The baseline distribution of malaria in the initial phase of elimination in Sabang Municipality, Aceh Province, Indonesia

ABSTRACT: BACKGROUND: Sabang Municipality, in Aceh Province, Indonesia, plans to initiate a malaria elimination programme in 2013. A baseline survey of the distribution of malaria in the municipality was conducted to lay the foundations for an evidence-based programme and to assess the island's readiness to begin the elimination process. METHODS: The entire population of the municipality was screened for malaria infection and G6PD deficiency. Specimens collected included blood slides, blots and tubes for selected households. Results and Discussion Samples were collected from 16,229 residents. Microscopic examination of the blood smears revealed 12 malaria infections; 10 with Plasmodium falciparum and 2 with Plasmodium vivax. To confirm the parasite prevalence, polymerase chain reaction (PCR) diagnosis was performed on the entire positive cases by microscopy and randomized 10% of the microscopically negative blood samples. PCR revealed an additional 11 subjects with malaria; one P. falciparum infection from the village of Paya Keunekai, and nine P. vivax infections and one mixed P. falciparum/P. vivax infection from the village of Batee Shok. The overall slide positivity rate was 0.074% (CI 95%: 0.070 - 0.078) and PCR corrected prevalence 0,590% (CI 95%: 0.582 - 0.597). Analysis of 937 blood samples for G6PD deficiency revealed two subjects (0.2%) of deficient G6PD. Analysis of several genes of the parasite, such as Pfdhfr, Pfdhps, Pfmdr1, Pfcrt, Pfmsp1, Pfmsp2, Pvdhfr, Pvdhps, Pvmdr1 and host gene, such as G6PD gene revealed that both P. falciparum and P. vivax carried the mutation associated with chloroquine resistance. CONCLUSION: Malariometric and host genetic analysis indicated that there is a low prevalence of both malaria and G6PD deficiency in the population of Sabang Municipality. Nevertheless, malaria cases were clustered in three rural villages and efforts for malaria elimination in Sabang should be particularly focused on those three villages.     

Exploring Indian Rosewood as a promising biogenic tool for the synthesis of metal nanoparticles with tailor-made morphologies

Stem bark extracts of Indian Rosewood, a traditionally used Indian medicinal plant, were used as highly efficient multifunctional green chemicals/biogenic agents in the rapid synthesis of stable, monometallic Ag and Au nanoparticles and their corresponding bimetallic alloy nanoparticles with interesting shapes and morphological characteristics. We determined that the high efficiency of these extracts is due to the presence of complex multifunctional molecules, such as polyphenolics and hydroxyflavonoids, which are involved in the reduction of Au^III and Ag^I ions to zerovalent metallic nanoparticles and the stabilization of their corresponding nanoparticles.     

High-throughput single nucleotide polymorphism genotyping for breeding applications in rice using the BeadXpress platform

Multiplexed single nucleotide polymorphism (SNP) markers have the potential to increase the speed and cost-effectiveness of genotyping, provided that an optimal SNP density is used for each application. To test the efficiency of multiplexed SNP genotyping for diversity, mapping and breeding applications in rice (Oryza sativa L.), we designed seven GoldenGate VeraCode oligo pool assay (OPA) sets for the Illumina BeadXpress Reader. Validated markers from existing 1536 Illumina SNPs and 44?K Affymetrix SNP chips developed at Cornell University were used to select subsets of informative SNPs for different germplasm groups with even distribution across the genome. A 96-plex OPA was developed for quality control purposes and for assigning a sample into one of the five O. sativa population subgroups. Six 384-plex OPAs were designed for genetic diversity analysis, DNA fingerprinting, and to have evenly-spaced polymorphic markers for quantitative trait locus (QTL) mapping and background selection for crosses between different germplasm pools in rice: Indica/Indica, Indica/Japonica, Japonica/Japonica, Indica/O. rufipogon, and Japonica/O. rufipogon. After testing on a diverse set of rice varieties, two of the SNP sets were re-designed by replacing poor-performing SNPs. Pilot studies were successfully performed for diversity analysis, QTL mapping, marker-assisted backcrossing, and developing specialized genetic stocks, demonstrating that 384-plex SNP genotyping on the BeadXpress platform is a robust and efficient method for marker genotyping in rice.     

Notes on the occurrence of the tropical eel Anguilla bicolor bicolor in Peninsular Malaysia, Malaysia

Previous studies indicated that a tropical freshwater eel Anguilla bicolor bicolor occurs in Africa, India, Sri Lanka, Bangladesh, Myanmar, Indonesia and Australia, but an intensive survey has indicated an extended distribution range for the species into Peninsular Malaysia. Thus, A. b. bicolor is a native subspecies of Malaysia.     

Clinico-Pathological Discrepancies in the Diagnosis of Causes of Maternal Death in Sub-Saharan Africa: Retrospective Analysis

Background

Maternal mortality is a major public-health problem in developing countries. Extreme differences in maternal mortality rates between developed and developing countries indicate that most of these deaths are preventable. Most information on the causes of maternal death in these areas is based on clinical records and verbal autopsies. Clinical diagnostic errors may play a significant role in this problem and might also have major implications for the evaluation of current estimations of causes of maternal death.

Methods and Findings

A retrospective analysis of clinico-pathologic correlation was carried out, using necropsy as the gold standard for diagnosis. All maternal autopsies (n = 139) during the period from October 2002 to December 2004 at the Maputo Central Hospital, Mozambique were included and major diagnostic discrepancies were analyzed (i.e., those involving the cause of death). Major diagnostic errors were detected in 56 (40.3%) maternal deaths. A high rate of false negative diagnoses was observed for infectious diseases, which showed sensitivities under 50%: HIV/AIDS-related conditions (33.3%), pyogenic bronchopneumonia (35.3%), pyogenic meningitis (40.0%), and puerperal septicemia (50.0%). Eclampsia, was the main source of false positive diagnoses, showing a low predictive positive value (42.9%).

Conclusions

Clinico-pathological discrepancies may have a significant impact on maternal mortality in sub-Saharan Africa and question the validity of reports based on clinical data or verbal autopsies. Increasing clinical awareness of the impact of obstetric and nonobstetric infections with their inclusion in the differential diagnosis, together with a thorough evaluation of cases clinically thought to be eclampsia, could have a significant impact on the reduction of maternal mortality.     

From autoinhibition to inhibition in trans: the Raf-1 regulatory domain inhibits Rok-α kinase activity

The activity of Raf-1 and Rok-α kinases is regulated by intramolecular binding of the regulatory region to the kinase domain. Autoinhibition is relieved upon binding to the small guanosine triphosphatases Ras and Rho. Downstream of Ras, Raf-1 promotes migration and tumorigenesis by antagonizing Rok-α, but the underlying mechanism is unknown. In this study, we show that Rok-α inhibition by Raf-1 relies on an intermolecular interaction between the Rok-α kinase domain and the cysteine-rich Raf-1 regulatory domain (Raf-1reg), which is similar to Rok-α''s own autoinhibitory region. Thus, Raf-1 mediates Rok-α inhibition in trans, which is a new concept in kinase regulation. This mechanism is physiologically relevant because Raf-1reg is sufficient to rescue all Rok-α–dependent defects of Raf-1–deficient cells. Downstream of Ras and Rho, the Raf-1–Rok-α interaction represents a novel paradigm of pathway cross talk that contributes to tumorigenesis and cell motility.     

Development of submergence-tolerant rice cultivars: the Sub1 locus and beyond

Background and Aims

Submergence is a recurring problem in the rice-producing rainfed lowlands of south and south-east Asia. Developing rice cultivars with tolerance of submergence and with agronomic and quality traits acceptable to farmers is a feasible approach to address this problem. The objectives of this study were to (a) develop mega varieties with Sub1 introgression that are submergence tolerant, (b) assess the performance of Sub1 in different genetic backgrounds, (c) determine the roles of the Sub1A and Sub1C genes in conferring tolerance, and (d) assess the level of tolerance in F₁ hybrids heterozygous for the Sub1A-1-tolerant allele.

Methods

Tolerant varieties were developed by marker-assisted backcrossing through two or three backcrosses, and their performance was evaluated to determine the effect of Sub1 in different genetic backgrounds. The roles of Sub1A and Sub1C in conferring the tolerant phenotype were further investigated using recombinants identified within the Sub1 gene cluster based on survival and gene expression data.

Key Results

All mega varieties with Sub1 introgression had a significantly higher survival rate than the original parents. An intolerant Sub1C allele combined with the tolerant Sub1A-1 allele did not significantly reduce the level of tolerance, and the Sub1C-1 expression appeared to be independent of the Sub1A allele; however, even when Sub1C-1 expression is completely turned off in the presence of Sub1A-2, plants remained intolerant. Survival rates and Sub1A expression were significantly lower in heterozygotes compared with the homozygous tolerant parent.

Conclusions

Sub1 provided a substantial enhancement in the level of tolerance of all the sensitive mega varieties. Sub1A is confirmed as the primary contributor to tolerance, while Sub1C alleles do not seem important. Lack of dominance of Sub1 suggests that the Sub1A-1 allele should be carried by both parents for developing tolerant rice hybrids.Key words: Oryza sativa, Sub1, marker-assisted backcrossing, mega varieties, submergence tolerance, recombinant, hybrid, abiotic stress     

Isolation of Cronobacter spp. (formerly Enterobacter sakazakii) from infant food, herbs and environmental samples and the subsequent identification and confirmation of the isolates using biochemical, chromogenic assays, PCR and 16S rRNA sequencing

Background  

Cronobacter spp. (formerly Enterobacter sakazakii), are a group of Gram-negative pathogens that have been implicated as causative agents of meningitis and necrotizing enterocolitis in infants. The pathogens are linked to infant formula; however, they have also been isolated from a wide range of foods and environmental samples.     

Bloom DNA Helicase Facilitates Homologous Recombination between Diverged Homologous Sequences

Bloom syndrome caused by inactivation of the Bloom DNA helicase (Blm) is characterized by increases in the level of sister chromatid exchange, homologous recombination (HR) associated with cross-over. It is therefore believed that Blm works as an anti-recombinase. Meanwhile, in Drosophila, DmBlm is required specifically to promote the synthesis-dependent strand anneal (SDSA), a type of HR not associating with cross-over. However, conservation of Blm function in SDSA through higher eukaryotes has been a matter of debate. Here, we demonstrate the function of Blm in SDSA type HR in chicken DT40 B lymphocyte line, where Ig gene conversion diversifies the immunoglobulin V gene through intragenic HR between diverged homologous segments. This reaction is initiated by the activation-induced cytidine deaminase enzyme-mediated uracil formation at the V gene, which in turn converts into abasic site, presumably leading to a single strand gap. Ig gene conversion frequency was drastically reduced in BLM^−/− cells. In addition, BLM^−/− cells used limited donor segments harboring higher identity compared with other segments in Ig gene conversion event, suggesting that Blm can promote HR between diverged sequences. To further understand the role of Blm in HR between diverged homologous sequences, we measured the frequency of gene targeting induced by an I-SceI-endonuclease-mediated double-strand break. BLM^−/− cells showed a severer defect in the gene targeting frequency as the number of heterologous sequences increased at the double-strand break site. Conversely, the overexpression of Blm, even an ATPase-defective mutant, strongly stimulated gene targeting. In summary, Blm promotes HR between diverged sequences through a novel ATPase-independent mechanism.The RecQ helicases, a subfamily of DNA helicases, carry out the unwinding of duplex DNA in the 3′ to 5′ direction. Homologs of RecQ have been identified in a wide range of organisms, from budding yeast to humans (reviewed in Ref. 1). There are five human RecQ family proteins: Blm, Wrn, RecQ1, RecQ4, and RecQ5. The BLM, WRN, and RECQ4 genes are mutated in Bloom syndrome, Werner syndrome, and Rothmund-Thomson syndrome, respectively (1–3). A hallmark of Bloom syndrome cells is the drastic increase in the level of sister chromatid exchange (SCE),⁴ which results from homologous recombination (HR) associated with cross-over of the DNA damage caused during DNA replication (4, 5). It is therefore believed that Blm acts as an anti-recombination factor and inhibits aberrant recombination. This idea is supported by the observation that Sgs1, the yeast ortholog of Blm, facilitates the resolution of aberrant joint molecules during meiotic HR (6, 7) and following replication blockage (8).HR plays a critical role in the maintenance of genome stability by repairing DNA double-strand breaks (DSBs) and releasing replication blockages at damaged template strands (9, 10). The current model for HR-mediated DSB repair is that DSBs are processed to produce a 3′ single-stranded overhang, along which Rad51 is polymerized (11, 12). The resulting Rad51-DNA filament undergoes homology search and strand invasion into intact homologous duplex DNA, leading to the formation of the D-loop structure. DNA synthesis from the invading strand followed by dissociation from the homologous duplex DNA and subsequent re-annealing of the newly synthesized strand with the other end of the DSB completes the repair. This type of HR, referred to as synthesis-dependent strand anneal (SDSA), results in sequence transfer from the intact template sequence (donor) to the damaged DNA (recipient), and accounts for the majority of mitotic HR (11, 13). Extensive strand exchange of the D-loop, on the other hand, leads to the generation of Holliday junction (HJ) intermediates. SDSA does not cause cross-overs, whereas HR involving the Holliday junction often causes cross-overs, such as SCE and meiotic HR. An increase in the level of SCE in Bloom syndrome cells therefore supports the idea that Blm suppresses the formation of HJ as well as recombinogenic DNA lesions. This idea is supported by the biochemical evidence of the Blm-dependent resolution of Holliday junctions (14). On the other hand, in Drosophila, DmBlm is known to facilitate the repair of DSB by promoting SDSA (15, 16). However, the role of Blm in SDSA in the other higher eukaryotic cells has not been defined.BLM^−/− cells established from the chicken DT40 B lymphocyte line exhibit a marked increase in the frequency of both SCE and targeted integration (17–19), as do human Bloom syndrome cells (20, 21). In this study, using the chicken DT40 cells, we investigated the role of Blm in SDSA induced by defined DNA damage. To this end, we evaluated this type of SDSA using two phenotypic assays designed to analyze Ig gene conversion and DSB-induced gene targeting. Ig gene conversion diversifies the Ig variable (V) gene through HR during in vitro passage. This reaction is initiated by activation-induced cytidine deaminase-mediated uracil formation at the functional rearranged V-region (22–24). Uracil is converted to an abasic site, probably leading to a single-strand gap (25). This lesion in the functional rearranged VJ_λ stimulates the nonreciprocal sequence transfer of a single nucleotide to several hundred nucleotides, from an array of “pseudo-V_λ” regions (donor), located upstream from the functional rearranged VJ_λ, to the rearranged V region (recipient) (26–28) (see Fig. 1A). Because donor and recipient segments have an ∼10% sequence divergence, sequential Ig gene conversion events are able to substantially diversify Ig V segments. Ig gene conversion is raised only by SDSA without the formation of a Holliday junction. Hence, phenotypic analysis of Ig gene conversion provides a unique opportunity to selectively examine the role of Blm in activation-induced cytidine deaminase-induced SDSA. Moreover, nucleotide sequence analysis of Ig gene conversion products can evaluate the accuracy of HR. Like Ig gene conversion, DSB-induced gene targeting is mediated only by SDSA. The induction of DSBs by a rare-cutting endonuclease, I-SceI, at the endogenous locus, increases the frequency of gene targeting by 3 orders of magnitudes, and the frequency of gene targeting can be evaluated by measuring the reconstitution of a marker gene (29) (see Fig. 1B).Open in a separate windowFIGURE 1.Schematic diagram of assay systems used in this study. A, principle of the Ig gene conversion assay. The predominantly sIgM-negative DT40 clone contains a frameshift in its rearranged V-J_λ segments, which can be repaired by pseudogene-templated conversion events. The rate of Ig gene conversion can be measured in subclones by flow cytometric analysis of sIgM staining. B, phenotypic assays of Ig gene conversion and DSB-induced gene targeting. Pseudo-V genes and the targeting fragment act as donors for the rearranged V_λ segment and S2neo, respectively.We here show that the loss of Blm drastically reduces the rate of Ig gene conversion without compromising its accuracy or affecting the length of the gene conversion tracts, indicating that Blm plays a role in the promotion of SDSA. This is an unexpected result, because Blm is in fact believed to suppress general HR reactions, particularly recombination between diverged homologous sequences. To understand the function of Blm in SDSA, we analyzed the effect of heterologous sequences near a DSB site on HR-dependent DSB repair. The data demonstrate that Blm can promote SDSA when there is sequence divergence between the damaged recipient DNA and the homologous donor sequence. Thus, Blm has both positive and negative effects on HR, depending upon the type of DNA damage and the step of the HR reaction.