Microgeographically diverse Plasmodium vivax populations at the Thai-Myanmar border.

Malaria transmission along international borders of the Greater Mekong Subregion is a big challenge for regional malaria elimination. At the Thai-Myanmar border, Plasmodium falciparum cases have dropped dramatically; however, increasing P. vivax prevalence and the emerging reports on hidden malaria burden due to asymptomatic infections demand attention. We conducted cross-sectional surveys to detect asymptomatic malaria infections in a small village located at Thai-Myanmar border and genotyped P. vivax infections in order to understand the level of genetic diversity on such a microgeographic scale. PCR/RFLP and DNA sequencing identified high levels of genetic polymorphisms at both Pvmsp3α and Pvmsp3ß loci among P. vivax infections. Combining the PCR/RFLP patterns of Pvmsp3α and Pvmsp3ß, a total of 10 genotypes were observed among 17 samples, while concatenated DNA sequences of Pvmsp3α and 3ß generated 14 haplotypes with haplotype diversity of 0.97. These markedly diverse parasites on a microgeographic scale suggest the circulation of a considerably large parasite population at the international border.

An efficient lactamisation/N-acyliminium Pictet-Spengler domino strategy for the diasteroselective synthesis of polyhydroxylated quinoxalinone, ß-carboline and quinazolinone derivatives.

A novel cascade strategy has been developed for the synthesis of polyhydroxylated tetrahydroindolo[1,2-a]pyrrolo[2,1-c]quinoxaline, tetrahydrodipyrrolo[1,2-a:2',1'-c]quinoxaline, hexahydro-1H-indolizino[8,7-b]indole, hexahydrobenzo[6,7]pyrrolo[1',2':1,2]azepino[3,4-b]indole, tetrahydrobenzo[4,5]imidazo[1,2-c]pyrrolo[1,2-a]quinazoline, and tetrahydropyrrolo[1,2-a]tetrazolo[1,5-c]quinazoline scaffolds. The key step is a lactamisation/Pictet-Spengler condensation of a bifunctional sugar-derived hydroxy-γ-lactone component with an appropriate bifunctional aromatic amine component. This modular approach features the in situ-generation of a cyclic N-acyliminium intermediate that allows the diastereoselective assembly of these diverse polycyclic systems efficiently under mild and operationally simple conditions.

A bioinformatic survey of RNA-binding proteins in Plasmodium.

BACKGROUND: The malaria parasites in the genus Plasmodium have a very complicated life cycle involving an invertebrate vector and a vertebrate host. RNA-binding proteins (RBPs) are critical factors involved in every aspect of the development of these parasites. However, very few RBPs have been functionally characterized to date in the human parasite Plasmodium falciparum. METHODS: Using different bioinformatic methods and tools we searched P. falciparum genome to list and annotate RBPs. A representative 3D models for each of the RBD domain identified in P. falciparum was created using I-TESSAR and SWISS-MODEL. Microarray and RNAseq data analysis pertaining PfRBPs was performed using MeV software. Finally, Cytoscape was used to create protein-protein interaction network for CITH-Dozi and Caf1-CCR4-Not complexes. RESULTS: We report the identification of 189 putative RBP genes belonging to 13 different families in Plasmodium, which comprise 3.5% of all annotated genes. Almost 90% (169/189) of these genes belong to six prominent RBP classes, namely RNA recognition motifs, DEAD/H-box RNA helicases, K homology, Zinc finger, Puf and Alba gene families. Interestingly, almost all of the identified RNA-binding helicases and KH genes have cognate homologs in model species, suggesting their evolutionary conservation. Exploration of the existing P. falciparum blood-stage transcriptomes revealed that most RBPs have peak mRNA expression levels early during the intraerythrocytic development cycle, which taper off in later stages. Nearly 27% of RBPs have elevated expression in gametocytes, while 47 and 24% have elevated mRNA expression in ookinete and asexual stages. Comparative interactome analyses using human and Plasmodium protein-protein interaction datasets suggest extensive conservation of the PfCITH/PfDOZI and PfCaf1-CCR4-NOT complexes. CONCLUSIONS: The Plasmodium parasites possess a large number of putative RBPs belonging to most of RBP families identified so far, suggesting the presence of extensive post-transcriptional regulation in these parasites. Taken together, in silico identification of these putative RBPs provides a foundation for future functional studies aimed at defining a unique network of post-transcriptional regulation in P. falciparum.

Molecular Evolution of PvMSP3α Block II in Plasmodium vivax from Diverse Geographic Origins.

Block II of Plasmodium vivax merozoite surface protein 3α (PvMSP3α) is conserved and has been proposed as a potential candidate for a malaria vaccine. The present study aimed to compare sequence diversity in PvMSP3a block II at a local microgeographic scale in a village as well as from larger geographic regions (countries and worldwide). Blood samples were collected from asymptomatic carriers of P. vivax in a village at the western border of Thailand and PvMSP3α was amplified and sequenced. For population genetic analysis, 237 PvMSP3α block II sequences from eleven P. vivax endemic countries were analyzed. PvMSP3α sequences from 20 village-level samples revealed two length variant types with one type containing a large deletion in block I. In contrast, block II was relatively conserved; especially, some non-synonymous mutations were extensively shared among 11 parasite populations. However, the majority of the low-frequency synonymous variations were population specific. The conserved pattern of nucleotide diversity in block II sequences was probably due to functional/structural constraints, which were further supported by the tests of neutrality. Notably, a small region in block II that encodes a predicted B cell epitope was highly polymorphic and showed signs of balancing selection, signifying that this region might be influenced by the immune selection and may serve as a starting point for designing multi-antigen/stage epitope based vaccines against this parasite.

Mating competitiveness and life-table comparisons between transgenic and Indian wild-type Aedes aegypti L.

BACKGROUND: OX513A is a genetically engineered strain of Aedes aegypti carrying a repressible, dominantly inherited transgene that confers lethality in immature heterozygous progeny. Released male OX513A adults have proven to be effective for the localised suppression of wild Ae. aegypti, highlighting its potential in vector control. Mating and life-table assessments were used to compare OX513A with reared Ae. aegypti strains collected from New Delhi and Aurangabad regions in India. RESULTS: Mating proportions of New Delhi females versus males of OX513A or New Delhi strains were 0.52 and 0.48 respectively, indicating no discrimination by females against either strain, and males of both strains were equally competitive. Developmental time from first instar to adult emergence was significantly longer for OX513A (10.7 ± 0.04 days) than for New Delhi (9.4 ± 0.04 days) and Aurangabad strains (9.1 ± 0.04 days). Differences in mean longevities, female reproductive parameters and population growth parameters between the strains were non-significant. CONCLUSIONS: The laboratory study demonstrates that only minor life-table variations of limited biological relevance exist between OX513A and Indian Ae. aegypti populations, and males had equal potential for mating competitiveness. Thus, results support the OX513A strain as a suitable candidate for continued evaluation towards sustainable management of Ae. aegypti populations in India.

Prioritising public health guidance topics in the National Institute for Health and Care Excellence using the Analytic Hierarchy Process.

OBJECTIVES: The Centre for Public Health (CPH), at the United Kingdom's National Institute for Health and Care Excellence (NICE) is responsible for producing national guidance relating to the promotion of good health and the prevention and treatment of disease. Given the challenges of developing guidance in this area, choosing the most appropriate topics for further study is of fundamental importance. This paper explores the current prioritisation process and describes how the Analytic Hierarchy Process (AHP), a multi criteria decision analysis (MCDA) technique, might be used to do so. STUDY DESIGN: A proposed approach is outlined, which was tested in a proof of concept pilot. This consisted of eight participants with experience of related NICE committees building scores for each topic together in a 'decision conference' setting. METHODS: Criteria were identified and subsequently weighted to indicate the relative importance of each. Participants then collaboratively estimated the performance of each topic on each criterion. RESULTS: Total scores for each topic were calculated, which could be ranked and used as the basis for better informed discussion for prioritising topics to recommend to the Minister for future guidance. Sensitivity analyses of the dataset found it to be robust. CONCLUSIONS: Choosing the right topics for guidance at the earliest possible time is of fundamental importance to public health guidance, and judgement is likely to play an important part in doing so. MCDA techniques offer a potentially useful approach to structuring the problem in a rational and transparent way. NICE should consider carefully whether such an approach might be worth pursuing in the future.

Analysis of population genetic structure of Indian Anopheles culicifacies species A using microsatellite markers.

BACKGROUND: Anopheles culicifacies sensu lato is an important vector of malaria in Southeast Asia contributing to almost 70% of malaria cases in India. It exists as morphologically similar sibling species A, B, C, D and E with varied geographical distribution patterns. Vector control measures have been difficult for this important vector as the sibling species have developed varying levels of resistance to the currently used insecticides. In view of the importance of this vector, we developed and validated a set of microsatellite markers and the same were used to analyze the population genetic structure of five different geographical populations of An. culicifacies A. METHODS: Anopheles culicifacies A samples were collected from different localities across India, and genotyping was performed using eight microsatellite markers on ABI Prism 310 Genetic Analyzer. Several statistical analyses were performed to ascertain the genetic diversity that exists within and between the populations. RESULTS: The markers were found to be moderately polymorphic in the populations. Genetic analysis indicated significant genetic differentiation between the majority of the population pairs analyzed and was not found to be related to the geographical distances between populations. CONCLUSION: This is the first and successful attempt to test the microsatellite markers developed for population genetic analysis of An. culicifacies A. Host feeding and breeding habits of species A suggest that factors other than ecological and geographical barriers were responsible for the genetic differentiation that has been observed between the populations.

Fight against dengue in India: progresses and challenges.

At the end of the last century, India has faced resurgence of many infectious diseases, of which dengue is one of the most important in terms of morbidity and mortality. The National Vector Borne Disease Control Program data show that dengue is established in India and is becoming endemic to many areas (dengue cases have increased steadily from ∼450 to ∼50,000 from 2000 to 2012). Despite extensive efforts being made in developing the effective dengue control measures, the number of dengue cases, their severity, and geographical boundaries are expanding alarmingly and posing dengue as one of the deadly disease. Recently, the increasing burden of dengue in the country has attracted the scientific as well as Indian Government's administrative attention; however, a lot remain to be achieved for managing the disease under threshold level. Like other vector-borne diseases, better management of the dengue needs balanced approach involving various aspects like disease prevention, cure/treatment, and the vector control, simultaneously. We have briefly discussed here the situation of dengue in India and have tried to highlight the worrying facets of dengue control and its implementation in Indian perspective. The review on various aspects of dengue control has revealed an urgent need for permanent surveillance programs, coupled with improvised disease diagnostics, effective anti-dengue treatment measures, and controlling the disease transmission by following an effective implementation of vector control programs.

Culex genome is not just another genome for comparative genomics.

Formal publication of the Culex genome sequence has closed the human disease vector triangle by meeting the Anopheles gambiae and Aedes aegypti genome sequences. Compared to these other mosquitoes, Culex quinquefasciatus possesses many specific hallmark characteristics, and may thus provide different angles for research which ultimately leads to a practical solution for controlling the ever increasing burden of insect-vector-borne diseases around the globe. We argue the special importance of the cosmopolitan species- Culex genome sequence by invoking many interesting questions and the possible of potential of the Culex genome to answer those.

In silico analysis of glutathione S-transferase supergene family revealed hitherto unreported insect specific δ- and ε-GSTs and mammalian specific µ-GSTs in Ixodes scapularis (Acari: Ixodidae).

The availability of whole genome sequence information of Ixodes scapularis (Acari: Ixodidae), an important disease vector of veterinary and public health importance, has opened up new opportunities to explore the vector species at genomic level. Use of acaricides is the mainstay in controlling the disease vector, as effective vaccines are not available for most of the diseases that are transmitted by ticks. The glutathione S-transferase (GST) enzymes are one of the important supergene families that are involved in protecting the organism from oxidative stress and xenobiotics including the acaricides. The analysis of GST supergene family from Ixodes identified all the three broad GST classes, viz. canonical, mitochondrial, and microsomal forms. In total, 35 GST genes belong to five different canonical GST classes, namely Delta (7 genes), Epsilon (5), Mu (14), Omega (3), and Zeta (3 genes) GST classes, and two mitochondrial Kappa class GST genes, and a single microsomal GST gene were found. Interestingly, Delta- and Epsilon-class members, which are thought to be specific to the class Insecta, were also identified in Ixodes. Further, vertebrate/mammalian specific Mu-GSTs (14 genes) were also identified in Ixodes. Analyses of the intron-exon organization revealed higher frequency of phase '0' and phase '2' introns. The comprehensive listing of the GST supergene family members from Ixodes may help in understanding molecular mechanisms of the acaricide resistance in mites and ticks. Cumulatively, these findings may provide an in-depth understanding of the complex evolution of GST supergene family, one of the oldest supergene families that exist in all the domains of life.

In silico characterization and comparative genomic analysis of the Culex quinquefasciatus glutathione S-transferase (GST) supergene family.

The glutathione S-transferases (GSTs) are phase II class of detoxification enzymes that are involved both directly and indirectly in insecticide resistance mechanisms. The Culex quinquefasciatus GST superfamily was analyzed by utilizing the public domain Culex genome sequence. In total, 35 cytosolic (seven classes) and 5 microsomal putatively active GSTs were retrieved, classified, and annotated. The study revealed the presence of three unclassified GSTs. Of 35 cytosolic GSTs, 65% contributed by insect specific Delta-Epsilon classes. Gene cluster analysis revealed that most of the genes of Delta, Epsilon, and Theta classes were organized into gene clusters. The gene organization analysis revealed the dominance of phase "0" introns in the Culex GST family. The studies on intron loss and gain events revealed that the Delta GSTs have experienced a higher number of loss and gains during their evolution. A positive correlation was observed between the phylogenetic relationship of members of the GST superfamily and their corresponding exon-intron organization. In addition, the genes within the gene clusters revealed the monophyletic phylogenetic relationship implying the importance of gene duplication events in the gene families' evolution. Finally, the comparative genomic analysis has shown a complex evolutionary scenario associated with the GST supergene family evolution in insects.

Malaria vector control: from past to future.

Malaria is one of the most common vector-borne diseases widespread in the tropical and subtropical regions. Despite considerable success of malaria control programs in the past, malaria still continues as a major public health problem in several countries. Vector control is an essential part for reducing malaria transmission and became less effective in recent years, due to many technical and administrative reasons, including poor or no adoption of alternative tools. Of the different strategies available for vector control, the most successful are indoor residual spraying and insecticide-treated nets (ITNs), including long-lasting ITNs and materials. Earlier DDT spray has shown spectacular success in decimating disease vectors but resulted in development of insecticide resistance, and to control the resistant mosquitoes, organophosphates, carbamates, and synthetic pyrethroids were introduced in indoor residual spraying with needed success but subsequently resulted in the development of widespread multiple insecticide resistance in vectors. Vector control in many countries still use insecticides in the absence of viable alternatives. Few developments for vector control, using ovitraps, space spray, biological control agents, etc., were encouraging when used in limited scale. Likewise, recent introduction of safer vector control agents, such as insect growth regulators, biocontrol agents, and natural plant products have yet to gain the needed scale of utility for vector control. Bacterial pesticides are promising and are effective in many countries. Environmental management has shown sufficient promise for vector control and disease management but still needs advocacy for inter-sectoral coordination and sometimes are very work-intensive. The more recent genetic manipulation and sterile insect techniques are under development and consideration for use in routine vector control and for these, standardized procedures and methods are available but need thorough understanding of biology, ethical considerations, and sufficiently trained manpower for implementation being technically intensive methods. All the methods mentioned in the review that are being implemented or proposed for implementation needs effective inter-sectoral coordination and community participation. The latest strategy is evolution-proof insecticides that include fungal biopesticides, Wolbachia, and Denso virus that essentially manipulate the life cycle of the mosquitoes were found effective but needs more research. However, for effective vector control, integrated vector management methods, involving use of combination of effective tools, is needed and is also suggested by Global Malaria Control Strategy. This review article raises issues associated with the present-day vector control strategies and state opportunities with a focus on ongoing research and recent advances to enable to sustain the gains achieved so far.

Multiplex PCR assay and phylogenetic analysis of sequences derived from D2 domain of 28S rDNA distinguished members of the Anopheles culicifacies complex into two groups, A/D and B/C/E.

A multiplex PCR assay was developed using the sequences of the D2 region of 28S ribosomal DNA (rDNA) to discriminate the five members of the Anopheles culicifacies complex provisionally designated as species A, B, C, D and E. Two minus strand primers derived from sequence differences in the D2 variable region and a universal plus strand primer derived from the conserved 28S (rDNA) has delimited five members into species A and D (group 1) and species B, C and E (group 2) in a PCR diagnostic assay. The complete 28S rDNA-D2 region sequence of A. culicifacies sibling species is reported for the first time. Inter-specific sequence divergence was greater than the intra-specific divergence. The phylogenetic relationships inferred from maximum likelihood, maximum parsimony and the neighbor joining analysis confirmed the presence of two unambiguous monophyly clades one consisting of species A and D and the other of species B, C and E and that the A. culicifacies sibling species diverged relatively recently in evolutionary terms despite their considerable differences in bionomics.

Regulation of cytokine gene expression in experimental autoimmune encephalomyelitis.

We previously reported that recovery of Lewis rats from experimental autoimmune encephalomyelitis (EAE) is associated with the appearance of suppressor T cells (Ts). These Ts secrete TGF-beta which down-regulates the production of inflammatory cytokines by the effector T cells that mediate this disease. In the present study, we immunized Lewis rats with myelin basic protein (MBP)+CFA, and evaluated purified T cells and MBP-activated spleen cells (SpC) during the paralytic phase (day 12) and after recovery (days 30-33) for TGF-beta and interferon (IFN)-gamma mRNA. We used reverse transcriptase-polymerase chain reaction (RT-PCR), quantitated on the basis of beta-actin mRNA. Abundant IFN-gamma mRNA was present in MBP-activated SpC obtained on day 12. In contrast, only trace IFN-gamma mRNA was detected in day 30 activated SpC, and no IFN-gamma mRNA was present in purified, nonactivated T cells obtained at either time. The level of IFN-gamma mRNA correlated with secretion of IFN-gamma as determined by ELISA on SpC culture supernatants, and with severity of adoptively transferred EAE by the activated SpC. Thus, it appears that IFN-gamma mRNA is both transcribed and translated in response to antigen activation, resulting in secretion of IFN-gamma by the disease-inducing Te. In contrast, when we used RT-PCR to investigate the expression of TGF-beta mRNA, we found the transcript present in isolated T cells and MBP-activated SpC obtained from rats at both days 12 and 30. The presence of TGF-beta mRNA at time points corresponding to both clinical EAE and recovery suggests post-transcriptional regulation of the production of this immunoregulatory cytokine.

Evaluation of astemizole purity by HPLC.

A liquid chromatographic method is described for the evaluation of Astemizole purity. The method is also applicable for the analysis of pharmaceutical dosage forms of Astemizole using indole as internal standard. Separation was achieved with a micro bondapak C(18) column and phosphate buffer (pH 6.0):acetonitrile (50:50 v/v) as eluent, at a flow rate of 1.5 ml/min with UV detection at 280 nm. The method is suitable for estimating the possible impurities if any present in Astemizole up to 0.05% for 1a and 0.25% for 1b. The method is simple, rapid and accurate for the analysis of Astemizole.

Antihepatotoxic activity of eclipta alba, tephrosia purpurea and boerhaavia diffusa.

Alcoholic and chloroform extracts of E. albaT. purpurea and B. diffusa were screened for antihepatotoxic activity. The extracts were given after the liver was damaged with CCl4. Liver function was assessed based on liver to boy weight ratio, pentobarbitone sleep time, serum levels of transaminase (SGPT, SGOT), alkaline phosphatase (SALP) and bilirubin. Alcoholic extract of E. alba was found to have good antihepatotoxic activity.

Left ventricular performance in coronary artery disease evaluated with systolic time intervals and echocardiography.

Simultaneous determinations of systolic time intervals (preejection period index [PEPI], left ventricular ejection time index [LVETI] and ratio of preejection period to left ventricular ejection time [PEP/LVET]) and echographic measures of left ventricular performance (percent change in minor axis diameter [%delta D], circumferential shortening rate [Vcf] and end-diastolic diameter [Dd]) were obtained in 25 normal subjects and 37 patients with previously documented transmural myocardial infarction. The group with previous infarction demonstrated significant (P less than 0.001) differences from the normal group in each of the noninvasive measures. PEP/LVET and %deltaD were the most sensitive measures of left ventricular dysfunction. Deviation from the normal range in these measures occurred, respectively, in 70 and 65 percent of patients without dyspnea or fatigability (20 patients) and 85 percent of those without angina pectoris (13 patients). Abnormalities in systolic time interval and echocardiographic measures were related to the severity of dyspnea and fatigability but not to that of angina. Neither the presence of phonocardiographically documented third or fourth sound gallops nor an abnormal cardiothoracic ratio by chest roentgenogram reliably detected patients with abnormal left ventricular performance. The range of abnormality in left ventricular performance did not differ between patients with prior anterior or diaphragmatic myocardial infarction. The frequency of abnormal performance was greatest among patients with combined sites of prior infarction. Among 26 patients studied by coronary arteriography, abnormal left ventricular performance as determined by values for PEP/LVET and %deltaD occurred in fewer than 30 percent of those with 70 percent or greater obstruction of one coronary artery and in more than 80 percent of those with two or three vessel involvement. There was a high correlation between systolic time intervals, %delta D and Vcf, the closest correlation occurring between PEP/LVET and %deltaD (r = -0.93). These data document the sensitivity of the noninvasive systolic time intervals and echographic measures and their superiority over current clinical bedside methods in evaluating left ventricular performance in patients with prior myocardial infarction.

Left ventricular performance in coronary artery disease by systolic time intervals and echocardiography.

Determinations of systolic time intervals and echocardiographic measures of left ventricular performance were made in twenty-five normal subjects and 37 patients who had convalesced from a previous transmural myocardial infarction. On group analysis the patients with previous myocardial infarction demonstrated significant differences from the normals in each of the noninvasive measures. Among the noninvasive measures the PEP/LVET and deltaD% proved to be the most sensitive indicators of left ventricular dysfunction. Deviations from the normal range in PEP/LVET and deltaD% occurred in 65% to 70% of patients asymptomatic for dyspnea and fatigability (20 patients) and in 85% of patients asymptomatic for angina pectoris (13 patients). Neither the presence of phonocardiographically documented S3 or S4 or the finding of an abnormal C/T ratio on standard chest x-ray reliably detected patients with abnormal left ventricular performance. Among 26 patients studied by coronary arteriography, abnormal left ventricular performance by PEP/LVET and deltaD% occurred in less than 30% of those with obstruction (70% or greater) of one coronary artery and in over 80% of those with obstruction of two or three major arteries. A close correlation existed between the level of left ventricular performance measured by the PEP/LVET and the deltaD% (r = 0.93). These studies document the high degree of sensitivity of the noninvasive measures and demonstrate their superiority over clinical methods for detecting abnormal left ventricular performance in patients with previous myocardial infarction.