Cell-type specific effects of mineralocorticoid receptor gene expression suggest intercellular communication regulating fibrosis in skeletal muscle disease.

Introduction: Duchenne muscular dystrophy (DMD) is a fatal striated muscle degenerative disease. DMD is caused by loss of dystrophin protein, which results in sarcolemmal instability and cycles of myofiber degeneration and regeneration. Pathology is exacerbated by overactivation of infiltrating immune cells and fibroblasts, which leads to chronic inflammation and fibrosis. Mineralocorticoid receptors (MR), a type of nuclear steroid hormone receptors, are potential therapeutic targets for DMD. MR antagonists show clinical efficacy on DMD cardiomyopathy and preclinical efficacy on skeletal muscle in DMD models. Methods: We have previously generated myofiber and myeloid MR knockout mouse models to dissect cell-specific functions of MR within dystrophic muscles. Here, we compared skeletal muscle gene expression from both knockouts to further define cell-type specific signaling downstream from MR. Results: Myeloid MR knockout increased proinflammatory and profibrotic signaling, including numerous myofibroblast signature genes. Tenascin C was the most highly upregulated fibrotic gene in myeloid MR-knockout skeletal muscle and is a component of fibrosis in dystrophic skeletal muscle. Surprisingly, lysyl oxidase (Lox), canonically a collagen crosslinker, was increased in both MR knockouts, but did not localize to fibrotic regions of skeletal muscle. Lox localized within myofibers, including only a region of quadriceps muscles. Lysyl oxidase like 1 (Loxl1), another Lox family member, was increased only in myeloid MR knockout muscle and localized specifically to fibrotic regions. Discussion: This study suggests that MR signaling in the dystrophic muscle microenvironment involves communication between contributing cell types and modulates inflammatory and fibrotic pathways in muscle disease.

Gene therapy delivered micro-dystrophins co-localize with transgenic utrophin in dystrophic skeletal muscle fibers.

Duchenne muscular dystrophy (DMD) is a devastating muscle disease caused by the absence of functional dystrophin. There are multiple ongoing clinical trials for DMD that are testing gene therapy treatments consisting of adeno-associated viral (AAV) vectors carrying miniaturized versions of dystrophin optimized for function, termed micro-dystrophins (µDys). Utrophin, the fetal homolog of dystrophin, has repeatedly been reported to be upregulated in human DMD muscle as a compensatory mechanism, but whether µDys displaces full-length utrophin is unknown. In this study, dystrophin/utrophin-deficient mice with transgenic overexpression of full-length utrophin in skeletal muscles were systemically administered low doses of either AAV6-CK8e-Hinge3-µDys (µDysH3) or AAV6-CK8e-µDys5 (µDys5). We used immunofluorescence to qualitatively assess the localization of µDys with transgenic utrophin and neuronal nitric oxide synthase (nNOS) in quadriceps muscles. µDys protein resulting from both gene therapies co-localized at myofiber membranes with transgenic utrophin. We also confirmed the sarcolemmal co-localization of nNOS with µDys5, but not with transgenic utrophin expression or µDysH3. Transgenic utrophin expression and µDys proteins produced from both therapies stabilize the dystrophin-glycoprotein complex as observed by sarcolemmal localization of ß-dystroglycan. This study suggests that µDys gene therapy will likely not inhibit any endogenous compensation by utrophin in DMD muscle.

The Interaction of Duchenne Muscular Dystrophy and Insulin Resistance.

Duchenne muscular dystrophy (DMD), caused by deficiency of functional dystrophin protein, is a fatal, progressive muscle disease that frequently includes metabolic dysregulation. Herein, we explore the physiologic consequences of dystrophin deficiency within the context of obesity and insulin resistance. We hypothesized that dystrophin deficiency increases the frequency of insulin resistance, and insulin resistance potentiates muscle pathology caused by dystrophin deficiency.

A fat- and sucrose-enriched diet causes metabolic alterations in mdx mice.

Duchenne muscular dystrophy (DMD), a progressive muscle disease caused by the absence of functional dystrophin protein, is associated with multiple cellular, physiological, and metabolic dysfunctions. As an added complication to the primary insult, obesity/insulin resistance (O/IR) is frequently reported in patients with DMD; however, how IR impacts disease severity is unknown. We hypothesized a high-fat, high-sucrose diet (HFHSD) would induce O/IR, exacerbate disease severity, and cause metabolic alterations in dystrophic mice. To test this hypothesis, we treated 7-wk-old mdx (disease model) and C57 mice with a control diet (CD) or an HFHSD for 15 wk. The HFHSD induced insulin resistance, glucose intolerance, and hyperglycemia in C57 and mdx mice. Of note, mdx mice on CD were also insulin resistant. In addition, visceral adipose tissue weights were increased with HFHSD in C57 and mdx mice though differed by genotype. Serum creatine kinase activity and histopathological analyses using Masson's trichrome staining in the diaphragm indicated muscle damage was driven by dystrophin deficiency but was not augmented by diet. In addition, markers of inflammatory signaling, mitochondrial abundance, and autophagy were impacted by disease but not diet. Despite this, in addition to disease signatures in CD-fed mice, metabolomic and lipidomic analyses demonstrated a HFHSD caused some common changes in C57 and mdx mice and some unique signatures of O/IR within the context of dystrophin deficiency. In total, these data revealed that in mdx mice, 15 wk of HFHSD did not overtly exacerbate muscle injury but further impaired the metabolic status of dystrophic muscle.

Indicators of increased ER stress and UPR in aged D2-mdx and human dystrophic skeletal muscles.

Duchenne muscular dystrophy (DMD) is a progressive muscle disease that results in muscle wasting, wheelchair dependence, and eventual death due to cardiac and respiratory complications. In addition to muscle fragility, dystrophin deficiency also results in multiple secondary dysfunctions, which may lead to the accumulation of unfolded proteins causing endoplasmic reticulum (ER) stress and the unfolded protein response (UPR). The purpose of this investigation was to understand how ER stress and the UPR are modified in muscle from D2-mdx mice, an emerging DMD model, and from humans with DMD. We hypothesized that markers of ER stress and the UPR are upregulated in D2-mdx and human dystrophic muscles compared to their healthy counterparts. Immunoblotting in diaphragms from 11-month-old D2-mdx and DBA mice indicated increased ER stress and UPR in dystrophic diaphragms compared to healthy, including increased relative abundance of ER stress chaperone CHOP, canonical ER stress transducers ATF6 and pIRE1α S724, and transcription factors that regulate the UPR such as ATF4, XBP1s, and peIF2α S51. The publicly available Affymetrix dataset (GSE38417) was used to analyze the expression of ER stress and UPR-related transcripts and processes. Fifty-eight upregulated genes related to ER stress and the UPR in human dystrophic muscles suggest pathway activation. Further, based on analyses using iRegulon, putative transcription factors that regulate this upregulation profile were identified, including ATF6, XBP1, ATF4, CREB3L2, and EIF2AK3. This study adds to and extends the emerging knowledge of ER stress and the UPR in dystrophin deficiency and identifies transcriptional regulators that may be responsible for these changes and be of therapeutic interest.

Recapitulating whole genome based population genetic structure for Indian wild tigers through an ancestry informative marker panel.

Identification of genetic structure within wildlife populations have implications in their conservation and management. Accurately inferring population genetic structure requires whole-genome data across the geographical range of the species, which can be resource-intensive. A cheaper strategy is to employ a subset of markers that can efficiently recapitulate the population genetic structure inferred by the whole genome data. Such ancestry informative markers (AIMs), have rarely been developed for endangered species such as tigers utilizing single nucleotide polymorphisms (SNPs). Here, we first identify the population structure of the Indian tiger using whole-genome sequences and then develop an AIMs panel with a minimum number of SNPs that can recapitulate this structure. We identified four population clusters of Indian tigers with North-East, North-West, and South Indian tigers forming three separate groups, and Terai and Central Indian tigers forming a single cluster. To evaluate the robustness of our AIMs, we applied it to a separate dataset of tigers from across India. Out of 92 SNPs present in our AIMs panel, 49 were present in the new dataset. These 49 SNPs were sufficient to recapitulate the population genetic structure obtained from the whole genome data. To the best of our knowledge, this is the first-ever SNP-based AIMs panel for big cats, which can be used as a cost-effective alternative to whole-genome sequencing for detecting the biogeographical origin of Indian tigers. Our study can be used as a guideline for developing an AIMs panel for the management of other endangered species where obtaining whole genome sequences are difficult.

Indices of Defective Autophagy in Whole Muscle and Lysosome Enriched Fractions From Aged D2-mdx Mice.

Duchenne muscular dystrophy (DMD) is a fatal, progressive muscle disease caused by the absence of functional dystrophin protein. Previous studies in mdx mice, a common DMD model, identified impaired autophagy with lysosomal insufficiency and impaired autophagosomal degradation as consequences of dystrophin deficiency. Thus, we hypothesized that lysosomal abundance would be decreased and degradation of autophagosomes would be impaired in muscles of D2-mdx mice. To test this hypothesis, diaphragm and gastrocnemius muscles from 11 month-old D2-mdx and DBA/2J (healthy) mice were collected. Whole muscle protein from diaphragm and gastrocnemius muscles, and protein from a cytosolic fraction (CF) and a lysosome-enriched fraction (LEF) from gastrocnemius muscles, were isolated and used for western blotting. Initiation of autophagy was not robustly activated in whole muscle protein from diaphragm and gastrocnemius, however, autophagosome formation markers were elevated in dystrophic muscles. Autophagosome degradation was impaired in D2-mdx diaphragms but appeared to be maintained in gastrocnemius muscles. To better understand this muscle-specific distinction, we investigated autophagic signaling in CFs and LEFs from gastrocnemius muscles. Within the LEF we discovered that the degradation of autophagosomes was similar between groups. Further, our data suggest an expanded, though impaired, lysosomal pool in dystrophic muscle. Notably, these data indicate a degree of muscle specificity as well as model specificity with regard to autophagic dysfunction in dystrophic muscles. Stimulation of autophagy in dystrophic muscles may hold promise for DMD patients as a potential therapeutic, however, it will be critical to choose the appropriate model and muscles that most closely recapitulate findings from human patients to further develop these therapeutics.

Population genetic variation of SLC6A4 gene, associated with neurophysiological development.

The serotonin transporter 5-HTT is encoded by a single gene SLC6A4. Polymorphisms in SLC6A4 has been associated with a wide variety of neurological and psychiatric disorders including increased risk of posttraumatic stress disorder, higher likelihood for depression, obsessive-compulsive disorder (OCD), increased hostility and criminal behaviour. Genes associated with complex diseases often exhibit strong signatures of purifying selection compared to others. Further, discernible population specific variation in the signature of natural selection have been observed for several complex disease-related genes. In this project we aimed to investigate the population genetic variation of the serotonin transporter gene (SLC6A4), focussing on the single nucleotide polymorphisms (SNPs). To this end, we employed 2504 individuals around the globe available in 1000 Genome project Phase III data and classified them into five ethnic groups: Americans (AMR), Europeans (EUR), Africans (AFR), East Asians (EAS) and South Asians (SAS). Principal component analysis (PCA) performed on all annotated SNPs of SLC6A4 depicted clear clustering between Africans and the rest of the world along PC1, and East Asians and other non-African populations along PC2. Further, these SNPs were found to be under strong selection pressure especially among East Asian populations with significantly high positive cross-population extended haplotype homozygosity scores compared to Africans, indicating that SLC6A4 has likely undergone a strong selective sweep among the East Asians in the recent past. Our study can potentially explain the association between polymorphisms in SLC6A4, and major depression and suicidal tendencies among people of East Asian ancestry and the absence of such associations among people of European ancestry.

Antimicrobial Effect of Leaves of Phyllanthus niruri and Solanum nigrum on Caries Causing Bacteria: An In vitro Study.

INTRODUCTION: Solanum nigrum and Phyllanthus niruri are common herbs which are indigeneous to India. Solanum nigrum commonly called 'manathakkali Keerai' in Tamil, forms an indispensable part of South Indian diet. Phyllanthus niruri (keezhanelli in Tamil) is a widely used medicinal plant, the leaves of which have been used extensively in Ayurveda and native medicine to cure various liver ailments. The herbs Solanumnigrum and Phyllanthus niruri have been found to be effective against numerous enteropathogens in various in vitro studies. AIM: To assess and compare the antibacterial efficacy of the crude alcoholic extract of the leaves of Solanum nigrum and Phyllanthus niruri against five cariogenic organisms. MATERIALS AND METHODS: Standard strains of the micro-organisms were obtained from ATCC (American Type Culture Collection) and MTCC (Microbial Type Culture Collection) which comprised of Streptococcus mutans MTCC no. 890, Streptococcus oralis MTCC no 2696, Lactobacillus acidophillus MTCC no. 10307, Streptococcus sanguis ATCC no. 10556 and Streptococcus salivarius ATCC no. 13419. The organisms obtained were revived and lawn cultured on Trypticase Soy Agar-Blood Agar (TSA-BA) and de Man, Rogosa and Sharpe (MRS) agar media. The antibacterial effect of the dried and powdered leaves of Solanum nigrum and Phyllanthus niruri was tested using agar well diffusion method. The zones of inhibition obtained after incubation were measured and tabulated. The antibacterial activity for the two herbs was compared using the Mann-Whitney test. RESULTS: The antibacterial zones of inhibition obtained for the herb Solanum nigrum was in the range of 12.3-14.6 mm and ranged from 9.7-11.6 mm for the herb Phyllanthus niruri. When the zones of inhibition were compared for the herbs, Solanum nigrum showed significantly greater zones of inhibition compared to Phyllanthus niruri for the organisms Streptococcussanguis, Streptococcus salivarius, Streptococcus oralis and Streptococcus mutans (p-value<0.05). CONCLUSION: The alcoholic extract of leaves of Solanum nigrum and Phyllanthus niruri showed significant antibacterial activity against cariogenic organisms, with Solanum nigrum being more anti-cariogenic than Phyllanthus niruri.