Expression Of Different Isoforms Research Articles

The Impact of Spliceosome Inhibition in SF3B1-Mutated Uveal Melanoma.

Unfortunately, treatment of patients with uveal melanoma (UM) with metastatic disease is limited. Twenty percent of patients with UM harbor a mutation in the splicing factor gene SF3B1, suggesting that aberrant spliceosome function plays a vital role in tumorigenesis. Splicing inhibitors exploit the preferential sensitivity of spliceosome-compromised leukemic cells to these compounds. We studied the effect of the splicing inhibitor E7107 using two UM cell lines and ex vivo cultured SF3B1- and BAP1-mutated primary UM tumor slices. These UM cell lines and ex vivo tumor slices were exposed for 24 hours to different concentrations of E7107. Tumor slices were stained with hematoxylin and eosin (H&E) and incubated with BAP1, MelanA, MIB-1, and caspase-3 antisera. The E7107-exposed UM cell lines exhibited decreased cell viability and increased apoptosis, with the greatest effect on SF3B1-mutated UM cells. A similar effect on UM tumor slices was observed upon exposure to E7107. Additionally, RNA was isolated for differential isoform expression analysis. No significant difference in isoform usage was found genome-wide. However, specific genes were differentially expressed after E7107 treatment in the SF3B1-mutated samples. Moreover, E7107 had the greatest effect on intron retention. This study indicates/suggests that mutated SF3B1 UM cells are more sensitive to the splicing inhibitor E7107 than wild-type SF3B1 UM cells.

Estrogen receptors and extracellular matrix: the critical interplay in cancer development and progression.

Cancer remains a significant global health concern. Breast cancer is a multifaceted and prevalent disease influenced by several factors, among which estrogen receptors (ERs) and the extracellular matrix (ECM) play pivotal roles. ERs, encompassing ERα and ERβ, exert significant diversity on tumor behavior, cell signaling, invasion, and metastatic potential, thus guiding breast cancer prognosis. Understanding the multifunctional connections between ERs and ECM that mediate the dynamics of tumor microenvironment is vital for unraveling the complexity of breast cancer pathobiology and identifying novel therapeutic targets. This critical review delves into the intricate nature of ERs, emphasizing their structural isoforms and the consequential impact on breast cancer outcomes. A detailed examination of ER-mediated cell signaling pathways reveals how differential expression of ERα and ERβ isoforms influence breast cancer cell behavior. The functional ERs-matrix interactions emerge as a pivotal factor in modulating epigenetic mechanisms of breast cancer cells, orchestrating changes in cellular phenotype and expression patterns of matrix modulators. Specifically, ERα isoforms are shown to regulate ECM signaling cascades, while the effects of ECM components on ERα activity highlight a bidirectional regulatory axis. The diversity of ERβ isoforms is also highlighted, illustrating their distinct contribution to ECM-mediated cellular responses. This review underscores the complex interplay between ERα/β isoforms and the ECM, shedding light onto the potential therapeutic strategies targeting these interactions to improve breast cancer management.

Differential MYC and PROM1 mRNA isoform expression in breast invasive carcinoma as biomarkers for subtyping and prognosis

BackgroundCancer stem cells are a subpopulation of tumor cells with the ability to self-renew; evidence suggests that cancer stem cells are responsible for recurrence, metastasis, and resistance to therapy. MYC and CD133 (PROM1 gene) are clinical biomarkers for cancer stem cells, and their dysregulation is involved in the progression of many cancers. Alternative splicing of these genes may contribute to cancer stem cell differentiation. MethodsTranscriptional and clinical data of PROM1 and MYC mRNA isoforms in breast cancer samples were downloaded from the TCGA Splicing Variants Database site, a web-tool to explore mRNA alternative-splicing based on TCGA samples. Data include RSEM isoform expression, clinical sample types, survival data, and clinical receptor expression. Breast cancer subtypes (luminal A, luminal B, Her2 positive, triple negative) were assigned on the basis of estrogen, progesterone, and HER2 expression. ResultsExpression of MYC isoforms uc003ysh.1 and uc003ysi.3 was significantly greater in triple-negative breast cancer compared with all other breast cancer subtypes (P < .001). Isoform uc003ysi.3 was associated with greater 5-year survival in luminal A breast cancer (hazard ratio, 0.79; 95% confidence interval, 0.65–0.96; P = .02). PROM1 isoforms uc003gop.2, uc003goq.3, uc003gos.2, and uc003gou.2 were expressed greatest in triple-negative breast cancer (P < .001). PROM1 isoform uc003gou.2 was associated with better 5-year survival in luminal A breast cancer (hazard ratio, 0.79; 95% confidence interval, 0.65–0.97; P = .02). ConclusionsMYC and PROM1 isoforms are differentially expressed in breast cancer subtypes. Certain isoforms confer better survival prognosis. Further work should be done to study alternative splicing in cancer stem cells.

Long-read transcript sequencing identifies differential isoform expression in the entorhinal cortex in a transgenic model of tau pathology

Increasing evidence suggests that alternative splicing plays an important role in Alzheimer’s disease (AD) pathology. We used long-read sequencing in combination with a novel bioinformatics tool (FICLE) to profile transcript diversity in the entorhinal cortex of female transgenic (TG) mice harboring a mutant form of human tau. Our analyses revealed hundreds of novel isoforms and identified differentially expressed transcripts – including specific isoforms of Apoe, App, Cd33, Clu, Fyn and Trem2 – associated with the development of tau pathology in TG mice. Subsequent profiling of the human cortex from AD individuals and controls revealed similar patterns of transcript diversity, including the upregulation of the dominant TREM2 isoform in AD paralleling the increased expression of the homologous transcript in TG mice. Our results highlight the importance of differential transcript usage, even in the absence of gene-level expression alterations, as a mechanism underpinning gene regulation in the development of AD neuropathology.

Reverse engineering neuron type-specific and type-orthogonal splicing-regulatory networks using single-cell transcriptomes.

Cell type-specific alternative splicing (AS) enables differential gene isoform expression between diverse neuron types with distinct identities and functions. Current studies linking individual RNA-binding proteins (RBPs) to AS in a few neuron types underscore the need for holistic modeling. Here, we use network reverse engineering to derive a map of the neuron type-specific AS regulatory landscape from 133 mouse neocortical cell types defined by single-cell transcriptomes. This approach reliably inferred the regulons of 350 RBPs and their cell type-specific activities. Our analysis revealed driving factors delineating neuronal identities, among which we validated Elavl2 as a key RBP for MGE-specific splicing in GABAergic interneurons using an in vitro ESC differentiation system. We also identified a module of exons and candidate regulators specific for long- and short-projection neurons across multiple neuronal classes. This study provides a resource for elucidating splicing regulatory programs that drive neuronal molecular diversity, including those that do not align with gene expression-based classifications.

Expression of alternatively spliced BCMA RNA with skipping of transmembrane domain.

7514 Background: The TNFRSF17 gene, which encodes for the B cell maturation antigen (BCMA), is a small gene expressed mainly on the surface of plasma cells and some DLBCL cells. This gene is composed of three exons. Exon 1 is responsible for the extracellular domain, exon 2 for the transmembrane domain, and exon 3 for the TRAF binding domain. The BCMA is currently targeted by various types of immunotherapies as a major therapeutic approach for the treatment of multiple myeloma (MM). This includes CAR-T cells, bi-specific antibodies, and antibody-drug conjugates (ADC). However, emerging data indicates that alternative splicing in genes is an important mechanism for expression of different isoforms that may influence antibody-based therapies. We explored the potential of the presence of alternative splicing in BCMA transcripts via sequencing of BCMA RNA in patients with lymphoma or multiple myeloma. Methods: RNA was extracted from 587 fresh bone marrow samples with lymphoid/plasma cell neoplasms or from FFPE samples with lymphoma. In addition, cfRNA was extracted from 260 peripheral blood plasma samples from patients with lymphoma or multiple myeloma. RNA was sequenced using a hybrid capture-targeted RNA panel with analysis focused on TNFRSF17 (BCMA) gene transcript. Quantification of RNA transcript was done using Salmon algorithm. Results: Of the 587 lymphoma/plasma cell samples, 161 (27%) samples showed alternative splicing involving deletion of exon 2 (BCMA∆Ex2). Of the 260 cfRNA samples, 14 (6%) showed BCMA∆Ex2. The median percentage of BCMA∆Ex2 transcripts was 0.7% of total BCMA transcripts in cellular samples as compared with 9% in cfRNA samples. In cellular samples, there was a correlation between levels of BCMA and BCMA∆Ex2 (R=0.63). Cases with higher levels of BCMA had significantly higher levels of BCMA∆Ex2 (P&lt;0.0001, Kruskal-Wallis). In contrast, cfRNA showed no correlation between levels of BCMA and levels of BCMA∆Ex2 (R=021, Spearman) and mildly higher level of BCMA∆Ex2 in cases with higher levels of BCMA (P=0.002, Kruskal-Wallis). Conclusions: BCMA exon 2 skipping is detected in a significant number of patients with multiple myeloma and lymphoma. The percentage of skipping transcripts is low in cells and relatively higher in cfRNA. Since exon 2 skipping deletes the transmembrane domain, this BCMA protein may remain in the cytoplasm or perhaps is secreted as cell-free BCMA protein. The demonstration of relatively higher levels of BCMA∆Ex2 in cfRNA is likely reflecting higher turnover of cells and raises the possibility that cells with this isoform of BCMA are more aggressive than cells without the expression of this isoform. Further studies are needed to explore the clinical relevance of the expression of such abnormal BCMA protein on treatment with the various forms of anti-body-based therapy or CAR-T.

Expression of alternatively spliced PD-L1 RNA with skipping of transmembrane domain.

e14596 Background: Programmed death-ligand 1 (PD-L1) is a transmembrane ligand for the programmed cell death protein 1 (PD-1), a receptor that inhibits T-cell activity. The PD-L1/PD-1 immune checkpoint axis has been successfully targeted to enhance antitumor immune responses. This gene is composed of seven exons. Exons 1-4 are responsible for the extracellular domain, exon 5 for the transmembrane domain, and exons 6-7 for the intracellular domain. Emerging data indicates that alternative splicing in genes is an important mechanism for expression of different isoforms that may influence antibody-based therapies. We explored the potential of the presence of alternative splicing in PD-L1 transcripts via sequencing of PD-L1 RNA in patients with various type of solid tumor cancers. Methods: RNA was extracted from 3443 FFPE solid tumor samples including lung, colon, pancreas, breast, and other cancer types. In addition, cfRNA was extracted from 532 peripheral blood plasma samples from patients with solid tumor cancers. RNA was sequenced using a hybrid capture targeted RNA panel on FFPE samples and peripheral blood with analysis focused on PD-L1 gene transcript. Quantification of RNA transcript was done using Salmon algorithm. Results: Of the 3443 FFPE samples, 38 (1.2%) samples showed alternative splicing involving deletion of exon 5 (PD-L1∆Ex5). The alternative splicing was in 2.10% of lung cancer, 2% of colon cancer, 0.70% of pancreatic cancer, 1.40% of gastric cancer, 0.38% of breast cancer, 0.30% of ovarian cancer, 1.10% of sarcoma cases, 1.50% of glioblastomas, 3.30% of thyroid cancer and 2% of urothelial cancer. Of the 532 cfRNA samples, 20 (3.7%) showed PD-L1∆Ex5. The median percentage of PD-L1∆Ex5 transcripts was 2.2% of total PD-L1 transcripts in tissue samples as compared with 6.6% in cfDNA samples. In tissue samples, there was a correlation between levels of PD-L1 and PD-L1∆Ex5 (r = .567, p &lt; .001). In contrast, cfRNA showed no significant correlation between levels of PD-L1 and levels of PD-L1∆Ex5 (r = .268). Conclusions: PD-L1 exon 5 skipping was detected in a number of patients with different types of solid tumor cancers. The percentage of skipping transcripts is low in cells and relatively higher in cfRNA. Since exon 5 skipping deletes the transmembrane domain, the PD-L1 protein may remain in the cytoplasm or perhaps is secreted as cell-free PD-L1 protein. The demonstration of relatively higher levels of PD-L1∆Ex5 in cfRNA is likely reflecting higher turnover of cells and raises the possibility that cells with this isoform of PD-L1 are more aggressive than cells without the expression of this isoform. Further studies are needed to explore the clinical relevance of the expression of such abnormal PD-L1 protein on treatment with immune checkpoint inhibitors.

Differential expression of WNT5A long and short isoforms in non-muscle-invasive bladder urothelial carcinoma.

Wnt ligands belong to a family of secreted glycoproteins in which binding to a range of receptors/co-receptors activates several intracellular pathways. WNT5A, a member of the Wnt family, is classified as a non-canonical Wnt whose activation triggers planar cell polarity (PCP) and Ca+2 downstream pathways. Aberrant expression of WNT5A has been shown to play both protective and harmful roles in an array of conditions, such as inflammatory disease and cancer. In the present study, using histological, immunohistochemical, and molecular methods, we investigated the expression of two isoforms of WNT5A, WNT5A-Short (WNT5A-S) and WNT5A-Long (WNT5A-L) in bladder urothelial carcinoma (UC). Three UC cell lines (RT4, J82, and T24), as well as a normal urothelial cell line, and formalin-fixed, paraffin-embedded (FFPE) transurethral resection (TUR) tissue samples from 17 patients diagnosed with UC were included in the study. WNT5A-L was the predominantly expressed isoform in urothelial cells, although WNT5A-S was also detectable. Further, although no statistically significant difference was found between the percentage of WNT5A-S transcripts in low-grade versus high-grade tumors, we did find a difference between the percentage of WNT5A-S transcripts found in non-invasion versus invasion of the lamina propria, subgroups of non-muscle-invasive tumors. In conclusion, both WNT5A-S and WNT5A-L isoforms are expressed in UC, and the percentage of their expression levels suggests that a higher proportion of WNT5A-S transcription may be associated with lamina propria invasion, a process preceding muscle invasion.

Alternative isoform expression of key thermogenic genes in human beige adipocytes.

The beneficial effect of thermogenic adipocytes in maintaining body weight and protecting against metabolic disorders has raised interest in understanding the regulatory mechanisms defining white and beige adipocyte identity. Although alternative splicing has been shown to propagate adipose browning signals in mice, this has yet to be thoroughly investigated in human adipocytes. We performed parallel white and beige adipogenic differentiation using primary adipose stem cells from 6 unrelated healthy subjects and assessed differential gene and isoform expression in mature adipocytes by RNA sequencing. We find 777 exon junctions with robust differential usage between white and beige adipocytes in all 6 subjects, mapping to 562 genes. Importantly, only 10% of these differentially spliced genes are also differentially expressed, indicating that alternative splicing constitutes an additional layer of gene expression regulation during beige adipocyte differentiation. Functional classification of alternative isoforms points to a gain of function for key thermogenic transcription factors such as PPARG and CITED1, and enzymes such as PEMT, or LPIN1. We find that a large majority of the splice variants arise from differential TSS usage, with beige-specific TSSs being enriched for PPARγ and MED1 binding compared to white-specific TSSs. Finally, we validate beige specific isoform expression at the protein level for two thermogenic regulators, PPARγ and PEMT. These results suggest that differential isoform expression through alternative TSS usage is an important regulatory mechanism for human adipocyte thermogenic specification.

Leishmania Ribosomal Protein (RP) paralogous genes compensate each other's expression maintaining protein native levels.

In the protozoan parasite Leishmania, most genes encoding for ribosomal proteins (RPs) are present as two or more copies in the genome. However, their untranslated regions (UTRs) are predominantly divergent and might be associated with a distinct regulation of the expression of paralogous genes. Herein, we investigated the expression profiles of two RPs (S16 and L13a) encoded by duplicated genes in Leishmania major. The genes encoding for the S16 protein possess identical coding sequences (CDSs) and divergent UTRs, whereas the CDSs of L13a diverge by two amino acids and by their UTRs. Using CRISPR/Cas9 genome editing, we generated knockout (Δ) and endogenously tagged transfectants for each paralog of L13a and S16 genes. Combining tagged and Δ cell lines we found evidence of differential expression of both RPS16 and RPL13a isoforms throughout parasite development, with one isoform consistently more abundant than its respective copy. In addition, compensatory expression was observed for each paralog upon deletion of the corresponding isoform, suggesting functional conservation between these proteins. This differential expression pattern relates to post-translational processes, given compensation occurs at the level of the protein, with no alterations detected at transcript level. Ribosomal profiles for RPL13a indicate a standard behavior for these paralogues suggestive of interaction with heavy RNA-protein complexes, as already reported for other RPs in trypanosomatids. We identified paralog-specific bound to their 3'UTRs which may be influential in regulating paralog expression. In support, we identified conserved cis-elements within the 3'UTRs of RPS16 and RPL13a; cis-elements exclusive to the UTR of the more abundant paralog or to the less abundant ones were identified.

Alternative splicing and its regulation in the malaria vector Anopheles gambiae

IntroductionAlternative splicing (AS) is a highly conserved mechanism that allows for the expansion of the coding capacity of the genome, through modifications of the way that multiple isoforms are expressed or used to generate different phenotypes. Despite its importance in physiology and disease, genome-wide studies of AS are lacking in most insects, including mosquitoes. Even for model organisms, chromatin associated processes involved in the regulation AS are poorly known.MethodsIn this study, we investigated AS in the mosquito Anopheles gambiae in the context of tissue-specific gene expression and mosquito responses to a Plasmodium falciparum infection, as well as the relationship between patterns of differential isoform expression and usage with chromatin accessibility changes. For this, we combined RNA-seq and ATAC-seq data from A. gambiae midguts and salivary glands, infected and non-infected.ResultsWe report differences between tissues in the expression of 392 isoforms and in the use of 247 isoforms. Secondly, we find a clear and significant association between chromatin accessibility states and tissue-specific patterns of AS. The analysis of differential accessible regions located at splicing sites led to the identification of several motifs resembling the binding sites of Drosophila transcription factors. Finally, the genome-wide analysis of tissue-dependent enhancer activity revealed that approximately 20% of A. gambiae transcriptional enhancers annotate to a differentially expressed or used isoform, and that their activation status is linked to AS differences between tissues.ConclusionThis research elucidates the role of AS in mosquito vector gene expression and identifies regulatory regions potentially involved in AS regulation, which could be important in the development of novel strategies for vector control.

Full-length isoform concatenation sequencing to resolve cancer transcriptome complexity

BackgroundCancers exhibit complex transcriptomes with aberrant splicing that induces isoform-level differential expression compared to non-diseased tissues. Transcriptomic profiling using short-read sequencing has utility in providing a cost-effective approach for evaluating isoform expression, although short-read assembly displays limitations in the accurate inference of full-length transcripts. Long-read RNA sequencing (Iso-Seq), using the Pacific Biosciences (PacBio) platform, can overcome such limitations by providing full-length isoform sequence resolution which requires no read assembly and represents native expressed transcripts. A constraint of the Iso-Seq protocol is due to fewer reads output per instrument run, which, as an example, can consequently affect the detection of lowly expressed transcripts. To address these deficiencies, we developed a concatenation workflow, PacBio Full-Length Isoform Concatemer Sequencing (PB_FLIC-Seq), designed to increase the number of unique, sequenced PacBio long-reads thereby improving overall detection of unique isoforms. In addition, we anticipate that the increase in read depth will help improve the detection of moderate to low-level expressed isoforms.ResultsIn sequencing a commercial reference (Spike-In RNA Variants; SIRV) with known isoform complexity we demonstrated a 3.4-fold increase in read output per run and improved SIRV recall when using the PB_FLIC-Seq method compared to the same samples processed with the Iso-Seq protocol. We applied this protocol to a translational cancer case, also demonstrating the utility of the PB_FLIC-Seq method for identifying differential full-length isoform expression in a pediatric diffuse midline glioma compared to its adjacent non-malignant tissue. Our data analysis revealed increased expression of extracellular matrix (ECM) genes within the tumor sample, including an isoform of the Secreted Protein Acidic and Cysteine Rich (SPARC) gene that was expressed 11,676-fold higher than in the adjacent non-malignant tissue. Finally, by using the PB_FLIC-Seq method, we detected several cancer-specific novel isoforms.ConclusionThis work describes a concatenation-based methodology for increasing the number of sequenced full-length isoform reads on the PacBio platform, yielding improved discovery of expressed isoforms. We applied this workflow to profile the transcriptome of a pediatric diffuse midline glioma and adjacent non-malignant tissue. Our findings of cancer-specific novel isoform expression further highlight the importance of long-read sequencing for characterization of complex tumor transcriptomes.

Different expression levels of interleukin-36 in asthma phenotypes

Interleukin (IL)-36 family is closely associated with inflammation and consists of IL-36α, IL-36β, IL-36γ, and IL-36Ra. The role of IL-36 in the context of asthma and asthmatic phenotypes is not well characterized. We examined the sputum IL-36 levels in patients with different asthma phenotypes in order to unravel the mechanism of IL-36 in different asthma phenotypes. Our objective was to investigate the induced sputum IL-36α, IL-36β, IL-36γ, and IL-36Ra concentrations in patients with mild asthma, and to analyze the relationship of these markers with lung function and other cytokines in patients with different asthma phenotypes. Induced sputum samples were collected from patients with mild controlled asthma (n = 62, 27 males, age 54.77 ± 15.49) and healthy non-asthmatic controls (n = 16, 10 males, age 54.25 ± 14.60). Inflammatory cell counts in sputum were determined. The concentrations of IL-36 and other cytokines in the sputum supernatant were measured by ELISA and Cytometric Bead Array. This is the first study to report the differential expression of different isoforms of IL-36 in different asthma phenotypes. IL-36α and IL-36β concentrations were significantly higher in the asthma group (P = 0.003 and 0.031), while IL-36Ra concentrations were significantly lower (P < 0.001) compared to healthy non-asthmatic controls. Sputum IL-36α and IL-36β concentrations in the neutrophilic asthma group were significantly higher than those in paucigranulocytic asthma (n = 24) and eosinophilic asthma groups (n = 23). IL-36α and IL-36β showed positive correlation with sputum neutrophils and total cell count (R = 0.689, P < 0.01; R = 0.304, P = 0.008; R = 0.689, P < 0.042; R = 0.253, P = 0.026). In conclusion, IL-36α and IL-36β may contribute to asthma airway inflammation by promoting neutrophil recruitment in airways. Our study provides insights into the inflammatory pathways of neutrophilic asthma and identifies potential therapeutic target.

Casein Kinase 1 Phosphomimetic Mutations Negatively Impact Connexin-43 Gap Junctions in Human Pluripotent Stem Cell-Derived Cardiomyocytes.

The transplantation of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) has shown promise in preclinical models of myocardial infarction, but graft myocardium exhibits incomplete host-graft electromechanical integration and a propensity for pro-arrhythmic behavior. Perhaps contributing to this situation, hPSC-CM grafts show low expression of connexin 43 (Cx43), the major gap junction (GJ) protein, in ventricular myocardia. We hypothesized that Cx43 expression and function could be rescued by engineering Cx43 in hPSC-CMs with a series of phosphatase-resistant mutations at three casein kinase 1 phosphorylation sites (Cx43-S3E) that have been previously reported to stabilize Cx43 GJs and reduce arrhythmias in transgenic mice. However, contrary to our predictions, transgenic Cx43-S3E hPSC-CMs exhibited reduced Cx43 expression relative to wild-type cells, both at baseline and following ischemic challenge. Cx43-S3E hPSC-CMs showed correspondingly slower conduction velocities, increased automaticity, and differential expression of other connexin isoforms and various genes involved in cardiac excitation-contraction coupling. Cx43-S3E hPSC-CMs also had phosphorylation marks associated with Cx43 GJ internalization, a finding that may account for their impaired GJ localization. Taken collectively, our data indicate that the Cx43-S3E mutation behaves differently in hPSC-CMs than in adult mouse ventricular myocytes and that multiple biological factors likely need to be addressed synchronously to ensure proper Cx43 expression, localization, and function.

Differential expression of various isoforms of superoxide dismutase in the cells of the human exocrine pancreas.

Superoxide dismutase (SOD) is an enzyme that plays acrucial role in protecting cells from oxidative damage. Our study aims to address the lack of papers simultaneously analyzing the immunoreactivity of all three distinct isoforms of SOD in human exocrine pancreas cells. Superoxide dismutases (SODs) facilitate the conversion of superoxide radicals into less harmful substances. By neutralizing superoxide radicals, SODs help prevent the formation of highly reactive and destructive species that can adversely affect manifold cellular components. The study analyzed immunoreactivity of SODs in samples of six healthy adult human pancreases, while using the indirect immunohistochemical method under alight microscope. SOD1 was predominantly found in centroacinar cells and epithelial cells of the duct system while SOD2 was mainly detected in the epithelial cells of interlobular ducts. Both enzymes were prominently present in the basal region of acinar cells near the cell nucleus. The expression of SOD3 was observed to be rare. Understanding the intracellular metabolism of SODs in healthy exocrine pancreas cells serves as abasis for determining the precise role of oxidative damage and SOD signaling in the pathogenesis of various pancreatic diseases, including chronic pancreatitis and pancreatic cancer (Fig. 6, Ref. 24). Text in PDF www.elis.sk Keywords: antioxidants, histology, immunohistochemistry, pancreas, superoxide dismutase.

Synaptopodin-2 Isoforms Have Specific Binding Partners and Display Distinct, Muscle Cell Type-Specific Expression Patterns.

Synaptopodin-2 (SYNPO2) is a protein associated with the Z-disc in striated muscle cells. It interacts with α-actinin and filamin C, playing a role in Z-disc maintenance under stress by chaperone-assisted selective autophagy (CASA). In smooth muscle cells, SYNPO2 is a component of dense bodies. Furthermore, it has been proposed to play a role in tumor cell proliferation and metastasis in many different kinds of cancers. Alternative transcription start sites and alternative splicing predict the expression of six putative SYNPO2 isoforms differing by extended amino- and/or carboxy-termini. Our analyses at mRNA and protein levels revealed differential expression of SYNPO2 isoforms in cardiac, skeletal and smooth muscle cells. We identified synemin, an intermediate filament protein, as a novel binding partner of the PDZ-domain in the amino-terminal extension of the isoforms mainly expressed in cardiac and smooth muscle cells, and demonstrated colocalization of SYNPO2 and synemin in both cell types. A carboxy-terminal extension, mainly expressed in smooth muscle cells, is sufficient for association with dense bodies and interacts with α-actinin. SYNPO2 therefore represents an additional and novel link between intermediate filaments and the Z-discs in cardiomyocytes and dense bodies in smooth muscle cells, respectively. In pathological skeletal muscle samples, we identified SYNPO2 in the central and intermediate zones of target fibers of patients with neurogenic muscular atrophy, and in nemaline bodies. Our findings help to understand distinct functions of individual SYNPO2 isoforms in different muscle tissues, but also in tumor pathology.

Insight of Oxidative Stress, Ultrastructural Transformations, and Elemental Variations in Rice Seedlings Exposed to Boron Toxicity: Unraveling the Role of Fe-SOD in Boron Tolerance

Rice (Oryza sativa L.) seedlings differing in boron tolerance were grown in hydroponics containing varying boron concentration. (as boric acid) for 8 days. Yoshida Nutrient Solution (Yoshida et al.1976) served as control, whereas 0.5 mM boron (moderately toxic) and 1.5 mM boron (highly toxic). Boron excess (B) caused marked reduction in length, biomass and relative water content of the seedlings, with more reductions in B-sensitive cv. Malaviya-36 as compared with B-tolerant cv. Brown Gora. B-sensitive seedlings showed higher B uptake in roots and shoots compared to the tolerant. Scanning electron microscopy (SEM) revealed ultrastructural damage to the guard cells with excess boron. Energy dispersive X-ray analysis (EDXA) of rice leaves showed decline in concentration of P, S, Ca and Mg in seedlings on B treatment. Increased production of reactive oxygen species O2˙−, H2O2, lipid peroxides, alteration in activity of antioxidative enzymes and increased membrane permeability was observed in B treated seedlings compared to controls. Real time PCR analysis of stress regulatory genes indicated differential expression of SOD isoforms in the two sets of cultivars with B treatment. Interestingly seedlings of B-tolerant cultivar were characterized by higher level of expression of Fe-SOD and its further increased expression on B treatment. Results suggest that B toxicity involves ultrastructural and elemental changes, increased generation of ROS and altered antioxidative enzyme activities in rice seedlings and increased expression Fe-SOD isoform appears to be associated with B tolerance.

Differential Expression of Insulin Growth Factor 1 (IGF-1) Isoforms in Different Types of Endometriosis: Preliminary Results of a Single-Center Study.

Endometriosis is a benign, estrogen-dependent gynecological condition with an uncertain exact pathogenetic mechanism. The aim of this study was to evaluate the potential differential expression of Insulin Growth Factor 1 (IGF-1) isoforms in deeply infiltrating endometriotic (DIE) lesions, in ovarian endometriomas, and in the eutopic endometrium of the same endometriosis patients and to compare their expression with that in the eutopic endometrium of women without endometriosis. A total of 39 patients were included: 28 with endometriosis, of whom 15 had endometriomas only, 7 had DIE nodules only, and 6 had both DIE and endometriomas, and 11 without endometriosis served as controls. We noticed a similar pattern of expression between IGF-1Ea and IGF-1Ec, which differed from that of the IGF-1Eb isoform, possibly implying differential biological actions of different isoforms in DIE subtypes. We observed a tendency of lower expression of IGF-1Ea and IGF-1Ec in endometriomas without DIE compared to endometriomas with concurrent DIE or in DIE nodules. In conclusion, differential expression of IGF-1 isoforms may indicate that DIE with its associated ovarian lesions and simple ovarian endometriosis should be considered as two forms of the disease developing under different molecular pathways.

Alternative Polyadenylation of Malic Enzyme 1 Is Essential for Accelerated Adipogenesis.

Understanding the mechanism of adipogenesis is an important basis for improving meat quality traits of livestock. Alternative polyadenylation (APA) is a vital mechanism to regulate the expression of eukaryotic genes. However, how the individual APA functions in adipogenesis remains elusive. This study was intended to investigate the effect of malic enzyme 1 (ME1) APA on adipogenesis. Here, intracellular lipid droplets were stained using Oil red O. 3' RACE was used to verify APA events of the ME1 gene. Interactions between ME1 3' untranslated region (3' UTR)-APA isoforms and miRNAs, as well as differential expression of isoforms, were examined using dual-luciferase reporter and molecular experiments. The mechanism of ME1 APA on adipogenesis was explored by gain and loss of function assays. In this study, two ME1 isoforms with different 3' UTR lengths were detected during adipogenesis. Moreover, the ME1 isoform with a short 3' UTR was significantly upregulated compared with the one with a long 3' UTR. Mechanistically, only the long ME1 isoform was targeted by miR-153-3p to attenuate adipogenesis, while the short one escaped the regulation of miR-153-3p to accelerate adipogenesis. Our results reveal a novel mechanism of ME1 APA in regulating adipogenesis.

HNF4α isoforms regulate the circadian balance between carbohydrate and lipid metabolism in the liver.

Hepatocyte Nuclear Factor 4α (HNF4α), a master regulator of hepatocyte differentiation, is regulated by two promoters (P1 and P2) which drive the expression of different isoforms. P1-HNF4α is the major isoform in the adult liver while P2-HNF4α is thought to be expressed only in fetal liver and liver cancer. Here, we show that P2-HNF4α is indeed expressed in the normal adult liver at Zeitgeber time (ZT)9 and ZT21. Using exon swap mice that express only P2-HNF4α we show that this isoform orchestrates a distinct transcriptome and metabolome via unique chromatin and protein-protein interactions, including with different clock proteins at different times of the day leading to subtle differences in circadian gene regulation. Furthermore, deletion of the Clock gene alters the circadian oscillation of P2- (but not P1-)HNF4α RNA, revealing a complex feedback loop between the HNF4α isoforms and the hepatic clock. Finally, we demonstrate that while P1-HNF4α drives gluconeogenesis, P2-HNF4α drives ketogenesis and is required for elevated levels of ketone bodies in female mice. Taken together, we propose that the highly conserved two-promoter structure of the Hnf4a gene is an evolutionarily conserved mechanism to maintain the balance between gluconeogenesis and ketogenesis in the liver in a circadian fashion.