A Multinational Analysis of Mutations and Heterogeneity in PZase, RpsA, and PanD Associated with Pyrazinamide Resistance in M/XDR Mycobacterium tuberculosis.

Pyrazinamide (PZA) is an important first-line drug in all existing and new tuberculosis (TB) treatment regimens. PZA-resistance in M. tuberculosis is increasing, especially among M/XDR cases. Noted issues with PZA Drug Susceptibility Testing (DST) have driven the search for alternative tests. This study provides a comprehensive assessment of PZA molecular diagnostics in M/XDR TB cases. A set of 296, mostly XDR, clinical M. tuberculosis isolates from four countries were subjected to DST for eight drugs, confirmatory Wayne's assay, and whole-genome sequencing. Three genes implicated in PZA resistance, pncA, rpsA, and panD were investigated. Assuming all non-synonymous mutations cause resistance, we report 90% sensitivity and 65% specificity for a pncA-based molecular test. The addition of rpsA and panD potentially provides 2% increase in sensitivity. Molecular heterogeneity in pncA was associated with resistance and should be evaluated as a diagnostic tool. Mutations near the N-terminus and C-terminus of PZase were associated with East-Asian and Euro-American lineages, respectively. Finally, Euro-American isolates are most likely to have a wild-type PZase and escape molecular detection. Overall, the 8-10% resistance without markers may point to alternative mechanisms of resistance. Confirmatory mutagenesis may improve the disconcertingly low specificity but reduce sensitivity since not all mutations may cause resistance

Construction Cost Sensitivity of a Lignocellulosic Ethanol Biorefinery

The technology has been developed to convert feedstock with cellulose content into ethanol. However, ethanol produced from cellulosic feedstock is the same as ethanol distilled from grain. The objective of research is to determine the price per gallon of ethanol needed so that producing lignocellulosic based ethanol become economically feasible.Environmental Economics and Policy, Production Economics,

Effects of Second Implant on Feedlot Gain and Carcass Traits

Two hundred eighteen steers were finished in a total confinement deep-bedded system at the Armstrong Research Farm, Lewis, IA during 2009. All steers were implanted with Synovex-Choice on day 1 and half the steers in each pen were implanted with Synovex-Choice on day 56. All steers were harvested on day 118. The 2nd implant resulted in an immediate and significant improvement in average daily gain. In the 76 day weigh period following reimplantation the group receiving the 2nd implant gained .66 lb/day more than the group not receiving an additional implant. The overall average daily gain of steers implanted once compared to the steers implanted twice was 3.81 vs. 4.10. The 2nd implant group produced significantly heavier carcasses. There were no significant differences in carcass fat cover or ribeye area. The twice implanted steers had a lower percentage low Choice or better (P=.0571) and a greater percentage Select (P=.0555). Implanting a second time resulted in an increase in carcass weights, an almost significant reduction in % Choice but still resulted in a numerical, non-significant increase in carcass value

Gene doctoring: a method for recombineering in laboratory and pathogenic Escherichia coli strains

Background: Homologous recombination mediated by the lambda-Red genes is a common method for making chromosomal modifications in Escherichia coli. Several protocols have been developed that differ in the mechanisms by which DNA, carrying regions homologous to the chromosome, are delivered into the cell. A common technique is to electroporate linear DNA fragments into cells. Alternatively, DNA fragments are generated in vivo by digestion of a donor plasmid with a nuclease that does not cleave the host genome. In both cases the lambda-Red gene products recombine homologous regions carried on the linear DNA fragments with the chromosome. We have successfully used both techniques to generate chromosomal mutations in E. coli K-12 strains. However, we have had limited success with these lambda-Red based recombination techniques in pathogenic E. coli strains, which has led us to develop an enhanced protocol for recombineering in such strains. \ud \ud Results: Our goal was to develop a high-throughput recombineering system, primarily for the coupling of genes to epitope tags, which could also be used for deletion of genes in both pathogenic and K-12 E. coli strains. To that end we have designed a series of donor plasmids for use with the lambda-Red recombination system, which when cleaved in vivo by the I-SceI meganuclease generate a discrete linear DNA fragment, allowing for C-terminal tagging of chromosomal genes with a 6xHis, 3xFLAG, 4xProteinA or GFP tag or for the deletion of chromosomal regions. We have enhanced existing protocols and technologies by inclusion of a cassette conferring kanamycin resistance and, crucially, by including the sacB gene on the donor plasmid, so that all but true recombinants are counter-selected on kanamycin and sucrose containing media, thus eliminating the need for extensive screening. This method has the added advantage of limiting the exposure of cells to the potential damaging effects of the lambda-Red system, which can lead to unwanted secondary alterations to the chromosome. \ud \ud Conclusion: We have developed a counter-selective recombineering technique for epitope tagging or for deleting genes in E. coli. We have demonstrated the versatility of the technique by modifying the chromosome of the enterohaemorrhagic O157:H7 (EHEC), uropathogenic CFT073 (UPEC), enteroaggregative O42 (EAEC) and enterotoxigenic H10407 (ETEC) E. coli strains as well as in K-12 laboratory strains

Hemoglobin genotype has minimal influence on the physiological response of juvenile atlantic cod (Gadus morhua) to environmental challenges

Hemoglobin (Hb) polymorphism in cod is associated with temperature‐related differences in biogeographical distribution, and several authors have suggested that functional characteristics of the various hemoglobin isoforms (HbIs) directly influence phenotypic traits such as growth rate. However, no study has directly examined whether Hb genotype translates into physiological differences at the whole animal level. Thus, we generated a family of juvenile Atlantic cod consisting of all three main Hb genotypes (HbI‐1/1, HbI‐2/2, and HbI‐1/2) by crossing a single pair of heterozygous parents, and we compared their metabolic and cortisol responses to an acute thermal challenge (10&deg;C to their critical thermal maximum [CTM] or 22&deg;C, respectively) and tolerance of graded hypoxia. There were no differences in routine metabolism (at 10&deg;C), maximum metabolic rate, metabolic scope, CTM (overall mean 22.9&deg; &plusmn; 0.2&deg;C), or resting and poststress plasma cortisol levels among Hb genotypes. Further, although the HbI‐1/1 fish grew more (by 15%&ndash;30% during the first 9 mo) when reared at 10&deg; &plusmn; 1&deg;C and had a slightly enhanced hypoxia tolerance at 10&deg;C (e.g., the critical O2 levels for HbI‐1/1, HbI‐2/2, and HbI‐1/2 cod were 35.56% &plusmn; 1.24%, and 40.20% &plusmn; 1.99% air saturation, respectively), these results are contradictory to expectations based on HbI functional properties. Thus, our findings (1) do not support previous assumptions that growth rate differences among cod Hb genotypes result from a more efficient use of the oxygen supply&mdash;that is, reduced standard metabolic rates and/or increased metabolic capacity&mdash;and (2) suggest that in juvenile cod, there is no selective advantage to having a particular Hb genotype with regards to the capacity to withstand ecologically relevant environmental challenges.<br /

Direct Interaction between AR and PAK6 in Androgen-Stimulated PAK6 Activation

A p21-activated kinase 6 (PAK6) was previously identified to be an androgen receptor (AR) interacting protein through a yeast two-hybrid screening. We used hormone responsive prostate cancer LAPC4 and LNCap cell lines as models to study the signaling events associated with androgen stimulation and PAK6. An androgen-stimulated PAK6 kinase activation was observed in LAPC4 cells expressing endogenous PAK6 and in LNCap cells ectopically expressing a wild type PAK6. This activation was likely mediated through a direct interaction between AR and PAK6 since siRNA knock-down of AR in LAPC4 cells downregulated androgen-stimulated PAK6 activation. In addition, LNCap cells expressing a non-AR-interacting PAK6 mutant exhibited dampened androgen-stimulated kinase activation. As a consequence of androgen-stimulated activation, PAK6 was phosphorylated at multiple serine/threonine residues including the AR-interacting domain of PAK6. Furthermore, androgen-stimulation promoted prostate cancer cell motility and invasion were demonstrated in LNCap cells ectopically expressing PAK6-WT. In contrast, LNCap expressing non-AR-interacting mutant PAK6 did not respond to androgen stimulation with increased cell motility and invasion. Our results demonstrate that androgen-stimulated PAK6 activation is mediated through a direct interaction between AR and PAK6 and PAK6 activation promotes prostate cancer cells motility and invasion

Seasonal and Spatial Variation of the Bacterial Mutagenicity of Fine Organic Aerosol in Southern California

The bacterial mutagenicity of a set of 1993 urban particulate air pollution samples is examined using the Salmonella typhimurium TM677 forward mutation assay. Ambient fine particulate samples were collected for 24 hr every sixth day throughout 1993 at four urban sites, including Long Beach, central Los Angeles, Azusa, and Rubidoux, California, and at an upwind background site on San Nicolas Island. Long Beach and central Los Angeles are congested urban areas where air quality is dominated by fresh emissions from air pollution sources; Azusa and Rubidoux are located farther downwind and receive transported air pollutants plus increased quantities of the products of atmospheric chemical reactions. Fine aerosol samples from Long Beach and Los Angeles show a pronounced seasonal variation in bacterial mutagenicity per cubic meter of ambient air, with maximum in the winter and a minimum in the summer. The downwind smog receptor site at Rubidoux shows peak mutagenicity (with postmitochondrial supernatant but no peak without postmitochondrial supernatant) during the September-October periods when direct transport from upwind sources can be expected. At most sites the mutagenicity per microgram of organic carbon from the aerosol is not obviously higher during the summer photochemical smog period than during the colder months. Significant spatial variation in bacterial mutagenicity is observed: mutagenicity per cubic meter of ambient air, on average, is more than an order of magnitude lower at San Nicolas Island than within the urban area. The highest mutagenicity values per microgram of organics supplied to the assay are found at the most congested urban sites at central Los Angeles and Long Beach. The highest annual average values of mutagenicity per cubic meter of air sampled occur at central Los Angeles. These findings stress the importance of proximity to sources of direct emissions of bacterial mutagens and imply that if important mutagen-forming atmospheric reactions occur, they likely occur in the winter and spring seasons as well as the photochemically more active summer and early fall periods

Geothermal exploration in the Fell Sandstone Formation (Mississippian) beneath the city centre of Newcastle upon Tyne, UK: the Newcastle Science Central Deep Geothermal Borehole

The postulate that geothermal energy might be recoverable from strata laterally equivalent to the Fell Sandstone Formation (Carboniferous: Mississippian) beneath Newcastle upon Tyne has been examined by the drilling and testing of the 1821 m deep Newcastle Science Central Deep Geothermal Borehole. This proved 376.5 m of Fell Sandstone Formation below 1400 m, much of which resembled braided river deposits found at outcrop, although some lower portions were reddened and yielded grains of aeolian affinity. Downhole logging after attainment of thermal equilibrium proved a temperature of 73°C at 1740 m, and allowed estimation of heat flow at about 88 mW m−2. This relatively high value probably reflects deep convective transfer of heat over a distance of >8 km from the North Pennine Batholith, along the Ninety Fathom Fault. The Fell Sandstone traversed by the borehole proved to be of low hydraulic conductivity (c. 7×10−5 m d−1). The water that entered the well was highly saline, with a Na–(Ca)–Cl signature similar to other warm waters encountered in the region. It remains for future directional drilling to establish whether sufficient natural fracture permeability can be encountered, or wells stimulated, to support commercial heat production

Determination of Carcass and Live Body Weight of Finishing Cattle from Front Body Weights Taken at a Scale—Electronic Identification Equipped Water Fountain

A water fountain oriented cattle weight monitoring system was designed to automatically identify cattle and weigh cattle when they approached the in-pen water fountain to drink. This system provides a labor free means to monitor cattle in terms of daily weight gain and also provides an objective method to evaluate the health of cattle based on frequency of drinking and deviations from their normal pattern