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Giardia intestinalis is a human parasite that causes a diarrheal disease in developing countries. G. intestinalis has a cytoskeleton (CSK) composed of microtubules and microfilaments, and the Giardia genome does not code for the canonical CSK-binding proteins described in other eukaryotic cells. To identify candidate actin and tubulin cross-linking proteins, we performed a BLAST analysis of the Giardia genome using a spectraplakins consensus sequence as a query. Based on the highest BLAST score, we selected a 259-kDa sequence designated as a cytoskeleton linker protein (CLP259). The sequence was cloned in three fragments and characterized by immunoprecipitation, confocal microscopy, and mass spectrometry (MS). CLP259 was located in the cytoplasm in the form of clusters of thick rods and colocalized with actin at numerous sites and with tubulin in the median body. Immunoprecipitation followed by mass spectrometry revealed that CLP259 interacts with structural proteins such as giardins, SALP-1, axonemal, and eight coiled-coils. The vesicular traffic proteins detected were Mu adaptin, Vacuolar ATP synthase subunit B, Bip, Sec61 alpha, NSF, AP complex subunit beta, and dynamin. These results indicate that CLP259 in trophozoites is a CSK linker protein for actin and tubulin and could act as a scaffold protein driving vesicular traffic.

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.

During infection with Dengue virus (DENV), host cells undergo a series of detrimental functional changes, including profound remodelling and redistribution of cell membrane structures,1,4 which giv...

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.

The organic fraction of municipal solid waste (OFMSW), mainly composed of lignocellulosic polymers, is extremely complex. Therefore, it is necessary to apply pretreatments to remove the lignin content and decrease the cellulose crystallinity in order to use the OFMSW for gas biofuels production in the context of biorefineries from waste. This work focused on critically reviewing the conventional pretreatments applied to OFMSW, with the goal of improving the H2 production, as well as other biofuels in modern biorefineries. There are a wide variety of pretreatments that have successfully been used, mainly alkaline, milling and dilute acid. In addition, some research has focused on the recovery and reutilization of the alkali, acid or solvents after the pretreatment, to be incorporated into new cycles of production, minimizing the environmental impacts. Moreover, it would be necessary to incorporate analytical tools, in order to determine the sustainability of the biorefinery project. It is concluded that waste pretreatments could significantly contribute to increased yields of biogas fuels in organic waste-based biorefineries. Therefore, establishing preliminary stages for conditioning biomass or wastes is essential to improve the degradation of wastes and bio-product generation. © 2016 Society of Chemical Industry

A modified clay cup (cantarito) microbial fuel cell (C-MFCs) was designed to digest the biomass effluent from a nopal biogas (NBE). To improve the process, commercial acrylic varnish (AV) was applied to the C-MFCs. The experiment was performed as:Both-C-MFCs, painting of AV on both sides of the clay cup; In-C-MFCs, painting of AV on the internal side, and Out-C-MFCs painting of AV on the external side. The order for the maximum volumetric power densities were Both-C-MFCs (1841.99 mW/m3)>Out-C-MFCs (1023.74 mW/m3) >In-C-MFCs (448.90 mW/m3). The control experiment without applied varnish did not show a stable potential, supporting the idea that the acryloyl group in varnish could favor the performance. Finally, a 4-digits clock was powered with two, Both-C-MFCs connected in series; the microbial diversity in this format was explored and a well-defined bacterial community including members of the phyla Actinobacteria, Bacteroidetes, Firmicutes, Proteobacteria, Synergistetes and candidate division TM7 was found.

In microbial fuel cells (MFCs) efficient extracellular electron transfer microbes, also known as anode-respiring bacteria, play an important role on cell performance. This type of microbes can be developed by application of enrichment procedures. The objective of this study was to compare a chemical (only C, final terminal electron acceptor Fe(III)), an electrochemical (only E), and a hybrid method (H, i.e., E followed by 3 serial transfers in iron (III) citrate medium) enrichment methods departing from a saline–sodic soil inoculum. In the electrochemical enrichment procedure in an electrolysis cell, the inoculum was subjected to a continuous electrical stress continually by posing the cell at −150 mV/SCE (+94 mV/SHE). The only C enrichment method delivered powers superior to the only E one (higher values of PAn,max = 49 mW m−2 and PV,max = 558 mW m−3 of C compared to 33 and 379 of only E). Interestingly, overall resistance as determined by EIS was lower for only E (1240 Ω) than for only C (1632 Ω). Yet, the hybrid H method, showed electrochemical characteristics consistently superior to both only C and only E methods (higher PAn,max and PV,max, lower internal resistance). Further detailed electrochemical studies of only E-method showed that the anodic resistance decreased with the time of operation of the electrolysis cell that would be consistent with the adaptability/enrichment purpose of the method. Also, Cyclic voltammetry peaks with values close to those reported for bacterial cytochromes appeared with time of cell operation. To the best of our knowledge, this is the first time that it is reported that serial transfers with Fe(III) as electron acceptor to an inoculum previously enriched in an electrolysis cell, leads to improved characteristics of MFC and increased Fe(III)-reducing capability of the inoculum.

A mutation coding for the amino acid change E335 to K is frequently found in the hemagglutinin-neuraminidase (HN) gene of Urabe AM9 mumps viruses isolated during post-vaccination meningitis cases. To identify if this mutation modifies the biological activities of the HN glycoprotein, two variants of Urabe AM9 vaccine differing at amino acid 335 (HN-E335 and HN-K335) were isolated and their receptor-binding specificity was determined by means of competence assays. Pre-incubation of the viruses with sialic acids inhibited both syncytia formation in Vero cells and replication in SH-SY5Y cells. Thus, HN-K335 showed higher affinity towards sialylalpha2,6lactose, whereas HN-G335 preferred sialylalpha2,3lactose. These results are relevant because a high expression of sialylalpha2,6lactose in nerve cells was confirmed by means of Sambucus nigra lectin-cytochemistry. In addition, kinetics assays showed that HN-K335 and HN-E335 also differ in their hydrolysis rate (Vmax values of 37.5 vs. 3.5 nmol min-1mg-1, respectively). Therefore, HN-K335 variant presented a neuraminidase activity level 11-fold higher than that of HN-E335 variant. In conclusion, the mutation affects the receptor-binding and neuraminidase activities of Urabe AM9 mumps virus variants.

Mesenchymal stem cells (MSCs) have a differentiation potential towards osteoblastic lineage when they are stimulated with soluble factors or specific biomaterials. This work presents a novel option for the delivery of MSCs from human amniotic membrane (AM-hMSCs) that employs bovine bone matrix Nukbone (NKB) as a scaffold. Thus, the application of MSCs in repair and tissue regeneration processes depends principally on the efficient implementation of the techniques for placing these cells in a host tissue. For this reason, the design of biomaterials and cellular scaffolds has gained importance in recent years because the topographical characteristics of the selected scaffold must ensure adhesion, proliferation and differentiation into the desired cell lineage in the microenvironment of the injured tissue. This option for the delivery of MSCs from human amniotic membrane (AM-hMSCs) employs bovine bone matrix as a cellular scaffold and is an efficient culture technique because the cells respond to the topographic characteristics of the bovine bone matrix Nukbone (NKB), i.e., spreading on the surface, macroporous covering and colonizing the depth of the biomaterial, after the cell isolation process. We present the procedure for isolating and culturing MSCs on a bovine matrix.

A high rate of post-vaccinal aseptic meningitis for Urabe AM9 mumps virus strain is well documented. This strain is composed of two virus variants differing at the nt 1081 (A/G) region in the hemagglutinin-neuraminidase (HN) gene. An association of HN-A(1081) variant with neurovirulence has been proposed. In order to test for neurotropism we isolated the HN-A(1081) and HN-G(1081) virus variants from Urabe AM9 mumps virus vaccine. Sequential passages were performed in monkey kidney Vero cells and human neuroblastoma SH-SY5Y cells. Viral replication was determined by conventional and real-time RT-PCR. The results show that clone HN-A(1081) can replicate efficiently in both cell types. However, a defective replication of clone HN-G(1081), lacking its genetic marker, was observed after the third passage in neuroblastoma cells. Kinetics assays showed that clone HN-A(1081) replicates faster than clone HN-G(1081). Viral clones were also inoculated into the brains of newborn rats. Clone HN-A(1081) replicated 14 times, while clone HN-G(1081) merely duplicated its level over the initial inoculum. These results suggest that there is a selective replication of HN-A(1081) mumps virus variants in cells of nervous origin.

The type II voltage-dependent sodium chan- nel is present in neuronal cells, where it mediates the prop- agationofnerveimpulses.RestrictedexpressionofthetypeII sodium channel gene to neurons is due, at least in part, to binding of the repressor protein REST (also termed NRSF or XBR) to the RE1 (also called NRSE) sequence in the type II sodium channel gene. Previous studies have shown that a domain in REST containing eight GL1-Kru ̈ppel zinc finger motifs mediates DNA binding. Deletional and GAL4-fusion geneanalysesnowrevealrepressordomainsthatlieoutsideof the DNA-binding domain in both the amino and carboxyl termini of REST. Mutational analysis further identifies a single zinc finger motif in the carboxyl-terminal domain as beingessentialforrepressingtypeIIsodiumchannelreporter genes. These studies reveal two domains in REST that may mediate interactions with other proteins involved in restrict- ingexpressionofalargesetofgenestothevertebratenervous system.

Expression of the type II voltage-dependent sodium channel gene is restricted to neurons by a silencer element active in nonneuronal cells. We have cloned cDNA coding for a transcription factor (REST) that binds to this silencer element. Expression of a recombinant REST protein confers the ability to silence type II reporter genes in neuronal cell types lacking the native REST protein, whereas expression of a dominant negative form of REST in nonneuronal cells relieves silencing mediated by the native protein. REST transcripts in developing mouse embryos are detected ubiquitously outside of the nervous system. We propose that expression of the type II sodium channel gene in neurons reflects a default pathway that is blocked in nonneuronal cells by the presence of REST.

The hallmark of Alzheimer's disease is the pathological aggregation of tau proteins into paired helical filaments and neurofibrillary tangles. This paper evaluates the abnormal expression and localization of chimeric tau molecules at the plasma membrane of COS-7 cells and its relationship with tau polymerization. Overexpression of these proteins, in combination with either tau441 or tau391, induces tau to assemble into beta-pleated sheets that are recognized by Thiazin red. Immunoelectromicroscopy analysis revealed the presence of filaments close to the plasma membrane resembling those found in Alzheimer's disease. The capacity of plasma membrane-associated chimeric tau proteins to capture full length tau was increased in the presence of H(2)O(2) or okadaic acid treatments. This suggests that hyperphosphorylation or an oxidative environment could both influence the biochemical properties of the cell that lead to assembly of paired helical filaments. The altered localization of tau protein at the plasma membrane could play a key role in the assembly of pathological tau.

Objective: This study has aimed to study different culture systems that might stimulate an increase in cell proliferation of normal and osteoarthritis chondrocytes from articular cartilage in rat model. Material and Methods: Three culture systems using chondrocytes embedded in alginate beads were tested: chondrocytes cultured in Dulbecco’s modified Eagle’s medium (DMEM) as control, a co-culture system consisting of a monolayer of de-differentiated chondrocytes as a source of mitotic factors, and an enriched medium containing culture medium obtained from a monolayer of chondrocytes and DMEM. Normal and osteoarthritis chondrocytes were stained with 5-carboxyfluorescein diacetate succinimidyl ester and were cultured in each of the three systems. After 5 days of culture cell, proliferation was detected by flow cytometry. Chondrocyte phenotype was confirmed by collagen type II and MMP-3 expression. To determine possible molecules released into the medium by the cultured chondrocyte monolayer and which would probably be involved in cell proliferation, a study of mRNA and expression of transforming growth factor- β 1 (TGF- β 1), fibroblastic growth factor-2 (FGF-2), epidermal growth factor (EGF), platelet derived growth factor-A (PDGF-A) and insulin-like growth factor-1 (IGF-1) proteins was conducted. Results and Conclusions: Chondrocytes in the co-culture system or in enriched medium showed an increase in proliferation; only when osteoarthritis chondrocytes were cultured in enriched medium would they display a statistically significant increase in their proliferation rate and in their viability. When chondrocytes from the monolayer were analysed, differential mRNA expression of TGF- β 1 and IGF-1 was found during all passages, which suggests that these two growth factors might be involved in chondrocyte proliferation.

A point mutation from guanine (G) to adenine (A) at nucleotide position 1081 in the hemagglutinin-neuraminidase (HN) gene has been associated with neurovirulence of Urabe AM9 mumps virus vaccine. This mutation corresponds to a glutamic acid (E) to lysine (K) change at position 335 in the HN glycoprotein. We have experimentally demonstrated that two variants of Urabe AM9 strain (HN-A1081 and HN-G1081) differ in neurotropism, sialic acidbinding affinity and neuraminidase activity. In the present study, we performed a structure-function analysis of that amino acid substitution; the structures of HN protein of both Urabe AM9 strain variants were predicted. Based on our analysis, the E/K mutation changes the protein surface properties and to a lesser extent their conformations, which in turn reflects in activity changes. Our modeling results suggest that this E/K interchange does not affect the structure of the sialic acid binding motif; however, the electrostatic surface differs drastically due to an exposed short alpha helix. Consequently, this mutation may affect the accessibility of HN to substrates and membrane receptors of the host cells. Our findings appear to explain the observed differences in neurotropism of these vaccine strains.

Urabe AM9 mumps virus vaccine causes post-vaccination meningitis. Two variants of Urabe AM9 virus differ in their replication efficiency in human nerve cells, HN-A 1081 variant being more neurotropic than HN-G 1081 . The effect of interferon (IFN) on viral replication and transcription was analyzed. Priming of nerve cells with IFN reduced more significantly the replication of HN-G 1081 variant (from 10 2.5 to 10 1.3 TCID 50 ) than that of HN-A 1081 (from 10 3.5 to 10 2.6 TCID 50 ). IFN-priming also reduced the transcription of HN-G 1081 genes, but not of HN-A 1081 . The effect of viral infection on the transcription of cellular IFN responsive genes was analyzed. HN-A 1081 virus reduced the transcription of STAT1, STAT2, p48 and MxA in both unprimed and IFN-primed cells; whereas HN-G 1081 virus just reduced MxA transcription. Since rubulavirus V protein inhibits IFN signaling, the V mRNA was cloned and sequenced, finding that HN-G 1081 but not HN-A 1081 presented three extra G in the P/V edition site, producing the insertion of Gly156 in the V protein. Our results suggest that the replication efficiency of Urabe AM9 mumps virus variants is influenced by their sensitivity to interferon and their capacity to reduce the antiviral response.

RE1 silencing transcription factor/neuron-restrictive silencing factor (REST/NRSF) mediates transcriptional repression in many neuron-specific genes by interaction with the repressor element 1/neuron-restrictive silencing element (RE1/NRSE). This element has been identified at least in 20 neuron specific genes. REST/NRSF is highly expressed in non-neuronal tissues, where it is thought to repress gene transcription. We performed a BLAST search to look for the presence of RE1/NRSE elements in the rat cytochrome P450 genes. We identified the presence of RE1/NRSE element in the cytochrome P450 genes CYP1A1, 2A2, 2E1 and 3A2. Electrophoretic mobility shift assay and supershift assays were carried out to prove functionality of these sites and detect the interaction of REST/NRSF with this sequence. Cotransfection studies in PC12 cells with a plasmid containing the RE1 element of the CYP genes, cloned upstream of the minimal type II sodium channel promoter, in the presence of REST/NRSF, showed a marked expression inhibition of the CAT reporter gene. These data suggest that the RE1 elements that exist in these four CYP genes might be a target for the REST/NRSF transcription factor and such an interaction might play a role in the negative regulation of these genes.