Novel mutations in G protein-coupled receptor gene (P2RY5) in families with autosomal recessive hypotrichosis (LAH3)

Autosomal recessive hypotrichosis (LAH3) is a rare hair disorder characterized by sparse hair on scalp and the rest of the body of affected individuals. Recently mutations in a G protein-coupled receptor gene, P2RY5, located at LAH3 locus, have been reported in several families with autosomal recessive hypotrichosis simplex and woolly hair. For the present study, 22 Pakistani families with autosomal recessive hypotrichosis were enrolled. Genotyping using microsatellite markers linked to three autosomal recessive forms of hypotrichosis (LAH1, LAH2, LAH3) showed the linkage of 2 families to the LAH2 locus and 14 to the LAH3 locus. The remaining 6 families were not linked to any of the three loci. Families linked to LAH3 locus were further subjected to screening of the P2RY5 gene with direct DNA sequencing. Three previously reported variants, c.69insCATG (p.24insHfs52), c.188A > T (p.D63V) and c.565G > A (p.E189K) were observed in eight families. Four novel nonsynonymous sequence variants, c.8G > C (p.S3T), c.36insA (p.D13RfsX16), c.160insA (p.N54TfsX58) and c.436G > A (p.G146R) were found to segregate within six families. Z. Azeem, M. Jelani, G. Naz, M. Tariq, N. Wasif, S. Kamran-ul-Hassan Naqvi contributed equally to this work.     

Function and structure of lipid storage droplet protein 1 studied in lipoprotein complexes

Triglycerides (TG) stored in lipid droplets (LDs) are the main energy reserve in all animals. The mechanism by which animals mobilize TG is complex and not fully understood. Several proteins surrounding the LDs have been implicated in TG homeostasis such as mammalian perilipin A and insect lipid storage proteins (Lsd). Most of the knowledge on LD-associated proteins comes from studies using cells or LDs leaving biochemical properties of these proteins uncharacterized. Here we describe the purification of recombinant Lsd1 and its reconstitution with lipids to form lipoprotein complexes suitable for functional and structural studies. Lsd1 in the lipid bound state is a predominately α-helical protein. Using lipoprotein complexes containing triolein it is shown that PKA mediated phosphorylation of Lsd1 promoted a 1.7-fold activation of the main fat body lipase demonstrating the direct link between Lsd1 phosphorylation and activation of lipolysis. Serine 20 was identified as the Lsd1-phosphorylation site triggering this effect.     

Pharmacological activities of crude acetone extract and purified constituents of Salvia moorcraftiana Wall.

The crude acetone extract of aerial parts of Salvia moorcraftiana Wall. was screened for various biological activities including Lemna bioassay, antifungal, antibacterial, leishmanicidal, insecticidal activities and brine shrimp cytotoxicity. It was found to possess strong phytotoxic activity against Lemna aequinoctials Welve. and moderate antifungal activity against animal and plant pathogens. The purified chemical constituents were tested for enzyme inhibition activity. Two constituents (compounds 3 and 8) were found to be effective inhibitors of alpha-glucosidase.     

A Constitutive Expression System for Cellulase Secretion in Escherichia coli and Its Use in Bioethanol Production

The production of biofuels from lignocellulosic biomass appears to be attractive and viable due to the abundance and availability of this biomass. The hydrolysis of this biomass, however, is challenging because of the complex lignocellulosic structure. The ability to produce hydrolytic cellulase enzymes in a cost-effective manner will certainly accelerate the process of making lignocellulosic ethanol production a commercial reality. These cellulases may need to be produced aerobically to generate large amounts of protein in a short time or anaerobically to produce biofuels from cellulose via consolidated bioprocessing. Therefore, it is important to identify a promoter that can constitutively drive the expression of cellulases under both aerobic and anaerobic conditions without the need for an inducer. Using lacZ as reporter gene, we analyzed the strength of the promoters of four genes, namely lacZ, gapA, ldhA and pflB, and found that the gapA promoter yielded the maximum expression of the β-galactosidase enzyme under both aerobic and anaerobic conditions. We further cloned the genes for two cellulolytic enzymes, β-1,4-endoglucanase and β-1,4-glucosidase, under the control of the gapA promoter, and we expressed these genes in Escherichia coli, which secreted the products into the extracellular medium. An ethanologenic E. colistrain transformed with the secretory β-glucosidase gene construct fermented cellobiose in both defined and complex medium. This recombinant strain also fermented wheat straw hydrolysate containing glucose, xylose and cellobiose into ethanol with an 85% efficiency of biotransformation. An ethanologenic strain that constitutively secretes a cellulolytic enzyme is a promising platform for producing lignocellulosic ethanol.     

Novel mutation G324C in <Emphasis Type="Italic">WNT1</Emphasis> mapped in a large Pakistani family with severe recessively inherited <Emphasis Type="Italic">Osteogenesis Imperfecta</Emphasis>

Introduction

Osteogenesis imperfecta (OI) is a clinically and genetically heterogeneous disease with skeletal fragility and variable extra-skeletal manifestations. To date several point mutations in 18 different genes causing different types of OI have been identified. Mutations in WNT1 compromise activity of the osteoblasts leading to disturbed bone mass accrual, fragility fractures and progressive skeletal abnormalities. The present study was conducted to determine the underlying genetic cause of an autosomal recessive skeletal dysplasia in a large consanguineous family from Chinute, Pakistan.

Materials and methods

Blood was collected from 24 individuals of affected family along with clinical data. Homozygosity mapping was performed to confirm consanguinity. SNPs were identified, followed by whole exome and Sanger sequencing. In silico characterization of WNT1 mutation was performed using multiple platforms.

Results

Nine affected family members exhibited severe bone deformities, recurrent fractures, short stature and low bone mineral density. SNP array data revealed homozygous segments >?1 Mb in length accounting for 2.1–12.7% of the genome in affected individuals and their siblings and a single 6,344,821 bp homozygous region in all affected individuals on chromosome 12q12-q13. This region includes two potential OI candidate genes WNT1 and VDR. We did whole-exome sequencing for both genes in two patients and identified a novel damaging missense mutation in exon 4 of WNT1: c.1168G?>?T (NM_005430) resulting in p.G324C. Sanger sequencing confirmed segregation of mutation with the disease in family.

Conclusion

We report a novel mutation responsible for OI and our investigation expands the spectrum of disease-causing WNT1 mutations and the resulting OI phenotypes.

     

Bioremediation of hexachlorocyclohexane (HCH) in soil using spent mushroom compost of Pleurotus ostreatus

Abstract

Hexachlorocyclohexane (HCH), a highly chlorinated pesticide, was used worldwide in the 1950s and 1960s. HCH toxic residues are still detected in environmental compartments. Thus, effective, viable and eco-friendly strategy is required for its remediation. In this study, degradation of four HCH isomers was evaluated by amending contaminated soil using four treatments of spent mushroom compost (SMC) of Pleurotus ostraetus. The soil was incubated for 5 weeks and was sampled every seven days. Quantitative attenuation in HCH was calculated using gas chromatography–electron capture detector (GC-ECD) and metabolite was identified using gas chromatography–mass selective detector (GC-MSD). Maximum reduction 58%, 26%, 45%, and 64% for α-, β-, γ- and δ-HCH isomers, respectively, using SMC and soil (both unsterilized) showed that this treatment was the best for bioremediation of HCH in soil. However, when one of the factors, either soil or SMC, was sterilized, a significant reduction in HCH degradation was noticed. The second most reduction of isomers was seen during treatment where unsterilized SMC was added in sterilized soil followed by treatment where SMC was sterilized but soil was not. Abiotic control did not remove any significant quantities of HCH. Simple first-order (SFO) kinetic confirmed that SMC reduced the half-live manifolds as compared to biotic control. Only one metabolite δ-PCCH was identified during the course of study.     

Nano Copper Induces Apoptosis in PK-15 Cells via a Mitochondria-Mediated Pathway

Nano-sized copper particles are widely used in various chemical, physical, and biological fields. However, earlier studies have shown that nano copper particles (40–100 μg/mL) can induce cell toxicity and apoptosis. Therefore, this study was conducted to investigate the role of nano copper in mitochondrion-mediated apoptosis in PK-15 cells. The cells were treated with different doses of nano copper (20, 40, 60, and 80 μg/mL) to determine the effects of apoptosis using acridine orange/ethidium bromide (AO/EB) fluorescence staining and a flow cytometry assay. The levels of malondialdehyde (MDA) and superoxide dismutase (SOD) in the PK-15 cells were examined using commercially available kits. Moreover, the mRNA levels of the Bax, Bid, Caspase-3, and CYCS genes were assessed by real-time PCR. The results revealed that nano copper exposure induced apoptosis and changed the mitochondrial membrane potential. In addition, nano copper significantly altered the levels of the Bax, Bid, Caspase-3, and CYCS genes at a concentration of 40 μg/mL. To summarize, nano copper significantly (P < 0.05) decreased the level of SOD and increased the level of MDA in PK-15 cells. Altogether, these results suggest that nano copper can play an important role in inducing the apoptotic pathway in PK-15 cells, which may be the mechanism by which nano copper induces nephrotoxicity.     

Sequence Analysis and Comparative Bioinformatics Study of Camelysin Gene (<Emphasis Type="Italic">calY</Emphasis>) Isolated from <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis>

Bacillus strains have been widely used for the production of fibrinolytic enzymes having role in the treatment of cardiovascular disorders. Purification and overproduction of such enzymes has increased their usage in medical fields including metalloproteinases with the ability to degrade extracellular matrix (ECM). Camelysin, a neutral metalloproteinase has been isolated from different species of bacteria like Bacillus cereus, Bacillus anthracis, and Bacillus thuringiensis with fibrinolytic, collagenolytic and actin degradation activity. This project successfully demonstrated the presence of 734-bp coding DNA sequence (CDS) encoding a 20.72331 kDa camelysin gene in local strain of Bacillus thuringiensis containing a signal peptide with cleavage site between residues 19 and 20. The sequence was submitted to GenBank (KT023597) and the sequence showed high homology with the camelysin protein of closely related Bacillus species. The alignment of related proteins through ClustalW displayed difference of four amino acids (“Q” replaced by “P” at position 169 and at position 182–184, “NQE” replaced by “HLK”) in the isolated protein. Comparison including structural and functional analysis of camelysin sequences isolated from different Bacillus species was carried out using different bioinformatics tools and software. The information would help in better understanding the properties of camelysin protein and its role in pathogenicity and clinical treatments.