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El proceso de ser hombre o mujer está inmerso en una trama, una red de relaciones, representaciones, discursos, prácticas, poderes, dominios, siempre atravesados por tiempo y espacio (diferenciados genéricamente). En este trabajo la construcción de género se sustenta y mantiene gracias a la relación que se da entre la «acción-discurso» y la «acción-práctica» Una acción discursiva que crea una «norma» de ser mujer, de comportarse, una «norma» que dispone cómo y cuándo deben ocuparse los espacios, en qué, cómo y dónde deben trabajar las mujeres y sobre todo cómo deben ser sus relaciones con los hombres. Y una acción práctica que por un lado mantiene esa «norma», y por otro la contradice constantemente, dejando huecos y grietas por los que algunas de estas mujeres se introducen, creando estrategias improvisadas de escape sobre los símbolos culturales más estáticos y férreos que mantienen las normas y los modelos. Lo que me propongo analizar en este artículo es cómo las mujeres «morenas» de la Costa Chica de Oaxaca, México, constituyen estrategias (conscientes e inconscientes) de escape de los mecanismos de normativización y control impuestos por las prácticas patriarcales que se ejercen en esta zona, siendo una de estas el establecimiento de relaciones «a-normales» dentro de las que se incluyen las «no-relaciones» con los hombres. Etre un homme ou une femme est un processus résultant d’une insertion dans un tissu social et un réseau de relations, de représentations, de discours, de pratiques, de rapports de pouvoir et de domination, toujours traversés par le temps et l’espace (différenciés en fonction du genre). Dans ce travail, la construction du genre est envisagée à partir de la relation qui existe entre « l’action-discours » et "l’action-pratique". L’action discursive crée une « norme » conditionnant la façon d’être une femme, de se comporter, la manière d’habiter l’espace et la temporalité de l’occupation de l’espace, le travail auquel les femmes peuvent accéder (en fonction du secteur, du lieu et du type d’activité) et surtout le rapport femmes-hommes. L’action pratique maintient cette « norme » tout en la remettant constamment en cause, laissant des creux et des interstices dans lesquels certaines femmes s’insinuent grâce à des stratégies improvisées. Cela leur permet d’échapper aux symboles culturels les mieux établis et les plus rigides qui sous-tendent ces normes et ces modèles. Cet article se propose d’analyser la manière dont les femmes à la peau foncée de la Costa Chica de l’Etat de Oaxaca au Mexique, mettent en place des stratégies (conscientes et inconscientes) pour s’affranchir des mécanismes de construction de normes et de contrôles imposés par les pratiques patriarcales en vigueur dans cette région. Parmi les relations « a-normales » entre hommes et femmes se trouve celle qui consiste à ne pas avoir de relations avec les hommes. Being a man or a woman is a process which results from belonging to a social fabric and to a network of relationships, representations, discourses, practices, and power relations which are always defined by time, space, and gender. In this article, gender construction is apprehended through the link between “discourse action” and “practice action”. Discourse action gives birth to a “norm” which defines what a woman is, how she behaves, how and when she can inhabit space, what type of work she can have access to (depending on the type and location and this professional activity), and, most importantly, the power relations between men and women. Practice action keeps this “norm” alive while constantly questioning it, which creates cracks through which some women enter thanks to improvised strategies. This allows them to escape the most established and rigid cultural symbols which frame these norms and models. This article studies the way dark-skinned women from the Costa Choca in the state of Oaxaca in Mexico come up with (both conscious and unconscious) strategies to free themselves from the mechanisms sustaining these norms which are imposed by the patriarchal practices of the region. Among “abnormal” relationships between men and women are these consisting in not having any relationship with men. O processo de ser homem ou de ser mulher está imerso em uma trama, uma rede de relações, representações, discursos, práticas, poderes, domínios, sempre atravessado pelo tempo e pelo espaço (diferenciados genericamente). Neste trabalho a construção de gênero se sustenta e se mantém graças à relação que existe entre a “ação-discurso” e a “ação-prática”. Uma ação discursiva que cria uma norma para o ser mulher, um comportamento, uma norma que indica como e quando devem se ocupados os espaços, em que, como e onde devem trabalhar as mulheres e, sobretudo, como devem ser suas relações com os homens. Uma ação prática que por um lado mantem a norma e por outro a contradiz constantemente, deixando vazios e fendas, nas quais algumas destas mulheres se introduzem, criando estratégias improvisadas de fuga por símbolos culturais mais estáticos e inflexíveis onde se mantem normas e modelos. O que proponho aqui é analisar como as mulheres “morenas” da Costa Chica de Oaxaca, México, formam estratégias (conscientes e inconscientes) para esquivar os mecanismos de normatização e controle impostos pelas práticas patriarcais que se exercem nesta zona, sendo uma delas o estabelecimento de relações “a-normais” dentre as quais se incluem as “não-relações” com homens.

Coffee is one of the most consumed beverages around the world, and as a consequence, spent coffee grounds are a massively produced residue that is causing environmental problems. Reusing them is a major focus of interest presently. We extracted mannooligosaccharides (MOS) from spent coffee grounds and submitted them to an in vitro fermentation with human feces. Results obtained suggest that MOS are able to exert a prebiotic effect on gut microbiota by stimulating the growth of some beneficial genera, such as Barnesiella, Odoribacter, Coprococcus, Butyricicoccus, Intestinimonas, Pseudoflavonifractor, and Veillonella. Moreover, short-chain fatty acids (SCFA) production also increased in a dose-dependent manner. However, we observed that 5-(hydroxymethyl)furfural, furfural, and polyphenols (which are either produced or released from the spent coffee grounds matrix during hydrolysis) could have an inhibitory effect on other beneficial genera, such as Faecalibacterium, Ruminococcus, Blautia, Butyricimonas, Dialister, Collinsella, and Anaerostipes, which could negatively affect the prebiotic activity of MOS.

The objective of this paper was to determine the water use efficiency (WUE) and light use efficiency (LUE) values of garlic (Allium sativum L.) and to analyze the normalization of these efficiencies for the atmospheric conditions. We analyzed the use of daily reference evapotranspiration (ETo) and the vapor pressure deficit (VPD) for these purposes. WUE and LUE were valued based on the experimental measurements of dry biomass production and the estimation of transpiration and radiation interception based on time series of Remote Sensing satellite images. The experimental data were obtained in two commercial fields each planted with a different variety of garlic (var. morado and var. spring) growing in different weather conditions in the province of Albacete (in the south-east of Spain). The correlation between the accumulated values of biomass and the estimated values of crop transpiration and radiation absorption resulted in strong linear relationships. The slope of these correlations resulted in values of WUE and LUE, WUE = 5.51 g m−2 mm−1 and LUE = 2.26 g·MJ−1 for both analyzed varieties. The results demonstrated that the inclusion of temperature stress coefficient is required when the WUE and LUE parameters are normalized using the ETo and the VPD variables. However, atmospheric normalization did not conclusively improve results in this study. Results obtained in this paper provided empirical evidence regarding the feasibility of estimating biomass production in garlic based on the same physiological principles used for other crops.

Cooking modifies food composition due to chemical reactions. Additionally, food composition shapes the human gut microbiota. Thus, the objective of this research was to unravel the effect of different food cooking methods on the structure and functionality of the gut microbiota. Common culinary techniques were applied to five foods, which were submitted to in vitro digestion-fermentation. Furosine, 5-(hydroxymethyl)furfural, and furfural were used as Maillard reaction indicators to control the heat treatment. Short-chain fatty acids production was quantified as indicator of healthy metabolic output. Gut microbial community structure was analyzed through 16S rRNA. Both food composition and cooking methods modified the microbiota composition and released short-chain fatty acids. In general, intense cooking technologies (roasting and grilling) increased the abundance of beneficial bacteria like Ruminococcus spp. or Bifidobacterium spp. compared to milder treatments (boiling). However, for some foods (banana or bread), intense cooking decreased the levels of healthy bacteria.

cope The purpose of the study was to evaluate the effect, regarding the metabolic and microbial profile of feces, of diet supplementation of healthy adults with pomegranate juice (PJ). Methods and results Twelve healthy adults were recruited to the study, which consisted of the intake of 200 mL/day of PJ during 4 weeks. Feces were collected before and after the supplementation with PJ. Metabolites (phenolic catabolites, short-chain fatty acids, and fecal steroids) and microbial profile were analyzed at baseline and at 4 weeks. Fecal phenolic metabolites, 3-phenylpropionic acid, catechol, hydroxytyrosol, and urolithin A, showed a significant increase in their concentration after supplementation with PJ. Among fecal steroids, parallel to the significant increase of cholesterol concentration, a significant decrease of coprostanol was observed. Although no significant changes in the microbiota profile were observed, different relationships between initial microbiota and the metabolites produced were found. Catechol showed positive and negative correlation with Oscillospora and Paraprevotella genera, respectively, and 3-phenylpropionic acid was positively correlated with Odoribacter genus. Conclusion Inclusion of PJ in the diet did not significantly alter the gut microbiota composition in healthy adults, but the individual bacterial composition could contribute to the generation of potential health-promoting phenolic metabolites.

Hepatotoxicity is the number one cause for agencies not approving and withdrawing drugs for the market. Drug-induced human hepatotoxicity frequently goes undetected in preclinical safety evaluations using animal models. Human-derived in vitro models represent a common alternative to in vivo tests to detect toxic effects during preclinical testing. Most current in vitro toxicity assays rely on the measurement of nonspecific or low sensitive endpoints, which result in poor concordance with human liver toxicity. Therefore, making more accurate predictions of the potential hepatotoxicity of new drugs remains a challenge. Metabolomics, whose aim is to globally assess all the metabolites present in a biological sample, may represent an alternative in the search for sensitive sublethal markers of drug-induced hepatotoxicity. To this end, a comprehensive LC-MS-based untargeted metabolite profiling analysis of HepG2 cells, exposed to a set of well-described model hepatotoxins and innocuous compounds, was performed. It allowed to determine meaningful metabolic changes triggered by a toxic insult and gave a first estimation of the main toxicity-related pathways. Based on these metabolic patterns, a partial least squares-discriminant analysis model, able to discriminate between nontoxic and hepatotoxic compounds, was constructed. The approach described herein may provide an alternative for animal testing in preclinical stages of drug development and a controlled experimental approach to gain a better understanding of the underlying causes of hepatotoxicity.

Drug-induced liver injury (DILI) has a considerable impact on human health and is a major challenge in drug safety assessments. DILI is a frequent cause of liver injury and a leading reason for post-approval drug regulatory actions. Considerable variations in the expression levels of both cytochrome P450 (CYP) and conjugating enzymes have been described in humans, which could be responsible for increased susceptibility to DILI in some individuals. We herein explored the feasibility of the combined use of HepG2 cells co-transduced with multiple adenoviruses that encode drug-metabolising enzymes, and a high-content screening assay to evaluate metabolism-dependent drug toxicity and to identify metabolic phenotypes with increased susceptibility to DILI. To this end, HepG2 cells with different expression levels of specific drug-metabolism enzymes (CYP1A2, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4, GSTM1 and UGT2B7) were exposed to nine drugs with reported hepatotoxicity. A panel of pre-lethal mechanistic parameters (mitochondrial superoxide production, mitochondrial membrane potential, ROS production, intracellular calcium concentration, apoptotic nuclei) was used. Significant differences were observed according to the level of expression and/or the combination of several drug-metabolism enzymes in the cells created ad hoc according to the enzymes implicated in drug toxicity. Additionally, the main mechanisms implicated in the toxicity of the compounds were also determined showing also differences between the different types of cells employed. This screening tool allowed to mimic the variability in drug metabolism in the population and showed a highly efficient system for predicting human DILI, identifying the metabolic phenotypes associated with increased DILI risk, and indicating the mechanisms implicated in their toxicity. Electronic supplementary material The online version of this article (doi:10.1007/s00204-017-2036-4) contains supplementary material, which is available to authorized users.

HIV infection causes a disruption of gut-associated lymphoid tissue, driving a shift in the composition of gut microbiota. A deeper understanding of the metabolic changes and how they affect the interplay with the host is needed. Here, we assessed functional modifications of HIV-associated microbiota by combining metagenomic and metatranscriptomic analyses. The transcriptionally active microbiota was well-adapted to the inflamed environment, overexpressing pathways related to resistance to oxidative stress. Furthermore, gut inflammation was maintained by the Gram-negative nature of the HIV-associated microbiota and underexpression of anti-inflammatory processes, such as short chain fatty acid biosynthesis or indole production. We performed co-occurrence and metabolic network analyses that showed relevance in the microbiota structure of both taxonomic and metabolic HIV-associated biomarkers. The Bayesian network revealed the most determinant pathways for maintaining the structure stability of the bacterial community. In addition, we identified the taxa's contribution to metabolic activities and their interactions with host health., This work was supported by grants to AM from the Spanish Ministry of Economy and Competitiveness (projects SAF 2012-31187, SAF2013-49788-EXP, and SAF2015-65878-R), the Carlos III Institute of Health (projects PIE14/00045 and AC15/00022), and the Generalitat Valenciana (project PrometeoII/2014/065) and was co-financed by FEDER. This work was supported by the Instituto de Salud Carlos III (Plan Estatal de IþDþi 2013–2016, project PI15/00345 and the Spanish AIDS Research Network (RD16/0025/0001 project) and co-financed by the European Development Regional Fund “A way to achieve Europe” (ERDF). JFV-C was supported by a CONACYT-SECITI fellowship, México. C.B.; DR and MF were funded by the Spanish Ministry of Economy and Competitiveness (CTQ2014-55279-R and BIO2014- 54494-R). SSV was supported by a grant from the Spanish Ministry of Science and Innovation (Contratos Juan Rodés, ECC/1051/2013). SM was funded by the Hospital Universitario Ramón y Cajal. VE was funded by the Hospital Clínico San Carlos. MJG was supported by a grant from the Fomento de la Investigación Sanitaria y Biomédica de la Comunidad Valenciana (FISABIO) (UGP-14-116).

We report a direct and fast synthesis route to grow boron-carbon-nitrogen layers based on microwave-assisted plasma enhanced chemical vapour deposition (PECVD) by using methylamine borane as a single source molecular precursor. This easy and inexpensive method allows controlled and reproducible growth of B-C-N layers onto thin Cu foils. Their morphological, structural, chemical, optical and transport properties have been thoroughly characterized by a number of different microscopies, transport and spectroscopic techniques. Though disorder and segregation into C-rich and h-BN-rich domains have been observed in ultrathin flat few layers, high doping levels have been reached, inducing strong modifications of the electronic, optical and transport properties of C-rich and h-BN-rich phases. This synthesis procedure can open new routes towards the achievement of homogeneous highly mixed ternary B-C-N phases

The irritable bowel syndrome (IBS) is a functional gastrointestinal disorder with a largely unknown aetiology and a wide range of symptoms. Most cross-sectional studies carried out so far suggest subtle alterations in the structure of the intestinal microbiota that are barely reproduced, partly because of the high inter-subject variation in the community composition and disorder-specific features. We performed a longitudinal study to explore the within-subject variation in the faecal microbiota in two patients with IBS classified into the diarrhoea subtype and the healthy spouse of one of them. Faecal communities were monitored over 6-8 weeks and analysed through metagenomic and metatranscriptomic approaches. We found a higher temporal instability in the fraction of active microbiota related to the IBS condition and fluctuating symptoms. Strong and quick shifts in the distribution of the active microbiota and changes in the global pattern of gene expression were detected in association with acute diarrhoea, whereas microbial composition and encoded functions were more stable. The specific alterations in the microbiota were barely reproduced within and between patients. Further research is needed to assess whether these changes are a consequence of the abnormal gut function in acute diarrhoeic episodes and the potential usefulness of tackling them.

Electron tomography (ET) is an indispensable high-resolution tool for three dimensional (3D) imaging in cell biology. When applied to immuno-labeled cells, ET can provide essential insights in both the cellular architecture and the dynamics. Current protocols for 3D immuno-labeling of intracellular antigens include permeabilization steps that cause random, extensive cell membrane disruption. This permeabilization results in a poor cell ultrastructure, limiting the usefulness of the specimens for high-resolution studies. Here we describe a novel method, based on a well-controlled permeabilization by targeted laser cell perforation, that allows for the 3D immuno-localization of cytoplasmic antigens in cultured cells. The approach is unique since it is applicable to both chemically and cryo-fixed cells and leads to a superior ultrastructural preservation for electron microscopy and tomography.

In the last decade, an extensive effort has been made to characterize the human intestinal microbiota by means of SSU rRNA gene sequence and metagenomic analysis. Relatively few studies have followed intestinal bacterial communities over time to assess their stability in the absence of perturbation. In this study, we have monitored the faecal bacteria of three healthy subjects during 15 consecutive days. The global community structure was analysed through SSU rRNA gene sequencing. In agreement with previous studies, we found that the between-subject variation in community structure was larger than within. The composition was fairly stable throughout, although daily fluctuations were detected for all genera and phylotypes at 97% of sequence identity. While the core shared between subjects was very small, each subject harboured a stable high-abundance core composed of a small number of bacterial groups (9% of the phylotypes accounted for between 74% and 93% of the sequences). This may suggest that studies aimed at linking the microbiota composition with disease risk should be limited to the numerically most dominant phylotypes, as the rest appears transient. Networks of potential interactions between co-occurring genera were also subject-specific, even for the same bacterial genus, which might be reflecting host-specific selective pressures and historic events.

Summary Irritable bowel syndrome (IBS) is the most common functional gastrointestinal disorder in western coun- tries. Previous studies on IBS, mostly based on faecal samples, suggest alterations in the intestinal micro- biota. However, no consensus has been reached regarding the association between specific bacteria and IBS. We explore the alterations of intestinal bac- terial communities in IBS using massive sequencing of amplified 16S rRNA genes. Mucosal biopsies of the ascending and descending colon and faeces from 16 IBS patients and 9 healthy controls were analysed. Strong inter-individual variation was observed in the composition of the bacterial communities in both patients and controls. These communities showed less diversity in IBS cases. There were larger differ- ences in the microbiota composition between biop- sies and faeces than between patients and controls. We found a few over-represented and under

Gut microbiota is the most complex bacterial community in the human body and its study may give important clues to the etiology of different intestinal diseases. Most studies carried out so far have used fecal samples, assuming that these samples have a similar distribution to the communities present throughout the colon. The present study was designed to test this assumption by comparing samples from the rectal mucosa and feces of nine healthy volunteers by sequencing libraries of 16S rRNA genes. At the family taxonomic level, where rarefaction curves indicate that the observed number of taxa is close to the expected one, we observe under different statistical analyses that fecal and mucosal samples cluster separately. The same is found at the level of species considering phylogenetic information. Consequently, it cannot be stated that both samples from a given individual are of similar composition. We believe that the evidence in support of this statement is strong and that it would not change by increasing the number of individuals and/or performing massive sequencing. We do not expect clinicians to stop using feces for research, but we think it is important to caution them on their potential lack of representativeness with respect to the bacterial biofilm on the rectal mucosa.

Caveolae are invaginations of the plasma membrane involved in multiple cellular processes, including transcytosis. In this paper we present an extensive 3-D electron tomographic study of the endothelial caveolar system in situ. Analysis of large cellular volumes of (high-pressure frozen, freeze-substituted and epon-embedded) human umbilical vein endothelial cells (HUVECs) provided a notable view on the architecture of the caveolar system that comprises – as confirmed by 3-D immunolabeling for caveolin of ‘intact’ cells –bona fide caveolae, free plasmalemmal vesicles, racemose invaginations and free multi-caveolar bodies. Application of template matching to tomograms allowed the 3-D localization of caveolar membrane coatings in a robust manner. In this way we observed that bona fide endothelial caveolae, cryofixed and embedded in their cellular context, show a spiral organization of the coating as shown in the past for chemically fixed and freeze-etched caveolae from fibroblasts. Meticulous 3-D analysis further revealed that the coatings are distributed in triads of spirals over the caveolar bulb and neck. Remarkably, this coating distribution is consistently present over the membranes of the other members of the caveolar system in HUVECs. The novel observations that we present clarify the ultrastructural complexity of the ‘intact’ caveolar system, setting a detailed morphological basis for its functional diversity.

article i nfo Article history: Bacillus thuringiensis Cry toxins are used in the control of insect pests. They are pore-forming toxins with a complex mechanism that involves the sequential interaction with receptors. They are produced as protoxins, which are activated by midgut proteases. Activated toxin binds to cadherin receptor, inducing an extra cleavage including helix α-1, facilitating the formation of a pre-pore oligomer. The toxin oligomer binds to secondary receptors such as aminopeptidase and inserts into lipid rafts forming pores and causing larval death. The primary threat to efficacy of Bt-toxins is the evolution of insect resistance. Engineered Cry1AMod toxins, devoid of helix α-1, could be used for the control of resistance in lepidopterans by bypassing the altered cadherin receptor, killing resistant insects affected in this receptor. Here we analyzed the mechanism of action of Cry1AbMod. We found that alkaline pH and the presence of membrane lipids facilitates the oligomerization of Cry1AbMod. In addition, tryptophan fluorescence emission spectra, ELISA binding to pure aminopeptidase receptor, calcein release assay and analysis of ionic-conductance in planar lipid bilayers, indicated that the secondary steps in mode of action that take place after interaction with cadherin receptor such as oligomerization, receptor binding and pore formation are similar in the Cry1AbMod and in the wild type Cry1Ab. Finally, the membrane-associated structure of Cry1AbMod oligomer was analyzed by electron crystallography showing that it forms a complex with a trimeric organization.

Hepatitis C virus (HCV) is a major health problem worldwide, infecting an estimated 170 million people. In this study, we have employed a large data set of sequences (14,654 sequences from between 25 and 100 clone sequences per analyzed region and per patient) from 67 patients infected with HCV genotype 1 (23 subtype 1a and 44 subtype 1b). For all patients, a sample prior to combined therapy with alpha interferon plus ribavirin was available, whereas for some patients additional samples after 6 or 12 months of treatment were also available. Twenty-seven patients responded to treatment (12 subtype 1a and 15 subtype 1b) and forty patients did not respond to treatment (11 subtype 1a vs. 29 subtype 1b). Two regions of the HCV genome were analyzed, one compressing the hypervariable regions (HVR1, HVR2 and HVR3) of the envelope 2 glycoprotein and another one including the interferon sensitive determining region (ISDR) and the V3 domain of the NS5A protein. Previously (Cuevas, J.M., Torres-Puente, M., Jiménez-Hernández, N., Bracho, M.A., García-Robles, I., Wrobel, B., Carnicer, F., del Olmo, J., Ortega, E., Moya, A., González-Candelas, F., 2008b. Genetic variability of hepatitis C virus before and after combined therapy of interferon plus ribavirin. Plos One 3 (8), e3058), several amino acid positions in both regions analyzed were detected to be under positive selection. Here, we have compared the amino acid composition of each positively selected position between responder and non-responder patients for both subtypes. If we exclude some non-conclusive cases, no clear differences were detected in any case. In conclusion, identifying specific positions as completely discriminatory of treatment response seems to be a difficult task. Our results, in concordance with previous studies, suggest that HCV evasion strategies are more likely based on a global increased variability, which would yield combinations of mutations with an increased resistance, than on the fixation of specific amino acids conferring resistance to antiviral treatment or immune response. In this sense, the particular systemic response from each patient could play an essential role in determining the outcome of the antiviral treatment.

In this technical note we report a tannic acid-mediated osmium impregnation method that, applied after freeze-substitution, increases membrane contrast in cells for transmission electron microscopy and tomography studies. The general staining that is achieved allows visualization of organelles, plasma membrane and associated specializations (e.g. caveolae) in non-post-stained plastic sections by conventional transmission electron microscopy. In combination with electron tomography it results in membranes with a proper contrast and equal staining pattern through the depth of the tomograms. The protocol that we contribute can serve as starting point for those willing to improve the membrane contrast of their specimens or to make 3D studies on the architecture of membranous compartments by electron tomography.

Different medicinal plants are widely used in Cuba and Mexico to treat several disorders. This paper reports in vitro inhibitory effects on the P450 system of herbal products commonly used by people in Cuba and Mexico in traditional medicine for decades. Experiments were conducted in human liver microsomes. The catalytic activities of CYP1A1/2, 2D6, and 3A4 were measured using specific probe substrates. The Heliopsis longipes extract exhibited a concentration-dependent inhibition of the three enzymes, and similar effects were produced by affinin (an alkamide isolated from the H. longipes extract) and two catalytically reduced alkamides. Mangifera indica L. and Thalassia testudinum extracts, two natural polyphenol-rich extracts, diminished CYP1A1/2 and 3A4 activities, but not the CYP2D6 activity. These results suggest that these herbs inhibit the major human P450 enzymes involved in drug metabolism and could induce potential herbal-drug interactions.

The envelope 2 protein of hepatitis C virus (HCV) presents three hypervariable regions, named HVR1, HVR2 and HVR3, in which the presence of antigenic sites has been described. Genetic variability in these regions may reflect the generation of escape mutants as a consequence of the immune response. Therefore, these regions would tend to accumulate amino acid changes along the infection process, an effect that could be accelerated by antiviral treatments. In this study, we have analyzed the E1–E2 region of 23 HCV patients non-responders to antiviral treatment, 7 of which were infected with subtype 1a, 15 with subtype 1b, and 1 with a new HCV-1 subtype, before and after 6 and/or 12 months of peg-interferon + ribavirin treatment. We have sequenced about 100 clones from each sample, analyzing a total of 4906 sequences. A detailed analysis of the evolutionary forces acting along the genome region studied confirmed the existence of the three hypervariable regions, characterized by significant changes in amino acid composition between samples taken at different times from the same patient and a high number of sites evolving under positive selection. Moreover, for the recently described HVR3, our results suggest that its location could be restricted to residues 434–450, instead of the originally postulated 431–466.

The gastrointestinal tract (GIT) microbiota has been identified as an important reservoir of antibiotic resistance genes (ARGs) that can be horizontally transferred to pathogenic species. Maternal GIT microbes can be transmitted to the offspring, and recent work indicates that such transfer starts before birth. We have used culture-independent genetic screenings to explore whether ARGs are already present in the meconium accumulated in the GIT during fetal life and in feces of 1-week-old infants. We have analyzed resistance to β-lactam antibiotics (BLr) and tetracycline (Tcr), screening for a variety of genes conferring each. To evaluate whether ARGs could have been inherited by maternal transmission, we have screened perinatal fecal samples of the 1-week-old babies’ mothers, as well as a mother–infant series including meconium, fecal samples collected through the infant’s 1st year, maternal fecal samples and colostrum. Our results reveal a high prevalence of BLr and Tcr in both meconium and early fecal samples, implying that the GIT resistance reservoir starts to accumulate even before birth. We show that ARGs present in the mother may reach the meconium and colostrum and establish in the infant GIT, but also that some ARGs were likely acquired from other sources. Alarmingly, we identified in both meconium and 1-week-olds’ samples a particularly elevated prevalence of mecA (>45%), six-fold higher than that detected in the mothers. The mecA gene confers BLr to methicillin-resistant Staphylococcus aureus, and although its detection does not imply the presence of this pathogen, it does implicate the young infant’s GIT as a noteworthy reservoir of this gene.

Summary. Hepatitis C virus (HCV) is a major health problem worldwide, infecting an estimated 170 million people. The high genetic variability of HCV contributes to the chronicity of hepatitis C. Here, we report results from a large-scale sequence analysis of 67 patients infected with HCV genotype 1, 23 with subtype 1a and 44 with subtype 1b. Two regions of the HCV genome were analysed in samples prior to combined therapy with alpha interferon plus ribavirin, one compressing the hypervariable regions (HVR1, HVR2 and HVR3) of the E2 glycoprotein and another one including the interferon-sensitive determining region (ISDR) and the V3 domain of the NS5A protein. Genetic diversity measures showed a clear tendency to higher genetic variability levels in nonresponder patients to antiviral treatment than in responder patients, although highly disperse values were present within each response group for both subtypes. A more detailed analysis of amino acid composition revealed the presence of several subtype-specific variants in a few positions, but no discriminating positions between responder and nonresponder patients were detected. Our results also revealed that most amino acid positions were highly conserved, especially for subtype 1a. We conclude that the outcome of the antiviral treatment might depend not only on the nature of one or a few independent positions, but more likely on the combination of several positions along the HCV genome. Moreover, the own host’s ability to generate an appropriate systemic response, in combination with the action of antivirals, is also likely to be essential for treatment outcome.

Little is known about the potential effects of insertions and deletions (indels) on the evolutionary dynamics of hepatitis C virus (HCV). In fact, the consequences of indels on antiviral treatment response are a field of investigation completely unexplored. Here, an extensive sequencing project was undertaken by cloning and sequencing serum samples from 25 patients infected with HCV subtype 1a and 48 patients with subtype 1b. For 23 patients, samples obtained after treatment with alpha interferon plus ribavirin were also available. Two genome fragments containing the hypervariable regions in the envelope 2 glycoprotein and the PKR-BD domain in NS5A were sequenced, yielding almost 16 000 sequences. Our results show that insertions are quite rare, but they are often present in biologically relevant domains of the HCV genome. Moreover, their frequency distributions between different time samples reflect the quasispecies dynamics of HCV populations. Deletions seem to be subject to negative selection.

Pore-forming toxins are biological weapons produced by a variety of living organisms, particularly bacteria but also by insects, reptiles, and invertebrates. These proteins affect the cell membrane of their target, disrupting permeability and leading eventually to cell death. The pore-forming toxins typically transform from soluble, monomeric proteins to oligomers that form transmembrane channels. The Cry toxins produced by Bacillus thuringiensis are widely used as insecticides. These proteins have been recognized as pore-forming toxins, and their primary action is to lyse midgut epithelial cells in their target insect. To exert their toxic effect, a prepore oligomeric intermediate is formed leading finally to membrane-inserted oligomeric pores. To understand the role of Cry oligomeric pre-pore formation in the insecticidal activity we isolated point mutations that affected toxin oligomerization but not their binding with the cadherin-like, Bt-R(1) receptor. We show the helix alpha-3 in domain I contains sequences that could form coiled-coil structures important for oligomerization. Some single point mutants in this helix bound Bt-R(1) receptors with similar affinity as the wild-type toxin, but were affected in oligomerization and were severally impaired in pore formation and toxicity against Manduca sexta larvae. These data indicate the pre-pore oligomer and the toxin pore formation play a major role in the intoxication process of Cry1Ab toxin in insect larvae.

En este articulo presento al pequeno comercio como una practica utilizada por las mujeres para ocupar espacios y feminizarlos, a la vez que estar presentes en la esfera productiva. Esto les permite resistir a las nuevas formas de socializacion (mucho mas individualistas) impuestas y fomentadas por el capitalismo neoliberal que empujan a las mujeres a nuevas formas de “domesticacion” y reclusion. La resistencia, a estas estrategias, realizada de forma consciente o inconsciente, se apoya necesariamente en la formacion y mantenimiento de redes de cooperacion y solidaridad femenina. Tambien en la participacion de las mujeres en, practicamente, todas las esferas rituales y festivas de la comunidad. Esta participacion sera posible gracias a la amplia red de relaciones comunitarias generadas alrededor de la institucion del compadrazgo y del comadrazgo (con este termino me refiero a las relaciones entre comadres, las cuales, en el caso que nos ocupa son de gran importancia y diferentes a las que se dan entre compadres ). Palabras-clave : Etnografia. Pequeno comercio. Redes femeninas. Comadrazgo. Espacios femeninos. Feminine solidarities and community spaces : f eminine practices on the Costa Chica of Oaxaca Abstract In this article I present small trade as a practice used by women to occupy spaces and feminize them, simultaneously making it possible for them to be present in the productive sphere. This allows them to resist to the new forms of socialization (much more individualistic) imposed and promoted by neoliberal capitalism which push the women toward new forms of "domestication" and imprisonment. Resistance, to these strategies, carried out consciously or unconsciously, rests necessarily on the formation and maintenance of networks of cooperation and feminine solidarity, as well as on women’s participation in, practically all the ritual and festive spheres of the community. This participation is possible thanks to the broad network of community relations generated around the institution of compadrazgo and of comadrazgo. Key words : Ethnography. Small trade. Feminine network. Comadrazgo. Women’s spaces.

Hepatotoxicity is the number one cause for agencies not approving and withdrawing drugs for the market. Drug-induced human hepatotoxicity frequently goes undetected in preclinical safety evaluations using animal models. Human-derived in vitro models represent a common alternative to in vivo tests to detect toxic effects during preclinical testing. Most current in vitro toxicity assays rely on the measurement of nonspecific or low sensitive endpoints, which result in poor concordance with human liver toxicity. Therefore, making more accurate predictions of the potential hepatotoxicity of new drugs remains a challenge. Metabolomics, whose aim is to globally assess all the metabolites present in a biological sample, may represent an alternative in the search for sensitive sublethal markers of drug-induced hepatotoxicity. To this end, a comprehensive LC-MS-based untargeted metabolite profiling analysis of HepG2 cells, exposed to a set of well-described model hepatotoxins and innocuous compounds, was performed. It allowed to determine meaningful metabolic changes triggered by a toxic insult and gave a first estimation of the main toxicity-related pathways. Based on these metabolic patterns, a partial least squares-discriminant analysis model, able to discriminate between nontoxic and hepatotoxic compounds, was constructed. The approach described herein may provide an alternative for animal testing in preclinical stages of drug development and a controlled experimental approach to gain a better understanding of the underlying causes of hepatotoxicity.

Several cryopreservation protocols for hepatocytes have been proposed over the past few years, but their effectiveness varies greatly as a function of the characteristics of the method used. One factor in the success of cryopreservation is the quality of cells before freezing. The results suggest that the cryopreservation of hepatocytes in a medium containing polyvinylpyrrolidone (PVP), in addition to DMSO, constitutes a convenient means of long-term storage of hepatocytes for preparing primary cultures to be used in drug metabolism studies. The combined use of the two cryoprotectants is particularly critical for low-viability cell suspensions. An interesting alternative to increase cell viability is the preculture of hepatocytes before cryopreservation. By the use of this procedure, high-quality cells, estimated in terms of post-thaw recovery, viability, adaptation of hepatocytes to culture, drug-metabolizing capability and cytochrome P450 induction, are obtained. Therefore, cryopreserved hepatocytes can provide a regular source of metabolically competent cells for in vitro investigations of the metabolic profile of new drugs and drug-drug interactions in pharmaco-toxicological research.

Resumen Objetivo Valorar los conocimientos y las actitudes de los profesionales de enfermeria sobre la infeccion del virus de la inmunodeficiencia humana (VIH) en el Hospital Mutua de Terrassa, en el ano 2002, y analizar las diferencias y similitudes con el estudio realizado en 1992 en el Hospital de Bellvitge. Ambos hospitales se encuentran en la provincia de Barcelona. Metodo Desarrollado en el ano 2002 en el Hospital Mutua de Terrassa, se trata de un estudio descriptivo, analitico, transversal y comparativo respecto del estudio realizado en el Hospital de Bellvitge en 1992. Se realizo mediante la administracion de un cuestionario de autocumplimentacion. Se selecciono una muestra representativa aleatoria del personal de enfermeria (diplomados de enfermeria y auxiliares de clinica). Resultados Se recogieron 216 cuestionarios (80% de la muestra) en 2002 y 170 cuestionarios (100% de la muestra) en 1992. Destaca que ha mejorado el conocimiento sobre el tratamiento del VIH. Asimismo han aumentado los conocimientos especificos: un 12% mas dijo saber que son las precauciones universales y el 62% disponia de tiempo y de material para aplicarlas. El 99% de los profesionales considero que no se debe reencapuchar la aguja. En las preguntas de opinion se observa una disminucion del 26% del miedo al contagio y del 9% del miedo al rechazo social. En el ano 2002 al 29% le gustaba trabajar con pacientes con el VIH. Conclusiones Los profesionales de enfermeria han mejorado, en algunos aspectos, sus conocimientos respecto a la enfermedad del VIH, y su actitud ante el sida, su motivacion y su aceptacion son mas favorables.

FK506 is a potent immunosuppressive drug used for the prevention of graft rejection in organ transplantation. Experimental and clinical studies have shown correlations between apoptosis and graft rejection, and apoptosis also plays a role in cell death after ischemia-reperfusion injury in the rat liver. Fas-mediated apoptosis is very likely involved in allograft rejection and experimental evidence has shown a decrease of FasR expression in mouse hepatocytes produced by the drugs. On the basis of these findings we have investigated the protective effect of FK506 in comparison with cyclosporine A (CsA) on Fas-induced apoptosis, by analysing the activation of downstream effector caspases in human hepatocytes. Apoptosis was induced by treatment with agonistic antibodies against FasR, which resulted in a significant activation of caspase-3 after 12 h. Prevention of the downstream activation of the caspase cascade and apoptosis was observed when hepatocytes were pre-treated for 3 h with immunosuppressant drugs. A significant reduction (ca. 30-40%) of caspase-3 activation by 5 microM FK506 and CsA was observed. Along with less activation of caspase-3 a decrease of apoptotic DNA fragmentation was found. In addition, FK506 significantly reduced not only caspase-8 but also caspase-9 activation, to a similar extent as CsA, thus suggesting a protective effect at the mitochondrial level of this drug, as has already been reported for CsA. These effects of FK506 help to explain its strong anti-rejection properties and suggest promising benefits of pharmacological preconditioning on ischemia-reperfusion injury following liver transplantation.

Suitability of human liver grafts preserved in Celsior solution (CS) for preparing metabolically competent hepatocyte cultures has been examined. To this end, basal and induced activity and mRNA levels of major hepatic cytochrome P450 (P450) enzymes have been measured. By 24 h in culture, measurable levels of the 10 P450 mRNAs studied were found in all hepatocyte preparations examined, with CYP2E1, CYP2C9, and CYP3A4 mRNAs being the most abundant. Compared with hepatocytes obtained from surgical liver resections (SLRs), lower content of each P450 mRNA was found in hepatocytes from the CS group; however, the relative distribution of individual P450 mRNAs was similar. Similar results were observed after measuring P450 activities. CYP1A2, CYP2A6, CYP2C9, CYP2C19, CYP2E1, and CYP3A4 activities in hepatocytes from CS-flushed grafts were lower than but comparable with those of cultures prepared from SLRs. No differences in the metabolite profile of testosterone were found. Treatment of hepatocytes from CS-preserved grafts with model P450 inducers shows that 2 microM methylcholanthrene only increased CYP1A1 and CYP1A2 mRNAs (100-fold over control), 1 mM phenobarbital markedly increased CYP2A6, CYP2B6, and CYP3A4 mRNA content (7-fold), and 50 microM rifampicin highly increased CYP3A4 mRNA levels (10-fold), whereas minor effects (3-fold) were observed in CYP2A6, CYP2B6, and CYP2C9 mRNAs. This induction pattern of P450s was similar, in terms of magnitude, reproducibility, and specificity, to that shown in primary hepatocytes from surgical biopsies. Overall, our results indicate that, cold-preserved in CS, liver grafts constitute a valuable source of human hepatocytes for drug metabolism studies.

Most of the locally advanced and metastatic prostate carcinomas (PCs) treated with antiandrogenic therapy eventually become refractory to this treatment. Locally produced factors may control prostate tumor biology after androgen withdrawal. Adrenomedullin (AM) is expressed in the prostate and could control cell growth in androgen-independent conditions. AM needs to be amidated by the enzyme peptidylglycine alpha-amidating monooxygenase (PAM) to become fully active. The objective of the present study was to analyze whether the expression of preproadrenomedullin (preproAM) and PAM in PC is regulated by androgens. For this purpose, human in vitro and in vivo PC models were grown in the presence or absence of androgens, and the expression of AM and PAM was examined by immunohistochemistry, Western blotting, RT-PCR, and Northern blotting. Furthermore, immunohistochemical analysis of AM in clinical specimens was performed to test if its expression is related to Gleason score and antiandrogenic therapy. In PC cell lines and xenografts, mRNA and protein AM levels were similar in the presence or absence of androgens. PAM expression seemed to be induced by androgen-withdrawal. Our results in clinical samples showed no relationship between AM expression and Gleason score or antiandrogenic treatment. In conclusion, our results demonstrate that preproAM and PAM expression in the human prostate is androgen-independent. In addition, we also report for the first time the expression of a novel PAM transcript in PC, which has not been previously described in other tissues.

There is increasing evidence for the important role played by regulatory peptides in the physiology of the normal and neoplastic prostate. Adrenomedullin (AM) and pro-adrenomedullin N-terminal 20 peptide (PAMP) are recently discovered regulatory peptides widely expressed in the normal prostate and in prostate carcinoma. AM is produced in secretory, stroma, and endothelial cells and in neurons of the prostate ganglia. PAMP is only produced by neuroendocrine cells. The expression of AM mRNA is regulated by androgens in the rat prostate. The number of neuroendocrine cells expressing PAMP is increased in prostate carcinoma after androgen deprivation, which shows that this peptide could regulate androgen-independent prostate tumor growth. However, the roles of AM and PAMP in the normal prostate and in prostate carcinoma are yet to be elucidated.

Most PCs show NE differentiation. Several studies have tried to correlate NE expression with disease status, but the reported findings have been contradictory. Prostatic NE cells synthesize peptides with a wide spectrum of potential functions. Some of these active peptides, such as PAMP, are amidated. PAM is the only carboxy-terminal peptide-amidating enzyme identified. We studied expression of PAMP and PAM in normal prostate and prostatic tumors (clinical specimens and human xenograft models) with or without prior androgen-deprivation therapy and found a wide distribution of both molecules in NE subpopulations of all kinds. Although the correlation of either marker to tumor grade, clinical progression or disease prognosis did not reach statistical significance, PAMP- or PAM-immunoreactive cells were induced after androgen-blockade therapy. In the PC-310 and PC-295 androgen-dependent models, PAMP or PAM NE differentiation was induced after castration in different ways, being higher in PC-310, which might explain its long-term survival after androgen deprivation. We show induction of expression of 2 new NE markers in clinical specimens and xenografted PC after endocrine therapy.

Electron tomography (ET) is a very important high-resolution tool for 3D imaging in cell biology. By combining the technique with immunolabeling, ET can provide essential insights into both cellular architecture and dynamics. We recently developed a protocol to achieve 3D immunolabeling of intracellular antigens without the need for uncontrolled permeabilization steps that cause random, extensive cell membrane disruption. Here we describe this novel method based on well-controlled permeabilization by targeted laser cell perforation. Mechanical permeabilization of the plasma membrane can be applied at specific sites without affecting other parts of the plasma membrane and intracellular membranes. Despite the relatively small opening created in the plasma membrane, the method allows specific 3D immunolocalization of cytoplasmic antigens in cultured cells by a pre-embedment protocol. The approach is unique and leads to a superior ultrastructural preservation for transmission electron microscopy and electron tomography.

Irritable bowel syndrome (IBS) is the most common functional gastrointestinal disorder in western countries. Previous studies on IBS, mostly based on faecal samples, suggest alterations in the intestinal microbiota. However, no consensus has been reached regarding the association between specific bacteria and IBS. We explore the alterations of intestinal bacterial communities in IBS using massive sequencing of amplified 16S rRNA genes. Mucosal biopsies of the ascending and descending colon and faeces from 16 IBS patients and 9 healthy controls were analysed. Strong inter-individual variation was observed in the composition of the bacterial communities in both patients and controls. These communities showed less diversity in IBS cases. There were larger differences in the microbiota composition between biopsies and faeces than between patients and controls. We found a few over-represented and under-represented taxa in IBS cases with respect to controls. The detected alterations varied by site, with no changes being consistent across sample types.

Human umbilical vein endothelial cells (HUVECs) cultured in vitro are a commonly used experimental system. When properly differentiated they acquire the so-called cobblestone phenotype; thereby mimicking an endothelium in vivo that can be used to shed light on multiple endothelial-related processes. In the present paper we report a simple, flexible, fast and reproducible method for an efficient isolation of viable HUVECs. The isolation is performed by sequential short trypsinization steps at room temperature. As umbilical cords are often damaged during labor, it is noteworthy that this new method can be applied even to short pieces of cord with success. In addition, we describe how to culture HUVECs as valid cobblestone cells in vitro on different types of extracellular matrix (basement membrane matrix, fibronectin and gelatin). We also show how to recognize mature cobblestone HUVECs by ordinary phase contrast microscopy. Our HUVEC model is validated as a system that retains important features inherent to the human umbilical vein endothelium in vivo. Phase contrast microscopy, immuno-fluorescence and electron microscopy reveal a tight cobblestone monolayer. Therein cells show Weibel-Palade bodies, caveolae and junctional complexes (comparable to the in vivo situation, as also shown in this study) and can internalize human low density lipoprotein. Isolation and culture of HUVECs as reported in this paper will result in an endothelium-mimicking experimental model convenient for multiple research goals. Electronic supplementary material The online version of this article (doi:10.1007/s10616-012-9459-9) contains supplementary material, which is available to authorized users.

In the present study, we developed a cell-based protocol for the identification of drugs able to induce steatosis. The assay measures multiple markers of toxicity in a 96-well plate format using high-content screening (HCS) technology. After treating HepG2 cells with increasing concentrations of the tested compounds, toxicity parameters were analyzed using fluorescent probes: BODIPY493/503 (lipid content), 2',7'-dihydrodichlorofluorescein diacetate (reactive oxygen species [ROS] generation), tetramethyl rhodamine methyl ester (mitochondrial membrane potential), propidium iodide (cell viability), and Hoechst 33342 (nuclei staining). A total of 16 drugs previously reported to induce liver steatosis through different mechanisms (positive controls) and six nonsteatotic compounds (negative controls) were included in the study. All the steatosis-positive compounds significantly increased BODIPY493/503 fluorescence in HepG2 cells, whereas none of the negative controls induced lipid accumulation. In addition to effects on fat levels, increased ROS generation was produced by certain compounds, which could be indicative of increased risk of liver damage. Our results suggest that this in vitro approach is a simple, rapid, and sensitive screening tool for steatosis-inducing drugs. This conclusion should be confirmed by testing a larger number of steatosis-positive and -negative inducers.

Electron tomography (ET) is an indispensable high-resolution tool for three dimensional (3D) imaging in cell biology. When applied to immuno-labeled cells, ET can provide essential insights in both the cellular architecture and the dynamics. Current protocols for 3D immuno-labeling of intracellular antigens include permeabilization steps that cause random, extensive cell membrane disruption. This permeabilization results in a poor cell ultrastructure, limiting the usefulness of the specimens for high-resolution studies. Here we describe a novel method, based on a well-controlled permeabilization by targeted laser cell perforation, that allows for the 3D immuno-localization of cytoplasmic antigens in cultured cells. The approach is unique since it is applicable to both chemically and cryo-fixed cells and leads to a superior ultrastructural preservation for electron microscopy and tomography.

The human gut is the natural habitat for a large and dynamic bacterial community that has a great relevance for health. Metagenomics is increasing our knowledge of gene content as well as of functional and genetic variability in this microbiome. However, little is known about the active bacteria and their function(s) in the gastrointestinal tract. We performed a metatranscriptomic study on ten healthy volunteers to elucidate the active members of the gut microbiome and their functionality under conditions of health. First, the microbial cDNAs obtained from each sample were sequenced using 454 technology. The analysis of 16S transcripts showed the phylogenetic structure of the active microbial community. Lachnospiraceae, Ruminococcaceae, Bacteroidaceae, Prevotellaceae, and Rickenellaceae were the predominant families detected in the active microbiota. The characterization of mRNAs revealed a uniform functional pattern in healthy individuals. The main functional roles of the gut microbiota were carbohydrate metabolism, energy production and synthesis of cellular components. In contrast, housekeeping activities such as amino acid and lipid metabolism were underrepresented in the metatranscriptome. Our results provide new insights into the functionality of the complex gut microbiota in healthy individuals. In this RNA-based survey, we also detected small RNAs, which are important regulatory elements in prokaryotic physiology and pathogenicity.

Aclar, a copolymer film with properties very similar to those of tissue culture plastic, is a versatile substrate to grow cells for light (including fluorescence) and electron microscopic applications in combination with both chemical fixation and cryoimmobilization. In this paper, we describe complete procedures to perform correlative light and electron microscopy using Aclar as substrate for the culture of cell monolayers to be finally embedded in plastic. First, we developed straightforward, efficient and flexible ways to mark the surface of the Aclar to create substrates to locate cells first at the light microscopy and then the electron microscopy level. All the methods enable the user to self-design gridded Aclar pieces, according to the purpose of the experiments, and create a large number of substrates in a short time. Second, we confirmed that marked Aclar supports the normal growth and morphology of cells. Third, we validated the correlative light and electron microscopy procedure using Aclar. This validation was done for the high-resolution analysis of endothelial cells using transmission electron microscopy and focused ion beam-scanning electron microscopy in combination with the use of fluorescence, phase contrast and/or bright field microscopy to map areas of interest at low resolution. The methods that we present are diverse, easy to implement and highly reproducible, and emphasize the versatility of Aclar as a cell growth substrate for diverse microscopic applications.

Hepatitis C virus (HCV) presents several regions involved potentially in evading antiviral treatment and host immune system. Two regions, known as PKR-BD and V3 domains, have been proposed to be involved in resistance to interferon. Additionally, hypervariable regions in the envelope E2 glycoprotein are also good candidates to participate in evasion from the immune system. In this study, we have used a cohort of 22 non-responder patients to combined therapy (interferon alpha-2a plus ribavirin) for which samples obtained just before initiation of therapy and after 6 or/and 12 months of treatment were available. A range of 25-100 clones per patient, genome region and time sample were obtained. The predominant amino acid sequences for each time sample and patient were determined. Next, the sequences of the PKR-BD and V3 domains and the hypervariable regions from different time samples were compared for each patient. The highest levels of variability were detected at the three hypervariable regions of the E2 protein and,to a lower extent, at the V3 domain of the NS5A protein. However, no clear patterns of adaptation to the host immune system or to antiviral treatment were detected. In summary, although high levels of variability are correlated to viral adaptive response, antiviral treatment does not seem to promote convergent adaptive changes. Consequently, other regions must be involved in evasion strategies likely based on a combination of multiple mechanisms, in which pools of changes along the HCV genome could confer viruses the ability to overcome strong selective pressures. J. Med. Virol. 81:650656, 2009. (C) 2009 Wiley-Liss, Inc.

Drugs are capable of inducing hepatic lipid accumulation. When fat accumulates, lipids are primarily stored as triglycerides which results in steatosis and provides substrates for lipid peroxidation. An in vitro multiparametric flow cytometry assay was performed in HepG2 cells by using fluorescent probes to analyze cell viability (propidium iodide, PI), lipid accumulation (BODIPY493/503), mitochondrial membrane potential (tetramethyl rhodamine methyl ester, TMRM) and reactive oxygen species generation (ROS) (2',7'-dihydrochlorofluorescein diacetate, DHCF-DA) as functional markers. All the measurements were restricted to live cells by gating the cells that excluded PI or those that exhibited the typical forward and side scatter features of live cells. The assay was qualified by analyzing a number of selected model drugs with a well documented induction of steatosis in vivo using different mechanisms as positive controls and several non-steatosic compounds as negative controls. For the cytometric screening assay, the concentrations tested were up to the corresponding IC10 value determined by the MTT assay. Among the parameters analyzed. increased BODIPY fluorescence was the most sensitive and selective marker of drug-induced steatosis. However, a more consistent predictive approach was the combination of two end-points: lipid accumulation and ROS generation. The assay correctly identified 100% of steatosis-positive and steatosis-negative compounds, and a high steatosis risk was predicted for amiodarone, doxycycline, tetracycline and valproate treatments at therapeutic doses. The results suggest that this cell-based assay may be a useful approach to identify the potential of drug candidates to induce steatosis. (C) 2009 Elsevier Ireland Ltd. All rights reserved.

Hepatocyte transplantation has been proposed as a method to support patients with liver insufficiency. Key factors for clinical cell transplantation to progress is to prevent hepatocyte damage, loss of viability and cell functionality, factors that depend on the nature of the tissue used for isolation to a large extent. The main sources of tissue for hepatocyte isolation are marginal livers that are unsuitable for transplantation, and segments from reduced cadaveric grafts. Hepatocellular transplantation requires infusing human hepatocytes in Suspension over a period of minutes to hours. The beneficial effect of hypothermic preservation of hepatocytes in infusion medium has been reported, but how critical issues towards the success of cell transplantation, such as the composition of infusion medium and duration of hepatocyte storage will affect hepatocyte quality for clinical cell infusion has not been systematically investigated. Infusion media composition is phosphate-buffered saline containing anticoagulants and human serum albumin. The supplementation of infusion media with glucose or N-acetyl-cystein, or with both component, at the same time, has been investigated. After isolation, hepatocytes were Suspended in each infusion medium and a sample at the 0 time point was harvested for cell viability and functional assessment. Thereafter, cells were incubated in different infusion media agitated on a rocker platform to simulate the clinical infusion technique. The time course of hepatocyte viability, funtionality (drug-metabolizing enzymes, urcogenic capability, ATP, glycogen, and GSH levels), apoptosis (caspase-3 activation), and attachment and monolayer formation were analyzed. The optimal preservation of cell viability, attaching capacity, and functionality, particularly GSH and glycogen levels, as well as drug-metabolizing cytochrome P450 enzymes, was found in infusion media supplemented with 2 mM N-acetyl-cystein and 15 mM glucose.

We present an analysis of the selective forces acting on two hepatitis C virus genome regions previously postulated to be involved in the viral response to combined antiviral therapy. One includes the three hypervariable regions in the envelope E2 glycoprotein, and the other encompasses the PKR binding domain and the V3 domain in the NS5A region. We used a cohort of 22 non-responder patients to combined therapy (interferon alpha-2a plus ribavirin) for which samples were obtained before initiation of therapy and after 6 or/and 12 months of treatment. A range of 25-100 clones per patient, genome region and time sample were sequenced. These were used to detect general patterns of adaptation, to identify particular adaptation mechanisms and to analyze the patterns of evolutionary change in both genome regions. These analyses failed to detect a common adaptive mechanism for the lack of response to antiviral treatment in these patients. On the contrary, a wide range of situations were observed, from patients showing no positively selected sites to others with many, and with completely different topologies in the reconstructed phylogenetic trees. Altogether, these results suggest that viral strategies to evade selection pressure from the immune system and antiviral therapies do not result from a single mechanism and they are likely based on a range of different alternatives, in which several different changes, or their combination, along the HCV genome confer viruses the ability to overcome strong selective pressures. cuevast@uv.es; mtorresp@uv.es; njimenez@uv.es; bracho@uv.es; garciai@uv.es; wrobel@uv.es; moya@uv.es; gonzalef@uv.es

Summary. Hepatitis C virus (HCV) infects approximately 3% of the world population. The chronicity of hepatitis C seems to depend on the level of genetic variability. We have recently (Torres-Puente et al., J Viral Hepat, 2008; 15: 188) reported genetic variability estimates from a large-scale sequence analysis of 67 patients infected with HCV subtypes 1a (23 patients) and 1b (44 patients) and related them to response, or lack of, to alpha-interferon plus ribavirin treatment.. Two HCV genome regions were analysed in samples prior to antiviral therapy, one compressing the three hypervariable regions of the E2 glycoprotein and another one including the interferon sensitive determining region and the V3 domain of the NS5A protein. Haplotype and nucleotide diversity measures showed a clear tendency to higher genetic variability levels in nonresponder than in responder patients. Here, we have refined the analysis of genetic variability (haplotype and nucleotide diversity, number of haplotypes and mutations) by considering their distribution in each of the biologically meaningful subregions mentioned above, as well as in their surrounding and intervening regions. Variability levels are very heterogeneous among the different subregions, being higher for nonresponder patients. Interestingly, significant differences were detected in the biologically relevant regions, but also in the surrounding regions, suggesting that the level of variability of the whole HCV genome, rather than exclusively that from the hypervariable regions, is the main indicator of the treatment response. Finally, the number of haplotypes and mutations seem to be better discriminators than haplotype and nucleotide diversity, especially in the NS5A region.

Background The repeats in toxin (Rtx) are an important pathogenicity factor involved in host cells invasion of Legionella pneumophila and other pathogenic bacteria. Its role in escaping the host immune system and cytotoxic activity is well known. Its repeated motives and modularity make Rtx a multifunctional factor in pathogenicity. Results The comparative analysis of rtx gene among 6 strains of L. pneumophila showed modularity in their structures. Among compared genomes, the N-terminal region of the protein presents highly dissimilar repeats with functionally similar domains. On the contrary, the C-terminal region is maintained with a fashionable modular configuration, which gives support to its proposed role in adhesion and pore formation. Despite the variability of rtx among the considered strains, the flanking genes are maintained in synteny and similarity. Conclusion In contrast to the extracellular bacteria Vibrio cholerae, in which the rtx gene is highly conserved and flanking genes have lost synteny and similarity, the gene region coding for the Rtx toxin in the intracellular pathogen L. pneumophila shows a rapid evolution. Changes in the rtx could play a role in pathogenicity. The interplay of the Rtx toxin with host membranes might lead to the evolution of new variants that are able to escape host cell defences.