Outcome of protease inhibitor substitution with nevirapine in HIV-1 infected children

<p>Abstract</p> <p>Background</p> <p>Protease inhibitors (PIs) have been associated with metabolic complications. There is a trend to switch to simpler therapy to improve these disturbances. We report a case-series describing the effects in metabolic abnormalities in seven HIV-infected children, previously treated with protease inhibitor (PI) after switching to nevirapine.</p> <p>Methods</p> <p>Seven children with stable PI-containing regimen and a long lasting HIV-1 RNA < 50 copies/ml were switched to nevirapine. All patients were naïve to non nucleoside reverse transcriptase inhibitor. PIs were switched to nevirapine. Preentry nucleoside reverse transcriptase inhibitors were maintained. The substitution of PIs with nevirapine was made when the patient showed hyperlipidemia or lipodystrophy or the physician and/or the patient's willingness to simplify. Clinical, laboratory data and anthropometric parameters were assessed every 3 months. Dual-energy X-Ray absorptiometry scans (DXA) was performed at baseline and at 12 months.</p> <p>Results</p> <p>Seven HIV-infected children were enrolled. Median age: 130 months (99,177). Median baseline CD4%: 32%. All had HIV-1 RNA < 50 copies/ml. Median length of preentry PI-therapy was 47 months (28, 91). Median age at the beginning of nevirapine was 120 months (99,177). Median decrease in cholesterol in 7.2 mmol/L was observed (P = 0.09), from baseline to 12 months. HDL-cholesterol increased in 5.1 mmol/L (P = 0.03) throughout the study period. No significant changes were observed in DXA with regard to body fat, but changes in total body bone mineral content and lean body content were significant. CD4% remained stable. All patients but one maintained viral load < 50 copies/ml at 12 months. The patient with virologic failure referred bad adherence. Children referred to take medication more easily.</p> <p>Conclusion</p> <p>PI substitution with nevirapine improved lipid profile in our patients, although this strategy did not show significant changes in body fat or lipodystrophy.</p

Analysis of a periplasmic thiol oxidoreductase in Rhizobium leguminosarum

In this work the expression and cellular localization of a predicted periplasmic thiol oxidoreductase encoded by Rhizobium leguminosarum 3841, ORF RL1083, were analysed. Based on the homology of the encoded protein with DsbA proteins from other bacteria we named it dsbA. The genetic organization of dsbA region showed that it is a monocystronic gene. A putative σ70 promoter was predicted upstream dsbA gene. The promoter region fused to gusA reporter gene revealed that dsbA is expressed in free-living conditions in different media and also, although at a lower level, in pea bacteroids. R. leguminosarum DsbA contains a potential Tat-dependent signal peptide. To localize this protein in different cellular fractions the protein was labelled by means of a C-terminal Strep tag. The DsbA-Strep protein was localized in the periplasmic fraction. At present three type of experiments are in progress: first, the study of DsbA Tat-dependence by using a tat mutant strain harbouring dsbA-Strep; second, the construction of a dsbA mutant and third the evaluation of periplasmic disulfide oxidoreductase activity of different strains: wild-type, tat mutant and dsbA mutant

Relevance of bacterial secretion systems Type III and Type VI in the Bradyrhizobium-Lupinus simbiosis

One of the most studied models in plant-microbe interaction is the symbiosis Rhizobium-legume. This symbiosis is highly specific and depends on several molecular signals produced by both partners. Some of these signals are bacterial proteins named effectors that are translocated into the plant cells by secretion systems similar to contractile nanomachines (injectisomes). The injectosomes puncture and deliver the effectors into the target cell. The two main injectiosomes are the secretion system type III (T3SS) and the secretion system type VI (T6SS). The genome of many rhizobia encodes T3SS and/or T6SS but their role in symbiosis is mostly unknown. The aim of this work is to study the symbiotic relevance of T3SS and T6SS of Bradyrhizobia that nodulate lupins that thrive in alkaline (Lupinus mariae-josephae) and acid soils (L. angustifolius) in the Iberian Peninsul

Functional roles of HypC and HupK in the biosynthesis of [NiFe] hydrogenase in Rhizobium leguminosarum.

Some rhizobia induce a hydrogen (H2)-uptake system with a [NiFe] hydrogenase along with nitrogenase to recover part of the energy lost as H2. Biosynthesis of NiFe hydrogenases is a process that ocurrs in the cytoplasm, where a number of auxiliary proteins (products of hup and hyp genes) are required to synthesize and insert the metal cofactors into the enzyme structural units. Although HypC is expressed in all hydrogenase systems, HupF and HupK are found only in bacteria that express the hydrogenase in the presence of oxygen (O2). Co-purification experiments have demonstrated HypC-HupK and HypC-HupL interactions. Results have shown that some conserved residues from HypC and HupK play a protective role of hydrogenase against the presence of O2

ARQUEOTOPOS Project. Harbour of Cartagena an approach from the Paleotopography and Geoarcheology

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Caracterización del sistema de secreción de tipo VI en Rhizobium etli Mim1

La simbiosis rizobio-leguminosa es altamente específica. La translocación de proteínas denominadas efectores desde el citoplasma bacteriano a la célula vegetal es un elemento relacionado con dicha especificidad. Los efectores pueden ser translocados a través de diferentes sistemas de secreción. El análisis de genomas de rizobios ha permitido identificar en algunos la presencia de sistemas de secreción de tipo VI (T6SS). El T6SS tiene como componente principal una nanoestructura similar a las que utilizan los bacteriófagos1 para inyectar su ADN y que las bacterias usan para secretar proteínas. Los genes implicados en la formación de T6SS están agrupados y los que codifican para componentes estructurales del sistema presentan mayor grado de conservación entre rizobios y frente a otras bacterias en comparación a los genes que codifican para efectores y reguladores del sistema. En nuestro grupo se está estudiando el T6SS de Rhizobium etli bv mimosae Mim12 aislada de nódulos de Mimosa affinis y capaz de nodular además Phaseolus vulgaris y Leucaena leucocephala. La cepa Mim1 contiene una agrupación de 28 genes en el plásmido f no simbiótico, relacionados con la formación de un T6SS, presentando una organización similar a la descrita en Agrobacterium tumefaciens C583 que consiste en dos operones divergentes. Se ha descrito para varios microorganismos que cuando el T6SS está activo, las proteínas Hcp y VgrG que forman parte del aparato de secreción pueden detectarse en el medio extracelular3. Los genes que codifican proteínas estructurales en las dos bacterias presentan una gran similitud, así Hcp muestra un 94% de identidad entre ambas permitiendo que los anticuerpos que detectan Hcp de Agrobacterium3 también reaccionen con Hcp de Mim1. Utilizando anticuerpos contra Hcp de Agrobacterium se ha identificado esta proteína en el medio extracelular de cultivos de Mim1 en fase estacionaria y débilmente en fase exponencial. También se ha demostrado su presencia en nódulos de judía y en cultivos crecidos en presencia de exudados de L. leucocephala, P. vulgaris y Pisum sativum. Además, con el fin de conocer en qué condiciones se activa el T6SS de Mim1, se analizó una región de ADN presumiblemente promotora comprendida entre las dos agrupaciones de genes orientados de forma divergente de Mim1. Esta región se fusionó transcripcionalmente a un gen b-gal delator sin promotor del vector pMP220 en las dos posibles orientaciones, una de las orientaciones (P1) controlaría la expresión de genes como hcp y posibles efectores y la otra (P2) de otros genes estructurales. Los resultados mostraron que ambas orientaciones se expresaban a altas DO600 (0,8-1) aunque los valores de P1 fueron entre dos y tres veces superiores a los de P2. Sin embargo a bajas DO600 (0,1-0,2) la actividad de P1 ser redujo a la mitad y la de P2 a niveles del control sin promotor. Con el objetivo de conocer el papel del T6SS en simbiosis se han realizado 3 mutantes que afectan a genes estructurales del T6SS de Mim1, uno en el gen hcp, otro en tssM y el tercero es una deleción de todos los genes presumiblemente dependientes de P2. Se examinó el fenotipo producido en P. vulgaris y L. leucocephala y se observó que los tres mutantes produjeron nódulos blancos y plantas con un porte similar a plantas no inoculadas, con menor tamaño que las inoculadas con la cepa parental y con un color más amarillento. En este trabajo se ha mostrado por primera vez que la presencia de un T6SS en rizobios tiene un efecto beneficioso en la simbiosis con varios hospedadores. En estos momentos se esta trabajando en la caracterización de posibles efectores. Referencias. 1. Records AR. 2011 The type VI secretion system: a multipurpose delivery sustem with a phage-like machinery. Mol Plant Microbe Interact 24: 751-757. 2. Rogel MA et al. 2014. Genomic basis of symbiovar mimosae in Rhizobium etli. BMC Genomics 15: 575 3. Wu, HY et al. 2012. Acid-induced type VI secretion system is regulated by ExoR-ChvG/Chv

Characterization of type VI secretion systems (T6SS) of endosymbionts from mimosa or lupine

The T6SS is a nanosyringe that injects proteins into prokaryotic or eukaryotic cells, and it is encoded in the genomes of more than 25% of Gram-negative bacteria (1). We are studying the T6SS of Rhizobium etli Mim1 and Bradyrhizobium sp. LmicA16, symbionts of Phaseolus vulgaris/Leucaena leucocephala and Lupinus micranthus/Lupinus angustifolius/Spartium junceum, respectively. R. etli Mim1 contains a T6SS gene cluster organized in two divergent operons. When the T6SS is active, Hcp, a constituent of the secretory apparatus, can be detected in the extracellular medium (2). Hcp has been immunologically detected in the supernatant of Mim1 cultures. This protein was also detected in bean nodule extracts and in cultures grown in the presence of different legumes exudates. The putative divergent promoters located between the two T6SS gene clusters were analysed by ?- gal fusions. The results showed high levels of expression of the two promoters at high OD and low values at lower ODs. Mutants affected in structural genes induced white nodules with P. vulgaris and L. leucocephala. On the other hand, mutagenesis of T6SS structural genes from LmicA16 strain produced different symbiotic phenotypes. An LmicA16 tssC mutant showed reduced levels of nitrogen fixation on L. micranthus, whereas the same mutant induced the formation of few white, non-fixing nodules on L. angustifolius and S. junceum. (1) Ho et al. (2013) Cell Host Microbe 15:9-21. (2) Wu et al. (2012) PLoS Pathog. 8:1-18 Funded by grants BIO2013-43040-P (MINECO), CGL2011-26932 (MICINN) and AL16-PID-06 (UPM)

New diagnoses of human immunodeficiency virus infection in the Spanish pediatric HIV Cohort (CoRISpe) from 2004 to 2013

Vertical human immunodeficiency virus (HIV) infection has decreased in industrialized countries in recent decades, but there are no studies on the mechanisms of HIV transmission among infected children in Spain. Our aim was to study the characteristics and trends of diagnoses of vertically HIV-infected children in Spain from 2004 to 2013. Vertically HIV-infected children were selected if they were diagnosed from 2004 to 2013, were aged 0 to 18 years old, and were included in the Cohort of the Spanish Pediatric HIV Network (CoRISpe). Demographic, clinical, immunological, and virological data at diagnosis were obtained. The rate of diagnoses of vertically HIV-infected children was calculated as the number of cases per 100,000 inhabitants. Obstetric data of mothers of Spanish children and prophylaxis at childbirth and postpartum were obtained. A total of 218 HIV-infected children were included in the study. Of this sample, 182 children (83.5%) were perinatally HIV infected, and 125 out of those 182 children (68.7%) were born in Spain. The vertically HIV-infected Spanish children were diagnosed earlier and were in better clinical and immunological condition at diagnosis than were foreign children. The rate of vertically HIV-infected children declined from 0.09 in 2004 to 0.03 in 2013 due to the decrease in the rate of children born in Spain (0.08 in 2004 vs 0.01 in 2013). A total of 60 out of 107 mothers (56.1%) of Spanish children were diagnosed at or after childbirth. However, this number declined between 2004 and 2013. The rate of new HIV diagnoses of vertically HIV-infected children decreased significantly between 2004 and 2013 from 0.09 to 0.03 per 100,000 inhabitants

Type VI secretion systems of Bradyhizobium nodulating lupines

The Rhizobium-legume symbiosis is highly specific and depends on several molecular signals produced by both partners. Some of these signals are bacterial proteins named effectors that are translocated into the plant cells by secretion systems similar to contractile nanomachines also called injectisomes (Deakin and Broughton, 2009). The injectisomes puncture and deliver the effectors into the target cell. One of these nanomachines, known as type VI secretion system (T6SS), was discovered recently and is reminiscent of phage injection machinery (Records, 2011). The role of these systems in legume endosymbiotic bacteria is mostly unknown, and this work presents the initial study of T6SSs from different bradyrhizobia. T6SSs have been identified in draft genomic sequences from Bradyrhizobium strains isolated from Lupinus spp. thriving in the Iberian Peninsula. In all cases, the genes encoding T6SSs were grouped and showed, in most cases, a high degree of conservation among genes encoding the structural components of the system. Bradyrhizobium sp. strain ISLU101 isolated from L. angustifolius, contains two clusters of genes involved in the formation of T6SS. One of such systems, designated as T6SS-1, contains 17 genes and shows a high degree of conservation regarding genes of B. diazoefficiens USDA110. The other one, T6SS-2, contains 16 genes flanked by insertion element sequences. Amino acid similarity between equivalent proteins encoded in both clusters is only about 40-50 %. A phylogenetic analysis based on the concatenation of sequences of several T6SS proteins was performed, and results indicate a clear separation of T6SS-2 from most rhizobial T6SSs. ISLU101 T6SS mutant derivatives in genes impO, impC1 and impC2 were generated by single homologous recombination of amplified internal fragments from the respective genes cloned into the suicide vector pK18mobsac. The symbiotic behaviour of mutants was examined with L. angustifolius. Results showed no effect of impC1 and impC2 mutations, while the impO mutant generated smaller plants with a mixture of white/red nodules. These results suggest that T6SSs may play a role in the Bradyrhizobium-lupines symbiose