Needles in the EST Haystack: Large-Scale Identification and Analysis of Excretory-Secretory (ES) Proteins in Parasitic Nematodes Using Expressed Sequence Tags (ESTs)

Excretory-secretory (ES) proteins are an important class of proteins in many organisms, spanning from bacteria to human beings, and are potential drug targets for several diseases. In this study, we first developed a software platform, EST2Secretome, comprised of carefully selected computational tools to identify and analyse ES proteins from expressed sequence tags (ESTs). By employing EST2Secretome, we analysed 4,710 ES proteins derived from 0.5 million ESTs for 39 economically important and disease-causing parasites from the phylum Nematoda. Several known and novel ES proteins that were either parasite- or nematode-specific were discovered, focussing on those that are either absent from or very divergent from similar molecules in their animal or plant hosts. In addition, we found many nematode-specific protein families of domains “transthyretin-like” and “chromadorea ALT,” considered vaccine candidates for filariasis in humans. We report numerous C. elegans homologues with loss-of-function RNAi phenotypes essential for parasite survival and therefore potential targets for parasite intervention. Overall, by developing freely available software to analyse large-scale EST data, we enabled researchers working on parasites for neglected tropical diseases to select specific genes and/or proteins to carry out directed functional assays for demystifying the molecular complexities of host–parasite interactions in a cell

Drug Repositioning and Pharmacophore Identification in the Discovery of Hookworm MIF Inhibitors

SummaryThe screening of bioactive compound libraries can be an effective approach for repositioning FDA-approved drugs or discovering new pharmacophores. Hookworms are blood-feeding, intestinal nematode parasites that infect up to 600 million people worldwide. Vaccination with recombinant Ancylostoma ceylanicum macrophage migration inhibitory factor (rAceMIF) provided partial protection from disease, thus establishing a “proof-of-concept” for targeting AceMIF to prevent or treat infection. A high-throughput screen (HTS) against rAceMIF identified six AceMIF-specific inhibitors. A nonsteroidal anti-inflammatory drug (NSAID), sodium meclofenamate, could be tested in an animal model to assess the therapeutic efficacy in treating hookworm disease. Furosemide, an FDA-approved diuretic, exhibited submicromolar inhibition of rAceMIF tautomerase activity. Structure-activity relationships of a pharmacophore based on furosemide included one analog that binds similarly to the active site, yet does not inhibit the Na-K-Cl symporter (NKCC1) responsible for diuretic activity

Duplex real-time reverse transcriptase PCR to determine cytokine mRNA expression in a hamster model of New World cutaneous leishmaniasis

<p>Abstract</p> <p>Background</p> <p>The Syrian hamster, <it>Mesocricetus auratus</it>, has distinct immunological features and is uniquely susceptible to intracellular pathogens. Studies in hamsters are limited by the relative unavailability of tools to conduct immunological studies. To address this limitation we developed duplex real-time reverse transcriptase (RT) PCR assays for the relative quantification of the mRNAs of hamster cytokines, chemokines, and related immune response molecules.</p> <p>Results</p> <p>Real-time RT-PCR primers and probes were synthesized for analysis of interleukin (IL)-4, IFN-γ, TNF-α, IL-10, IL-12p40, TGF-β, IL-13, IL-21, chemokine ligand (CCL) 22, CCL17, Chemokine (C-C motif) receptor 4 and FoxP3 expression. Standard curves and validation experiments were performed for each real-time RT-PCR assay, allowing us to use the comparative Ct (2^-ΔΔCt) method to calculate changes in gene expression. Application of the real-time RT PCR assays to a biological model was demonstrated by comparing mRNA expression in skin and lymph node tissues between uninfected and <it>Leishmania panamensis </it>infected hamsters.</p> <p>Conclusions</p> <p>The duplex real-time RT PCR assays provide a powerful approach for the quantification of cytokine transcription in hamsters, and their application to a model of cutaneous leishmaniasis suggests that a balanced type 1 and type 2 cytokine response contributes to the chronic, nonprogressive course of disease. These new molecular tools will further facilitate investigation into the mechanisms of disease in the hamster, not only for models of leishmaniasis, but also for other viral, bacterial, fungal, and parasitic infections.</p

Comparison of percutaneous vs oral infection of hamsters with the hookworm Ancylostoma ceylanicum: Parasite development, pathology and primary immune response

Background Hundreds of millions of people in poor countries continue to suffer from disease caused by bloodfeeding hookworms. While mice and rats are not reliably permissive hosts for any human hookworm species, adult Golden Syrian hamsters are fully permissive for the human and animal pathogen Ancylostoma ceylanicum. Similar to humans, hamsters may be infected with A. ceylanicum third-stage larvae orally or percutaneously. Oral infection typically leads to consistent worm yields in hamsters but may not accurately reflect the clinical and immunological manifestations of human infection resulting from skin penetration. Methodology/Principal findings In this study we compared host responses following percutaneous infection to those utilizing an established oral infection protocol. Infected hamsters exhibited a dose-dependent pathology, with 1000 percutaneous larvae (L3) causing anemia and adult worm recovery comparable to that of 50 orally administered L3. A delayed arrival and maturity of worms in the intestine was observed, as was variation in measured cellular immune responses. A long-term study found that the decline in blood hemoglobin was more gradual and did not reach levels as low, with the nadir of disease coming later in percutaneously infected hamsters. Both groups exhibited moderate growth delay, an effect that was more persistent in the percutaneously infected group. Fecal egg output also peaked later and at lower levels in the percutaneously infected animals. In contrast to orally infected hamsters, antibody titers to larval antigens continued to increase throughout the course of the experiment in the percutaneous group. Conclusions/Significance These results demonstrate that the route of infection with A. ceylanicum impacts disease pathogenesis, as well as humoral and cellular immune responses in an experimental setting. These data further validate the utility of the Golden Syrian hamster as a model of both oral and percutaneous infection with human hookworms

The Impact of Dynamic Emissivity−Temperature Trends on Spaceborne Data: Applications to the 2001 Mount Etna Eruption

Spaceborne detection and measurements of high-temperature thermal anomalies enable monitoring and forecasts of lava flow propagation. The accuracy of such thermal estimates relies on the knowledge of input parameters, such as emissivity, which notably affects computation of temperature, radiant heat flux, and subsequent analyses (e.g., effusion rate and lava flow distance to run) that rely on the accuracy of observations. To address the deficit of field and laboratory-based emissivity data for inverse and forward modelling, we measured the emissivity of ‘a’a lava samples from the 2001 Mt. Etna eruption, over the wide range of temperatures (773 to 1373 K) and wavelengths (2.17 to 21.0 µm). The results show that emissivity is not only wavelength dependent, but it also increases non-linearly with cooling, revealing considerably lower values than those typically assumed for basalts. This new evidence showed the largest and smallest increase in average emissivity during cooling in the MIR and TIR regions (~30% and ~8% respectively), whereas the shorter wavelengths of the SWIR region showed a moderate increase (~15%). These results applied to spaceborne data confirm that the variable emissivity-derived radiant heat flux is greater than the constant emissivity assumption. For the differences between the radiant heat flux in the case of variable and constant emissivity, we found the median value is 0.06, whereas the 25th and the 75th percentiles are 0.014 and 0.161, respectively. This new evidence has significant impacts on the modelling of lava flow simulations, causing a dissimilarity between the two emissivity approaches of ~16% in the final area and ~7% in the maximum thickness. The multicomponent emissivity input provides means for ‘best practice’ scenario when accurate data required. The novel approach developed here can be used to test an improved version of existing multi-platform, multi-payload volcano monitoring systems

Моделирование формирования структуры металломатричных композитов в процессе синтеза с оценкой эффективных свойств

Работа посвящена моделированию процесса кристаллизации композита с металлической матрицей и твердыми включениями с учетом условий синтеза (давление, скорость охлаждения), моделированию процесса формирования переходной зоны между частицами и матрицей и расчету эффективных свойств получаемых композитов.The work is devoted to modeling the crystallization process of metal matrix composite with solid inclusions, taking into account the synthesis conditions (pressure, cooling rate), to modeling the formation of the transition zone between particles and matrix, and calculating the effective properties of the resulting composites

Biological properties of extracellular vesicles and their physiological functions

In the past decade, extracellular vesicles (EVs) have been recognized as potent vehicles of intercellular communication, both in prokaryotes and eukaryotes. This is due to their capacity to transfer proteins, lipids and nucleic acids, thereby influencing various physiological and pathological functions of both recipient and parent cells. While intensive investigation has targeted the role of EVs in different pathological processes, for example, in cancer and autoimmune diseases, the EV-mediated maintenance of homeostasis and the regulation of physiological functions have remained less explored. Here, we provide a comprehensive overview of the current understanding of the physiological roles of EVs, which has been written by crowd-sourcing, drawing on the unique EV expertise of academia-based scientists, clinicians and industry based in 27 European countries, the United States and Australia. This review is intended to be of relevance to both researchers already working on EV biology and to newcomers who will encounter this universal cell biological system. Therefore, here we address the molecular contents and functions of EVs in various tissues and body fluids from cell systems to organs. We also review the physiological mechanisms of EVs in bacteria, lower eukaryotes and plants to highlight the functional uniformity of this emerging communication system

TLR7 gain-of-function genetic variation causes human lupus

Although circumstantial evidence supports enhanced Toll-like receptor 7 (TLR7) signalling as a mechanism of human systemic autoimmune disease evidence of lupus-causing TLR7 gene variants is lacking. Here we describe human systemic lupus erythematosus caused by a TLR7 gain-of-function variant. TLR7 is a sensor of viral RNA and binds to guanosine. We identified a de novo, previously undescribed missense TLR7Y264H variant in a child with severe lupus and additional variants in other patients with lupus. The TLR7Y264H variant selectively increased sensing of guanosine and 2',3'-cGMP1 and was sufficient to cause lupus when introduced into mice. We show that enhanced TLR7 signalling drives aberrant survival of B cell receptor (BCR)-activated B cells, and in a cell-intrinsic manner, accumulation of CD11c+ age-associated B cells and germinal centre B cells. Follicular and extrafollicular helper T cells were also increased but these phenotypes were cell-extrinsic. Deficiency of MyD88 (an adaptor protein downstream of TLR7) rescued autoimmunity, aberrant B cell survival, and all cellular and serological phenotypes. Despite prominent spontaneous germinal-centre formation in Tlr7Y264H mice, autoimmunity was not ameliorated by germinal-centre deficiency, suggesting an extrafollicular origin of pathogenic B cells. We establish the importance of TLR7 and guanosine-containing self-ligands for human lupus pathogenesis, which paves the way for therapeutic TLR7 or MyD88 inhibition

First Colombian Multicentric Newborn Screening for Congenital Toxoplasmosis

Congenital toxoplasmosis can result in permanent sequel as blindness or neurological damage in children and it seems to be more severe in South America than in other continents. There is a lack of information about this frequency in Colombia, where no control program is established, although it is a recognized cause of potentially preventable congenital blindness. We propose the first Colombian multicentric study to determine the frequency and impact of congenital toxoplasmosis. More than 15,000 newborns in seven cities were studied. Newborns were tested at birth by doing a cord blood test for toxoplasmosis. Additionally, children from mothers with history of toxoplasmosis acquired during pregnancy were recalled for a follow-up. The program identified fifteen children otherwise undiagnosed; three of these children died as consequence of congenital toxoplasmosis. The frequency of the congenital infection varied significantly between cities, being higher in Armenia and Florencia, intermediate in Bogota, Bucaramanga and Barranquilla and very low in western cities such as Cucuta and Riohacha. For the first time a significant correlation was found between mean rainfall at the city and the incidence of this congenital infection