Forward genetic screen using a gene-breaking trap approach identifies a novel role of grin2bb-associated RNA transcript (grin2bbART) in zebrafish heart function

LncRNA-based control affects cardiac pathophysiologies like myocardial infarction, coronary artery disease, hypertrophy, and myotonic muscular dystrophy. This study used a gene-break transposon (GBT) to screen zebrafish (Danio rerio) for insertional mutagenesis. We identified three insertional mutants where the GBT captured a cardiac gene. One of the adult viable GBT mutants had bradycardia (heart arrhythmia) and enlarged cardiac chambers or hypertrophy; we named it “bigheart.” Bigheart mutant insertion maps to grin2bb or N-methyl D-aspartate receptor (NMDAR2B) gene intron 2 in reverse orientation. Rapid amplification of adjacent cDNA ends analysis suggested a new insertion site transcript in the intron 2 of grin2bb. Analysis of the RNA sequencing of wild-type zebrafish heart chambers revealed a possible new transcript at the insertion site. As this putative lncRNA transcript satisfies the canonical signatures, we called this transcript grin2bb associated RNA transcript (grin2bbART). Using in situ hybridization, we confirmed localized grin2bbART expression in the heart, central nervous system, and muscles in the developing embryos and wild-type adult zebrafish atrium and bulbus arteriosus. The bigheart mutant had reduced Grin2bbART expression. We showed that bigheart gene trap insertion excision reversed cardiac-specific arrhythmia and atrial hypertrophy and restored grin2bbART expression. Morpholino-mediated antisense downregulation of grin2bbART in wild-type zebrafish embryos mimicked bigheart mutants; this suggests grin2bbART is linked to bigheart. Cardiovascular tissues use Grin2bb as a calcium-permeable ion channel. Calcium imaging experiments performed on bigheart mutants indicated calcium mishandling in the heart. The bigheart cardiac transcriptome showed differential expression of calcium homeostasis, cardiac remodeling, and contraction genes. Western blot analysis highlighted Camk2d1 and Hdac1 overexpression. We propose that altered calcium activity due to disruption of grin2bbART, a putative lncRNA in bigheart, altered the Camk2d-Hdac pathway, causing heart arrhythmia and hypertrophy in zebrafish

Role of PLEXIND1/TGFβ signaling axis in pancreatic ductal adenocarcinoma progression correlates with the mutational status of KRAS

PLEXIND1 is upregulated in several cancers, including pancreatic ductal adenocarcinoma (PDAC). It is an established mediator of semaphorin signaling, and neuropilins are its known coreceptors. Herein, we report data to support the proposal that PLEXIND1 acts as a transforming growth factor beta (TGFβ) coreceptor, modulating cell growth through SMAD3 signaling. Our findings demonstrate that PLEXIND1 plays a pro-tumorigenic role in PDAC cells with oncogenic KRAS (KRASmut). We show in KRASmut PDAC cell lines (PANC-1, AsPC-1,4535) PLEXIND1 downregulation results in decreased cell viability (in vitro) and reduced tumor growth (in vivo). Conversely, PLEXIND1 acts as a tumor suppressor in the PDAC cell line (BxPC-3) with wild-type KRAS (KRASwt), as its reduced expression results in higher cell viability (in-vitro) and tumor growth (in vivo). Additionally, we demonstrate that PLEXIND1-mediated interactions can be selectively disrupted using a peptide based on its C-terminal sequence (a PDZ domain-binding motif), an outcome that may possess significant therapeutic implications. To our knowledge, this is the first report showing that (1) PLEXIND1 acts as a TGFβ coreceptor and mediates SMAD3 signaling, and (2) differential roles of PLEXIND1 in PDAC cell lines correlate with KRASmut and KRASwt status

A multicentric consortium study demonstrates that dimethylarginine dimethylaminohydrolase 2 is not a dimethylarginine dimethylaminohydrolase

Dimethylarginine dimethylaminohydrolase 1 (DDAH1) protects against cardiovascular disease by metabolising the risk factor asymmetric dimethylarginine (ADMA). However, the question whether the second DDAH isoform, DDAH2, directly metabolises ADMA has remained unanswered. Consequently, it is still unclear if DDAH2 may be a potential target for ADMA-lowering therapies or if drug development efforts should focus on DDAH2's known physiological functions in mitochondrial fission, angiogenesis, vascular remodelling, insulin secretion, and immune responses. Here, an international consortium of research groups set out to address this question using in silico, in vitro, cell culture, and murine models. The findings uniformly demonstrate that DDAH2 is incapable of metabolising ADMA, thus resolving a 20-year controversy and providing a starting point for the investigation of alternative, ADMA-independent functions of DDAH2

Zebrafish as a Model for Cardiovascular and Metabolic Disease: The Future of Precision Medicine

The zebrafish (Danio rerio) has emerged as an appreciated and versatile model organism for studying cardiovascular and metabolic diseases, offering unique advantages for both basic research and drug discovery. The genetic conservation between zebrafish and humans and their high fecundity and transparent embryos allow for efficient large-scale genetic and drug-oriented screening studies. Zebrafish possess a simplified cardiovascular system that shares similarities with mammals, making them particularly suitable for modeling various aspects of heart development, function, and disease. The transparency of zebrafish embryos enables the real-time visualization of cardiovascular dynamics, offering insights into early embryonic events and facilitating the study of heart-related anomalies. In metabolic research, zebrafish provide a cost-effective platform for modeling obesity, type 2 diabetes, hyperlipidemia, and other metabolic disorders. Their high reproductive rate allows for the generation of large cohorts for robust statistical analyses, while advanced genetic tools, such as CRISPR/Cas9, enable precise gene editing with which to model specific genetic mutations associated with human diseases. Zebrafish metabolic models have been instrumental in elucidating the molecular mechanisms underlying metabolic diseases, studying the effects of environmental factors, and identifying potential therapeutic targets. Additionally, the permeability of zebrafish embryos to small molecules facilitates drug discovery and screening, offering a rapid and economical approach to identifying compounds with therapeutic potential. In conclusion, zebrafish cardiovascular and metabolic disease models continue to contribute significantly to our perception of disease pathogenesis, providing a platform for translational research and developing novel therapeutic interventions. The versatility, scalability, and genetic manipulability of zebrafish position them as an invaluable asset in unraveling the complexities of cardiovascular and metabolic diseases. This review presents an overview of the zebrafish model’s key features and contributions to investigating cardiovascular and metabolic disorders. We discuss the benefits and drawbacks of using zebrafish models to study human disease and the critical findings revealed by the progress in this endeavor to date

Advances in Glioblastoma Therapy: An Update on Current Approaches

Glioblastoma multiforme (GBM) is a primary malignant brain tumor characterized by a high grade of malignancy and an extremely unfavorable prognosis. The current efficacy of established treatments for GBM is insufficient, necessitating the prompt development of novel therapeutic approaches. The progress made in the fundamental scientific understanding of GBM is swiftly translated into more advanced stages of therapeutic studies. Despite extensive efforts to identify new therapeutic approaches, GBM exhibits a high mortality rate. The current efficacy of treatments for GBM patients is insufficient due to factors such as tumor heterogeneity, the blood–brain barrier, glioma stem cells, drug efflux pumps, and DNA damage repair mechanisms. Considering this, pharmacological cocktail therapy has demonstrated a growing efficacy in addressing these challenges. Towards this, various forms of immunotherapy, including the immune checkpoint blockade, chimeric antigen receptor T (CAR T) cell therapy, oncolytic virotherapy, and vaccine therapy have emerged as potential strategies for enhancing the prognosis of GBM. Current investigations are focused on exploring combination therapies to mitigate undesirable side effects and enhance immune responses against tumors. Furthermore, clinical trials are underway to evaluate the efficacy of several strategies to circumvent the blood–brain barrier (BBB) to achieve targeted delivery in patients suffering from recurrent GBM. In this review, we have described the biological and molecular targets for GBM therapy, pharmacologic therapy status, prominent resistance mechanisms, and new treatment approaches. We also discuss these promising therapeutic approaches to assess prospective innovative therapeutic agents and evaluated the present state of preclinical and clinical studies in GBM treatment. Overall, this review attempts to provide comprehensive information on the current status of GBM therapy

A genome-wide map of circular RNAs in adult zebrafish

Circular RNAs are a new addition to the growing list of diverse species of RNAs that are formed by covalent linked 3' and 5' end forming a closed loop structure. Circular RNAs are characteristically resistant to exonuclease treatment and are relatively stable to linear transcripts. Circular RNAs are formed by alternate splicing mechanism but do not follow the canonical order of exons. Backsplice junctions are unique to circRNAs.  CircRNAs are shown to possess potential to act as miRNA sponges and control transcription of mRNAs. CircRNAs are also reported as biomarkers for the disease like Alzheimer's, Parkinson's and cancer. A huge number of circRNA transcripts have been identified in model organisms including C.elegans, mouse, Drosophila as well as human. But there are no circular RNAs reported in zebrafish that is a very good model to study developmental stages, cardiovascular and blood-related disorders. In order to use zebrafish as a model organism and study the role of circRNAs in disease, we have used in-house generated RNA-sequencing data for five tissues including blood, brain, muscle, gills and heart. We discarded the reads mapped contiguously and full length over reference genome and identified back-splice junctions for putative circRNA transcripts. We identified a total of 3428 circRNA junctions out of which 78% were tissue specific. We validated 22 selected candidates for 5 tissues based on literature significance. We quantitatively analysed 5 tissue-enriched candidates using Real-time PCR. We also observed that major proportion of circRNAs is originating from protein coding loci. These circRNAs could be used to further study their role in hematopoietic and cardiovascular diseases

Reverse Genetics Screen in Zebrafish Identifies a Role of miR-142a-3p in Vascular Development and Integrity

<div><p>MicroRNAs are a well-studied class of non-coding RNA and are known to regulate developmental processes in eukaryotes. Their role in key biological processes such as vasculature development has attracted interest. However, a comprehensive understanding of molecular regulation of angiogenesis and vascular integrity during development remains less explored. Here we identified miRNAs involved in the development and maintenance of vasculature in zebrafish embryos using a reverse genetics approach. Using a combination of bioinformatics predictions and literature based evidences we mined over 701 Human and 329 Zebrafish miRNAs to derive a list of 29 miRNAs targeting vascular specific genes in zebrafish. We shortlisted eight miRNAs and investigated their potential role in regulating vascular development in zebrafish transgenic model. In this screen we identified three miRNAs, namely miR-1, miR-144 and miR-142a-3p that have the potential to influence vascular development in zebrafish. We show that miR-142a-3p mediates vascular integrity and developmental angiogenesis <em>in vivo.</em> Overexpression of miR-142a-3p results in loss of vascular integrity, hemorrhage and vascular remodeling during zebrafish embryonic development, while loss of function of miR-142a-3p causes abnormal vascular remodeling. MiR-142a-3p functions in part by directly repressing <em>cdh5</em> (<em>VE-cadherin).</em> The vascular abnormalities that results from modulation of miR-142a-3p are reminiscent of <em>cdh5</em> perturbation in zebrafish embryos. We also demonstrate that the action of miR-142a on <em>cdh5</em> is potentially regulated by Lmo2, an important transcription factor, known for its role in vasculature development. The miR142a-3p mediated control of <em>cdh5</em> constitutes an additional layer of regulation for maintaining vascular integrity and developmental angiogenesis. These findings have implications in development, wound repair and tumor growth.</p> </div

<i>In silico</i> pipeline for selection of miRNAs involved in vasculature development.

<p><i>In silico</i> pipeline for selection of miRNAs involved in vasculature development.</p

Overexpression of miR-142a-3p in zebrafish embryos induces vascular defects.

<p>A – D - Confocal images (GFP and GFP/RFP merged) of 10 µM miR-142a-3p duplex injected Tg<i>(fli1:EGFP, gata1a: dsRed)</i> embryos at 2 dpf (Dorsal View of head) depicting dorsal midbrain junction (DMJ) and central arteries (CtA) (10× magnification). A, B - non-injected control embryos and C, D - 10 µM miR-142a-3p duplex injected embryos. A, C – GFP, B, D – GFP/RFP merged. Asterisk sign indicate the site of vascular defect.</p