Optimized Effective Potentials in Finite Basis Sets

The finite basis optimized effective potential (OEP) method within density functional theory is examined as an ill-posed problem. It is shown that the generation of nonphysical potentials is a controllable manifestation of the use of unbalanced, and thus unsuitable, basis sets. A modified functional incorporating a regularizing smoothness measure of the OEP is introduced. This provides a condition on balanced basis sets for the potential, as well as a method to determine the most appropriate OEP potential and energy from calculations performed with any finite basis set.Comment: 23 pages, 28 figure

Characterisation of the sarcomeric myosin heavy chain multigene family in the laboratory guinea pig

BACKGROUND:Several chronic conditions leading to skeletal muscle dysfunction are known to be associated with changes in the expression of myosin heavy chain (MHC) isoforms at both the mRNA and protein level. Many of these conditions are modelled, pre-clinically, in the guinea pig due to similar disease onset and progression to the human condition, and their generally well-characterised anatomy. MHC composition is amenable to determination by protein and mRNA based methodologies, the latter quantifying the expression of MHC isoform-specific gene transcripts allowing the detection of earlier, and more subtle changes. As such, the MHC mRNAs, and specific oligonucleotide primers of all common laboratory species have been available for some time. However, due to incomplete genomic annotation, assessment of guinea pig MHC mRNA expression has not been previously possible, precluding the full characterisation of early changes in skeletal muscle in response to disease and disease modulation.The purpose of this study was to characterise the multigenic structure of the sarcomeric MHC family in the guinea pig, and to design and validate specific oligonucleotide primers to enable the assessment of the predominant adult-muscle associated MHC mRNAs in relevant disease models.RESULTS:Using a combination of ligase-mediated rapid amplification of 5' and 3' cDNA ends (RACE) and bioinformatics, mRNAs to the four main skeletal-muscle isoforms of MHC were determined. Specific oligonucleotide primers were designed, and following verification of their specificity, found to successfully determine the expression of each MHC mRNA independently.CONCLUSIONS:Because of their utilisation in the in vivo modelling of disease, there is a requirement to develop molecular methods that accurately differentiate the different MHC mRNAs in the guinea pig to enable rapid profiling of muscle composition in appropriate disease models. The methods developed here are suitable for the characterisation of muscle MHC expression at the molecular level from animal tissue samples and biopsy material. The publication of these specific oligonucleotide primers for the guinea pig MHC variants will enable researchers to rapidly and accurately quantify acute changes in MHC mRNA expression in either developmental or in guinea pig disease models where a marker of altered skeletal muscle function is required

Improving efficiency in meat production

Selective breeding and improved nutritional management over the past 20–30 years has resulted in dramatic improvements in growth efficiency for pigs and poultry, particularly lean tissue growth. However, this has been achieved using high-quality feed ingredients, such as wheat and soya that are also used for human consumption and more recently biofuels production. Ruminants on the other hand are less efficient, but are normally fed poorer quality ingredients that cannot be digested by human subjects, such as grass or silage. The challenges therefore are to: (i) maintain the current efficiency of growth of pigs and poultry, but using more ingredients not needed to feed the increasing human population or for the production of biofuels; (ii) improve the efficiency of growth in ruminants; (iii) at the same time produce animal products (meat, milk and eggs) of equal or improved quality. This review will describe the use of: (a) enzyme additives for animal feeds, to improve feed digestibility;(b) known growth promoting agents, such as growth hormone, β-agonists and anabolic steroids, currently banned in the European Union but used in other parts of the world; (c) recent transcriptomic studies into molecular mechanisms for improved growth efficiency via low residual feed intake. In doing so, the use of genetic manipulation in animals will also be discussed

Can we normalise developmentally appropriate health care for young people in UK hospital settings? An ethnographic study

OBJECTIVE: The WHO has argued that adolescent-responsive health systems are required. Developmentally appropriate healthcare (DAH) for young people is one approach that could underpin this move. The aim of this study was to explore the potential for DAH to become normalised, to become a routine, taken-for-granted, element of clinical practice. DESIGN: Qualitative ethnographic study. Analyses were based on procedures from first-generation grounded theory and theoretically informed by normalisation process theory. SETTING: Two tertiary and one secondary care hospital in England. PARTICIPANTS: 192 participants, health professionals (n=121) and managers (n=71) were recruited between June 2013 and January 2015. Approximately 1600 hours of non-participant observations in clinics, wards and meeting rooms were conducted, alongside 65 formal qualitative interviews. RESULTS: We observed diverse values and commitments towards the care of young people and provision of DAH, including a distributed network of young person-orientated practitioners. Informal networks of trust existed, where specific people, teams or wards were understood to have the right skill-mix, or mindset, or access to resources, to work effectively with young people. As young people move through an organisation, the preference is to direct them to other young person-orientated practitioners, so inequities in skills and experience can be self-sustaining. At two sites, initiatives around adolescent and young adult training remained mostly within these informal networks of trust. At another, through support by wider management, we observed a programme that sought to make the young people's healthcare visible across the organisation, and to get people to reappraise values and commitment. CONCLUSION: To move towards normalisation of DAH within an organisation, we cannot solely rely on informal networks and cultures of young person-orientated training, practice and mutual referral and support. Organisation-wide strategies and training are needed, to enable better integration and consistency of health services for all young people

Active vitamin D increases myogenic differentiation in C2C12 cells via a vitamin D response element on the myogenin promoter

Background Skeletal muscle development during embryogenesis depends on proliferation of myoblasts followed by differentiation into myotubes/ multinucleated myofibers. Vitamin D (VD) has been shown to affect these processes, but there is conflicting evidence within the current literature on the exact nature of these effects due to a lack of time course data. With 20-40% of pregnant women worldwide being VD deficient, it is crucial that a clearer understanding of the impact of VD on myogenesis is gained.Methods A detailed 8-day differentiation time course was used where C2C12 cells were differentiated in control media (2% horse serum or 0.1% ethanol) or with different concentrations of active VD, 1,25(OH)2D3 (10-13M, 10-11M, 10-9M or 10-7M), and measurements were taken at 6 time points. DNA, creatine kinase and protein assays were carried out as well as quantitative PCR to determine expression of Myf5, MyoD, myogenin, MHC I, MHC neonatal, MHC embryonic, MHC IIa, MHC IIx and MHC IIb mRNAs. Transfections were carried out using one vector containing the myogenin promoter and another containing the same promoter with a 3 base mutation within a putative vitamin D response element (VDRE) to determine effects of 1,25(OH)2D3 on myogenin transcription. Finally, a ChIP assay was performed to determine whether the VD receptor (VDR) binds to the putative VDRE.Results 1,25(OH)2D3 caused an inhibition of proliferation and an increase in differentiation in C2C12 cells. Myf5, myogenin, MHC I, MHC neonatal, MHC embryonic, MHC IIa, MHC IIx and MHC IIb expression were all increased by 1,25(OH)2D3. Myotube size was also increased by VD. When the putative VDRE on the myogenin promoter was mutated, the increase in expression by VD was lost. ChIP analysis revealed that the VDR does bind to the putative VDRE on the myogenin promoter.Conclusions Active VD directly increases myogenin transcription via a functional VDRE on the myogenin promoter, resulting in increased myogenic differentiation, increased expression of both the early and late MHC isoforms, and also increased myotube size. These results highlight the importance of VD status during pregnancy for normal myogenesis to occur, but further in vivo work is needed

Leucine and mTORc1 act independently to regulate 2-deoxyglucose uptake in L6 myotubes

© 2020, The Author(s). Chronic mTORc1 hyperactivation via obesity-induced hyperleucinaemia has been implicated in the development of insulin resistance, yet the direct impact of leucine on insulin-stimulated glucose uptake in muscle cells remains unclear. To address this, differentiated L6 myotubes were subjected to various compounds designed to either inhibit mTORc1 activity (rapamycin), blunt leucine intracellular import (BCH), or activate mTORc1 signalling (3BDO), prior to the determination of the uptake of the glucose analogue, 2-deoxyglucose (2-DG), in response to 1mM insulin. In separate experiments, L6 myotubes were subject to various media concentrations of leucine (0–0.8mM) for 24h before 2-DG uptake in response to insulin was assessed. Both rapamycin and BCH blunted 2-DG uptake, irrespective of insulin administration, and this occurred in parallel with a decline in mTOR, 4E-BP1, and p70S6K phosphorylation status, but little effect on AKT phosphorylation. In contrast, reducing leucine media concentrations suppressed 2-DG uptake, both under insulin- and non-insulin-stimulated conditions, but did not alter the phosphorylation state of AKT-mTORc1 components examined. Unexpectedly, 3BDO failed to stimulate mTORc1 signalling, but, nonetheless, caused a significant increase in 2-DG uptake under non-insulin-stimulated conditions. Both leucine and mTORc1 influence glucose uptake in muscle cells independent of insulin administration, and this likely occurs via distinct but overlapping mechanisms

Effect of adeno-associated virus (AAV)-mediated overexpression of PEPCK-M (Pck2) on Clenbuterol-induced muscle growth

We previously identified PEPCK-M (encoded by the Pck2 gene) to be highly up-regulated in skeletal muscle of pigs treated with Ractopamine, an anabolic beta-adrenergic receptor agonist. To determine whether PEPCK-M had a causative role in modulating the skeletal muscle growth response to Ractopamine, we used adeno-associated virus 1 (AAV1) to over-express Pck2 (AAV-Pck2) in murine skeletal muscle. A contralateral limb design was employed, such that each mouse served as its own control (injected with a GFP-only expressing AAV1, labelled AAV-GFP). Daily injections of Clenbuterol (1 mg/kg for 21 days) or vehicle control were also carried out to assess the effects of AAV-Pck2 overexpression on the anabolic response to a beta-adrenergic agonist. AAV-Pck2 overexpression in leg muscles of male C57BL6/J mice for 4 weeks (6–10 weeks of age) increased Pck2 mRNA (~100-fold), protein (not quantifiable) and enzyme activity (~3-fold). There was a trend (p = 0.0798) for AAV-Pck2 overexpression to reduce TA muscle weights, but there was no significant effect on muscle fibre diameters or myosin heavy chain isoform (MyHC) mRNA expression. When skeletal muscle growth was induced by daily administration of Clenbuterol (for 21 days), overexpression of AAV-Pck2 had no effect on the growth response, nor did it alter the expression of Phosphoserine Aminotransferase-1 (Psat1) or Asparagine Synthetase (Asns) mRNA or the Clenbuterol-induced decreases in MyHC IIa and IIx mRNA expression (p = 0.0065 and p = 0.0267 respectively). However AAV-Pck2 overexpression reduced TA muscle weights (p = 0.0434), particularly in the Control (vehicle treated) mice (p = 0.059 for AAV x Clenbuterol interaction) and increased the expression of Seryl-tRNA Synthetase (Sars) mRNA (p = 0.0477). Hence, contrary to the original hypothesis, AAV-Pck2 overexpression reduced TA muscle weights and did not mimic or alter the muscle hypertrophic effects of the beta-adrenergic agonist, Clenbuterol

98 The effect of mezquite pod flour in a wheat-based diet on broiler chicken growth performance

Application Inclusion of Mezquite pod flour in broiler chicken diets significantly reduced weight gain, this could inform a recommendation against the use of non-processed Mezquite in broiler chicken diets. Introduction Mezquite is an under-utilised crop and a possible alternative to soybean. Mezquite is part of the genus Prosopis, a group of leguminous trees. Their pods, containing seeds, are valued for their nutrition and are often ground into flour with 7–17% protein [1]. There are few studies looking at feeding pods to monogastric species, but Girma et al. [2] found that a 30% inclusion reduced growth and feed intake in broiler chickens, while a 10 or 20% inclusion had no significant effect. This study aimed to assess the impact of a 15% inclusion of Mezquite pod flour on broiler chicken growth and performance. Materials and Methods This trial was approved by the Animal Welfare & Ethical Review Body at the University of Nottingham. Eighty, one day old Ross 308 Broiler chickens were group housed for 6 days before being split into 20 pens of 4. Birds were fed either ad-libitum wheat-soya based diet (CON n = 40), or 15% inclusion of non-processed ground Mezquite pod (Prosopis juliflora) (MEZQ n = 40); collected from the Baringo area, Kenya. Both diets were balanced for equivalent energy and protein content, predominantly replacing wheat. Feed intake and weights were recorded twice weekly, before culling on days 35 and 36. The right breast muscle and liver were dissected and weighed. Data was analysed by one-way (treatment) or two-way (time × treatment) ANOVA (Genstat 19th edition), with significance taken as p < 0.05. Results A 15% inclusion of Mezquite pod flour in the diet negatively affected weight gain and FCR. A two-way ANOVA showed a significant interaction between time and feed for weight gain (data not shown), due to significantly higher growth of CON broilers. Mezquite inclusion therefore significantly decreased the final bodyweight (P < 0.001, one-way ANOVA). Total feed intake was similar, resulting in a significantly higher FCR for the MEZQ group (p < 0.001, one-way ANOVA). CON breast muscle weights were significantly higher (p < 0.001, one-way ANOVA). Liver weights were taken as an indication of effects on metabolic active tissues, but these were similar (Table 1). Conclusion These results suggest that Mezquite pod inclusion affects feed utilisation. This could be due to antinutritional factors, such as tannins or trypsin inhibitors, inhibiting digestion. Further study is needed to identify if antinutritional factors are responsible, and whether post-harvest processing or the use of exogenous enzymes reduces the negative impact