Triticum durum metallothionein. Isolation of the gene and structural characterization of the protein using solution scattering and molecular modeling

A novel gene sequence, with two exons and one intron, encoding a metallothionein (MT) has been identified in durum wheat Triticum durum cv. Balcali85 genomic DNA. Multiple alignment analyses on the cDNA and the translated protein sequences showed that T. durum MT (dMT) can be classified as a type 1 MT. dMT has three Cys-X-Cys motifs in each of the N- and C-terminal domains and a 42-residue-long hinge region devoid of cysteines. dMT was overexpressed in Escherichia coli as a fusion protein (GSTdMT), and bacteria expressing the fusion protein showed increased tolerance to cadmium in the growth medium compared with controls. Purified GSTdMT was characterized by SDS- and native-PAGE, size exclusion chromatography, and matrix-assisted laser desorption ionization time-of-flight mass spectrometry. It was shown that the recombinant protein binds 4 +/- 1 mol of cadmium/mol of protein and has a high tendency to form stable oligomeric structures. The structure of GSTdMT and dMT was investigated by synchrotron x-ray solution scattering and computational methods. X-ray scattering measurements indicated a strong tendency for GSTdMT to form dimers and trimers in solution and yielded structural models that were compatible with a stable dimeric form in which dMT had an extended conformation. Results of homology modeling and ab initio solution scattering approaches produced an elongated dMT structure with a long central hinge region. The predicted model and those obtained from x-ray scattering are in agreement and suggest that dMT may be involved in functions other than metal detoxification.     

Effects of selenium on altered mechanical and electrical cardiac activities of diabetic rat

Since selenium compounds can restore some metabolic parameters and structural alterations of diabetic rat heart, we were tempted to investigate whether these beneficial effects extend to the diabetic rat cardiac dysfunctions. Diabetes was induced by streptozotocin (50mg/kg body weight) and rats were then treated with sodium selenite (5 micromol/kg body weight/day) for four weeks. Electrically stimulated isometric contraction and intracellular action potential in isolated papillary muscle strips and transient (I(to)) and steady state (I(ss)) outward K(+) currents in isolated cardiomyocytes were recorded. Sodium selenite treatment could reverse the prolongation in both action potential duration and twitch duration of the diabetic rats, and also cause significant increases in the diminished amplitudes of the two K(+) currents. Treatment of rats with sodium selenite also markedly increased the depressed acid-soluble sulfhydryl levels of the hearts. Our data suggest that the beneficial effects of sodium selenite treatment on the mechanical and electrical activities of the diabetic rat heart appear to be due to the restoration of the diminished K(+) currents, partially, related to the restoration of the cell glutathione redox cycle.     

Mutations in SLC34A2 cause pulmonary alveolar microlithiasis and are possibly associated with testicular microlithiasis

Pulmonary alveolar microlithiasis (PAM) is a rare disease characterized by the deposition of calcium phosphate microliths throughout the lungs. We first identified a PAM locus by homozygosity mapping to 4p15, then identified, by a candidate-gene approach, the gene responsible for the disease as SLC34A2 (the type IIb sodium-phosphate cotransporter gene), which is involved in phosphate homeostasis in several organs. We identified six homozygous exonic mutations in the seven unrelated patients with PAM we studied. Three of the mutations were frameshifts, one was a chain termination, one was an amino acid substitution, and one was a deletion spanning the minimal promoter and the first exon. Absence of functional protein product of the gene is compatible with calcium phosphate deposition in alveolar airspaces. We show that impaired activity of the phosphate transporter is presumably responsible for the microliths and that PAM is a recessive monogenic disease with full penetrance. Testicular microlithiasis (TM) is a disease that is more common than PAM. It is often associated with cancer and infertility. Since the gene we identified is also expressed in testis, we searched for mutations in subjects with TM. In 2 of the 15 subjects with TM we studied, we identified two rare variants, one synonymous and the other noncoding, that are possibly associated with the condition.     

Improved thermostability of Clostridium thermocellum endoglucanase Cel8A by using consensus-guided mutagenesis

The use of thermostable cellulases is advantageous for the breakdown of lignocellulosic biomass toward the commercial production of biofuels. Previously, we have demonstrated the engineering of an enhanced thermostable family 8 cellulosomal endoglucanase (EC 3.2.1.4), Cel8A, from Clostridium thermocellum, using random error-prone PCR and a combination of three beneficial mutations, dominated by an intriguing serine-to-glycine substitution (M. Anbar, R. Lamed, E. A. Bayer, ChemCatChem 2:997-1003, 2010). In the present study, we used a bioinformatics-based approach involving sequence alignment of homologous family 8 glycoside hydrolases to create a library of consensus mutations in which residues of the catalytic module are replaced at specific positions with the most prevalent amino acids in the family. One of the mutants (G283P) displayed a higher thermal stability than the wild-type enzyme. Introducing this mutation into the previously engineered Cel8A triple mutant resulted in an optimized enzyme, increasing the half-life of activity by 14-fold at 85°C. Remarkably, no loss of catalytic activity was observed compared to that of the wild-type endoglucanase. The structural changes were simulated by molecular dynamics analysis, and specific regions were identified that contributed to the observed thermostability. Intriguingly, most of the proteins used for sequence alignment in determining the consensus residues were derived from mesophilic bacteria, with optimal temperatures well below that of C. thermocellum Cel8A.     

Somatic mosaic activating mutations in PIK3CA cause CLOVES syndrome

Congenital lipomatous overgrowth with vascular, epidermal, and skeletal anomalies (CLOVES) is a sporadically occurring, nonhereditary disorder characterized by asymmetric somatic hypertrophy and anomalies in multiple organs. We hypothesized that CLOVES syndrome would be caused by a somatic mutation arising during early embryonic development. Therefore, we employed massively parallel sequencing to search for somatic mosaic mutations in fresh, frozen, or fixed archival tissue from six affected individuals. We identified mutations in PIK3CA in all six individuals, and mutant allele frequencies ranged from 3% to 30% in affected tissue from multiple embryonic lineages. Interestingly, these same mutations have been identified in cancer cells, in which they increase phosphoinositide-3-kinase activity. We conclude that CLOVES is caused by postzygotic activating mutations in PIK3CA. The application of similar sequencing strategies will probably identify additional genetic causes for sporadically occurring, nonheritable malformations.     

Confidence-based Somatic Mutation Evaluation and Prioritization

Next generation sequencing (NGS) has enabled high throughput discovery of somatic mutations. Detection depends on experimental design, lab platforms, parameters and analysis algorithms. However, NGS-based somatic mutation detection is prone to erroneous calls, with reported validation rates near 54% and congruence between algorithms less than 50%. Here, we developed an algorithm to assign a single statistic, a false discovery rate (FDR), to each somatic mutation identified by NGS. This FDR confidence value accurately discriminates true mutations from erroneous calls. Using sequencing data generated from triplicate exome profiling of C57BL/6 mice and B16-F10 melanoma cells, we used the existing algorithms GATK, SAMtools and SomaticSNiPer to identify somatic mutations. For each identified mutation, our algorithm assigned an FDR. We selected 139 mutations for validation, including 50 somatic mutations assigned a low FDR (high confidence) and 44 mutations assigned a high FDR (low confidence). All of the high confidence somatic mutations validated (50 of 50), none of the 44 low confidence somatic mutations validated, and 15 of 45 mutations with an intermediate FDR validated. Furthermore, the assignment of a single FDR to individual mutations enables statistical comparisons of lab and computation methodologies, including ROC curves and AUC metrics. Using the HiSeq 2000, single end 50 nt reads from replicates generate the highest confidence somatic mutation call set.     

SEASONAL VARIATION IN THE EFFECT OF A FIXED DOSE OF HEPARIN ON ACTIVATED CLOTTING TIME IN PATIENTS PREPARED FOR OPEN-HEART SURGERY *

We investigated the effect of an injected bolus of 5 mg kg^- heparin at one circadian stage (08:30 to 11:00) on blood coagulation during different months of the year. Activated clotting times (ACTs) were assessed before and 5 min after heparin dosing to ensure extracorporeal circulation during open-heart surgery. The ACT data of 1083 presumably day-active Turkish patients (816 men and 267 women, mostly older than 46 years) who underwent coronary bypass surgery between 08:30 and 11:00 in the years from 1994 to 1997 were analyzed for annual rhythmicity. The ACT values obtained just before and 5 min after heparinization were subjected to cosinor analysis using a 365.25-day period to assess seasonality in basal ACT level and heparin effect. A small-amplitude annual rhythm with a wintertime peak was documented in the morning ACT in the group of 1083 patients. Rhythms of similar magnitude and staging were also detected in heparin effect on ACT in the 1083 patients and in subgroups categorized by gender. Circannual rhythmicity in the heparin effect on ACT was also documented in the elderly (≥ 45 years old), but not young (18-45 years old) patients. The annual mean effect of heparin on the ACT was statistically significantly greater in younger than older patients. The relatively low-amplitude circannual rhythm in heparin effect on ACT (∼10% of the annual mean) is not viewed as being meaningful in patient preparation for bypass surgery for the 5 mg kg^-1 level of heparin dosing. (Chronobiology International, 18(5), 865-873, 2001)