Biology subjects

Uapinyoying, P.

Publications and source records attributed to Uapinyoying, P..

4 recordsLinked to original sources

bioRxiv2024

Failure to Resolve Inflammation Contributes to Juvenile-Onset Cardiomyopathy in a Mouse Model of Duchenne Muscular Dystrophy

Absence of dystrophin protein causes cardiac dysfunction in patients with Duchenne muscular dystrophy (DMD). Unlike boys with DMD, the common mouse model of DMD (B10-mdx) does not manifest cardiac deficits until late adulthood. This has limited our understanding of the mechanism and therapeutic approaches to target the pediatric onset of cardiac pathology in DMD. Here we show that the mdx mouse model on the DBA/2J genetic background (D2-mdx) displays juvenile-onset cardiac degeneration. Molecular and histological analysis revealed that cardiac damage in this model is linked to increased leukocyte chemotactic signaling and an inability to resolve inflammation. These deficiencies result in chronic inflammation and fibrotic conversion of the extracellular matrix (ECM) in the juvenile D2-mdx heart. To address these pathologies, we tested the utility of pro-resolution therapy to clear chronic cardiac inflammation. Use of an N-formyl peptide receptor (FPR) agonist helped physiologically resolve inflammation and mitigate the downstream events that lead to fibrotic degeneration of cardiomyocytes, preventing juvenile onset cardiac muscle loss. These results establish the utility of D2-mdx model to study events associated with pediatric-onset cardiac damage and demonstrates pro-resolution therapy as an alternate to anti-inflammatory therapy for treating cardiac degenerative pathology responsible for cardiomyopathy in DMD patients.

cell biology↗

bioRxiv2024

Allele-specific CRISPR/Cas9 editing inactivates a single nucleotide variant associated with collagen VI muscular dystrophy

The application of allele-specific gene editing tools can expand the therapeutic options for dominant genetic conditions, either via gene correction or via allelic gene inactivation in situations where haploinsufficiency is tolerated. Here, we used allele-targeted CRISPR/Cas9 guide RNAs (gRNAs) to introduce inactivating frameshifting indels at a single nucleotide variant in the COL6A1 gene (c.868G>A; G290R), a variant that acts as dominant negative and that is associated with a severe form of congenital muscular dystrophy. We expressed spCas9 along with allele-targeted gRNAs, without providing a repair template, in primary fibroblasts derived from four patients and one control subject. Amplicon deep-sequencing for two gRNAs tested showed that single nucleotide deletions accounted for the majority of indels introduced. While activity of the two gRNAs was greater at the G290R allele, both gRNAs were also active at the wild-type allele. To enhance allele-selectivity, we introduced deliberate additional mismatches to one gRNA. One of these optimized gRNAs showed minimal activity at the WT allele, while generating productive edits and improving collagen VI matrix in cultured patient fibroblasts. This study strengthens the potential of gene editing to treat dominant-negative disorders, but also underscores the challenges in achieving allele selectivity with gRNAs.

molecular biology↗

bioRxiv2018

Impaired ureagenesis due to arginine-insensitive N-acetylglutamate synthase

The urea cycle protects the central nervous system from ammonia toxicity by converting ammonia to non-toxic urea. N-acetylglutamate synthase (NAGS) is an enzyme that catalyzes the formation of N-acetylglutamate (NAG), an allosteric activator of carbamylphosphate synthetase 1 (CPS1), the rate limiting enzyme of the urea cycle. Enzymatic activity of mammalian NAGS doubles in the presence of L-arginine but the physiological significance of NAGS activation by L-arginine is unknown. Previously, we have described the creation of a NAGS knockout (Nags-/-) mouse, which develops hyperammonemia without N-carbamylglutamate and L-citrulline supplementation (NCG+Cit). In order to investigate the effect of L-arginine on ureagenesis in vivo, we used adeno associated virus (AAV) mediated gene transfer to deliver either wild-type or E354A mutant mouse NAGS (mNAGS), which is not activated by L-arginine, to Nags-/- mice. The ability of the E354A mNAGS mutant protein to rescue Nags-/- mice was determined by measuring their activity on the voluntary wheel following NCG+Cit withdrawal. The Nags-/- mice that received E354A mNAGS remained apparently healthy and active but had elevated plasma ammonia concentration despite similar expression levels of the E354A mNAGS and control wild-type NAGS proteins. The corresponding mutation in human NAGS (NP 694551.1:p.E360D) that abolishes binding and activation by L-arginine was also identified in a patient with hyperammonemia due to NAGS deficiency. Taken together, our results suggest that L-arginine binding to the NAGS enzyme is essential for normal ureagenesis.

molecular biology↗

bioRxiv2017

Interactive analysis of Long-read RNA isoforms with Iso-Seq Browser

BackgroundLong-read RNA sequencing, such as Pacific Biosciences Iso-Seq method, enables generation of sequencing reads that are 10 kilobases or even longer. These reads are ideal for discovering splice junctions and resolving full-length gene transcripts without time-consuming and error-prone techniques such as transcript assembly and junction inference.\n\nResultsIso-Seq Browser is a Web-based visual analytics tool for long-read RNA sequencing data produced by Pacific Biosciences isoform sequencing (Iso-Seq) techniques. Key features of the Iso-Seq Browser are: 1) an exon-only web-based interface with zooming and exon highlighting for exploring reference gene transcripts and novel gene isoforms, 2) automated grouping of transcripts and isoforms by similarity, 3) many customization features for data exploration and creating publication ready figures, and 4) exporting selected isoforms into fasta files for further analysis. Iso-Seq Browser is written in Python using several scientific libraries. The application and analyses described in this paper are freely available to both academic and commercial users at https://github.com/goeckslab/isoseq-browser\n\nConclusionsIso-Seq Browser enables interactive genome-wide visual analysis of long RNA sequence reads. Through visualization, highlighting, clustering, and filtering of gene isoforms, ISB makes it simple to identify novel isoforms and novel isoform features such as exons, introns and untranslated regions.

bioinformatics↗