RNA and β-Hemolysin of Group B Streptococcus Induce Interleukin-1β (IL-1β) by Activating NLRP3 Inflammasomes in Mouse Macrophages

The inflammatory cytokine IL-1β is critical for host responses against many human pathogens. Here, we define Group B Streptococcus (GBS)-mediated activation of the Nod-like receptor-P3 (NLRP3) inflammasome in macrophages. NLRP3 activation requires GBS expression of the cytolytic toxin, β-hemolysin, lysosomal acidification, and leakage. These processes allow the interaction of GBS RNA with cytosolic NLRP3. The present study supports a model in which GBS RNA, along with lysosomal components including cathepsins, leaks out of lysosomes and interacts with NLRP3 to induce IL-1β production.     

Food availability affects circadian clock-controlled activity and Zugunruhe in the night migratory male blackheaded bunting (Emberiza melanocephala)

This study investigated the functional linkage between food availability and activity behavior in the Palaearctic Indian night migratory blackheaded bunting (Emberiza melanocephala) subjected to artificial light-dark (LD) cycles. Two experiments were performed on photosensitive birds. In the first one, birds were exposed to short days (LD 10/14; Experiment 1A), long days (LD 13/11; Experiment 1B), or increasing daylengths (8 to 13?h light/d; Experiment 1C) and presented with food either for the whole or a restricted duration of the light period. In Experiments 1A and 1B, illumination of the light and dark periods or of the dark period, alone, was changed to assess the influence of the light environment on direct and circadian responses to food cycles. In the second experiment, birds were exposed to LD 12/12 or LD 8/16 with food availability overlapping with the light (light and food presence in phase) or dark period (light and food presence in antiphase). Also, birds were subjected to constant dim light (LL(dim)) to examine the phase of the activity rhythms under synchronizing influence of the food cycles. Similarly, the presentation of food ad libitum (free food; FF) during an experiment examined the effects of the food-restriction regimes on activity rhythms. A continuous measurement of the activity-rest pattern was done to examine both the circadian and direct effects of the food and LD cycles. Measurement of activity at night enabled assessment of the migratory phenotype, premigratory restlessness, or Zugunruhe. The results show that (i) light masked the food effects if they were present together; (ii) birds had a higher anticipatory activity and food intake during restricted feeding conditions; and (iii) food at night alone reduced both the duration and amount of Zugunruhe as compared to food during the day alone. This suggests that food affects both the daily activity and seasonal Zugunruhe, and food cycles act as a synchronizer of circadian rhythms in the absence of dominant natural environmental synchronizers, such as the light-dark cycle.     

Molecular Studies on the Microbial Diversity Associated with Mining-Impacted Coeur d’Alene River Sediments

Mass mortalities of larval cultures of Chilean scallop Argopecten purpuratus have repeatedly occurred in northern Chile, characterized by larval agglutination and accumulation in the bottom of rearing tanks. The exopolysaccharide slime (EPS) producing CAM2 strain was isolated as the primary organism from moribund larvae in a pathogenic outbreak occurring in a commercial hatchery producing larvae of the Chilean scallop Argopecten purpuratus located in Bahía Inglesa, Chile. The CAM2 strain was characterized biochemically and was identified by polymerase chain reaction amplification of 16S rRNA as Halomonas sp. (Accession number DQ885389.1). Healthy 7-day-old scallop larvae cultures were experimentally infected for a 48-h period with an overnight culture of the CAM2 strain at a final concentration of ca. 10⁵ cells per milliliter, and the mortality and vital condition of larvae were determined by optical and scanning electron microscopy (SEM) to describe the chronology of the disease. Pathogenic action of the CAM2 strain was clearly evidenced by SEM analysis, showing a high ability to adhere and detach larvae velum cells by using its “slimy” EPS, producing agglutination, loss of motility, and a posterior sinking of scallop larvae. After 48 h, a dense bacterial slime on the shell surface was observed, producing high percentages of larval agglutination (63.28 ± 7.87%) and mortality (45.03 ± 4.32%) that were significantly (P < 0.05) higher than those of the unchallenged control cultures, which exhibited only 3.20 ± 1.40% dead larvae and no larval agglutination. Furthermore, the CAM2 strain exhibited a high ability to adhere to fiberglass pieces of tanks used for scallop larvae rearing (1.64 × 10⁵ cells adhered per square millimeters at 24 h postinoculation), making it very difficult to eradicate it from the culture systems. This is the first report of a pathogenic activity on scallop larvae of Halomonas species, and it prompts the necessity of an appraisal on biofilm-producing bacteria in Chilean scallop hatcheries.     

Simultaneous and comparative assessment of parent and mutant strain of Rhizobium meliloti for nutrient limitation and enhanced polyhydroxyalkanoate (PHA) production using optimization studies

Nutrient limitation conditions, optimization and comparison of polyhydroxyalkanoate (PHA) yields and biomass production by parent and mutant strains of Rhizobium meliloti were investigated. Complex interactions among concentrations of sucrose (5–55 g/l), urea (0.05–0.65 g/l) inoculum (10–250 ml/l) and K₂HPO₄ (0.5–2 g/l), were studied using central composite rotatable design (CCRD) experiments. Phosphate-limiting medium (0.33 g K₂HPO₄/l) in the presence of excess carbon (sucrose 42.5 g/l) results in more production of PHA (2.2 g/l) in the parent strain. In comparison, the mutant strain required moderate levels of sucrose (30 g/l), along with excess of phosphate (1 g/l) for high PHA content of cell biomass (80%) and PHA yield (3.3 g/l). Optimised PHA production (biomass 4.8 g/l and PHA 3.09 g/l) by the parent strain occurred at: sucrose 51.58 g/l, urea 0.65 g/l, K₂HPO₄ 0.48 g/l and inoculum 10 ml/l. In the mutant strain, higher yields of biomass (9.05 g/l) and PHA (5.66 g/l) were obtained in Optimised medium containing: sucrose 55 g/l, urea 0.65 g/l, K₂HPO₄ 1.0 g/l and inoculum 150.58 ml/l.     

A data-mining approach to spacer oligonucleotide typing of Mycobacterium tuberculosis

MOTIVATION: The Direct Repeat (DR) locus of Mycobacterium tuberculosis is a suitable model to study (i) molecular epidemiology and (ii) the evolutionary genetics of tuberculosis. This is achieved by a DNA analysis technique (genotyping), called sp acer oligo nucleotide typing (spoligotyping ). In this paper, we investigated data analysis methods to discover intelligible knowledge rules from spoligotyping, that has not yet been applied on such representation. This processing was achieved by applying the C4.5 induction algorithm and knowledge rules were produced. Finally, a Prototype Selection (PS) procedure was applied to eliminate noisy data. This both simplified decision rules, as well as the number of spacers to be tested to solve classification tasks. In the second part of this paper, the contribution of 25 new additional spacers and the knowledge rules inferred were studied from a machine learning point of view. From a statistical point of view, the correlations between spacers were analyzed and suggested that both negative and positive ones may be related to potential structural constraints within the DR locus that may shape its evolution directly or indirectly. RESULTS: By generating knowledge rules induced from decision trees, it was shown that not only the expert knowledge may be modeled but also improved and simplified to solve automatic classification tasks on unknown patterns. A practical consequence of this study may be a simplification of the spoligotyping technique, resulting in a reduction of the experimental constraints and an increase in the number of samples processed.     

A spectrophotometric method to monitor the catalytic activity of microsomal cytochrome P-450 IIB1/2: comparison with fluorometric assay

A simple spectrophotometric method to monitor the catalytic activity of microsomal cytochrome P-450 IIB1/2 has been developed. The method employs measurement of utilization of NADPH, consumption of the substrate, pentoxyresorufin (PRF) and formation of the product, resorufin (RF) in the same reaction mixture containing hepatic microsomes from phenobarbital treated rats. The velocity of NADPH utilization (16.36 nmole/min/nmole P-450), PRF consumption (1.58 nmole/min/nmole P-450) and RF formation (1.57 nmole/min/nmole P-450) suggested a stoichiometry of 1:1 between the substrate and the product alongwith utilization of 10 molecules of NADPH. However, the Km for the enzyme activity (nmole RF formed/min/nmole P-450) using varying concentrations of PRF and NADPH as substrates were found to be 11.6 and 20.2 microM, respectively. The spectrophotometric method was compared with fluorometric method in terms of linearity with time, P-450 content and Vmax, Km values observed for the reaction. Inhibition studies with metyrapone and SKF 525A in the utilization of NADPH, consumption of PRF and formation of RF suggested that the method could be useful in monitoring the effect of various inhibitors on the P-450 IIB1/2 reaction.     

Selective toxicity of engineered lentivirus lytic peptides in a CF airway cell model

Lentivirus lytic peptides (LLPs) are derived from HIV-1 and have antibacterial properties. LLP derivatives (eLLPs) were engineered for greater potency against Staphylococcus aureus (SA) and Pseudomonas aeruginosa (PA). Minimum bactericidal concentration (MBC) was determined in low and physiologic salt concentrations. MBC was decreased against SA and equivalent against PA in physiologic salt when compared to the parent compound LLP1. In a novel cystic fibrosis (CF) airway cell model, one derivative, WLSA5, reduced the number of adherent PA and only moderately affected CF cell viability. Overall, eLLPs are selectively toxic to bacteria and may be useful against CF airway infections.     

New alkaloids from Glycosmis mauritiana

Chemical investigation of the roots of G. mauritiana resulted in the isolation of two new alkaloids; 1-hydroxy-3-methoxy-2-(3-methylbut-2-enyl)-N-methylacridan-9-one (1) and 4,8-dimethoxy-3-(3-methylbut-2-enyl)-N-methyl-2-quinolone (6). The structures of these new bases have been established by chemical and spectroscopic methods and confirmed in the case of 6 by its synthesis. Interestingly, the formic acid-catalysed cyclisation of 1 gave the dealkylated product 3 along with the pyrano-[2, 3-a]-acridine (4).