PCR reveals significantly higher rates of Trypanosoma cruzi infection than microscopy in the Chagas vector, Triatoma infestans: High rates found in Chuquisaca, Bolivia

<p>Abstract</p> <p>Background</p> <p>The Andean valleys of Bolivia are the only reported location of sylvatic <it>Triatoma infestans</it>, the main vector of Chagas disease in this country, and the high human prevalence of <it>Trypanosoma cruzi </it>infection in this region is hypothesized to result from the ability of vectors to persist in domestic, peri-domestic, and sylvatic environments. Determination of the rate of <it>Trypanosoma </it>infection in its triatomine vectors is an important element in programs directed at reducing human infections. Traditionally, <it>T. cruzi </it>has been detected in insect vectors by direct microscopic examination of extruded feces, or dissection and analysis of the entire bug. Although this technique has proven to be useful, several drawbacks related to its sensitivity especially in the case of small instars and applicability to large numbers of insects and dead specimens have motivated researchers to look for a molecular assay based on the polymerase chain reaction (PCR) as an alternative for parasitic detection of <it>T. cruzi </it>infection in vectors. In the work presented here, we have compared a PCR assay and direct microscopic observation for diagnosis of <it>T. cruzi </it>infection in <it>T. infestans </it>collected in the field from five localities and four habitats in Chuquisaca, Bolivia. The efficacy of the methods was compared across nymphal stages, localities and habitats.</p> <p>Methods</p> <p>We examined 152 nymph and adult <it>T. infestans </it>collected from rural areas in the department of Chuquisaca, Bolivia. For microscopic observation, a few drops of rectal content obtained by abdominal extrusion were diluted with saline solution and compressed between a slide and a cover slip. The presence of motile parasites in 50 microscopic fields was registered using 400× magnification. For the molecular analysis, dissection of the posterior part of the abdomen of each insect followed by DNA extraction and PCR amplification was performed using the TCZ1 (5' – CGA GCT CTT GCC CAC ACG GGT GCT – 3') and TCZ2 (5' – CCT CCA AGC AGC GGA TAG TTC AGG – 3') primers. Amplicons were chromatographed on a 2% agarose gel with a 100 bp size standard, stained with ethidium bromide and viewed with UV fluorescence.</p> <p>For both the microscopy and PCR assays, we calculated sensitivity (number of positives by a method divided by the number of positives by either method) and discrepancy (one method was negative and the other was positive) at the locality, life stage and habitat level. The degree of agreement between PCR and microscopy was determined by calculating Kappa (<it>k</it>) values with 95% confidence intervals.</p> <p>Results</p> <p>We observed a high prevalence of <it>T. cruzi </it>infection in <it>T. infestans </it>(81.16% by PCR and 56.52% by microscopy) and discovered that PCR is significantly more sensitive than microscopic observation. The overall degree of agreement between the two methods was moderate (Kappa = 0.43 ± 0.07). The level of infection is significantly different among communities; however, prevalence was similar among habitats and life stages.</p> <p>Conclusion</p> <p>PCR was significantly more sensitive than microscopy in all habitats, developmental stages and localities in Chuquisaca, Bolivia. Overall we observed a high prevalence of <it>T. cruzi </it>infection in <it>T. infestans </it>in this area of Bolivia; however, microscopy underestimated infection at all levels examined.</p

Mechanism of and Threshold Biomechanical Conditions for Falsetto Voice Onset

The sound source of a voice is produced by the self-excited oscillation of the vocal folds. In modal voice production, a drastic increase in transglottal pressure after vocal fold closure works as a driving force that develops self-excitation. Another type of vocal fold oscillation with less pronounced glottal closure observed in falsetto voice production has been accounted for by the mucosal wave theory. The classical theory assumes a quasi-steady flow, and the expected driving force onto the vocal folds under wavelike motion is derived from the Bernoulli effect. However, wavelike motion is not always observed during falsetto voice production. More importantly, the application of the quasi-steady assumption to a falsetto voice with a fundamental frequency of several hundred hertz is unsupported by experiments. These considerations suggested that the mechanism of falsetto voice onset may be essentially different from that explained by the mucosal wave theory. In this paper, an alternative mechanism is submitted that explains how self-excitation reminiscent of the falsetto voice could be produced independent of the glottal closure and wavelike motion. This new explanation is derived through analytical procedures by employing only general unsteady equations of motion for flow and solids. The analysis demonstrated that a convective acceleration of a flow induced by rapid wall movement functions as a negative damping force, leading to the self-excitation of the vocal folds. The critical subglottal pressure and volume flow are expressed as functions of vocal fold biomechanical properties, geometry, and voice fundamental frequency. The analytically derived conditions are qualitatively and quantitatively reasonable in view of reported measurement data of the thresholds required for falsetto voice onset. Understanding of the voice onset mechanism and the explicit mathematical descriptions of thresholds would be beneficial for the diagnosis and treatment of voice diseases and the development of artificial vocal folds

Modulation of Wnt/β-catenin signaling and proliferation by a ferrous iron chelator with therapeutic efficacy in genetically engineered mouse models of cancer

Using a screen for Wnt/β-catenin inhibitors, a family of 8-hydroxyquinolone derivatives with in vivo anti-cancer properties was identified. Analysis of microarray data for the lead compound N-((8-hydroxy-7-quinolinyl) (4-methylphenyl)methyl)benzamide (HQBA) using the Connectivity Map database suggested that it is an iron chelator that mimics the hypoxic response. HQBA chelates Fe2+ with a dissociation constant of ∼10−19 , with much weaker binding to Fe3+ and other transition metals. HQBA inhibited proliferation of multiple cell lines in culture, and blocked the progression of established spontaneous cancers in two distinct genetically engineered mouse models of mammary cancer, MMTV-Wnt1 and MMTV-PyMT mice, without overt toxicity. HQBA may inhibit an iron-dependent factor that regulates cell-type-specific β-catenin-driven transcription. It inhibits cancer cell proliferation independently of its effect on β-catenin signaling, as it works equally well in MMTV-PyMT tumors and diverse β-catenin-independent cell lines. HQBA is a promising specific intracellular Fe2+ chelator with activity against spontaneous mouse mammary cancers

Biochemical Properties of Highly Neuroinvasive Prion Strains

Infectious prions propagate from peripheral entry sites into the central nervous system (CNS), where they cause progressive neurodegeneration that ultimately leads to death. Yet the pathogenesis of prion disease can vary dramatically depending on the strain, or conformational variant of the aberrantly folded and aggregated protein, PrPSc. Although most prion strains invade the CNS, some prion strains cannot gain entry and do not cause clinical signs of disease. The conformational basis for this remarkable variation in the pathogenesis among strains is unclear. Using mouse-adapted prion strains, here we show that highly neuroinvasive prion strains primarily form diffuse aggregates in brain and are noncongophilic, conformationally unstable in denaturing conditions, and lead to rapidly lethal disease. These neuroinvasive strains efficiently generate PrPSc over short incubation periods. In contrast, the weakly neuroinvasive prion strains form large fibrillary plaques and are stable, congophilic, and inefficiently generate PrPSc over long incubation periods. Overall, these results indicate that the most neuroinvasive prion strains are also the least stable, and support the concept that the efficient replication and unstable nature of the most rapidly converting prions may be a feature linked to their efficient spread into the CNS

Visual Methods for the Design of Shape-Changing Interfaces

Shape-changing interfaces use physical change in shape as input and/or output. As the field matures, it will move from technology-driven design toward more formal processes. However, this is challenging: end-users are not aware of the capabilities of shape-change, devices are difficult to demonstrate, and presenting single systems can ‘trap’ user-thinking into particular forms. It is crucial to ensure this technology is developed with requirements in mind to ensure successful end-user experiences. To address this challenge, we developed and tested (n = 50) an approach that combines low-fidelity white-box prototypes and high-fidelity video footage with end-user diagram and scenario sketching to design context dependent devices. We analysed the outputs of our test process and identified themes in device design requirements, and from this constructed a shape-change stack model to support practitioners in developing, classifying, and synthesising end-user requirements for this novel technology

Phylogeography of the Patagonian otter Lontra provocax: adaptive divergence to marine habitat or signature of southern glacial refugia?

<p>Abstract</p> <p>Background</p> <p>A number of studies have described the extension of ice cover in western Patagonia during the Last Glacial Maximum, providing evidence of a complete cover of terrestrial habitat from 41°S to 56°S and two main refugia, one in south-eastern Tierra del Fuego and the other north of the Chiloé Island. However, recent evidence of high genetic diversity in Patagonian river species suggests the existence of aquatic refugia in this region. Here, we further test this hypothesis based on phylogeographic inferences from a semi-aquatic species that is a top predator of river and marine fauna, the huillín or Southern river otter (<it>Lontra provocax</it>).</p> <p>Results</p> <p>We examined mtDNA sequences of the control region, ND5 and Cytochrome-b (2151 bp in total) in 75 samples of <it>L. provocax </it>from 21 locations in river and marine habitats. Phylogenetic analysis illustrates two main divergent clades for <it>L. provocax </it>in continental freshwater habitat. A highly diverse clade was represented by haplotypes from the marine habitat of the Southern Fjords and Channels (SFC) region (43°38' to 53°08'S), whereas only one of these haplotypes was paraphyletic and associated with northern river haplotypes.</p> <p>Conclusions</p> <p>Our data support the hypothesis of the persistence of <it>L. provocax </it>in western Patagonia, south of the ice sheet limit, during last glacial maximum (41°S latitude). This limit also corresponds to a strong environmental change, which might have spurred <it>L. provocax </it>differentiation between the two environments.</p

Molecular Insights into Reprogramming-Initiation Events Mediated by the OSKM Gene Regulatory Network

Somatic cells can be reprogrammed to induced pluripotent stem cells by over-expression of OCT4, SOX2, KLF4 and c-MYC (OSKM). With the aim of unveiling the early mechanisms underlying the induction of pluripotency, we have analyzed transcriptional profiles at 24, 48 and 72 hours post-transduction of OSKM into human foreskin fibroblasts. Experiments confirmed that upon viral transduction, the immediate response is innate immunity, which induces free radical generation, oxidative DNA damage, p53 activation, senescence, and apoptosis, ultimately leading to a reduction in the reprogramming efficiency. Conversely, nucleofection of OSKM plasmids does not elicit the same cellular stress, suggesting viral response as an early reprogramming roadblock. Additional initiation events include the activation of surface markers associated with pluripotency and the suppression of epithelial-to-mesenchymal transition. Furthermore, reconstruction of an OSKM interaction network highlights intermediate path nodes as candidates for improvement intervention. Overall, the results suggest three strategies to improve reprogramming efficiency employing: 1) anti-inflammatory modulation of innate immune response, 2) pre-selection of cells expressing pluripotency-associated surface antigens, 3) activation of specific interaction paths that amplify the pluripotency signal