Preferences for the selection of unique tRNA primers revealed from analysis of HIV-1 replication in peripheral blood mononuclear cells

BACKGROUND: All human immunodeficiency virus (HIV-1) uses a host tRNA(Lys,3 )as the primer for reverse transcription. The tRNA(Lys,3 )is bound to a region on the HIV-1 genome, the primer-binding site (PBS), that is complementary to the 18 terminal nucleotides of tRNA(Lys,3). How HIV-1 selects the tRNA from the intracellular milieu is unresolved. RESULTS: HIV-1 tRNA primer selection has been investigated using viruses in which the primer-binding site (PBS) and a sequence within U5 were altered so as to be complementary to tRNA(Met), tRNA(Pro )or tRNA(Ile). Analysis of the replication of these viruses in human peripheral blood mononuclear cells (PBMC) revealed preferences for the selection of certain tRNAs. HIV-1 with the PBS altered to be complementary to tRNA(Met), with and without the additional mutation in U5 to be complementary to the anticodon of tRNA(Met), stably maintains the PBS complementary to tRNA(Met )following extended in vitro culture in PBMC. In contrast, viruses with either the PBS or PBS and U5 mutated to be complementary to tRNA(Ile )were unstable during in vitro replication in PBMC and reverted to utilize tRNA(Lys,3). Viruses with the PBS altered to be complementary to tRNA(Pro )replicated in PBMC but reverted to use tRNA(Lys,3); viruses with mutations in both the U5 and PBS complementary to tRNA(Pro )maintained this PBS, yet replicated poorly in PBMC. CONCLUSION: The results of these studies demonstrate that HIV-1 has preferences for selection of certain tRNAs for high-level replication in PBMC

Greater Omentectomy Improves Insulin Sensitivity in Nonobese Dogs

Visceral adiposity is strongly associated with insulin resistance; however, little evidence directly demonstrates that visceral fat per se impairs insulin action. Here, we examine the effects of the surgical removal of the greater omentum and its occupying visceral fat, an omentectomy (OM), on insulin sensitivity (SI) and β-cell function in nonobese dogs. Thirteen male mongrel dogs were used in this research study; animals were randomly assigned to surgical treatment with either OM (n = 7), or sham-surgery (SHAM) (n = 6). OM failed to generate measurable changes in body weight (+2%; P = 0.1), or subcutaneous adiposity (+3%; P = 0.83) as assessed by magnetic resonance imaging (MRI). The removal of the greater omentum did not significantly reduce total visceral adipose volume (−7.3 ± 6.4%; P = 0.29); although primary analysis showed a trend for OM to increase SI when compared to sham operated animals (P = 0.078), further statistical analysis revealed that this minor reduction in visceral fat alleviated insulin resistance by augmenting SI of the periphery (+67.7 ± 35.2%; P = 0.03), as determined by the euglycemic-hyperinsulinemic clamp. Insulin secretory response during the hyperglycemic step clamp was not directly influenced by omental fat removal (presurgery 6.82 ± 1.4 vs. postsurgery: 6.7 ± 1.2 pmol/l/mg/dl, P = 0.9). These findings provide new evidence for the deleterious role of visceral fat in insulin resistance, and suggest that a greater OM procedure may effectively improve insulin sensitivity

Precision Primordial 4^4He Measurement with CMB Experiments

Big bang nucleosynthesis (BBN) and the cosmic microwave background (CMB) are two major pillars of cosmology. Standard BBN accurately predicts the primordial light element abundances ($^4$He, D, $^3$He and $^7$Li), depending on one parameter, the baryon density. Light element observations are used as a baryometers. The CMB anisotropies also contain information about the content of the universe which allows an important consistency check on the Big Bang model. In addition CMB observations now have sufficient accuracy to not only determine the total baryon density, but also resolve its principal constituents, H and $^4$He. We present a global analysis of all recent CMB data, with special emphasis on the concordance with BBN theory and light element observations. We find $\Omega_{B}h^{2}=0.025+0.0019-0.0026$ and $Y_{p}=0.250+0.010-0.014$ (fraction of baryon mass as $^4$He) using CMB data alone, in agreement with $^4$He abundance observations. With this concordance established we show that the inclusion of BBN theory priors significantly reduces the volume of parameter space. In this case, we find $\Omega_{B}h^2=0.0244+0.00137-0.00284$ and $Y_p = 0.2493+0.0006-0.001$. We also find that the inclusion of deuterium abundance observations reduces the $Y_p$ and $\Omega_{B}h^2$ ranges by a factor of $\sim$2. Further light element observations and CMB anisotropy experiments will refine this concordance and sharpen BBN and the CMB as tools for precision cosmology.Comment: 7 pages, 3 color figures made minor changes to bring inline with journal versio

Multidisciplinary lab – Does it work?

As part of the restructure of the BSc program of The University of Newcastle, a multidisciplinary laboratory course (SCIE1002) was introduced for the first time this year as a core course. Students are given the opportunity to develop their capacity to engage and understand the perspectives of multiple disciplines while addressing scientific challenges. They also learn essential/foundational laboratory skills required in their chosen majors and across a range of other diverse science disciplines. The course was developed by several disciplines within the Faculty of Science: Biological Sciences, Chemistry, Earth Sciences, Environmental Science and Management, Geography, Physics and Psychology. Explored using multiple disciplinary approaches, the laboratory sessions focus on two practical research questions: Water – Would you drink it? and Energy – How much does it cost? Initial students’ feedback are positive; students engage with the online contextual pre-lab materials and, particularly, with the laboratory active learning as they provide real world relevance. The laboratory setting generated a highly interactive environment with student peers and staff enhancing students’ learning and building staff-students relationships. This presentation will discuss the development and implementation of the course, challenges encountered and planned improvements guided by both students and staff feedback

Teleological role of L-2-hydroxyglutarate dehydrogenase in the kidney

L-2-hydroxyglutarate (L-2HG) is an oncometabolite found elevated in renal tumors. However, this molecule might have physiological roles that extend beyond its association with cancer, as L-2HG levels are elevated in response to hypoxia and during Drosophila larval development. L-2HG is known to be metabolized by L-2HG dehydrogenase (L2HGDH), and loss of L2HGDH leads to elevated L-2HG levels. Despite L2HGDH being highly expressed in the kidney, its role in renal metabolism has not been explored. Here, we report our findings utilizing a novel CRISPR/Cas9 murine knockout model, with a specific focus on the role of L2HGDH in the kidney. Histologically, L2hgdh knockout kidneys have no demonstrable histologic abnormalities. However, GC-MS metabolomics demonstrates significantly reduced levels of the TCA cycle intermediate succinate in multiple tissues. Isotope labeling studies with [U-13C] glucose demonstrate that restoration of L2HGDH in renal cancer cells (which lowers L-2HG) leads to enhanced incorporation of label into TCA cycle intermediates. Subsequent biochemical studies demonstrate that L-2HG can inhibit the TCA cycle enzyme α-ketoglutarate dehydrogenase. Bioinformatic analysis of mRNA expression data from renal tumors demonstrates that L2HGDH is co-expressed with genes encoding TCA cycle enzymes as well as the gene encoding the transcription factor PGC-1α, which is known to regulate mitochondrial metabolism. Restoration of PGC-1α in renal tumor cells results in increased L2HGDH expression with a concomitant reduction in L-2HG levels. Collectively, our analyses provide new insight into the physiological role of L2HGDH as well as mechanisms that promote L-2HG accumulation in disease states

Nucleosynthesis Constraints on a Massive Gravitino in Neutralino Dark Matter Scenarios

The decays of massive gravitinos into neutralino dark matter particles and Standard Model secondaries during or after Big-Bang nucleosynthesis (BBN) may alter the primordial light-element abundances. We present here details of a new suite of codes for evaluating such effects, including a new treatment based on PYTHIA of the evolution of showers induced by hadronic decays of massive, unstable particles such as a gravitino. We also develop an analytical treatment of non-thermal hadron propagation in the early universe, and use this to derive analytical estimates for light-element production and in turn on decaying particle lifetimes and abundances. We then consider specifically the case of an unstable massive gravitino within the constrained minimal supersymmetric extension of the Standard Model (CMSSM). We present upper limits on its possible primordial abundance before decay for different possible gravitino masses, with CMSSM parameters along strips where the lightest neutralino provides all the astrophysical cold dark matter density. We do not find any CMSSM solution to the cosmological Li7 problem for small m_{3/2}. Discounting this, for m_{1/2} ~ 500 GeV and tan beta = 10 the other light-element abundances impose an upper limit m_{3/2} n_{3/2}/n_\gamma < 3 \times 10^{-12} GeV to < 2 \times 10^{-13} GeV for m_{3/2} = 250 GeV to 1 TeV, which is similar in both the coannihilation and focus-point strips and somewhat weaker for tan beta = 50, particularly for larger m_{1/2}. The constraints also weaken in general for larger m_{3/2}, and for m_{3/2} > 3 TeV we find a narrow range of m_{3/2} n_{3/2}/n_\gamma, at values which increase with m_{3/2}, where the Li7 abundance is marginally compatible with the other light-element abundances.Comment: 74 pages, 40 Figure

Conditional genome engineering reveals canonical and divergent roles for the Hus1 component of the 9-1-1 complex in the maintenance of the plastic genome of Leishmania.

Leishmania species are protozoan parasites whose remarkably plastic genome limits the establishment of effective genetic manipulation and leishmaniasis treatment. The strategies used by Leishmania to maintain its genome while allowing variability are not fully understood. Here, we used DiCre-mediated conditional gene deletion to show that HUS1, a component of the 9-1-1 (RAD9- RAD1-HUS1) complex, is essential and is required for a G2/M checkpoint. By analyzing genome wide instability in HUS1 ablated cells, HUS1 is shown to have a conserved role, by which it preserves genome stability, and also a divergent role, by which it promotes genome variability. These roles of HUS1 are related to distinct patterns of formation and resolution of single-stranded DNA and γH2A, throughout the cell cycle. Our findings suggest that Leishmania 9-1-1 subunits have evolved to co-opt canonical genomic maintenance and genomic variation functions. Hence, this study reveals a pivotal function of HUS1 in balancing genome stability and transmission in Leishmania. These findings may be relevant to understanding the evolution of genome maintenance and plasticity in other pathogens and eukaryote

Primordial Nucleosynthesis for the New Cosmology: Determining Uncertainties and Examining Concordance

Big bang nucleosynthesis (BBN) and the cosmic microwave background (CMB) have a long history together in the standard cosmology. The general concordance between the predicted and observed light element abundances provides a direct probe of the universal baryon density. Recent CMB anisotropy measurements, particularly the observations performed by the WMAP satellite, examine this concordance by independently measuring the cosmic baryon density. Key to this test of concordance is a quantitative understanding of the uncertainties in the BBN light element abundance predictions. These uncertainties are dominated by systematic errors in nuclear cross sections. We critically analyze the cross section data, producing representations that describe this data and its uncertainties, taking into account the correlations among data, and explicitly treating the systematic errors between data sets. Using these updated nuclear inputs, we compute the new BBN abundance predictions, and quantitatively examine their concordance with observations. Depending on what deuterium observations are adopted, one gets the following constraints on the baryon density: OmegaBh^2=0.0229\pm0.0013 or OmegaBh^2 = 0.0216^{+0.0020}_{-0.0021} at 68% confidence, fixing N_{\nu,eff}=3.0. Concerns over systematics in helium and lithium observations limit the confidence constraints based on this data provide. With new nuclear cross section data, light element abundance observations and the ever increasing resolution of the CMB anisotropy, tighter constraints can be placed on nuclear and particle astrophysics. ABRIDGEDComment: 54 pages, 20 figures, 5 tables v2: reflects PRD version minor changes to text and reference