Probing RNA recognition by human ADAR2 using a high-throughput mutagenesis method.

Adenosine deamination is one of the most prevalent post-transcriptional modifications in mRNA. In humans, ADAR1 and ADAR2 catalyze this modification and their malfunction correlates with disease. Recently our laboratory reported crystal structures of the human ADAR2 deaminase domain bound to duplex RNA revealing a protein loop that binds the RNA on the 5' side of the modification site. This 5' binding loop appears to be one contributor to substrate specificity differences between ADAR family members. In this study, we endeavored to reveal detailed structure-activity relationships in this loop to advance our understanding of RNA recognition by ADAR2. To achieve this goal, we established a high-throughput mutagenesis approach which allows rapid screening of ADAR variants in single yeast cells and provides quantitative evaluation for enzymatic activity. Using this approach, we determined the importance of specific amino acids at 19 different positions in the ADAR2 5' binding loop and revealed six residues that provide essential structural elements supporting the fold of the loop and key RNA-binding functional groups. This work provided new insight into RNA recognition by ADAR2 and established a new tool for defining structure-function relationships in ADAR reactions

Why do we invest ethically?

Analysis in this paper has proposed three potential motives for ethical investment - financial returns, non-wealth returns and social change. The motives are developed from the literature and illustrated in the context of a 'best of sector' fund and a socially screened fund. We find that the proposed motives are neither exhaustive nor exclusive and one single motive will not explain the behaviour of all ethical investors. There may be a trade-off between financial and psychic returns for some investors. The trade-off for consumption-investors is expected to be close to zero (total utility is maximised with small levels of ethical investment in the fun of participation model) and is expected to vary with the ethical intensity of investment-investors, as shown when we include ethical intensity into the investor's utility function. Psychic return can also be viewed as an increase in happiness and this approach would lend itself to empirical testing to increase our understanding of why we invest ethically

DNA editing in DNA/RNA hybrids by adenosine deaminases that act on RNA.

Adenosine deaminases that act on RNA (ADARs) carry out adenosine (A) to inosine (I) editing reactions with a known requirement for duplex RNA. Here, we show that ADARs also react with DNA/RNA hybrid duplexes. Hybrid substrates are deaminated efficiently by ADAR deaminase domains at dA-C mismatches and with E to Q mutations in the base flipping loop of the enzyme. For a long, perfectly matched hybrid, deamination is more efficient with full length ADAR2 than its isolated deaminase domain. Guide RNA strands for directed DNA editing by ADAR were used to target six different 2΄-deoxyadenosines in the M13 bacteriophage ssDNA genome. DNA editing efficiencies varied depending on the sequence context of the editing site consistent with known sequence preferences for ADARs. These observations suggest the reaction within DNA/RNA hybrids may be a natural function of human ADARs. In addition, this work sets the stage for development of a new class of genome editing tools based on directed deamination of 2΄-deoxyadenosines in DNA/RNA hybrids

The influence of single base triplet changes on the stability of a Pur·Pur·Pyr triple helix determined by affinity cleaving

The influence of sixteen base triplet changes at a single position within a pur·pur·pyr triple helix was examined by affinity cleaving. For the 15 base pair target site studied here, G·GC, A·AT and T·AT triplets stabilize a triple helix to a greater extent than the other 13 natural triplets (pH = 7.4, 25°C). Weaker interactions were detected for the C·AT, A·GC and T·CG triplets. The absence of specific, highly stabilizing interactions between third strand bases and the CG or TA base pairs demonstrates a current sequence limitation to formation of this structure. Models for the two dimensional base triplet interactions for all possible 16 natural triplets are presented

Fermented Liquid Feed: The potential for eliminating enteropathogens from feed

The aim of this study was to determine the effect of temperature on the survival of Salmonella and E. coli in fermented liquid pig feed (FLF). Liquid feed, fermented with Lactobacillus plantarum, was challenged with Salmonella or E. coli (six serovars of each) at 20, 30 or 37 C. Temperature significantly affected the survival of Salmonella and E. coli. In FLF containing ca 230 mmol L-1 lactic acid the mean decimal reduction time (D) for Salmonella was reduced from 157 (±10) min at 20 C to 12 (±1.5) min at 30 C and \u3c 5 min at 37 C. Likewise, the mean D for E. coli was significantly reduced from \u3e180 min at 20 C to 30 (±12.7) min at 30 oC and 21 (±6.2) min at 37 C. These studies suggest that successful elimination of potential pathogens from liquid feed can be achieved through fermentation with appropriate lactic acid bacteria and temperature control

The effect on biological performance and faecal microbiology of feeding finishing pigs on liquid diets fermented with lactic bacteria

Thirty-two Hermitage-Seaborough, Hybrid pigs, were allocated at 16 weeks of age to a randomized block design with four treatments and four replicates

The effect of copper, zinc and iron on the death rate of Salmonella under acid conditions

Liquid pig feed (LPF) and skimmed milk (SM) acidified to pH 4 with lactic or hydrochloric acid (HCl) were treated with 0, 2.5, 5. 12.5, 25 or 50 ppm Cu2+ and challenged with 107 c.f.u ml-1 Salmonella Typhimurium DT104:30. The decimal reduction time (D) was determined by enumeration of viable Salmonellae at appropriate time intervals after inoculation

The influence of single base triplet changes on the stability of a Pur·Pur·Pyr triple helix determined by affinity cleaving

The influence of sixteen base triplet changes at a single position within a pur·pur·pyr triple helix was examined by affinity cleaving. For the 15 base pair target site studied here, G·GC, A·AT and T·AT triplets stabilize a triple helix to a greater extent than the other 13 natural triplets (pH = 7.4, 25°C). Weaker interactions were detected for the C·AT, A·GC and T·CG triplets. The absence of specific, highly stabilizing interactions between third strand bases and the CG or TA base pairs demonstrates a current sequence limitation to formation of this structure. Models for the two dimensional base triplet interactions for all possible 16 natural triplets are presented

Effect of micro-organism and particle size on fermentation of sorghum and maize for poultry feed

A study was conducted to investigate the effect of particle size and micro-organism on fermentation of sorghum and maize for poultry feed. Sorghum (Sorghum bicolour L. Moench) and maize were milled in a hammer mill and separated into coarse, medium, fine and very fine particles sizes with a stack of sieves of apertures 2.5 mm, 850 μm and 500 μm, from the first to the last sieve and ending in a pan with the very fine particles. Samples were weighed into 100 g sachets and irradiated using 60Co at 25 kGy γ-radiation. Grains were fermented with sterile distilled water for 24 h at a ratio of 1 feed:1.4 water and inoculated with 0.01 ml of an overnight culture of De Man, Rogosa and Sharpe (MRS) broth containing Pediococcus acidilactici (PA1) or Lactobacillus plantarum (SLP) (ca 109 cfu/ml). The medium was incubated at 30°C simultaneously with a control treatment without lactic acid bacteria (LAB). Subsamples were collected aseptically at the beginning of the fermentation (0 h) and at 4, 8, 24 h after fermentation for pH, sugar and organic acids analysis. Significant reductions in the pH of maize and sorghum for LAB treatments (PA1 and SLP) were evident after 8 hours of fermentation. Twenty four hour lactic acid concentrations from coarse particle size fermentations were not significantly different from concentrations in the medium and fine particle size fermentations. The choice of LAB did not affect the concentration of lactic acid for any particle size. However, acetic acid production from fermentation with PA1 was significantly higher (P<0.01) than the concentration obtained with SLP. Results suggest that moderate grain processing may be enough to permit production of biosafe levels of lactic acid in fermented feed for poultry birds.Keywords: Fermentation, lactic acid bacteria, maize, particle size, sorghum African Journal of Biotechnology Vol. 12(26), pp. 4147-415

EVALUATION OF RELATIONSHIPS BETWEEN REPORTED RESILIENCE AND SOLDIER OUTCOMES, Report #2: Positive Performance Outcomes in Officers (Promotions, Selections, & Professions)

This technical report presents an analysis of reported resilience and psychological health among the U.S. Army’s Officer Corps. The focus of the current report is on linking resilience and psychological health (hereafter referred to as R/PH) to objective outcomes associated with high job performance. Specifically, this report examines the statistical relationships between officer R/PH - as measured by the Army’s Global Assessment Tool (GAT) - and promotions to Brigadier General, early (below zone) Field Grade Officer promotions, selections for command / key billet assignments, and officers who serve in career fields that require terminal professional degrees (e.g., medical doctors, dentists, lawyers, etc). Results show that officers who have been promoted to Brigadier General are more emotionally and socially fit than their peers who have not received a promotion to Brigadier General. These officers are more engaged with their work, have higher levels of organizational trust and friendship, report lower levels of loneliness, are more optimistic, and report higher levels of positive affect and lower levels of negative affect. Each of these findings is in line with our expectations regarding R/PH and job performance, and the findings comport with a substantial body of work in the academic literature. Additionally, analysis of R/PH for officers promoted early resulted in findings similar to above. In particular, those who have been promoted below zone report higher levels of work engagement, friendship, organizational trust, optimism, and coping abilities. In short, these officers score higher on the GAT dimensions of Emotional and Social Fitness than their peers who were not promoted early (“due course” officers). Similar results were found for officers selected for command for key billet assignments. They are also more emotionally and socially fit than their peers who were not selected for command (more engaged with their work, have higher levels of organizational trust and friendship, are less lonely, are more optimistic, and report higher levels of positive affect and lower levels of negative affect). There are no practical differences in R/PH between officers serving in career fields that require terminal professional degrees and other officers serving in “line” career fields (rank-matched analysis, Captain - Colonel). In light of academic literature on the subject, this finding is somewhat surprising as it suggests that advanced professional education / training alone may not influence (or be influenced by) R/PH. When taken together, the findings above strongly suggest there is a relationship between reported resilience and psychological health and outcomes associated with high job performance, but we are currently unable to determine causality. Stated differently, we do not know if the reported R/PH contributed to the performance outcomes, or if the high job performance outcomes contributed to the reported R/PH. Further data collection and analysis over the next 12-24 months will broaden our understanding of the relationships