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The Salmonella enterica serovar Typhimurium RcsCDB system regulates the synthesis of colanic acid and the flagellum as well as the expression of virulence genes. We previously demonstrated that the rcsC11 mutant, which constitutively activates the RcsB regulator, attenuates Salmonella virulence in an animal model. This attenuated phenotype was also produced by deletion of the slyA gene. In this work, we investigated if this antagonistic behavior is produced by modulating the expression of both regulator-encoding genes. We demonstrated that SlyA overproduction negatively regulates rcsB transcription. A bioinformatics analysis enabled us to identify putative SlyA binding sites on both promoters, P(rcsDB) and P(rcsB), which control rcsB transcriptional levels. We also determined that SlyA is able to recognize and bind to these predicted sites to modulate the activity of both rcsB promoters. According to these results, SlyA represses rcsB transcription by direct binding to specific sites located on the rcsB promoters, thus accounting for the attenuated/virulence antagonistic behaviors. Moreover, we showed that the opposite effect between both regulators also physiologically affects the Salmonella motility phenotype. In this sense, we observed that under SlyA overproduction, P(rcsB) is repressed, and consequently, bacterial motility is increased. On the basis of these results, we suggest that during infection, the different RcsB levels produced act as a switch between the virulent and attenuated forms of Salmonella. Thereby, we propose that higher concentrations of RcsB tilt the balance toward the attenuated form, while absence or low concentrations resulting from SlyA overproduction tilt the balance toward the virulent form. IMPORTANCE The antagonistic behavior of RcsB and SlyA on virulence gene expression led us to hypothesize that there is interplay between both regulators in a regulatory network and these could be considered coordinators of this process. Here, we report that the SlyA virulence factor influences motility behavior by controlling rcsB transcription from the P(rcsB) promoter. We also demonstrate that SlyA negatively affects the expression of the rcsB gene by direct binding to P(rcsDB) and P(rcsB) promoters. We suggest that different levels of RcsB act as a switch between the virulent and attenuated forms of Salmonella, where high concentrations of the regulator tend to tilt the balance toward the attenuated form and low concentrations or its absence tilt it toward the virulent form.

Las aguas de desecho dispuestas en una corriente superficial, lagos, ríos, mar, sin ningún tratamiento, ocasionan graves inconvenientes de contaminación que afectan la flora y la fauna. Estas aguas residuales, antes de ser vertidas en las masas receptoras, deben recibir un tratamiento adecuado, capaz de modificar sus condiciones físicas, químicas y microbiológicas, para evitar que su disposición cause la alteración y degradación de los ecosistemas asociados y problemas de salud pública. La investigación tuvo como objetivo evaluar el efecto de mezclas de quitosano y extracto acuoso de la cascara de naranja a diferentes concentraciones en el proceso de coagulación-floculación de aguas residuales. Para lo cual, en realizo una prueba de jarras con agitación rápida y lenta para evaluar turbidez (NTU), demanda química de oxígeno (DQO), demanda bioquímica de oxígeno (DBO), sólidos suspendidos totales (SST) y SS (sólidos sedimentables). Los resultados mostraron que todos los tratamientos presentaron diferencias significativas (p

Molars are highly integrated biological structures that have been used for inferring evolutionary relationships among taxa. However, parallel and convergent morphological traits can be affected by developmental and functional constraints. Here, we analyze molar shapes of platyrrhines in order to explore if platyrrhine molar diversity reflects homogeneous patterns of molar variation and covariation. We digitized 30 landmarks on mandibular first and second molars of 418 extant and 11 fossil platyrrhine specimens to determine the degree of integration of both molars when treated as a single module. We combined morphological and phylogenetic data to investigate the phylogenetic signal and to visualize the history of molar shape changes. All platyrrhine taxa show a common shape pattern suggesting that a relatively low degree of phenotypic variation is caused by convergent evolution, although molar shape carries significant phylogenetic signal. Atelidae and Pitheciidae show high levels of integration with low variation between the two molars, whereas the Cebinae/Saimiriinae, and especially Callitrichinae, show greater variation between molars and trend toward a modular organization. We hypothesize that biomechanical constraints of the masticatory apparatus, and the dietary profile of each taxon are the main factors that determine high covariation in molars. In contrast, low molar shape covariation may result from the fact that each molar exhibits a distinct ecological signal, as molars can be exposed to distinct occlusal loadings during food processing, suggesting that different selective pressures on molars can reduce overall molar integration.

Tooth morphology has been widely used to infer taxonomic affinities. Both morphological and genetic analyses have revealed significant differences among capuchins, suggesting that two distinct monophyletic groups exist: the gracile capuchins (Cebus) and the robust capuchins (Sapajus). We developed a geometric morphometrics (GM) model to determine if the two groups also show distinct molar shapes and to explore the influence of size, phylogeny, and biogeography in shaping molar morphology. We characterized first and second molar crown shape variability in seven species representative of the two genera (Cebus albifrons, C. olivaceus, Sapajus apella, S. robustus, S. libidinosus, S. nigritus, and S. xanthosternos), using two-dimensional (2D) and three-dimensional (3D) GM. The results showed that 2D GM discriminated the two groups better than 3D GM, possibly because it accounted for cusp position and crown contour, but not cusp height, which may be a useful trait for inferring adaptive foraging ecology but presents a risk of homoplasy. In addition, the presence of a phylogenetic signal in the first molar shape (2D) is likely to reflect similarity to the ancestral condition and provides evidence of gradual evolution of molar robustness in the robust clade. We suggest that the shape of the first molar is informative about phylogenetic affinities, whereas the shape of the second molar is more informative about biogeographic variability. However, molar shape similarities may be affected by convergent evolution, as environmental factors in different biogeographical regions may have a significant effect on molar morphology, as seen in the closely related capuchins.

Morphometric variation of biological structures has been widely used to determine taxonomic affinities among taxa, and teeth are especially informative for both deep phylogenetic relationships and specific ecological signals. We report 2-dimensional geometric morphometrics (GM) analyses of occlusal crown surfaces of lower molars (M1, n = 141; M2, n = 158) of cercopithecoid primate species. A 12-landmark configuration, including cusp tips and 8 points of the molar crown contour, were used to evaluate patterns of variation in lower molar shape among cercopithecoid primates and to predict the taxonomic attribution of 2 archaeological macaques from Roman time periods. The results showed that the lower molar shape of cercopithecoid primates reflects taxonomic affinities, mostly at a subfamily level and close to a tribe level. Thus, the cusp positions and crown contour were important elements of the pattern related to interspecific variation. Additionally, the archaeological specimens, attributed to Macaca sylvanus based on osteological information, were classified using the GM molar shape variability of the cercopithecoid primates studied. The results suggest that their molar shape resembled both M. sylvanus and M. nemestrina, and species attribution varied depending on the comparative sample used.

Dps, the most abundant protein during the stationary growth phase, in Salmonella enterica is required for resistance to reactive oxygen species produced by the host during infection. It has been reported that in Salmonella dps expression is controlled by RpoS and Fur proteins. However, the regulation and function of Dps remain to be resolved. In the present work we demonstrate that activation of the complex RcsCDB regulatory system increases dps expression during exponential growth of Salmonella. In addition, we show that such dps upregulation produces high levels of H2O2 resistance. This phenotype allows the bacteria to avoid reactive oxygen species killing at early stages of growth, thus protecting its genetic material. Fil: Farizano, Juan Vicente. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina Fil: Torres, Mariela Analía. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina Fil: Pescaretti, María de Las Mercedes. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina Fil: Delgado, Monica Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina

The purpose of this study was to compare the life situations and level of therapeutic adherence of a sample of 29 women and 47 men from the Mexican state of San Luis Potosi living with HIV/AIDS. A semi-structured interview was administered. All participants live under very difficult conditions, particularly women: they have poor schooling, live alone and in charge of their children, with economic strains and poor family support. Statistically signifi cant differences were found between male and female participants regarding transmission mode (p ≤ 0.00), alcohol use (p ≤ 0.03) and family support (p ≤ 0.00), among others. Women and men showed insufficient levels of therapeutic adherence to guarantee a healthy life style.

The Salmonella enterica serovar Typhimurium lipopolysaccharide consisting of covalently linked lipid A, non-repeating core oligosaccharide, and the O-antigen polysaccharide is the most exposed component of the cell envelope. Previous studies demonstrated that all of these regions act against the host immunity barrier. The aim of this study was to define the role and interaction of PmrAB-dependent gene products required for the lipopolysaccharide component synthesis or modification mainly during the Salmonella infection. The PmrAB two-component system activation promotes a remodeling of lipid A and the core region by addition of 4-aminoarabinose and/or phosphoethanolamine. These PmrA-dependent activities are produced by activation of ugd, pbgPE, pmrC, cpta, and pmrG transcription. In addition, under PmrA-regulator activation, the expression of wzzfepE and wzzst genes is induced, and their products are required to determine the O-antigen chain length. Here we report for the first time that Wzzst protein is necessary to maintain the balance of 4-aminoarabinose and phosphoethanolamine lipid A modifications. Moreover, we demonstrate that the interaction of the PmrA-dependent pbgE2 and pbgE3 gene products is important for the formation of the short O-antigen region. Our results establish that PmrAB is the global regulatory system that controls lipopolysaccharide modification, leading to a coordinate regulation of 4-aminoarabinose incorporation and O-antigen chain length to respond against the host defense mechanisms. Fil: Farizano, Juan Vicente. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina Fil: Pescaretti, María de Las Mercedes. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina Fil: Lopez, Fabian E.. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina Fil: Fong-Fu, Hsu. Washington University in St. Louis; Estados Unidos Fil: Delgado, Monica Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina. University of Washington; Estados Unidos

The SbmA protein is involved in the transport of MccB17-, MccJ25-, bleomycin- and proline-rich peptides into the Escherichia coli cytoplasm. sbmA gene homologues were found in a variety of bacteria. However, the physiological role of this protein still remains unknown. Previously, we found that a combination of sbmA and tolC mutations in Tn10-carrying E. coli K-12 strains results in hypersusceptibility to tetracycline. In this work, we studied sbmA expression in a tolC mutant background and observed an increased expression throughout growth. We ruled out the global transcriptional regulator RpoS and the small RNA micF as intermediates in this regulation. The tolC mutation induced the expression of other well-characterized strong σ(E) -dependent promoters in E. coli. We observed that the increase in σ(E) activity led to a greater sbmA expression, conversely eliminating σ(E) prevented expression of sbmA. We also observed that the sbmA upregulation in a tolC mutant context was abolished in an rpoE-null strain. These results suggest a σ(E) -dependent positive regulation on sbmA by the tolC mutation. We hypothesize that this mechanism might be part of a compensatory cell envelope stress response.

Aim: This study focuses on the production, purification and characterization of serraticin A, a novel cold-active antimicrobial produced by Serratia proteamaculans 136. Methods and Results: A Ser. proteamaculans strain producing a novel cold-active antimicrobial was isolated from Isla de los Estados, Argentina. Antimicrobial production was optimized in a BIOFLO 101 bioreactor under batch culture mode, with temperature, pH and dissolved oxygen controlled conditions. A purification protocol was developed including activated charcoal adsorption, solid-phase C18 extraction (SPE) and semi-preparative HPLC. The molecular weight was determined by LC/QTOF/MS/MS mass analysis. Conclusions: Serratia proteamaculans 136 produces a cold-active low molecular bacteriocin-like compound named serraticin A. In this work, it has been laboratory-scale produced, purified and partially characterized. Cross-immunity test revealed that serraticin A is very different from other well-known microcins assayed, with a wide inhibitory spectrum, showing an interesting biotechnology potential to be applied as a control agent against pathogenic bacteria. Significance and Impact of the Study: The present study is the first report of a cold-active compound with antimicrobial activity from Ser. proteamaculans. The work also highlights that cold environments could be a suitable source of micro-organisms with ability to produce cold-active biomolecules of biotechnological interest.

Autophagy is a major intracellular pathway for the lysosomal degradation of long-lived cytoplasmic macromolecules and damaged or surplus organelles. More recently, autophagy has also been linked with innate and adaptive immune responses against intracellular pathogens, including Mycobacterium tuberculosis, which can survive within macrophages by blocking fusion of the phagosome with lysosomes. Induction of autophagy by the Th1 cytokine IFN-gamma enables infected macrophages to overcome this phagosome maturation block and inhibit the intracellular survival of mycobacteria. Conversely, the Th2 cytokines IL-4 and IL-13 inhibit autophagy in murine and human macrophages. We discuss how differential modulation of autophagyby Th1 and Th2 cytokines may represent an important feature of the host response to mycobacteria. (C) 2008 Elsevier B.V. All rights reserved.

Transcription factors (TFs) modulate gene expression as a consequence of internal or exogenous changes in cell signaling. TFs can bind to DNA either with their effector bound (holo conformation), or as free proteins (apo conformation). With the aim of contributing to the understanding of the evolutionary fitness and organizational principles behind the different TF conformations, we inquire into the origins of these conformational differences by analyzing these two TF conformations from the perspective of Savageau's demand theory. For the control of a gene whose function is in high demand, we found that evolutionary constraints are responsible for activator TFs binding to DNA mainly in holo conformation whereas apo activation is under-represented. The mathematically controlled comparison of the apo and holo conformations reveals formal and evolutionary arguments in favor of this TF control asymmetry, which suggests that evolution favors holo activation under environmental conditions commonly found by E. coli in the human digestive tract. Specifically, the sensibility analysis performed for the holo conformation, in the positive mode of regulation, shows that the wild-type is more robust for situations where realizable changes in the model's parameters favored a better performance under non-stressful environmental conditions commonly found by E. coli in the human digestive tract. By contrast, the positive apo conformation is better adapted to adverse situations. On the other hand, the sensibility analysis performed for the negative mode of regulation showing none of the TF active conformations presents an advantage.

Pseudomonas aeruginosais a critical colonizer of the respiratory tract in cystic fibrosis. The chronic infections with this microorganism contribute to excessive inflammation and progressive lung damage in cystic fibrosis patients. The full repertoire ofPseudomonasproducts that promote inflammation in the cystic fibrosis lung is not known. Here we show thatP. aeruginosaDNA released from the bacterium, but not human DNA from epithelial cells orEscherichia coliDNA, displays proinflammatory properties and induces human respiratory epithelial cells to secrete interleukin-8 (IL-8), a key chemokine causing excessive neutrophil infiltration in the cystic fibrosis lung. IL-8 secretion was not due to an increase in NF-κB- or activator protein-1-dependent IL-8 promoter transcription, but instead depended on p38 and Erk mitogen-activated protein kinases. No secretion of IL-8 was observed using conventional Toll-like receptor 9 ligands (CpG oligonucleotides), although it could be demonstrated that parts of the Toll-like receptor 9-signaling pathway were functional, since class B and C CpG oligonucleotide ligands stimulated production of RANTES chemokine. The IL-8 secretion in response toP. aeruginosaDNA was decreased by treatments that inhibit acidification of intracellular organelles, using chloroquine, a pH-neutralizing compound, or bafilomycin A1, an inhibitor of vacuolar H+-ATPase. These data indicate that DNA released fromP. aeruginosaduring chronic infections may significantly contribute to the proinflammatory processes in cystic fibrosis. Our findings also show that treatments with drugs diminishing organellar acidification may reduce the inflammatory response in cystic fibrosis.

Summary Bacterial pathogens have the ability to sense their presence in host tissues and to promote expression of their virulence factors in a time- and location-dependent manner. However, little is known about those genes whose expression is detrimental and thus suppressed during infection. Here we report that constitutive activation of the RcsC/YojN/RcsB system resulting from a mutation in the rcsC sensor gene dramatically attenuates Salmonella virulence. Mutation of the cognate response regulator gene rcsB restored full virulence to the rcsC constitutive mutant, indicating that virulence attenuation results from aberrant expression of RcsB-regulated genes. The virulence attenuation phenotype was partially dependent on the regulatory gene rcsA, which is necessary for transcription of certain RcsB-regulated genes, and on the RcsB- and RcsA-dependent colanic acid capsule synthesis cps operon. The rcsC constitutive mutant was phagocytized less efficiently by macrophages and it was defective for invasion of non-phagocytic cells and survival within macrophages; but it could protect mice upon challenge with wild-type Salmonella. Our results suggest that a successful infection demands that pathogens turn off expression of products that might interfere with virulence functions.

Dissertação de mestrado integrado em Arquitectura (área de especialização em Cidade e Território), A ideia que inicialmente motiva este trabalho de investigação assenta na simplificação e semelhança gráfica imposta pela cartografia normativa, resultando representações abstractas difíceis de identificar com os lugares reais. Esta problemática incita, por um lado, uma pesquisa historicamente desconstrutiva sobre a prática cartográfica, avaliando os sucessivos processos e códigos de que se apropriou para transformar a realidade de forma a conseguir ser lida e incorporada no mapa; Por outro, é introduzida a acção de caminhar como uma ferramenta que possibilita, por si só, uma aproximação e um tipo de leitura e conhecimento directo, minucioso e complexo de um lugar, mas também, na sua relação com o mapa, confirmando, reprovando e complementando, ao percorrer a realidade, as informações que fornece. O contraste entre a escala cartográfica e pedonal será explorado através de um exercício de atravessamento concreto – a actual linha de fronteira entre os distritos de Braga e Viana do Castelo, uma linha que à partida só existe no mapa, mas que, caminhando sobre ela no território real, pode adquirir espessuras que comprovam a sua existência e que compreendem os motivos do seu traçado, aparentemente abstracto no mapa. Para concretizar a linha de pensamento exposta o trabalho divide-se em quatro partes: a primeira incorpora quatro capítulos e corresponde a uma pesquisa teórica que explora, por um lado, os processos inerentes à construção de um mapa e, por outro, as múltiplas relações que o homem estabeleceu com o território a partir do atravessamento pedonal, de forma a reunir as ferramentas que a acção de caminhar oferece para explorar e conhecer o território. Depois de contextualizadas teoricamente as duas escalas, introduz-se o exercício prático de atravessar a linha-limite na parte II, estruturada em três capítulos, começando por apresentar o caso de estudo e definir uma estratégia para a sua análise/acção, passando por explicar a concretização do percurso e, por fim, ler e interpretar in situ os motivos para o traçado da linha-limite. O mapa é enquadrado quer na preparação/execução do percurso quer na descodificação da passagem da linha no território real, praticando constantemente o território a partir do percurso e do mapa. Por fim, na terceira parte estabelecem-se conclusões e reflexões sobre os temas abordados e, na última parte apresentam-se as referências bibliográficas e os anexos, onde se incorpora um arquivo do território a partir de situações captadas pelo caminhante durante o atravessamento e organiza-se um dossier com documentação referente à prática do caminhar., The idea that initially promotes this investigation relies on the simplification and graphical resemblance imposed by the mapping rules, resulting in abstract representations, difficult to identify with the real places. This problem incites on the one hand, a historically deconstructive research of the cartographic practice, evaluating the successive processes and codes used to transform reality in order to be readable and built-in the map; On the other, the action of walking is introduced as a tool that allows, by itself, an approach and a direct, detailed and complex reading and knowledge of a place, but also in its relationship with the map, confirming, reproving and completing, by crossing the reality, the information it provides. The contrast between the cartographic and pedestrian scale will be explored through a real walking exercise - exploring the current boundary line between the districts of Braga and Viana do Castelo, a line which initially only exists on the map. But walking on it in the real territory allows it acquire thicknesses, proving its existence and informing the motives of its layout, apparently abstract on the map. To materialize the line of thought exposed, the dissertation is divided into four parts: the first one incorporates four chapters and corresponds to a theoretical research that explores, on the one hand, the necessary processes to construct a map, and, on the other, the multiple relationships man has established with the territory from the pedestrian crossing, in order to gather the tools that the action of walking offers to explore and know the territory. After a theoretical contextualization of the two scales, we introduce the practical exercise of crossing the limit-line in Part II, structured in three chapters, beginning with the presentation of case study and setting a strategy for its analysis/action, then explaining the realization of the path, and finally, reading and interpreting the reasons for the limit-line layout. The map is used either in the preparation/realization of the route, either in the process of decoding the passage of the line in the actual territory. In this way, the walking exercise constantly practices the territory from the route and the map. Finally, in the third part are established conclusions and reflections on the topics addressed and, in the last part, we present the references and attachments, where an archive includes situations from the crossed territory, captured by the walker during the traversing, and is organized a dossier containing documentation related to the practice of walking.

The RcsCDB system of Salmonella enterica serovar Typhimurium is implicated in the control of capsule and flagella synthesis. The hybrid sensor RcsC, the phosphotransferase RcsD and the regulator RcsB, constitute the main components of the RcsCDB system. The proposed Rcs signaling cascade involves the autophosphorylation of RcsC and the transfer of the phosphate group to RcsB, mediated by RcsD. We previously reported that the overexpression of rcsB repress the transcription of rcsD by an autoregulation mechanism. Moreover, we demonstrated that during the rcsD repression, the RcsB-dependent flagellar modulation remained active. These results suggest that the Rcs phosphorelay mechanism occurs even in the absence of RcsD. In this work, we established the existence of two alternative phosphorelay pathways driving activation of this system. We demonstrated that RcsC and RcsD can act as histidine kinase proteins which, after autophosphorylated, are able to independently transfer the phosphate to RcsB. Our results suggest that these pathways could be activated by different environmental signals, leading different levels of RcsB-phosphorylated to produce a differential gene modulation. These findings contribute to a better understanding of the complexity and importance of the Rcs system activation, where more than one phosphate flow pathway increases the possibilities to exert gene regulation for a quick environmental changes response. Fil: Pescaretti, María de Las Mercedes. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina Fil: Farizano, Juan Vicente. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina Fil: Morero, Roberto Dionisio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina Fil: Delgado, Monica Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina

Background Microcin J25 (MccJ25) is a plasmid-encoded antibiotic peptide produced by Escherichia coli (E. coli). MccJ25 enters into the sensitive E. coli strains by the outer membrane receptor FhuA and the inner membrane proteins TonB, ExbB, ExbD and SbmA. The resistance of Salmonella enterica serovar Typhimurium (S. Typhimurium) to MccJ25 is attributed to the inability of its FhuA protein to incorporate the antibiotic into the cell. Results In this work we demonstrate that S. Typhimurium becomes notably susceptible to MccJ25 when replicating within macrophages. In order to determine the possible cause of this phenomenon, we studied the sensitivity of S. Typhimurium to MccJ25 at conditions resembling those of the internal macrophage environment, such as low pH, low magnesium and iron deprivation. We observed that the strain was only sensitive to the antibiotic at low pH, leading us to attribute the bacterial sensitization to this condition. A MccJ25-resistant E. coli strain in which fhuA is deleted was also inhibited by the antibiotic at low pH. Then, we could assume that the MccJ25 sensitivity change observed in both E. coli fhuA and S. Typhimurium is mediated by a MccJ25 uptake independent of the FhuA receptor. Moreover, low pH incubation also sensitized S. Typhimurium to the hydrophobic antibiotic novobiocin, which does not affect enteric bacteria viability because it is unable to penetrate the bacterial outer membrane. This observation supports our hypothesis about low pH producing a modification in the bacterial membrane permeability that allows an unspecific MccJ25 uptake. On the other hand, MccJ25 inhibited S. Typhimurium when cells were preincubated in acidic pH medium and then treated at neutral pH with the antibiotic. Conclusions Our results suggest that acidic condition does not alter MccJ25 hydrophobicity but irreversibly modifies bacterial membrane permeability. This would allow an unspecific antibiotic uptake into the cell. From our data it is possible to infer that intracellular pathogenic strains, which are in vitro resistant to MccJ25, could become susceptible ones in vivo. Therefore, the MccJ25 action spectrum would be broader than what in vitro experiments indicate.

Morphology has been widely used for inferring the phylogenies of numerous taxonomic groups. Recent molecular studies performed on extant non-human primates, however, have cast doubt on the reliability of cranial and postcranial characters for characterizing evolutionary affinities. Because molecular evidence is often not available for fossil specimens, detecting phylogenetic signals in anatomical features is of great relevance. Here we have analyzed molar (M1 and M2) crown shape by means of geometric morphometrics in a large sample of both extant and fossil Miocene catarrhine primates to detect the phylogenetic signal in molar morphometry. Results support that molar shape carries a strong phylogenetic signal, mostly at the superfamily level but also to some extent at the family level. Dietary factors, however, appear to have less influence, especially for M2. The Miocene Pliopithecoidea, Cercopithecoidea, and Hominoidea superfamilies clearly grouped according to the expected taxonomic affinities with the extant groups, although some discrepancies were found depending on the tooth considered. Our findings suggest that although molar crown shape can be used as a reliable proxy for establishing taxonomic affinities of catarrhine fossil primates with extant groups, a significant amount of interspecific variation exists, indicative of derived adaptations at the genus or species level. This research was funded by the Spanish grants CGL2011-22999 (Generalitat of Catalonia) and SGR2009-884 (Ministry of Economy and Competitiveness) to APP.

The genus Salmonella is the most common agent causative of foodborne diseases. Although genus Salmonella members are genetically close microorganisms, they show wide variations in host-specificity, virulence and disease manifestations. Salmonellosis caused by contaminated water or food is usually present as two clinical forms: typhoid fever and nontyphoidal diseases. The latter producing gastroenteritis is frequently caused by Salmonella Typhimurium and Salmonella Enteritidis. The nontyphoidal S. Typhimurium infection involves the following steps: bacterial adhesion, invasion, SCV maturation and replication. During these steps there is a strong interaction between host and pathogen, in which the pathogen must resist different host defense mechanisms. In each of these stages the bacteria modulate the expression of diverse groups of genes, most of which are encoded in Salmonella pathogenicity islands (SPI). This modulation is in general under control of the so-called two-component systems (TCS). The TCSs are capable of sensing different environmental conditions and trigger a physiological response. Currently, the risk of contracting salmonellosis disease has been considerably increased due to the emergence of new serovars showing multiple-drug resistance that presents a high risk to human health. In this work, we summarize the new advances in the study of host-pathogen interactions during the Salmonella infection that leads to the establishment of the disease. This finding highlights the role of the S. Typhimurium secretion systems and effectors during infection. In addition, we mentioned some strategies that could be explored in order to take control of Salmonella infections. © 2011 Elsevier Ltd. Fil: Lopez, Fabian Enrique. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Departamento de Bioquímica de la Nutrición | Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas. Departamento de Bioquímica de la Nutrición; Argentina. Instituto de Química Biologica “Dr. Bernabe Bloj”; Argentina Fil: Pescaretti, María de Las Mercedes. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Departamento de Bioquímica de la Nutrición | Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas. Departamento de Bioquímica de la Nutrición; Argentina. Instituto de Química Biologica “Dr. Bernabe Bloj”; Argentina Fil: Morero, Roberto Dionisio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Departamento de Bioquímica de la Nutrición | Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas. Departamento de Bioquímica de la Nutrición; Argentina. Instituto de Química Biologica “Dr. Bernabe Bloj”; Argentina Fil: Delgado, Monica Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Departamento de Bioquímica de la Nutrición | Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas. Departamento de Bioquímica de la Nutrición; Argentina. Instituto de Química Biologica “Dr. Bernabe Bloj”; Argentina

The degree of polymerization of O-antigen from Salmonella enterica serovar Typhimurium is controlled by the products of the wzz st and wzz fepE genes. In the present study we investigated the role of the PmrA/PmrB regulatory system in wzz fepE transcription. We report that the direct binding of the PmrA regulator to a specific promoter site induces the expression of the wzz fepE gene. This effect increases the amount of very long (VL) O-antigen, which is required for the resistance of Salmonella to serum human complement and polymyxin B, and for the replication of the bacteria within macrophages. The results obtained here highlight functional differences between Wzz fepE and Wzz st, although the genes for both proteins are regulated in a PmrA-dependent way. Fil: Pescaretti, María de Las Mercedes. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina Fil: Lopez, Fabian Enrique. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina Fil: Morero, Roberto Dionisio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina Fil: Delgado, Monica Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina

Introducción: El dolor postoperatorio es un tipo especial de dolor agudo, de gran repercusión en el sector de la salud. Un control óptimo del mismo en procederes proctológicos y del periné es un verdadero reto. Su tratamiento correcto puede aliviar el sufrimiento y permitir la movilización temprana y acortar la estadía hospitalaria. Objetivos: Identificar la utilidad de opioides en analgesia postoperatoria en cirugía proctológica. Método: Se realizó un estudio experimental, a simple ciegas, comparativo y prospectivo, para seleccionar la dosis de morfina intratecal a utilizar para la analgesia postoperatoria en pacientes a quienes se les realizó cirugía proctológica en el Servicio de Anestesia y Reanimación del Hospital Docente Clínico Quirúrgico Dr. Salvador Allende durante el 2009. La muestra estuvo constituida por 210 pacientes, seleccionados de forma aleatoria. Se agruparon en tres grupos según la dosis de morfina administrada: Grupo I (74 pacientes) se administró 3 µg/Kg; Grupo II (68 pacientes) 4 µg/Kg y en el Grupo III (68 pacientes) 5 µg/Kg. Resultados: El valor medio de la duración de la analgesia en horas en el Grupo I fue de 11,97 ± 1,67 DS, en el grupo II fue de 21,54 ± 2,29 y en el III de 21,4 ± 2,03. El prurito fue el efecto adverso con mayor incidencia, seguido por la retención urinaria, las nauseas y los vómitos. Conclusiones: La dosis de 4 µg/Kg parece ser la más recomendable para la analgesia postoperatoria con morfina intratecal en cirugía proctológica si se toma en cuenta la duración de la analgesia y los efectos adversos encontrados en nuestro estudio.

The antibiotic peptide microcin J25 (MccJ25), produced by an Escherichia coli strain, is ribosomally synthesized and consists of 21 amino acid residues (G1-G-A-G-H5-V-P-E-Y-F10-V-G-I-G-T15-P-I-S-F-Y20-G) (4, 12). MccJ25 is a lasso peptide (1, 10, 17), contains a lactam linkage between the α-amino group of Gly1 and the γ-carboxyl of Glu8, forming an 8-residue ring (Gly1 to Glu8), which is termed a lariat ring. The “tail” (Tyr9 to Gly21) passes through the ring, with Phe19 and Tyr20 straddling each side of the tail, sterically trapping the tail within the ring. MccJ25 amino acids F10 to P16 form a β-hairpin structure comprising two β-strands (F10-V11 and T15-P16) and a β-turn (V11 to G14). The uptake of MccJ25 into the E. coli periplasmic space depends on the outer membrane receptor FhuA and the inner membrane proteins TonB, ExbD, and ExbB (11, 13). Additionally, the inner membrane protein SbmA transports MccJ25 from the periplasmic to the cytoplasmic space (13). Once inside the sensitive cell, the peptide is able to inhibit E. coli RNA polymerase (RNAP) and membrane respiratory chain, which represent the MccJ25 targets (2, 5, 7, 18). Several Salmonella enterica serovars showed high sensitivity against MccJ25, while others, like Salmonella enterica serovar Typhimurium, S. enterica serovar Derby, and some S. enterica serovar Enteritidis strains were completely resistant (16). Since introduction of the E. coli fhuA allele cloned in a multicopy plasmid into these bacteria rendered them hypersensitive to the antibiotic, we concluded that this intrinsic resistance is due to the inability of the FhuA receptor protein to mediate the penetration of MccJ25. In fact, MccJ25 was able to inhibit both intracellular targets in the transformed strains (16). The polianionic lipopolysaccharide (LPS) component of the outer membrane is stabilized by divalent cation bridges (15). It was suggested that many cationic peptides are able to bind to LPS and disrupt these bridges, resulting in an increased bacterial membrane permeabilization. Vaara and Porro (15) characterized a series of synthetic peptides that were able to sensitize Gram-negative bacteria to hydrophobic and amphipathic antibiotics. One of them, KFFKFFKFFK [(KFF)3K], a peptide rich in cationic lysine and hydrophobic phenylalanine residues, showed a potent effect on outer membrane disorganization and weak damage to the cytoplasmic membrane (15). In this work, we have shown that the (KFF)3K peptide allows MccJ25 uptake independently of the FhuA and SbmA receptors, turning in vitro microcin-resistant strains into susceptible ones. Moreover, we have demonstrated that (KFF)3K was able to exert the same inhibitory effect in vivo on S. Typhimurium replicating within eukaryotic cells.

The SbmA protein is involved in the transport of MccB17-, MccJ25-, bleomycin- and proline-rich peptides into the Escherichia coli cytoplasm. sbmA gene homologues were found in a variety of bacteria. However, the physiological role of this protein still remains unknown. Previously, we found that a combination of sbmA and tolC mutations in Tn10-carrying E. coli K-12 strains results in hypersusceptibility to tetracycline. In this work, we studied sbmA expression in a tolC mutant background and observed an increased expression throughout growth. We ruled out the global transcriptional regulator RpoS and the small RNA micF as intermediates in this regulation. The tolC mutation induced the expression of other well-characterized strong σ(E) -dependent promoters in E. coli. We observed that the increase in σ(E) activity led to a greater sbmA expression, conversely eliminating σ(E) prevented expression of sbmA. We also observed that the sbmA upregulation in a tolC mutant context was abolished in an rpoE-null strain. These results suggest a σ(E) -dependent positive regulation on sbmA by the tolC mutation. We hypothesize that this mechanism might be part of a compensatory cell envelope stress response.

IRGM, a human immunity-related GTPase, confers autophagic defence against intracellular pathogens by an unknown mechanism. Here, we report an unexpected mode of IRGM action. IRGM demonstrated differential affinity for the mitochondrial lipid cardiolipin, translocated to mitochondria, affected mitochondrial fission and induced autophagy. Mitochondrial fission was necessary for autophagic control of intracellular mycobacteria by IRGM. IRGM influenced mitochondrial membrane polarization and cell death. Overexpression of IRGMd, but not IRGMb splice isoforms, caused mitochondrial depolarization and autophagy-independent, but Bax/Bak-dependent, cell death. By acting on mitochondria, IRGM confers autophagic protection or cell death, explaining IRGM action both in defence against tuberculosis and in the damaging inflammation caused by Crohn's disease.

En 2006 se llevó a cabo una prospección en la Cueva del Reguerillo. Se pretendía autentificar o negar la existencia de manifestaciones artísticas paleolíticas, como venía reflejándose en la bibliografía precedente. La documentación de los paneles grabados nos permitió definir e inventariar otro tipo de representaciones postpaleolíticas (retículas, marañas, bandas o haces de líneas…), que no dejan de tener un interés relevante., In 2006 we carried out a survey in the cave of El Reguerillo. We pretended to authenticate or to deny the existence of Palaeolithic artistic expressions, which presence had been considered in the previous bibliography. The documentation of the engraved panels allowed us to define and to make up the inventory of another type of interesting postpalaeolithic representations (reticles, tangles, stripes or bundles of lines…).

International audience; The majority of studies on autophagy, a cytoplasmic homeostatis pathway of broad biological and medical significance, have been hitherto focused on the phosphatidylinositol 3-kinases as the regulators of autophagy. Here, we addressed the reverse process driven by phosphoinositide phosphatases and uncovered a key negative regulatory role in autophagy of a phosphatidylinositol 3-phosphate (PI3P) phosphatase Jumpy (MTMR14). Jumpy associated with autophagic isolation membranes and early autophagosomes, defined by the key factor Atg16 necessary for proper localization and development of autophagic organelles. Jumpy orchestrated orderly succession of Atg factors by controlling recruitment to autophagic membranes of the sole mammalian Atg factor that interacts with PI3P, WIPI-1 (Atg18), and by affecting the distribution of Atg9 and LC3, the two Atg factors controlling organization and growth of autophagic membranes. A catalytically inactive Jumpy mutant, R336Q, found in congenital disease centronuclear myopathy, lost the ability to negatively regulate autophagy. This work reports for the first time that initiation of autophagy is controlled not only by the forward reaction of generating PI3P through a lipid kinase but that its levels are controlled by a specific PI3P phosphatase, which when defective can lead to human disease.

Autophagy is a cell biological process, enabling cells to autodigest their own cytosol when starved, remove cytoplasmic protein aggregates too large for proteasomal degradation, eliminate aberrant or over-proliferated organelles, and sanitize the cytoplasm by killing intracellular microbes. The role of autophagy has been expanded in recent years to include diverse immunological effector and regulatory functions. In this review, we summarize the multiple immunological roles of autophagy uncovered to date and focus primarily on details of induction of autophagy by pattern recognition receptors, as a newly established Toll-like receptor output. Taken together with other links between autophagy and innate and adaptive immunity processes, this cell-autonomous antimicrobial defense may be evolutionarily positioned at the root of immunity with the multiple innate and adaptive immunity connections uncovered to date reflecting a co-evolution of this ancient cell-defense mechanism and more advanced immunological systems in metazoans.

Introducción: La ventilación controlada por presión es un modo de ventilación ampliamente utilizado en el fallo respiratorio severo, donde ha demostrado que mejora la oxigenación arterial. Objetivo: Comparar la ventilación controlada por volumen comúnmente utilizado durante la ventilación unipulmonar con tres estrategias ventilatorias de ventilación controlada por presión. Método: Se realizó un estudio comparativo y prospectivo de 100 pacientes tratados por cirugía torácica divididos en cuatro grupos según modo ventilatorio utilizado. Grupo I VCV, con volumen minuto 100 mL/kg. Grupo II VCP con Vot de 10 mL/kg, Grupo III VCP con Vot de 8 mL/kg, y Grupo IV VCP con 5cm de H2O de PEEP y un Vot de 8 mL/kg. Se comparó PaO2, SatO2, Shunt Intrapulmonar, Presión Pico y Meseta a los 30 minutos de la ventilación unipulmonar y los episodios de desaturación arterial. Se utilizó Chi Cuadrado y ANOVA para el análisis estadístico. Resultados: La PaO2 presentó valores similares en los Grupos I y II, 148,28 ± 68,21 y 146,8 ± 67,8 mmHg, respectivamente, disminuyó en el Grupo III a 117,2 ± 51,0 mmHg y aumentó significativamente en el Grupo IV 189,0 ± 49,2 mmHg. La SatO2 se incrementó y el shunt intrapulmonar disminuyó con significación estadística en el Grupo IV. Las presiones en la vía aérea fueron menores durante la VCP. Conclusión: La ventilación controlada por presión "per se" no mejoró las variables de oxigenación durante la ventilación unipulmonar, pero sí, permite alcanzar menores presiones en la vía aérea. La administración de PEEP durante la ventilación controlada por presión evidenció mejoría significativa de la oxigenación.

SummaryAutophagy allows cells to self-digest portions of their own cytoplasm for a multitude of physiological purposes, including innate and adaptive immunity functions. In one of its innate immunity manifestations, autophagy, is known to contribute to the killing of intracellular microbes, including Mycobacterium tuberculosis, although the molecular mechanisms have been unclear. Here, we delineated sequential steps of the autophagic pathway necessary to control intracellular M. tuberculosis and found that in addition to autophagy initiation and maturation, an accessory autophagy-targeting molecule p62 (A170 or SQSTM1) was required for mycobactericidal activity. The p62 adaptor protein delivered specific ribosomal and bulk ubiquitinated cytosolic proteins to autolysosomes where they were proteolytically converted into products capable of killing M. tuberculosis. Thus, p62 brings cytosolic proteins to autolysosomes where they are processed from innocuous precursors into neo-antimicrobial peptides, explaining in part the unique bactericidal properties of autophagic organelles.

The recognition of autophagy as an immune mechanism has been affirmed in recent years. One of the model systems that has helped in the development of our current understanding of how autophagy and more traditional immunity systems cooperate in defense against intracellular pathogens is macrophage infection with Mycobacterium tuberculosis. M. tuberculosis is a highly significant human pathogen that latently infects billions of people and causes active disease in millions of patients worldwide. The ability of the tubercle bacillus to persist in human populations rests upon its macrophage parasitism. One of the initial reports on the ability of autophagy to act as a cell-autonomous innate immunity mechanism capable of eliminating intracellular bacteria was on M. tuberculosis. This model system has further contributed to the recognition of multiple connections between conventional immune regulators and autophagy. In this chapter, we will review how these studies have helped to establish the following principles: (1) autophagy functions as an innate defense mechanism against intracellular microbes; (2) autophagy is under the control of pattern recognition receptors (PRR) such as Toll-like receptors (TLR), and it acts as one of the immunological output effectors of PRR and TLR signaling; (3) autophagy is one of the effector functions associated with the immunity-regulated GTPases, which were initially characterized as molecules involved in cell-autonomous defense, but whose mechanism of function was unknown until recently; (4) autophagy is an immune effector of Th1/Th2 T cell response polarization—autophagy is activated by Th1 cytokines (which act in defense against intracellular pathogens) and is inhibited by Th2 cytokines (which make cells accessible to intracellular pathogens). Collectively, the studies employing the M. tuberculosis autophagy model system have contributed to the development of a more comprehensive view of autophagy as an immunological process. This work and related studies by others have led us to propose a model of how autophagy, an ancient innate immunity defense, became integrated over the course of evolution with other immune mechanisms of ever-increasing complexity.

Tuberculosis is one of the world's most prevalent infectious diseases. The causative agent, M. tuberculosis, asymptomatically infects more than 30% of the world population and causes 8 million cases of active disease and 2 million deaths annually. Its pathogenic success stems from its ability to block phagolysosome biogenesis and subsequent destruction in the host macrophages. Recently, our laboratory has uncovered autophagy as a new means of overcoming this block and promoting the killing of mycobacteria. Here we describe the methods to study autophagy during M. tuberculosis infection of macrophages. The described assays can be used to investigate and identify factors important for autophagic elimination of mycobacteria that could potentially provide new therapeutic targets to defeat this disease.

Se propone la restauración – reconstrucción – recreación virtual del arte rupestre prehistórico como alternativa económica y no destructiva a las intervenciones directas. Mediante montaje fotográfico, se pretende recrear el aspecto que debieron tener los emplazamientos con arte prehistórico en origen, a partir de un método inocuo, reversible y modificable.Virtual restoration - reconstruction - recreation of rock art would be an economical and non-destructive alternative to direct intervention. By using photomontage, an innocuous as well as reversible method,we expect to recreate how rock art sites would have looked like originally.

Autophagy is a physiologically and immunologically controlled intracellular homeostatic pathway that sequesters and degrades cytoplasmic targets including macromolecular aggregates, cellular organelles such as mitochondria, and whole microbes or their products. Recent advances show that autophagy plays a role in innate immunity in several ways: (i) direct elimination of intracellular microbes by digestion in autolysosomes, (ii) delivery of cytosolic microbial products to pattern recognition receptors (PRRs) in a process referred to as topological inversion, and (iii) as an anti-microbial effector of Toll-like receptors and other PRR signaling. Autophagy eliminates pathogens in vitro and in vivo but, when aberrant due to mutations, contributes to human inflammatory disorders such as Crohn's disease. In this review, we examine these relationships and propose that autophagy is one of the most ancient innate immune defenses that has possibly evolved at the time of alpha-protobacteria-pre-eukaryote relationships, leading up to modern eukaryotic cell-mitochondrial symbiosis, and that during the metazoan evolution, additional layers of immunological regulation have been superimposed and integrated with this primordial innate immunity mechanism.

The physiologic importance of autophagy proteins for control of mammalian bacterial and parasitic infection in vivo is unknown. Using mice with granulocyte- and macrophage-specific deletion of the essential autophagy protein Atg5, we show that Atg5 is required for in vivo resistance to the intracellular pathogens Listeria monocytogenes and Toxoplasma gondii. In primary macrophages, Atg5 was required for interferongamma (IFN-gamma)/LPS-induced damage to the T. gondii parasitophorous vacuole membrane and parasite clearance. While we did not detect classical hallmarks of autophagy, such as autophagosomes enveloping T. gondii, Atg5 was required for recruitment of IFN-gamma-inducible p47 GTPase IIGP1 (Irga6) to the vacuole membrane, an event that mediates IFN-gamma-mediated clearance of T. gondii. This work shows that Atg5 expression in phagocytic cells is essential for cellular immunity to intracellular pathogens in vivo, and that an autophagy protein can participate in immunity and intracellular killing of pathogens via autophagosome-independent processes such as GTPase trafficking.

Nava, Alejandro. La inquieta superficie terrestre. México: (SEP; FCE; CONACYT), 1993. (La Ciencia para Todos; 113).

Autophagy is a newly recognized innate defense mechanism, acting as a cell-autonomous system for elimination of intracellular pathogens. The signals and signalling pathways inducing autophagy in response to pathogen invasion are presently not known. Here we show that autophagy is controlled by recognizing conserved pathogen-associated molecular patterns (PAMPs). We screened a PAMP library for effects on autophagy in RAW 264.7 macrophages and found that several prototype Toll-like receptor (TLR) ligands induced autophagy. Single-stranded RNA and TLR7 generated the most potent effects. Induction of autophagy via TLR7 depended on MyD88 expression. Stimulation of autophagy with TLR7 ligands was functional in eliminating intracellular microbes, even when the target pathogen was normally not associated with TLR7 signalling. These findings link two innate immunity defense systems, TLR signalling and autophagy, provide a potential molecular mechanism for induction of autophagy in response to pathogen invasion, and show that the newly recognized ability of TLR ligands to stimulate autophagy can be used to treat intracellular pathogens.

Interconversions of phosphoinositides play a pivotal role during phagocytosis and at the subsequent stages of phagosomal maturation into the phagolysosome. Several model systems have been used to study the role of phosphoinositides in phagosomal membrane remodelling. These include phagosomes formed by inanimate objects such as latex beads, or pathogenic bacteria, e.g. Mycobacterium tuberculosis. The latter category provides naturally occurring tools to dissect membrane trafficking processes governing phagolysosome biogenesis. M. tuberculosis persists in infected macrophages by blocking Rab conversion and affecting Rab effectors. One of the major Rab effectors involved in this process is the type III phosphatidylinositol 3-kinase hVPS34. The lipid kinase hVPS34 and its enzymatic product PtdIns3 P are critical for the default pathway of phagosomal maturation into phagolysosomes. Mycobacteria block PtdIns3 P production and thus arrest phagosomal maturation. PtdIns3 P is also critical for the process of autophagy, recently recognized as an effector of innate immunity defenses. Induction of autophagy by pharmacological, physiological, or immunological means, overcomes mycobacterial phagosome maturation block in a PtdIns3 P generation dependent manner and eliminates intracellular M. tuberculosis. PtdIns3 P and PtdIns3 P -dependent processes represent an important cellular nexus where fundamental trafficking processes, disease causing host–pathogen interactions, and innate and adaptive immunity defense mechanisms meet.

Endosomal hyperacidification in cystic fibrosis (CF) respiratory epithelial cells is secondary to a loss of sodium transport control owing to a defective form of the CF transmembrane conductance regulator CFTR. Here, we show that endosomal hyperacidification can be corrected by activating the signalling cascade controlling sodium channels through cyclic GMP. Nitric oxide (NO) donors corrected the endosomal hyperacidification in CF cells. Stimulation of CF cells with guanylate cyclase agonists corrected the pH in endosomes. Exposure of CF cells to an inhibitor of cGMP-specific phosphodiesterase PDE5, Sildenafil, normalized the endosomal pH. Treatment with Sildenafil reduced secretion by CF cells of the proinflammatory chemokine interleukin 8 following stimulation with Pseudomonas aeruginosa products. Thus, the endosomal hyperacidification and excessive proinflammatory response in CF are in part due to deficiencies in NO- and cGMP-regulated processes and can be pharmacologically reversed using PDE5 inhibitors.

The lipopolysaccharide (LPS) is the outermost component of the cell envelope in Gram-negative bacteria. It consists of the hydrophobic lipid A, a short non-repeating core oligosaccharide and a distal polysaccharide termed O-antigen. We report here that the PmrA/PmrB and RcsC/YojN/RcsB two-component systems of Salmonella enterica serovar Typhimurium independently promote transcription of the wzzst gene, which encodes a protein that determines the chain length of the O-antigen. We show that the regulatory proteins PmrA and RcsB footprint partially overlapping regions of the wzz st promoter stimulating transcription from the same start site. Induction of the PmrA/PmrB or RcsC/YojN/RcsB systems increased the fraction of LPS molecules containing 16-35 O-antigen subunits, leading to heightened resistance to serum. The LPS of a rcsB null mutant exhibited an altered mobility in the O-antigen subunits attached to the lipid A-core region when separated on a SDS/PAGE gel, suggesting that RcsB may regulate additional LPS genes. Inactivation of the wzzst gene eliminated the enhanced swarming behaviour exhibited by the rcsB mutant. That multiple regulatory systems control wzzst expression suggests that the Wzzst protein is required under different environmental conditions. Fil: Delgado, Monica Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina Fil: Mouslim, Chakib. University of Washington; Estados Unidos Fil: Groisman, Eduardo A.. University of Washington; Estados Unidos

In the present study, we showed that yojI, an Escherichia coli open reading frame with an unknown function, mediates resistance to the peptide antibiotic microcin J25 when it is expressed from a multicopy vector. Disruption of the single chromosomal copy of yojI increased sensitivity of cells to microcin J25. The YojI protein was previously assumed to be an ATP-binding-cassette-type exporter on the basis of sequence similarities. We demonstrate that YojI is capable of pumping out microcin molecules. Thus, one obvious explanation for the protective effect against microcin J25 is that YojI action keeps the intracellular concentration of the peptide below a toxic level. The outer membrane protein TolC in addition to YojI is required for export of microcin J25 out of the cell. Microcin J25 is thus the first known substrate for YojI. Fil: Delgado, Monica Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia. Instituto de Química Biológica; Argentina Fil: Vincent, Paula Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia. Instituto de Química Biológica; Argentina Fil: Farias, Ricardo N.. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia. Instituto de Química Biológica; Argentina Fil: Salomon, Raul Armando. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia. Instituto de Química Biológica; Argentina

Adhesion to the extracellular matrix (ECM) is critical for epithelial tissue survival and function, because detachment from the ECM induces metabolic stress and programmed cell death via anoikis. Recently, it was shown that macroautophagy (hereafter called autophagy) is also induced by detachment from the ECM in different cell types, including human and murine epithelial cells and fibroblasts. Initiation of autophagy upon ECM detachment appears to be involved in cell survival, as inhibition of autophagy enhances cell death by anoikis. The BMF (Bcl2 modifying factor) protein contains the BCL2 homology region 3, and thereby is a member of the BCL2 protein family. BMF is implicated in regulating cell death following detachment of cells from the ECM. During ECM detachment BMF mRNA is upregulated to allow anoikis in human breast epithelial cells, and suppressing BMF expression disrupts anoikis. BMF is attached to the cytoskeleton through the DYNLL2/DLC2 (dynein, light chain, LCB-type 2) that connects it with MYO5/myosin V and the actin filaments. Loss of attachment of a transformed cell line (MCF-7) releases BMF from the cytoskeleton, and the released BMF interacts with mitochondrial BCL2 to induce apoptosis. We found that BMF also has a role as a regulator of autophagy. When BMF levels are suppressed in IFNγ-treated human or mouse airway epithelial cells, LC3-II conversion and BECN1 expression are increased. Furthermore, primary airway epithelial cells or embryonic fibroblasts from bmf−/− mice present more LC3-II and more autophagosome structures when analyzed by electron microscopy. The conclusion regarding the formation of autophagosomes is also supported by the observed change from a diffuse to punctate fluorescence of cherry-LC3 in bmf−/− cells. In contrast, restoring BMF levels in IFNγ-treated cells decreases the levels of LC3-II and BECN1, supporting the idea that BMF inhibits autophagy. We also demonstrated that BMF co-immunoprecipitates with BECN1 and BCL2, and that the interaction between BECN1 and BCL2 decreases in the absence of BMF, suggesting that BMF may inhibit initiation of autophagy by stabilizing the BECN1-BCL2 protein complex. However, the molecular mechanisms of this interaction, whether other proteins are part of this inhibitory complex, and whether other pathways impinge on this role of BMF remain to be elucidated. Because BMF is involved in detachment-induced cell death, and our observations show that BMF regulates autophagy, together with the recent reports on autophagy being involved in ECM detachment and cell death, led us to postulate that detachment-induced autophagy may involve the BMF protein. Detachment from the ECM may cause the release of BMF from the cytoskeleton allowing the autophagy inhibitory complex BECN1-BCL2 to be released and BECN1 to localize to the ER to initiate autophagosome formation. Other pathways that are initiated by ECM detachment may also be involved in inducing autophagy. Suppression of the PI3K-AKT-MTORC1 pathway activation is the major regulator of autophagy induction in detached mouse fibroblasts. However, in human mammary epithelial cells deprived of ECM contact, activation of the IKK complex (the catalytic subunits CHUK/IKKα and IKBKB/IKKβ, and the regulatory subunit IKBKG/IKKγ) is crucial in promoting autophagy and is independent of MTOR signaling. AMP-activated protein kinase (AMPK) is robustly activated during ECM detachment, but its role in activating autophagy after ECM detachment still needs to be verified. In this context, because BMF has been reported to be upregulated by AMPK activation, upregulated BMF following detachment from the ECM may lose its capacity to inhibit autophagy, because it is no longer attached to the cytoskeleton. This idea is also consistent with the upregulation of BMF mRNA to allow anoikis after ECM detachment in human breast epithelial cells. After cell detachment, there is also increased phosphorylation of EIF2S1/eIF2α (eukaryotic translation initiation factor 2, subunit 1 α, 35 kDa), a stress-regulated transcriptional suppressor that upregulates autophagy during nutritional starvation and ER stress. The various pathways may either occur simultaneously or may interact to enhance autophagy. For example, IKK-stimulated autophagy is controlled by the canonical AMPK-MTOR pathway and MAPK8/JNK1 activation in HeLa cells, although in certain cell types the IKK pathway induces autophagy independent of MTOR. All these pathways, including BMF released from the cytoskeleton, may participate in ECM detachment-induced autophagy depending on cell type and the nutritional or stressed conditions of the cells. Therefore, we can speculate that when BMF is released from the cytoskeleton in response to ECM detachment, BMF may interact with one or more autophagy-activating pathways. As autophagy is a survival mechanism that protects cells from dying after ECM detachment, the intensity of autophagy induction may determine why some cell types die within a few hours while others survive for several days following ECM detachment. We would propose that following detachment-induced expression, BMF would have a role in inhibiting autophagy or enhancing anoikis depending on whether BMF is attached to DYNLL2/DLC2 or is released into the cytoplasm (Fig. 1). Figure 1. Proposed pathway for BMF involvement in anoikis and autophagy. ECM detachment is a topic of high interest as the survival of oncogenic cells lacking matrix contact is considered to be a critical feature for metastasis. Therefore, detailed understanding of the pathways involved in regulating detachment-induced autophagy could help develop better therapies for a variety of cancers.

Escherichia coli microcin J25 (MccJ25) is a 2107-Da peptide antibiotic whose uptake into E. coli is mediated by the outer-membrane receptor FhuA and the inner membrane proteins TonB, ExbB, ExbD, and SbmA. A survey of the sensitivity of several Salmonella enterica serovars showed that the antibiotic was highly active against some serovars, while S. Typhimurium, S. Derby, and some S. Enteritidis strains were completely resistant. Resistant strains became hypersensitive to MccJ25 when given the fhuA gene of E. coli, indicating that insensitivity is due to the inability of the FhuA protein to mediate penetration of MccJ25. Whereas in E. coli MccJ25 targets RNA polymerase, in S. Typhimurium it inhibits not only RNA synthesis but also cell respiration. Fluorescence viability staining showed that S. Typhimurium cells exposed to MccJ25 remain viable but are unable to form colonies. Fil: Vincent, Paula Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia. Instituto de Química Biológica; Argentina Fil: Delgado, Monica Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia. Instituto de Química Biológica; Argentina Fil: Farias, Ricardo Norberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia. Instituto de Química Biológica; Argentina Fil: Salomon, Raul Armando. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia. Instituto de Química Biológica; Argentina

Escherichia coli microcin J25 (MccJ25) is a 2107-Da peptide antibiotic whose uptake into E. coli is mediated by the outer-membrane receptor FhuA and the inner membrane proteins TonB, ExbB, ExbD, and SbmA. A survey of the sensitivity of several Salmonella enterica serovars showed that the antibiotic was highly active against some serovars, while S. Typhimurium, S. Derby, and some S. Enteritidis strains were completely resistant. Resistant strains became hypersensitive to MccJ25 when given the fhuA gene of E. coli, indicating that insensitivity is due to the inability of the FhuA protein to mediate penetration of MccJ25. Whereas in E. coli MccJ25 targets RNA polymerase, in S. Typhimurium it inhibits not only RNA synthesis but also cell respiration. Fluorescence viability staining showed that S. Typhimurium cells exposed to MccJ25 remain viable but are unable to form colonies.

The Escherichia coli plasmid pTUC100 encodes production of, and immunity to, the peptide antibiotic microcin J25. In the present study, an approximately 8-kb fragment immediately adjacent to the previously sequenced microcin region was isolated and its DNA sequence was determined. The main features of the newly characterized region are: (i) a basic replicon which is almost identical to that of the RepFIIA plasmid R100; (ii) two ORFs with 96% identity to two ORFs of unknown function on pO157, a large plasmid harbored by enterohemorragic E. coli, and a large ORF which does not show significant homology to any other reported nucleotide or protein sequence; and (iii) two intact insertion sequences, IS1294 and IS1. Sequence analysis, as well as that of the G + C content of both the 8-kb fragment and the previously sequenced microcin locus, lead us to propose that plasmid pTUC100 is a composite structure assembled from DNA elements from various sources. Fil: Delgado, Monica Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia. Instituto de Química Biológica; Argentina Fil: Salomon, Raul Armando. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia. Instituto de Química Biológica; Argentina

A mutation in the conserved segment of the rpoC gene, which codes for the largest RNA polymerase (RNAP) subunit, beta', was found to make Escherichia coli cells resistant to microcin J25 (MccJ25), a bactericidal 21-amino acid peptide active against Gram-negative bacteria (Delgado, M. A., Rintoul, M. R., Farias, R. N., and Salomon, R. A. (2001) J. Bacteriol. 183, 4543-4550). Here, we report that mutant RNAP prepared from MccJ25-resistant cells, but not the wild-type RNAP, is resistant to MccJ25 in vitro, thus establishing that RNAP is a true cellular target of MccJ25. We also report the isolation of additional rpoC mutations that lead to MccJ25 resistance in vivo and in vitro. The new mutations affect beta' amino acids in evolutionarily conserved segments G, G', and F and are exposed into the RNAP secondary channel, a narrow opening that connects the enzyme surface with the catalytic center. We also report that previously known rpoB (RNAP beta subunit) mutations that lead to streptolydigin resistance cause resistance to MccJ25. We hypothesize that MccJ25 inhibits transcription by binding in RNAP secondary channel and blocking substrate access to the catalytic center.

Escherichia coli microcin J25 (MccJ25) is a plasmid-encoded, cyclic peptide antibiotic consisting of 21 unmodified amino acid residues. It is primarily active on gram-negative bacteria related to the producer strain, inducing cell filamentation in an SOS-independent way. A mutation causing resistance to MccJ25 was isolated. Genetic analysis indicated that it resided in the rpoC gene, encoding the β′ subunit of RNA polymerase, at 90 rain on the E. coli genetic map. The mutation was genetically crossed on to a plasmid containing the wild-type rpoC gene. The presence of the recombinant plasmid conferred complete resistance to otherwise sensitive strains. Nucleotide sequencing of the plasmid-borne, mutant rpoC gene revealed a ACC (Thr)-to-ATC (Ile) change at codon 931, within homology block G, an evolutionarily conserved region in the large subunits of all RNA polymerases. MccJ25 decreased RNA synthesis both in vivo and in vitro. These results point to the RNA polymerase as the target of microcin action. We favor the possibility that the filamentous phenotype induced by MccJ25 results from impaired transcription of genes coding for cell division proteins. As far as we know, MccJ25 is the first peptide antibiotic shown to affect RNA polymerase. Fil: Delgado, Monica Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina Fil: Rintoul, Maria Regina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina Fil: Farias, Ricardo Norberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina Fil: Salomon, Raul Armando. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina

Microcin J25 is a 2,107-Da, plasmid-encoded, cyclopeptide antibiotic produced by Escherichia coli. We have isolated lacZ fusions to mcjA (encoding the 58-amino-acid microcin precursor) and mcjB and mcjC (which are required for microcin maturation), and the regulation of these fusions was used to identify factors that control the expression of these genes. The mcjA gene was found to be dramatically induced as cells entered the stationary phase. Expression of mcjA could be induced by resuspending uninduced exponential-phase cells in spent supernatant obtained from an early-stationary-phase culture. Induction of mcjA expression was not dependent on high cell density, pH changes, anaerobiosis, or the buildup of some inducer. A starvation for carbon and inorganic phosphate induced mcjA expression, while under nitrogen limitation there was no induction at all. These results taken together suggest that stationary-phase induction of mcjA is triggered by nutrient depletion. The mcjB and mcjC genes were also regulated by the growth phase of the culture, but in contrast to mcjA , they showed substantial expression already during exponential growth. Induction of the microcin genes was demonstrated to be independent of RpoS, the cyclic AMP-Crp complex, OmpR, and H-NS. Instead, we found that the growth-phase-dependent expression of mcjA , mcjB , and mcjC may be explained by the concerted action of the positively acting transition state regulators ppGpp, Lrp, and integration host factor. Measurements of microcin J25 production by strains defective in these global regulators showed a good correlation with the reduced expression of the fusions in such mutant backgrounds.

Objetivo. Se analiza la relación entre algunos indicadores de enfermedades mentales y la adherencia terapéutica en pacientes con VIH/SIDA. Participantes. Muestra de 76 pacientes mexicanos infectados por VIH/SIDA. Procedimiento. Entrevista a profundidad a pacientes de un hospital público de la ciudad de San Luis Potosí, México. Resultados. 80% de los participantes presentó indicadores de enfermedades mentales correspondientes a: 28% conducta antisocial; 20% depresión clínica; 13% síndrome de estrés postraumático, 12% psicosis orgánica y 8% discapacidad intelectual. El porcentaje de pacientes infectados con VIH/SIDA con indicadores de enfermedad mental concuerda con los resultados de investigaciones realizadas en otros países. Solamente 7% de los participantes mostró un nivel de adherencia apropiado. La adherencia no guarda una relación lineal con la presencia de indicadores de enfermedad mental. La adherencia se relaciona con la escolaridad, la utilización de estrategias para recordar la toma del medicamento, confiar el estatus a los familiares y apoyo familiar.