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.     

Application of Cyanidin-3-Glucosides as a functional food ingredient in rice-based bakery products

BackgroundBlack pericarp rice has recently become popular among rice consumers for its diverse health benefits specially anti-cancer effect. Cyanidin-3-Glucosides (C3G), an prominant bioactive component of anthocyanins which is abundantly present in black pericarp rice.ObjectivesWe investigated, how effectively it can be used to fortify Cyanidin-3-Glucosides (C3G) content in red and white pericarp polished rice or rice based bakery products for more nutritional value.MethodIn the present study, we have characterized several black pericarp rice cultivars along with some red pericarp and white pericarp rice cultivars by physicochemical including mineral profiling, and quantified the C3G by UFLC and LCMS.ResultsC3G content was significantly reduced from raw rice to cooked rice condition. All the black pericarp rice cultivars synthesized C3G, while this content was not detected in red and white pericarp rice cultivars. However, when 25% of black pericarp rice were mixed with 75% red or white pericarp polished rice, C3G content was significantly retained in cooked rice conditions. Formulation of rice-based bakery food product using black pericarp rice powder was also remarkably retained the C3G content as compared to that of cooking. Black rice is harder in texture, difficult to digest and needs higher energy for cooking. Therefore, we tried to circumvent these challenges by fortifying 25% of black pericarp rice with white or red pericarp rice.ConclusionFortification of C3G enriched black rice (25%) with red or white pericarp rice (75%) might bring a better nutritional quality in both cooking and baking condition. This may lead a way to the effective management of the non-communicable disease such as cancer for common rice consuming population.     

Insight into the interaction of antitubercular and anticancer compound clofazimine with human serum albumin: spectroscopy and molecular modelling

The binding of clofazimine to human serum albumin (HSA) was investigated by applying optical spectroscopy and molecular docking methods. Fluorescence quenching data revealed that clofazimine binds to protein with binding constant in the order of 10⁴ M^?1, and with the increase in temperature, Stern–Volmer quenching constants gradually decreased indicating quenching mode to be static. The UV–visible spectra showed increase in absorbance upon interaction of HSA with clofazimine which further reveals formation of the drug–albumin complex. Thermodynamic parameters obtained from fluorescence data indicate that the process is exothermic and spontaneous. Forster distance (R_o) obtained from fluorescence resonance energy transfer is found to be 2.05 nm. Clofazimine impelled rise in α-helical structure in HSA as observed from far-UV CD spectra while there are minor alterations in tertiary structure of the protein. Clofazimine interacts strongly with HSA inducing secondary structure in the protein and slight alterations in protein topology as suggested by dynamic light scattering results. Moreover, docking results indicate that clofazimine binds to hydrophobic pocket near to the drug site II in HSA.     

Lasioglossins LLIII affect the morphogenesis of Candida albicans and reduces the duration of experimental vaginal candidiasis in mice

Lasioglossins are a group of peptides with identified antimicrobial activity. The inhibitory effects of two synthetic lasioglossin derivatives, LLIII and D‐isomeric variant LLIII‐D, on morphological changes in Candida albicans in vitro and the effect of local administration of LLIII during experimental murine candidiasis were investigated. C. albicans blastoconidia were grown in the presence of lasioglossin LLIII or LLIII‐D at concentrations of 11.5 μM and 21 μM, respectively, for 1, 2 and 3 days and their viability determined by flow cytometry using eosin Y staining. Morphological changes were examined by light and fluorescent microscopy. The Candida‐inhibitory effect of daily intravaginal administration of 0.7 or 1.4 μg of LLIII was assessed in mice with experimentally‐induced vaginal candidiasis. LLIII and LLIII‐D lasioglossins exhibited candidacidal activity in vitro (>76% after 24 hr and >84% after 48 hr of incubation). After 72 hr incubation of Candida with low concentration of lasioglossins, an increase in viability was detected, probably due to a Candida antimicrobial peptides evasion strategy. Furthermore, lasioglossins inhibited temperature‐induced morphotype changes toward hyphae and pseudohyphae with sporadic occurrence of atypical cells with two or enlarged nuclei, suggesting interference with mitosis or cytokinesis. Local application of LLIII reduced the duration of experimental candidiasis with no evidence of adverse effects. Lasioglossin LLIII is a promising candidate for development as an antimicrobial drug for treating the vaginal candidiasis.

     

Production enhancement and refolding of caprine growth hormone expressed in Escherichia coli

This study describes comparison between IPTG and lactose induction on expression of caprine growth hormone (cGH), enhancing cell densities of Escherichia coli cultures and refolding the recombinant cGH, produced as inclusion bodies, to biologically active state. 2–3 times higher cell densities were obtained in shake flask cultures when induction was done with lactose showing almost same level of expression as in case of IPTG induction. With lactose induction highest cell densities were achieved in TB (OD₆₀₀ 16.3) and M9NG (OD₆₀₀ 16.1) media, producing 885 and 892 mg cGH per liter of the culture, respectively. Lactose induction done at mid-exponential stage resulted in a higher cell density and thus higher product yield. cGH over-expressed as inclusion bodies was solubilized in 50 mM Tris–Cl buffer (pH 12.5) containing 2 M urea, followed by dilution and lowering the pH in a step-wise manner to obtain the final solution in 50 mM Tris–Cl (pH 9.5). The cGH was purified by Q-Sepharose chromatography followed by gel filtration with a recovery yield of 39% on the basis of total cell proteins. The product thus obtained showed a single band by SDS–PAGE analysis. MALDI-TOF analysis showed a single peak with a mass of 21,851 dalton, which is very close to its calculated molecular weight. A bioassay based on proliferation of Nb2 rat lymphoma cells showed that the purified cGH was biologically active.     

Synthesis of bis-Schiff bases of isatins and their antiglycation activity

Bis-Schiff bases 1–27 have been synthesized and their in vitro antiglycation potential has been evaluated. Compounds 21 (IC₅₀ = 243.95 ± 4.59 μM), 20 (IC₅₀ = 257.61 ± 5.63 μM), and 7 (IC₅₀ = 291.14 ± 2.53 μM) showed an excellent antiglycation activity better than the standard (rutin, IC₅₀ = 294.46 ± 1.50 μM). This study has identified a series of potential molecules as antiglycation agents. A structure–activity relationship has been studied, and all the compounds were characterized by spectroscopic techniques.