Search

Nonlinear electrokinetics (EK), specifically electrophoresis of the second kind, dielectrophoresis (DEP) and electrorotation (EROT), have gained significant interest recently for their flexibility and labeless discriminant manner of operation. The current applications of these technologies are a clear advancement from what they were when first discovered, but also still show strong signs of future growth. The present review article presents a discussion of the current uses of microscale nonlinear EK technologies as analytical, sensing, and purification tools for microorganisms. The discussion is focused on some of the latest discoveries with various nonlinear EK microfluidic techniques, such as DEP particle trapping and EROT for particle assessments, for the analysis of microorganisms ranging from viruses to parasites. Along the way, special focus was given to key research articles from within the past two years to provide the most up-to-date knowledge on the current state-of-the-art within the field of microscale EK, and from there, an outlook on where the future of the field is headed is also included., (© 2020 Wiley-VCH GmbH.)

Very long chain polyunsaturated fatty acids (VLCPUFAs) such as docosahexaenoic acid (DHA, 22:6n-3), arachidonic acid (ARA, 20:4n-6) and eicosapentaenoic acid (EPA, 20:5-n3) are nutritionally important for humans and animals. De novo biosynthesis of these fatty acids mainly occurs in microorganisms and goes through either an aerobic pathway catalyzed by type I/II fatty acid synthase, desaturases and elongases or an anaerobic pathway catalyzed by a polyunsaturated fatty acid synthase. After synthesis, VLCPUFAs must be incorporated into glycerolipids for storage through acyl assembly processes. Understanding the mechanisms for the biosynthesis of VLCPUFAs and their incorporation into glycerolipids is important not only for developing a renewable, sustainable and environment-friendly source of these fatty acids in microorganisms, but also, for designing effective strategies for metabolic engineering of these fatty acids in heterologous systems. This review highlights recent findings which have increased our understanding of biosynthesis of VLCPUFAs and their incorporation into glycerolipids in microorganisms. Future directions in improving the production of VLCPUFAs in native microbial producers are also discussed along with transgenic production of these fatty acids in oleaginous microorganisms and oilseed crops for food and feed uses., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The transient receptor potential cation channel subfamily V member 1 (TRPV1) receptor is an important mediator of nociception and its expression is enriched in nociceptive neurons. TRPV1 signaling has been implicated in bladder pain and is a potential analgesic target. Resiniferatoxin is the most potent known agonist of TRPV1. Acute exposure of the rat bladder to resiniferatoxin has been demonstrated to result in pain-related freezing and licking behaviors that are alleviated by virally encoded IL-4. The interleukin-4-inducing principle of Schistosoma mansoni eggs (IPSE) is a powerful inducer of IL-4 secretion, and is also known to alter host cell transcription through a nuclear localization sequence-based mechanism. We previously reported that IPSE ameliorates ifosfamide-induced bladder pain in an IL-4- and nuclear localization sequence-dependent manner. We hypothesized that pre-administration of IPSE to resiniferatoxin-challenged mice would dampen pain-related behaviors. IPSE indeed lessened resiniferatoxin-triggered freezing behaviors in mice. This was a nuclear localization sequence-dependent phenomenon, since administration of a nuclear localization sequence mutant version of IPSE abrogated IPSE's analgesic effect. In contrast, IPSE's analgesic effect did not seem IL-4-dependent, since use of anti-IL-4 antibody in mice given both IPSE and resiniferatoxin did not significantly affect freezing behaviors. RNA-Seq analysis of resiniferatoxin- and IPSE-exposed bladders revealed differential expression of TNF/NF-κb-related signaling pathway genes. In vitro testing of IPSE uptake by urothelial cells and TRPV1-expressing neuronal cells showed uptake by both cell types. Thus, IPSE's nuclear localization sequence-dependent therapeutic effects on TRPV1-mediated bladder pain may act on TRPV1-expressing neurons and/or may rely upon urothelial mechanisms.

Parasite infections modulate immunologic responses, and the loss of parasite infections in the last two to three decades might explain the increased prevalence of allergic diseases in developed countries. However, parasites can enhance allergic responses. Parasites contain or release allergen-like molecules that induce the specific immunoglobulin, IgE, and trigger type-2 immune responses. Some parasites and their proteins, such as Anisakis and Echinococcus granulosus allergens, act as typical allergens. A number of IgE-binding proteins of various helminthic parasites are cross-reactive to other environmental allergens, which cause allergic symptoms or hamper accurate diagnosis of allergic diseases. The cross-reactivity is based on the fact that parasite proteins are structurally homologous to common environmental allergens. In addition, IgE-binding proteins of parasites might be useful for developing vaccines to prevent host re-infection. This review discusses the functions of the IgE-biding proteins of parasites., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Introduction: Over 300 species of parasites can possibly be passed on humans. Most of the parasitic infections are defined based on their pathogenicity; however, some positive effects of a parasite existence within the human body have recently been suggested. Beneficial outcomes of parasite infections might result from the production and release of metabolites, modification of host immune response or products uptake of the host., Objective: The aim of the study was a comprehensive analysis of a wide range of effects of parasites on the human body, including an overview of the toxic and positive effects., State of Knowledge: In the light of the latest research presenting the unconventional use of parasites in medicine, the widely understood of their impact on the human body can also be considered in a positive context. Clinical cases from diseases caused by the toxic effects of parasites, as described in recent years, indicate that the problem of parasitic infections still persists. Despite a great deal of knowledge about the toxic effects of parasites on the human organism and, above all, despite the improvement in sanitary conditions, there is a resurgence of parasitic infections, as evidenced, e.g. by the examples presented in this review., Conclusions: The examples of positive effects of parasites presented so far give hope for the future in terms of fighting many diseases for which pharmacological treatment has not yet brought a positive effect. A better understanding of those processes might lead to the development of new methods of unconventional medical treatment.

The investigation of the glycan repertoire of several organisms has revealed a wide variation in terms of structures and abundance of glycan moieties. Among the parasites, it is possible to observe different sets of glycoconjugates across taxa and developmental stages within a species. The presence of distinct glycoconjugates throughout the life cycle of a parasite could relate to the ability of that organism to adapt and survive in different hosts and environments. Carbohydrates on the surface, and in excretory-secretory products of parasites, play essential roles in host-parasite interactions. Carbohydrate portions of complex molecules of parasites stimulate and modulate host immune responses, mainly through interactions with specific receptors on the surface of dendritic cells, leading to the generation of a pattern of response that may benefit parasite survival. Available data reviewed here also show the frequent aspect of parasite immunomodulation of mammalian responses through specific glycan interactions, which ultimately makes these molecules promising in the fields of diagnostics and vaccinology.

Glycosylphosphatidylinositol (GPI) is a complex glycolipid structure that acts as a membrane anchor for many cell-surface proteins of eukaryotes. GPI-anchored proteins are particularly abundant in protozoa and represent the major carbohydrate modification of many cell-surface parasite proteins. A minimal GPI-anchor precursor consists of core glycan (ethanolamine-PO 4 -Manα1-2Manα1-6Manα1-4GlcNH 2 ) linked to the 6-position of the D-myo-inositol ring of phosphatidylinositol. Although the GPI core glycan is conserved in all organisms, many differences in additional modifications to GPI structures and biosynthetic pathways have been reported. The preassembled GPI-anchor precursor is post-translationally transferred to a variety of membrane proteins in the lumen of the endoplasmic reticulum in a transamidase-like reaction during which a C-terminal GPI attachment signal is released. Increasing evidence shows that a significant proportion of the synthesized GPIs are not used for protein anchoring, particularly in protozoa in which a large amount of free GPIs are being displayed at the cell surface. The characteristics of GPI biosynthesis are currently being explored for the development of parasite-specific inhibitors. Especially this pathway, at least for Trypanosoma brucei, has been validated as a drug target. Furthermore, thanks to an increase of new innovative strategies to produce pure synthetic carbohydrates, a novel era in the use of GPIs in diagnostic, anti-GPI antibody production, as well as parasitic protozoa GPI-based vaccine approach is developing fast.

Background: Complex network approach allows the representation and analysis of complex systems of interacting agents in an ordered and effective manner, thus increasing the probability of discovering significant properties of them. In the present study, we defined and built for the first time a complex network based on data obtained from Immune Epitope Database for parasitic organisms. We then considered the general topology, the node degree distribution, and the local structure (triadic census) of this network. In addition, we calculated 9 node centrality measures for observed network and reported a comparative study of the real network with three theoretical models to detect similarities or deviations from these ideal networks., Result: The results obtained corroborate the utility of the complex network approach for handling information and data mining within the database under study., Conclusion: They confirm that this type of approach can be considered a valuable tool for preliminary screening of the best experimental conditions to determine whether the amino acid sequences being studied are true epitopes or not., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

Optical trapping has become an optimal choice for biological research at the microscale due to its non-invasive performance and accessibility for quantitative studies, especially on the forces involved in biological processes. However, reliable force measurements depend on the calibration of the optical traps, which is different for each experiment and hence requires high control of the local variables, especially of the trapped object geometry. Many biological samples have an elongated, rod-like shape, such as chromosomes, intracellular organelles (e.g., peroxisomes), membrane tubules, certain microalgae, and a wide variety of bacteria and parasites. This type of samples often requires several optical traps to stabilize and orient them in the correct spatial direction, making it more difficult to determine the total force applied. Here, we manipulate glass microcylinders with holographic optical tweezers and show the accurate measurement of drag forces by calibration-free direct detection of beam momentum. The agreement between our results and slender-body hydrodynamic theoretical calculations indicates potential for this force-sensing method in studying protracted, rod-shaped specimens.

Toxic microalgae have their own pathogens, and understanding the way in which these microalgae respond to antagonistic attacks may provide information about their capacity to persist during harmful algal bloom events. Here, we compared the effects of the physical presence of the parasite Amoebophrya sp. and exposure to waterborne cues from cultures infected with this parasite, on gene expression by the toxic dinoflagellates, Alexandrium fundyense. Compared with control samples, a total of 14,882 Alexandrium genes were differentially expressed over the whole-parasite infection cycle at three different time points (0, 6 and 96 h). RNA sequencing analyses indicated that exposure to the parasite and parasitic waterborne cues produced significant changes in the expression levels of Alexandrium genes associated with specific metabolic pathways. The observed upregulation of genes associated with glycolysis, the tricarboxylic acid cycle, fatty acid β-oxidation, oxidative phosphorylation and photosynthesis suggests that parasite infection increases the energy demand of the host. The observed upregulation of genes correlated with signal transduction indicates that Alexandrium could be sensitized by parasite attacks. This response might prime the defence of the host, as indicated by the increased expression of several genes associated with defence and stress. Our findings provide a molecular overview of the response of a dinoflagellate to parasite infection., (© 2016 Alfred-Wegener-Institut Helmholtz-Zentrum für Polar- und Meeresforschung. Molecular Ecology published by John Wiley & Sons Ltd.)

Recently, parasite infections or parasite-derived products have been suggested as a therapeutic strategy with suppression of immunopathology, which involves the induction of regulatory T cells or/and T helper type 2 (Th2) responses. In a recent study, researchers reported that constructed recombinant galectin (rTl-gal) isolated from an adult worm of the gastrointestinal nematode parasite Toxascaris leonina attenuated clinical symptoms of inflammatory bowel disease in mice treated with dextran sulphate sodium. Noting the role of rTl-gal in inflammatory disease, we attempted to investigate the effect of the parasite via its rTl-gal on neuronal autoimmune disease using experimental autoimmune encephalomyelitis (EAE), a mouse inflammatory and demyelinating autoimmune disease model of human multiple sclerosis. In this model, rTl-gal-treated experimental autoimmune encephalomyelitis (EAE) mice failed to recover after the peak of the disease, leading to persistent central nervous system (CNS) damage, such as demyelination, gliosis and axonal damage. Further, rTl-gal-treated EAE mice markedly increased the number of CD45R/B220(+) B cells in both infiltrated inflammation and the periphery, along with the increased production of autoantibody [anti-myelin oligodendrocyte glycoprotein (MOG)35-55 ] in serum at chronic stage. Upon antigen restimulation, rTl-gal treatment affected the release of overall cytokines, especially interferon (IFN)-γ and tumour necrosis factor (TNF)-α. Our results suggest that galectin isolated from a gastrointestinal parasite can deliver a harmful effect to EAE contrary to its beneficial effect on inflammatory bowel disease., (© 2015 British Society for Immunology.)

Cryptosporidiosis is an infectious disease caused by protozoan parasites of the Cryptosporidium genus. Infection is associated with mild to severe diarrhea that usually resolves spontaneously in healthy human adults, but may lead to severe complications in young children and in immunocompromised patients. The genome of C. parvum contains a gene, CUTA_CRYPI, that may play a role in regulating the intracellular concentration of copper, which is a toxic element in excess. Here, the crystal structure of this CutA1 protein, Cp-CutA1, is reported at 2.0 Å resolution. As observed for other CutA1 structures, the 117-residue protein is a trimer with a core ferrodoxin-like fold. Circular dichroism spectroscopy shows little, in any, unfolding of Cp-CutA1 up to 353 K. This robustness is corroborated by (1)H-(15)N HSQC spectra at 333 K, which are characteristic of a folded protein, suggesting that NMR spectroscopy may be a useful tool to further probe the function of the CutA1 proteins. While robust, Cp-CutA1 is not as stable as the homologous protein from a hyperthermophile, perhaps owing to a wide β-bulge in β2 that protrudes Pro48 and Ser49 outside the β-sheet.

The human genome encodes a gene for an enzymatically active chitinase (CHIT1) located in a single copy on Chromosome 1, which is highly expressed by activated macrophages and in other cells of the innate immune response. Several dysfunctional mutations are known in CHIT1, including a 24-bp duplication in Exon 10 causing catalytic deficiency. This duplication is a common variant conserved in many human populations, except in West and South Africans. Thus it has been proposed that human migration out of Africa and the consequent reduction of exposure to chitin from environmental factors may have enabled the conservation of dysfunctional mutations in human chitinases. Our data obtained from 85 indigenous Amerindians from Peru, representative of populations characterized by high prevalence of chitin-bearing enteroparasites and intense entomophagy, reveal a very high frequency of the 24-bp duplication (47.06%), and of other single nucleotide polymorphisms which are known to partially affect enzymatic activity (G102S: 42.7% and A442G/V: 25.5%). Our finding is in line with a founder effect, but appears to confute our previous hypothesis of a protective role against parasite infection and sustains the discussion on the redundancy of chitinolytic function., (Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Purpose: The therapeutic efficacy of a synthetic parasite-derived peptide GK1, an immune response booster, was evaluated in a mouse melanoma model. This melanoma model correlates with human stage IIb melanoma, which is treated with wide surgical excision; a parallel study employing a surgical treatment was carried out as an instructive goal., Experimental Design: C57BL/6 mice were injected subcutaneously in the flank with 2×10(5) B16-F10 murine melanoma cells. When the tumors reached 20 mm3, mice were separated into two different groups; the GK1 group, treated weekly with peritumoral injections of GK1 (10 μg/100 μL of sterile saline solution) and the control group, treated weekly with an antiseptic peritumoral injection of 100 μL of sterile saline solution without further intervention. All mice were monitored daily for clinical appearance, tumor size, and survival. Surgical treatment was performed in parallel when the tumor size was 20 mm3 (group A), 500 mm3 (group B), and >500 mm3 (group C)., Results: The GK1 peptide effectively increased the mean survival time by 9.05 days, corresponding to an increase of 42.58%, and significantly delayed tumor growth from day 3 to 12 of treatment. In addition, tumor necrosis was significantly increased (p<0.05) in the treated mice. The overall survival rates obtained with surgical treatment at 6 months were 83.33% for group A, 40% for group B, and 0% for group C, with significant differences (p<0.05) among the groups., Conclusions: The GK1 peptide demonstrated therapeutic properties in a mouse melanoma model, as treatment resulted in a significant increase in the mean survival time of the treated animals (42.58%). The potential for GK1 to be used as a primary or adjuvant component of chemotherapeutic cocktails for the treatment of experimental and human cancers remains to be determined, and surgical removal remains a challenge for any new experimental treatment of melanoma in mouse models.

Protists of the genus Leishmania are obligatory intracellular parasites that cause a wide range of cutaneous, mucocutaneous, and visceral diseases in humans. They cycle between phagolysosomes of mammalian macrophages and the sand fly midgut, proliferating as intracellular amastigotes and extracellular promastigotes, respectively. Exposure to a lysosomal environment, i.e. acidic pH and body temperature, signals promastigotes to differentiate into amastigotes. Time course analyses indicated that Leishmania differentiation is a highly regulated and coordinated process. However, the role of posttranslational events such as protein phosphorylation in this process is still unknown. Herein, we analyzed and compared the phosphoproteomes of L. donovani amastigotes and promastigotes using an axenic host-free system that simulates parasite differentiation. Shotgun phosphopeptide analysis revealed 1614 phosphorylation residues (p-sites) corresponding to 627 proteins. The analysis indicated that the majority of the p-sites are stage-specific. Serine phosphorylation in a previously identified trypanosomatid-specific "SF" motif was significantly enriched in amastigotes. We identified a few phosophotyrosines (pY), mostly in proteins known to participate in signal transduction pathways. The analysis indicated that Leishmania contains proteins with multiple p-sites that are phosphorylated at distinct stages of the life cycle. For over half of the phosphorylation events, changes in phosphoprotein abundance did not positively correlate with changes in protein abundance, suggesting functional regulation. This study compares, for the first time, the phosphoproteins of L. donovani axenic promastigotes and amastigotes and provides the largest data set of the Leishmania phosphoproteome to date.

Dead-end ultrafiltration (DEUF) has been reported to be a simple, field-deployable technique for recovering bacteria, viruses, and parasites from large-volume water samples for water quality testing and waterborne disease investigations. While DEUF has been reported for application to water samples having relatively low turbidity, little information is available regarding recovery efficiencies for this technique when applied to sampling turbid water samples such as those commonly found in lakes and rivers. This study evaluated the effectiveness of a DEUF technique for recovering MS2 bacteriophage, enterococci, Escherichia coli, Clostridium perfringens, and Cryptosporidium parvum oocysts in surface water samples having elevated turbidity. Average recovery efficiencies for each study microbe across all turbidity ranges were: MS2 (66%), C. parvum (49%), enterococci (85%), E. coli (81%), and C. perfringens (63%). The recovery efficiencies for MS2 and C. perfringens exhibited an inversely proportional relationship with turbidity, however no significant differences in recovery were observed for C. parvum, enterococci, or E. coli. Although ultrafilter clogging was observed, the DEUF method was able to process 100-L surface water samples at each turbidity level within 60 min. This study supports the use of the DEUF method for recovering a wide array of microbes in large-volume surface water samples having medium to high turbidity., (Published by Elsevier B.V.)

Advances in lipid separation methods and mass spectrometry technologies allow the fine characterization of the lipidome of parasites, ranging from unicellular protists to worms, which cause threatening infections in vertebrates, including humans. Specific lipid structures or lipid metabolic pathways can inspire the development of novel antiparasitic drugs. Changes in the lipid balance in membranes of parasites can also provide clues on the dynamics of drugs and some mechanisms of drug resistance. This review highlights recent trends in parasite lipidomics, combined with functional analyses, for the discovery of novel targets and the development of novel drugs., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Chronic lymphatic filarial (LF) infection is associated with suppression of parasite-specific T cell responses that persist even following elimination of infection. While several mechanisms have been implicated in mediating this T cell specific downregulation, a role for alterations in the homeostasis of T effector and memory cell populations has not been explored. Using multiparameter flow cytometry, we investigated the role of persistent filarial infection on the maintenance of T cell memory in patients from the filarial-endemic Cook Islands. Compared to filarial-uninfected endemic normals (EN), microfilaria (mf) positive infected patients (Inf) had a reduced CD4 central memory (T(CM)) compartment. In addition, Inf patients tended to have more effector memory cells (T(EM)) and fewer effector cells (T(EFF)) than did ENs giving significantly smaller T(EFF):T(EM) ratios. These contracted T(CM) and T(EFF) populations were still evident in patients previously mf+ who had cleared their infection (CLInf). Moreover, the density of IL-7Rα, necessary for T memory cell maintenance (but decreased in T effector cells), was significantly higher on memory cells of Inf and CLInf patients, although there was no evidence for decreased IL-7 or increased soluble IL7-Rα, both possible mechanisms for signaling defects in memory cells. However, effector cells that were present in Inf and CLInf patients had lower percentages of HLA-DR suggesting impaired function. These changes in T cell populations appear to reflect chronicity of infection, as filarial-infected children, despite the presence of active infection, did not show alterations in the frequencies of these T cell phenotypes. These data indicate that filarial-infected patients have contracted T(CM) compartments and a defect in effector cell development, defects that persist even following clearance of infection. The fact that these global changes in memory and effector cell compartments do not yet occur in infected children makes early treatment of LF even more crucial.

According to World Health Organization estimates, infectious organisms are responsible for approximately one in four deaths worldwide. Animal models play an essential role in the development of vaccines and therapeutic agents but large numbers of animals are required to obtain quantitative microbiological data by tissue sampling. Biophotonic imaging (BPI) is a highly sensitive, nontoxic technique based on the detection of visible light, produced by luciferase-catalysed reactions (bioluminescence) or by excitation of fluorescent molecules, using sensitive photon detectors. The development of bioluminescent/fluorescent microorganisms therefore allows the real-time noninvasive detection of microorganisms within intact living animals. Multiple imaging of the same animal throughout an experiment allows disease progression to be followed with extreme accuracy, reducing the number of animals required to yield statistically meaningful data. In the study of infectious disease, the use of BPI is becoming widespread due to the novel insights it can provide into established models, as well as the impact of the technique on two of the guiding principles of using animals in research, namely reduction and refinement. Here, we review the technology of BPI, from the instrumentation through to the generation of a photonic signal, and illustrate how the technique is shedding light on infection dynamics in vivo., (© 2010 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

Carbohydrate-protein interactions are involved in various intracellular functions and play an essential role in biological system, particularly at the level of cell-cell recognition, cell adhesion, and cell signaling processes. The importance of carbohydrate-protein binding is now recognized as a major mode of interaction between microbial pathogens and animal cells. Using innovative synthetic methods for oligosaccharide assembly an increasing number of synthetic carbohydrates of biomedical importance is available. Here, we illustrate using some case studies that show the design and the use of a glycosylphosphatidylinositol oligosaccharide library. We discuss the importance of parasite glycosylphosphatidylinositol-protein interactions including receptors, enzymes, and antibodies. Furthermore, glycosylphosphatidylinositol epitope mapping studies are of interest in the field of parasitic diseases, and provide a promising platform to understand structure-function relationships of glycosylphosphatidylinositols.

Pathogens interact with their hosts at different spatial and temporal scales. Studying these interactions therefore requires a wide range of imaging tools and approaches that bridge physics and biology, as shown by this Minireview focusing on recent studies of the causative agents of malaria, toxoplasmosis, and sleeping sickness., (Copyright 2010 Elsevier Inc. All rights reserved.)

Two invasive fish species, the round goby Apollonia melanostoma syn. Neogobius melanostomus (Pallas 1814) and the bighead goby Neogobius kessleri (Günther, 1861), have established a firm population in Austrian waters during the past 15 years. As there have been no records of the parasite fauna from these populations, a total of 79 specimens of A. melanostoma and 12 specimens of N. kessleri were examined for parasites between May and October 2007 from three different sampling sites from the Danube River in Austria. In total 12 parasite taxa were recovered. The protozoans Trichodina sp. and Ichthyophthirius multifiliis from the gills and skin; two crustacean species, Paraergasilus brevidigitus and Ergasilus sieboldi, from the gills; and the two monogeneans Gyrodactylus sp. and Dactylogyrus sp., from the skin and gills respectively, all occurred at low prevalence and intensities. Furthermore, cystacanths of the acanthocephalan Acanthocephalus lucii were found in the body cavity. Metacercariae of the digeneans Diplostomum sp. and Tylodelphys clavata were found in the lens of the eye and the vitreous humour, respectively. Adults of two digeneans, Nicolla skrjabini and Bunodera nodulosa, were found in the intestine. In addition, during this survey metacercariae of the Holarctic digenean Bucephalus polymorphus, encysted in the skin and fins, with prevalence up to 78%, were recorded for the first time in Austria.

Tubulin is a highly conserved, negatively charged protein that is found in essentially all eukaryotic cells. These properties ensure that isolation protocols successful in one system will likely work, with a few modifications, in most systems. Tubulin has been isolated most frequently from mammalian brain, and the main difference encountered in other systems versus brain is that tubulin is much less abundant in nearly all other sources than it is in brain. This means that attempting to purify tubulin by direct polymerization from a homogenate will often fail or be quite inefficient. However, the conservation of negative charge on tubulin means that an initial ion exchange step can be used to both purify and concentrate the protein from most systems. Polymerization-competent tubulin can usually be obtained by inducing polymerization in the salt eluate from the ion exchange step. We describe protocols for this procedure and describe its application to a number of vertebrate, fungal, protozoal, and plant sources., (Copyright 2010 Elsevier Inc. All rights reserved.)

The development of methods that can predict the metal-mediated biological activity based only on the 3D structure of metal-unbound proteins has become a goal of major importance. This work is dedicated to the amino terminal Cu(II)- and Ni(II)-binding (ATCUN) motifs that participate in the DNA cleavage and have antitumor activity. We have calculated herein, for the first time, the 3D electrostatic spectral moments for 415 different proteins, including 133 potential ATCUN antitumor proteins. Using these parameters as input for Linear Discriminant Analysis, we have found a model that discriminates between ATCUN-DNA cleavage proteins and nonactive proteins with 91.32% Accuracy (379 out of 415 of proteins including both training and external validation series). Finally, the model has predicted for the first time the DNA cleavage function of proteins from the pathogen parasites. We have predicted possible ATCUN-like proteins with a probability higher than 99% in nine parasite families such as Trypanosoma, Plasmodium, Leishmania, or Toxoplasma. The distribution by biological function of the ATCUN proteins predicted has been the following: oxidoreductases 70.5%, signaling proteins 62.5%, lyases 58.2%, membrane proteins 45.5%, ligases 44.4%, hydrolases 41.3%, transferases 39.2%, cell adhesion proteins 34.5%, metal binders 33.5%, translation proteins 25.0%, transporters 16.7%, structural proteins 9.1%, and isomerases 8.2%. The model is implemented at http://miaja.tic.udc.es/Bio-AIMS/ATCUNPred.php.

Alpha-14 giardin (annexin E1), a member of the alpha giardin family of annexins, has been shown to localize to the flagella of the intestinal protozoan parasite Giardia lamblia. Alpha giardins show a common ancestry with the annexins, a family of proteins most of which bind to phospholipids and cellular membranes in a Ca(2+)-dependent manner and are implicated in numerous membrane-related processes including cytoskeletal rearrangements and membrane organization. It has been proposed that alpha-14 giardin may play a significant role during the cytoskeletal rearrangement during differentiation of Giardia. To gain a better understanding of alpha-14 giardin's mode of action and its biological role, we have determined the three-dimensional structure of alpha-14 giardin and its phospholipid-binding properties. Here, we report the apo crystal structure of alpha-14 giardin determined in two different crystal forms as well as the Ca(2+)-bound crystal structure of alpha-14 giardin, refined to 1.9, 1.6 and 1.65 A, respectively. Although the overall fold of alpha-14 giardin is similar to that of alpha-11 giardin, multiwavelength anomalous dispersion phasing was required to solve the alpha-14 giardin structure, indicating significant structural differences between these two members of the alpha giardin family. Unlike most annexin structures, which typically possess N-terminal domains, alpha-14 giardin is composed of only a core domain, followed by a C-terminal extension that may serve as a ligand for binding to cytoskeletal protein partners in Giardia. In the Ca(2+)-bound structure we detected five bound calcium ions, one of which is a novel, highly coordinated calcium-binding site not previously observed in annexin structures. This novel high-affinity calcium-binding site is composed of seven protein donor groups, a feature rarely observed in crystal structures. In addition, phospholipid-binding assays suggest that alpha-14 giardin exhibits calcium-dependent binding to phospholipids that coordinate cytoskeletal disassembly/assembly during differentiation of the parasite.

Infection by pathogens is generally initiated by the specific recognition of host epithelia surfaces and subsequent adhesion is essential for invasion. In their infection strategy, microorganisms often use sugar-binding proteins, that is lectins and adhesins, to recognize and bind to host glycoconjugates where sialylated and fucosylated oligosaccharides are the major targets. The lectin/glycoconjugate interactions are characterized by their high specificity and most of the time by multivalency to generate higher affinity of binding. Recent crystal structures of viral, bacterial, and parasite receptors in complex with human histo-blood group epitopes or sialylated derivatives reveal new folds and novel sugar-binding modes. They illustrate the tight specificity between tissue glycosylation and lectins.

Purified RNA transcripts from venom glands dissected from the parasitoid wasp Microctonus hyperodae were copied, cloned and sequenced using traditional dideoxy sequencing methods. Using mass spectrometry analysis of the trypsinised PAGE gel protein bands we identified the RNA transcripts for the 3 most abundant proteins found in the venom and hence obtained their full protein sequence. Other abundant transcripts were also further sequenced. To reduce the effort required to obtain transcript information we dissected venom glands from a second parasitoid, Microctonus aethiopoides (Morocco biotype). The RNA transcripts were purified and reverse transcribed but instead of cloning the cDNA it was directly sequenced using Roche GS20 pyrosequencing. Results from a single GS20 sequencing run provided data similar to that obtained by the traditional methods used in analysing transcripts from M. hyperodae in a fraction of the time and cost. Comparing the transcripts between the two species showed that a similar range of genes are expressed with the putative orthologs of seven of the eight full length genes characterised from M. hyperodae being found in M. aethiopoides. Pyrosequencing should provide a valuable new method for rapidly sampling transcripts from a wide range of specialised insect tissues.

The transfer of genes from an endosymbiont to its host typically requires acquisition of targeting signals by the gene product to ensure its return to the endosymbiont for function. Many hundreds of plastid-derived genes must have acquired transit peptides for successful relocation to the nucleus. Here, we explore potential evolutionary origins of plastid transit peptides in the malaria parasite Plasmodium falciparum. We show that exons of the P. falciparum genome could serve as transit peptides after exon shuffling. We further demonstrate that numerous randomized peptides and even whimsical sequences based on English words can also function as transit peptides in vivo. Thus, facile acquisition of transit peptides from existing sequence likely expedited endosymbiont integration through intracellular gene transfer.

As more facts emerge regarding the ways in which parasite-derived molecules modulate the host immune response, it is possible to envisage how a lack of infection by agents that once infected humans commonly might contribute to the rise in autoimmune disease. Through effects on cells of both the innate and adaptive arms of the immune response, parasites can orchestrate a range of outcomes that are beneficial not only to parasites, in terms of facilitating their life cycles, but also to their host, in limiting pathology.

Carotenoid compositions of two freshwater Gammarus species (Crustacea: Amphipoda) and of their common acanthocephalan parasite Polymorphus minutus were characterized. The effect of carotenoid uptake by the parasite was addressed by comparing the carotenoid content of uninfected and infected female hosts. Using high-pressure liquid chromatography (HPLC), co-chromatography of reference pigments and electron ionization mass spectrometry of collected HPLC fractions (EI-MS), several xanthophylls and non-polar compounds were identified. Seven kinds of carotenoids, mainly xanthophylls, were identified in gammarids. Astaxanthin was predominant, amounting to 40 wt.% of total carotenoid in both uninfected G. pulex and G. roeseli. By contrast, we found only non-polar compounds with a predominance of esterified forms of astaxanthin in P. minutus larvae. No significant effect of infection on carotenoid content was evidenced in G. pulex and G. roeseli females. Our study highlights the use of a Matrix Solid Phase Dispersion as an efficient extraction method of both xanthophylls and non-polar pigments in small samples, including lipid-rich ones as P. minutus parasite. We discuss on the presumptive pathway leading to the formation of free astaxanthin in gammarids via hydroxy compounds, and on the accumulation of esters of astaxanthin in parasites.

We study the dynamics of a model that describes the competitive interaction between an invading species (a parasite) and its antibodies in an living being. This model was recently used to examine the dynamical competition between Tripanosoma cruzi and its antibodies during the acute phase of Chagas' disease. Depending on the antibody properties, the model yields three types of outcomes, corresponding, respectively, to healing, chronic disease, and host death. Here, we study the dynamics of the parasite-antibody interaction with the help of simulations, obtaining phase trajectories and phase diagrams for the system. We show that, under certain conditions, the size of the parasite inoculation can be crucial for the infection outcome and that a retardation in the stimulated production of an antibody species may result in the parasite gaining a definitive advantage. We also find a criterion for the relative sizes of the parameters that are required if parasite-generated decoys are indeed to help the invasion. Decoys may also induce a qualitatively different outcome: a limit cycle for the antibody-parasite population phase trajectories.

A palladium(II)-catalyzed hydroxycyclization-carbonylation-lactonization sequence with appropriate pent-4-ene-1,3-diols provides efficient access to the bicyclic gamma-lactones, 5-n-butyl- and 5-n-hexyltetrahydrofuro-[3,2-b]furan-2(3H)-ones (3) and (4), respectively, in both racemic and enantiomeric forms. Some of the substrate pent-4-ene-1,3-diols of high enantiomeric excess (ee) have been derived from racemic terminal epoxides by hydrolytic kinetic resolution (HKR) using cobalt (III)-salen complexes. (9Z,12R)-(+)-Ricinoleic acid also serves as a "chiral pool" source of other pent-4-ene-1,3-diols. These syntheses and enantioselective gas chromatography confirm the structures and absolute stereochemistry of the lactones in some species of parasitic wasps (Hymenoptera: Braconidae). The highly abundant 5-n-hexyltetrahydrofuro-[3,2-b]furan-2(3H)-one (4) in Diachasmimorpha kraussii and D. longicaudata is of high ee (>99%) with (3aR,5R,6aR) stereochemistry.

Parasitic helminth infections are characterized by eosinophilia and markedly elevated levels of circulating antigen-nonspecific immunoglobulin E (IgE), responses from which concern helminth protection. We previously purified a factor from Dirofilaria immitis that induces antigen-nonspecific IgE in mice and rats. Recombinant DiAg (rDiAg) has various biological activities. It is also known that parasitic helminth infection generates tremendous Th2 responses. The nonobese diabetic (NOD) mouse spontaneously develops Th1 cell-dependent autoimmune diabetes. Here we investigated the effects of rDiAg on the initiation and progression of this disease. rDiAg treatment of 6-week-old NOD females (the age at which insulitis typically begins) completely prevented insulitis and diabetes. Thus, rDiAg impaired the islet Ag-specific Th1 cell response in vivo, and the prevention of diabetes by rDiAg was associated with switching of the response from a Th1 to a Th2 profile. Since rDiAg clearly prevented insulitis by inhibiting the development and further accumulation of pathogenic Th1 cells to islets of Langerhans, we conclude that DiAg is a native Th2 inducer in filarial helminth and that Th1 responses are required for early events in the development of spontaneous autoimmune diabetes. In conclusion, the presence of parasitic helminth infections may play an important role as an immunomodulator in some autoimmune diseases or allergies., (Copyright 2001 Academic Press.)

Glycoconjugates are abundant and ubiquitious on the surface of many protozoan parasites. Their tremendous diversity has implicated their critical importance in the life cycle of these organisms. This review highlights our current knowledge of the major glycoconjugates, with particular emphasis on their structures, of representative protozoan parasites, including Leishmania, Trypanosoma, Giardia, Plasmodia, and others.

Now in the time especially intensive development of industry the question of environment pollution became very important. According to review of the problem heavy metals are one of the most hazardous constituents of xenobiotics. Their toxic effects, among other, are manifested directly on the elements of the immune system as well on the modification of the immune response. The influence of heavy metals on the host-parasite system is rather poorly recognized and here we are discused about it, according to literature and own experience. Special attention is paying to their impact on the immune response of infected and treated with lead, cadmium or mercury host and on the parasite. The definitive host eg. fishes and their some parasite can be used as potential accumulation indicators of aquatic environment of heavy metal pollution.

Prostaglandins have been already pretty well recognized as metabolic regulators in vertebebrata tissues mainly in mammals. I.ess reports concerned the occurrence of prostaglandins in invertebrates. In the present review we summarise literature data about the presence of prostaglandins and other eicosanoids in various groups of parasites and their possible role in host-parasite interaction. Prostaglandins have also been found in very primitive organisms as bacteria, varions plants and protozoa. We summarise that prostaglandins seem to be a very ancient group, going back to the roots of evolution. They are as universal in cell physiology as DNA in genetics. In host-parasiter eicosanoids also parasitic origin, play an important role as a modulators of hosts immune responsiveness.

The serpin superfamily of proteins has expanded rapidly in recent years as represented by the ovalbumin-type serpin subfamily. PCR methods have been used to identify new ovalbumin-type serpins, including leupin or SCCA-2, a close relative of SCCA. Although in vitro inhibition of proteases by these serpins has been shown, and we have evidence that leupin can protect cells against apoptosis, the exact role of these serpins is not well understood. Finding relevant targets is a major challenge in serpin biology, and we have investigated the yeast-2-hybrid trap for identification of new interactions. Preliminary studies suggest that serpins are at the upper limit for bait size, but this technique may be useful for identifying interactions where full-length serpin protein is not required.

A little over a decade ago, novel immunostimulating complexes (ISCOMs) were described. This review examines the position and progress that ISCOM technology has achieved in the fields of vaccine research and medicine over this period. Much of the work on ISCOMs has remained in the area of vaccine research where there is still an urgent need for improved adjuvants to help combat important diseases such as AIDS, malaria and influenza. Currently the only widely licensed adjuvants for human use are the aluminium salts, but with the trend towards highly purified subunit vaccines, which are inherently less immunogenic than some of the older vaccines, potent adjuvants capable of promoting specific immune responses are required. ISCOMs are one such technology that offers many of these requirements and as their use in vaccines enters its second decade clinical trials are commencing that will establish whether these submicron, non-living particles composed of saponin, cholesterol, phospholipid and in many cases protein, are useful components for a range of human vaccines.

Ubiquinone (coenzyme Q, CoQ) was analyzed and individual homologues quantified in 11 species of parasitic and free-living protozoa by a combination of thin-layer chromatography and high performance liquid chromatography. Fast atom bombardment ionization-mass spectrometry was used for the first time to confirm the identity of the fractionated CoQ homologues and proved to be a fast, gentle and convenient method for ubiquinone identification. Ubiquinone was detected in all organisms including those devoid of identifiable mitochondria. However, significantly lower levels of CoQ were present in those organisms lacking this respiratory organelle (5- to 50-fold lower in Entamoeba histolytica (CoQ9) and 15- to 350-fold for Giardia lamblia (CoQ9) and Tritrichomonas foetus (CoQ10)). Coenzyme Q9 was the predominant homologue in promastigotes of Leishmania donovani and Leishmania major. Lower amounts of CoQ8 and CoQ10 were also detected in L. donovani, and CoQ8 in L. major. Comparison of the in vitro cultivated promastigote and amastigote forms of Leishmania pifanoi and Leishmania amazonensis revealed CoQ9 to be the sole detectable ubiquinone homologue in the amastigote (macrophage) stage, whereas CoQ8 and CoQ10 were also present in the promastigotes (life cycle stage found in the insect gut) of L. pifanoi, and CoQ7 and CoQ8 in promastigotes of L. amazonensis. Interestingly, the total amounts of CoQ were similar in both forms of these organisms. The free-living ciliates, Tetrahymena thermophila and Paramecium tetraurelia contained CoQ8 as the predominant ubiquinone species and this homologue was also present in the isolated cilia from P. tetraurelia. The marine ciliate, Parauronema acutum contained CoQ7 as well as CoQ8. Comparison of xenosome-containing P. acutum with organisms lacking the symbiont revealed that twice the level of CoQ8 was present in cells grown with this cytoplasmic gram-negative bacterium. Results suggest that CoQ is ubiquitous amongst the protozoa, regardless of the presence of mitochondria, and may function in alternative roles to that of mitochondrial electron transport chain component.