scholarly journals Early pathological signs in young dysf−/− mice are improved by halofuginone

2019 ◽  
Author(s):  
Hila Barzilai-Tutsch ◽  
Olga Genin ◽  
Mark Pines ◽  
Orna Halevy
Keyword(s):  
Late Onset ◽  
Disease Onset ◽  
Inhibitory Effect ◽  
Muscle Performance ◽  
Continuous Treatment ◽  
Wild Type ◽  
Muscle Fibrosis ◽  
Early Stages ◽  
Primary Myoblasts ◽  
Collagen Level

AbstractDysferlinopathies are a non-lethal group of late-onset muscular dystrophies. Here, we evaluated the fusion ability of primary myoblasts from young dysf−/− mice and the muscle histopathology prior to, and during early stages of disease onset. The ability of primary myoblasts of 5-weekold dysf−/− mice to form large myotubes was delayed compared to their wild-type counterparts, as evaluated by scanning electron microscopy. However, their fusion activity, as reflected by the presence of actin filaments connecting several cells, was enhanced by the antifibrotic drug halofuginone. Early dystrophic signs were already apparent in 4-week-old dysf−/− mice; their collagen level was double that in wild-type mice and continued to rise until 5 months of age. Continuous treatment with halofuginone from 4 weeks to 5 months of age reduced muscle fibrosis in a phosphorylated-Smad3 inhibition-related manner. Halofuginone also enhanced myofiber hypertrophy, reduced the percentage of centrally nucleated myofibers, and increased muscle performance. Together, the data suggest an inhibitory effect of halofuginone on the muscle histopathology at very early stages of dysferlinopathy, and better generation of force and muscle performance. These results offer new opportunities for early pharmaceutical treatment in dysferlinopathies with favorable outcomes at later stages of life.

scholarly journals The GNAQ T96S Mutation Affects Cell Signaling and Enhances the Oncogenic Properties of Hepatocellular Carcinoma

2021 ◽  
Vol 22 (6) ◽  
pp. 3284
Author(s):  
Eugene Choi ◽  
Sung Jean Park ◽  
Gunhee Lee ◽  
Seung Kew Yoon ◽  
Minho Lee ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Expression Vector ◽  
Signal Transduction Pathway ◽  
Inhibitory Effect ◽  
The Cancer Genome Atlas ◽  
Treatment Modalities ◽  
Protein Signaling ◽  
Wild Type ◽  
Anchorage Independent Growth ◽  
Mapk Pathways

Hepatocellular carcinoma (HCC), the most common malignant tumor in the liver, grows and metastasizes rapidly. Despite advances in treatment modalities, the five-year survival rate of HCC remains less than 30%. We sought genetic mutations that may affect the oncogenic properties of HCC, using The Cancer Genome Atlas (TCGA) data analysis. We found that the GNAQ T96S mutation (threonine 96 to serine alteration of the Gαq protein) was present in 12 out of 373 HCC patients (3.2%). To examine the effect of the GNAQ T96S mutation on HCC, we transfected the SK-Hep-1 cell line with the wild-type or the mutant GNAQ T96S expression vector. Transfection with the wild-type GNAQ expression vector enhanced anchorage-independent growth, migration, and the MAPK pathways in the SK-Hep-1 cells compared to control vector transfection. Moreover, cell proliferation, anchorage-independent growth, migration, and the MAPK pathways were further enhanced in the SK-Hep-1 cells transfected with the GNAQ T96S expression vector compared to the wild-type GNAQ-transfected cells. In silico structural analysis shows that the substitution of the GNAQ amino acid threonine 96 with a serine may destabilize the interaction between the regulator of G protein signaling (RGS) protein and GNAQ. This may reduce the inhibitory effect of RGS on GNAQ signaling, enhancing the GNAQ signaling pathway. Single nucleotide polymorphism (SNP) genotyping analysis for Korean HCC patients shows that the GNAQ T96S mutation was found in only one of the 456 patients (0.22%). Our data suggest that the GNAQ T96S hotspot mutation may play an oncogenic role in HCC by potentiating the GNAQ signal transduction pathway.

scholarly journals Transcriptome analyses of 7-day-old zebrafish larvae possessing a familial Alzheimer’s disease-like mutation in psen1 indicate effects on oxidative phosphorylation, ECM and MCM functions, and iron homeostasis

BMC Genomics ◽  
2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Yang Dong ◽  
Morgan Newman ◽  
Stephen M. Pederson ◽  
Karissa Barthelson ◽  
Nhi Hin ◽  
...  
Keyword(s):  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
Oxidative Phosphorylation ◽  
Transcriptome Analysis ◽  
Iron Homeostasis ◽  
Late Onset ◽  
Wild Type ◽  
Familial Alzheimer’S Disease ◽  
Zebrafish Larvae ◽  
Familial Alzheimer's Disease

Abstract Background Early-onset familial Alzheimer’s disease (EOfAD) is promoted by dominant mutations, enabling the study of Alzheimer’s disease (AD) pathogenic mechanisms through generation of EOfAD-like mutations in animal models. In a previous study, we generated an EOfAD-like mutation, psen1Q96_K97del, in zebrafish and performed transcriptome analysis comparing entire brains from 6-month-old wild type and heterozygous mutant fish. We identified predicted effects on mitochondrial function and endolysosomal acidification. Here we aimed to determine whether similar effects occur in 7 day post fertilization (dpf) zebrafish larvae that might be exploited in screening of chemical libraries to find ameliorative drugs. Results We generated clutches of wild type and heterozygous psen1Q96_K97del 7 dpf larvae using a paired-mating strategy to reduce extraneous genetic variation before performing a comparative transcriptome analysis. We identified 228 differentially expressed genes and performed various bioinformatics analyses to predict cellular functions. Conclusions Our analyses predicted a significant effect on oxidative phosphorylation, consistent with our earlier observations of predicted effects on ATP synthesis in adult heterozygous psen1Q96_K97del brains. The dysregulation of minichromosome maintenance protein complex (MCM) genes strongly contributed to predicted effects on DNA replication and the cell cycle and may explain earlier observations of genome instability due to PSEN1 mutation. The upregulation of crystallin gene expression may be a response to defective activity of mutant Psen1 protein in endolysosomal acidification. Genes related to extracellular matrix (ECM) were downregulated, consistent with previous studies of EOfAD mutant iPSC neurons and postmortem late onset AD brains. Also, changes in expression of genes controlling iron ion transport were observed without identifiable changes in the prevalence of transcripts containing iron responsive elements (IREs) in their 3′ untranslated regions (UTRs). These changes may, therefore, predispose to the apparent iron dyshomeostasis previously observed in 6-month-old heterozygous psen1Q96_K97del EOfAD-like mutant brains.

scholarly journals PCR-based genotyping assays to detect germline APC variant associated with hereditary gastrointestinal polyposis in Jack Russell terriers

2021 ◽  
Vol 17 (1) ◽  
Author(s):  
Kyoko Yoshizaki ◽  
Akihiro Hirata ◽  
Hiroyuki Matsushita ◽  
Naohito Nishii ◽  
Mifumi Kawabe ◽  
...  
Keyword(s):  
Mutant Allele ◽  
False Negative ◽  
Disease Onset ◽  
Pcr Amplification ◽  
Buccal Swab ◽  
Wild Type ◽  
Gastrointestinal Polyposis ◽  
Pcr Rflp ◽  
Formalin Fixed Paraffin Embedded ◽  
Rflp Assay

Abstract Background The prevalence of gastrointestinal (GI) neoplastic polyps in Jack Russell terriers (JRTs) has increased in Japan since the late 2000s. Recently, we demonstrated that JRTs with GI polyps harbor identical germline variant in the APC gene (c.[462_463delinsTT]) in the heterozygous state. Thus, this disease is an autosomal dominant hereditary disorder. Although the affected JRTs have distinct features, such as the development of multiple GI polyps and an early age of disease onset, genetic testing is indispensable for a definitive diagnosis. Here, polymerase chain reaction (PCR)-based assays capable of detecting germline APC variant were designed and validated using synthetic wild-type and mutant DNAs and genomic DNAs from carrier and non-carrier dogs. Result First, the PCR-restriction fragment length polymorphism (PCR-RFLP) assay was developed by taking advantage of the germline APC variant creating a new restriction site for MseI. In the PCR-RFLP assay, the 156-bp region containing the variant site was amplified by PCR and subsequently digested with MseI, yielding diagnostic 51 and 58 bp fragments from the mutant allele and allowing determination of the APC genotypes. It was possible to determine the genotypes using genomic DNA extracted from the peripheral blood, buccal swab, or formalin-fixed paraffin-embedded tissue. Next, a TaqMan duplex real-time PCR assay was developed, where a 78-bp region flanking the variant was amplified in the presence of wild-type allele- and mutant allele-specific fluorescent probes. Using blood-derived DNA, altogether 40 cycles of PCR amplification determined the APC genotypes of all examined samples by measuring the fluorescence intensities. Importantly, false-positive and false-negative errors were never detected in both assays. Conclusion In this study, we developed highly reliable genetic tests for hereditary GI polyposis in JRTs, providing accurate assessment of the presence of the causative germline APC variant. The genotyping assays could contribute to the diagnosis and prevention of hereditary GI polyposis in dogs.

ELFIN, the United Kingdom preterm lactoferrin trial: interpretation and future questions

2020 ◽  
Author(s):  
Janet Elizabeth Berrington ◽  
William McGuire ◽  
NIcholas David Embleton
Keyword(s):  
United Kingdom ◽  
Preterm Infants ◽  
Late Onset ◽  
Disease Onset ◽  
Intervention Group ◽  
Control Group ◽  
Bovine Lactoferrin ◽  
The United Kingdom ◽  
Late Onset Sepsis ◽  
The Uk

Previous studies suggested that supplemental bovine lactoferrin (BLF) given to preterm infants (<32 weeks gestation) may reduce late onset sepsis (LOS) and necrotising enterocolitis (NEC), but have been underpowered. The Enteral Lactoferrin in Neonates (ELFIN) study, performed in the United Kingdom (UK), aimed to further address this issue with a well powered double blinded placebo controlled trial of >2200 preterm infants. ELFIN did not demonstrate a reduction in LOS or NEC, or several other clinically important measures. 316 (29%) of 1093 infants in the intervention group developed late-onset sepsis versus 334 (31%) of 1089 in the control group with an adjusted risk ratio of 0·95 (95% CI 0·86–1·04; p=0· 233). Reasons for the differences in ELFIN trial results and other studies may include population differences, the routine use of antifungals in the UK, timing of administration of the lactoferrin in relation to disease onset, or specific properties of the lactoferrin used in different trials. Further exploration is being undertaken in the UK NIHR funded Mechanisms Affecting the Guts of Preterm Infants in Enteral feeding trials (MAGPIE) study, for which results should be available soon.

The phosphoenolpyruvate: sugar phosphotransferase system of Streptococcus salivarius. Identification of a IIIman protein

1987 ◽  
Vol 33 (2) ◽  
pp. 118-122 ◽  
Author(s):  
Christian Vadeboncoeur ◽  
Lucie Gauthier
Keyword(s):  
Type Strain ◽  
Wild Type Strain ◽  
Deae Cellulose ◽  
Reaction Medium ◽  
Inhibitory Effect ◽  
Spontaneous Mutant ◽  
Streptococcus Salivarius ◽  
Wild Type ◽  
Phosphotransferase System ◽  
Growth Inhibitory

A double-spontaneous mutant resistant to the growth inhibitory effect of α-methylglucoside and 2-deoxyglucose was isolated from Streptococcus salivarius. This mutant strain, called αS3L11, did not grow on mannose and grew poorly on 5 mM fructose and 5 mM glucose. Isolated membranes of strain αS3L11 were unable to catalyse the phosphoenolpyruvate-dependent phosphorylation of mannose in the presence of purified enzyme I and HPr. Addition of dialysed membrane-free cellular extract of the wild-type strain to the reaction medium restored the activity. The factor that restored the phosphoenolpyruvate–mannose phosphotransferase activity to membranes of strain αS3L11 was called IIIman. This factor was partially purified from the wild-type strain by DEAE-cellulose chromatography, DEAE-TSK chromatography, and molecular seiving on a column of Ultrogel AcA 34. This partially purified preparation also enhanced the phosphoenolpyruvate-dependent phosphorylation of glucose, fructose, and 2-deoxyglucose in strain αS3L11.

scholarly journals Yeasts as Complementary Model Systems for the Study of the Pathological Repercussions of Enhanced Synphilin-1 Glycation and Oxidation

2021 ◽  
Vol 22 (4) ◽  
pp. 1677
Author(s):  
David Seynnaeve ◽  
Daniel P. Mulvihill ◽  
Joris Winderickx ◽  
Vanessa Franssens
Keyword(s):  
Lewy Bodies ◽  
Inhibitory Effect ◽  
Model Systems ◽  
Wild Type ◽  
Glyoxalase System ◽  
Inclusion Formation ◽  
Growth Defects ◽  
Yeast Strains ◽  
Autophagic Degradation ◽  
Detrimental Impact

Synphilin-1 has previously been identified as an interaction partner of α-Synuclein (αSyn), a primary constituent of neurodegenerative disease-linked Lewy bodies. In this study, the repercussions of a disrupted glyoxalase system and aldose reductase function on Synphilin-1 inclusion formation characteristics and cell growth were investigated. To this end, either fluorescent dsRed-tagged or non-tagged human SNCAIP, which encodes the Synphilin-1 protein, was expressed in Saccharomyces cerevisiae and Schizosaccharomyces pombe yeast strains devoid of enzymes Glo1, Glo2, and Gre3. Presented data shows that lack of Glo2 and Gre3 activity in S. cerevisiae increases the formation of large Synphilin-1 inclusions. This correlates with enhanced oxidative stress levels and an inhibitory effect on exponential growth, which is most likely caused by deregulation of autophagic degradation capacity, due to excessive Synphilin-1 aggresome build-up. These findings illustrate the detrimental impact of increased oxidation and glycation on Synphilin-1 inclusion formation. Similarly, polar-localised inclusions were observed in wild-type S. pombe cells and strains deleted for either glo1+ or glo2+. Contrary to S. cerevisiae, however, no growth defects were observed upon expression of SNCAIP. Altogether, our findings show the relevance of yeasts, especially S. cerevisiae, as complementary models to unravel mechanisms contributing to Synphilin-1 pathology in the context of neurodegenerative diseases.

Abstract 17081: End Stage Mitochondrial Cardiomyopathy And Heart Transplantation Due To Pathogenic Biallelic C1QBP Variants

Circulation ◽  
2021 ◽  
Vol 144 (Suppl_2) ◽  
Author(s):  
Nicholas S Wilcox ◽  
Stuart Prenner ◽  
Marisa Cevasco ◽  
Courtney Condit ◽  
Amy Goldstein ◽  
...  
Keyword(s):  
Mitochondrial Genome ◽  
Heart Transplantation ◽  
Genome Sequencing ◽  
Large Scale ◽  
De Novo ◽  
Late Onset ◽  
Genetic Disorder ◽  
Disease Onset ◽  
Serum Lactate ◽  
Metabolic Decompensation

Case Presentation: A 29-year-old male with LVH diagnosed in childhood was admitted with acute HF. TTE showed LVEF 5-10% and LV thrombi for which he was anticoagulated. He received inappropriate ICD shocks due to T wave oversensing, leading to cardiogenic shock requiring VA-ECMO support. Serum lactate peaked at 17 mmol/L due to cardiac and metabolic decompensation. He underwent heart transplantation (HT) on hospital day (HD) 8 and tolerated standard immunosuppression. First endomyocardial biopsy showed acute cellular rejection requiring pulse steroids. He was discharged on HD 33. Trio whole exome and mitochondrial genome sequencing revealed biallelic variants in complement component 1Q subcomponent-binding protein ( C1QBP ), due to a maternally inherited likely pathogenic variant c.612C>G (p.F204L in exon 5) and an apparently de novo deletion of 17p13.2, spanning exons 4-6 of C1QBP and exon 6 of the RPAIN gene. Mitochondrial genome sequencing of the explanted heart revealed multiple large-scale mitochondrial DNA deletions at 33% heteroplasmy. Discussion: C1QBP variants are associated with mitochondrial and multi-organ dysfunction. Only 12 patients exhibiting biallelic C1QBP variants are reported. Four died in the peripartum period due to fetal hydrops or HF; 5 exhibited early-onset cardiomyopathy (CM); 3 others had late-onset ophthalmoplegia without CM. The p.F204L variant has been reported in 1 patient with compound C1QBP p.F204L/p.C186S heterozygosity who died from hydrops fetalis and a second with p.F204L homozygosity with late-onset ophthalmoplegia and skeletal myopathy without CM. Differences in the size, heteroplasmy, and tissue distribution of mitochondrial genome secondary deletions may explain variability in disease onset and progression. We present the first patient with biallelic pathogenic C1QBP gene variants with mitochondrial CM to undergo HT and highlight the diagnosis and management of an exceptionally uncommon genetic disorder.

Activation and inhibition of erythropoietin receptor function: role of receptor dimerization

1994 ◽  
Vol 14 (6) ◽  
pp. 3535-3549
Author(s):  
S S Watowich ◽  
D J Hilton ◽  
H F Lodish
Keyword(s):  
Signal Transduction ◽  
Growth Hormone Receptor ◽  
Initial Step ◽  
Inhibitory Effect ◽  
Erythropoietin Receptor ◽  
Interface Region ◽  
Wild Type ◽  
Receptor Dimerization ◽  
Dimer Interface ◽  
Constitutively Active

Members of the cytokine receptor superfamily have structurally similar extracellular ligand-binding domains yet diverse cytoplasmic regions lacking any obvious catalytic domains. Many of these receptors form ligand-induced oligomers which are likely to participate in transmembrane signaling. A constitutively active (factor-independent) mutant of the erythropoietin receptor (EPO-R), R129C in the exoplasmic domain, forms disulfide-linked homodimers, suggesting that the wild-type EPO-R is activated by ligand-induced homodimerization. Here, we have taken two approaches to probe the role EPO-R dimerization plays in signal transduction. First, on the basis of the crystal structure of the ligand-bound, homodimeric growth hormone receptor (GH-R) and sequence alignment between the GH-R and EPO-R, we identified residues of the EPO-R which may be involved in intersubunit contacts in an EPO-R homodimer. Residue 129 of the EPO-R corresponds to a residue localized to the GH-R dimer interface region. Alanine or cysteine substitutions were introduced at four other residues of the EPO-R predicted to be in the dimer interface region. Substitution of residue E-132 or E-133 with cysteine renders the EPO-R constitutively active. Like the arginine-to-cysteine mutation at position 129 in the exoplasmic domain (R129C), E132C and E133C form disulfide-linked homodimers, suggesting that constitutive activity is due to covalent dimerization. In the second approach, we have coexpressed the wild-type EPO-R with inactive mutants of the receptor missing all or part of the cytosolic domain. These truncated receptors have a dominant inhibitory effect on the proliferative action of the wild-type receptor. Taken together, these results strengthen the hypothesis that an initial step in EPO- and EPO-R-mediated signal transduction is ligand-induced receptor dimerization.

Abstract 1805: Chromosome 9p21.3 Genetic Variation is Associated with Angiographic CAD but not with CAD Disease Burden

Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
Chrissa P Mower ◽  
Jeffrey L Anderson ◽  
Benjamin D Horne ◽  
James J Park ◽  
Jesse L Coleman ◽  
...  
Keyword(s):  
Genetic Variation ◽  
Disease Burden ◽  
Disease Onset ◽  
Cardiac Risk ◽  
Wild Type ◽  
Chi Square ◽  
High Risk Disease ◽  
Chi Square Test ◽  
Male Frequency ◽  
Cad Risk

Genetic variation at the 9p21.3 locus rs2383206 is associated with coronary heart disease (CHD) phenotypes. In a comparison of patients with and without angiographically confirmed CHD, the G allele of rs2383206 was present more frequently in diseased vs controls (normal angiograms). However, the pathophysiologic impact, whether it affects initiation, severity, or triggers an event, of the 9p21.3 locus remains unknown. We sought to determine whether 9p21.3 variation affects disease severity (promotion) by assessing its association with CAD burden. Methods: Genotyping for rs2383206 using 5′exonuclease chemistry (Taqman) was performed on 1759 subjects. Subjects were grouped as homozygous wild-type (low risk), heterozygous (intermediate risk) or homozygous risk-associated genotype (high risk). Disease burden was assessed by 1, 2, or 3 vessels; ≥70% stenosis and the validated Duke CAD Index (DCI). Comparison used a chi-square test (single vs multivessel disease) and analysis of variance (ANOVA) for the DCI comparison. Results: Average age 51.1± 7.4 years, 64.0% male. Frequency of the CAD risk allele did not differ among groups with 1, 2, or 3 vessel disease. There was no difference among groups with respect to the DCI. After adjustment for standard cardiac risk factors, the rs2383206 genotype was associated with CAD compared to controls (OR (CI)=1.73(1.26–1.85), p=0.001). Conclusion: The rs2383206 polymorphism was not associated with CAD disease burden. Findings suggest the rs2383206 polymorphism, although associated with disease onset is not likely involved in its progression. These findings will aid in refining the application of 9p21.3 for risk assessment and developing novel preventive and therapeutic strategies.

Extracellular pH alkalinization by Cl−/HCO3− exchanger is crucial for TASK2 activation by hypotonic shock in proximal cell lines from mouse kidney

2007 ◽  
Vol 292 (2) ◽  
pp. F628-F638 ◽  
Author(s):  
S. L'Hoste ◽  
H. Barriere ◽  
R. Belfodil ◽  
I. Rubera ◽  
C. Duranton ◽  
...  
Keyword(s):  
Cell Lines ◽  
Regulatory Volume Decrease ◽  
Inhibitory Effect ◽  
Buffering Capacity ◽  
Mouse Kidney ◽  
Wild Type ◽  
Cytosolic Ph ◽  
Hypotonic Shock ◽  
External Ph ◽  
Volume Decrease

We have previously shown that K+-selective TASK2 channels and swelling-activated Cl− currents are involved in a regulatory volume decrease (RVD; Barriere H, Belfodil R, Rubera I, Tauc M, Lesage F, Poujeol C, Guy N, Barhanin J, Poujeol P. J Gen Physiol 122: 177–190, 2003; Belfodil R, Barriere H, Rubera I, Tauc M, Poujeol C, Bidet M, Poujeol P. Am J Physiol Renal Physiol 284: F812–F828, 2003). The aim of this study was to determine the mechanism responsible for the activation of TASK2 channels during RVD in proximal cell lines from mouse kidney. For this purpose, the patch-clamp whole-cell technique was used to test the effect of pH and the buffering capacity of external bath on Cl− and K+ currents during hypotonic shock. In the presence of a high buffer concentration (30 mM HEPES), the cells did not undergo RVD and did not develop outward K+ currents (TASK2). Interestingly, the hypotonic shock reduced the cytosolic pH (pHi) and increased the external pH (pHe) in wild-type but not in cftr −/− cells. The inhibitory effect of DIDS suggests that the acidification of pHi and the alkalinization of pHe induced by hypotonicity in wild-type cells could be due to an exit of HCO3−. In conclusion, these results indicate that Cl− influx will be the driving force for HCO3− exit through the activation of the Cl−/HCO3− exchanger. This efflux of HCO3− then alkalinizes pHe, which in turn activates TASK2 channels.

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