Hydrogen sulfide induces direct radical-associated DNA damage

Hydrogen sulfide (H(2)S) is produced by indigenous sulfate-reducing bacteria in the large intestine and represents an environmental insult to the colonic epithelium. Clinical studies have linked the presence of either sulfate-reducing bacteria or H(2)S in the colon with chronic disorders such as ulcerative colitis and colorectal cancer, although at this point, the evidence is circumstantial and underlying mechanisms remain undefined. We showed previously that sulfide at concentrations similar to those found in the human colon induced genomic DNA damage in mammalian cells. The present study addressed the nature of the DNA damage by determining if sulfide is directly genotoxic or if genotoxicity requires cellular metabolism. We also questioned if sulfide genotoxicity is mediated by free radicals and if DNA base oxidation is involved. Naked nuclei from untreated Chinese hamster ovary cells were treated with sulfide; DNA damage was induced by concentrations as low as 1 micromol/L. This damage was effectively quenched by cotreatment with butylhydroxyanisole. Furthermore, sulfide treatment increased the number of oxidized bases recognized by formamidopyrimidine [fapy]-DNA glycosylase. These results confirm the genotoxicity of sulfide and strongly implicate that this genotoxicity is mediated by free radicals. These observations highlight the possible role of sulfide as an environmental insult that, given a predisposing genetic background, may lead to genomic instability or the cumulative mutations characteristic of colorectal cancer.     

CRISPR/Cas9-Mediated Transgenesis of the Masu Salmon (Oncorhynchus masou) elovl2 Gene Improves n-3 Fatty Acid Content in Channel Catfish (Ictalurus punctatus)

Marine Biotechnology - Omega-3 polyunsaturated fatty acids (n-3 PUFAs), particularly eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3), play a very important role in...     

Ecology,conservation, and phylogenetic position of the Madagascar Jacana Actophilornis albinucha

The Madagascar Jacana Actophilornis albinucha (Jacanidae) is an endemic shorebird found in the threatened wetlands of western Madagascar. This species is presumed to exhibit classical polyandry; however, few data are available to support that assumption. More generally, a lack of basic understanding of this species hinders conservation efforts. We conducted the most extensive study of the Madagascar Jacana to date, and report on its: 1) distribution, population size and density; 2) degree of sexual size dimorphism; and 3) phylogenetic position. The surveys were conducted at 54 lakes, between January and October in 2016. Madagascar Jacana were found at 22 lakes, and within these were distributed at a mean density of 3.5 ± 0.74 [SE] individuals per hectare of surveyed habitat. We estimate the global population size to be between 975 and 2 064 individuals, and habitat destruction appears to be the main threat to the species. Females were significantly larger than males, consistent with reports for other Jacanidae species. Using a mitochondrial DNA fragment, we expanded the Jacanidae genetic phylogeny, and confirmed that Madagascar Jacana is the sister species to the African Jacana Actophilornis africanus. Further studies are urgently needed to thoroughly re-assess the threat status and population trend of the Madagascar Jacana.     

p53-Independent and -Dependent Requirements for E1B-55K in Adenovirus Type 5 Replication

The adenovirus type 5 mutant dl1520 was engineered previously to be completely defective for E1B-55K functions. Recently, this mutant (also known as ONYX-015) has been suggested to replicate preferentially in p53(-) and some p53(+) tumor cell lines but to be attenuated in primary cultured cells (C. Heise, A. Sampson-Johannes, A. Williams, F. McCormick, D. D. F. Hoff, and D. H. Kirn, Nat. Med. 3:639-645, 1997). It has been suggested that dl1520 might be used as a "magic bullet" that could selectively lyse tumor cells without harm to normal tissues. However, we report here that dl1520 replication is independent of p53 genotype and occurs efficiently in some primary cultured human cells, indicating that the mutant virus does not possess a tumor selectivity. Although it was not the sole host range determinant, p53 function did reduce dl1520 replication when analyzed in a cell line expressing temperature-sensitive p53 (H1299-tsp53) (K. L. Fries, W. E. Miller, and N. Raab-Traub, J. Virol. 70:8653-8659, 1996). As found earlier for other E1B-55K mutants in HeLa cells (Y. Ho, R. Galos, and J. Williams, Virology 122:109-124, 1982), dl1520 replication was temperature dependent in H1299 cells. When p53 function was restored at low temperature in H1299-tsp53 cells, it imposed a modest defect in viral DNA replication and accumulation of late viral cytoplasmic mRNA. However, in both H1299 and H1299-tsp53 cells, the defect in late viral protein synthesis appeared to be much greater than could be accounted for by the modest defects in late viral mRNA levels. We therefore propose that in addition to countering p53 function and modulating viral and cellular mRNA nuclear transport, E1B-55K also stimulates late viral mRNA translation.     

Quantitative data on training new world primates to urinate

This study assessed the effectiveness of operant conditioning in training three species of captive callitrichid primates (Leontopithecus rosalia, Callithrix geoffroyi, and Saguinus imperator) to urinate on demand. There were three goals to the study: 1) to develop a system for quantitatively assessing positive reinforcement training; 2) to ascertain whether or not positive reinforcement techniques can be used to train callitrichid monkeys to urinate on demand, and if so, how many training sessions are required; and 3) to determine the effect on urination behavior of the trainer entering the cage to collect a urine sample. Positive reinforcement with a continuous reinforcement schedule was used to capture a natural behavior: urination. Training sessions (30 min each) were conducted at dawn thrice weekly during five consecutive phases: habituation, control, training (animals were rewarded for urinating), maintenance (animals had reached a defined training criteria and continued to be rewarded for urinating), and collection (animals were rewarded for urinating, and the trainer entered the cage to collect the sample). The numbers of 30-min training sessions required to train the three monkey species (L. rosalia, C. geoffroyi, and S. imperator) were five, six, and eight, respectively. For the three species, the mean number of urinations per animal was significantly greater during the training, maintenance, and collection phases compared to the control phase. However, the three species differed significantly in the manner in which the rates of urination changed across the five phases. A higher proportion of subjects urinated during the training, maintenance, and collection phases compared to the control phase. Latency to first urination varied significantly across the five phases, with significantly reduced latencies to urinate during the training, maintenance, and collection phases compared to the control phase. The entry of the trainer into the cage to collect the urine sample did not appear to alter urination behavior. We demonstrate that operant conditioning techniques, which typically incur minimal cost, time investment, and disturbance, can be used to increase the quantity of urine samples collected for physiological analysis, the proportion of animals that urinate, and the speed of sample collection.     

A mammalian artificial chromosome engineering system (ACE System) applicable to biopharmaceutical protein production, transgenesis and gene-based cell therapy

Mammalian artificial chromosomes (MACs) provide a means to introduce large payloads of genetic information into the cell in an autonomously replicating, non-integrating format. Unique among MACs, the mammalian satellite DNA-based Artificial Chromosome Expression (ACE) can be reproducibly generated de novo in cell lines of different species and readily purified from the host cells' chromosomes. Purified mammalian ACEs can then be re-introduced into a variety of recipient cell lines where they have been stably maintained for extended periods in the absence of selective pressure. In order to extend the utility of ACEs, we have established the ACE System, a versatile and flexible platform for the reliable engineering of ACEs. The ACE System includes a Platform ACE, containing >50 recombination acceptor sites, that can carry single or multiple copies of genes of interest using specially designed targeting vectors (ATV) and a site-specific integrase (ACE Integrase). Using this approach, specific loading of one or two gene targets has been achieved in LMTK⁻ and CHO cells. The use of the ACE System for biological engineering of eukaryotic cells, including mammalian cells, with applications in biopharmaceutical production, transgenesis and gene-based cell therapy is discussed.     

Type I IFN-beta gene therapy suppresses cardiac CD8+ T-cell infiltration during autoimmune myocarditis

Gene therapy using DNA encoding type I IFN subtypes IFNA6, IFNA9 and IFNB suppresses murine cytomegalovirus (MCMV)-myocarditis, a predominantly cell-mediated disease in BALB/c mice. CD8(+) T cells are the principal cell type within the inflamed myocardium. As such, we investigated the effects of IFN subtype treatment on this T-cell subset and other cell types in the cardiac infiltrate. In the acute phase of disease, IFNA6 and IFNA9 treatments significantly reduced the number of CD8(+) T cells within the foci of cellular infiltration in the heart. During the chronic phase, which is primarily autoimmune in nature, IFNB treatment significantly reduced CD8(+) T cells. B-cell and neutrophil numbers in the cardiac infiltrate were also reduced following IFNB immunotherapy. Although early inflammatory responses are important for resolution of virus infection, high numbers of lymphocytes persisting in the myocardium may lead to exacerbation of disease. Our data suggests that type I IFN DNA therapy regulates cardiac cellular infiltration. Thus, treatment with IFN-beta administered prophylactically to high-risk patients in acquiring CMV infection may reduce the development of chronic autoimmune myocarditis.     

Determination of the absolute configuration of Anisotome irregular diterpenes: application of CD and NMR methods

Several Anisotome diterpene derivatives were synthesized in an attempt to obtain a crystalline compound for X-ray analysis. Although we were unable to obtain a suitable crystal, the absolute configuration of the irregular diterpene skeleton was determined using two other techniques: a circular dichroism (CD) protocol based on a tetraarylporphyrin molecular tweezer that allowed prediction of the absolute stereochemistry on a microscale level, and a method employing differences in NMR shifts from derivatization of the naturally occurring acid 1 with enantiomers of a phenylglycine methyl ester (PGME) chiral anisotropic reagent. The excellent agreement between the CD and NMR methods led to the assignment of a 2S-absolute configuration for anisotomenoic acid 1.