DIET INDUCED OBESITY MAY AFFECT THE FORCE-VELOCITY RELATIONSHIP IN RAT SOLEUS

INTRODUCTION Obesity is associated with chronic, low-grade inflammation that has been shown to affect several musculoskeletal tissues [1].  Previously, we observed that diet induced obesity (DIO) using a high-fat, high sugar diet results in molecular and morphological alterations in muscles, including an increase in intramuscular fat. However, it remains unclear if these alterations affect muscle function, as few studies have characterized the functional properties of muscles in obese individuals [2]. Arguably, one of the most important functional properties of skeletal muscle is the force-velocity relationship (FVR).  When muscles are shortening at increasing velocities, force decays exponentially as the proportion of attached cross bridges and the average force per cross-bridge decreases. As fat infiltration reduces the contractile material and structure of obese muscles, the functional properties of muscles, specifically the FVR may be affected. Therefore, the purpose of this study was to characterize the FVR in rat soleus muscles obtained from obese and normal rats. We hypothesized that soleus muscles of obese rats would produce lower absolute and relative forces at any given shortening velocity compared with muscles from control animals. METHODS Outbred, individually housed male Sprague-Dawley rats, aged 10-12 weeks, were randomized to a high fat, high sugar diet (DIO, 40% fat 45% sucrose, n=9) or a standard chow diet (n=5, chow, 12% fat, 0% sucrose) for 12 weeks. Prior to surgery, animals were sedated, weighed, and body composition was quantified using dual energy X-ray absorptiometry. The right soleus muscle was exposed and a custom cuff-type electrode was implanted on the tibial nerve. The soleus tendon was isolated from the Achilles with the calcaneus attached and fixed to a motor along the muscle’s natural alignment. The muscle was then stretched to its optimum length and electrically stimulated at 35Hz at 2.5x the motor unit threshold [3]. An isometric force reading was acquired over 2.5 s stimulation. The soleus was then stretched past its optimum length and shortened at increasing velocities, a force reading was collected at optimal length using Windaq Software. Data were collected at 1000Hz. Forces during shortening were measured at the same length and time point as the isometric reference force. This process was repeated for shortening velocities increasing from 2 mm/s to 70 mm/s. Animals were sacrificed and soleus muscles were harvested and weighed. Data were processed using a custom Matlab ® zero-phase filter program. Instantaneous forces were normalized to the peak isometric force for each animal. Comparisons were made using a Student’s t-tests, α=0.05. RESULTS On average, DIO animals had higher body mass and body fat compared to the control rats (p<0.001). Soleus mass was similar between DIO animals and chow (p=0.321), as was peak active isometric force (DIO: 2.48±0.10 N, chow: 2.08±0.33 p=0.183). FVR relationships were statistically different at shortening velocities between 3 and 35 mm/s (DIO > Chow at each given velocity; p<0.05, Fig. 1). DISCUSSION The results opposed the expected outcomes of this study and, therefore, the hypothesis was not satisfied. These findings could be due to a higher proportion of fast twitch fibers in the DIO or longer fascicle lengths in the DIO rats, but these speculations remain to be tested. Another possible explanation involves a potential increase in sensitivity to stimulation in the DIO soleus muscle resulting in increased force. Future work will examine other structural levels and muscle contractile proteins to understand these preliminary findings

THE EFFECTS OF DIET INDUCED OBESITY ON THE FORCE-LENGTH RELATIONSHIP IN RAT SOLEUS

INTRODUCTION Obesity is associated with chronic inflammation, which has been shown to affect the integrity of musculoskeletal tissues [1]. Previous data from our group suggests that obesity can result in intramuscular fat deposition [1]. It is unclear if this structural alteration has functional consequences, as the implications of obesity on muscle mechanics are not well understood. Therefore, the purpose of this study was to quantify the active force produced by soleus muscles of obese and non-obese rats at a range of muscle lengths. As the inclusion of fat into the muscle fibers will leave less room for contractile proteins, we hypothesized that obese rats will produce lower forces normalized to muscle mass at every length than non-obese control rats.   METHODS Fourteen rats were randomly allocated to a 12-week diet: either an obesity-inducing high fat high sucrose diet (DIO, 40% fat, 45% sucrose, n=8) or a standard chow diet (chow, 12% fat 0% sucrose, n=6). Prior to surgery, body composition was evaluated using dual energy X-ray absorptiometry. Custom-made tibial nerve cuffs were surgically attached to the right tibial nerve of each animal. The soleus was exposed, mechanically isolated, and clamped to a force transducer. The muscle was then stretched to a predetermined length and electrically stimulated at 3 times the motor unit threshold (50Hz) and the force output was measured [3]. Force tracings were digitized using WINDAQ® software. Passive, active, and total forces produced by the soleus were normalized to the maximum in vivo length of each animal. Forces were averaged into 5% length intervals within each animal. Students t-tests or a two-way ANOVA were conducted between groups, and a Bonferroni correction was used as needed, α=0.05. RESULTS DIO rats had increased body mass (DIO 816.4 ± 30.1g, chow 645.0 ± 28.3g; p<0.05) and body fat (DIO 39.2 ± 1.3%, chow 21.8 ± 2.1%; p<0.05) compared to chow-fed rats. Soleus mass (DIO: 0.28 ± 0.01 g, chow: 0.26 ± 0.11 g, p=0.32), was similar between the two groups. Absolute peak isometric force was similar between the two groups (DIO: 2.58 ± 0.10 N, chow: 2.18 ± 0.34 N, p=0.23). Active isometric force normalized to soleus mass was significantly higher in DIO group rats at every muscle length (Figure 1, p<0.05). DISCUSSION AND CONCLUSIONS On average, DIO rats produced more active force at a given normalized length and soleus mass than chow rats, a finding that refutes our original hypothesis. Since optimal length occurs at the same relative muscle length for both groups, and since the decline in force from maximum is similar between groups, it appears that fascicle length, and an associated shift in the force-length relationship cannot explain our results. Results of differences in the force-velocity relationship (not shown here) suggest that the DIO rats may have a higher proportion of fast twitch fibres, but the relative force among slow and fast fibres is similar, and thus also should not affect these results. The results suggest that the force per cross-sectional area is higher in muscles from obese compared to lean rats, a finding that defies explanation at this time and needs thorough investigation in the future. Histology and tests looking at fibre and cell level muscle structures may provide more insight

Integrated farm management for sustainable agriculture:lessons for knowledge exchange and policy

As a response to the environmentally and socially destructive practices of post-war mechanization and intensification, the concept of sustainable agriculture has become prominent in research, policy, and practice. Sustainable agriculture aims to balance the economic, environmental, and social aspects of farming, creating a resilient farming system in the long-term. Over the last few decades, various concepts have been used in research and policy to encourage the adoption of sustainable practices. Within such a congested space, this paper assesses the value of ‘integrated farm management’ as a concept for the promotion of sustainable agriculture. The concept is the subject of renewed policy interest in England and Wales and it is also being promoted in Europe. Previous research, however, has suggested that integrated farm management may not be well understood or widely practised. There are also criticisms that it can be impractical and poorly differentiated from similar ideas. As such, renewed insights are required into how useful the concept might be for encouraging sustainable agriculture. Using a mixed methods approach, we gathered the views of farmers, farm advisors, and industry representatives about integrated farm management in England and Wales, and interpreted these through a theoretical framework to judge the strength of the concept. Overall, the general principles of Integrated Farm Management were found to be coherent and familiar to most of our respondents. However, the concept performed poorly in terms of its resonance, simplicity of message, differentiation from other similar terms and theoretical utility. We reflect on our findings in the context of other ways to promote sustainable agriculture, drawing out messages for policy and knowledge exchange in England and Wales, as well as elsewhere.This research was funded as part of Defra's Sustainable Intensification Platform (Project Code LM0201). In addition, WJS was funded by Arcadia, LVD was funded by the UK Natural Environment Research Council partly under the Biodiversity and Ecosystem Service Sustainability (BESS) programme, grant codes NE/K015419/1 and NE/N014472/1

4-Oxocyclo­hexa­neacetic acid: catemeric hydrogen bonding and spontaneous resolution of a single conformational enanti­omer in an achiral ∊-keto acid

The asymmetric unit of the title compound, C8H12O3, consists of a single conformational enanti­omer, which aggregates in the catemeric acid-to-ketone hydrogen-bonding mode [O⋯O = 2.682 (4) Å and O—H⋯O = 172 (6)°]. Four hydrogen-bonding chains of translationally related mol­ecules pass through the cell orthogonal to the 43 screw axis along c, alternating in the 110 and the 10 direction, with alignment with respect to this axis of + + − −. Successive chains are rotated by 90° around the c axis. One C—H⋯O=C close contact, involving the carboxyl group, exists

Vaccine-elicited human T cells recognizing conserved protein regions inhibit HIV-1

Virus diversity and escape from immune responses are the biggest challenges to the development of an effective vaccine against HIV-1. We hypothesized that T-cell vaccines targeting the most conserved regions of the HIV-1 proteome, which are common to most variants and bear fitness costs when mutated, will generate effectors that efficiently recognize and kill virus-infected cells early enough after transmission to potentially impact on HIV-1 replication and will do so more efficiently than whole protein-based T-cell vaccines. Here, we describe the first-ever administration of conserved immunogen vaccines vectored using prime-boost regimens of DNA, simian adenovirus and modified vaccinia virus Ankara to uninfected UK volunteers. The vaccine induced high levels of effector T cells that recognized virus-infected autologous CD4+ cells and inhibited HIV-1 replication by up to 5.79 log10. The virus inhibition was mediated by both Gag- and Pol- specific effector CD8+ T cells targeting epitopes that are typically subdominant in natural infection. These results provide proof of concept for using a vaccine to target T cells at conserved epitopes, showing that these T cells can control HIV-1 replication in vitro

Structurally diverse mitochondrial branched chain aminotransferase (BCATm) leads with varying binding modes identified by fragment screening

Inhibitors of mitochondrial branched chain aminotransferase (BCATm), identified using fragment screening, are described. This was carried out using a combination of STD-NMR, thermal melt (Tm), and biochemical assays to identify compounds that bound to BCATm, which were subsequently progressed to X-ray crystallography, where a number of exemplars showed significant diversity in their binding modes. The hits identified were supplemented by searching and screening of additional analogues, which enabled the gathering of further X-ray data where the original hits had not produced liganded structures. The fragment hits were optimized using structure-based design, with some transfer of information between series, which enabled the identification of ligand efficient lead molecules with micromolar levels of inhibition, cellular activity, and good solubility

Human umbilical cord perivascular cells improve human pancreatic islet transplant function by increasing vascularization

Islet transplantation is an efficacious therapy for type 1 diabetes; however, islets from multiple donor pancreata are required, and a gradual attrition in transplant function is seen. Here, we manufactured human umbilical cord perivascular mesenchymal stromal cells (HUCPVCs) to Good Manufacturing Practice (GMP) standards. HUCPVCs showed a stable phenotype while undergoing rapid ex vivo expansion at passage 2 (p2) to passage 4 (p4) and produced proregenerative factors, strongly suppressing T cell responses in the resting state and in response to inflammation. Transplanting an islet equivalent (IEQ):HUCPVC ratio of 1:30 under the kidney capsule in diabetic NSG mice demonstrated the fastest return to normoglycemia by 3 days after transplant: Superior glycemic control was seen at both early (2.7 weeks) and later stages (7, 12, and 16 weeks) versus ratios of 1:0, 1:10, and 1:50, respectively. Syngeneic islet transplantation in immunocompetent mice using the clinically relevant hepatic portal route with a marginal islet mass showed that mice transplanted with an IEQ:HUCPVC ratio of 1:150 had superior glycemic control versus ratios of 1:0, 1:90, and 1:210 up to 6 weeks after transplant. Immunodeficient mice transplanted with human islets (IEQ:HUCPVC ratio of 1:150) exhibited better glycemic control for 7 weeks after transplant versus islet transplant alone, and islets transplanted via the hepatic portal vein in an allogeneic mouse model using a curative islet mass demonstrated delayed rejection of islets when cotransplanted with HUCPVCs (IEQ:HUCPVC ratio of 1:150). The immunosuppressive and proregenerative properties of HUCPVCs demonstrated long-term positive effects on graft function in vivo, indicating that they may improve long-term human islet allotransplantation outcomes

The circadian clock protein REVERBα inhibits pulmonary fibrosis development

Pulmonary inflammatory responses lie under circadian control; however, the importance of circadian mechanisms in the underlying fibrotic phenotype is not understood. Here, we identify a striking change to these mechanisms resulting in a gain of amplitude and lack of synchrony within pulmonary fibrotic tissue. These changes result from an infiltration of mesenchymal cells, an important cell type in the pathogenesis of pulmonary fibrosis. Mutation of the core clock protein REVERBα in these cells exacerbated the development of bleomycin-induced fibrosis, whereas mutation of REVERBα in club or myeloid cells had no effect on the bleomycin phenotype. Knockdown of REVERBα revealed regulation of the little-understood transcription factor TBPL1. Both REVERBα and TBPL1 altered integrinβ1 focal-adhesion formation, resulting in increased myofibroblast activation. The translational importance of our findings was established through analysis of 2 human cohorts. In the UK Biobank, circadian strain markers (sleep length, chronotype, and shift work) are associated with pulmonary fibrosis, making them risk factors. In a separate cohort, REVERBα expression was increased in human idiopathic pulmonary fibrosis (IPF) lung tissue. Pharmacological targeting of REVERBα inhibited myofibroblast activation in IPF fibroblasts and collagen secretion in organotypic cultures from IPF patients, thus suggesting that targeting of REVERBα could be a viable therapeutic approach