Epitope tagging of chromosomal genes in <i>Salmonella</i>

We have developed a simple and efficient procedure for adding an epitope-encoding tail to one or more genes of interest in the bacterial chromosome. The procedure is a modification of the gene replacement method of Datsenko and Wanner [Datsenko, K. A. &amp; Wanner, B. L. (2000) Proc. Natl. Acad. Sci. USA 97, 6640–6645]. A DNA module that begins with the epitope-encoding sequence and includes a selectable marker is amplified by PCR with primers that carry extensions (as short as 36 nt) homologous to the last portion of the targeted gene and to a region downstream from it. Transformation of a strain expressing bacteriophage ʎ red functions yields recombinants carrying the targeted gene fused to the epitope-encoding sequence. The resulting C-terminal-tagged protein can be identified by standard immuno-detection techniques. In an initial application of the method, we have added the sequences encoding the FLAG and 3xFLAG and influenza virus hemagglutinin epitopes to various genes of Salmonella enterica serovar Typhimurium, including putative and established pathogenic determinants present in prophage genomes. Epitope fusion proteins were detected in bacteria growing in vitro, tissue culture cells, and infected mouse tissues. This work identified a prophage locus specifically expressed in bacteria growing intracellularly. The procedure described here should be applicable to a wide variety of Gram-negative bacteria and is particularly suited for the study of intracellular pathogens

Petrography and porosity of hydrocarbon bearing sandstones: Bajo Barreal formation (Cretaceous), Golfo San Jorge basin, Argentina

Con el objeto de profundizar en el conocimiento sobre la migración de los fluidos y las posibilidades de recuperación de hidrocarburos, se realizaron estudios petrográ- ficos y de porosidad en areniscas fluviales cretácicas de la sección superior de la Formación Bajo Barreal en el yacimiento Cerro Guadal Norte, cuenca Golfo San Jorge, Santa Cruz, Argentina. La sección superior de la Formación Bajo Barreal está compuesta por areniscas finas y medias con estratificación cruzada o planar, maciza y laminación ondulítica. En el tope de los cuerpos pueden desarrollarse niveles limolíticos con laminación paralela. Los depósitos están bien expuestos alrededor de la laguna La Sin Nombre, donde se describieron dos perfiles estratigráficos. En los cuerpos con exposición lateral completa se realizaron descripciones arquitecturales y perfiles estratigráficos de detalle cada 10 m. Se tomaron 49 muestras orientadas (al norte magnético) de areniscas medias. A partir de cada muestra se prepararon dos cortes petrográficos, perpendiculares entre sí, impregnados con resina epoxi azul. Las areniscas son principalmente litarenitas feldespáticas. También se identificaron litarenitas y grauvacas. Casi todas las areniscas son submaduras. La porosidad media es aproximadamente del 18%, de distribución homogénea a heterogénea. Contienen proporciones variables de cementos arcillosos, ceolíticos, silíceos y carbonáticos. Se observa un incremento del contenido de cemento ceolítico hacia el techo de los cuerpos, mientras que los cementos arcillosos son predominantes hacia la base. La distribución de la porosidad tiene un diseño más o menos constante; disminuye lateralmente desde los canales principales hacia las barras adyacentes; también decrece desde la base hacia el techo de las barras donde son frecuentes los depósitos de canalizaciones menores o «cross bar-channel» y en la dirección aguas abajo.Petrography and porosity studies were performed to improve the knowledge about fluid migrations and additional hydrocarbon recovery projects throughout Cretaceous fluvial sandstones of the Bajo Barreal Formation Upper section in the Cerro Guadal Norte oil field (Golfo San Jorge Basin, Santa Cruz, Argentina). The Upper section of the Bajo Barreal Formation is composed of fine and medium sandstones which have mainly cross but planar stratification, massive and ripple lamination were also recorded. Occasionally, light brown siltstones with parallel lamination are observed at the top of the sandstone bodies. Extensive fluvial channel belt deposits are well exposed around La Sin Nombre pond, where two stratigraphic logs were described. In addition, sedimentary architectural descriptions and short detailed lateral logs, separated by tens of meters each, were recorded. Forty nine north-oriented medium sandstones were sampled, and two blue epoxy impregnated petrographic thin sections were prepared for every sample, one parallel and the other normal to the North. The sandstones are mainly feldspar litharenites although litharenites and feldspar wackes were also identified. Most of them are submature and the mean porosity is around 18 % varying from homogeneous to heterogeneous. Clayey, zeolitic, siliceous and carbonatic cements are present in different proportions. A comparison among petrography, porosity and architectural studies indicates an increase of zeolitic cements towards the top of the sandstone bodies, whereas the clayey cements are predominant at the base of the sandstone deposits. Furthermore, porosity decreases from main channel to adjacent bars and cross over bar-channel. In the bar-channel deposit, porosity decreases in both downstream and upward.Fil: Ibañez, Lucía Marina. Universidad Nacional de Tucumán. Facultad de Ciencias Naturales e Instituto Miguel Lillo; Argentina. Fundación Miguel Lillo; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Ovejero, Ramona. Universidad Nacional de Tucumán. Facultad de Ciencias Naturales e Instituto Miguel Lillo; ArgentinaFil: Georgieff, Sergio Miguel. Universidad Nacional de Tucumán. Facultad de Ciencias Naturales e Instituto Miguel Lillo; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Ferreira, Leonor. YPF; ArgentinaFil: Bossi, Gerardo Eugenio. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Arquitectura y paleoambientes de los depósitos fluviales gravosos de la Formación Las Cumbres (Neógeno), en Villa Mervil, La Rioja, Argentina

Un corte de la ruta nacional 60, 500 m al oeste de Villa Mervil (Sierras de Mazán, La Rioja) muestra el contacto entre las Formaciones Salicas y Las Cumbres, con excelente exposición de la arquitectura fluvial que pudo ser extendida en tres dimensiones y por más de 700 m en sentido N-S, a ambos lados de la ruta. El corte muestra el contacto entre las Formaciones Salicas (Plioceno) y Las Cumbres (Plio-Pleistoceno). Pueden ser reconocidos cinco estadios de sedimentación: A-S, el inferior, constituido por areniscas friables fluviales y eólicas; F, de capas tabulares consistentes en limos de barreal y areniscas de planicie de distributarios (escorrenteras y barras efímeras) con abundantes calcretos y rizoconcreciones; B, el mejor preservado y que ha permitido el análisis 3D, de conglomerados arenosos, que corta profundamente los dos estadios inferiores. El Estadio B está organizado en una serie de mesoformas de canal, con barrancas izquierdas (al Oeste) bien definidas. En su etapa final de abandono (Estadio L), fue rellenado por capas tabulares de arenas finas limos pardos, que contienen una arcilita verde y una capa de micrita blanca, que pueden asignarse a un depósito lacustre somero. Finalmente yace sobre todo el conjunto anterior, el Estadio C, de conglomerados finos arenosos que forman un extenso sistema de canales y planicie aluvial pero mal representado en estos afloramientos. Las particularidades de estos depósitos pueden ser comparadas con expresiones locales del ambiente actual: el río Pituil (al oeste de la Sierra de Velasco) la llanura arenosa del Bolsón de Pipanaco y el barreal del Señor de la Peña, donde impera un clima árido con una escasas lluvias estivales, indicado condiciones climáticas similares

130 purification of large scale mrna encoding zfn nucleases by dhplc technology

A novel strategy of targeted gene correction of the interleukin-2 receptor common gamma chain (IL2RG) gene for the treatment of X-linked Severe Combined Immunodeficiency (SCID-X1) is achieved by the combination of a pair of IL2RG-specific Zinc Finger Nucleases (ZFN) and the correct-gene template DNA delivered by integration-defective lentiviral vector (IDLV).The transient expression of the ZFN pair targeting the disease-causing gene is obtained by the electroporation of the two corresponding mRNAs, produced by in vitro transcription starting from plasmid DNA template. A major limitation of the mRNA transcribed in vitro is the presence of residual contaminants such as short RNAs and double stranded (ds)RNAs that may affect the function and spectrophotometric quantification of the product hampering therefore the delivery of high quality and precise amount of mRNA to target cells. Moreover, dsRNA contaminants represent a possible risk in terms of immunogenicity of the product, leading to activation of unwanted innate immune response with consequent reduction/abrogation of mRNA translation as well as potential alteration of the properties of the transfected cells. To improve nuclease expression while decreasing cellular innate response to mRNA transfection we combined different strategies: (i) inclusion of UTRs and polyA tails in the DNA template used for mRNA production; (ii) use of modified nucleotides during mRNA production and (iii) purification of the mRNAs by dHPLC with a reverse phase column made of non-porous matrix consisting of polystyrene-divinylbenzene copolymer beads alkylated with C-18 chains (Transgenomic, LTD.). In particular, the purification of in vitro transcribed mRNAs by means of dHPLC has been shown to strongly improve the translation of mRNA and significantly reduce the contaminant presence thus preventing innate immunity and eventually increasing modified cells persistence in vivo. We have developed feasible and reproducible, small and large scale mRNA production and downstream purification processes of the ZFN pairs obtaining accurate RNA quantification and reduced risk of immunogenicity. The full process achieved a 60% yield, loading with a 500µg RNA for each run with a single clean chromatographic peak. Furthermore, the level of residual organic solvent (i.e. Acetonitrile) used in the purification process is compatible with that applicable into clinic. The highly translatable non-immunogenic dHPLC-purified mRNA can be delivered without toxicity and represents a powerful and safe tool for the application of gene therapy protocols

Seroepidemiology of Toxoplasma gondii infection in psychiatric inpatients in a northern Mexican city

BACKGROUND: Patients with psychiatric disorders were found to show a high seroprevalence of Toxoplasma gondii infection. There is scarce information about the epidemiology of T. gondii infection in psychiatric patients in Mexico. Therefore, we sought to determine the prevalence of T. gondii infection and associated socio-demographic, clinical and behavioural characteristics in a population of psychiatric patients in Durango City, Mexico. Seroprevalence in patients was compared with that obtained in a control population. METHODS: One hundred and thirty seven inpatients of a public psychiatric hospital and 180 controls were examined for the presence of IgG and IgM antibodies against T. gondii by enzyme-linked immunoassay (Diagnostic Automation Inc., Calabasas, CA, USA). The control population consisted of blood donors of a public blood bank and elderly persons attending a senior center in the same city. Age in controls (42 years +/- 20.2) was comparable with that of the psychiatric patients (43.7 years +/-13.8) (p = 0.42). Socio-demographic, clinical and behavioral characteristics from the patients were also obtained. RESULTS: Anti-T. gondii IgG antibodies indicating latent infection with T. gondii was found in 25 (18.2%) of 137 psychiatric inpatients and 16 (8.9%) of 180 controls (p = 0.02). Ten (26.3%) of 38 schizophrenic patients had latent infection and this prevalence was also significantly higher than that observed in controls (p = 0.005). Prevalence of anti-T. gondii IgM antibodies was comparable among patients and controls (4.4% vs 2.2%, respectively, p = 0.22). Multivariate analysis showed that T. gondii infection in inpatients was positively associated with sexual promiscuity (adjusted OR = 15.8; 95% CI: 3.8–64.8), unwashed raw fruit consumption (adjusted OR = 5.19; 95% CI: 2.3–11.3), and a history of surgery (adjusted OR = 6.5; 95% CI: 2.6–16), and negatively associated with lamb meat consumption (adjusted OR = 0.26; 95% CI: 0.10–0.63). CONCLUSION: In the present study, psychiatric inpatients in Durango, Mexico, in general and schizophrenia inpatients in particular had a significantly higher prevalence of T. gondii infection than the control group. Results suggest that unwashed raw fruit consumption might be the most important route of T. gondii transmission in our psychiatric inpatients while lamb meat consumption the less important. Additional studies will have to elucidate the causative relation between infection with T. gondii and psychiatric disorders

Refinement of the diagnostic approach for the identification of children and adolescents affected by familial hypercholesterolemia: Evidence from the LIPIGEN study

Background and aims: We aimed to describe the limitations of familiar hypercholesterolemia (FH) diagnosis in childhood based on the presence of the typical features of FH, such as physical sings of cholesterol accumulation and personal or family history of premature cardiovascular disease or hypercholesterolemia, comparing their prevalence in the adult and paediatric FH population, and to illustrate how additional information can lead to a more effective diagnosis of FH at a younger age.Methods: From the Italian LIPIGEN cohort, we selected 1188 (&gt;= 18 years) and 708 (&lt;18 years) genetically-confirmed heterozygous FH, with no missing personal FH features. The prevalence of personal and familial FH features was compared between the two groups. For a sub-group of the paediatric cohort (N = 374), data about premature coronary heart disease (CHD) in second-degree family members were also included in the evaluation.Results: The lower prevalence of typical FH features in children/adolescents vs adults was confirmed: the prevalence of tendon xanthoma was 2.1% vs 13.1%, and arcus cornealis was present in 1.6% vs 11.2% of the cohorts, respectively. No children presented clinical history of premature CHD or cerebral/peripheral vascular disease compared to 8.8% and 5.6% of adults, respectively. The prevalence of premature CHD in first-degree relatives was significantly higher in adults compared to children/adolescents (38.9% vs 19.7%). In the sub-cohort analysis, a premature CHD event in parents was reported in 63 out of 374 subjects (16.8%), but the percentage increased to 54.0% extending the evaluation also to second-degree relatives.Conclusions: In children, the typical FH features are clearly less informative than in adults. A more thorough data collection, adding information about second-degree relatives, could improve the diagnosis of FH at younger age

Refinement of the diagnostic approach for the identification of children and adolescents affected by familial hypercholesterolemia: Evidence from the LIPIGEN study

Background and aims: We aimed to describe the limitations of familiar hypercholesterolemia (FH) diagnosis in childhood based on the presence of the typical features of FH, such as physical sings of cholesterol accumulation and personal or family history of premature cardiovascular disease or hypercholesterolemia, comparing their prevalence in the adult and paediatric FH population, and to illustrate how additional information can lead to a more effective diagnosis of FH at a younger age. Methods: From the Italian LIPIGEN cohort, we selected 1188 (≥18 years) and 708 (&lt;18 years) genetically-confirmed heterozygous FH, with no missing personal FH features. The prevalence of personal and familial FH features was compared between the two groups. For a sub-group of the paediatric cohort (N = 374), data about premature coronary heart disease (CHD) in second-degree family members were also included in the evaluation. Results: The lower prevalence of typical FH features in children/adolescents vs adults was confirmed: the prevalence of tendon xanthoma was 2.1% vs 13.1%, and arcus cornealis was present in 1.6% vs 11.2% of the cohorts, respectively. No children presented clinical history of premature CHD or cerebral/peripheral vascular disease compared to 8.8% and 5.6% of adults, respectively. The prevalence of premature CHD in first-degree relatives was significantly higher in adults compared to children/adolescents (38.9% vs 19.7%). In the sub-cohort analysis, a premature CHD event in parents was reported in 63 out of 374 subjects (16.8%), but the percentage increased to 54.0% extending the evaluation also to second-degree relatives. Conclusions: In children, the typical FH features are clearly less informative than in adults. A more thorough data collection, adding information about second-degree relatives, could improve the diagnosis of FH at younger age