Virtual screening studies to identify novel inhibitors for Sigma F protein of Mycobacteriumm tuberculosis

Tuberculosis [TB] is one of the oldest threats to public health. TB is caused by the pathogen Mycobacterium tuberculosis [MTB]. The Sigma factors are essential for the survival of MTB. The Sigma factor Sigma F [SigF] regulates genes expression under stress conditions. The SigF binds to RNA polymerase and forms a holoenzyme, which initiates the transcription of various genes. The Usfx, an anti-SigF protein, binds to SigF and alters the transcription initiation and gene expression. In the present work, virtual screening studies are taken up to identify the interactions between SigF and small molecular inhibitors which can inhibit the formation of holoenzyme. The studies reveal that ARG 104 and ARG 224 amino acid residues of SigF protein are forming important binding interactions with the ligands. The in silico ADME properties for the ligand data set are calculated to check the druggability of the molecules

Occurrence of Lutzomyia longipalpis (Phlebotominae) and canine visceral leishmaniasis in a rural area of Ilha Solteira, SP, Brazil / Ocorrência de Lutzomyia longipalpis (Phlebotominae) e leishmaniose visceral canina em uma área rural de Ilha Solteira, SP, Brasil

This study aimed to investigate the occurrence of Lutzomyia longipalpis and also the canine visceral leishmaniasis (CVL) in a rural area of Ilha Solteira, state of São Paulo. Blood samples were collected from 32 dogs from different rural properties (small farms) and were analyzed by ELISA and the indirect immunofluorescence antibody test (IFAT) in order to diagnose CVL. From these serological tests, 31.25% of the dogs were positive for CVL and these were distributed in 66.7% (8/12) of the rural properties, which were positive for L. longipalpis. CDC (Center for Disease Control and Prevention) light traps were installed in 12 properties (one per property) and insects were caught on three consecutive days per month for one year. L. longipalpis was present on 100% of the rural properties visited, at least once during the twelve-month interval, totaling 64 males and 25 females. The insects were more numerous after the peak of the rain, but the association between prevalence of peridomestic vectors and the climatic data (precipitation, relative air humidity and temperature) and the occurrences of CVL among dogs on each rural property were not statistical significant (p <0.05). However, the occurrence of CVL cases in dogs and the presence of L. longipalpis indicate that more attention is necessairy for the control of this disease in the rural area studied.

O objetivo desse trabalho foi o estudo da prevalência de Lutzomyia longipalpis e da leishmaniose visceral canina (LVC) em uma área rural do município de Ilha Solteira do estado de São Paulo. Amostras de sangue foram coletadas de 32 cães provenientes de pequenas propriedades rurais e analisadas por meio dos métodos sorológicos ELISA (imunoensaio enzimático indireto) e RIFI (reação de imunofluorescência indireta) para o diagnóstico da LVC. Pelos exames sorológicos, dos 32 cães avaliados, 31,25% foram diagnosticados positivos para LVC, os quais estavam diostribuídos em 66,67% (8/12) das propriedades positivas para Lutzomyia longipalpis. Armadilhas luminosas do tipo CDC (Center for Disease Control and Prevention) foram instaladas em 12 propriedades, sendo uma por propriedade, e as coletas dos insetos foram realizadas três dias consecutivos a cada mês, durante um ano. O inseto L. longipalpis foi encontrado em 100% das propriedades visitadas, pelo menos uma vez no ano, totalizando 65 machos e 25 fêmeas. A maior quantidade de insetos foi observada principalmente após a ocorrência dos maiores picos de precipitação pluvial, mas a associação entre a prevalência dos vetores peridomiciliares e os dados climáticos (precipitação, umidade relativa do ar e temperatura) assim como a ocorrência da CVL em cães em cada propriedade não foi estatisticamente significante (p<0.05). No entanto, alerta-se que pela presença dos casos de LVC nos cães amostrados e também de L. longipalpis, maior atenção deve ser dada durante as investigações epidemiológicas para o controle dessa doença nessa área rural estudada.

Infant and young child feeding in four departments in Haiti: mixed-method study on prevalence of recommended practices and related attitudes, beliefs, and other determinants / Alimentación de los lactantes y niños pequeños en cuatro departamentos de Haití: un estudio con diseño de método mixto sobre la prevalencia de las prácticas recomendadas y las actitudes, las creencias y otros determinantes relacionados

OBJECTIVE: To determine and describe the prevalence and patterns of three recommended practices for infant and young child feeding-exclusive breastfeeding (EB), continued breastfeeding (CB), and achievement of minimum dietary diversity-in four regions in Haiti, and to identify the attitudes and beliefs that inform these practices and any other factors that may facilitate or impede their implementation. METHODS: This study utilized a mixed-methods approach consisting of 1) a cross-sectional survey (n = 310) and 2) 12 focus group discussions among women ≥18 years old with children ≤ 2 years old. Multivariable logistic regression analyses were conducted to identify factors associated with 1) EB during the first six months of life, 2) CB for children ≥ 2 years old, and 3) receipt of a diverse variety of complementary foods. Qualitative data were recorded, transcribed verbatim, and analyzed for common themes. Data were collected in June and July 2013 in four departments in Haiti: Artibonite, Nippes, Ouest, and Sud-Est. RESULTS: Prevalence of EB, CB, and achievement of minimum dietary diversity was 57.0%, 11.9%, and 21.2% respectively. EB was statistically significantly associated with infant's age when controlling for annual household income, location of most recent birth, or receipt of CB counseling (odds ratio (OR) = 0.67 (95% CI: 0.47-0.97)). CB was not statistically significantly associated with rural place of residence, receipt of CB counseling, parity, or infant's age. Meeting minimum dietary diversity was not significantly associated with parity, receipt of postnatal care, rural place of residence, location of most recent birth, receipt of infant and young child feeding counseling, or level of schooling. Beliefs surrounding the relationship between the mother's health and her diet on the quality of breast milk may prohibit EB and CB. Qualitative data revealed that dietary diversity may be low because 1) mothers often struggle to introduce complementary foods and 2) those that are traditionally introduced are not varied and primarily consist of grains and starches. CONCLUSIONS: Prevalence of the three recommended infant and young child feeding practices examined in this study is suboptimal, particularly CB and achievement of minimum dietary diversity. Future communication and programming efforts should address the misunderstandings and concerns identified through the qualitative methods used in this research.

OBJETIVO: Determinar y describir la prevalencia y los modelos de tres prácticas recomendadas para la alimentación de los lactantes y los niños pequeños (la lactancia materna exclusiva [LME], la lactancia materna continuada [LMC] y el logro de una diversidad alimentaria mínima, en cuatro regiones de Haití, y determinar las actitudes y creencias en las que se basan estas prácticas y otros factores que puedan facilitar o impedir su implantación. MÉTODOS: Este estudio utilizó un diseño de método mixto que consistió en 1) una encuesta transversal (n = 310) y 2) 12 grupos de discusión formados por mujeres de ≥ 18 años de edad o mayores con niños de ≤ 2 años de edad o menores. Se llevaron a cabo análisis de regresión logística multivariable para determinar los factores asociados con 1) la LME durante los seis primeros meses de vida, 2) la LMC en niños de ≥ 2 años de edad o mayores, y 3) el aporte de una amplia variedad de alimentos complementarios. Se registraron, se transcribieron al pie de la letra y se analizaron los datos cualitativos referentes a temas comunes. Estos datos se recopilaron en junio y julio del 2013, en cuatro departamentos de Haití: Artibonite, Nippes, Oeste y Sudeste. RESULTADOS: Las prevalencias de la LME, la LMC y el logro de una diversidad alimentaria mínima fueron de 57,0, 11,9 y 21,2%, respectivamente. La LME se asoció de manera estadísticamente significativa con la edad del lactante si se controlaban las variables de ingresos familiares anuales, ubicación del parto más reciente, o provisión de orientación en materia de LMC (razón de posibilidades [OR] = 0,67 [IC de 95%:0.47-0.97]). La LMC no se asoció de una manera estadísticamente significativa con la residencia en un entorno rural, la provisión de orientación en materia de LMC, la paridad o la edad de lactante. El logro de una diversidad alimentaria mínima no se asoció significativamente con la paridad, la provisión de atención posnatal, la residencia en un entorno rural, la ubicación del parto más reciente, la provisión de orientación en materia de alimentación de los lactantes y los niños pequeños, o el nivel de escolarización. Las creencias con respecto a la relación entre la salud de la madre y su régimen alimentario con la calidad de la leche materna pueden limitar la LME y la LMC. Los datos cualitativos revelaron que la diversidad alimentaria puede ser escasa como consecuencia de que 1) las madres a menudo se esfuerzan por introducir los alimentos complementarios, y 2) los que se introducen tradicionalmente no son variados y consisten principalmente en cereales y féculas. CONCLUSIONES: Las prevalencias de las tres prácticas de alimentación de los lactantes y los niños pequeños recomendadas analizadas en este estudio son subóptimas, en particular las correspondientes a la LMC y al logro de una diversidad alimentaria mínima. Las futuras iniciativas de comunicación y programación deberían abordar los malentendidos y las inquietudes detectadas mediante los métodos cualitativos utilizados en esta investigación.

Autonomia do graduando em enfermagem na (re)construção do conhecimento mediado pela aprendizagem baseada em problemas / Autonomy of nursing undergraduate student in the (re)construction of knowledge mediated by problem-based learning / La autonomía del estudiante de enfermería en la (re)construcción del conocimiento mediada por el aprendizaje basado en problemas

Estudo qualitativo, método Bricolagem, que objetivou analisar como a Aprendizagem Baseada em Problemas (ABP) promove o desenvolvimento da autonomia do aluno no processo de aprender a aprender. Os sujeitos foram 16 alunos e dois tutores envolvidos na disciplina. A coleta dos dados combinou entrevista semiestruturada, observação participante, registro em portfólios, fichas de avaliação, e gravação em áudio das tutorias. A análise dos dados seguiu estratégias de interpretação definidas pelas autoras: leituras iniciais e aprofundada; construção e reunião de mapas de significados; elaboração, descrição e análise de categorias empíricas, à luz do referencial teórico. A ABP favorece a (re)construção de conhecimentos pela utilização de saberes e experiências prévias, que são compartilhados no pequeno grupo; pelo processo de teorização; e pela via do conhecimento pertinente - aquele passível de aplicação à prática. Concluímos que a ABP estimula o aprendizado contínuo, desenvolvendo no aluno autonomia no processo de aprender a aprender.

This is a qualitative study, using the ‘Do it yourself’ method, which aimed to analyze how Problem-Based Learning (PBL) promotes the development of learner’s autonomy in the process of learning to learn. The subjects were 16 students and two tutors involved in the discipline. Data collection techniques combined semi-structured interviews, participant observation, log in portfolios, evaluation forms, and audio recording of the tutorials. Data analysis followed interpretation strategies defined by the authors: initial and in depth readings; construction and assembly of meanings’ maps; development, description and analysis of empirical categories, in the light of the theoretical framework. The PBL favors the (re)construction of knowledge by the use of prior knowledge and experiences that are shared in small group; through the process of theorization; and by means of relevant knowledge - one that can be applied to practice. We conclude that PBL encourages continuous learning, developing in the student the autonomy in the process of learning to learn.

Estudio cualitativo, con método Bricolage, que tuvo como objetivo analizar cómo el Aprendizaje Basado en Problemas (ABP) promueve el desarrollo de la autonomía del alumno en el proceso de aprender a aprender. Los sujetos fueron 16 alumnos y dos tutores que participan en la disciplina. Para recolección de datos se combinaran entrevistas estructuradas, observación participante, registro en portfolios, formularios de evaluación y grabación de audio de los tutoriales. El análisis de los datos siguió las estrategias de interpretación definidos por los autores: lecturas iniciales y profundadas; construcción y montaje de mapas de significados; el desarrollo, descripción y análisis de categorías empíricas, a la luz de lo referencial teórico. El ABP favorece la construcción de conocimientos mediante el uso de saberes y experiencias que se comparten en grupos pequeños, a través del proceso de teorización, y por medio de los conocimientos pertinentes - uno que se puede aplicar a la práctica. Llegamos a la conclusión que el ABP promueve el aprendizaje continuo, el desarrollo de los estudiantes, la autonomía en el proceso de aprender a aprender.

Characterization of an Extracytoplasmic Chaperone Spy in Protecting Salmonella against Reactive Oxygen/Nitrogen Species

Antimicrobial actions of reactive oxygen/nitrogen species (ROS/RNS) derived from products of NADPH oxidase and inducible nitric oxide (NO) synthase in host phagocytes inactivate various bacterial macromolecules. To cope with these cytotoxic radicals, pathogenic bacteria have evolved to conserve systems necessary for detoxifying ROS/RNS and repairing damages caused by their actions. In response to these stresses, bacteria also induce expression of molecular chaperones to aid in ameliorating protein misfolding. In this study, we explored the function of a newly identified chaperone Spy, that is localized exclusively in the periplasm when bacteria exposed to conditions causing spheroplast formation, in the resistance of Salmonella Typhimurium to ROS/RNS. A spy deletion mutant was constructed in S. Typhimurium by a PCR-mediated method of one-step gene inactivation with lambda Red recombinase, and subjected to ROS/RNS stresses. The spy mutant Salmonella showed a modest decrease in growth rate in NO-producing cultures, and no detectable difference of growth rate in H2O2 containing cultures, compared with that of wild type Salmonella. Quantitative RT-PCR analysis showed that spy mRNA levels were similar regardless of both stresses, but were increased considerably in Salmonella mutants lacking the flavohemoglobin Hmp, which are incapable of NO detoxification, and lacking an alternative sigma factor RpoS, conferring hypersusceptibility to H2O2. Results demonstrate that Spy expression can be induced under extreme conditions of both stresses, and suggest that the protein may have supportive roles in maintaining proteostasis in the periplasm where various chaperones may act in concert with Spy, thereby protecting bacteria against toxicities of ROS/RNS.

Caracterização da superexpressão do fator sigma ECF σx em Pseudomonas aeruginosa PA14 / Characterization ofthe ECF sigma fator σx overexpression in Pseudomonas aeruginosa PA14

Pseudomonas aeruginosa é uma proteobactéria do grupo gama muito versátil, capaz de colonizar ambientes variados e infectar hospedeiros filogeneticamente distintos, incluindo humanos imunocomprometidos. Os fatores sigma de função extracitoplasmática (ECF) são membros de sistemas de sinalização de superfície celular (CSS), abundantes em P. aeruginosa. Vinte genes codificando fatores sigma ECF estão presentes nos genomas sequenciados de P. aeruginosa, a maioria fazendo parte de sistemas TonB relacionados à captação de ferro. Neste trabalho, seis fatores sigma pobremente caracterizados foram superexpressos na linhagem PA14 a partir de um promotor induzível por arabinose para investigar seu papel na expressão dos sistemas de dois componentes PvrSR e RcsCB, que atuam na regulação da fímbria CupD, além de sua influência no crescimento de culturas de P. aeruginosa. Não foi observado efeito positivo de nenhum dos fatores sigma testados na expressão dos sistemas de dois componentes e a superexpressão de cinco deles tampouco levou a qualquer alteração no crescimento, porém a produção de piocianina foi alterada na superexpressão de PA14_55550 e a superexpressão de PA14_26600 e PA14_46810 levou a um discreto aumento no início da formação de biofilme em PA14. Por outro lado, culturas superexpressando σx (ALB04) apresentaram um perfil alterado de lipopolissacarídeo e uma curva de crescimento bifásica, alcançando precocemente uma fase estacionária seguida de uma recuperação do crescimento até uma segunda fase estacionária. Durante a primeira fase estacionária, a maior parte das células aumenta de tamanho e morre, mas as células remanescentes retornam à morfologia selvagem e seguem para a segunda fase de crescimento exponencial. Isso não acontece devido a mutações compensatórias, uma vez que células coletadas de pontos tardios da curva e diluídas em meio novo repetem este comportamento. Apesar de trabalhos com a linhagem PAO1 associarem σx à transcrição de oprF, que codifica a principal porina não específica de Pseudomonas, nas condições dos nossos ensaios em PA14 a expressão dessa porina não foi induzida pela superexpressão de σx. Assim, os efeitos observados nessa superexpressão também não podem ser atribuídos a OprF. A transcrição de oprF em PA14 mostrou-se majoritariamente dependente da região promotora a que se atribui a ligação de σ70, ao contrário dos relatos na literatura da dependência da região de ligação a σx. Análises proteômicas foram realizadas para investigar os elementos envolvidos nesses efeitos de superexpressão de σx, o que revelou a indução de diversas enzimas envolvidas na via de biossíntese de ácidos graxos. As células superexpressando σx apresentam uma maior proporção de ácidos hexadecanoico (C16) e hexadecenoico (C16:1) e dados de anisotropia mostram uma maior fluidez da(s) membrana(s). Este trabalho é o primeiro relato de um fator sigma ECF envolvido em biossíntese de lipídeos em P. aeruginosa

Pseudomonas aeruginosa is a very versatile gammaproteobacteria, able to colonize different environments and to infect phylogenetically distinct hosts, including immunocompromised humans. The extracytoplasmic function sigma factors (ECFs) are members of cell signaling systems (CSS), abundant in P. aeruginosa. Twenty genes coding for ECF sigma factors are present in the sequenced genomes of P. aeruginosa, most of them being part of TonB systems related to iron uptake. In this work, six poorly characterized sigma factors were overexpressed in strain PA14 from an arabinose inducible promoter to investigate their role in the expression of the two-component systems PvrSR and RcsCB, which regulates CupD fimbria, and their influence in P. aeruginosa cultures growth. None of the tested sigma factors led to two-component systems upregulation and overexpression of five of them caused no change in the growth profile, but pyocyanin production was altered in PA14_55550 overexpression and PA14_26600 and PA14_46810 overexpression led to a slight increase in biofilm initiation in PA14. By the other side, cultures overexpressing σx (ALB04) presented an altered lipopolysaccharide profile and a biphasic growth curve, reaching an early stationary phase followed by a growth resuming untill a second stationary phase. During the early stationary phase, most cells swells and dies, but the remaining cells return to wild type morphology and proceed to the second exponential phase of growth. This is not due to compensatory mutations, since cells collected from late points of the curve and diluted in fresh medium repeat this behavior. Although studies with strain PAO1 associate σx with transcription of oprF, encoding the major nonspecific porin of Pseudomonas, under our experiments conditions with PA14, this porin expression is not induced by σx overexpression. Thus, the effects observed in this overexpression cannot be attributed to OprF. Transcription of oprF in PA14 proved to be mainly controlled by the σ70-dependent promoter region instead of the σx-dependent promoter region reported in the literature. Proteomic analyses were performed to investigate the elements involved in these effects of σx overexpression, which revealed the induction of several enzymes involved in fatty acids biosynthesis. Cells overexpressing σx exhibit a greater proportion of hexadecanoic (C16) and hexadecenoic (C16: 1) acids and anisotropy data show higher fluidity of the membrane (s). This work is the first report of an ECF sigma factor involved in lipid biosynthesis in P. aeruginosa

Effects of exogenous carbon monoxide-releasing molecules 2 on the vitality and toxicity of E.coli / 中华烧伤杂志

<p><b>OBJECTIVE</b>To explore the effects of exogenous carbon monoxide-releasing molecules 2 (CORM-2) on the vitality and toxicity of E. coli ATCC 25922, and to analyze the potential mechanism.</p><p><b>METHODS</b>(1) In vitro experiments. Standard strains of E. coli ATCC 25922 were divided into groups A (without addition), B, C, D, and E according to the random number table, and then the latter 4 groups were respectively cultured with 1.2 mmol/L CORM-2, 1.6 mmol/L CORM-2, 1.2 mmol/L inactive CORM-2 (iCORM-2), 1.6 mmol/L iCORM-2, with six samples in each group. After being cultured for 0, 3, 5, 8, 10, 12, 16, 20, 24, 27, 30, 48 hours, proliferative vitality of E. coli was examined (denoted as absorption value under 600 nm wavelength), and bacteria number was counted. Other standard strains of E. coli ATCC 25922 were divided into groups F (without addition) and G (cultured with 0.8 mmol/L CORM-2), the expressions of genes fliA, dnaK, marA, and waaQ related to E. coli were detected by quantitative real-time (qRT)-PCR. (2) In vivo experiments. Other standard strains of E. coli ATCC 25922 were grouped as A', B', C', D', and E' and treated with the same method as that in groups A, B, C, D, and E, and 0.5 mL bacterial liquid of each group were collected when the absorption value of bacterial liquid in group A' was equal to 0.4 (under 600 nm wavelength). Seventy-two C57BL/6 mice were divided into groups, namely blank control (without treatment), H, I, J, K, and L according to the random number table, with 12 mice in each group. The mice in the latter 5 groups were intraperitoneally injected with 0.5 mL bacterial suspension of groups A', B', C', D', and E' respectively. After injection, general condition of mice in groups H, I, J, K, and L was observed. The serum levels of TNF-α and IL-6 were determined at post injection hour (PIH) 6, 12. The liver and lung samples were harvested for determination of myeloperoxidase (MPO) activity at PIH 12. The same process was carried out in blank control group. Data were processed with repeated measure analysis of variance (ANOVA), factorial design ANOVA, one-way ANOVA, and t test.</p><p><b>RESULTS</b>(1) In vitro experiments. Compared with those of groups A and D, the proliferative vitality and bacteria number of E. coli in group B were all decreased (with F values respectively 1170.80, 217.52, P values all below 0.01). Compared with those of groups A and E, the proliferative vitality and bacteria number of E. coli in group C were also obviously decreased (with F values respectively 7948.34, 14 432.85, P values all below 0.01). Compared with those in group F, the expression of fliA was downregulated, while the expressions of dnaK, marA, and waaQ were upregulated in group G (with t values 30.28, -165.54, -168.88, -187.28, P values all below 0.01). (2) In vivo experiments. Symptoms including listlessness and tachypnea were observed in mice in groups H, K, and L, and they were ameliorated or not obvious in groups I and J. At PIH 6, the serum levels of TNF-α and IL-6 in groups H and K were respectively (647.3 ± 3.8) pg/mL, (3.44 ± 0.22) ng/mL and (639.3 ± 0.8) pg/mL, (2.47 ± 0.32) ng/mL, which were obviously higher than those in group I [(124.6 ± 10.7) pg/mL, (1.03 ± 0.16) ng/mL, with t values from 15.22 to 84.03, P values all below 0.01]. The serum levels of TNF-α and IL-6 in group J at PIH 6, 12 were also obviously decreased as compared with those in groups H and L (with t values from 19.27 to 245.34, P values all below 0.01). MPO activity of liver and lung tissues were significantly attenuated in group I at PIH 12 as compared with those in groups H and K, and it was also attenuated in group J when compared with those in groups H and L (with t values respectively from 17.36 to 18.92 and 2.35 to 3.61, P values all below 0.01).</p><p><b>CONCLUSIONS</b>CORM-2 can obviously inhibit the vitality and toxicity of E. coli, which might be attributable to regulation of expressions of genes fliA, dnaK, marA, and waaQ of E. coli.</p>

Metabolism of Salmonella enterica serovar typhi influenced by RpoE and RpoS under hyperosmosis / 中华预防医学杂志

<p><b>OBJECTIVE</b>To study the role of RpoE and RpoS on the influence of the metabolism and growth of bacterial under hyperosmotic stress.</p><p><b>METHODS</b>The rpoS/rpoE double deletion mutant of Salmonella enterica serovar typhi (S. typhi) was prepared by homologous recombination through the suicide plasmid mediated. The recombination was visualized by PCR. Growth curves were drawn by using photometric value A600 as the ordinate and cultivation time as abscissa. The survival abilities of bacterial were compared under hyperosmotic stress. Statistical differences of early logarithmic growth stage (4 h) and laters logarithmic growth stage (12 h) were analyzed by one-way ANOVA. The expression difference of metabolism related genes of wild-type and mutant strains of S. Typhi incubated under hyperosmotic stress were investigated by Salmonella genomic DNA microarray. Real-time quantitative PCR (qRT-PCR) was performed to validate the results of microarray assay in some selected genes.</p><p><b>RESULTS</b>The rpoS/rpoE double deletion mutant of S. Typhi was successfully generated. The analysis of growth curve showed that the 4-hour and 12-hour A600 values were separately 0.503 ± 0.018 and 2.060 ± 0.112 in rpoS deletion mutant strains, 0.293 ± 0.053 and 1.933 ± 0.115 in rpoE deletion mutant strains, and 0.051 ± 0.007 and 0.963 ± 0.111 in rpoS/rpoE double deletion mutant strains; all of which were lower than the values of wild-type strains, who were 0.725 ± 0.097 and 2.496 ± 0.171, respectively. The difference were statistically significant (P < 0.05). The genomic DNA microarray revealed that 42 genes relevant with bacterial metabolism were influenced by RpoE and RpoS. Results of qRT-PCR showed that the expression values of rpsE, rbsK, nusG and etuB in rpoS deletion mutant strains were (1.86 ± 0.14)×10(6), (1.37 ± 0.11)×10(6), (2.72 ± 0.58)×10(6) and (8.27 ± 1.01)×10(6) copies/µg, respectively; while those in rpoE deletion mutant strains were (2.19 ± 0.17)×10(6), (1.51 ± 0.12)×10(6), (2.73 ± 0.57)×10(6) and (9.63 ± 1.42)×10(6) copies/µg, respectively. Compared with the values in wild-type strains, which were separately (1.94 ± 0.10)×10(6), (1.52 ± 0.11)×10(6), (2.39 ± 0.52)×10(6) and (10.83 ± 1.52)×10(6) copies/µg, the differences was not statistical significance (P > 0.05). However, compared with the values in rpoS/rpoE double mutant strains, which were separately (5.64 ± 0.59)×10(6), (4.17 ± 0.40)×10(6), (9.44 ± 1.22)×10(6) and (2.95 ± 0.88)×10(6) copies/µg, the difference was significant (P < 0.05).</p><p><b>CONCLUSION</b>RpoE and RpoS could influence the expression of lots of metabolism genes. Together, they regulated the metabolism and growth of S. Typhi under hyperosmotic stress.</p>

Aspectos biológicos, clínicos y epidemiológicos de la tuberculosis latente: [revision] / Biological, clinical and epidemiological aspects of latent tuberculosis: [review]

Mycobacterium tuberculosis afecta a la humanidad desde hace más de 20 000 años. Su morbimortalidad es elevada, por lo que repercute económicamente en los países en desarrollo. La infección latente, caracterizada por la presencia de bacilos vivos en tejidos del huésped, con ausencia de signos y síntomas clínicos, es una característica de esta enfermedad, ya que la micobacteria puede adaptar su metabolismo para mantenerse viva con baja o nula replicación, dificultando su eliminación de los tejidos por los fármacos antituberculosos y permaneciendo inadvertida al reconocimiento y eliminación por el sistema inmunológico. Varias son las interrogantes de esta forma de tuberculosis (TB): la falta de conocimiento del metabolismo del bacilo en estado durmiente, su relación con la inmunidad del hospedero y la identificación de antígenos como marcadores diagnósticos de infección subclínica durante la latencia. Este artículo resume los aspectos biológicos, clínicos y epidemiológicos más importantes de esta forma de tuberculosis.

Mycobacterium tuberculosis, the causal agent of tuberculosis, has affected humankind for approximately 20 000 years. Tuberculosis is a devastating disease, particularly in developing countries. One of its most notable characteristics is latent infection, in which live bacilli persist in the host tissues without clinical manifestations. Thus, the tuberculous bacilli adapt their metabolism to remain viable with low or no replication, avoiding their elimination by the immune system or conventional chemotherapy. Among the several problems that are particularly important to the understanding of this form of tuberculosis, and are not well-known, are the key metabolic steps that allow mycobacteria to remain in a dormant state and its interaction with host immunity. This article reviews some of the most significant biological, clinical and epidemiological aspects of this form of tuberculosis.

Study of biological characteristics of Pseudomonas aeruginosa strains isolated from patients with cystic fibrosis and from patients with extra-pulmonary infections

A total of 120 strains of Pseudomonas aeruginosa, isolated from cystic fibrosis (CF) patients (n = 80) and from patients having extra-pulmonary infections (n = 40) were studied regarding the presence of some virulence factors (hemolysin, gelatinase and elastase production) and presence of the algD and algU genes as detected by polymerase chain reaction-PCR. There was not a significant difference for the production of gelatinase and hemolysin between non-mucoid strains from CF patients and other isolates from extra-pulmonary infections and mucoid strains. The production of elastase was found to be significant among these strains. The algD gene was detected by PCR in all studied strains but the algU gene was detected only in 25 percent of the mucoid strains. Conclusion withdrawn from the results were: (i) hemolysin and gelatinase production although present in many strains of P aeruginosa should not be considered as general virulence factors for the mucoid phenotype but could help in the pathogenic process; (ii) elastase production could be a necessary virulence factor for the initial pathogenesis process; (iii) mucoid and non-mucoid phenotypes could also be expressed according to the host's tissues or environment, and finally, (iv) more than one regulator system for alginate production is probably present in each strain.

Basal transcription machinery: role in regulation of stress response in eukaryotes.

The holoenzyme of prokaryotic RNA polymerase consists of the core enzyme, made of two alpha, beta, beta' and omega subunits, which lacks promoter selectivity and a sigma (sigma) subunit which enables the core enzyme to initiate transcription in a promoter dependent fashion. A stress sigma factor sigma(s), in prokaryotes seems to regulate several stress response genes in conjunction with other stress specific regulators. Since the basic principles of transcription are conserved from simple bacteria to multicellular complex organisms, an obvious question is: what is the identity of a counterpart of sigma(s), that is closest to the core polymerase and that dictates transcription of stress regulated genes in general? In this review, we discuss the logic behind the suggestion that like in prokaryotes,eukaryotes also have a common functional unit in the transcription machinery through which the stress specific transcription factors regulate rapid and highly controlled induction of gene expression associated with generalized stress response and point to some candidates that would fit the bill of the eukaryotic sigma(s).

Condiciones ambientales que afectan la expresiín del gen suoM: un factor sigma de estrés en M. smegmatis / Environment conditions affect the expression of the suoM gene: an stress sigma factor in M. smegmatis

Los factores sigmas se asocian a la ARN polimerasa y le confieren especificidad de reconocimiento de regiones promotoras. Esto provee un nivel de regulación transcripcional, que determina que un grupo de genes sea expresado de acuerdo a las necesidades fisiológicas de la célula bacteriana. El análisis de secuencia de la región del origen de replicación de Mycobacterium smegmatis reveló la presencia de un marco de lectura con capacidad codificante para un sigma de la subfamilia ECF (extracytoplasmic function), los cuales se carcaterizan por dirigir la transcripción de genes en respuesta a cambios en las condiciones ambientales. Con el objeto de identificar las condiciones ambientales que estimulan la expresión del gen aislado, designado suoM (sigma unido al origen en microbacterias), se contruyeron fusiones transcripcionales PsuoM-lacZ' sobre el plásmido de fusión de operones pJEM15. Las construcciones se introdujeron por electroporación en M.smegmatis y las células transformadas se sometieron a varias condiciones de estrés ambiental a fin de estudiar la actividad promotora del gen suoM mediante ensayos ß-galactosidasa. Se observó un incremento de 2,0-3,5 veces en la actividad promotora, al transferir las células a 45°C y cuando el cultivo alcanza fase estacionaria de crecimiento. Los resultados sugieren que suoM podría estar involucrado en la regulación de la expresión de genes en condiciones de elevada temperatura y en fase estacionaria de crecimiento, condiciones inductoras que podrían tener algún significado en el potencial patogénico de micobacterias y en la habilidad de M.tuberculosis para causar infección latente.

Factores sigma y respuesta a estrés en micobacterias (revisión) / Sigma factors and response a stress in mycobacterium (review)

La presencia de múltiples factores sigma de respuesta a condiciones de estrés confiere una gran flexibilidad a la maquinaria transcripcional micobacteriana, sugiriendo que puede existir solapamiento a cooperación de funciones entre varios miembros ECF para defender las micobacterias de potenciales daños que se puedan generar en condiciones de estrés y ofrece un nuevo modelo para investigar estrategias regulatorias utilizadas por M.tuberculosis para el crecimiento intracelular, evasión del sistema inmune del hospedador y su contribución a la impresionante capacidad de adaptación y supervivencia de las micobacterias.

Análisis de ARNm de un factor de supervivencia en micobacterias / mRNA analysis of a survival factor in mycobacterias

Las bacterias patógenas diseñan estrategias de regulación de genes para defender de microambientes adversos encontrados en tejidos del hospedador. Una de las estrategias exitosas de regulación se basa en la existencia de múltiples factores sigma alternativos de la subfamilia ECF ("extracytoplamic function") que dirigen a la ARN polimerasa bacteriana al reconocimiento de promotores de genes, cuya expresión promueve cambios adaptativos para defenderse de daño potenciales. En este trabajo se reportan datos preliminares del patrón de expresión del factor sigma ECF micobacteriano SuoM ("sigma unido al orígen de replicación en micobacterias") de M. tuberculosis y M. bovis BCG en respuesta a cambios en condiciones ambientales. A tal fin, se contruyeron fusiones transcripcionales reporteras de suoM-lacZ` de M. tuberculosis y se ensayó la actividad promotora de este gen a través ensayos de actividad ß-galactosidasa en células de M. bovis BCG sometidas a diversos tratamientos. Se observó un incremento en la expresión suoM en condiciones de choque térmico y en fase estacionaria de crecimiento, lo cual fué confirmado al analizar directamente los ARNm de suoM en M. bovis BCG por "northern blot" e hibridización. Los resultados obtenidos, así como la identidad de los genes entre M. bovis BCG y M. tuberculosis sugiere que SuoM pudiera regular procesos celulares equivalentes en ambos especies micobacterianas relacionadas con funciones de adaptación y supervivencia frente a condiciones de estrés térmico y cuando cesa el crecimiento exponencial. Los estudios de regulación de la expresión de genes de supervivencia pueden contribuir a dilucidar mecanismos de virulencia bacteriana

Caracterización de la expresión y función sigM de un factor sigma ECF en micobacterias / Characterization of the expression and function sigM of an ECF sigma factor in mycobacteriums

La supervivencia del bacilo Micobacteria tuberculosis dentro del macrófago depende de su capacidad de responder al stress oxidativa, y los factores sigma, subfamilia ECF, probablemente tienen un rol importante. Investigamos un factor sigma, sigM, cuyos gen se encuentran cerca del origen de replicación. Tanto en M.smegmatis como en M.bovis BCG, la expresión del gen esta inducida en temperaturas altas y en fase estacionaria. Cepas de M.smegmatis sin una copia activa de sigM muestran un defecto en la supervivencia en el stress oxidativo y también en la inducción de la actividad de thioredoxina reductasa, que reduce puentes disulfuricos formado en stress oxidativo. Estos datos sugieren que SigM regula los tioredoxinas y constituye parte de unas respuestas protectivas muy complejos de la bacteria

Vólvulo de sigma / Volvulo of sigma

Expone que el volvulo de sigma es un cuadro obstructivo de tipo mecánico, provocado por la torsión del mesosigma, a lo que se añade trastornos hidroelectrolíticos y metabólicos; su gravedad es directamente proporcional a su duración del cuadro; su etiología no es clara, se presenta generalmente en la quinta o sexta décadas de vida. Su tratamiento debe ser inmediato, inciciándose con la estabilización de los signos vitales seguido de la reducción del vólvulo. El caso que se presenta tuvo un desenlace fatal debido a múltiples factores: deterioro del estado clínico por la idiosincracia propia de la paciente, por las limitaciones del Hospital y por la magnitud del vólvulo determinada al momento de la cirugía. Amerita un mejor conocimiento de la sintomatología y del manejo clínico para n el futuro evitar complicaciones adversas.

Tratamiento quirurgico del volvulo / Surgical treatment of volvulus

El volvulo es una patologia frecuente en nuestra poblacion campesina, existen diversos procedimientos quirurgicos para su tratamiento; es importante elegir un metodo efectivo acorde a las condiciones socioeconomicas del universo afectado y que permita una rehabilitacion optima y precoz del paciente. Se revisaron 1878 historias clinicas de pacientes atendidos en el Servicio de Cirugia del Hospital santa Barbara entre el 1 de enero de 1990 y el 31 de noviembre de 1993; se selcccionaron las historias correspondientes a los pacientes tratados por presentar volvulo (n=144), estudiandose las variables: edad, sexo, raza, procednecia y ocupacion de los pacientes; localizacion topografica del volvulo; metodo terapeutico practicado; complicaciones postquirurgicas; mortalidad. La prevalencia de la patologia en el universo estudiado es del 7.7 por ciento ; los mas afectados son los hombres en la 6ta. decada de la vida, mestizos de procedencia rural y agricultores; el volvulo mas frecuente diagnosticado es el sigma; la cirugia mas realizada es la colectomia y anastomosis primaria inmediata; las complicaciones se presentan en el 14.5 por ciento de los operados, siendo la mas observada el absceso de pared; la mortalidad es de 4.2 por ciento . La recesion del segmento volvulado y la anastomosis primaria inmediata son medidas terapeuticas afectivas; constituyen un metodo que presenta escasas complicaciones, ademas minima mortalidad; favorece la actividad precoz del paciente, no siendo necesaria una reintervencion quirurgica posterior, como se requiere en otras cirugias alternativas (como la operacion de Hartamann) del tratamiento quirurgico del volvulo.

Los receptores sigma y PCP/NMDA: su rol fisiopatológico en algunos cuadros psicóticos y su tratamiento farmacológico / Sigma and PCP/NMDA receptors: its physiopathological role in some psychotic pictures and its pharmacological treatment

En este trabajo se analiza el conocimiento actual sobre los receptores sigma y PCP/NMDA. Se enfatiza que el haloperidol y muchos otros antipsicóticos similares presentan una afinidad relativamente alta por el receptor sigma. Esto sugiere que el efecto farmacológico de estas drogas pudiera ser debido a un efecto no tan solo en el receptor D2, sino que también a través del receptor sigma. Además se analizan los posibles mecanismos fisiopatológicos de algunos cuadros como las psicosis esquizomorfas epilépticas, el efecto de hormonas esteroidales y estimulantes cerebrales y la implicancia de este conocimiento para su manejo terapéutico. La conclusión más importante de este trabajo es que los receptores sigma y PCP/NMDA parecen estar involucrados en la génesis de los síntomas psicóticos productivos, lo que permite una mejor clasificación de estos cuadros y el desarrollo de antipsicóticos específicos para cada disfunción