Evaluation of a simple,rapid and field-adapted diagnostic assay for enterotoxigenic E. coli and Shigella

Understanding the global burden of enterotoxigenic E. coli (ETEC) and Shigella diarrhea as well as estimating the cost effectiveness of vaccines to control these two significant pathogens have been hindered by the lack of a diagnostic test that is rapid, simple, sensitive, and can be applied to the endemic countries. We previously developed a simple and rapid assay, Rapid Loop mediated isothermal amplification based Diagnostic Test (RLDT) for the detection of ETEC and Shigella spp. (Shigella). In this study, the RLDT assay was evaluated in comparison with quantitative PCR (qPCR), culture and conventional PCR for the detection of ETEC and Shigella. This validation was performed using previously collected stool samples from endemic countries, from the travelers to the endemic countries, as well as samples from a controlled human infection model study of ETEC. The performance of RLDT from dried stool spots was also validated. RLDT resulted in excellent sensitivity and specificity compared to qPCR (99% and 99.2% respectively) ranging from 92.3 to 100% for the individual toxin genes of ETEC and 100% for Shigella. Culture was less sensitive compared to RLDT. No significant differences were noted in the performance of RLDT using samples from various sources or stool samples from moderate to severe diarrhea or asymptomatic infections. RLDT performed equally well in detection of ETEC and Shigella from the dried stool samples on filter papers. This study established that RLDT is sufficiently sensitive and specific to be used as a simple and rapid diagnostic assay to detect ETEC and Shigella in endemic countries to determine disease burden of these pathogens in the national and subnational levels. This information will be important to guide public health and policy makers to prioritize resources for accelerating the development and introduction of effective preventative and/or treatment interventions against these enteric infections.     

Inactivation of platelet-derived growth factor receptor by the tumor suppressor PTEN provides a novel mechanism of action of the phosphatase

PTEN, mutated in a variety of human cancers, is a dual specificity protein phosphatase and also possesses D3-phosphoinositide phosphatase activity on phosphatidylinositol 3,4,5-tris-phosphate (PIP(3)), a product of phosphatidylinositol 3-kinase. This PIP(3) phosphatase activity of PTEN contributes to its tumor suppressor function by inhibition of Akt kinase, a direct target of PIP(3). We have recently shown that Akt regulates PDGF-induced DNA synthesis in mesangial cells. In this study, we demonstrate that expression of PTEN in mesangial cells inhibits PDGF-induced Akt activation leading to reduction in PDGF-induced DNA synthesis. As a potential mechanism, we show that PTEN inhibits PDGF-induced protein tyrosine phosphorylation with concomitant dephosphorylation and inactivation of tyrosine phosphorylated and activated PDGF receptor. Recombinant as well as immunopurified PTEN dephosphorylates autophosphorylated PDGF receptor in vitro. Expression of phosphatase deficient mutant of PTEN does not dephosphorylate PDGF-induced tyrosine phosphorylated PDGF receptor. Rather its expression increases tyrosine phosphorylation of PDGF receptor. Furthermore, expression of PTEN attenuated PDGF-induced signal transduction including phosphatidylinositol 3-kinase and Erk1/2 MAPK activities. Our data provide the first evidence that PTEN is physically associated with platelet-derived growth factor (PDGF) receptor and that PDGF causes its dissociation from the receptor. Finally, we show that both the C2 and tail domains of PTEN contribute to binding to the PDGF receptor. These data demonstrate a novel aspect of PTEN function where it acts as an effector for the PDGF receptor function and negatively regulates PDGF receptor activation.     

Shared genetic basis of resistance to Bt toxin Cry1ac in independent strains of pink bollworm

Classical and molecular genetic analyses show that two independently derived resistant strains of pink bollworm, Pectinophora gossypiella (Saunders), share a genetic locus at which three mutant alleles confer resistance to Bacillus thuringiensis (Bt) toxin Cry1Ac. One laboratory-selected resistant strain (AZP-R) was derived from individuals collected in 1997 from 10 Arizona cotton fields, whereas the other (APHIS-98R) was derived from a long-term susceptible laboratory strain. Both strains were previously reported to show traits of "mode 1" resistance, the most common type of lepidopteran resistance to Cry1A toxins. Inheritance of resistance to a diagnostic concentration of Cry1Ac (10 microg per gram of diet) was recessive in both strains. In interstrain complementation tests for allelism, F1 progeny from crosses between the two strains were resistant to the diagnostic concentration of Cry1Ac. These results indicate that a major resistance locus is shared by the two strains. Analysis of DNA from the pink bollworm cadherin gene (BtR) using allele-specific polymerase chain reaction (PCR) tests showed that the previously identified resistance alleles (r1, r2, and r3) occurred in both strains, but their frequencies differed between strains. In conjunction with previous findings, the results reported here suggest that PCR-based detection of the three known cadherin resistance alleles might be useful for monitoring resistance to Cry1Ac-producing Bt cotton in field populations of pink bollworm.     

Role of linkage specific 9-O-acetylated sialoglycoconjugates in activation of the alternate complement pathway in mammalian erythrocytes

Substitution of the -OH group at C-9 of sialic acid by an O-acetyl ester has been suggested to modify various biological phenomena that are regulated by sialic acids. Amongst them, enhancement of erythrocyte lysis by 9-O-acetylated sialic acid determinants through modulation of the alternate pathway of complement has been extensively studied on murine erythrocytes [1]. A variable expression of linkage specific 9-O-acetylated sialoglycoconjugates as defined by the lectinogenic epitope of Achatinin-H namely 9-O-acetylated sialic acid 26Gal NAc was identified on rabbit, guinea pig, hamster, rat, mouse and human erythrocytes. This differential expression of linkage specific 9-O-acetylated sialoglycoconjugates strongly correlated with the susceptibility of mammalian erythrocytes to lysis by the alternate pathway of complement. Additionally, low levels of antibodies directed against O-acetylated sialic acids in these mammalian species suggested that these constitutively present determinants have low immunogenicity. Taken together, our results indicate that complement mediated hemolysis depends not simply upon the extent of surface 9-O-acetylated sialic acids present but more importantly upon the specific linkage.     

Two new predatory mites (Acari: Bdellidae,Phytoseiidae) collected from medicinal plants in West Bengal,India

Two new species of predatory mites, one each of Bdellodes Oudemans (Fam. Bdellidae) and Phytoseius Ribaga (Fam. Phytoseiidae) recorded for the first time from two medicinal plants viz. Ambroma augusta (L.) L.f. (Fam. Sterculiaceae) and Clerodendrum viscosum Vent (Fam. Verbenaceae), respectively, are described in this paper.     

Bamboo stumps as mosquito larval habitats in Darjeeling Himalayas, India: A spatial scale analysis

Bamboo stumps can be a congenial breeding habitat of the mosquitoes. In view of this, a preliminary assessment of the dipteran immatures inhabiting the stumps of bamboo groves in the Darjeeling Himalayas was carried out at a spatial scale. Of the 104 stumps of Dendrocalamus hamiltoni surveyed, 70 were found to host immatures of three dipteran species, the mosquitoes Aedes aegypti and Culex quinquefasciatus and the midges Chironomus sp. in varying densities. Though the stumps varied in diameter, in each stump on average 12. 1 immatures were found. The abundance of the immatures was positively correlated with the diameter of the stumps (r=+0.382; P < 0.001) but negatively with the pH of the water present in the stumps (r=–0.336; P < 0.01). The coefficient of association was found to be +8.4 for the Ae. aegypti and Chironomus immatures, while in the rest of the species pair the association seemed to be independent. Thus it can be concluded that the stumps in the bamboo groves of Darjeeling Himalayas provides a favourable habitat for the mosquito and chironomid immatures.     

N-Acetylcysteine Ameliorates Neurotoxic Effects of Manganese Intoxication in Rats: A Biochemical and Behavioral Study

Neurochemical Research - Clinical and experimental evidences reveal that excess exposure to manganese is neurotoxic and leads to cellular damage. However, the mechanism underlying manganese...     

Laboratory evaluation of the rapid diagnostic tests for the detection of Vibrio cholerae O1 using diarrheal samples

BackgroundCholera, an acute diarrheal disease is a major public health problem in many developing countries. Several rapid diagnostic tests (RDT) are available for the detection of cholera, but their efficacies are not compared in an endemic setting. In this study, we have compared the specificity and sensitivity of three RDT kits for the detection of Vibrio cholerae O1 and compared their efficiency with culture and polymerase chain reaction (PCR) methods.MethodsFive hundred six diarrheal stool samples collected from patients from two different hospitals in Kolkata, India were tested using SD Bioline Cholera, SMART-II Cholera O1 and Crystal-VC RDT kits. All the stool samples were screened for the presence of V. cholerae by direct and enrichment culture methods. Stool DNA-based PCR assay was made to target the cholera toxin (ctxAB) and O1 somatic antigen (rfb) encoding genes. Statistical evaluation of the RDTs has been made using STATA software with stool culture and PCR results as the gold standards. The Bayesian latent class model (LCM) was used to evaluate the diagnostic tests in the absence of the gold standard.ResultsInvolving culture technique as gold standard, the sensitivity and specificity of the cholera RDT kits in the direct testing of stools was highest with SAMRT-II (86.1%) and SD-Cholera (94.4%), respectively. The DNA based PCR assays gave very high sensitivity (98.4%) but the specificity was comparatively low (75.3%). After enrichment, the high sensitivity and specificity was detected with SAMRT-II (78.8%) and SD-Cholera (99.1%), respectively. Considering PCR as the gold standard, the sensitivity and specificity of the RDTs remained between 52.3–58.2% and 92.3–96.8%, respectively. In the LCM, the sensitivity of direct and enrichment testing was high in SAMRT-II (88% and 92%, respectively), but the specificity was high in SD cholera for both the methods (97% and 100%, respectively). The sensitivity/specificity of RDTs and direct culture have also been analyzed considering the age, gender and diarrheal disease severity of the patients.ConclusionOverall, the performance of the RDT kits remained almost similar in terms of specificity and sensitivity. Performance of PCR was superior to the antibody-based RDTs. The RTDs are very useful in identifying cholera cases during outbreak/epidemic situations and for making them as a point-of-care (POC) testing tool needs more improvement.