Rab1b-GBF1-ARF1 Secretory Pathway Axis Is Required for Birnavirus Replication.

Birnaviruses are members of the Birnaviridae family, responsible for major economic losses to poultry and aquaculture. The family is composed of nonenveloped viruses with a segmented double-stranded RNA (dsRNA) genome. Infectious bursal disease virus (IBDV), the prototypic family member, is the etiological agent of Gumboro disease, a highly contagious immunosuppressive disease in the poultry industry worldwide. We previously demonstrated that IBDV hijacks the endocytic pathway for establishing the viral replication complexes on endosomes associated with the Golgi complex (GC). Here, we report that IBDV reorganizes the GC to localize the endosome-associated replication complexes without affecting its secretory functionality. By analyzing crucial proteins involved in the secretory pathway, we showed the essential requirement of Rab1b for viral replication. Rab1b comprises a key regulator of GC transport and we demonstrate that transfecting the negative mutant Rab1b N121I or knocking down Rab1b expression by RNA interference significantly reduces the yield of infectious viral progeny. Furthermore, we showed that the Rab1b downstream effector Golgi-specific BFA resistance factor 1 (GBF1), which activates the small GTPase ADP ribosylation factor 1 (ARF1), is required for IBDV replication, since inhibiting its activity by treatment with brefeldin A (BFA) or golgicide A (GCA) significantly reduces the yield of infectious viral progeny. Finally, we show that ARF1 dominant negative mutant T31N overexpression hampered IBDV infection. Taken together, these results demonstrate that IBDV requires the function of the Rab1b-GBF1-ARF1 axis to promote its replication, making a substantial contribution to the field of birnavirus-host cell interactions. IMPORTANCE Birnaviruses are unconventional members of the dsRNA viruses, with the lack of a transcriptionally active core being the main differential feature. This structural trait, among others that resemble those of the plus single-stranded (+ssRNA) viruses features, suggests that birnaviruses might follow a different replication program from that conducted by prototypical dsRNA members and the hypothesis that birnaviruses could be evolutionary links between +ssRNA and dsRNA viruses has been argued. Here, we present original data showing that IBDV-induced GC reorganization and the cross talk between IBDV and the Rab1b-GBF1-ARF1 mediate the intracellular trafficking pathway. The replication of several +ssRNA viruses depends on the cellular protein GBF1, but its role in the replication process is not clear. Thus, our findings make a substantial contribution to the field of birnavirus-host cell interactions and provide further evidence supporting the proposed evolutionary connection role of birnaviruses, an aspect which we consider especially relevant for researchers working in the virology field.

A Novel Single Domain Antibody Targeting FliC Flagellin of Salmonella enterica for Effective Inhibition of Host Cell Invasion.

The enteric pathogen, Salmonella enterica is a major cause of human gastroenteritis globally and with increasing bacterial resistance to antibiotics, alternative solutions are urgently needed. Single domain antibodies (sdAbs), the smallest antibody fragments that retain antigen binding specificity and affinity, are derived from variable heavy-chain only fragments (VHH) of camelid heavy-chain-only immunoglobulins. SdAbs typically contain a single disulfide bond simplifying recombinant protein production in microbial systems. These factors make sdAbs ideally suited for the development of effective anti-bacterial therapeutics. To this end, we generated an anti-Salmonella VHH library from which we screened for high affinity sdAbs. We present a novel sdAb (Abi-Se07) that targets the Salmonella virulence factor, FliC, required for bacterial motility and invasion of host cells. We demonstrate that Abi-Se07 bound FliC with a K D of 16.2 ± 0.1 nM. In addition, Abi-Se07 exhibited cross-serovar binding to whole cells of S. enterica serovar Typhimurium, Heidelberg, and Hadar. Abi-Se07 significantly inhibited bacterial motility and significantly reduced S. enterica colonization in a more native environment of chicken jejunum epithelium. Taken together, we have identified a novel anti-Salmonella sdAb and discuss future efforts toward therapeutic development.

Human Adipose-Derived Mesenchymal Stem/Stromal Cells Handling Protocols. Lipid Droplets and Proteins Double-Staining.

Human Adipose-derived mesenchymal stem/stromal cells (hASCs) are of great interest because of their potential for therapeutic approaches. The method described here covers every single step necessary for hASCs isolation from subcutaneous abdominal adipose tissue, multicolor phenotyping by flow cytometry, and quantitative determination of adipogenic differentiation status by means of lipid droplets (LDs) accumulation, and Western blot analysis. Moreover, to simultaneously analyze both LDs accumulation and cellular proteins localization by fluorescence microscopy, we combined Oil Red O (ORO) staining with immunofluorescence detection. For LDs quantification we wrote a program for automatic ORO-stained digital image processing implemented in Octave, a freely available software package. Our method is based on the use of the traditional low cost neutral lipids dye ORO, which can be imaged both by bright-field and fluorescence microscopy. The utilization of ORO instead of other more expensive lipid-specific dyes, together with the fact that the whole method has been designed employing cost-effective culture reagents (standard culture medium and serum), makes it affordable for tight-budget research laboratories. These may be replaced, if necessary or desired, by defined xeno-free reagents for clinical research and applications.

Infectious bursal disease virus uptake involves macropinocytosis and trafficking to early endosomes in a Rab5-dependent manner.

Infectious bursal disease virus (IBDV) internalization is sparsely known in terms of molecular components of the pathway involved. To describe the cell biological features of IBDV endocytosis, we employed perturbants of endocytic pathways such as pharmacological inhibitors and overexpression of dominant-negative mutants. Internalization analysis was performed quantifying infected cells by immunofluorescence and Western blot detection of the viral protein VP3 at 12 h post-infection reinforced by the analysis of the capsid protein VP2 localization after virus uptake at 1 h post-infection. We compared IBDV infection to the internalization of well-established ligands with defined endocytic pathways: transferrin, cholera-toxin subunit B and dextran. To describe virus endocytosis at the morphological level, we performed ultrastructural studies of viral internalization kinetics in control and actin dynamics-blocked cells. Our results indicate that IBDV endocytic internalization was clathrin- and dynamin-independent, and that IBDV uses macropinocytosis as the primary entry mechanism. After uptake, virus traffics to early endosomes and requires exposure to the low endocytic pH as well as a functional endocytic pathway to complete its replication cycle. Moreover, our results indicate that the GTPase Rab5 is crucial for IBDV entry supporting the participation of the early endosomal pathway in IBDV internalization and infection of susceptible cells.

Relación entre actividad antioxidante y contenido de fenoles y flavonoides totales en semillas de Cucurbita spp / Relationship between antioxidant activity and content of phenols and total flavonoids in seeds of Cucurbita spp

El propósito de este trabajo fue determinar la actividad antioxidante en extractos de semillas de cuatro variedades de Cucurbita spp: Tetsukabuto, un híbrido entre C. moschata y C. maxima Duchesne ex Lam. ("zapallo japonés"); C. mixta Pangalo ("calabaza rayada"); C. moschata (Duchesne ex Lam.) Duchesne ex Poir. ("coreanito") y C. maxima Duchesne ("calabaza plomo") con respecto a su contenido de fenoles totales y flavonoides. Se obtuvieron extractos de polaridad decreciente utilizando los siguientes solventes: agua acidificada, metanol, acetona y acetato de etilo; los fenoles totales y los flavonoides se cuantificaron con el método de Folin-Ciocalteu, y el de formación de complejos con AlCl3 al 5 % respectivamente. Para determinar la actividad antioxidante de los extractos se utilizó la técnica de decoloración del radical libre 2,2- difenil-1-picrilhidracilo (DPPH•). El contenido de fenoles totales fue: Tetsukabuto (165,48 ± 0,94); C. mixta Pangalo (293,07 ± 6,86); C. moschata (118,79 ± 3,72); C. maxima Duchesne (194,91 ± 7,51) (µmol GAE/g de extracto). Los valores de flavonoides oscilaron entre (32,18 ± 1,39) y (96,63 ± 1,14) mg de quercetina/g de extracto. La actividad antioxidante expresada como concentración inhibidora media (IC50) mostró los siguientes resultados: Tetsukabuto (117,69 ± 5,81), C. mixta Pangalo (77,75 ± 3,64), C. moschata (110,67 ± 1,70), C. maxima Duchesne (87,39 ± 8,64) (mg/ml). Un análisis unidireccional de varianza mostró que hay diferencias significativas (p < 0,05) entre las variedades estudiadas. De los resultados obtenidos se observa una clara relación entre el contenido de fenoles totales y flavonoides con actividad antioxidante. C. mixta Pangalo fue la variedad que mostró mayor actividad antioxidante, que puede ser considerada fuente de antioxidantes naturales para producir un efecto benéfico para la salud. (AU)

Multivariate characterization of argentinean mistletoe,Ligaria cuneifolia (Loranthaceae) according to theirmineral nutrient composition / Caracterización multivariable de muérdago criollo, Ligaria cuneifolia (Loranthaceae) de acuerdo a su composición mineral

The aim of this study was to characterize samples of Ligaria cuneifolia collected from three areas of the north-east region of Argentina, during two sampling periods. In this work, the authors propose a mathematical model for searching associations among mineral contents and other factors such us geographic origin or sampling period. Mineral monitoring as a pattern recognition method is a promising tool in the characterization and/or standardization of phytomedicines. In the present work measurable amounts of Al, Ca, Co, Cr, Cu, Fe, K, Li, Mg, Mn, Na, P, Sr, and Zn were detected in phytopharmaceutical samples of L. cuneifolia by inductively coupled plasma optical emission spectrometry (ICP-OES). Finally, this methodology allows reliable determinations of mineral content in pharmaceutical quality control of medicinal plants.

El objetivo de este estudio fue caracterizar muestras de Ligaria cuneifolia recolectadas en tres zonas de la región noreste de Argentina, durante dos períodos de muestreo. En este trabajo los autores proponen un modelo matemático para la búsqueda de asociaciones entre el contenido mineral y otros factores como el origen geográfico o el periodo de muestreo. El monitoreo de las concentraciones de los elementos minerales, como método para el reconocimiento de patrones, es una herramienta prometedora en la caracterización y/o estandarización de fitofármacos. En el presente trabajo se pudieron detectar cantidades medibles de Al, Ca, Co, Cr, Cu, Fe, K, Li, Mg, Mn, Na, P, Sr y Zn que fueron detectadas en muestras fitomedicinales de L. cuneifolia por espectroscopía de emisión óptica de plasma acoplado inductivamente (ICP-OES). Finalmente, esta metodología permitió realizar determinaciones confiables del contenido mineral en el control de calidad farmacéutica de plantas medicinales.

Natural contamination with arsenic and other trace elements in groundwater of the Central-West region of Chaco, Argentina.

This study covered the central agricultural region of the Chaco province, which lacks a permanent river networks. However, during the rainy period there is localized groundwater recharge. About 84 groundwater samples were taken during the period April-December 2007. These groundwater samples were collected from two different depths: 62 samples from shallow wells (4 to 20 m) and 24 samples from deep wells (20 to 100 m). Chemical variables were determined: pH, specific conductance, total dissolved solid, hardness, alkalinity, HCO(3)-, CO(3)(2-), SO(4)(2-), Cl-, NO(3)-, NO(2) -, NH(4)+, F-, As((tot)), Na+, K+, Ca2+, Mg2+, Fe, Cu, Ni, Pb and Zn. The chemical composition of groundwater in the study area is dominantly sodium bicarbonate and sodium chloride bicarbonate, comprising more than 60% (52/86) of shallow and deep groundwater samples. Of the 86 analyzed groundwater samples, 88% exceeded the WHO (World Health Organization) and CAA (Código Alimentario Argentino) standards (10 µg/L) for As (arsenic) and 9% exceeded the WHO standard (1.5 mg/L) for F(-).Groundwater highly contaminated with As (max. 1,073 µg/L) and F- (max. 4.2 mg/L) was found in shallow aquifer. The contaminated groundwater is characterized by high pH (max. 8.9), alkalinity (max. HCO(3)- 1,932 mg/L), SO(4)(2-) (max. 11,862 mg/L), Na(+) (max. 3,158 mg/L), Cl(-) (max. 10,493 mg/L) and electric conductivity greater than 33.3 µS/cm. Other associated elements (Ni, Pb, Cu and Zn) are present in low concentrations, except for Fe that in 32% of samples exceeded the guideline value of 0.3 mg/L suggested by the CAA.

Arsénico total en agua de consumo en el centro-oeste de la provincia del Chaco

Muestra los resultados encontrados en la zona centro-oeste de la provinci del Chaco a partir de un relevamiento realizado con el fin de obtener información ordenada y sistematizada sobre los niveles de arsénico en aguas de consumo

Arsénico total en agua de consumo en el centro-oeste de la provincia del Chaco

Muestra los resultados encontrados en la zona centro-oeste de la provinci del Chaco a partir de un relevamiento realizado con el fin de obtener información ordenada y sistematizada sobre los niveles de arsénico en aguas de consumo