5-Methyl-Tetrahydrofolate and the S-Adenosylmethionine Cycle in C57BL/6J Mouse Tissues: Gender Differences and Effects of Arylamine N-Acetyltransferase-1 Deletion

Folate catabolism involves cleavage of the C⁹-N¹⁰ bond to form p-aminobenzoylgluamate (PABG) and pterin. PABG is then acetylated by human arylamine N-acetyltransferase 1 (NAT1) before excretion in the urine. Mice null for the murine NAT1 homolog (Nat2) show several phenotypes consistent with altered folate homeostasis. However, the exact role of Nat2 in the folate pathway in vivo has not been reported. Here, we examined the effects of Nat2 deletion in male and female mice on the tissue levels of 5-methyl-tetrahydrofolate and the methionine-S-adenosylmethionine cycle. We found significant gender differences in hepatic and renal homocysteine, S-adenosylmethionine and methionine levels consistent with a more active methionine-S-adenosylmethionine cycle in female tissues. In addition, methionine levels were significantly higher in female liver and kidney. PABG was higher in female liver tissue but lower in kidney compared to male tissues. In addition, qPCR of mRNA extracted from liver tissue suggested a significantly lower level of Nat2 expression in female animals. Deletion of Nat2 affected liver 5- methyl-tetrahydrofolate in female mice but had little effect on other components of the methionine-S-adenosylmethionine cycle. No N-acetyl-PABG was observed in any tissues in Nat2 null mice, consistent with the role of Nat2 in PABG acetylation. Surprisingly, tissue PABG levels were similar between wild type and Nat2 null mice. These results show that Nat2 is not required to maintain tissue PABG homeostasis in vivo under normal conditions.     

Novel Binding Partners and Differentially Regulated Phosphorylation Sites Clarify Eps8 as a Multi-Functional Adaptor

Eps8 is involved in both cell signalling and receptor trafficking. It is a known phosphorylation substrate for two proteins involved in the fibroblast growth factor receptor (FGFR) signalling pathway: the receptor itself and Src. Here we report a differential proteomic analysis of Eps8 aimed to identify specific FGFR and Src family kinase dependent phosphosites and co-associated phosphodependent binding partners. This study reveals a total of 22 Eps8 pTyr and pSer/Thr phosphorylation sites, including those that are dependent on Src family and FGFR kinase activity. Peptide affinity purification of proteins that bind to a selection of the pTyr phosphosites has identified a range of novel Eps8 binding partners including members of the intracellular vesicle trafficking machinery (clathrin and AP-2), proteins which have been shown to regulate activated receptor trafficking (NBR1 and Vav2), and proteins involved in receptor signalling (IRS4 and Shp2). Collectively this study significantly extends the understanding of Eps8 post-translational modification by regulated phosphorylation, identifies novel Eps8 binding partners implicated in receptor trafficking and signalling, and confirms the functions of Eps8 at the nexus of receptor signalling and vesicular trafficking.     

Did the lateral enamel of Neandertal anterior teeth grow differently from that of modern humans?

The formation of lateral enamel in Neandertal anterior teeth has been the subject of recent studies. When compared to the anterior teeth of modern humans from diverse regions (Point Hope, Alaska; Newcastle upon Tyne, England; southern Africa), Neandertal anterior teeth appear to fall within the modern human range of variation for lateral enamel formation time. However, the lateral enamel growth curves of Neandertals are more linear than those of these modern human samples. Other researchers have found that the lateral enamel growth curves of Neandertals are more linear than those of Upper Paleolithic and Mesolithic modern humans as well. The statistical significance of this apparent difference between Neandertal and modern human lateral enamel growth curves is analyzed here. The more linear Neandertal enamel growth curves result from the smaller percentage of total perikymata located in the cervical halves of their teeth. The percentage of total perikymata in the cervical halves of teeth is therefore compared between the Neandertal sample (n=56 teeth) and each modern human population sample: Inuit (n=65 teeth), southern African (n=114 teeth), and northern European (n=115 teeth). There are 18 such comparisons (6 tooth types, Neandertals vs. each of the three modern human populations). Eighteen additional comparisons are made among the modern human population samples. Statistically significant differences are found for 16 of the 18 Neandertal vs. modern human comparisons but for only two of the 18 modern human comparisons. Statistical analyses repeated for subsamples of less worn teeth show a similar pattern. Because surface curvature is thought to affect perikymata spacing, we also conducted measurements to assess surface curvature in thirty teeth. Our analysis shows that surface curvature is not a factor in this lateral enamel growth difference between Neandertals and modern humans.     

Regulatory mechanisms to control tissue alpha-tocopherol

To test the hypothesis that hepatic regulation of alpha-tocopherol metabolism would be sufficient to prevent overaccumulation of alpha-tocopherol in extrahepatic tissues and that administration of high doses of alpha-tocopherol would up-regulate extrahepatic xenobiotic pathways, rats received daily subcutaneous injections of either vehicle or 0.5, 1, 2, or 10 mg alpha-tocopherol/100 g body wt for 9 days. Liver alpha-tocopherol increased 15-fold in rats given 10 mg alpha-tocopherol/100 g body wt (mg/100 g) compared with controls. Hepatic alpha-tocopherol metabolites increased with increasing alpha-tocopherol doses, reaching 40-fold in rats given the highest dose. In rats injected with 10 mg/100 g, lung and duodenum alpha-tocopherol concentrations increased 3-fold, whereas alpha-tocopherol concentrations of other extrahepatic tissues increased 2-fold or less. With the exception of muscle, daily administration of less than 2 mg/100 g failed to increase alpha-tocopherol concentrations in extrahepatic tissues. Lung cytochrome P450 3A and 1A levels were unchanged by administration of alpha-tocopherol at any dose. In contrast, lung P-glycoprotein (MDR1) levels increased dose dependently and expression of this xenobiotic transport protein was correlated with lung alpha-tocopherol concentrations (R(2)=0.88, p<0.05). Increased lung MDR1 may provide protection from exposure to environmental toxins by increasing alveolar space alpha-tocopherol.     

Inferring Clonal Composition from Multiple Sections of a Breast Cancer

Cancers arise from successive rounds of mutation and selection, generating clonal populations that vary in size, mutational content and drug responsiveness. Ascertaining the clonal composition of a tumor is therefore important both for prognosis and therapy. Mutation counts and frequencies resulting from next-generation sequencing (NGS) potentially reflect a tumor''s clonal composition; however, deconvolving NGS data to infer a tumor''s clonal structure presents a major challenge. We propose a generative model for NGS data derived from multiple subsections of a single tumor, and we describe an expectation-maximization procedure for estimating the clonal genotypes and relative frequencies using this model. We demonstrate, via simulation, the validity of the approach, and then use our algorithm to assess the clonal composition of a primary breast cancer and associated metastatic lymph node. After dividing the tumor into subsections, we perform exome sequencing for each subsection to assess mutational content, followed by deep sequencing to precisely count normal and variant alleles within each subsection. By quantifying the frequencies of 17 somatic variants, we demonstrate that our algorithm predicts clonal relationships that are both phylogenetically and spatially plausible. Applying this method to larger numbers of tumors should cast light on the clonal evolution of cancers in space and time.     

Optimal Behavioral Hierarchy

Human behavior has long been recognized to display hierarchical structure: actions fit together into subtasks, which cohere into extended goal-directed activities. Arranging actions hierarchically has well established benefits, allowing behaviors to be represented efficiently by the brain, and allowing solutions to new tasks to be discovered easily. However, these payoffs depend on the particular way in which actions are organized into a hierarchy, the specific way in which tasks are carved up into subtasks. We provide a mathematical account for what makes some hierarchies better than others, an account that allows an optimal hierarchy to be identified for any set of tasks. We then present results from four behavioral experiments, suggesting that human learners spontaneously discover optimal action hierarchies.     

Activation of extrinsic apoptosis pathway in HCV monoinfected and HIV–HCV coinfected patients,irrespective of liver disease severity

Chronic hepatitis C virus (HCV) infection is associated with increased levels of peripheral T cell apoptosis. We aimed to study whether T cell apoptosis markers indicate pathways that may contribute to clinical progression in HCV monoinfected and HIV–HCV coinfected patients. Activation of the extrinsic apoptosis pathways was measured by levels of death receptor Fas, initiator caspase 8 and effector caspases 3 and 7 activity and Annexin V binding on peripheral CD4 and CD8 T cells of HCV monoinfected and HIV/HCV coinfected patients, as well as healthy controls and HIV-infected, hepatitis B virus-infected and primary biliary cirrhosis disease controls. Association with liver fibrosis was assessed by biopsy or by transient elastography. HCV monoinfected and HIV–HCV coinfected patients displayed enhanced peripheral CD4 and CD8 T cell apoptosis. Caspase 8 activity was highest in HIV–HCV coinfection, without enhanced downstream activity of caspases 3 and 7. Level of peripheral T cell apoptosis was independent of liver fibrosis or other disease parameters in all disease groups. The extrinsic apoptosis pathway is upregulated in HCV monoinfection and HIV–HCV coinfection, but this is independent of liver disease severity.     

Grip Strength across the Life Course: Normative Data from Twelve British Studies

Introduction

Epidemiological studies have shown that weaker grip strength in later life is associated with disability, morbidity, and mortality. Grip strength is a key component of the sarcopenia and frailty phenotypes and yet it is unclear how individual measurements should be interpreted. Our objective was to produce cross-sectional centile values for grip strength across the life course. A secondary objective was to examine the impact of different aspects of measurement protocol.

Methods

We combined 60,803 observations from 49,964 participants (26,687 female) of 12 general population studies in Great Britain. We produced centile curves for ages 4 to 90 and investigated the prevalence of weak grip, defined as strength at least 2.5 SDs below the gender-specific peak mean. We carried out a series of sensitivity analyses to assess the impact of dynamometer type and measurement position (seated or standing).

Results

Our results suggested three overall periods: an increase to peak in early adult life, maintenance through to midlife, and decline from midlife onwards. Males were on average stronger than females from adolescence onwards: males’ peak median grip was 51 kg between ages 29 and 39, compared to 31 kg in females between ages 26 and 42. Weak grip strength, defined as strength at least 2.5 SDs below the gender-specific peak mean, increased sharply with age, reaching a prevalence of 23% in males and 27% in females by age 80. Sensitivity analyses suggested our findings were robust to differences in dynamometer type and measurement position.

Conclusion

This is the first study to provide normative data for grip strength across the life course. These centile values have the potential to inform the clinical assessment of grip strength which is recognised as an important part of the identification of people with sarcopenia and frailty.     

Cost-Effectiveness of HIV Drug Resistance Testing to Inform Switching to Second Line Antiretroviral Therapy in Low Income Settings

Background

To guide future need for cheap resistance tests for use in low income settings, we assessed cost-effectiveness of drug resistance testing as part of monitoring of people on first line ART - with switching from first to second line ART being conditional on NNRTI drug resistance mutations being identified.

Methods

An individual level simulation model of HIV transmission, progression and the effect of ART which accounts for adherence and resistance development was used to compare outcomes of various potential monitoring strategies in a typical low income setting in sub-Saharan Africa. Underlying monitoring strategies considered were based on clinical disease, CD4 count or viral load. Within each we considered a strategy in which no further measures are performed, one with a viral load measure to confirm failure, and one with both a viral load measure and a resistance test. Predicted outcomes were assessed over 2015–2025 in terms of viral suppression, first line failure, switching to second line regimen, death, HIV incidence, disability-adjusted-life-years averted and costs. Potential future low costs of resistance tests ($30) were used.

Results

The most effective strategy, in terms of DALYs averted, was one using viral load monitoring without confirmation. The incremental cost-effectiveness ratio for this strategy was $2113 (the same as that for viral load monitoring with confirmation). ART monitoring strategies which involved resistance testing did not emerge as being more effective or cost effective than strategies not using it. The slightly reduced ART costs resulting from use of resistance testing, due to less use of second line regimens, was of similar magnitude to the costs of resistance tests.

Conclusion

Use of resistance testing at the time of first line failure as part of the decision whether to switch to second line therapy was not cost-effective, even though the test was assumed to be very inexpensive.     

Comparative analysis of vertebrate <Emphasis Type="Italic">Shh</Emphasis> genes identifies novel conserved non-coding sequence

Abstract The puffer fish Takifugu rubripes (Fugu), with its compact genome, is an ideal model organism for comparative genomics. Sonic hedgehog (Shh) is a key protein in the patterning of differentiating cells during embryonic development. We have sequenced the Fugu Shh gene and compared it with the mammalian and zebrafish orthologs, identifying a number of novel conserved, non-coding sequences upstream of exon one and within the two introns. Additional conserved sequences serve to delineate activator regions and enhancers previously characterized through functional analysis. Control elements can thus be rapidly and effectively predicted by comparative methodology in its own right as well as complementing other, functional methods. This work demonstrates the value of using Fugu in comparative genomics, which has allowed identification of new putative regulatory elements, as well as corroborating enhancers identified by the more traditional deletion mapping method.