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The infection caused by Entamoeba histolytica is still a serious public health problem, especially in developing countries. The goal of this study was to evaluate the effect of terfenadine against Entamoeba histolytica. The trophozoites were exposed to 1, 2, 3, and 4 μM of terfenadine, for 24 and 48 h. Consequently, the viability of cells was determined by trypan blue exclusion test. The effect of terfenadine on adhesion of Entamoeba histolytica was evaluated in Caco-2 cells. In addition, the effect of terfenadine on the erythrophagocytic capacity of the parasite was investigated. The results show that terfenadine affects the growth and cell viability in a time- and dose-dependent manner. The higher inhibitory effects were observed with 4 µM at 48 h; 91.6% of growth inhibition and only 22.5% of trophozoites were viable. Additionally, we demonstrate that terfenadine is highly selective for the parasite and has low toxicity on Caco-2 cells. Furthermore, adhesion to Caco-2 cells and erythrophagocytic capacity were significantly inhibited. These findings demonstrate that terfenadine exerts significant effects on the virulence of Entamoeba histolytica. This is the first study demonstrating the amoebicidal activity of terfenadine and the results suggest it may be effective in the treatment of amoebiasis.

Porcine reproductive and respiratory syndrome (PRRS) is an infectious disease characterized by severe reproductive failure in sows, acute respiratory disorders in growing pigs, and high mortality in piglets. The causative agent of this syndrome is the PRRS virus (PRRSV), an RNA virus belonging to the Arteriviridae family. To date, several quantitative approaches of proteomics have been applied to analyze the gene expression profiles during PRRSV infection in PAMs and MARC-145 cells, and few proteins have been consistent among independent studies, probably due to the differences in the levels of virulence of different PRRSV strains used and/or due to analytical conditions. In this study, total proteins isolated from noninfected and infected MARC-145 cells with a Mexican PRRSV strain were relatively quantified using label-free based DIA approach in combination with ion-mobility separation. As a result, 1456 quantified proteins were found to be shared between the control and infected samples. Afterward, these proteins were filtered, and 699 of them were considered without change. Also, 17 proteins were up-regulated and 19 proteins were down-regulated during the PRSSV infection. Bioinformatic analysis revealed that many of the differentially expressed proteins are involved in processes like antigen processing, presentation of antigens, response to viruses, response to IFNs, and innate immune response, among others. The present work is the first one which provides a detailed proteomic analysis through label-free based DIA approach in MARC-145 cells during the infection with a Mexican PRRSV strain.

Objective: To describe the management strategies of controlled suckling and the male effect to reduce postpartum anestrus, and to show the participation of kisspeptin to regulate the effect of both factors. Design/Methodology/Approach: A review of scientific publications was conducted, in order to show the importance of suckling and the male effect as strategies to reduce postpartum anestrus in the ewe, as well as the relation of kisspeptin with both factors. Results: Seasonal anestrus can be avoided with the use of breeds adapted to the local environment, such as Pelibuey. Postpartum anestrus occurs mainly as a result of suckling, since the latter inhibits the pulsating secretion of the gonadotropin-releasing hormone (GnRH) and the luteinizing hormone (LH). The exact path of this inhibition is unknown, although it seems that endogenous opioid peptides and kisspeptin are intermediaries. Controlled suckling and the male effect are management strategies that improve the reproductive behavior of postpartum ewes. Kisspeptin regulates the influence of the male effect through the secretion of GnRH/LH. Study Limitations/Implications: To understand the impacts of suckling and the male effect on the duration of postpartum anestrus, as well as the participation of kisspeptin in the regulation of both effects, will allow designing management strategies to improve the reproductive efficiency of the ewes. Findings/Conclusions: Controlled suckling and the male effect reduce postpartum anestrus and improve the reproductive behavior of the ewes; advancing knowledge of the kisspeptin effect could improve the effectiveness of both techniques.

Extracellular vesicles, EVs, are a heterogeneous complex of lipidic membranes, secreted by any cell type, in any fluid such as urine. EVs can be of different sizes ranging from 40-100 nm in diameter such as in exosomes to 100-1000 nm in microvesicles. They can also contain different molecules that can be used as biomarkers for the prognosis and diagnosis of many diseases. Many techniques have been developed to characterize these vesicles. One of these is flow cytometry. However, there are no existing reports to show how to quantify the concentration of EVs and differentiate them by size, along with biomarker detection. This work aims to describe a procedure for the isolation, quantification, and phenotypification of urinary extracellular vesicles, uEVs, using a conventional cytometer for the analysis without any modification to its configuration. The method's limitations include staining a maximum of four different biomarkers per sample. The method is also limited by the amount of EVs available in the sample. Despite these limitations, with this protocol and its subsequent analysis, we can obtain more information on the enrichment of EVs markers and the abundance of these vesicles present in urine samples, in diseases involving kidney and brain damage.

B lymphocytes are recognized for their crucial role in the adaptive immunity since they represent the only leukocyte lineage capable of differentiating into Ab-secreting cells. However, it has been demonstrated that these lymphocytes can exert several Ab-independent functions, including engulfing and processing Ags for presentation to T cells, secreting soluble mediators, providing co-stimulatory signals, and even participating in lymphoid tissues development. Beyond that, several reports claiming the existence of multiple B cell subsets contributing directly to innate immune responses have appeared. These “innate-like” B lymphocytes, whose phenotype, development pathways, tissue distribution, and functions are in most cases notoriously different from those of conventional B cells, are crucial to early protective responses against pathogens by exerting “crossover” defensive strategies that blur the established boundaries of innate and adaptive branches of immunity. Examples of these mechanisms include the rapid secretion of the polyspecific natural Abs, increased susceptibility to innate receptors-mediated activation, cytokine secretion, downstream priming of other innate cells, usage of specific variable immunoglobulin gene-segments, and other features. As these new insights emerge, it is becoming preponderant to redefine the functionality of B cells beyond their classical adaptive-immune tasks.

Objectives This study was undertaken to investigate the amoebicidal potential of curcumin on Entamoeba histolytica, as well as its synergistic effect with metronidazole. Methods Entamoeba histolytica trophozoites were exposed to 100, 200 and 300 μm of curcumin, for 6, 12 and 24 h. Consequently, the viability of cells was determined by trypan blue exclusion test. All specimens were further analysed by scanning electron microscopy. For drug combination experiment, the Chou-Talalay method was used. Key findings Curcumin affected the growth and cell viability in a time- and dose-dependent manner. The higher inhibitory effects were observed with 300 μm at 24 h; 65.5% of growth inhibition and only 28.8% of trophozoites were viable. Additionally, curcumin also altered adhesion and the morphology of the trophozoites. Scanning electron microscopy revealed treated trophozoites with damages on the membrane, size alterations and parasites with loss of cellular integrity. In addition, the combination of curcumin + metronidazole exhibited a synergistic effect; the activity of both drugs was improved. Conclusions This is the first report evaluating the effectiveness of curcumin against E. histolytica. Our results suggest that CUR could be considered for evaluation in future pharmacological studies as a promising amoebicidal agent or as complementary therapy.

This study tested whether the intravenous application of kisspeptin can stimulate the pulsatile secretion of LH in suckling ewes during postpartum anestrus. Ten days after lambing, Pelibuey ewes were allocated among two groups: (1) continuous suckling (n = 8), where the lambs remained with their mothers, and (2) restricted suckling (n = 8), where the mothers suckled their lambs twice daily for 30 min. On Day 19 postpartum, the ewes were individually penned with ad libitum access to water and feed and given an indwelling catheter in each jugular vein. On Day 20, 4 mL of blood was sampled every 15 min from 08:00 to 20:00 h to determine LH pulse frequency. At 14:00 h, four ewes in each group received 120 μg of kisspeptin diluted in 3 mL of saline as a continuous infusion for 6 h, the remaining four ewes in each group received only saline. The interaction between kisspeptin and suckling type did not affect LH pulse frequency (p &gt, 0.05). Before kisspeptin administration, pulse frequency was similar in all groups (1.50 ± 0.40 pulses per 6 h, p &gt, 0.05). With the application of kisspeptin, pulse frequency increased to 3.50 ± 0.43 pulses per 6 h (p ≤ 0.014), so the concentration of LH (1.11 ± 0.14 ng mL−1) was greater in kisspeptin-treated ewes than in saline-treated ewes (0.724 ± 0.07 ng mL−1, p ≤ 0.040). The frequency of LH pulses was greater with restricted suckling than with continuous suckling (2.44 ± 0.29 versus 1.69 ± 0.29 pulses per 6 h, p ≤ 0.040). We conclude that intravenous application of kisspeptin increases the pulsatile secretion of LH in suckling ewes and that suckling might reduce kisspeptin neuronal activity, perhaps explaining the suppression of ovulation. Moreover, the effects of kisspeptin and suckling on pulsatile LH secretion appear to be independent, perhaps operating through different neural pathways.

Infection with the enteric protozoan Entamoeba histolytica is still a serious public health problem, especially in developing countries. Amoebic liver abscess (ALA) is the most common extraintestinal manifestation of the amoebiasis, and it can lead to serious and potentially life-threatening complications in some people. ALA can be cured by metronidazole (MTZ), however, because it has poor activity against luminal trophozoites, 40&ndash, 60% of treated patients get repeated episodes of invasive disease and require repeated treatments that can induce resistance to MTZ, this may emerge as an important public health problem. Anti-virulence strategies that impair the virulence of pathogens are one of the novel approaches to solving the problem. In this study, we found that low doses of curcumin (10 and 50 &mu, M) attenuate the virulence of E. histolytica without affecting trophozoites growth or triggering liver injury. Curcumin (CUR) decreases the expression of genes associated with E. histolytica virulence (gal/galnac lectin, ehcp1, ehcp5, and amoebapore), and is correlated with significantly lower amoebic invasion. In addition, oxidative stress is critically involved in the etiopathology of amoebic liver abscess, our results show no changes in mRNA expression levels of superoxide dismutase (SOD) and catalase (CAT) after E. histolytica infection, with or without CUR. This study provides clear evidence that curcumin could be an anti-virulence agent against E. histolytica, and makes it an attractive potential starting point for effective treatments that reduce downstream amoebic liver abscess.

B lymphocytes are a leukocyte subset capable of developing several functions apart from differentiating into antibody-secreting cells. These processes are triggered by external activation signals that induce changes in the plasma membrane properties, regulated by the formation of different lipid-bilayer subdomains that are associated with the underlying cytoskeleton through different linker molecules, thus allowing the functional specialization of regions within the membrane. Among these, there are tetraspanin-enriched domains. Tetraspanins constitute a superfamily of transmembrane proteins that establish lateral associations with other molecules, determining its activity and localization. In this study, we identified TSPAN33 as an active player during B-lymphocyte cytoskeleton and plasma membrane-related phenomena, including protrusion formation, adhesion, phagocytosis, and cell motility. By using an overexpression model of TSPAN33 in human Raji cells, we detected a specific distribution of this protein that includes membrane microvilli, the Golgi apparatus, and extracellular vesicles. Additionally, we identified diminished phagocytic ability and altered cell adhesion properties due to the aberrant expression of integrins. Accordingly, these cells presented an enhanced migratory phenotype, as shown by its augmented chemotaxis and invasion rates. When we evaluated the mechanic response of cells during fibronectin-induced spreading, we found that TSPAN33 expression inhibited changes in roughness and membrane tension. Contrariwise, TSPAN33 knockdown cells displayed opposite phenotypes to those observed in the overexpression model. Altogether, our data indicate that TSPAN33 represents a regulatory element of the adhesion and migration of B lymphocytes, suggesting a novel implication of this tetraspanin in the control of the mechanical properties of their plasma membrane.

There are several drugs for the treatment of giardiasis; however, there is a tendency for patients to abandon treatment because of drug-related adverse effects, resulting in relapses, acquired resistance, and higher rates of treatment failure. Recently, we reported some podophyllotoxin-type lignans from Bursera fagaroides var. fagaroides showing antigiardial activity. In the present work, we demonstrated that 5'-desmethoxy-peltatin-A-methylether (5-DES), acetylpodophyllotoxin (APOD), and podophyllotoxin (POD) affect the distribution and staining pattern of microtubular structures on Giardia trophozoites. Virtual screening results revealed that the lignans act via binding in a hydrophobic pocket in the heterodimer interface of tubulin in Giardia. This study provides useful insight to understand the action mechanism of 5DES, APOD, and POD on Giardia lamblia. The optimization of these podophyllotoxin-type lignans will lead to promising candidates for antigiardial drugs.

The wild type huntingtin protein (Htt), supports the production of brain-derived neurotrophic factor (BDNF), a survival factor for striatal neurons, through cytoplasmic sequestering of RE-1silencing transcription factor (REST). In Huntington´s Disease an inherited degenerative disease, caused by a CAG expansion in the 5´coding region of the gene, the mutant huntingtin protein (mHtt), causes that REST enters pathologically into the nucleus of cells, resulting in the repression of neuronal genes including BDNF, resulting in the progressive neuronal death. It has been reported that Htt associates with Hsp90 and this interaction is involved in regulation of huntingtin aggregation. Discovering mechanisms to reduce the cellular levels of mutant huntingtin and REST provide promising strategies for treating Huntington disease. Here, we use the yeast two-hybrid system to show that N-terminus or REST interacts with the heat shock protein 90 (Hsp90) and identifies REST as an Hsp90 Client Protein. To assess the effects of Hsp90 we used antisense oligonucleotide, and evaluated the levels mHtt and REST levels. Our results show that direct knockdown of endogenous Hsp90 significantly reduces the levels of REST and mutant Huntingtin, decreased the percentage of cells with mHtt in nucleus and rescued cells from mHtt-induced cellular cytotoxicity. Additionally Hsp90-specific inhibitors geldanamicyn and PUH71 dramatically reduced mHtt and REST levels, thereby providing neuroprotective activity. Our data show that Hsp90 is necessary to maintain the levels of REST and mHtt, which suggests that the interactions between Hsp90-REST and Hsp90-Huntingtin could be potential therapeutic targets in Huntington's disease.

Giardia lamblia is a flagellated protozoan responsible for giardiasis, a worldwide diarrheal disease. The adverse effects of the pharmacological treatments and the appearance of drug resistance have increased the rate of therapeutic failures. In the search for alternative therapeutics, drug repositioning has become a popular strategy. Acetylsalicylic acid (ASA) exhibits diverse biological activities through multiple mechanisms. However, the full spectrum of its activities is incompletely understood. In this study we show that ASA displayed direct antigiardial activity and affected the adhesion and growth of trophozoites in a time-dose-dependent manner. Electron microscopy images revealed remarkable morphological alterations in the membrane, ventral disk, and caudal region. Using mass spectrometry and real-time quantitative reverse transcription (qRT-PCR), we identified that ASA induced the overexpression of heat shock protein 70 (HSP70). ASA also showed a significant increase of five ATP-binding cassette (ABC) transporters (giABC, giABCP, giMDRP, giMRPL and giMDRAP1). Additionally, we found low toxicity on Caco-2 cells. Taken together, these results suggest an important role of HSPs and ABC drug transporters in contributing to stress tolerance and protecting cells from ASA-induced stress.

Objectives Lung metastasizing leiomyomatosis (LML) is an infrequently diagnosed pathology developed after sexual maturation, commonly preceded by uterine myomas. Symptoms can include difficulties to breathe, cough, dyspnea and pain, because of mechanical obstruction exerted by expanding local growing leiomyomas. Lung leiomyomas are normally detected by imaging studies, but nowadays the precise diagnosis demands histological characterization of biopsies obtained from the affected tissues. The purpose of the present study was to determine the presence of genomic alterations in circulating cells of LML. Methods Immunohistochemical characterization of a lung biopsy extracted by thoracoscopy was performed. Pathologic proliferative smooth muscle cells were observed in a major lung metastasizing nodule, with a growing pattern similar to a uterine myoma. The presence of cellular linages different to smooth muscle cells was discarded by testing the presence of a battery of molecular markers. Also, a normal karyotype was determine by GTG-banding cytogenetic study, but a high density microarray analysis revealed six submicroscopic chromosomal regions displaying genomic abnormalities: microduplications were detected on chromosomes 4, 14, 17 and 22; and microdeletions on chromosomes 8 and 10. Conclusion This study remarks the relevance of submicroscopic chromosomal analysis of unusual pathologic conditions such as Benign Metastasizing Leiomyomatosis. This propitiate a better understanding of the molecular basis on the development of the pathology, in order to reckon on minimally invasive diagnostic methods, and to design appropriate treatments.

After a joint lesion, high-impact exercise is a risk factor for the development of osteoarthritis (OA). The degradation of articular cartilage in OA has been associated with the activation of inflammatory cytokine signaling pathways. However, differences in cytokine expression in healthy and injured cartilage after exercise have not yet been analyzed. We used immunofluorescence and Western blot to study the expression of IL-1β and IL-10 in the articular cartilage of normal (N), sham-operated (S), and menisectomized (OA) rats subjected or not to high-impact exercise (E) for 3, 6, and 10 days (N, NE, S, SE, and OA groups). Cartilage integrity and proteoglycan content were only affected in the OA groups. Exercise increased the amount of IL-1β and IL-10 positive chondrocytes in NE and SE groups compared with non-exercised groups (N and S). The expression of IL-1β was up-regulated over time in the NE and OA groups, although in the late stages the increase was higher in the OA groups. In contrast, the expression of anti-inflammatory IL-10 was low in the OA group, whereas in the NE groups expression levels were higher at each time point analyzed. These results suggest that anti- and pro-inflammatory molecules in the cartilage might be tightly regulated to maintain the integrity of the tissue and that when this equilibrium is broken (when the meniscus is removed), the pro-inflammatory cytokines take over and OA develops.

Giardiasis, a diarrheal disease, is highly prevalent in developing countries. Several drugs are available for the treatment of this parasitosis; unfortunately, all of them have variable efficacies and adverse effects. Bursera fagaroides has been known for its anti-inflammatory and antidiarrheal properties in Mexican traditional medicine. We investigated the in vitro anti-giardial activities of four podophyllotoxin-type lignans from Bursera fagaroides var. fagaroides, namely, 5′-desmethoxy-β-peltatin-A-methylether (5-DES), acetylpodophyllotoxin (APOD), burseranin (BUR), and podophyllotoxin (POD). All lignans affected the Giardia adhesion and electron microscopy images revealed morphological alterations in the caudal region, ventral disk, membrane, and flagella, to different extents. Only 5-DES, APOD, and POD caused growth inhibition. Using the Caco-2 human cell line as a model of the intestinal epithelium, we demonstrated that APOD displayed direct antigiardial killing activity and low toxicity on Caco-2 cells. This finding makes it an attractive potential starting point for new antigiardial drugs.

B lymphocytes develop different immune-related functions apart from differentiating into antibody-secreting cells. These processes are triggered by external stimuli that activate them and induce changes in the physicochemical properties of their plasma membrane. These changes are regulated by the formation of discrete lipid-bilayer compartments, including the tetraspanin-enriched microdomains. Tetraspanins compose a superfamily of transmembrane proteins that establish lateral associations with other molecules, determining its activity. In this study, we identified TSPAN33 as an active player during B lymphocyte cytoskeleton and plasma membrane-related phenomena. By using an overexpression model of TSPAN33 in human Raji cells, we located this protein on different membrane organelles including Golgi and intracellular vesicles as observed by confocal microscopy. Additionally, these cells displayed diminished phagocytic and cell adhesion properties, due to aberrant surface expression of integrins. This phenotype also prove to enhance 2D and 3D migration in response to CCL21. Strikingly, when we evaluated the mechanical plasma membrane response of the cells using atomic force microscopy, during fibronectin-induced spreading, we found that TSPAN33 expression inhibited normal changes in membrane tension and roughness parameters. For that reason, we generate TSPAN33 knock-down cells that showed enhanced adhesive and phagocytic capacities when compared with control cells. Altogether, our data indicate that TSPAN33 represents an inhibitory element of the adhesion and migration of B cells, suggesting also a novel implication of this protein in the regulation of mechanical properties of the plasma membrane.

Giardia lamblia is a worldwide protozoan responsible for a significant number of intestinal infections. There are several drugs for the treatment of giardiasis, but they often cause side effects. Curcumin, a component of turmeric, has antigiardial activity; however, the molecular target and mechanism of antiproliferative activity are not clear. The effects of curcumin on cellular microtubules have been widely investigated. Since tubulin is the most abundant protein in the cytoskeleton of Giardia, to elucidate whether curcumin has activity against the microtubules of this parasite, we treated trophozoites with curcumin and the cells were analyzed by scanning electron microscopy and confocal microscopy. Curcumin inhibited Giardia proliferation and adhesion in a time-concentration-dependent mode. The higher inhibitory concentrations of curcumin (3 and 15μM) disrupted the cytoskeletal structures of trophozoites; the damage was evident on the ventral disk, flagella and in the caudal region, also the membrane was affected. The immunofluorescence images showed altered distribution of tubulin staining on ventral disk and flagella. Additionally, we found that curcumin caused a clear reduction of tubulin expression. By docking analysis and molecular dynamics we showed that curcumin has a high probability to bind at the interface of the tubulin dimer close to the vinblastine binding site. All the data presented indicate that curcumin may inhibit Giardia proliferation by perturbing microtubules.

Breast cancer is the principal cancer in women worldwide. Although there are serum tumor markers such as CEA and HER2, they are detected in advanced stages of the disease and used as progression and recurrence markers. Therefore, there is a necessity for the identification of new markers that might lead to an early detection and also provide evidence of an effective treatment. The aim of this work was to determine the differential protein expression profiles of four breast cancer cell lines in comparison to a normal control cell line by iTRAQ labelling and tandem mass spectrometry, in order to identify putative biomarkers of the disease. We identified 1,020 iTRAQ-labelled polypeptides with at least one peptide identified with more than 95% in confidence. Overexpressed polypeptides in all cancer cell lines were 78, whilst the subexpressed were 128. We categorised them with PANTHER program into biological processes, being the metabolic pathways the most affected. We detected six groups of proteins with the STRING program involved in DNA topology, glycolysis, translation initiation, splicing, pentose pathway, and proteasome degradation. The main subexpressed protein network included mitochondrial proteins involved in oxidative phosphorylation. We propose BAG6, DDX39, ANXA8 and COX4 as putative biomarkers in breast cancer. Biological significance We report a set of differentially expressed proteins in the MCF7 and T47D (Luminal A), MDA-MB-231 (Claudin low) and SK-BR-3 (HER2(+)) breast cancer cell lines that have not been previously reported in breast cancer disease. From these proteins, we propose BAG6, DDX39, ANXA8 and COX4 as putative biomarkers in breast cancer. On the other hand, we propose sets of unique polypeptides in each breast cancer cell line that can be useful in the classification of different subtypes of breast cancer. (C) 2015 Elsevier B.V. All rights reserved.

Background and Aims Helicobacter pylori iceA1 and iceA2 gene amplification is usually performed to identify mixed populations as both genes are apparently reportedly exclusive. However, some strains isolated from Mexico show both iceA genes. The aim of this study was to establish the frequency of these genes in Mexican isolates and genomic diversity of the H. pylori strains. Methods One hundred thirty six biopsies were obtained from 68 patients (39 children and 29 adults). The presence of H. pylori was confirmed in 3/18 children and 6/19 adults by culture. There were 93 clinical strains isolated from nine patients. Additionally, we studied 37 strains from a strain collection isolated from 10 patients. The strains were genotyped and dual iceA genes were identified by polymerase chain reaction (PCR) and amplicons were sequenced. In addition, an enterobacterial repetitive intergenic consensus PCR (ERIC-PCR) assay was performed as fingerprinting method. Results The genotypification of the H. pylori isolates indicated that all strains were vacA +; 86% babA2+ , 86% cagA+ , 82% vacA s1m1+ , 19% iceA1+, 9% iceA2+, and 72% of them carried both iceA1 and iceA2 genes . The ERIC-PCR profiling revealed that the strains clustered in eight genetic groups depending on the presence of iceA1, iceA2 or both. A basic local multiple alignment analysis of the nucleotide sequences revealed that the iceA1 and iceA2 genes exhibited no relevant similarity. Conclusion The results here showed the presence of triple-positive strains ( babA, cagA, vacA ) of H. pylori and strains carrying simultaneously both iceA1 and iceA2 genes.