OSTRFPD: multifunctional tool for genome-wide short tandem repeat analysis for DNA, transcripts, and amino acid sequences with integrated primer designer

Microsatellite mining is a common outcome of the in silico approach to genomic studies. The resulting short tandemly repeated DNA could be used as molecular markers for studying polymorphism, genotyping and forensics. The omni short tandem repeat finder and primer designer (OSTRFPD) is among the few versatile, platform-independent open-source tools written in Python that enables researchers to identify and analyse genome-wide short tandem repeats in both nucleic acids and protein sequences. OSTRFPD is designed to run either in a user-friendly fully featured graphical interface or in a command line interface mode for advanced users. OSTRFPD can detect both perfect and imperfect repeats of low complexity with customisable scores. Moreover, the software has built-in architecture to simultaneously filter selection of flanking regions in DNA and generate microsatellite-targeted primers implementing the Primer3 platform. The software has built-in motif-sequence generator engines and an additional option to use the dictionary mode for custom motif searches. The software generates search results including general statistics containing motif categorisation, repeat frequencies, densities, coverage, guanine–cytosine (GC) content, and simple text-based imperfect alignment visualisation. Thus, OSTRFPD presents users with a quick single-step solution package to assist development of microsatellite markers and categorise tandemly repeated amino acids in proteome databases. Practical implementation of OSTRFPD was demonstrated using publicly available whole-genome sequences of selected Plasmodium species. OSTRFPD is freely available and open-sourced for improvement and user-specific adaptation

Genome-wide microsatellite characteristics of five human plasmodium species, focusing on plasmodium malariae and P. ovale curtisi

Microsatellites can be utilized to explore genotypes, population structure, and other genomic features of eukaryotes. Systematic characterization of microsatellites has not been a focus for several species of Plasmodium, including P. malariae and P. ovale, as the majority of malaria elimination programs are focused on P. falciparum and to a lesser extent P. vivax. Here, five human malaria species (P. falciparum, P. vivax, P. malariae, P. ovale curtisi, and P. knowlesi) were investigated with the aim of conducting in-depth categorization of microsatellites for P. malariae and P. ovale curtisi. Investigation of reference genomes for microsatellites with unit motifs of 1–10 base pairs indicates high diversity among the five Plasmodium species. Plasmodium malariae, with the largest genome size, displays the second highest microsatellite density (1421 No./Mbp; 5% coverage) next to P. falciparum (3634 No./Mbp; 12% coverage). The lowest microsatellite density was observed in P. vivax (773 No./Mbp; 2% coverage). A, AT, and AAT are the most commonly repeated motifs in the Plasmodium species. For P. malariae and P. ovale curtisi, microsatellite-related sequences are observed in approximately 18–29% of coding sequences (CDS). Lysine, asparagine, and glutamic acids are most frequently coded by microsatellite-related CDS. The majority of these CDS could be related to the gene ontology terms “cell parts,” “binding,” “developmental processes,” and “metabolic processes.” The present study provides a comprehensive overview of microsatellite distribution and can assist in the planning and development of potentially useful genetic tools for further investigation of P. malariae and P. ovale curtisi epidemiology

Polymorphic markers for identification of parasite population in Plasmodium malariae

Background Molecular genotyping in Plasmodium serves many aims including providing tools for studying parasite population genetics and distinguishing recrudescence from reinfection. Microsatellite typing, insertion-deletion (INDEL) and single nucleotide polymorphisms is used for genotyping, but only limited information is available for Plasmodium malariae, an important human malaria species. This study aimed to provide a set of genetic markers to facilitate the study of P. malariae population genetics. Methods Markers for microsatellite genotyping and pmmsp1 gene polymorphisms were developed and validated in symptomatic P. malariae field isolates from Myanmar (N = 37). Fragment analysis was used to determine allele sizes at each locus to calculate multiplicity of infections (MOI), linkage disequilibrium, heterozygosity and construct dendrograms. Nucleotide diversity (π), number of haplotypes, and genetic diversity (Hd) were assessed and a phylogenetic tree was constructed. Genome-wide microsatellite maps with annotated regions of newly identified markers were constructed. Results Six microsatellite markers were developed and tested in 37 P. malariae isolates which showed sufficient heterozygosity (0.530–0.922), and absence of linkage disequilibrium (I AS =0.03, p value > 0.05) (N = 37). In addition, a tandem repeat (VNTR)-based pmmsp1 INDEL polymorphisms marker was developed and assessed in 27 P. malariae isolates showing a nucleotide diversity of 0.0976, haplotype gene diversity of 0.698 and identified 14 unique variants. The size of VNTR consensus repeat unit adopted as allele was 27 base pairs. The markers Pm12_426 and pmmsp1 showed greatest diversity with heterozygosity scores of 0.920 and 0.835, respectively. Using six microsatellites markers, the likelihood that any two parasite strains would have the same microsatellite genotypes was 8.46 × 10−4 and was further reduced to 1.66 × 10−4 when pmmsp1 polymorphisms were included. Conclusions Six novel microsatellites genotyping markers and a set of pmmsp1 VNTR-based INDEL polymorphisms markers for P. malariae were developed and validated. Each marker could be independently or in combination employed to access genotyping of the parasite. The newly developed markers may serve as a useful tool for investigating parasite diversity, population genetics, molecular epidemiology and for distinguishing recrudescence from reinfection in drug efficacy studies

Utility of Plasmodium falciparum DNA from rapid diagnostic test kits for molecular analysis and whole genome amplification

Background Rapid diagnostic tests (RDTs) have become the most common diagnostic tool for detection of Plasmodium falciparum malaria, in particular in remote areas. RDT blood spots provide a source of parasite DNA for molecular analysis. In this study, the utility of RDTs for molecular analysis and the performance of different methods for whole genome amplification were investigated. Methods Positive P. falciparum RDTs were collected from Kayin, Myanmar from August 2014 to January 2016. The RDT samples were stored for 6 months, 9 months, 20 months, 21 months, and 32 months before DNA extraction and subsequent molecular analysis of P. falciparum kelch 13 (pfkelch13) mutations, P. falciparum multidrug resistance 1 (pfmdr1), and P. falciparum plasmepsin 2 (pfplasmepsin2) gene amplification. In addition, performance of four whole genome amplification (WGA) kits were compared, including REPLI-g®, MALBACTM, PicoPLEX®, and GenomePlex®, for which DNA quantity and quality were compared between original DNA and post-WGA products. Results The proportion of successful amplification of the different molecular markers was similar between blood spots analysed from RDTs stored for 6, 9, 20, 21, or 32 months. Successful amplification was dependent on the molecular markers fragment length (p value  Conclusions Blood spots from RDTs are a good source for molecular analysis of P. falciparum, even after storage up to 32 months. WGA of RDT-derived parasite DNA reliably increase DNA quantity with sufficient quality for molecular analysis of resistance markers.</p

Molecular characterization of Plasmodium falciparum antifolate resistance markers in Thailand between 2008 and 2016

Background Resistance to anti-malarials is a major threat to the control and elimination of malaria. Sulfadoxine–pyrimethamine (SP) anti-malarial treatment was used as a national policy for treatment of uncomplicated falciparum malaria in Thailand from 1973 to 1990. In order to determine whether withdrawal of this antifolate drug has led to restoration of SP sensitivity, the prevalence of genetic markers of SP resistance was assessed in historical Thai samples. Methods Plasmodium falciparum DNA was collected from the Thailand–Myanmar, Thailand–Malaysia and Thailand–Cambodia borders during 2008–2016 (N = 233). Semi-nested PCR and nucleotide sequencing were used to assess mutations in Plasmodium falciparum dihydrofolate reductase (pfdhfr), P. falciparum dihydropteroate synthase (pfdhps). Gene amplification of Plasmodium falcipaurm GTP cyclohydrolase-1 (pfgch1) was assessed by quantitative real-time PCR. The association between pfdhfr/pfdhps mutations and pfgch1 copy numbers were evaluated. Results Mutations in pfdhfr/pfdhsp and pfgch1 copy number fluctuated overtime through the study period. Altogether, 14 unique pfdhfr–pdfhps haplotypes collectively containing quadruple to octuple mutations were identified. High variation in pfdhfr–pfdhps haplotypes and a high proportion of pfgch1 multiple copy number (51% (73/146)) were observed on the Thailand–Myanmar border compared to other parts of Thailand. Overall, the prevalence of septuple mutations was observed for pfdhfr–pfdhps haplotypes. In particular, the prevalence of pfdhfr–pfdhps, septuple mutation was observed in the Thailand–Myanmar (50%, 73/146) and Thailand–Cambodia (65%, 26/40) border. In Thailand–Malaysia border, majority of the pfdhfr–pfdhps haplotypes transaction from quadruple (90%, 9/10) to quintuple (65%, 24/37) during 2008–2016. Within the pfdhfr–pfdhps haplotypes, during 2008–2013 the pfdhps A/S436F mutation was observed only in Thailand–Myanmar border (9%, 10/107), while it was not identified later. In general, significant correlation was observed between the prevalence of pfdhfr I164L (ϕ = 0.213, p-value = 0.001) or pfdhps K540E/N (ϕ = 0.399, p-value ≤ 0.001) mutations and pfgch1 gene amplification. Conclusions Despite withdrawal of SP as anti-malarial treatment for 17 years, the border regions of Thailand continue to display high prevalence of antifolate and anti-sulfonamide resistance markers in falciparum malaria. Significant association between pfgch1 amplification and pfdhfr (I164L) or pfdhps (K540E) resistance markers were observed, suggesting a compensatory mutation

Molecular characterization of Plasmodium falciparum antifolate resistance markers in Thailand between 2008 and 2016

Background Resistance to anti-malarials is a major threat to the control and elimination of malaria. Sulfadoxine–pyrimethamine (SP) anti-malarial treatment was used as a national policy for treatment of uncomplicated falciparum malaria in Thailand from 1973 to 1990. In order to determine whether withdrawal of this antifolate drug has led to restoration of SP sensitivity, the prevalence of genetic markers of SP resistance was assessed in historical Thai samples. Methods Plasmodium falciparum DNA was collected from the Thailand–Myanmar, Thailand–Malaysia and Thailand–Cambodia borders during 2008–2016 (N = 233). Semi-nested PCR and nucleotide sequencing were used to assess mutations in Plasmodium falciparum dihydrofolate reductase (pfdhfr), P. falciparum dihydropteroate synthase (pfdhps). Gene amplification of Plasmodium falcipaurm GTP cyclohydrolase-1 (pfgch1) was assessed by quantitative real-time PCR. The association between pfdhfr/pfdhps mutations and pfgch1 copy numbers were evaluated. Results Mutations in pfdhfr/pfdhsp and pfgch1 copy number fluctuated overtime through the study period. Altogether, 14 unique pfdhfr–pdfhps haplotypes collectively containing quadruple to octuple mutations were identified. High variation in pfdhfr–pfdhps haplotypes and a high proportion of pfgch1 multiple copy number (51% (73/146)) were observed on the Thailand–Myanmar border compared to other parts of Thailand. Overall, the prevalence of septuple mutations was observed for pfdhfr–pfdhps haplotypes. In particular, the prevalence of pfdhfr–pfdhps, septuple mutation was observed in the Thailand–Myanmar (50%, 73/146) and Thailand–Cambodia (65%, 26/40) border. In Thailand–Malaysia border, majority of the pfdhfr–pfdhps haplotypes transaction from quadruple (90%, 9/10) to quintuple (65%, 24/37) during 2008–2016. Within the pfdhfr–pfdhps haplotypes, during 2008–2013 the pfdhps A/S436F mutation was observed only in Thailand–Myanmar border (9%, 10/107), while it was not identified later. In general, significant correlation was observed between the prevalence of pfdhfr I164L (ϕ = 0.213, p-value = 0.001) or pfdhps K540E/N (ϕ = 0.399, p-value ≤ 0.001) mutations and pfgch1 gene amplification. Conclusions Despite withdrawal of SP as anti-malarial treatment for 17 years, the border regions of Thailand continue to display high prevalence of antifolate and anti-sulfonamide resistance markers in falciparum malaria. Significant association between pfgch1 amplification and pfdhfr (I164L) or pfdhps (K540E) resistance markers were observed, suggesting a compensatory mutation

Molecular characterization of mercury resistant bacteria inhabiting polluted water bodies of different geographical locations in India

Item does not contain fulltextMercury pollution is a major environmental problem that arises as a result of natural processes as well as from anthropogenic sources. In response to toxic mercury compounds, microbes have developed astonishing array of resistance systems to detoxify them. To address this challenge, this study was aimed in screening bacterial isolates for their tolerance against varied concentrations of phenylmercuric acetate. Mercury transformation by bacteria being sensitive to factors such as available carbon source, etc. that affect mer-mediated transformation, screened mercury tolerant bacteria were also studied for their tolerance to different antimicrobials and carbon sources, followed by identification using biochemical as well as 16S rRNA approach. Following identification, gene encoding organomercurial lyase catalyzing protonolytic cleavage of C-Hg bond of organic mercury was amplified using gene specific primers, cloned in pGEMT((R)) easy vector and sequenced. Microbe-based approach using organomercurial lyase encoded by merB gene being potentially economic, provides foundation to facilitate genetic manipulation of this environmentally important enzyme to remove high concentrations of obstinate mercury using holistic, multifaceted approach for use in bioremediation through generation of transgenics or as catalyst for use in bioreactors

Involvement of Akt/NF-κB pathway in antitumor effects of parthenolide on glioblastoma cells in vitro and in vivo

<p>Abstract</p> <p>Background</p> <p>Glioblastoma is the most common and most aggressive form of malignant glioma and is very difficult to treat. Controlling tumour cell invasion and angiogenesis is essential to improve the prognosis of glioblastoma patients. Since constitutive activation of nuclear factor-κB (NF-κB) is necessary for tumour progression, NF-κB may be an important pharmacological target for this disease. Our study aimed to evaluate the antitumour effects of parthenolide, a NF-κB inhibitor, in two human glioblastoma cell lines (U87MG and U373) and in glioblastoma xenografts. Furthermore, we aimed to investigate the molecular mechanisms underlying these effects.</p> <p>Methods</p> <p>The anti-invasive and anti-angiogenic effects of parthenolide were analysed using in vitro invasion and angiogenesis assays. Parthenolide-induced growth inhibition of glioblastoma cells in vitro was determined using the MTT (methyl thiazolyl tetrazolium) assay. In addition, the effect of parthenolide on orthotropic implantation in vivo was evaluated using an intracerebral human glioblastoma xenograft model.</p> <p>Results</p> <p>We found that parthenolide suppresses proliferation, invasion, and tumour- induced angiogenesis of glioblastoma cells. Molecular studies demonstrated that parthenolide suppresses gene and protein expression of angiogenic factors. Furthermore, parthenolide reduced Akt phosphorylation and activated mitochondrial signalling, suggesting that the antitumour function of parthenolide may be mediated not only by the inhibition of NF-κB but also by the inhibition of Akt signalling and the activation of apoptotic proteins. Parthenolide suppressed neovascularity and tumour growth in glioblastoma xenografts.</p> <p>Conclusion</p> <p>The present study identified parthenolide as a new therapeutic agent for glioblastomas.</p

Distinct phenotypes of human prostate cancer cells associate with different adaptation to hypoxia and pro-inflammatory gene expression

Hypoxia and inflammation are strictly interconnected both concurring to prostate cancer progression. Numerous reports highlight the role of tumor cells in the synthesis of pro-inflammatory molecules and show that hypoxia can modulate a number of these genes contributing substantially to the increase of cancer aggressiveness. However, little is known about the importance of the tumor phenotype in this process. The present study explores how different features, including differentiation and aggressiveness, of prostate tumor cell lines impact on the hypoxic remodeling of pro-inflammatory gene expression and malignancy. We performed our studies on three cell lines with increasing metastatic potential: the well differentiated androgen-dependent LNCaP and the less differentiated and androgen-independent DU145 and PC3. We analyzed the effect that hypoxic treatment has on modulating pro-inflammatory gene expression and evaluated the role HIF isoforms and NF-kB play in sustaining this process. DU145 and PC3 cells evidenced a higher normoxic expression and a more complete hypoxic induction of pro-inflammatory molecules compared to the well differentiated LNCaP cell line. The role of HIF1a and NF-kB, the master regulators of hypoxia and inflammation respectively, in sustaining the hypoxic proinflammatory phenotype was different according to cell type. NF-kB was observed to play a main role in DU145 and PC3 cells in which treatment with the NF-kB inhibitor parthenolide was able to counteract both the hypoxic pro-inflammatory shift and HIF1a activation but not in LNCaP cells. Our data highlight that tumor prostate cell phenotype contributes at a different degree and with different mechanisms to the hypoxic pro-inflammatory gene expression related to tumor progression