PCR-based methods for the detection of L1014 kdr mutation in Anopheles culicifacies sensu lato

<p>Abstract</p> <p>Background</p> <p><it>Anopheles culicifacies s.l</it>., a major malaria vector in India, has developed widespread resistance to DDT and is becoming resistant to pyrethroids–the only insecticide class recommended for the impregnation of bed nets. Knock-down resistance due to a point mutation in the voltage gated sodium channel at L1014 residue (<it>kdr</it>) is a common mechanism of resistance to DDT and pyrethroids. The selection of this resistance may pose a serious threat to the success of the pyrethroid-impregnated bed net programme. This study reports the presence of <it>kdr </it>mutation (L1014F) in a field population of <it>An. culicifacies s.l</it>. and three new PCR-based methods for <it>kdr </it>genotyping.</p> <p>Methods</p> <p>The IIS4-IIS5 linker to IIS6 segments of the para type voltage gated sodium channel gene of DDT and pyrethroid resistant <it>An. culicifacies s.l</it>. population from the Surat district of India was sequenced. This revealed the presence of an A-to-T substitution at position 1014 leading to a leucine-phenylalanine mutation (L1014F) in a few individuals. Three molecular methods viz. Allele Specific PCR (AS-PCR), an Amplification Refractory Mutation System (ARMS) and Primer Introduced Restriction Analysis-PCR (PIRA-PCR) were developed and tested for <it>kdr </it>genotyping. The specificity of the three assays was validated following DNA sequencing of the samples genotyped.</p> <p>Results</p> <p>The genotyping of this <it>An. culicifacies s.l</it>. population by the three PCR based assays provided consistent result and were in agreement with DNA sequencing result. A low frequency of the <it>kdr </it>allele mostly in heterozygous condition was observed in the resistant population. Frequencies of the different genotypes were in Hardy-Weinberg equilibrium.</p> <p>Conclusion</p> <p>The Leu-Phe mutation, which generates the <it>kdr </it>phenotype in many insects, was detected in a pyrethroid and DDT resistant <it>An. culicifacies s.l</it>. population. Three PCR-based methods were developed for <it>kdr </it>genotyping. All the three assays were specific. The ARMS method was refractory to non-specific amplification in non-stringent amplification conditions. The PIRA-PCR assay is able to detect both the codons for the phenylalanine mutation at <it>kdr </it>locus, i.e., TTT and TTC, in a single assay, although the latter codon was not found in the population genotyped.</p

Subcellular distribution of glutathione and cysteine in cyanobacteria

Glutathione plays numerous important functions in eukaryotic and prokaryotic cells. Whereas it can be found in virtually all eukaryotic cells, its production in prokaryotes is restricted to cyanobacteria and proteobacteria and a few strains of gram-positive bacteria. In bacteria, it is involved in the protection against reactive oxygen species (ROS), osmotic shock, acidic conditions, toxic chemicals, and heavy metals. Glutathione synthesis in bacteria takes place in two steps out of cysteine, glutamate, and glycine. Cysteine is the limiting factor for glutathione biosynthesis which can be especially crucial for cyanobacteria, which rely on both the sufficient sulfur supply from the growth media and on the protection of glutathione against ROS that are produced during photosynthesis. In this study, we report a method that allows detection and visualization of the subcellular distribution of glutathione in Synechocystis sp. This method is based on immunogold cytochemistry with glutathione and cysteine antisera and computer-supported transmission electron microscopy. Labeling of glutathione and cysteine was restricted to the cytosol and interthylakoidal spaces. Glutathione and cysteine could not be detected in carboxysomes, cyanophycin granules, cell walls, intrathylakoidal spaces, periplasm, and vacuoles. The accuracy of the glutathione and cysteine labeling is supported by two observations. First, preadsorption of the antiglutathione and anticysteine antisera with glutathione and cysteine, respectively, reduced the density of the gold particles to background levels. Second, labeling of glutathione and cysteine was strongly decreased by 98.5% and 100%, respectively, in Synechocystis sp. cells grown on media without sulfur. This study indicates a strong similarity of the subcellular distribution of glutathione and cysteine in cyanobacteria and plastids of plants and provides a deeper insight into glutathione metabolism in bacteria

Phosphatidylserine is a critical modulator for Akt activation

Association of Akt with phosphatidylserine enhances binding to PIP3, inducing conformational changes in Akt that promote its phosphorylation-mediated activation

Transgenic technologies to induce sterility

The last few years have witnessed a considerable expansion in the number of tools available to perform molecular and genetic studies on the genome of Anopheles mosquitoes, the vectors of human malaria. As a consequence, knowledge of aspects of the biology of mosquitoes, such as immunity, reproduction and behaviour, that are relevant to their ability to transmit disease is rapidly increasing, and could be translated into concrete benefits for malaria control strategies. Amongst the most important scientific advances, the development of transgenic technologies for Anopheles mosquitoes provides a crucial opportunity to improve current vector control measures or design novel ones. In particular, the use of genetic modification of the mosquito genome could provide for a more effective deployment of the sterile insect technique (SIT) against vector populations in the field. Currently, SIT relies on the release of radiation sterilized males, which compete with wild males for mating with wild females. The induction of sterility in males through the genetic manipulation of the mosquito genome, already achieved in a number of other insect species, could eliminate the need for radiation and increase the efficiency of SIT-based strategies. This paper provides an overview of the mechanisms already in use for inducing sterility by transgenesis in Drosophila and other insects, and speculates on possible ways to apply similar approaches to Anopheles mosquitoes

Transgenic technologies to induce sterility

The last few years have witnessed a considerable expansion in the number of tools available to perform molecular and genetic studies on the genome of Anopheles mosquitoes, the vectors of human malaria. As a consequence, knowledge of aspects of the biology of mosquitoes, such as immunity, reproduction and behaviour, that are relevant to their ability to transmit disease is rapidly increasing, and could be translated into concrete benefits for malaria control strategies. Amongst the most important scientific advances, the development of transgenic technologies for Anopheles mosquitoes provides a crucial opportunity to improve current vector control measures or design novel ones. In particular, the use of genetic modification of the mosquito genome could provide for a more effective deployment of the sterile insect technique (SIT) against vector populations in the field. Currently, SIT relies on the release of radiation sterilized males, which compete with wild males for mating with wild females. The induction of sterility in males through the genetic manipulation of the mosquito genome, already achieved in a number of other insect species, could eliminate the need for radiation and increase the efficiency of SIT-based strategies. This paper provides an overview of the mechanisms already in use for inducing sterility by transgenesis in Drosophila and other insects, and speculates on possible ways to apply similar approaches to Anopheles mosquitoes

An Estimate of the Incidence of Prostate Cancer in Africa: A Systematic Review and Meta-Analysis

Prostate cancer (PCa) is rated the second most common cancer and sixth leading cause of cancer deaths among men globally. Reports show that African men suffer disproportionately from PCa compared to men from other parts of the world. It is still quite difficult to accurately describe the burden of PCa in Africa due to poor cancer registration systems.We systematically reviewed the literature on prostate cancer in Africa and provided a continentwide incidence rate of PCa based on available data in the regio

Global, regional, and national incidence, prevalence, and years lived with disability for 310 diseases and injuries, 1990-2015:a systematic analysis for the Global Burden of Disease Study 2015

Background Non-fatal outcomes of disease and injury increasingly detract from the ability of the world's population to live in full health, a trend largely attributable to an epidemiological transition in many countries from causes affecting children, to non-communicable diseases (NCDs) more common in adults. For the Global Burden of Diseases, Injuries, and Risk Factors Study 2015 (GBD 2015), we estimated the incidence, prevalence, and years lived with disability for diseases and injuries at the global, regional, and national scale over the period of 1990 to 2015.Methods We estimated incidence and prevalence by age, sex, cause, year, and geography with a wide range of updated and standardised analytical procedures. Improvements from GBD 2013 included the addition of new data sources, updates to literature reviews for 85 causes, and the identification and inclusion of additional studies published up to November, 2015, to expand the database used for estimation of non-fatal outcomes to 60 900 unique data sources. Prevalence and incidence by cause and sequelae were determined with DisMod-MR 2.1, an improved version of the DisMod-MR Bayesian meta-regression tool first developed for GBD 2010 and GBD 2013. For some causes, we used alternative modelling strategies where the complexity of the disease was not suited to DisMod-MR 2.1 or where incidence and prevalence needed to be determined from other data. For GBD 2015 we created a summary indicator that combines measures of income per capita, educational attainment, and fertility (the Socio-demographic Index [SDI]) and used it to compare observed patterns of health loss to the expected pattern for countries or locations with similar SDI scores.Findings We generated 9.3 billion estimates from the various combinations of prevalence, incidence, and YLDs for causes, sequelae, and impairments by age, sex, geography, and year. In 2015, two causes had acute incidences in excess of 1 billion: upper respiratory infections (17.2 billion, 95% uncertainty interval [UI] 15.4-19.2 billion) and diarrhoeal diseases (2.39 billion, 2.30-2.50 billion). Eight causes of chronic disease and injury each affected more than 10% of the world's population in 2015: permanent caries, tension-type headache, iron-deficiency anaemia, age-related and other hearing loss, migraine, genital herpes, refraction and accommodation disorders, and ascariasis. The impairment that affected the greatest number of people in 2015 was anaemia, with 2.36 billion (2.35-2.37 billion) individuals affected. The second and third leading impairments by number of individuals affected were hearing loss and vision loss, respectively. Between 2005 and 2015, there was little change in the leading causes of years lived with disability (YLDs) on a global basis. NCDs accounted for 18 of the leading 20 causes of age-standardised YLDs on a global scale. Where rates were decreasing, the rate of decrease for YLDs was slower than that of years of life lost (YLLs) for nearly every cause included in our analysis. For low SDI geographies, Group 1 causes typically accounted for 20-30% of total disability, largely attributable to nutritional deficiencies, malaria, neglected tropical diseases, HIV/AIDS, and tuberculosis. Lower back and neck pain was the leading global cause of disability in 2015 in most countries. The leading cause was sense organ disorders in 22 countries in Asia and Africa and one in central Latin America; diabetes in four countries in Oceania; HIV/AIDS in three southern sub-Saharan African countries; collective violence and legal intervention in two north African and Middle Eastern countries; iron-deficiency anaemia in Somalia and Venezuela; depression in Uganda; onchoceriasis in Liberia; and other neglected tropical diseases in the Democratic Republic of the Congo.Interpretation Ageing of the world's population is increasing the number of people living with sequelae of diseases and injuries. Shifts in the epidemiological profile driven by socioeconomic change also contribute to the continued increase in years lived with disability (YLDs) as well as the rate of increase in YLDs. Despite limitations imposed by gaps in data availability and the variable quality of the data available, the standardised and comprehensive approach of the GBD study provides opportunities to examine broad trends, compare those trends between countries or subnational geographies, benchmark against locations at similar stages of development, and gauge the strength or weakness of the estimates available. Copyright (C) The Author(s). Published by Elsevier Ltd.</p

Pan-cancer analysis of whole genomes

Cancer is driven by genetic change, and the advent of massively parallel sequencing has enabled systematic documentation of this variation at the whole-genome scale(1-3). Here we report the integrative analysis of 2,658 whole-cancer genomes and their matching normal tissues across 38 tumour types from the Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium of the International Cancer Genome Consortium (ICGC) and The Cancer Genome Atlas (TCGA). We describe the generation of the PCAWG resource, facilitated by international data sharing using compute clouds. On average, cancer genomes contained 4-5 driver mutations when combining coding and non-coding genomic elements; however, in around 5% of cases no drivers were identified, suggesting that cancer driver discovery is not yet complete. Chromothripsis, in which many clustered structural variants arise in a single catastrophic event, is frequently an early event in tumour evolution; in acral melanoma, for example, these events precede most somatic point mutations and affect several cancer-associated genes simultaneously. Cancers with abnormal telomere maintenance often originate from tissues with low replicative activity and show several mechanisms of preventing telomere attrition to critical levels. Common and rare germline variants affect patterns of somatic mutation, including point mutations, structural variants and somatic retrotransposition. A collection of papers from the PCAWG Consortium describes non-coding mutations that drive cancer beyond those in the TERT promoter(4); identifies new signatures of mutational processes that cause base substitutions, small insertions and deletions and structural variation(5,6); analyses timings and patterns of tumour evolution(7); describes the diverse transcriptional consequences of somatic mutation on splicing, expression levels, fusion genes and promoter activity(8,9); and evaluates a range of more-specialized features of cancer genomes(8,10-18).Peer reviewe

Global, regional, and national levels of maternal mortality, 1990-2015 : a systematic analysis for the Global Burden of Disease Study 2015

Background In transitioning from the Millennium Development Goal to the Sustainable Development Goal era, it is imperative to comprehensively assess progress toward reducing maternal mortality to identify areas of success, remaining challenges, and frame policy discussions. We aimed to quantify maternal mortality throughout the world by underlying cause and age from 1990 to 2015. Methods We estimated maternal mortality at the global, regional, and national levels from 1990 to 2015 for ages 10-54 years by systematically compiling and processing all available data sources from 186 of 195 countries and territories, 11 of which were analysed at the subnational level. We quantified eight underlying causes of maternal death and four timing categories, improving estimation methods since GBD 2013 for adult all-cause mortality, HIV-related maternal mortality, and late maternal death. Secondary analyses then allowed systematic examination of drivers of trends, including the relation between maternal mortality and coverage of specific reproductive health-care services as well as assessment of observed versus expected maternal mortality as a function of Socio-demographic Index (SDI), a summary indicator derived from measures of income per capita, educational attainment, and fertility. Findings Only ten countries achieved MDG 5, but 122 of 195 countries have already met SDG 3.1. Geographical disparities widened between 1990 and 2015 and, in 2015, 24 countries still had a maternal mortality ratio greater than 400. The proportion of all maternal deaths occurring in the bottom two SDI quintiles, where haemorrhage is the dominant cause of maternal death, increased from roughly 68% in 1990 to more than 80% in 2015. The middle SDI quintile improved the most from 1990 to 2015, but also has the most complicated causal profile. Maternal mortality in the highest SDI quintile is mostly due to other direct maternal disorders, indirect maternal disorders, and abortion, ectopic pregnancy, and/or miscarriage. Historical patterns suggest achievement of SDG 3.1 will require 91% coverage of one antenatal care visit, 78% of four antenatal care visits, 81% of in-facility delivery, and 87% of skilled birth attendance. Interpretation Several challenges to improving reproductive health lie ahead in the SDG era. Countries should establish or renew systems for collection and timely dissemination of health data; expand coverage and improve quality of family planning services, including access to contraception and safe abortion to address high adolescent fertility; invest in improving health system capacity, including coverage of routine reproductive health care and of more advanced obstetric care-including EmOC; adapt health systems and data collection systems to monitor and reverse the increase in indirect, other direct, and late maternal deaths, especially in high SDI locations; and examine their own performance with respect to their SDI level, using that information to formulate strategies to improve performance and ensure optimum reproductive health of their population.Peer reviewe