Myocardial infarction and intramyocardial injection models in swine

Sustainable and reproducible large animal models that closely replicate the clinical sequelae of myocardial infarction (MI) are important for the translation of basic science research into bedside medicine. Swine are well accepted by the scientific community for cardiovascular research, and they represent an established animal model for preclinical trials for US Food and Drug Administration (FDA) approval of novel therapies. Here we present a protocol for using porcine models of MI created with a closed-chest coronary artery occlusion-reperfusion technique. This creates a model of MI encompassing the anteroapical, lateral and septal walls of the left ventricle. This model infarction can be easily adapted to suit individual study design and enables the investigation of a variety of possible interventions. This model is therefore a useful tool for translational research into the pathophysiology of ventricular remodeling and is an ideal testing platform for novel biological approaches targeting regenerative medicine. This model can be created in approximately 8-10 h.     

Evidence of field-evolved resistance to organophosphates and pyrethroids in Chrysoperla carnea (Neuroptera: Chrysopidae)

The toxicity of some of the most commonly used insecticides in the organophosphate and pyrethroid classes were investigated against different Chrysoperla carnea (Stephens) (Neuroptera: Chrysopidae) populations collected over three consecutive years (2005-2007). The populations were tested using leaf dip bioassays for residual effects and topical applications to measure the response of larvae that would come into direct contact with field application of insecticides. In leaf dip assays, the LC50 (micrograms per milliliter; 120 h) values for chlorpyrifos and profenofos were in the range of 59.3-1,023 and 180.02-1,118 respectively. The LC50 values for lambda-cyhalthrin, alphamethrin, and deltamethrin were 359.08-2,677, 112.9-923.5, and 47.81-407.03, respectively. The toxicity for the above insecticides in topical application was similar to toxicity in leaf dip assays. The susceptibility of a laboratory population, which was locally developed and designated as (Lab-PK), to deltamethrin was comparable with another susceptible laboratory population. Resistance ratios for five field populations were generally low to medium for deltamethrin, but high to very high for chlorpyrifos, profenofos, lambda-cyhalthrin and alphamethrin compared with the Lab-PK population. Our data also suggested that the five field populations had multiple resistance to two classes of insecticides. The populations showed resistance to two organophosphates tested and to lambda-cyhalthrin and alphamethrin; however, resistance to deltamethrin was only found at two locations. This pattern indicates occurrence of two divergent patterns of resistance within pyrethroids. The resistance to the insecticides was stable across 3 yr, suggesting field selection for general fitness had also taken place in various populations of C. carnea. The broad spectrum of resistance and stability of resistance to insecticides in C. carnea in the current study suggested that it could be a prime candidate for mass releases and compatible with most spray programs.     

Cross-resistance and genetics of resistance to indoxacarb in Spodoptera litura (Lepidoptera: Noctuidae)

Bioassays (at generation G1) with a newly collected field population of Spodoptera litura (F.) (Lepidoptera: Noctuidae) from Multan, Pakistan, showed resistance ratios of 15, 23, 37, and 16 for indoxacarb, spinosad, abamectin, and emamectin, respectively, compared with a laboratory susceptible population, Lab-PK. At G1, the field population was selected with indoxacarb by using single pair crosses. For Indoxa-SEL, bioassay at G4 found that selection increased resistance ratio to 95 for indoxacarb compared with Lab-PK. Selection with indoxacarb significantly increased resistance to spinosad and emamectin; however, resistance to abamectin was observed to drop. A significant reduction in the resistance to indoxacarb was observed in Indoxa-SEL at G9, indicating unstable resistance to indoxacarb; however, it was stable for fipronil. Synergism tests with microsomal oxidase and esterase-specific inhibitors suggested that the indoxacarb resistance was associated with microsomal oxidase. Reciprocal genetic crosses between Indoxa-SEL and Lab-PK populations indicated that resistance was autosomal and incompletely dominant. Tests of monogenic inheritance suggested that resistance to indoxacarb was controlled by more than one locus.     

The Mating System in Saccharomyces

Many, but not all, Saccharomyces species are heterothallic.The mating types in heterothallic species are determined bytwo allelic genes. The mating-type genes occasionally mutatefrom one to the other. Vegetative cells of opposite mating typewere found in some single ascospore colonies. They show conjugationtubes, stimulated by the proximity of cells of the other matingtype. The cultures alao show zygotes, which on sporulation yieldplus(+) and minus(–) progenies. The zygotes bud off diploid vegetative cells which grow fasterthan the original haploid cells and so tend to replace them.If mutation occurs early, replacement is complete and the culturegives no mating reaction but sporulates. If it occurs late,the culture is a mixture of haploid cells giving a mating reactionand diploid cells that will sporulate.     

DNA photolyase of enterococci: possible explanation for its low sunlight inactivation rate

DNA photolyase is perhaps the most ancient and direct arsenal in curing the UV-induced dimers formed in the microbial genome. Out of two cofactors of the enzyme, catalytic and light harvesting, differences in the latter have provided basis for categorizing photolyases of prokaryotes as folate and deazaflavin types. In the present study, the homology modeling of DNA photolyase of Enterococcus faecalis was undertaken. The predicted models were structurally compared with the crystal structure coordinates of photolyases from Escherichia coli (folate type) and Anacystis nidulans (deazaflavin type). Discrepancies present in the multiple sequence alignment and tertiary structures, particularly at the light harvesting cofactor (methenyltetrahydrofolic acid, MTHF; 8-hydroxy-5-deazaflavin, 8-HDF) binding sites indicated the mechanistic nature of enterococcal photolyase. Concisely, despite the greater holistic homology with folate-type photolyase, enterococcal photolyase was characterized as deazaflavin-type. The presence of 8-HDF binding sites and groove architecture of substrate binding sites were also found supportive in this regard. The inter cofactor distance and/or orientation also implied to the efficient energy transfer in photolyase of Enterococcus in comparison with E. coli. In addition, we observed relatively high protein deformability in the enterococcal genome, which may favors the repair action of photolyase. The findings are expected to provide molecular insights into the difference in sunlight inactivation rate of two important fecal contamination indicators, namely Enterococcus and E. coli.     

Genome-Wide Relatedness of Treponema pedis,from Gingiva and Necrotic Skin Lesions of Pigs,with the Human Oral Pathogen Treponema denticola

Treponema pedis and T. denticola are two genetically related species with different origins of isolation. Treponema denticola is part of the human oral microbiota and is associated with periodontitis while T. pedis has been isolated from skin lesions in animals, e.g., digital dermatitis in cattle and necrotic ulcers in pigs. Although multiple Treponema phylotypes may exist in ulcerative lesions in pigs, T. pedis appears to be a predominant spirochete in these lesions. Treponema pedis can also be present in pig gingiva. In this study, we determined the complete genome sequence of T. pedis strain T A4, isolated from a porcine necrotic ear lesion, and compared its genome with that of T. denticola. Most genes in T. pedis were homologous to those in T. denticola and the two species were similar in general genomic features such as size, G+C content, and number of genes. In addition, many homologues of specific virulence-related genes in T. denticola were found in T. pedis. Comparing a selected pair of strains will usually not give a complete picture of the relatedness between two species. We therefore complemented the analysis with draft genomes from six T. pedis isolates, originating from gingiva and necrotic ulcers in pigs, and from twelve T. denticola strains. Each strain carried a considerable amount of accessory genetic material, of which a large part was strain specific. There was also extensive sequence variability in putative virulence-related genes between strains belonging to the same species. Signs of lateral gene-transfer events from bacteria known to colonize oral environments were found. This suggests that the oral cavity is an important habitat for T. pedis. In summary, we found extensive genomic similarities between T. pedis and T. denticola but also large variability within each species.     

The Interactive Approach of Rhizobacteria and l-tryptophan on Growth,Physiology, Tuber Characteristics,and Iron Concentration of Potato (Solanum tuberosum L.)

Iron deficiency is one of the most prevailing micronutrient deficiencies throughout the globe. Iron malnutrition affects billions of people around the world especially children and pregnant women. Its deficiencies can be overcome through microbial biofortification: a process of deliberately increasing desirable nutrients in crop plants. Plant growth-promoting rhizobacteria (PGPR) can improve iron content in edible plant tissues through different direct and indirect mechanisms. Adding plant growth regulators along with rhizobacteria makes it a novel fortification approach. In the current experiment, the interactive effect of two bacterial isolates (O-13 & K-10) alone and in consortium with l-tryptophan in the presence of iron sulfate was evaluated on growth, physiology, tuber characteristics, and iron concentration in potato (Solanum tuberosum L.). Results revealed that inoculation with PGPR and plant growth regulator (PGR) significantly improved the plant height, straw yield, and the number of tubers per plant. Potato (Solanum tuberosum L.) tuber characteristics (starch content, vitamin-C, relative water content) were also improved significantly. O-13, K-10, and l-tryptophan had significantly improved the iron concentration up to 20.59, 33.12, and 28.95%, respectively. However, inoculation with the microbial consortium and l-tryptophan showed a significant increase of up to one-fold in the iron concentration of potato (Solanum tuberosum L.) as compared with uninoculated control. The results suggest that rhizobacteria can help the plant to uptake nutrients from the soil. These findings concluded on the fact that the interactive effect of microbial assisted biofortification and plant growth regulator is a novel, promising, and cost-effective approach to mitigate micronutrient deficiencies especially in resource-limited countries.

     

Sequence Analysis and Comparative Bioinformatics Study of Camelysin Gene (<Emphasis Type="Italic">calY</Emphasis>) Isolated from <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis>

Bacillus strains have been widely used for the production of fibrinolytic enzymes having role in the treatment of cardiovascular disorders. Purification and overproduction of such enzymes has increased their usage in medical fields including metalloproteinases with the ability to degrade extracellular matrix (ECM). Camelysin, a neutral metalloproteinase has been isolated from different species of bacteria like Bacillus cereus, Bacillus anthracis, and Bacillus thuringiensis with fibrinolytic, collagenolytic and actin degradation activity. This project successfully demonstrated the presence of 734-bp coding DNA sequence (CDS) encoding a 20.72331 kDa camelysin gene in local strain of Bacillus thuringiensis containing a signal peptide with cleavage site between residues 19 and 20. The sequence was submitted to GenBank (KT023597) and the sequence showed high homology with the camelysin protein of closely related Bacillus species. The alignment of related proteins through ClustalW displayed difference of four amino acids (“Q” replaced by “P” at position 169 and at position 182–184, “NQE” replaced by “HLK”) in the isolated protein. Comparison including structural and functional analysis of camelysin sequences isolated from different Bacillus species was carried out using different bioinformatics tools and software. The information would help in better understanding the properties of camelysin protein and its role in pathogenicity and clinical treatments.