Whole genome sequencing for quantifying germline mutation frequency in humans and model species: cautious optimism

Factors affecting the type and frequency of germline mutations in animals are of significant interest from health and toxicology perspectives. However, studies in this field have been limited by the use of markers with low detection power or uncertain relevance to phenotype. Whole genome sequencing (WGS) is now a potential option to directly determine germline mutation type and frequency in family groups at all loci simultaneously. Medical studies have already capitalized on WGS to identify novel mutations in human families for clinical purposes, such as identifying candidate genes contributing to inherited conditions. However, WGS has not yet been used in any studies of vertebrates that aim to quantify changes in germline mutation frequency as a result of environmental factors. WGS is a promising tool for detecting mutation induction, but it is currently limited by several technical challenges. Perhaps the most pressing issue is sequencing error rates that are currently high in comparison to the intergenerational mutation frequency. Different platforms and depths of coverage currently result in a range of 10-10(3) false positives for every true mutation. In addition, the cost of WGS is still relatively high, particularly when comparing mutation frequencies among treatment groups with even moderate sample sizes. Despite these challenges, WGS offers the potential for unprecedented insight into germline mutation processes. Refinement of available tools and emergence of new technologies may be able to provide the improved accuracy and reduced costs necessary to make WGS viable in germline mutation studies in the very near future. To streamline studies, researchers may use multiple family triads per treatment group and sequence a targeted (reduced) portion of each genome with high (20-40 ×) depth of coverage. We are optimistic about the application of WGS for quantifying germline mutations, but caution researchers regarding the resource-intensive nature of the work using existing technology.     

Superoxide Dismutase Concentration and Activity in Familial Amyotrophic Lateral Sclerosis

Abstract: Some cases of autosomal-dominant familial amyotrophic lateral sclerosis (FALS) have been associated with mutations in SOD1 , the gene that encodes Cu/Zn superoxide dismutase (Cu/Zn SOD). We determined the concentrations (µg of Cu/Zn SOD/mg of total protein), specific activities (U/µg of total protein), and apparent turnover numbers (U/µmol of Cu/Zn SOD) of Cu/Zn SOD in erythrocyte lysates from patients with known SOD1 mutations. We also measured the concentrations and activities of Cu/Zn SOD in FALS patients with no identifiable SOD1 mutations, sporadic ALS (SALS) patients, and patients with other neurologic disorders. The concentration and specific activity of Cu/Zn SOD were decreased in all patients with SOD1 mutations, with mean reductions of 51 and 46%, respectively, relative to controls. In contrast, the apparent turnover number of the enzyme was not altered in these patients. For the six mutations studied, there was no correlation between enzyme concentration or specific activity and disease severity, expressed as either duration of disease or age of onset. No significant alterations in the concentration, specific activity, or apparent turnover number of Cu/Zn SOD were detected in the FALS patients with no identifiable SOD1 mutations, SALS patients, or patients with other neurologic disorders. That Cu/Zn SOD concentration and specific activity are equivalently reduced in erythrocytes from patients with SOD1 mutations suggests that mutant Cu/Zn SOD is unstable in these cells. That concentration and specific activity do not correlate with disease severity suggests that an altered, novel function of the enzyme, rather than reduction of its dismutase activity, may be responsible for the pathogenesis of FALS.     

The Deuterator: software for the determination of backbone amide deuterium levels from H/D exchange MS data

Background  

The combination of mass spectrometry and solution phase amide hydrogen/deuterium exchange (H/D exchange) experiments is an effective method for characterizing protein dynamics, and protein-protein or protein-ligand interactions. Despite methodological advancements and improvements in instrumentation and automation, data analysis and display remains a tedious process. The factors that contribute to this bottleneck are the large number of data points produced in a typical experiment, each requiring manual curation and validation, and then calculation of the level of backbone amide exchange. Tools have become available that address some of these issues, but lack sufficient integration, functionality, and accessibility required to address the needs of the H/D exchange community. To date there is no software for the analysis of H/D exchange data that comprehensively addresses these issues.     

Neural mitochondrial Ca2+ capacity impairment precedes the onset of motor symptoms in G93A Cu/Zn-superoxide dismutase mutant mice

Mitochondrial respiratory chain dysfunction, impaired intracellular Ca2+ homeostasis and activation of the mitochondrial apoptotic pathway are pathological hallmarks in animal and cellular models of familial amyotrophic lateral sclerosis associated with Cu/Zn-superoxide dismutase mutations. Although intracellular Ca2+ homeostasis is thought to be intimately associated with mitochondrial functions, the temporal and causal correlation between mitochondrial Ca2+ uptake dysfunction and motor neuron death in familial amyotrophic lateral sclerosis remains to be established. We investigated mitochondrial Ca2+ handling in isolated brain, spinal cord and liver of mutant Cu/Zn-superoxide dismutase transgenic mice at different disease stages. In G93A mutant transgenic mice, we found a significant decrease in mitochondrial Ca2+ loading capacity in brain and spinal cord, as compared with age-matched controls, very early on in the course of the disease, long before the onset of motor weakness and massive neuronal death. Ca2+ loading capacity was not significantly changed in liver G93A mitochondria. We also confirmed Ca2+ capacity impairment in spinal cord mitochondria from a different line of mice expressing G85R mutant Cu/Zn-superoxide dismutase. In excitable cells, such as motor neurons, mitochondria play an important role in handling rapid cytosolic Ca2+ transients. Thus, mitochondrial dysfunction and Ca2+-mediated excitotoxicity are likely to be interconnected mechanisms that contribute to neuronal degeneration in familial amyotrophic lateral sclerosis.     

Role of excitotoxicity in human neurological disease.

An increasing body of evidence has implicated excitoxicity as a mechanism of neuronal death in both acute and chronic neurological diseases. A major recent advance has been the successful cloning and expression of the non-NMDA, NMDA, and metabotropic glutamate receptors. The cellular mechanisms responsible for cell death following activation of these receptors are still being clarified. A recent advance in conceptualizing excitotoxicity is the notion that a slow excitotoxic process may occur as a consequence of either a receptor abnormality or an impairment of energy metabolism. It is possible that such a mechanism may occur in neurodegenerative illnesses. Recent therapeutic studies have focused on glycine site antagonists and on the efficacy of non-NMDA antagonists in ischemia.     

Salivary secretion during selective β-adrenoreceptor stimulation and blockade in the parotid gland of red kangaroos,<Emphasis Type="Italic"> Macropus rufus</Emphasis>

Intracarotid infusions of noradrenaline (0.3 nmol.kg(-1) x min(-1)) stimulated salivary fluid secretion and caused increases in salivary concentrations of protein, potassium. magnesium. chloride and phosphate, and decreases in bicarbonate. These effects of intracarotid noradrenaline were not reduced by simultaneous intracarotid infusion of phentolamine (3.0 nmol.kg(-1) x min(-1)) but were significantly greater than the responses accompanying intravenous noradrenaline infusion. Concomitant administration of the beta-antagonist, CGP20712A, were much more effective in blocking the noradrenaline-induced changes in salivary composition than equimolar infusions of the beta2-antagonist, ICI118551, thereby confirming the presence of beta1-adrenoreceptors. Intracarotid infusion of salbutamol at 0.6 nmol x kg(-1) x min(-1) and 6.0 nmol x kg(-1) x min(-1) caused increasing but qualitatively similar changes in salivary composition to intracarotid noradrenaline but was less effective than noradrenaline in augmenting salivary protein release. Equimolar intravenous infusions of salbutamol and noradrenaline were equally potent in altering salivary electrolyte concentrations but salbutamol by this route had less effect on protein release and fluid secretion. Concurrent intravenous and intracarotid infusions of beta1-(CGP) and beta2-(ICI) antagonists with intracarotid salbutamol showed that the beta2-antagonist was more potent than the beta1-antagonist by the intracarotid route thereby demonstrating the presence of glandular beta2-receptors and eliminating the possibility that the response to salbutamol was due totally by reflex increases in general sympathetic tone triggered by lowered blood pressure. It was concluded that the kangaroo parotid has functional beta1- and beta2-adrenoreceptor subtypes in endpieces whereas the data provide little support for either adrenoreceptor subtype being present in the excurrent duct system.     

Gut microbiome composition and function in experimental colitis during active disease and treatment-induced remission

Dysregulated immune responses to gut microbes are central to inflammatory bowel disease (IBD), and gut microbial activity can fuel chronic inflammation. Examining how IBD-directed therapies influence gut microbiomes may identify microbial community features integral to mitigating disease and maintaining health. However, IBD patients often receive multiple treatments during disease flares, confounding such analyses. Preclinical models of IBD with well-defined disease courses and opportunities for controlled treatment exposures provide a valuable solution. Here, we surveyed the gut microbiome of the T-bet^−/− Rag2^−/− mouse model of colitis during active disease and treatment-induced remission. Microbial features modified among these conditions included altered potential for carbohydrate and energy metabolism and bacterial pathogenesis, specifically cell motility and signal transduction pathways. We also observed an increased capacity for xenobiotics metabolism, including benzoate degradation, a pathway linking host adrenergic stress with enhanced bacterial virulence, and found decreased levels of fecal dopamine in active colitis. When transferred to gnotobiotic mice, gut microbiomes from mice with active disease versus treatment-induced remission elicited varying degrees of colitis. Thus, our study provides insight into specific microbial clades and pathways associated with health, active disease and treatment interventions in a mouse model of colitis.     

1H nuclear magnetic resonance assay of erythrocyte triosephosphate isomerase

A direct method for measuring the activity of erythrocyte triosephosphate isomerase using 1H NMR spectroscopy was developed. NMR spectroscopy allows the simultaneous monitoring of the substrate and the product of the reaction by virtue of the differences in the NMR spectrum of each chemical species. The assay conditions were based on a modification of a conventional spectrophotometric method. The enzymatic activity measured using NMR gave results comparable to those obtained in a standard assay. The results were used in the kinetic characterization of triosephosphate isomerase in hemolysates from subjects with homozygous or heterozygous deficiency of the enzyme. In general, NMR spectroscopy has the potential for wide application in the rapid development of new enzyme assays.