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

Axonal plasticity in response to active forces generated through magnetic nano-pulling

Mechanical force is crucial in guiding axon outgrowth before and after synapse formation. This process is referred to as "stretch growth." However, how neurons transduce mechanical input into signaling pathways remains poorly understood. Another open question is how stretch growth is coupled in time with the intercalated addition of new mass along the entire axon. Here, we demonstrate that active mechanical force generated by magnetic nano-pulling induces remodeling of the axonal cytoskeleton. Specifically, the increase in the axonal density of microtubules induced by nano-pulling leads to an accumulation of organelles and signaling vesicles, which, in turn, promotes local translation by increasing the probability of assembly of the "translation factories." Modulation of axonal transport and local translation sustains enhanced axon outgrowth and synapse maturation

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

Genomic-Bioinformatic Analysis of Transcripts Enriched in the Third-Stage Larva of the Parasitic Nematode Ascaris suum

Differential transcription in Ascaris suum was investigated using a genomic-bioinformatic approach. A cDNA archive enriched for molecules in the infective third-stage larva (L3) of A. suum was constructed by suppressive-subtractive hybridization (SSH), and a subset of cDNAs from 3075 clones subjected to microarray analysis using cDNA probes derived from RNA from different developmental stages of A. suum. The cDNAs (n = 498) shown by microarray analysis to be enriched in the L3 were sequenced and subjected to bioinformatic analyses using a semi-automated pipeline (ESTExplorer). Using gene ontology (GO), 235 of these molecules were assigned to ‘biological process’ (n = 68), ‘cellular component’ (n = 50), or ‘molecular function’ (n = 117). Of the 91 clusters assembled, 56 molecules (61.5%) had homologues/orthologues in the free-living nematodes Caenorhabditis elegans and C. briggsae and/or other organisms, whereas 35 (38.5%) had no significant similarity to any sequences available in current gene databases. Transcripts encoding protein kinases, protein phosphatases (and their precursors), and enolases were abundantly represented in the L3 of A. suum, as were molecules involved in cellular processes, such as ubiquitination and proteasome function, gene transcription, protein–protein interactions, and function. In silico analyses inferred the C. elegans orthologues/homologues (n = 50) to be involved in apoptosis and insulin signaling (2%), ATP synthesis (2%), carbon metabolism (6%), fatty acid biosynthesis (2%), gap junction (2%), glucose metabolism (6%), or porphyrin metabolism (2%), although 34 (68%) of them could not be mapped to a specific metabolic pathway. Small numbers of these 50 molecules were predicted to be secreted (10%), anchored (2%), and/or transmembrane (12%) proteins. Functionally, 17 (34%) of them were predicted to be associated with (non-wild-type) RNAi phenotypes in C. elegans, the majority being embryonic lethality (Emb) (13 types; 58.8%), larval arrest (Lva) (23.5%) and larval lethality (Lvl) (47%). A genetic interaction network was predicted for these 17 C. elegans orthologues, revealing highly significant interactions for nine molecules associated with embryonic and larval development (66.9%), information storage and processing (5.1%), cellular processing and signaling (15.2%), metabolism (6.1%), and unknown function (6.7%). The potential roles of these molecules in development are discussed in relation to the known roles of their homologues/orthologues in C. elegans and some other nematodes. The results of the present study provide a basis for future functional genomic studies to elucidate molecular aspects governing larval developmental processes in A. suum and/or the transition to parasitism

An integrative biostratigraphic, chemostratigraphic, and sequence stratigraphic perspective of the Ordovician–Silurian boundary on Anticosti Island (Canada)

Anticosti Island, Canada, has long been recognized as an exceptional OrdovicianâSilurian boundary succession with the potential to serve as one of the best records of climatic, oceanographic, and biological events associated with the Late Ordovician mass extinction. However, differing interpretations as to the position of the Hirnantian Stage within the stratigraphic succession due to the paucity of diagnostic graptolites, the apparent absence of a typical Hirnantia fauna within the Upper Ordovician Ellis Bay Formation, and lateral facies variability among outcrops has hindered the study of the OrdovicianâSilurian boundary on the island, particularly in the eastern half of the outcrop belt. Definitively identifying the stratigraphic position of the Hirnantian Stage within the succession is therefore critical for understanding this classic OrdovicianâSilurian boundary section, as well as for the integration of data from Anticosti into our global understanding of the Late Ordovician mass extinction. Here, we take an integrative approach to studying the Ellis Bay and lowermost Becscie formations, combining new paleobiological, geochemical, radiometric, and sequence stratigraphic constraints from ongoing fieldwork with existing biostratigraphic, geochemical, and palynological studies in the context of newly measured stratigraphic sections. These formations record six depositional sequences bounded by regionally traceable but subtle unconformities, often mantled by thin siliciclastic veneers reworked into transgressive lag facies. Many of these unconformities have gone unrecognized despite more than a century of work at certain localities. Furthermore, despite previous controversy, multiple lines of evidence favor a Hirnantian age for the entire Ellis Bay and lowermost Becscie formations, including newly recognized occurrences of Hirnantia and Hindella in the lower Ellis Bay Formation, a two-phased positive carbon isotope excursion, with the second phase reaching ~6â° in the Laframboise Member of the Ellis Bay Formation, and a U-Pb TIMS age of 443.61 ± 0.52 Ma from zircons in a bentonite from the mid-Ellis Bay Formation. While graptolite and conodont biostratigraphy support this age model, determination based on chitinozoan biozonation is more equivocal but may be controlled by facies preferences. Conodont, brachiopod, and chemostratigraphic data additionally suggest that the Hirnantian Stage may extend slightly into the lower Becscie Formation on the western end of Anticosti and well into the lower Becscie Formation in the eastern part of Anticosti. Our reappraisal of a classic OrdovicianâSilurian boundary section has important implications for understanding the sequence of climatic, environmental, and biological events throughout the Late Ordovician mass extinction. Given that the Ellis Bay and lowermost Becscie formations are indeed Hirnantian in age (encompassing ~2 My), these formations record six fourth-order depositional sequences of approximately ~333 ky. Furthermore, comparison of the Hirnantian of Anticosti to coeval exposures suggests that other regions may be incomplete at the level of the fourth-order cycles that occur in the Ellis Bay Formation. Resulting uncertainties in correlations based on unconformities and interpretations of stratigraphic architecture may therefore greatly complicate global correlation of Hirnantian records. Further study of this issue is critical, as stratigraphic architecture is expected to be an overarching control on the expression of oceanographic, climatic, and biotic events at a regional scale, complicating the interpretation of the pattern and drivers of the Late Ordovician mass extinction