Isolation of heavy metal-resistant Staphylococcus epidermidis strain TWSL_22 and evaluation of heavy metal bioremediation potential of recombinant E. coli cloned with isolated cadD.

A heavy metal-resistant bacterial strain, TWSL_22 was isolated from an industrial effluent sample and tested for heavy metal tolerance and resistance. The strain was molecularly characterized as Staphylococcus epidermidis based on 16S rDNA gene analysis and the sequence was deposited in the NCBI repository (accession number KT184893.1). Metal removal activity (P < .001) of TWSL_22 was 99.99 ± 0.001%, 74.43 ± 2.51%, and 51.16 ± 4.17% for Cd, Pb, and Cu, respectively. Highest MIC was observed for Cd. Antibiotic susceptibility assays revealed the strain TWSL_22 to be resistant to several antibiotics. The strain was screened for possible heavy metal-resistant genes and presence of cadA, copA, and cadD was confirmed by PCR. A DNA fragment containing complete sequence of cadD (618 bp) was isolated and cloned into pET 21a(+), transformed into E. coli BL21 and designated as E. coli/cadDET. E. coli/cadDET showed high metal tolerance capacity and could remove over 82% of heavy metals (Zn2+, Cd2+, Cu2+, and Cr3+) in the industrial effluent.

Compensatory Base Changes Reveal Sexual Incompatibility among Members of the Anopheles subpictus Sensu Lato (Diptera: Culicidae) Species Complex in Sri Lanka.

The mosquito Anopheles (Cellia) subpictus sensu lato (s.l.) is a major secondary vector of malaria in Sri Lanka. The sibling species composition in this species complex in Sri Lanka remains debatable. Compensatory base changes (CBCs) in the secondary structures of internal transcribed spacer 2 (ITS2) are reliable sources to predict sexual incompatibility among closely related species. The objective of the present study was to investigate the An. subpictus s.l. populations in Sri Lanka using the CBC analysis. Mosquito DNA was amplified and sequenced for the ITS2 region. The sequences were annotated using ITS2 Database. ITS2 secondary structures were constructed and analyzed for CBCs using various bioinformatics tools. The ITS2 regions consisted of two different lengths, 575 bp and 480 bp. The two CBCs and three hemi CBCs identified in the present study suggest that there may be at least two sexually incompatible sibling species. In conclusion, it is likely that there may be only two reproductively isolated sibling species in the An. subpictus species complex in Sri Lanka. However, due to high divergence of ITS2 in these species, it is reasonable to assume that they may be undergoing a speciation event to separate as a distinct species.

Genetic association between antipsychotic induced weight gain and FTO gene in a Sri Lankan population

Background: About 30% of patients treated with second generation antipsychotics (SGA) experience weight gain. Although there is evidence that the FTO gene is associated with obesity its role in antipsychotic induced weight gain is not so clear. Methods: A genetic association study was carried out to identify the association between FTO rs9939609 and antipsychotic induced weight gain. Sample consisted of 180 cases and 120 controls. Cases were patients diagnosed with schizophrenia or schizoaffective disorder, treated with second-generation antipsychotics for a minimum of 3 months, and had gained at least 10% of body weight. Controls were patients with schizophrenia treated with second-generation antipsychotics for a minimum of 3 months but had not gained ≥10% of body weight. Genomic DNA was extracted from whole blood. Polymerase chain reaction of the samples was done. Real-time quantitative PCR (qPCR) was carried out using BIO-RAD CFX96 Touch TM PCR detection system. Results: Females were significantly more among cases (58.3%) than controls (35%). Cases (52.4%) were significantly more likely to be overweight or obese than controls (13.8%). Genotype distribution was in Hardy-Weinberg equilibrium (p=0.43). Cochran-Armitage trend test was not significant. Risk of antipsychotic induced weight gain in the AA genotype [OR 1.69 (95% CI 0.74-3.86)] and AT genotype [OR 1.1 (95% CI 0.67-1.79)] were not significantly higher than the TT genotype. Recessive model showed that AA/AT genotypes were at significantly higher risk of being obese/overweight [OR 1.84 (95% CI 1.05-3.2)]. Conclusions: There was no significant association between FTO rs9939609 and antipsychotic induced weight gain. AA/AT genotypes had significantly higher risk of overweight/obesity.

Polycystic ovary syndrome (PCOS) and kisspeptin - A Sri Lankan study.

Context: Polycystic ovary syndrome (PCOS) is the commonest endocrine disorder affecting young women. Kisspeptins are a family of closely related peptides encoded by Kiss1 gene that controls the hypothalamic-pituitary-gonadal axis by binding to its receptor (GPR54) expressed in gonadotropin-releasing hormone (GnRH) neurons and releases GnRH. Since GnRH secretion is deregulated in PCOS, we hypothesized that dysregulated gonadotropin secretion in PCOS is reflected by kisspeptin levels. Aim: We aimed to measure serum kisspeptin levels of subjects with well-characterized PCOS versus controls and explore any correlation between kisspeptin and PCOS-related reproductive and metabolic disturbances. Materials and Methods: : Consecutive women with PCOS manifesting from adolescence (n = 55) and adult controls (n = 110) were recruited. Pre-treatment baseline clinical, anthropometry, and biochemical parameters were measured in all. Serum kisspeptin and testosterone levels were determined by enzyme-linked immunosorbent assay method. Results: : Serum kisspeptin and testosterone concentrations were significantly higher in women with PCOS (kisspeptin 4.873 nmol/L; testosterone 4.713 nmol/L) than controls (kisspeptin 4.127 nmol/L; testosterone 3.415 nmol/L; P < 0.05). Serum kisspeptin levels were positively associated with PCOS (odds ratio: 1.853; 95% confidence interval: 1.246-2.755; P = 0.002) in our studied population. Conclusion: Serum kisspeptin levels are higher in Sri Lankan women with PCOS manifesting from adolescence compared with controls regardless of body mass index. We propose serum kisspeptin concentration as a useful marker to recognize PCOS that manifests from adolescence.

Development of siRNA mediated RNA interference and functional analysis of novel parasitic nematode-specific protein of Setaria digitata.

Despite the differences of the host, parasitic nematodes may share commonalities in their parasitizing genes. Setaria digitata novel protein (SDNP) is such an entity which is parasitic nematode-specific and having sequence similarities with those of W. bancrofti, B. malayi, Loa loa and Onchocerca volvulus. Post-transcriptional gene silencing by siRNA mediated RNA interference (RNAi) is a widely used technique in functional genomics. Though the technique has been used in several free-living, plant and animal parasitic nematodes, it has not yet been tried out for the filarial worm S. digitata. In this study, we developed an effective siRNA delivery method by microinjection and utilized the siRNAi tool to knockdown SDNP to study the phenotypic and cellular changes associated with the interference. qPCR analysis revealed, a significant reduction of SDNP transcript levels following siRNA microinjection into S. digitata adult worms. Similarly, immunohistochemical staining indicated a reduction of SDNP protein expression. Furthermore, worms treated with siRNA showed a significant reduction of microfilariae release together with embryonic lethality by arresting an early developmental stage compared to non-treated worms. A distinct motility reduction was also observed in treated worms compared to non-treated counterparts. This is the first report of the amenability of S. digitata to the siRNA induced RNAi. The presence of inter-domain linkers of muscle-specific twitchin kinase and calcium-dependent protein kinase isoform CDPK1 together with what our results revealed suggest that SDNP is most likely a protein involved in muscle movement and growth and development of the nematode. Hence SDNP has the characteristics of a potential drug target.

Unveiling Members of Colletotrichum acutatum Species Complex Causing Colletotrichum Leaf Disease of Hevea brasiliensis in Sri Lanka.

Colletotrichum is an important fungal genus with great diversity, which causes anthracnose of a variety of crop plants including rubber trees. Colletotrichum acutatum and Colletotrichum gloeosporioides have been identified as the major causative agents of Colletotrichum leaf disease of rubber trees in Sri Lanka based on morphology, pathogenicity, and the analysis of internally transcribed spacer sequences of the nuclear ribosomal DNA. This study has been conducted to investigate the members of the C. acutatum species complex causing rubber leaf disease using a morphological and multi gene approach. For the first time in Sri Lanka, Colletotrichum simmondsii, Colletotrichum laticiphilum, Colletotrichum nymphaeae, and Colletotrichum citri have been identified as causative agents of Colletotrichum leaf disease in addition to C. acutatum s. str. Among them, C. simmondsii has been recognized as the major causative agent.

Identification of cattle, buffaloes and rodents as reservoir animals of Leptospira in the District of Gampaha, Sri Lanka.

BACKGROUND: Leptospirosis is an important emerging infectious disease in Sri Lanka. Rats are the most important reservoir of Leptospira but domestic and wild mammals may also act as important maintenance or accidental hosts. In Sri Lanka, knowledge of reservoir animals of leptospires is poor. The objective of this study was to identify potential reservoir animals of Leptospira in the District of Gampaha, Sri Lanka. FINDINGS: Blood and kidney samples were collected from 38 rodents and mid-stream urine samples were randomly collected from 45 cattle and five buffaloes in the District of Gampaha. Kidney and urine samples were tested by real-time polymerase chain reaction (PCR) and serum samples were tested by the microscopic agglutination test (MAT). Of the 38 rodent kidney samples, 11% (4/38) were positive by real-time PCR. The prevalence of leptospiral carriage was 11% (3/26) and 8% (1/12) in female and male rodents, respectively. Three rodent serum samples were positive by MAT. Of the 50 cattle/buffalo urine samples tested, 10% (5/50) were positive by real-time PCR. The prevalence of leptospiral carriage was 9% (4/45) and 20% (1/5) in cattle and buffaloes, respectively. CONCLUSION: Results of PCR and MAT showed that Leptospira were present in a significant proportion of the rodents and farm animals tested in this study and suggest that these (semi-) domestic animals form an infection reservoir for Leptospira. Therefore, there is a potential zoonotic risk to public health, most notably to farmers in this area.

Application of a real time Polymerase Chain Reaction (PCR) assay for the early diagnosis of human leptospirosis in Sri Lanka.

Leptospirosis has a major impact on health in Sri Lanka but is probably grossly under-recognized due to difficulties in clinical diagnosis and lack of diagnostic laboratory services. The objective of this study was to establish and evaluate a SYBR Green-based real-time Polymerase Chain Reaction (rt-PCR) assay for early, rapid and definitive laboratory diagnosis of leptospirosis in Sri Lanka. The rt-PCR assay was established and analytical specificity and sensitivity were determined using reference DNA samples. Evaluation of the assay for diagnosis of clinical samples was performed using two panels of serum samples obtained from 111 clinically suspected adult patients. Patients were confirmed as leptospirosis (n = 65) and non-leptospirosis (n = 30) by the Patoc - MAT. Other 16 samples gave ambiguous results. The analytical sensitivity of the rt-PCR was approximately 60 genome copies and no cross-reactivity was observed with saprophytic Leptospira spp. and other pathogenic microorganisms. Based on confirmation with Patoc-MAT on paired samples this corresponds to a diagnostic sensitivity and specificity of 67.7% (44/65) and 90.0% (27/30), respectively. This study showed that rt-PCR has the potential to facilitate rapid and definitive diagnosis of leptospirosis during early phase of infection in Sri Lanka.

Patterns of drug resistance and RFLP analysis of Mycobacterium tuberculosis strains isolated from recurrent tuberculosis patients in Sri Lanka.

The aim of the study was to determine drug sensitivity and DNA fingerprints of Mycobacterium tuberculosis strains from retreatment cases of pulmonary tuberculosis. The study population consisted of 131 culture positive, retreatment tuberculosis patients admitted to the Chest Hospital, Welisara, Sri Lanka who had taken anti-tuberculosis drugs previously. Forty-eight percent of the isolates were susceptible to all 12 drugs tested. Twenty isolates were resistant to first line drugs, 28 to both first and second line drugs and 17 to second line drugs. Forty-six percent were resistant to a single drug, 23% to two and 19% to 3 drugs, respectively. Resistance to p-aminosalicylic acid (15%) was most common followed by ethambutol (14%), isoniazid and pyrazinamide (12%). Multi-drug resistance was present in four isolates. Using RFLP analysis the copy number and IS 6110 element in M. tuberculosis strains varied from one to seven, the majority having 3 to 5 copies. The prevalence of acquired drug resistance to individual drugs was comparatively lower except resistance to ethambutol. The majority of retreatment patients belonged to the defaulter category and this stresses the importance of implementing directly observed treatment short course and susceptibility testing of isolates in retreatment TB patients to prevent the spread of drug resistance. By using the IS 6110 genetic marker it was possible to differentiate most of the M. tuberculosis isolates. However, for an unambiguous confirmation of the identities of strains, additional genetic markers should be employed in strain typing such as spoligotyping.

The cyclooxygenases.

Cyclooxygenases (COXs) catalyze the rate-limiting step in the production of prostaglandins, bioactive compounds involved in processes such as fever and sensitivity to pain, and are the target of aspirin-like drugs. COX genes have been cloned from coral, tunicates and vertebrates, and in all the phyla where they are found, there are two genes encoding two COX isoenzymes; it is unclear whether these genes arose from an early single duplication event or from multiple independent duplications in evolution. The intron-exon arrangement of COX genes is completely conserved in vertebrates and mostly conserved in all species. Exon boundaries largely define the four functional domains of the encoded protein: the amino-terminal hydrophobic signal peptide, the dimerization domain, the membrane-binding domain, and the catalytic domain. The catalytic domain of each enzyme contains distinct peroxidase and cyclooxygenase active sites; COXs are classified as members of the myeloperoxidase family. All COXs are homodimers and monotopic membrane proteins (inserted into only one leaflet of the membrane), and they appear to be targeted to the lumenal membrane of the endoplasmic reticulum, where they are N-glycosylated. In mammals, the two COX genes encode a constitutive isoenzyme (COX-1) and an inducible isoenzyme (COX-2); both are of significant pharmacological importance.

COX-3, a cyclooxygenase-1 variant inhibited by acetaminophen and other analgesic/antipyretic drugs: cloning, structure, and expression.

Two cyclooxygenase isozymes, COX-1 and -2, are known to catalyze the rate-limiting step of prostaglandin synthesis and are the targets of nonsteroidal antiinflammatory drugs. Here we describe a third distinct COX isozyme, COX-3, as well as two smaller COX-1-derived proteins (partial COX-1 or PCOX-1 proteins). COX-3 and one of the PCOX-1 proteins (PCOX-1a) are made from the COX-1 gene but retain intron 1 in their mRNAs. PCOX-1 proteins additionally contain an in-frame deletion of exons 5-8 of the COX-1 mRNA. COX-3 and PCOX mRNAs are expressed in canine cerebral cortex and in lesser amounts in other tissues analyzed. In human, COX-3 mRNA is expressed as an approximately 5.2-kb transcript and is most abundant in cerebral cortex and heart. Intron 1 is conserved in length and in sequence in mammalian COX-1 genes. This intron contains an ORF that introduces an insertion of 30-34 aa, depending on the mammalian species, into the hydrophobic signal peptide that directs COX-1 into the lumen of the endoplasmic reticulum and nuclear envelope. COX-3 and PCOX-1a are expressed efficiently in insect cells as membrane-bound proteins. The signal peptide is not cleaved from either protein and both proteins are glycosylated. COX-3, but not PCOX-1a, possesses glycosylation-dependent cyclooxygenase activity. Comparison of canine COX-3 activity with murine COX-1 and -2 demonstrates that this enzyme is selectively inhibited by analgesic/antipyretic drugs such as acetaminophen, phenacetin, antipyrine, and dipyrone, and is potently inhibited by some nonsteroidal antiinflammatory drugs. Thus, inhibition of COX-3 could represent a primary central mechanism by which these drugs decrease pain and possibly fever.

Trans-spliced leader RNA, 5S-rRNA genes and novel variant orphan spliced-leader of the lymphatic filarial nematode Wuchereria bancrofti, and a sensitive polymerase chain reaction based detection assay.

A genomic library of Wuchereria bancrofti was examined for the presence of the 22 nucleotide spliced leader (SL) which plays a vital role in the maturation of the 5' end of certain mRNAs through the addition of a small spliced leader (SL) exon and also in the generation of monocistronic mRNA from initial polycistronic transcripts in nematodes. Here, we report the characterization of three SL RNA genes (SLG1, SLG2 and SLG3), an internal copy of a novel variant SL1 sequence (SL1v) with 23 nucleotides within an open reading frame of 75 amino acid residues of an unknown gene and two 5S-rRNA genes (5SR2 and 5SR3) from two genomic clones (TZP/11, TZP/91) of W. bancrofti. Our results revealed that the genes for the spliced leader RNA of W. bancrofti (SL RNA) is reiterated within the 5S-rRNA gene cluster and are in the same orientation. The genes SLG1, SLG2 and SLG3 were identical in nucleotide sequence except for an additional nucleotide at position 43 on SLG2. Sequence analysis of the three genes indicated that the 22-nt sequence is invariably adjacent to the dinucleotide GT, characteristic of a potential spliced donor site. The Sm-binding sequence AATTTTGG was conserved in SLG1, SLG2 and SLG3. Further, both 5' and 3' flanking regions of genes SLG1, SLG2 and SLG3 shared considerable sequence similarity. Two 5S-rRNA genes characterized from the genomic clone TZP 11 were shown to have sequence heterogeneity. Genomic southern showed that the spliced leader sequence is multicopy within the W. bancrofti genome and is also encoded in the region of DNA unlinked to the 5S rRNA gene cluster. Primers designed to amplify intergenic regions between 5S-rRNA and SL RNA genes in a PCR assay were found to be specific for W. bancrofti and was sensitive enough to detect 1 pg of W. bancrofti DNA or 1/8th of a microfilariae in infected blood samples. The high specificity and sensitivity of the optimised PCR assay makes it an ideal diagnostic tool for the identification of W. bancrofti in both the host and the vector.

A polymerase chain reaction based method for the detection of Culex quinquefasciatus (Diptera: culicidae).

Culex quinquefasciatus Say is the major vector of the filarial parasite Wuchereria bancrofti (Cobbold) which causes lymphatic filariasis in humans. A repetitive DNA sequence from the genome of C. quinquefasciatus has been cloned and completely sequenced. The 693 bp cloned fragment had an A+T content of 72%. Dot matrix analysis of the fragment did not reveal any direct or inverted repeats within it. Southern blot analysis using a variety of restriction enzymes appeared to indicate that the cloned fragment was interspersed within the genome with a copy number of approximately 30,000. A search of the GenBank database did not reveal significant homologies to any previously cloned sequences. Although the probe was sensitive enough to detect picogram quantities of DNA, it was not specific for C. quinquefasciatus, as it hybridized with DNA from other mosquito species, Culex pseudovishnui Colless, Culex gelidus Theobald, Culex tritaeniorhynchus Giles, Anopheles vagus Dönitz and Mansonia uniformis (Theobald). However PCR primers derived from the cloned sequence, IpC, were found to be specific and amplified only C. quinquefasciatus DNA. The optimized PCR assay was found to be very sensitive and was capable of detecting DNA from all stages of C. quinquefasciatus thus making it an ideal diagnostic tool.

Molecular characterisation of the actin gene of the filarial parasite Wuchereria bancrofti.

Wuchereria bancrofti is the major cause of lymphatic filariasis in humans. Although it is responsible for this immensely morbid and debilitating disease, very little is known of the basic molecular biology of this parasite, and there is a vast lack of knowledge on its gene organisation. In this study, the actin gene of W. bancrofti has been characterised by sequencing a clone isolated from a genomic DNA library of this parasite. The 5' flanking region had a potential TATA box and a putative mRNA initiation site. The gene had five exons encoding 376 amino acids, and four introns ranging in size from 109 to 190bp. The 3' flanking region had a potential polyadenylation signal with the sequence ATTAAA which is a common natural variant of the conventional sequence AATAAA. The gene was AT-rich, with a GC content of 37.2%. Southern blot analysis of W. bancrofti genomic DNA indicated that the gene is possibly found as a single copy. The actin amino acid sequence of W. bancrofti showed a high degree of homology to the actin of many organisms of different taxonomic groups, but the highest homology was observed with the free-living nematode Plectus acuminatus. This suggests that P. acuminatus may bear a close evolutionary relationship to W. bancrofti.

Effects of the restriction of food and water intake on the distribution and retention of radioiodine in mice.

The effects of the restriction of food and water intakes on gastrointestinal absorption, distribution to organs and excretion of 131I were investigated in C3H/He mice. The animals were divided into four groups and administered orally 37 kBq carrier-free Na 131I in 0.25 ml normal saline. One group of animals was given food and water ad libitum throughout the experimental period. Food and water to the remaining groups were restricted before and/or after the administration of 131I. The animals in each group were sacrificed 4 h and 24 h after administration, and the activity of 131I in thyroid, blood, liver, kidney, gastrointestinal tract, urine, feces, and carcass was measured. There was a significant increase in the retention of 131I in the thyroid and the concentration of 131I in the blood due to the restriction of food and water after the administration of 131I. In contrast, a significant decrease in the urinary excretion was observed in these animals. In those animals, which fasted before administration only, the retention of 131I in the thyroid and other organs were decreased. Therefore, for an accurate diagnosis and effective therapy with radioiodine as well as effective radiation protection, the intake of food and water should be taken into account.

Molecular characterisation of a hsp70 gene from the filarial parasite Setaria digitata.

The filarial parasite Setaria digitata is the causative agent of cerebrospinal nematodiasis in its abnormal hosts such as sheep, goats and horses, and therefore is of significant veterinary importance. Since very little is currently known about the biology of this parasite at molecular level, we have cloned and characterised a hsp70 gene, the first gene to be reported from this parasite. The genomic clone isolated contained sequences from two hsp70 genes. One gene, hsp70-2, was completely sequenced and found to contain nine introns ranging in size from 78 to 195 bp. The region upstream of the initiation codon contained a putative TATA box, two CAAT box elements and three heat-shock elements. A putative transcription initiation site was also identified. The 5' untranslated region contained a splice acceptor sequence. The gene was typically AT rich, having a GC content of 44.5%. The deduced aa sequence potentially encoded a cytosolic protein of 645 aa, which had three consecutive repeats of a tetrapeptide motif, GGMP, at the carboxyl end. The gene appeared to be constitutively transcribed and was not significantly enhanced in response to heat shock in adult worms. Another hsp70 gene (hsp70-1) was located further upstream, arranged in direct tandem with hsp70-2. Southern blot analysis revealed the presence of two or three additional hsp70-related genes in the S. digitata genome.

A sensitive polymerase chain reaction based assay for the detection of Setaria digitata: the causative organism of cerebrospinal nematodiasis in goats, sheep and horses.

A sensitive PCR assay for the detection of Setaria digitata has been developed. Two oligonucleotide primers (17 nt) were designed from a previously cloned and characterized tandemly arranged repetitive sequence of Setaria digitata. Using these primers, it was possible to amplify small quantities (100 fg) of S. digitata genomic DNA. A simple procedure, using proteinase K and non-ionic detergent NP 40, was followed to process the host blood samples and mosquitoes harbouring L3 larvae. The sensitivity of the polymerase chain reaction based assay surpasses the microscopic detection and the previously reported oligonucleotide based chemiluminescent detection of microfilariae in infected host blood samples and L3 larvae in mosquitoes.

A simple and rapid non-radioactive oligonucleotide based hybridization assay for the detection of Wuchereria bancrofti.

Five biotin labeled oligonucleotides was designed based on a previously cloned and characterized repetitive DNA sequence specific for Wuchereria bancrofti. The oligonucleotide mix (containing five probes) when used in a hybridization assay, detected as little as 100 pg of purified W. bancrofti, microfilarial DNA, a single infective stage larva and a single microfilaria in 50 microl blood sample. A simple protocol was followed for the hybridization assay. Blood samples lysed with sterile distilled water and digested with proteinase K in the presence of a detergent were directly applied on to nylon membranes for dot blot assays. The DNA extract of mosquitos carrying infective stage larvae was eluted through sephadex G-50 minicolumns prior to blotting. The assay was also able to detect DNA extracted from microfilariae infected samples stored over five days at room temperature (28 degrees C). This simple and rapid oligonucleotide hybridization protocol with the highly sensitive chemiluminescent-based detection has significant potential for the development of a field kit to detect W. bancrofti infection.