Identification of long-chain alkane-degrading (LadA) monooxygenases in Aspergillus flavus via in silico analysis.

Efficient degradation of alkanes in crude oil by the isolated Aspergillus flavus MM1 alluded to the presence of highly active alkane-degrading enzymes in this fungus. A long-chain alkane-degrading, LadA-like enzyme family in A. flavus was identified, and possible substrate-binding modes were analyzed using a computational approach. By analyzing publicly available protein databases, we identified six uncharacterized proteins in A. flavus NRRL 3357, of which five were identified as class LadAα and one as class LadAß, which are eukaryotic homologs of bacterial long-chain alkane monooxygenase (LadA). Computational models of A. flavus LadAα homologs (Af1-Af5) showed overall structural similarity to the bacterial LadA and the unique sequence and structural elements that bind the cofactor Flavin mononucleotide (FMN). A receptor-cofactor-substrate docking protocol was established and validated to demonstrate the substrate binding in the A. flavus LadAα homologs. The modeled Af1, Af3, Af4, and Af5 captured long-chain n-alkanes inside the active pocket, above the bound FMN. Isoalloxazine ring of reduced FMN formed a π-alkyl interaction with the terminal carbon atom of captured alkanes, C16-C30, in Af3-Af5 and C16-C24 in Af1. Our results confirmed the ability of identified A. flavus LadAα monooxygenases to bind long-chain alkanes inside the active pocket. Hence A. flavus LadAα monooxygenases potentially initiate the degradation of long-chain alkanes by oxidizing bound long-chain alkanes into their corresponding alcohol.

Reduction of lag in crude oil degradation by Aspergillus when it is in synergy with Bacillus in biofilm mode.

A major hindrance to the effective use of fungi in bioremediation is their inherent slow growth. Despite this, Aspergillus spp. may be used effectively. Our experiments demonstrate that bacteria, although inefficient in hydrocarbon degradation, may be effectively used in a consortium to overcome the lag in fungal utilization of petroleum hydrocarbons. Crude petroleum oil (160 mg; at 8 g/L) in minimal medium was inoculated with a previously isolated biofilm-forming consortium (Aspergillus sp. MM1 and Bacillus sp. MM1) as well as monocultures of each organism and incubated at 30 ℃ under static conditions. Residual oil was analyzed by GC-MS. Crude oil utilization of Aspergillus-Bacillus biofilm was 24 ± 1.4% in 3 days, increased to 66 ± 7% by day 5 and reached 99 ± 0.2% in 7 days. Aspergillus sp. MM1 monoculture degraded only 14 ± 6% in 5 days. However, at the end of 7 days, it was able to utilize 98 ± 2%. Bacillus sp. MM1 monoculture utilized 20 ± 4% in 7 days. This study indicates that there is a reduction of the fungal lag in bioremediation when it is in association with the bacterium. Although in monoculture, Bacillus sp. MM1 is inefficient in crude oil degradation, it synergistically enhances the initial rate of crude petroleum oil degradation of the fungus in the consortium. The rapid initial removal of as much crude oil as possible from contaminated sites is vital to minimize detrimental impacts on biodiversity.

Genotyping Sri Lankan women with polycystic ovary syndrome (PCOS): towards a novel screening tool.

Introduction: Polycystic ovary syndrome (PCOS) is the multigenic, endocrine disorder of young women. Inheritance of PCOS is likely to be oligogenic and genetic basis remains largely unknown. Screening the candidate genes of PCOS and their SNPs individually is time consuming. Hence, developing a tool that would help in screening multiple candidate genes simultaneously is essential to determine the exact genetic basis of PCOS. Objectives: This study aimed to develop a simple and cost-effective genetic screening tool to simultaneously genotype 16 single nucleotide polymorphisms (SNPs) of PCOS. Methods: The genetic screening tool was developed using allele specific real time quantitative PCR (AS-qPCR) in 96 well PCR plate. Eight SNPs identified in our previous study as well as 8 SNPs identified from other reported studies that had a strong association in the etiology of PCOS were used to develop the tool. Samples from our previous study were reanalyzed using the developed genetic screening tool. Genetic screening tool results were validated with Sanger sequencing. Results: Totally 10 AS-qPCR runs (160 reactions = 16SNPs*10runs) were performed using the developed tool and all except 3 genotype results agreed with Sanger sequencing. The tool showed 100% specificity and 96% sensitivity. Conclusion: The developed genetic screening tool has excellent potential in determining the genotype of multiple SNPs of PCOS simultaneously. This tool is highly suitable for developing countries as a cost effective and accurate early genetic screening test for PCOS. Thus, provides a reliable, fast and user-friendly genotyping method facilitating a wider implication in clinical practice.

In depth analysis of the association of FTO SNP (rs9939609) with the expression of classical phenotype of PCOS: a Sri Lankan study.

BACKGROUND: PCOS is a common disorder of women due to genetic, endocrine and environmental effects that manifests from puberty. The rs9939609 variant of fat mass and obesity associated (FTO) gene is linked to metabolic derangement in PCOS. We previously identified FTO (rs9939609) as a susceptibility locus for PCOS among Sri Lankan women and also explored the role of kisspeptin. Associated factors of the FTO candidate gene among South Asians with PCOS are unknown. METHODS: This study aimed to determine the association between FTO (rs9939609) polymorphism with clinical (BMI, acanthosis nigricans, hirsutism) and biochemical (serum kisspeptin and testosterone levels) characteristics of PCOS in a cohort of Sri Lankan women. Genetic and clinical data including serum kisspeptin and testosterone concentrations of our previously reported cases (n = 55) and controls (n = 110) were re-analyzed, specifically for an association with rs9939609 variant of FTO gene. RESULTS: Logistic regression analysis (AA - OR = 5.7, 95% CI = 2.41-13.63, p < 0.05) and genetic inheritance analysis (AA - OR = 5.49, 95%CI = 2.34-12.88, p < 0.05) showed that FTO (rs9939609) polymorphism is significantly associated with PCOS and its metabolic manifestations. Serum testosterone was significantly higher in affected women with mutant genotypes (AA+AT) than with the normal allele (TT) (p < 0.05). Although serum kisspeptin was higher in subjects with PCOS and mutant alleles than controls, this difference was not significant (p > 0.05). CONCLUSION: FTO gene variant rs9939609 is associated with hyperandrogenemia and metabolic manifestations of PCOS among women of Sri Lankan descent with the well-characterized phenotype. Serum kisspeptin and the FTO genotypes lack a significant association when adjusted for confounders.

Biofilm mediated synergistic degradation of hexadecane by a naturally formed community comprising Aspergillus flavus complex and Bacillus cereus group.

BACKGROUND: The hydrophobic nature of hydrocarbons make them less bioavailable to microbes, generally leading to low efficiency in biodegradation. Current bioremediation strategies for hydrocarbon contamination, uses induced mixed microbial cultures. This in-vitro study demonstrates the utilization of naturally occurring communities in suitable habitats for achieving highly efficient, synergistic degradation of hydrocarbons in a simple community structure without additives. METHODS: Enrichment media supplemented with 1% (7652.53 mg/L) hexadecane (HXD) as the sole carbon source were inoculated with samples of soil with waste polythene, collected from a municipal landfill in order to isolate microbial communities. Gas Chromatography-Mass Spectrometry (GC-MS) analysis was performed on HXD grown co-cultures and individual counterparts after 14 days incubation and percentage degradation was calculated. Microbes were identified using 16S rRNA gene and Internal Transcribed Spacer region sequencing. Biofilm formation was confirmed through scanning electron microscopy, in the most efficient community. RESULTS: Three mixed communities (C1, C2 and C3) that demonstrated efficient visual disintegration of the HXD layer in the static liquid cultures were isolated. The C1 community showed the highest activity, degrading > 99% HXD within 14 days. C1 comprised of a single fungus and a bacterium and they were identified as a Bacillus sp. MM1 and an Apsergillus sp. MM1. The co-culture and individual counterparts of the C1 community were assayed for HXD degradation by GC-MS. Degradation by the fungal and bacterial monocultures were 52.92 ± 8.81% and 9.62 ± 0.71% respectively, compared to 99.42 ± 0.38% by the co-culture in 14 days. This proved the synergistic behavior of the community. Further, this community demonstrated the formation of a biofilm in oil-water interface in the liquid medium. This was evidenced from scanning electron microscopy (SEM) showing the Bacillus cells attached on to Aspergillus mycelia. CONCLUSIONS: This study demonstrates the utilization of naturally formed fungal-bacterial communities for enhanced biodegradation of hydrocarbons such as hexadecane and reports for the first time, synergistic degradation of hexadecane through biofilm formation, by a community comprising of Bacillus cereus group and Aspergillus flavus complex.

Identification of selected genetic polymorphisms in polycystic ovary syndrome in Sri Lankan women using low cost genotyping techniques.

BACKGROUND: Polycystic ovary syndrome (PCOS), the commonest endocrine disorder affecting young women, appears to be a multigenic trait with contributing genes being unclear. Hence, analysis of polymorphisms in multiple candidate genes is required. Currently available genotyping methods are expensive, time-consuming with limited analytical sensitivity. AIM: (i) Develop and validate high resolution melting (HRM) assay and allele-specific real-time quantitative PCR (AS-qPCR) for genotyping selected SNPs associated with PCOS. (ii) Identify selected SNPs and their association with a Sri Lankan cohort of well-characterized PCOS. METHODS: DNA was extracted from women with well-characterized PCOS from adolescence (n = 55) and ethnically matched controls (n = 110). FTO (Fat mass and obesity associated gene; rs9939609), FSHB (Follicle stimulating hormone beta subunit; rs6169), FSHR (Follicle stimulating hormone receptor; rs6165/rs6166), and INSR (Insulin receptor; rs1799817) genes were genotyped using HRM assay. GnRH1 (Gonadotropin releasing hormone; rs6185), LHB (Luteinizing hormone beta subunit; rs1800447/rs34349826) and LHCGR (Luteinizing hormone/choriogonadotropin receptor; rs2293275) genes were genotyped using AS-qPCR method. Genotyping results were validated using Sanger sequencing. RESULTS: A significant association was observed within FTO gene polymorphism (rs9939609) and PCOS. Genotype frequency of FTO gene (rs9939609)-cases versus controls were TT-36.4% vs.65.4% (p<0.05), AT-23.6% vs.20.9%, AA-40% vs.13.6% (p<0.05). Genotype frequencies of the SNPs GnRH1 (rs6185), FSHB (rs6169), FSHR (rs6165 & rs6166), LHB (rs1800447 & rs34349826), LHCGR (rs2293275) and INSR (rs1799817) were not significantly different between cases and controls (p>0.05). Only the mutant alleles were observed for LHB rs1800447 and rs34349826 SNPs in both groups. The HRM and AS-qPCR assay results had 100% concordance with sequencing results. CONCLUSIONS: FTO gene rs9939609 polymorphism is significantly more prevalent among Sri Lankan PCOS subjects while the other selected SNPs of HPG axis genes and INSR gene showed no association. HRM and AS-qPCR assays provide a reliable, fast and user-friendly genotyping method facilitating wider implication in clinical practice.

Prevalence of G6PD deficiency in selected populations from two previously high malaria endemic areas of Sri Lanka.

Glucose-6-Phosphate Dehydrogenase (G6PD) enzyme deficiency is known to offer protection against malaria and an increased selection of mutant genes in malaria endemic regions is expected. However, anti-malarial drugs such as primaquine can cause haemolytic anaemia in persons with G6PD deficiency. We studied the extent of G6PD deficiency in selected persons attending Teaching Hospitals of Anuradhapura and Kurunegala, two previously high malaria endemic districts in Sri Lanka. A total of 2059 filter-paper blood spots collected between November 2013 and June 2014 were analysed for phenotypic G6PD deficiency using the modified WST-8/1-methoxy PMS method. Each assay was conducted with a set of controls and the colour development assessed visually as well as with a microplate reader at OD450-630nm. Overall, 142/1018 (13.95%) and 83/1041 (7.97%) were G6PD deficient in Anuradhapura and Kurunegala districts respectively. The G6PD prevalence was significantly greater in Anuradhapura when compared to Kurunegala (P<0.0001). Surprisingly, females were equally affected as males in each district: 35/313 (11.18%) males and 107/705 (15.18%) females were affected in Anuradhapura (P = 0.089); 25/313 (7.99%) males and 58/728 (7.97%) females were affected in Kurunegala (P = 0.991). Prevalence was greater among females in Anuradhapura than in Kurunegala (P<0.05), while no such difference was observed between the males (P>0.05). Severe deficiency (<10% normal) was seen among 28/1018 (2.75%) in Anuradhapura (7 males; 21 females) and 17/1041 (1.63%) in Kurunegala (7 males; 10 females). Enzyme activity between 10-30% was observed among 114/1018 (11.20%; 28 males; 86 females) in Anuradhapura while it was 66/1041 (6.34%; 18 males; 48 females) in Kurunegala. Screening and educational programmes for G6PD deficiency are warranted in these high risk areas irrespective of gender for the prevention of disease states related to this condition.

Polymerase chain reaction optimization for amplification of Guanine-Cytosine rich templates using buccal cell DNA.

CONTEXT: Amplification of Guanine-Cytosine (GC) -rich sequences becomes important in screening and diagnosis of certain genetic diseases such as diseases arising due to expansion of GC-rich trinucleotide repeat regions. However, GC-rich sequences in the genome are refractory to standard polymerase chain reaction (PCR) amplification and require a special reaction conditions and/or modified PCR cycle parameters. AIM: Optimize a cost effective PCR assay to amplify the GC-rich DNA templates. SETTINGS AND DESIGN: Fragile X mental retardation gene (FMR 1) is an ideal candidate for PCR optimization as its GC content is more than 80%. Primers designed to amplify the GC rich 5' untranslated region of the FMR 1 gene, was selected for the optimization of amplification using DNA extracted from buccal mucosal cells. MATERIALS AND METHODS: A simple and rapid protocol was used to extract DNA from buccal cells. PCR optimization was carried out using three methods, (a) substituting a substrate analog 7-deaza-dGTP to dGTP (b) in the presence of a single PCR additive and (c) using a combination of PCR additives. All PCR amplifications were carried out using a low-cost thermostable polymerase. RESULTS: Optimum PCR conditions were achieved when a combination of 1M betaine and 5% dimethyl sulfoxide (DMSO) was used. CONCLUSIONS: It was possible to amplify the GC rich region of FMR 1 gene with reproducibility in the presence of betaine and DMSO as additives without the use of commercially available kits for DNA extraction and the expensive thermostable polymerases.