Reconstruction of a Genome-Scale Metabolic Model of Streptomyces albus J1074: Improved Engineering Strategies in Natural Product Synthesis

Streptomyces albus J1074 is recognized as an effective host for heterologous production of natural products. Its fast growth and efficient genetic toolbox due to a naturally minimized genome have contributed towards its advantage in expressing biosynthetic pathways for a diverse repertoire of products such as antibiotics and flavonoids. In order to develop precise model-driven engineering strategies for de novo production of natural products, a genome-scale metabolic model (GEM) was reconstructed for the microorganism based on protein homology to model species Streptomyces coelicolor while drawing annotated data from databases and literature for further curation. To demonstrate its capabilities, the Salb-GEM was used to predict overexpression targets for desirable compounds using flux scanning with enforced objective function (FSEOF). Salb-GEM was also utilized to investigate the effect of a minimized genome on metabolic gene essentialities in comparison to another Streptomyces species, S. coelicolor

Definition of a high-affinity Gag recognition structure mediating packaging of a retroviral RNA genome

All retroviral genomic RNAs contain a cis-acting packaging signal by which dimeric genomes are selectively packaged into nascent virions. However, it is not understood how Gag (the viral structural protein) interacts with these signals to package the genome with high selectivity. We probed the structure of murine leukemia virus RNA inside virus particles using SHAPE, a high-throughput RNA structure analysis technology. These experiments showed that NC (the nucleic acid binding domain derived from Gag) binds within the virus to the sequence UCUG-UR-UCUG. Recombinant Gag and NC proteins bound to this same RNA sequence in dimeric RNA in vitro; in all cases, interactions were strongest with the first U and final G in each UCUG element. The RNA structural context is critical: High-affinity binding requires base-paired regions flanking this motif, and two UCUG-UR-UCUG motifs are specifically exposed in the viral RNA dimer. Mutating the guanosine residues in these two motifs—only four nucleotides per genomic RNA—reduced packaging 100-fold, comparable to the level of nonspecific packaging. These results thus explain the selective packaging of dimeric RNA. This paradigm has implications for RNA recognition in general, illustrating how local context and RNA structure can create information-rich recognition signals from simple single-stranded sequence elements in large RNAs

Identification of Sare0718 As an Alanine-Activating Adenylation Domain in Marine Actinomycete Salinispora arenicola CNS-205

BACKGROUND: Amino acid adenylation domains (A domains) are critical enzymes that dictate the identity of the amino acid building blocks to be incorporated during nonribosomal peptide (NRP) biosynthesis. NRPs represent a large group of valuable natural products that are widely applied in medicine, agriculture, and biochemical research. Salinispora arenicola CNS-205 is a representative strain of the first discovered obligate marine actinomycete genus, whose genome harbors a large number of cryptic secondary metabolite gene clusters. METHODOLOGY/PRINCIPAL FINDINGS: In order to investigate cryptic NRP-related metabolites in S. arenicola CNS-205, we cloned and identified the putative gene sare0718 annotated "amino acid adenylation domain". Firstly, the general features and possible functions of sare0718 were predicted by bioinformatics analysis, which suggested that Sare0718 is a soluble protein with an AMP-binding domain contained in the sequence and its cognate substrate is L-Val. Then, a GST-tagged fusion protein was expressed and purified to further explore the exact adenylation activity of Sare0718 in vitro. By a newly mentioned nonradioactive malachite green colorimetric assay, we found that L-Ala but not L-Val is the actual activated amino acid substrate and the basic kinetic parameters of Sare0718 for it are K(m) = 0.1164±0.0159 (mM), V(max) = 3.1484±0.1278 (µM/min), k(cat) = 12.5936±0.5112 (min(-1)). CONCLUSIONS/SIGNIFICANCE: By revealing the biochemical role of sare0718 gene, we identified an alanine-activating adenylation domain in marine actinomycete Salinispora arenicola CNS-205, which would provide useful information for next isolation and function elucidation of the whole cryptic nonribosomal peptide synthetase (NRPS)-related gene cluster covering Sare0718. And meanwhile, this work also enriched the biochemical data of A domain substrate specificity in newly discovered marine actinomycete NRPS system, which bioinformatics prediction will largely depend on

Caracterización clínico patológica de la enfermedad Granulomatosa de Pterophyllum scalare (escalar) en confinamiento

RESUMEN: Para la caracterización clínico-patológica de la enfermedad granulomatosa  de peces escalar Pterophylum scalare en sistemas de cultivo se propuso: hacer el seguimiento de cuatro planteles establecidos, además de tener en cuenta los casos recibidos en el servicio de diagnóstico del IALL, se describieron signos y lesiones en animales clínicamente enfermos, identifican- do lesiones y posibles  agentes etiológicos con H-E tradicional y diferenciarlos con coloración Ziehl- Neelsen. De forma complementaria, a partir de animales enfermos, aislar y cultivar los agentes etiológicos.  Adicionalmente, el estudio pretendió  revisar y discutir factores de manejo asociados a la enfermedad en sistemas de producción de los Llanos de Colombia. Para  el efecto se observaron 92 animales durante 90 días en el Laboratorio  de Bioensayos del IALL en Villavicencio, Meta, Colombia, procedentes de 5 fuentes diferentes, en las cuales se presentaron casos de enfermedad evidente: De los animales observados 43  desarrollaron  signos clínicos; se tomaron 20 ejemplares adultos para estudios anatomopatológicos  y 19 para aislamientos microbiológicos. En los sistemas de producción se encontró: alta densidad de siembra, mala calidad del agua y uso indiscriminado de drogas  antibióticas, factores que pueden predisponer a la presencia de la enfermedad. Los animales  afectados  fueron única- mente adultos.  Los signos clínicos principales fueron: depresión, letar- go, nado errático, focos de descamación y úlceras en la piel. Macro y microscópicamente el órgano interno mas afectado  fue el bazo, pudiéndose establecer el mayor grado de severidad y extensión de la lesión granulomatosa  (3 en una escala de 0 a 3).  Los aislamientos fueron positivos al grupo de las Micobacterias; se aislaron colonias de tres  de las muestras  en medio Lowestein Jensen, dos de ellas por cultivo a 28 °C, con pigmento amarillo de crecimiento entre dos y tres meses y otra con crecimiento a 28°C con pigmento al mes de incubación, estudios de identificación por métodos genotípicos  y fenotípicos; se halló fenotípicamente Mycobacterium fortuitum en los dos aislamientos y genotípicamente; Mycobacterium fortuitum tipo 1 y Mycobacterium peregrinum tipo 2 respectivamente.  La incidencia de la infección micobacteriana  en planteles  de reproductores  de escalar es relativamente alta el 46% de los animales observados desarrolló signos clínicos posiblemente asociada  a factores estresantes como el hacinamiento, mala nutrición, fluctuaciones de temperatura, calidad del agua  y manipulación inadecuada. Mientras  se profundiza en aspectos epidemiológicos y se establece otro tipo de aproximación preventiva, se recomienda,  evitar  el uso de antibióticos de forma indiscriminada, mejorar condiciones de manejo: desinfección, calidad de agua, densidad y nutrición adecuadas,  ayudarían  a disminuir el estrés y la susceptibilidad de los animales Palabras Claves: Pterophyllum scalare, Micobacteriosis. Enfermedad granulomatosa de peces ornamentales.ABSTRACT:For the clinical-pathological characterization of the granulomatous disease of angel fish Pterophyllum  scalare  in culture systems intended: to make the pursuit  of four established  facilities, besides keeping in mind the cases received in the service of diagnosis of the IALL, signs and lesions were described in clinically sick animals,  identifying lesions and possible etiologic agents with H-E traditional and to differentiate them with Ziehl-Neelsen stain. In a  complementary  way, starting from sick animals,  to isolate and to cultivate  the etiologic agents. Additionally, the study sought to revise and  to  discuss handling factors associated to the disease in production systems of the Plains of Colombia. For the effect 92 animals  were observed during 90 days in the Laboratory of Bioassays of the IALL in Villavicencio, Meta, Colombia, coming from 5 different sources, in which cases of evident illness  were presented:  Of the obser ved animals 43 developed clinical signs; 20 mature specimens were taken for anatomopathologic studies and 19  for microbiologic isolations. In the production systems were: high sowing density, bad water quality and indiscriminate  use of antibiotic drugs, factors that  can predispose to the presence of the disease. The affected animals were only adults. The main clinical  signs were: depression,  lethargy, erratic swimming, flakeout focuses and skin ulcers. Macro and microscopically, the most affected internal  organ  was the  spleen, being able to establish the biggest degrees of severity and extension of the granulomatous lesion (3 in a scale of 0 at 3). The isolations were positive to  the  Mycobacterium group;  colonies of three  of the samples  were isolated  between Lowestein Jensen medium, two of them for cultivation to 28° C, colonies with yellow pigment  of growth  between two and three months and another with growth to 28° C with pigment to the month of incubation, typification studies characterized the cultivations like with Mycobacterium  fortuitum.The incidence of the  mycobacterium infection in facilities of reproducers  of scalar  is relatively high -46% of the observed animals developed clinical signs  possibly associated to stressant factors like the accumulation, bad nutrition, fluctuations of temperature, water quality  and inadequate  manipulation.  While we are  deepened inepidemic aspects and another type of preventive approach  settles down, is recommended, to avoid the use  of antibiotics drugs in an indiscriminate  form, to improve handling conditions: disinfection, water quality, densities and adapted nutrition, they would help to diminish the stress    and susceptibility of the animals.Key words: Pterophylum scalare, Micobacteriosis, granulomatous disease of ornamental  fishRESUMEN: Para la caracterización clínico-patológica de la enfermedad granulomatosa  de peces escalar Pterophylum scalare en sistemas de cultivo se propuso: hacer el seguimiento de cuatro planteles establecidos, además de tener en cuenta los casos recibidos en el servicio de diagnóstico del IALL, se describieron signos y lesiones en animales clínicamente enfermos, identifican- do lesiones y posibles  agentes etiológicos con H-E tradicional y diferenciarlos con coloración Ziehl- Neelsen. De forma complementaria, a partir de animales enfermos, aislar y cultivar los agentes etiológicos.  Adicionalmente, el estudio pretendió  revisar y discutir factores de manejo asociados a la enfermedad en sistemas de producción de los Llanos de Colombia. Para  el efecto se observaron 92 animales durante 90 días en el Laboratorio  de Bioensayos del IALL en Villavicencio, Meta, Colombia, procedentes de 5 fuentes diferentes, en las cuales se presentaron casos de enfermedad evidente: De los animales observados 43  desarrollaron  signos clínicos; se tomaron 20 ejemplares adultos para estudios anatomopatológicos  y 19 para aislamientos microbiológicos. En los sistemas de producción se encontró: alta densidad de siembra, mala calidad del agua y uso indiscriminado de drogas  antibióticas, factores que pueden predisponer a la presencia de la enfermedad. Los animales  afectados  fueron única- mente adultos.  Los signos clínicos principales fueron: depresión, letar- go, nado errático, focos de descamación y úlceras en la piel. Macro y microscópicamente el órgano interno mas afectado  fue el bazo, pudiéndose establecer el mayor grado de severidad y extensión de la lesión granulomatosa  (3 en una escala de 0 a 3).  Los aislamientos fueron positivos al grupo de las Micobacterias; se aislaron colonias de tres  de las muestras  en medio Lowestein Jensen, dos de ellas por cultivo a 28 °C, con pigmento amarillo de crecimiento entre dos y tres meses y otra con crecimiento a 28°C con pigmento al mes de incubación, estudios de identificación por métodos genotípicos  y fenotípicos; se halló fenotípicamente Mycobacterium fortuitum en los dos aislamientos y genotípicamente; Mycobacterium fortuitum tipo 1 y Mycobacterium peregrinum tipo 2 respectivamente.  La incidencia de la infección micobacteriana  en planteles  de reproductores  de escalar es relativamente alta el 46% de los animales observados desarrolló signos clínicos posiblemente asociada  a factores estresantes como el hacinamiento, mala nutrición, fluctuaciones de temperatura, calidad del agua  y manipulación inadecuada. Mientras  se profundiza en aspectos epidemiológicos y se establece otro tipo de aproximación preventiva, se recomienda,  evitar  el uso de antibióticos de forma indiscriminada, mejorar condiciones de manejo: desinfección, calidad de agua, densidad y nutrición adecuadas,  ayudarían  a disminuir el estrés y la susceptibilidad de los animales Palabras Claves: Pterophyllum scalare, Micobacteriosis. Enfermedad granulomatosa de peces ornamentales.ABSTRACT:For the clinical-pathological characterization of the granulomatous disease of angel fish Pterophyllum  scalare  in culture systems intended: to make the pursuit  of four established  facilities, besides keeping in mind the cases received in the service of diagnosis of the IALL, signs and lesions were described in clinically sick animals,  identifying lesions and possible etiologic agents with H-E traditional and to differentiate them with Ziehl-Neelsen stain. In a  complementary  way, starting from sick animals,  to isolate and to cultivate  the etiologic agents. Additionally, the study sought to revise and  to  discuss handling factors associated to the disease in production systems of the Plains of Colombia. For the effect 92 animals  were observed during 90 days in the Laboratory of Bioassays of the IALL in Villavicencio, Meta, Colombia, coming from 5 different sources, in which cases of evident illness  were presented:  Of the obser ved animals 43 developed clinical signs; 20 mature specimens were taken for anatomopathologic studies and 19  for microbiologic isolations. In the production systems were: high sowing density, bad water quality and indiscriminate  use of antibiotic drugs, factors that  can predispose to the presence of the disease. The affected animals were only adults. The main clinical  signs were: depression,  lethargy, erratic swimming, flakeout focuses and skin ulcers. Macro and microscopically, the most affected internal  organ  was the  spleen, being able to establish the biggest degrees of severity and extension of the granulomatous lesion (3 in a scale of 0 at 3). The isolations were positive to  the  Mycobacterium group;  colonies of three  of the samples  were isolated  between Lowestein Jensen medium, two of them for cultivation to 28° C, colonies with yellow pigment  of growth  between two and three months and another with growth to 28° C with pigment to the month of incubation, typification studies characterized the cultivations like with Mycobacterium  fortuitum.The incidence of the  mycobacterium infection in facilities of reproducers  of scalar  is relatively high -46% of the observed animals developed clinical signs  possibly associated to stressant factors like the accumulation, bad nutrition, fluctuations of temperature, water quality  and inadequate  manipulation.  While we are  deepened inepidemic aspects and another type of preventive approach  settles down, is recommended, to avoid the use  of antibiotics drugs in an indiscriminate  form, to improve handling conditions: disinfection, water quality, densities and adapted nutrition, they would help to diminish the stress    and susceptibility of the animals.Key words: Pterophylum scalare, Micobacteriosis, granulomatous disease of ornamental  fis

Internalisation du tritium sous forme organique chez les embryons et larves de poisson zèbre.

Les populations naturelles d'organismes aquatiques sont exposées à des radionucléides émetteurs de différents types de radionucléides (α, β, γ) [1]. Ces expositions peuvent mener à l'apparition d'effets délétères qui dépendent de plusieurs facteurs tels que le type de rayonnement, la dose absorbée, le temps d'exposition et la répartition subcellulaire du radionucléide dans l'organisme. La caractérisation à l'échelle subcellulaire de la distribution d'un radionucléide et la dose associée est donc cruciale pour déterminer les mécanismes associés aux effets induits [2]. Une étude a mis en avant qu'après une exposition au tritium libre, l'internalisation chez les embryons et larves de poisson zèbre augmente linéairement avec l'activité présente dans le milieu [1]. Deux autres études ont mis en avant que l'exposition du poisson zèbre au tritium libre pendant son développement entraîne une modification de l'expression de certains gènes et une augmentation du taux de dommages à l'ADN menant à des altérations musculaires et à une modification du comportement natatoire des larves [3]. Cette étude se concentre sur le tritium sous forme organique, plus spécifiquement la thymidine tritiée. Afin d'étudier la toxicité de la thymidine tritiée sur le poisson zèbre à ses stades précoces de développement, l'internalisation du tritium et la répartition subcellulaire de ses dépôts d'énergie ont été étudiés. Des embryons de poisson zèbre ont été exposés à de la thymidine tritiée à différentes activités (de 2,4 à 595 kBq/mL) et l'internalisation du tritium a été mesurée dans les embryons et larves de poisson zèbre (1 et 4 jours post fertilisation). Des simulations de microdosimétrie ont également été réalisées en utilisant les calculs Monte Carlo sur Geant4-DNA afin de représenter la répartition cellulaire des dépôts d'énergie. Les résultats expérimentaux ont montré que le tritium était majoritairement internalisé sous forme organique, et que son internalisation augmentait exponentiellement avec l'activité externe jusqu'à atteindre un point de saturation à environ 250 kBq/mL chez les œufs et les larves, respectivement. Les simulations de microdosimétrie ont montré que l'énergie moyenne déposée par le tritium augmentait linéairement avec le rayon de la cellule. Les simulations ont également mis en avant que les cellules aux plus faibles rayons étaient plus à risque d'être la cible des électrons de faible énergie. Ces électrons réalisant des dépôts d'énergie plus proches les uns des autres, un plus grand risque biologique est également estimé dans le cas des cellules de plus faible rayon. Le développement, le comportement natatoire, l'intégrité de l'ADN et l'expression génique de certains gènes d'intérêt sont en cours d'étude chez les individus exposés à de la thymidine tritiée à des débits de dose de 8 et 40 kBq/mL pendant 1 à 4 jours

Antiproliferative and palliative activity of flavonoids in colorectal cancer

Flavonoids are plant bioactive compounds of great interest in nutrition and pharmacology, due to their remarkable properties as antioxidant, anti-inflammatory, antibacterial, antifungal and antitumor drugs. More than 5000 different flavonoids exist in nature, with a huge structural diversity and a plethora of interesting pharmacological properties. In this work, five flavonoids were tested for their potential use as antitumor drugs against three CRC cell lines (HCT116, HT-29 and T84). These cell lines represent three different stages of this tumor, one of which is metastatic. Xanthohumol showed the best antitumor activity on the three cancer cell lines, even better than that of the clinical drug 5-fluorouracil (5-FU), although no synergistic effect was observed in the combination therapy with this drug. On the other hand, apigenin and luteolin displayed slightly lower antitumor activities on these cancer cell lines but showed a synergistic effect in combination with 5-FU in the case of HTC116, which is of potential clinical interest. Furthermore, a literature review highlighted that these flavonoids show very interesting palliative effects on clinical symptoms such as diarrhea, mucositis, neuropathic pain and others often associated with the chemotherapy treatment of CRC. Flavonoids could provide a double effect for the combination treatment, potentiating the antitumor effect of 5-FU, and simultaneously, preventing important side effects of 5-FU chemotherapy

Translational activation by the noncoding RNA DsrA involves alternative RNase III processing in the rpoS 5′-leader

The intricate regulation of the Escherichia coli rpoS gene, which encodes the stationary phase sigma-factor σS, includes translational activation by the noncoding RNA DsrA. We observed that the stability of rpoS mRNA, and concomitantly the concentration of σS, were significantly higher in an RNase III-deficient mutant. As no decay intermediates corresponding to the in vitro mapped RNase III cleavage site in the rpoS leader could be detected in vivo, the initial RNase III cleavage appears to be decisive for the observed rapid inactivation of rpoS mRNA. In contrast, we show that base-pairing of DsrA with the rpoS leader creates an alternative RNase III cleavage site within the rpoS/DsrA duplex. This study provides new insights into regulation by small regulatory RNAs in that the molecular function of DsrA not only facilitates ribosome loading on rpoS mRNA, but additionally involves an alternative processing of the target