CsA-induced side effects on the heart tissue.

Adipose tissue plasticity in pheochromocytoma patients suggests a role of the splicing machinery in human adipose browning

Adipose tissue from pheochromocytoma patients acquires brown fat features, making it a valuable model for studying the mechanisms that control thermogenic adipose plasticity in humans. Transcriptomic analyses revealed a massive downregulation of splicing machinery components and splicing regulatory factors in browned adipose tissue from patients, with upregulation of a few genes encoding RNA-binding proteins potentially involved in splicing regulation. These changes were also observed in cell culture models of human brown adipocyte differentiation, confirming a potential involvement of splicing in the cell-autonomous control of adipose browning. The coordinated changes in splicing are associated with a profound modification in the expression levels of splicing-driven transcript isoforms for genes involved in the specialized metabolism of brown adipocytes and those encoding master transcriptional regulators of adipose browning. Splicing control appears to be a relevant component of the coordinated gene expression changes that allow human adipose tissue to acquire a brown phenotype.

Mapping splice QTLs reveals distinct transcriptional and post-transcriptional regulatory variation of gene expression and identifies putative alternative splicing variation mediating complex trait variation in pigs

BackgroundAlternative splicing is an important step in gene expression, generating multiple isoforms for the same genes and greatly expanding the diversity of proteomes. Genetic variation in alternative splicing contributes to phenotypic diversity in natural populations. However, the genetic basis of variation in alternative splicing in livestock including pigs remains poorly understood.ResultsIn this study, using a Duroc x Pietrain F2 pig population, we performed genome-wide analysis of alternative splicing estimated from stranded RNA-Seq data in skeletal muscle. We characterized the genetic architecture of alternative splicing and compared its basic features with those of overall gene expression. We detected a large number of novel alternative splicing events that were not previously annotated. We found heritability of quantitative alternative splicing scores (percent spliced in or PSI) to be lower than that of overall gene expression. In addition, heritabilities showed little correlation between alternative splicing and overall gene expression. We mapped expression QTLs (eQTLs) and splice QTLs (sQTLs) and found them to be largely non-overlapping. Finally, we integrated sQTL mapping with phenotype QTL (pQTL mapping to identify potential mediator of pQTL effect by alternative splicing.ConclusionsOur results suggest that regulatory variation exists at multiple levels and that their genetic controls are distinct, offering opportunities for genetic improvement.

ASAS-EGB: A statistical framework for estimating allele-specific alternative splicing events using transcriptome data

Ubiquitous Alternative splicing (AS) in eukaryotes greatly increases the biodiversity of proteins and involves in disease and cancer. Allele-specific AS studies can facilitate the identification of cis-acting elements because both alleles share the same cellular environment. Due to the limited information provided on the exons defined by AS events, we propose a statistical framework and algorithm ASAS-EGB for ASAS analysis using the gene transcriptome. The framework obtains exclusively compatible sets of gene isoforms supporting each event isoform, and uses both phased and non-phased SNPs within the exons on the gene isoforms for inference. Using this strategy, we have demonstrated ASAS-EGB can yield better ASAS inferential performance than using event isoforms. ASAS-EGB supports both single-end and paired-end RNA-seq data, and we have proved its robustness using RNA-seq replicates of individual NA12878. ASAS-EGB builds Bayesian models for ASAS analysis, and the MCMC method is used to solve the problem. With more detailed annotations for individual genomes and transcriptomes appearing in the future, the algorithm proposed by the paper can provide better support for these data to reveal the regulatory mechanisms of individual genomes.

АНАЛИЗ ПАТОГЕННОСТИ НОВОЙ МИССЕНС-МУТАЦИИ В ГЕНЕ MYOF, ВЫЯВЛЕННОЙ У ПАЦИЕНТКИ С НАСЛЕДСТВЕННЫМ АНГИООТЕКОМ С НОРМАЛЬНЫМ УРОВНЕМ C1-ИНГИБИТОРА

Hereditary angioedema (HAE) is a genetically determined disorder accompanied by specific symptoms associated with sporadic subcutaneous and submucosal edema. The described cases of type III HAE not associated with mutations in the SERPING1 gene are characterized by the corresponding clinical picture with normal values and functional activity of the C1-inhibitor. Type III HAE is associated with mutations in the F12, PLG, ANGPT1, KNG1, MYOF, and HS3ST6 genes. Mutations in the MYOF and HS3ST6 genes remain the least studied as yet. And as for the MYOF gene, a single mutation, Arg217Ser, is known to be associated with the development of HAE. The purposes of this research were to study the new missense mutation NC_000010.10:g.95093020C>T in the MYOF gene and predictive assessment of its contribution to the HAE pathogenesis using the bioinformatics analysis. There was a blood sample obtained from a 14 y/o female patient with HAE clinical manifestations and without a decrease in the level and the lack in function of the C1-inhibitor. The research methods included sequencing of the patient's complete exome, bioinformatic analysis of the MYOF gene mutation using a number of databases and Internet resources with the purpose of assessing the conservation of the amino acid position of the substitution and predicting the effect of the mutation on the protein. Results: the girl had a previously undescribed missense mutation NC_000010.10:g.95093020C>T in exon 42 of the MYOF gene (isoform A) in the heterozygous state. The mutation resulted in the replacement of arginine with glutamine at position 1590 of the amino acid sequence (p.Arg1590Gln, rs201619869). The use of bioinformatics analysis allowed assuming the potential pathogenicity of the detected missense mutation, which could cause the observed edema. The possible pathways for the involvement of myoferlin with the detected mutation in the HAE pathogenesis are discussed in the Article. The use of in silico analysis allowed conducting the detailed study of the detected mutation considering its effect on the protein structure, which is the basis of its normal functioning. According to the results of the study, rare mutations in the MYOF gene can be involved in the HAE pathogenesis provoking edema through various cascades of biochemical reactions. In the course of the study, a new missense mutation in the MYOF gene, pathogenetically significant for the HAE development, was described for the first time.

Abstract 3560: Understanding alternative splicing in Ewing sarcoma progression and metastasis to inform novel therapy development

Abstract Introduction: Ewing sarcoma (ES) is the second most common pediatric bone cancer, and patients with metastases have five-year survival rates of <30%. Both local and metastatic ES tumors are characterized by the EWS-FLI1 chromosomal translocation, resulting in an aberrant transcription factor that increases ES cell proliferation and metastasis. EWS-FLI1 activity and ES transformation are reliant upon increased IGF-1 signaling, so initial therapies have centered on IGF-1R inhibition. However, compensatory upregulation of insulin receptor A (IR-A), an oncogenic spliced isoform of IR, provides a viable resistance mechanism to support ES growth and progression. IR has two spliced isoforms formed by the inclusion or exclusion of exon 11 (IR-B and IR-A respectively), and IR-A is differentially expressed in cancers due to its high affinity binding to IGF-II. We hypothesize that the pathogenesis and metastatic potential of ES is driven in part by alternative splicing of IR and other key genes. Furthermore, modulation of this splicing via antisense oligonucleotides (ASOs) aided by adeno-associated viral (AAV) delivery may be a novel therapeutic avenue for ES. Results: We performed long-read mRNA sequencing to identify differentially spliced isoforms between the matched ES cell lines CHLA-9 and CHLA-10 (derived from a localized and metastatic ES tumor respectively). Our analysis revealed global differences in alternative splicing between CHLA-9 and CHLA-10 and identified gene isoforms in the insulin receptor/IGF signaling pathway (IGFBP3) that are unique to CHLA-10. Additionally, we detected both previously reported and novel isoforms for CD99, RHOA, RACK1, splicing factors (PTBP1), and eukaryotic initiation factors, among other genes. To further understand IR alternative splicing, our labs have developed an IR ASO targeting the CUG-BP1 regulatory site upstream of exon 11, and we have previously validated its efficacy in HeLa cells transfected with an IR minigene and in pediatric sarcoma cell lines. In ES cell lines, we observed significant IR-A upregulation in a panel of ES cell lines and patient-derived xenografts derived from local and metastatic tumors, highlighting the opportunity for intervention with our ASO. To facilitate delivery, we have explored the use of AAV vectors, a clinically approved option for gene therapy, and shown successful transduction of AAV-8 over a time-course in ES cells. Conclusions: Our results indicate that alternative splicing is differential between local and metastatic ES and produces isoforms unique to the metastatic setting. Additionally, the oncogenic IR-A isoform and other spliced isoforms are viable targets for ASO therapy aided by AAV delivery. By better understanding splicing in ES, we can identify targetable interactions at the transcriptional level and develop innovative therapeutics that can improve the clinical outcomes of ES patients. Citation Format: Akila S. Venkataramany, Safiya Khurshid, Anthony R. Miller, Saranga Wijeratne, Pin-Yi Wang, Timothy P. Cripe, Dawn S. Chandler. Understanding alternative splicing in Ewing sarcoma progression and metastasis to inform novel therapy development. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3560.

Abstract 3529: EWSR1-WT1 isoform selectivity, DNA binding, and druggable targets: unpacking the biology of desmoplastic small round cell tumor

Abstract Desmoplastic small round cell tumor (DSRCT) is a rare pediatric cancer caused by the EWSR1-WT1 fusion gene. No targeted therapies have been established and multimodal therapy is ineffective with a 5-year survival rate of 15-25%. Many questions about the biology of DSRCT remain unanswered including the critical genes regulated by the EWSR1-WT1 fusion gene, the role of the - and +KTS fusion gene isoforms produced by alternative splicing, and the relationship between EWSR1-WT1 binding and gene expression alterations. To address these questions, we established four doxycycline-inducible DSRCT cell lines that selectively knockdown the expression of EWSR1-WT1. RNA-seq analysis on these cell lines with or without EWSR1-WT1 knockdown identified commonly altered genes and pathways. To illuminate the role of the EWSR1-WT1 -KTS and +KTS isoforms, the -KTS, +KTS, or both isoforms were overexpressed in a mesothelial cell line, a hypothesized cell of origin for DSRCT. Remarkably, different gene expression alterations were observed between the -KTS and +KTS isoforms. While overexpression of the -KTS isoform upregulated 765 genes and downregulated 366 genes, overexpression of the +KTS isoform upregulated 203 genes and downregulated 25 genes. Gene set enrichment analysis showed EWSR1-WT1 regulated gene signatures are recapitulated by overexpression of the -KTS isoform or both +/-KTS isoforms, but not the +KTS isoform alone, suggesting the -KTS isoform predominantly regulates gene expression in DSRCT. To interrogate the influence of direct and indirect gene regulation by EWSR1-WT1, ChIP-seq and CUT&RUN data were integrated into our RNA-seq analysis. Across each of our four DSRCT cell lines, approximately 25% of EWSR1-WT1 bound peaks led to upregulation, 10% led to downregulation, and 65% led to no gene expression alteration. EWSR1-WT1 binding accounted for 33% of EWSR1-WT1 commonly upregulated genes but only 4.2% of commonly downregulated genes. Upregulated EWSR1-WT1 bound genes were more likely to be bound in the promoter or first intron and coincide with Znf263 or Hoxb4 motifs. To evaluate the functional role of EWSR1-WT1 targets in DSRCT biology, a screen of the 24 druggable EWSR1-WT1 targets was conducted and a number of novel and known target genes were identified as important for DSRCT survival. Current work is verifying these targets with gene knockdown and evaluating their function in vivo. Citation Format: Justin W. Magrath, Ilon A. Goldberg, Alifiani B. Hartono, Sean B. Lee. EWSR1-WT1 isoform selectivity, DNA binding, and druggable targets: unpacking the biology of desmoplastic small round cell tumor. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3529.

Genomic patterns linked to gray matter alterations underlying working memory deficits in adults and adolescents with attention-deficit/hyperactivity disorder

Attention-deficit/hyperactivity disorder (ADHD) is a highly heritable neurodevelopmental disorder, with onset in childhood and a considerable likelihood to persist into adulthood. Our previous work has identified that across adults and adolescents with ADHD, gray matter volume (GMV) alteration in the frontal cortex was consistently associated with working memory underperformance, and GMV alteration in the cerebellum was associated with inattention. Recent knowledge regarding ADHD genetic risk loci makes it feasible to investigate genomic factors underlying these persistent GMV alterations, potentially illuminating the pathology of ADHD persistence. Based on this, we applied a sparsity-constrained multivariate data fusion approach, sparse parallel independent component analysis, to GMV variations in the frontal and cerebellum regions and candidate risk single nucleotide polymorphisms (SNPs) data from 341 unrelated adult participants, including 167 individuals with ADHD, 47 unaffected siblings, and 127 healthy controls. We identified one SNP component significantly associated with one GMV component in superior/middle frontal regions and replicated this association in 317 adolescents from ADHD families. The association was stronger in individuals with ADHD than in controls, and stronger in adults and older adolescents than in younger ones. The SNP component highlights 93 SNPs in long non-coding RNAs mainly in chromosome 5 and 21 protein-coding genes that are significantly enriched in human neuron cells. Eighteen identified SNPs have regulation effects on gene expression, transcript expression, isoform percentage, or methylation level in frontal regions. Identified genes highlight MEF2C, CADM2, and CADPS2, which are relevant for modulating neuronal substrates underlying high-level cognition in ADHD, and their causality effects on ADHD persistence await further investigations. Overall, through a multivariate analysis, we have revealed a genomic pattern underpinning the frontal gray matter variation related to working memory deficit in ADHD.

Recombinant Production and Purification of Inosine 5-Mono Phosphate Dehydrogenase 1 Retinal Isoforms for Functional Studies

Inosine 5-monophosphate dehydrogenase 1 (IMPDH1) is the rate-limiting enzyme in the de novo purine nucleotide biosynthesis. IMPDH1 catalyzes IMP-oxidation to XMP, which in continue is converted to guanine nucleotides. Like mammals, the mouse IMPDH1 (mH1) has retinal-specific isoforms named H1 (546) and H1 (603). Mutations in the IMPDH1 gene are believed to cause retinal degenerative disease, retinitis pigmentosa, mediated by retinal-specific gene variants. After RNA extraction from the mouse retina, RT-PCR was done using NdeI and XhoI harboring primers. Tree mH1 isoform genes were amplified and cloned into a pET26b+ vector separately. The recombinant expression vectors were then transformed in E. coli BL21 (DE3) strain, expressed under IPTG-induced conditions and purified with Ni-NTA agarose resin. Activity assay of recombinant proteins was done by using spectrophotometric methods. Here, we cloned and optimized the expression and the purification of recombinant mH1 canonical and retinal isoforms in E. coli to gain soluble and highly active protein for further functional assays. Recombinant protein production in prokaryotic hosts, especially E. coli, is the most common method in large-scale of protein production for functional and structural studies. However, maximal yield and activity of recombinant proteins require optimal conditions for expression and purification, which is what we showed in the present study for the mouse IMPDH1 recombinant isoforms.

Secretory Phospholipase A2 (sPLA2) Isozymes as Potential Targets in Tobacco Condensate- induced Colon Damage.

To find out the role of secretory phospholipase A2 (sPLA2) isozymes as potential targets in tobacco condensate-induced colon damage. The effects of cigarette smoke condensate (CSC) and the molecular mechanisms involved in the regulation of phospholipase A2 (PLA2) and its isozymes in colon cells, which are still unclear and emerging, are studied. The study aimed to check the effect of CSC on cell viability and reactive oxygen species (ROS) and superoxide. Also, the effect of CSC on gene expression of different secretory phospholipase A2 (sPLA2) was evaluated. Moreover, the impact of inhibition of sPLA2 on various cell properties i.e. cell viability, cell proliferation, membrane damage and free radicals' generation is also studied. CSC-induced changes were evaluated in cell viability by MTT assay, followed by the evaluation of membrane modulation by flow cytometry, free radical generation by fluorescent dyes, PLA2 isoforms gene expression patterns and their suppression by small interfering RNA (siRNA) studied in HCT-15 male and HT-29 female colon cells. Our results demonstrate that HCT-15 and HT-29 cells treated with CSC significantly reduced the cell viability by 50% within 48 h and significantly enhanced the total reactive oxygen species (ROS) by 2 to 10-fold, and mitochondrial ROS (mtROS) and superoxide radicals (SOR) by 2-fold each. Treatment with CSC significantly unregulated secretory phospholipase A2 (sPLA2) IID group and down-regulated IB and cytosolic phospholipase (cPLA2) IVA groups in HCT-15 cells without affecting them in HT-29 cells. Silencing the sPLA2 IID group results in an increase in cell viability and a decrease in ROS. Silencing the PLA2 IVA gene in the HCT-15 cells showed a reduced expression which had no impact on the CSC-induced cell proliferation, membrane damage and free radicals (ROS, mtROS, and SOR) generation. Therefore, identifying cell-specific sPLA2 isozymes seems to play a key role in controlling the ROSinduced damage by CSC and helps develop specific therapeutic strategies.

Alternatively Spliced Isoforms of MUC4 and ADAM12 as Biomarkers for Colorectal Cancer Metastasis.

There is a pertinent need to develop prognostic biomarkers for practicing predictive, preventive and personalized medicine (PPPM) in colorectal cancer metastasis. The analysis of isoform expression data governed by alternative splicing provides a high-resolution picture of mRNAs in a defined condition. This information would not be available by studying gene expression changes alone. Hence, we utilized our prior data from an exon microarray and found ADAM12 and MUC4 to be strong biomarker candidates based on their alternative splicing scores and pattern. In this study, we characterized their isoform expression in a cell line model of metastatic colorectal cancer (SW480 & SW620). These two genes were found to be good prognostic indicators in two cohorts from The Cancer Genome Atlas database. We studied their exon structure using sequence information in the NCBI and ENSEMBL genome databases to amplify and validate six isoforms each for the ADAM12 and MUC4 genes. The differential expression of these isoforms was observed between normal, primary and metastatic colorectal cancer cell lines. RNA-Seq analysis further proved the differential expression of the gene isoforms. The isoforms of MUC4 and ADAM12 were found to change expression levels in response to 5-Fluorouracil (5-FU) treatment in a dose-, time- and cell line-dependent manner. Furthermore, we successfully detected the protein isoforms of ADAM12 and MUC4 in cell lysates, reflecting the differential expression at the protein level. The change in the mRNA and protein expression of MUC4 and ADAM12 in primary and metastatic cells and in response to 5-FU qualifies them to be studied as potential biomarkers. This comprehensive study underscores the importance of studying alternatively spliced isoforms and their potential use as prognostic and/or predictive biomarkers in the PPPM approach towards cancer.

Nanopore long-read RNAseq reveals transcriptional variations in citrus species.

The number of studies on plant transcriptomes using ONT RNAseq technology is rapidly increasing in recent. It is a powerful method to decipher transcriptomic complexity, particularly alternative splicing (AS) event detection. Citrus plants are the most important widely grown fruit crops. Exploring different AS events in citrus contributes to transcriptome improvement and functional genome study. Here, we performed ONT RNAseq in 9 species (Atalantia buxifolia, Citrus clementina, C. grandis, C. ichangensis, C. reticulata, C. sinensis, Clausena lansium, Fortunella hindsii, and Poncirus trifoliata), accompanied with Illumina sequencing. Non-redundant full-length isoforms were identified between 41,957 and 76,974 per species. Systematic analysis including different types of isoforms, number of isoforms per gene locus, isoform distribution, ORFs and lncRNA prediction and functional annotation were performed mainly focused on novel isoforms, unraveling the capability of novel isoforms detection and characterization. For AS events prediction, A3, RI, and AF were overwhelming types across 9 species. We analyzed isoform similarity and evolutionary relationships in all species. We identified that multiple isoforms derived from orthologous single copy genes among different species were annotated as enzymes, nuclear-related proteins or receptors. Isoforms with extending sequences on 5', 3', or both compared with reference genome were filtered out to provide information for transcriptome improvement. Our results provide novel insight into comprehending complex transcriptomes in citrus and valuable information for further investigation on the function of genes with diverse isoforms.

Apolipoprotein E Gene Polymorphism, Serum Lipids, and Risk of Superficial Fungal Infections in Egyptian Patients - A Preliminary Case-Controlled Study.

Apolipoprotein E (APOE) gene isoforms have been found to affect the risk of superficial fungal infections (SFIs). However, the data only cover a few ethnicities. The present work intended to investigate the association of APOE gene polymorphism and serum lipids with the susceptibility of SFIs among a group of Egyptian patients. Standard laboratory methods were used to estimate the serum lipid profile, and polymerase chain reaction-restriction fragment length polymorphism was used to detect APOE gene polymorphism in deoxyribonucleic acid extracted from 150 SFI patients and an equal number of apparently healthy matched controls. Serum total cholesterol, triglycerides, and low-density lipoprotein cholesterol were significantly higher in the studied patients than in controls. The APOE gene ε2, ε4 alleles, and ε3/4 and ε3/2 genotypes were significantly distributed in the patients than in the controls. APOE ε3/3 genotype was predominant in dermatophytosis and tinea versicolour patients, and ε3/4 genotype was predominant in candidiasis. ApoE alleles ε2 and ε4, and genotypes ε2/3 and ε3/4 are linked to SFI and may be risk factors, whereas allele ε3 and genotype ε3/3 may be protective for SFI in the Egyptian population studied. The lipid profile results suggest that hyperlipidemia may provide evidence for SFI pathogenesis. However; further large-scale studies are still needed to validate our results.

Presynaptic Cytomatrix Proteins.

The Cytomatrix Assembled at the active Zone (CAZ) of a presynaptic terminal displays electron-dense appearance and defines the center of the synaptic vesicle release. The protein constituents of CAZ are multiple-domain scaffolds that interact extensively with each other and also with an ensemble of synaptic vesicle proteins to ensure docking, fusion, and recycling. Reflecting the central roles of the active zone in synaptic transmission, CAZ proteins are highly conserved throughout evolution. As the nervous system increases complexity and diversity in types of neurons and synapses, CAZ proteins expand in the number of gene and protein isoforms and interacting partners. This chapter summarizes the discovery of the core CAZ proteins and current knowledge of their functions.

Characterization and Analysis of the Full-Length Transcriptome Provide Insights into Fruit Quality Formation in Kiwifruit Cultivar Actinidia arguta cv. Qinziyu

Kiwifruit is an economically important horticultural crop with extremely high values in nutrition and health care. However, the molecular mechanisms underlying fruit quality formation remain largely limited for most kiwifruit varieties. Recently, a new kiwifruit cultivar with a high level of soluble solids, Actinidia arguta cv. Qinziyu (full-red flesh) was discovered through the introduction and propagation test. To provide new insights into fruit quality formation in a typical kiwifruit cultivar, we integrated full-length transcriptome surveys based on PacBio single-molecule real-time (SMRT) sequencing, key enzyme genes expression involved in carbohydrate and amino acids metabolism pathways, and bHLH gene family analysis to enhance the understanding of soluble sugar, organic acid, and anthocyanin biosynthesis in A. arguta cv. Qinziyu. A total of 175,913 CCSs were generated, of which 124,789 were identified as FLNC transcripts. In total, 45,923 (86.99%) transcripts were successfully annotated, and more than 76.05% of the transcripts were longer than 1 Kb. KEGG pathway analysis showed that 630 candidate genes encoding 55 enzymes were mainly involved in carbohydrate and amino acid biosynthesis pathways. Further analysis verified the expression of 12 key enzyme genes (e.g., pyruvate kinase (PK), enolase (ENO), hexokinase (HK), and phosphoglycerate kinase (PGK)) in flowers using quantitative real-time PCR. Furthermore, we also screened 10 AabHLH proteins’ function in anthocyanin biosynthesis and characterized the AabHLH gene family in A. arguta cv. Qinziyu. Overall, our research data generated by SMRT technology provide the first set of gene isoforms from a full-length transcriptome in A. arguta cv. Qinziyu and more comprehensive insights into the molecular mechanism of fruit quality formation.

The Pattern of Chromosome Aberrations and Molecular Markers in the Population of Hematological Patients Diagnosed at the University Clinical Center Tuzla

Introduction: Besides cardiovascular, malignant diseases are one of the leading causes of death in Bosnia and Herzegovina. At the top of this list are hematological diseases. This research aimed to identify cytogenetic and molecular biomarkers in patients treated for different types of hematological neoplasms. Methods: The retrospective study included 1600 samples of patients with different hematological diseases in the period from January 2006 to May 2022. The Polymerase Chain Reaction (RT-PCR) method was used to determine the presence of genetic rearrangements and to confirm the findings of conventional cytogenetic analysis. Results: Chromosomal aberrations were found in 739 (46,18%) patients. Using the RT-PCR technique, positive cases were increased by 1,5%. The BCR-ABL fusion gene was present in e14-a2 transcript form in 73% of samples, e13-a2 isoform in 21%, e1-a2 in 2%, while e14-a2/e1-a2 transcript coexpression was present in a percentage of 4% of the samples. The PML-RARa fusion gene was found in the form of bcr 1 transcripts in 21%, bcr2 32% and bcr3 59% of the samples. In twelve cases A type of the CBFB-MYH11 fusion transcript was detected. The MLL-AF4 fusion was found in only one case. Conclusion: The obtained percentages of frequency of individual molecular gene isoforms are in accordance with the results of most other researchers. This refers to the Balkan population and the Caucasian ethnic group.

Cloning, characterization and expression analysis of caspase-8 genes from the golden pompano (Trachinotus ovatus)

Bacterial infection triggers apoptosis and immunity by regulating the expression of caspases. In this article, we report two caspase-8 paralogs, termed ToCas8 and ToCas8-like, from an economically important teleost golden pompano (Trachinotus ovatus). Caspase-8 is a classic cysteine protease that is essential for the initiation and execution of apoptosis and is a critical regulator in initiating death receptor-induced programmed cell death. The two proteins encoded by ToCas8 and ToCas8-like were characterized by multiple sequence alignment and protein structure analysis showing similarity to their counterparts in vertebrates and exhibiting typical features of the Caspase-8 protein family. Gene synteny analysis further confirmed that two isoforms of caspase-8 genes exactly existed in golden pompano. Phylogenetic analysis showed that the Caspase-8 proteins of the selected species in the study were clustered together, and Cas8 and Cas8-like were clustered into two different subgroups. In addition, ToCas8 and ToCas8-like were widely distributed and both were expressed at high levels in gill, kidney and intestinal tissues. Transcript levels of ToCas8 and ToCas8-like were significantly upregulated in response to LPS and Poly(I:C) challenges, suggesting that caspase-8 genes are involved in the immune response to pathogens. In summary, we have identified two isoforms of caspase-8 in golden pompano that are involved in the immune response. These findings will expand our understanding of the evolutionary history and functional characteristics of caspase-8 genes in vertebrates.