Eight common genetic variants associated with serum dheas levels suggest a key role in ageing mechanisms

Dehydroepiandrosterone sulphate (DHEAS) is the most abundant circulating steroid secreted by adrenal glands-yet its function is unknown. Its serum concentration declines significantly with increasing age, which has led to speculation that a relative DHEAS deficiency may contribute to the development of common age-related diseases or diminished longevity. We conducted a meta-analysis of genome-wide association data with 14,846 individuals and identified eight independent common SNPs associated with serum DHEAS concentrations. Genes at or near the identified loci include ZKSCAN5 (rs11761528; p = 3.15×10-36), SULT2A1 (rs2637125; p = 2.61×10-19), ARPC1A (rs740160; p = 1.56×10-16), TRIM4 (rs17277546; p = 4.50×10-11), BMF (rs7181230; p = 5.44×10-11), HHEX (rs2497306; p = 4.64×10-9), BCL2L11 (rs6738028; p = 1.72×10-8), and CYP2C9 (rs2185570; p = 2.29×10-8). These genes are associated with type 2 diabetes, lymphoma, actin filament assembly, drug and xenobiotic metabolism, and zinc finger proteins. Several SNPs were associated with changes in gene expression levels, and the related genes are connected to biological pathways linking DHEAS with ageing. This study provides much needed insight into the function of DHEAS

Somatic mutations of CADM1 in aldosterone-producing adenomas and gap junction-dependent regulation of aldosterone production

Aldosterone-producing adenomas (APAs) are the commonest curable cause of hypertension. Most have gain-of-function somatic mutations of ion channels or transporters. Herein we report the discovery, replication and phenotype of mutations in the neuronal cell adhesion gene CADM1. Independent whole exome sequencing of 40 and 81 APAs found intramembranous p.Val380Asp or p.Gly379Asp variants in two patients whose hypertension and periodic primary aldosteronism were cured by adrenalectomy. Replication identified two more APAs with each variant (total, n = 6). The most upregulated gene (10- to 25-fold) in human adrenocortical H295R cells transduced with the mutations (compared to wildtype) was CYP11B2 (aldosterone synthase), and biological rhythms were the most differentially expressed process. CADM1 knockdown or mutation inhibited gap junction (GJ)-permeable dye transfer. GJ blockade by Gap27 increased CYP11B2 similarly to CADM1 mutation. Human adrenal zona glomerulosa (ZG) expression of GJA1 (the main GJ protein) was patchy, and annular GJs (sequelae of GJ communication) were less prominent in CYP11B2-positive micronodules than adjacent ZG. Somatic mutations of CADM1 cause reversible hypertension and reveal a role for GJ communication in suppressing physiological aldosterone production

Dominant Negative Mutants of Bacillus thuringiensis Cry1Ab Toxin Function as Anti-Toxins: Demonstration of the Role of Oligomerization in Toxicity

BACKGROUND:Bacillus thuringiensis Cry toxins, that are used worldwide in insect control, kill insects by a mechanism that depends on their ability to form oligomeric pores that insert into the insect-midgut cells. These toxins are being used worldwide in transgenic plants or spray to control insect pests in agriculture. However, a major concern has been the possible effects of these insecticidal proteins on non-target organisms mainly in ecosystems adjacent to agricultural fields. METHODOLOGY/PRINCIPAL FINDINGS:We isolated and characterized 11 non-toxic mutants of Cry1Ab toxin affected in different steps of the mechanism of action namely binding to receptors, oligomerization and pore-formation. These mutant toxins were analyzed for their capacity to block wild type toxin activity, presenting a dominant negative phenotype. The dominant negative phenotype was analyzed at two levels, in vivo by toxicity bioassays against susceptible Manduca sexta larvae and in vitro by pore formation activity in black lipid bilayers. We demonstrate that some mutations located in helix alpha-4 completely block the wild type toxin activity at sub-stoichiometric level confirming a dominant negative phenotype, thereby functioning as potent antitoxins. CONCLUSIONS/SIGNIFICANCE:This is the first reported case of a Cry toxin dominant inhibitor. These data demonstrate that oligomerization is a fundamental step in Cry toxin action and represent a potential mechanism to protect special ecosystems from the possible effect of Cry toxins on non-target organisms

Absence of CD34 on Murine Skeletal Muscle Satellite Cells Marks a Reversible State of Activation during Acute Injury

Background: Skeletal muscle satellite cells are myogenic progenitors that reside on myofiber surface beneath the basal lamina. In recent years satellite cells have been identified and isolated based on their expression of CD34, a sialomucin surface receptor traditionally used as a marker of hematopoietic stem cells. Interestingly, a minority of satellite cells lacking CD34 has been described. Methodology/Principal Findings: In order to elucidate the relationship between CD34+ and CD34- satellite cells we utilized fluorescence-activated cell sorting (FACS) to isolate each population for molecular analysis, culture and transplantation studies. Here we show that unless used in combination with a7 integrin, CD34 alone is inadequate for purifying satellite cells. Furthermore, the absence of CD34 marks a reversible state of activation dependent on muscle injury. Conclusions/Significance: Following acute injury CD34- cells become the major myogenic population whereas the percentage of CD34+ cells remains constant. In turn activated CD34- cells can reverse their activation to maintain the pool of CD34+ reserve cells. Such activation switching and maintenance of reserve pool suggests the satellite cell compartment is tightly regulated during muscle regeneration

Dynamics of Adrenal Steroids Are Related to Variations in Th1 and Treg Populations during Mycobacterium tuberculosis Infection in HIV Positive Persons

Tuberculosis (TB) remains the most frequent cause of illness and death from an infectious agent, and its interaction with HIV has devastating effects. We determined plasma levels of dehydroepiandrosterone (DHEA), its circulating form DHEA-suphate (DHEA-s) and cortisol in different stages of M. tuberculosis infection, and explored their role on the Th1 and Treg populations during different scenarios of HIV-TB coinfection, including the immune reconstitution inflammatory syndrome (IRIS), a condition related to antiretroviral treatment. DHEA levels were diminished in HIV-TB and HIV-TB IRIS patients compared to healthy donors (HD), HIV+ individuals and HIV+ individuals with latent TB (HIV-LTB), whereas dehydroepiandrosterone sulfate (DHEA-s) levels were markedly diminished in HIV-TB IRIS individuals. HIV-TB and IRIS patients presented a cortisol/DHEA ratio significantly higher than HIV+, HIV-LTB and HD individuals. A positive correlation was observed between DHEA-s and CD4 count among HIV-TB individuals. Conversely, cortisol plasma level inversely correlated with CD4 count within HIV-TB individuals. M. tuberculosis-specific Th1 lymphocyte count was increased after culturing PBMC from HIV-TB individuals in presence of DHEA. We observed an inverse correlation between DHEA-s plasma level and Treg frequency in co-infected individuals, and CD4+FoxP3+ Treg frequency was increased in HIV-TB and IRIS patients compared to other groups. Strikingly, we observed a prominent CD4+CD25-FoxP3+ population across HIV-TB and HIV-TB IRIS patients, which frequency correlated with DHEA plasma level. Finally, DHEA treatment negatively regulated FoxP3 expression without altering Treg frequency in co-infected patients. These data suggest an enhancing role for DHEA in the immune response against M. tuberculosis during HIV-TB coinfection and IRIS

Empirical Evaluation of Bone Extraction Protocols

The application of high-resolution analytical techniques to characterize ancient bone proteins requires clean, efficient extraction to obtain high quality data. Here, we evaluated many different protocols from the literature on ostrich cortical bone and moa cortical bone to evaluate their yield and relative purity using the identification of antibody-antigen complexes on enzyme-linked immunosorbent assay and gel electrophoresis. Moa bone provided an ancient comparison for the effectiveness of bone extraction protocols tested on ostrich bone. For the immunological part of this study, we focused on collagen I, osteocalcin, and hemoglobin because collagen and osteocalcin are the most abundant proteins in the mineralized extracellular matrix and hemoglobin is common in the vasculature. Most of these procedures demineralize the bone first, and then the remaining organics are chemically extracted. We found that the use of hydrochloric acid, rather than ethylenediaminetetraacetic acid, for demineralization resulted in the cleanest extractions because the acid was easily removed. In contrast, the use of ethylenediaminetetraacetic acid resulted in smearing upon electrophoretic separation, possibly indicating these samples were not as pure. The denaturing agents sodium dodecyl sulfate, urea, and guanidine HCl have been used extensively for the solubilization of proteins in non-biomineralized tissue, but only the latter has been used on bone. We show that all three denaturing agents are effective for extracting bone proteins. One additional method tested uses ammonium bicarbonate as a solubilizing buffer that is more appropriate for post-extraction analyses (e.g., proteomics) by removing the need for desalting. We found that both guanidine HCl and ammonium bicarbonate were effective for extracting many bone proteins, resulting in similar electrophoretic patterns. With the increasing use of proteomics, a new generation of scientists are now interested in the study of proteins from not only extant bone but also from ancient bone

Gene mutations that promote adrenal aldosterone production, sodium retention, and hypertension

Andreas G Moraitis,1 William E Rainey,1,2 Richard J Auchus1 1Division of Metabolism, Endocrinology, and Diabetes, Department of Internal Medicine, 2Department of Physiology, University of Michigan, Ann Arbor, MI, USA Abstract: Primary aldosteronism (PA) is the most common form of secondary hypertension, found in about 5% of all hypertension cases, and up to 20% of resistant hypertension cases. The most common forms of PA are an aldosterone-producing adenoma and idiopathic (bilateral) hyperaldosteronism. Rare genetic forms of PA exist and, until recently, the only condition with a known genetic mechanism was familial hyperaldosteronism type 1, also known as glucocorticoid-remediable aldosteronism (FHA1/GRA). FHA type 3 has now been shown to derive from germline mutations in the KCNJ5 gene, which encodes a potassium channel found on the adrenal cells. Remarkably, somatic mutations in KCNJ5 are found in about one-third of aldosterone-producing adenomas, and these mutations are likely to be involved in their pathogenesis. Finally, mutations in the genes encoding an L-type calcium channel (CACNA1D) and in genes encoding a sodium–potassium adenosine triphosphatase (ATP1A1) or a calcium adenosine triphosphatase (ATP2B3) are found in other aldosterone-producing adenomas. These findings provide a working model, in which adenoma formation and/or aldosterone production in many cases derives from increased calcium entry, which drives the pathogenesis of primary aldosteronism. Keywords: hyperaldosteronism, hereditary, potassium channel, calcium channe