Evaluation of qPCR-based assays for leprosy diagnosis directly in clinical specimens

The increased reliability and efficiency of the quantitative polymerase chain reaction (qPCR) makes it a promising tool for performing large-scale screening for infectious disease among high-risk individuals. To date, no study has evaluated the specificity and sensitivity of different qPCR assays for leprosy diagnosis using a range of clinical samples that could bias molecular results such as difficult-to-diagnose cases. In this study, qPCR assays amplifying different M. leprae gene targets, sodA, 16S rRNA, RLEP and Ag 85B were compared for leprosy differential diagnosis. qPCR assays were performed on frozen skin biopsy samples from a total of 62 patients: 21 untreated multibacillary (MB), 26 untreated paucibacillary (PB) leprosy patients, as well as 10 patients suffering from other dermatological diseases and 5 healthy donors. To develop standardized protocols and to overcome the bias resulted from using chromosome count cutoffs arbitrarily defined for different assays, decision tree classifiers were used to estimate optimum cutoffs and to evaluate the assays. As a result, we found a decreasing sensitivity for Ag 85B (66.1%), 16S rRNA (62.9%), and sodA (59.7%) optimized assay classifiers, but with similar maximum specificity for leprosy diagnosis. Conversely, the RLEP assay showed to be the most sensitive (87.1%). Moreover, RLEP assay was positive for 3 samples of patients originally not diagnosed as having leprosy, but these patients developed leprosy 5-10 years after the collection of the biopsy. In addition, 4 other samples of patients clinically classified as non-leprosy presented detectable chromosome counts in their samples by the RLEP assay suggesting that those patients either had leprosy that was misdiagnosed or a subclinical state of leprosy. Overall, these results are encouraging and suggest that RLEP assay could be useful as a sensitive diagnostic test to detect M. leprae infection before major clinical manifestations.     

Development of a molecular diagnostic system to discriminate Dreissena polymorpha (zebra mussel) and Dreissena bugensis (quagga mussel)

A 3-primer PCR system was developed to discriminate invasive zebra (Dreissena polymorpha) and quagga (Dreissena bugensis) mussel. The system is based on: 1) universal primers that amplifies a region of the nuclear 28s rDNA gene from both species and 2) a species-specific primer complementary to either zebra or quagga mussel. The species-specific primers bind to sequences between the binding sites for the universal primers resulting in the amplification of two products from the target species and one product from the nontarget species. Therefore, nontarget products are positive amplification controls. The 3-primer system accurately discriminated zebra and quagga mussels from seven geographically distinct populations.     

Classifying leukemia types with chromatin conformation data

Background

Although genetic or epigenetic alterations have been shown to affect the three-dimensional organization of genomes, the utility of chromatin conformation in the classification of human disease has never been addressed.

Results

Here, we explore whether chromatin conformation can be used to classify human leukemia. We map the conformation of the HOXA gene cluster in a panel of cell lines with 5C chromosome conformation capture technology, and use the data to train and test a support vector machine classifier named 3D-SP. We show that 3D-SP is able to accurately distinguish leukemias expressing MLL-fusion proteins from those expressing only wild-type MLL, and that it can also classify leukemia subtypes according to MLL fusion partner, based solely on 5C data.

Conclusions

Our study provides the first proof-of-principle demonstration that chromatin conformation contains the information value necessary for classification of leukemia subtypes.     

Chronic exposure to ammonia induces isoform-selective alterations in the intracellular distribution and NMDA receptor-mediated translocation of protein kinase C in cerebellar neurons in culture

Hyperammonemia is responsible for most neurological alterations in patients with hepatic encephalopathy by mechanisms that remain unclear. Hyperammonemia alters phosphorylation of neuronal protein kinase C (PKC) substrates and impairs NMDA receptor-associated signal transduction. The aim of this work was to analyse the effects of hyperammonemia on the amount and intracellular distribution of PKC isoforms and on translocation of each isoform induced by NMDA receptor activation in cerebellar neurons. Chronic hyperammonemia alters differentially the intracellular distribution of PKC isoforms. The amount of all isoforms (except PKC zeta) was reduced (17-50%) in the particulate fraction. The contents of alpha, beta1, and epsilon isoforms decreased similarly in cytosol (65-78%) and membranes (66-83%), whereas gamma, delta, and theta; isoforms increased in cytosol but decreased in membranes, and zeta isoform increased in membranes and decreased in cytosol. Chronic hyperammonemia also affects differentially NMDA-induced translocation of PKC isoforms. NMDA-induced translocation of PKC alpha and beta is prevented by ammonia, whereas PKC gamma, delta, epsilon, or theta; translocation is not affected. Inhibition of phospholipase C did not affect PKC alpha translocation but reduced significantly PKC gamma translocation, indicating that NMDA-induced translocation of PKC alpha is mediated by Ca2+, whereas PKC gamma translocation is mediated by diacylglycerol. Chronic hyperammonemia reduces Ca+2-mediated but not diacylglycerol-mediated translocation of PKC isoforms induced by NMDA.     

Malaria liver stage susceptibility locus identified on mouse chromosome 17 by congenic mapping

Host genetic variants are known to confer resistance to Plasmodium blood stage infection and to control malaria severity both in humans and mice. This work describes the genetic mapping of a locus for resistance to liver stage parasite in the mouse. First, we show that decreased susceptibility to the liver stage of Plasmodium berghei in the BALB/c mouse strain is attributable to intra-hepatic factors and impacts on the initial phase of blood stage infection. We used QTL mapping techniques to identify a locus controlling this susceptibility phenotype (LOD score 4.2) on mouse chromosome 17 (belr1 locus). Furthermore, analysis of congenic mouse strains delimited the belr1 locus boundaries distally to the H2 region. Quantification of parasites in the liver of infected congenic mice strongly suggested that the belr1 locus represents a genetic factor controlling the expansion of P. berghei in the hepatic tissue. The mapping of belr1 locus raises the hypothesis that host gene variation is able to control the progression of Plasmodium liver stage infection and opens the possibility that the human genomic region orthologue to belr1 may contain genes that confer resistance to the human malaria liver stage.     

Impact of osmotic stress on physiological and biochemical characteristics in drought-susceptible and drought-resistant wheat genotypes

In this study, the seedlings of two wheat cultivars were used: drought-resistant Chinese Spring (CS) and drought-susceptible (SQ1). Seedlings were subjected to osmotic stress in order to assess the differences in response to drought stress between resistant and susceptible genotype. The aim of the experiment was to evaluate the changes in physiological and biochemical characteristics and to establish the optimum osmotic stress level in which differences in drought resistance between the genotypes could be revealed. Plants were subjected to osmotic stress by supplementing the root medium with three concentrations of PEG 6000. Seedlings were grown for 21 days in control conditions and then the plants were subjected to osmotic stress for 7 days by supplementing the root medium with three concentrations of PEG 6000 (D1, D2, D3) applied in two steps: during the first 3 days of treatment ?0.50, ?0.75 and ?1.00 and next ?0.75, ?1.25 and ?1.5 MPa, respectively. Measurements of gas exchange parameters, chlorophyll content, height of seedlings, length of root, leaf and root water content, leaf osmotic potential, lipid peroxidation, and contents of soluble carbohydrates and proline were taken. The results highlighted statistically significant differences in most traits for treatment D2 and emphasized that these conditions were optimum for expressing differences in the responses to osmotic stress between SQ1 and CS wheat genotypes. The level of osmotic stress defined in this study as most suitable for differentiating drought resistance of wheat genotypes will be used in further research for genetic characterization of this trait in wheat through QTL analysis of mapping population of doubled haploid lines derived from CS and SQ1.     

Peroxisome proliferator-activated receptor gamma up-regulates the Bcl-2 anti-apoptotic protein in neurons and induces mitochondrial stabilization and protection against oxidative stress and apoptosis

Peroxisome proliferator-activated receptor gamma (PPARgamma) has been proposed as a therapeutic target for neurodegenerative diseases because of its anti-inflammatory action in glial cells. However, PPARgamma agonists preventbeta-amyloid (Abeta)-induced neurodegeneration in hippocampal neurons, and PPARgamma is activated by the nerve growth factor (NGF) survival pathway, suggesting a neuroprotective anti-inflammatory independent action. Here we show that the PPARgamma agonist rosiglitazone (RGZ) protects hippocampal and dorsal root ganglion neurons against Abeta-induced mitochondrial damage and NGF deprivation-induced apoptosis, respectively, and promotes PC12 cell survival. In neurons and in PC12 cells RGZ protective effects are associated with increased expression of the Bcl-2 anti-apoptotic protein. NGF-differentiated PC12 neuronal cells constitutively overexpressing PPARgamma are resistant to Abeta-induced apoptosis and morphological changes and show functionally intact mitochondria and no increase in reactive oxygen species when challenged with up to 50 microM H2O2. Conversely, cells expressing a dominant negative mutant of PPARgamma show increased Abeta-induced apoptosis and disruption of neuronal-like morphology and are highly sensitive to oxidative stress-induced impairment of mitochondrial function. Cells overexpressing PPARgamma present a 4- to 5-fold increase in Bcl-2 protein content, whereas in dominant negative PPARgamma-expressing cells, Bcl-2 is barely detected. Bcl-2 knockdown by small interfering RNA in cells overexpressing PPARgamma results in increased sensitivity to Abeta and oxidative stress, further suggesting that Bcl-2 up-regulation mediates PPARgamma protective effects. PPARgamma prosurvival action is independent of the signal-regulated MAPK or the Akt prosurvival pathways. Altogether, these data suggest that PPARgamma supports survival in neurons in part through a mechanism involving increased expression of Bcl-2.     

Evolutionary patchwork of an insecticidal toxin shared between plant-associated pseudomonads and the insect pathogens Photorhabdus and Xenorhabdus

Background

Root-colonizing fluorescent pseudomonads are known for their excellent abilities to protect plants against soil-borne fungal pathogens. Some of these bacteria produce an insecticidal toxin (Fit) suggesting that they may exploit insect hosts as a secondary niche. However, the ecological relevance of insect toxicity and the mechanisms driving the evolution of toxin production remain puzzling.

Results

Screening a large collection of plant-associated pseudomonads for insecticidal activity and presence of the Fit toxin revealed that Fit is highly indicative of insecticidal activity and predicts that Pseudomonas protegens and P. chlororaphis are exclusive Fit producers. A comparative evolutionary analysis of Fit toxin-producing Pseudomonas including the insect-pathogenic bacteria Photorhabdus and Xenorhadus, which produce the Fit related Mcf toxin, showed that fit genes are part of a dynamic genomic region with substantial presence/absence polymorphism and local variation in GC base composition. The patchy distribution and phylogenetic incongruence of fit genes indicate that the Fit cluster evolved via horizontal transfer, followed by functional integration of vertically transmitted genes, generating a unique Pseudomonas-specific insect toxin cluster.

Conclusions

Our findings suggest that multiple independent evolutionary events led to formation of at least three versions of the Mcf/Fit toxin highlighting the dynamic nature of insect toxin evolution.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1763-2) contains supplementary material, which is available to authorized users.