Effect of Process Parameters and Build Orientation on Microstructure and Impact Energy of Electron Beam Powder Bed Fused Ti-6Al-4V.

To fully exploit the benefits of additive manufacturing (AM), an understanding of its processing, microstructural, and mechanical aspects, and their interdependent characteristics, is necessary. In certain instances, AM materials may be desired for applications where impact toughness is a key property, such as in gas turbine fan blades, where foreign or direct object damage may occur. In this research, the impact energy of a series of Ti-6Al-4V specimens produced via electron beam powder bed fusion (EBPBF) was established via Charpy impact testing. Specimens were produced with five different processing parameter sets, in both the vertical and horizontal build orientation, with microstructural characteristics of prior ß grain area, prior ß grain width, and α lath width determined in the build direction. The results reveal that horizontally oriented specimens have a lower impact energy compared to those built in the vertical orientation, due to the influence of epitaxial grain growth in the build direction. Relationships between process parameters, microstructural characteristics, and impact energy results were evaluated, with beam velocity displaying the strongest trend in terms of impact energy results, and normalised energy density exhibiting the most significant influence across all microstructural measurements.

Defective fasting-induced PKA activation impairs adipose tissue glycogen degradation in obese Zucker rats.

BACKGROUND: Obesity is associated with development of insulin resistance in adipose tissue (AT). Human obesity has been associated with increased glycogen deposition in adipocytes. Adipocytes synthesise glycogen prior to the formation of lipids. The present study examined adipose glycogen content in obese Zucker rats and the effect of fasting on glycogen-metabolising enzymes. We hypothesised that obesity imposes a blunted response to fasting through impaired activation of glycogen-metabolizing enzymes, which dampens glycogen mobilization in obese Zucker rats. METHODS: We investigated the effect of 24h fasting on AT glycogen metabolism in 12-week old obese Zucker rats. Epididymal fat pads were collected from rats fed ad-libitum and fasted for 24h. Glycogen content, glycogen synthase and phosphorylase enzyme activity, and PKA activity were analysed as well as total and phosphorylated protein content for glycogen-metabolizing enzymes glycogen synthase and phosphorylase, glucose transporter GLUT4, and cAMP-dependent response element binding protein levels. RESULTS: Twelve-week old obese Zucker rats showed increased AT glycogen content (adipose glycogen content [mean ± SD], lean: 3.95 ± 2.78 to 0.75 + 0.69 µg.mg-1; p < 0.005 fed vs fasted, and obese: 5.23 ± 3.38 to 5.019 ± 1.99 µg.mg-1; p = ns fed and fasted and p < 0.005 lean vs obese), and impaired fasting-induced glycogen mobilization following a 24h fast. These defects were associated with dysfunctional glycogen-metabolizing enzymes, characterized by: (1) blunted phosphorylation-mediated activation and downregulated protein expression of glycogen phosphorylase, and (2) an impaired phosphorylation-mediated inactivation of glycogen synthase. Furthermore, these defects were related to impaired fasting-induced protein kinase A (PKA) activation. CONCLUSION: This study provides evidence of a defective glycogen metabolism in the adipose associated with impaired fasting-induced activation of the upstream kinase protein kinase A, which render a converging point to obesity-related primary alterations in carbohydrate and lipid metabolism in the AT.

The Influence of Process Parameters and Build Orientation on the Creep Behaviour of a Laser Powder Bed Fused Ni-based Superalloy for Aerospace Applications.

Additive Layer Manufacturing (ALM) is an innovative net shape manufacturing technology that offers the ability to produce highly intricate components not possible through traditional wrought and cast procedures. Consequently, the aerospace industry is becoming ever more attentive in exploiting such technology for the fabrication of nickel-based superalloys in an attempt to drive further advancements within the holistic gas turbine. Given this, the requirement for the mechanical characterisation of such material is rising in parallel, with limitations in the availability of material processed restricting conventional mechanical testing; particularly with the abundance of process parameters to evaluate. As such, the Small Punch Creep (SPC) test method has been deemed an effective tool to rank the elevated temperature performance of alloys processed through ALM, credited to the small volumes of material utilised in each test and the ability to sample material from discrete locations. In this research, the SPC test will be used to assess the influence of a number of key process variables on the mechanical performance of Laser Powder Bed Fused (LPBF) Ni-based superalloy CM247LC. This will also include an investigation into the influence of build orientation and post-build treatment on creep performance, whilst considering the structural integrity of the different experimental builds.

Mas Receptor Activation Slows Tumor Growth and Attenuates Muscle Wasting in Cancer.

Cancer cachexia is a multifactorial syndrome characterized by a progressive loss of skeletal muscle mass associated with significant functional impairment. Cachexia robs patients of their strength and capacity to perform daily tasks and live independently. Effective treatments are needed urgently. Here, we investigated the therapeutic potential of activating the "alternative" axis of the renin-angiotensin system, involving ACE2, angiotensin-(1-7), and the mitochondrial assembly receptor (MasR), for treating cancer cachexia. Plasmid overexpression of the MasR or pharmacologic angiotensin-(1-7)/MasR activation did not affect healthy muscle fiber size in vitro or in vivo but attenuated atrophy induced by coculture with cancer cells in vitro. In mice with cancer cachexia, the MasR agonist AVE 0991 slowed tumor development, reduced weight loss, improved locomotor activity, and attenuated muscle wasting, with the majority of these effects dependent on the orexigenic and not antitumor properties of AVE 0991. Proteomic profiling and IHC revealed that mechanisms underlying AVE 0991 effects on skeletal muscle involved miR-23a-regulated preservation of the fast, glycolytic fibers. MasR activation is a novel regulator of muscle phenotype, and AVE 0991 has orexigenic, anticachectic, and antitumorigenic effects, identifying it as a promising adjunct therapy for cancer and other serious muscle wasting conditions. SIGNIFICANCE: These findings demonstrate that MasR activation has multiple benefits of being orexigenic, anticachectic, and antitumorigenic, revealing it as a potential adjunct therapy for cancer.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/4/706/F1.large.jpg.See related commentary by Rupert et al., p. 699.

Predicting Receipt of Social Security Administration Disability Benefits Using Biomarkers and Other Physiological Measures: Evidence From the Health and Retirement Study.

OBJECTIVES: The objective of this study was to assess how well physiological measures, including biomarkers and genetic indicators, predict receipt of Social Security Administration (SSA) disability benefits among U.S. adults aged 51 to 65 years. METHOD: We used data from the 2006 to 2012 waves of the Health and Retirement Study (HRS), linked to SSA administrative data. Using logistic regression, we predicted benefit receipt (either Social Security Disability Insurance or Supplemental Security Income) using 19 distinct physiological markers, adjusting for age, sex, race, and select medication use. We then calculated the propensity (i.e., predicted probability) that each HRS respondent received benefits and assessed how well propensity score-based classifications could identify beneficiaries and nonbeneficiaries. RESULTS: Thirteen percent of respondents received benefits. Using the propensity score cut point that maximized the sum of sensitivity and specificity, the model correctly predicted 75.9% of beneficiaries and 73.5% of nonbeneficiaries. DISCUSSION: Physiological measures have moderate power to predict SSA disability benefit receipt.

No evidence of direct association between GLUT4 and glycogen in human skeletal muscle.

Previous studies have demonstrated that exercise increases whole body and skeletal muscle insulin sensitivity that is linked with increased GLUT4 at the plasma membrane following insulin stimulation and associated with muscle glycogen depletion. To assess the potential direct association between muscle glycogen and GLUT4, seven untrained, male subjects exercised for 60 min at ~75% VO2 peak, with muscle samples obtained by percutaneous needle biopsy immediately before and after exercise. Exercise reduced muscle glycogen content by ~43%. An ultracentrifugation protocol resulted in a ~2-3-fold enriched glycogen fraction from muscle samples for analysis. Total GLUT4 content was unaltered by exercise and we were unable to detect any GLUT4 in glycogen fractions, either with or without amylase treatment. In skinned muscle fiber segments, there was very little, if any, GLUT4 detected in wash solutions, except following exposure to 1% Triton X-100. Amylase treatment of single fibers did not increase GLUT4 in the wash solution and there were no differences in GLUT4 content between fibers obtained before or after exercise for any of the wash treatments. Our results indicate no direct association between GLUT4 and glycogen in human skeletal muscle, before or after exercise, and suggest that alterations in GLUT4 translocation associated with exercise-induced muscle glycogen depletion are mediated via other mechanisms.

Early Identification of Potential SSDI Entrants in California: The Predictive Value of State Disability Insurance and Workers' Compensation Claims.

Purpose Examine the potential for using information in short-term disability claims to identify workers at high risk of leaving the workforce and entering Social Security Disability Insurance (SSDI). Methods We analyze state-wide California data on claimants of State Disability Insurance (SDI) and Workers' Compensation (WC) and present statistics on: (1) characteristics (primary diagnosis, sex, age, geography, wage level) by claim duration (0-3, 4-6, 7-12, 12 + months); and (2) the ability of initial claim characteristics to predict duration of at least 12 months. All data are for claims with disability lasting more than 1 week. Results 22.2% of SDI claims last longer than 6 months and 12.5% last 12 months. More WC claims reach these durations: 33.7 and 18.6%, respectively. Long-term SDI and WC claimants are similar to SSDI awardees, nationwide, but differ in age distribution; they are typically younger. Conclusions Characteristics of SDI and WC claims can help predict claims likely to last 12 months, but more information is needed to effectively target early intervention services. Waiting longer to intervene improves targeting but risks missing opportunities where early intervention could be more effective. Collecting additional information at SDI or WC entry or soon thereafter could improve both the efficiency and timing of interventions.

Mail order pharmacy use and adherence to secondary prevention drugs among stroke patients.

BACKGROUND AND PURPOSE: Mail order pharmacies (MOP) are increasingly being used to deliver medications for chronic disease management. Their use is linked to similar or even greater medication adherence than local pharmacy (LP) use. We are unaware of any studies that have evaluated the association of mail order pharmacy use with drug adherence among stroke patients. METHODS: We conducted cross-sectional analyses of patients discharged with ischemic stroke from 24 hospitals in a managed care network, who received a new anticoagulant, antiplatelet, anti-glycemic, antihypertensive, and/or lipid-lowering medication between January 1, 2007 and June 30, 2015. We defined good adherence as medication availability ≥80% of the time, and compared adherence between mail-order users (≥66% of refills by mail) and local pharmacy users (all refills in person). Relationship between delivery method and adherence was evaluated using multivariate regression models. RESULTS: A total of 44,658 eligible patients refilled an index medication. Of these, 13,295 in the LP and 6801 in MOP groups met inclusion criteria. Patients in the MOP group were more likely to be white, and less likely to have hypertension, diabetes, and smoke tobacco. Continuous Medication Gap (CMG) adherence was 0.28 in the LP group and 0.11 in the MOP group (p < 0.001). At 90-days there were 893 hospital readmissions for the LP group and 375 for the MOP group for a rate of 0.07 vs 0.06 (p < 0.001). In the multivariable analysis, adherence was associated with MOP use, (OR 0.12, 95% CI 0.11-0.14) and decreased readmission at 90 days (OR 0.62, 95% CI 0.55-0.71). CONCLUSIONS: Stroke patients who use MOP vs. LP are more likely to have good medication adherence. Future studies should examine the impact of mail-order pharmacy use on vascular risk marker control and events after stroke.

Unravelling the Carbohydrate-Binding Preferences of the Carbohydrate-Binding Modules of AMP-Activated Protein Kinase.

The ß subunit of adenosine monophosphate (AMP)-activated protein kinase (AMPK), which exists as two isoforms (ß1 and ß2) in humans, has a carbohydrate-binding module (CBM) that interacts with glycogen. Although the ß1- and ß2-CBMs are structurally similar, with strictly conserved ligand-contact residues, they show different carbohydrate affinities. ß2-CBM shows the strongest affinity for both branched and unbranched oligosaccharides and it has recently been shown that a Thr insertion into ß2-CBM (Thr101) forms a pocket to accommodate branches. This insertion does not explain why ß2-CBM binds all carbohydrates with stronger affinity. Herein, it is shown that residue 134 (Val for ß2 and Thr for ß1), which does not come into contact with a carbohydrate, appears to account for the affinity difference. Characterisation by NMR spectroscopy, however, suggests that mutant ß2-Thr101Δ/Val134Thr differs from that of ß1-CBM, and mutant ß1-Thr101ins/Thr134Val differs from that of ß2-CBM. Furthermore, these mutants are less stable to chemical denaturation, relative to that of wild-type ß-CBMs, which confounds the affinity analyses. To support the importance of Thr101 and Val134, the ancestral CBM has been constructed. This CBM retains Thr101 and Val134, which suggests that the extant ß1-CBM has a modest loss of function in carbohydrate binding. Because the ancestor bound carbohydrate with equal affinity to that of ß2-CBM, it is concluded that residue 134 plays an indirect role in carbohydrate binding.

Employment Support for the Transition to Retirement.

As workers near retirement, many experience a medical event that limits the ability to work. Public programs provide health insurance and income support for these individuals, but that support is often not adequate to protect against poverty following the onset of a new health condition. Moreover, these policies generally are not designed to encourage continuing work rather than premature retirement. In this article, we propose a new type of program-Employment Support for the Transition to Retirement-designed to encourage older workers with health limitations to remain in the workforce, reducing their reliance on federal disability and early retirement benefits. We illustrate that a simple version of this type of program could significantly reduce poverty in the target population, would cost less per participant than existing programs, and could potentially contribute to greater economic security and reduced growth in federal expenditures as an element of social security policy reforms.

Using the Health and Retirement Study for Disability Policy Research: A Review.

The Health and Retirement Study (HRS) is a preeminent data source for research related to the experiences of workers nearing retirement, including the large share of those workers who experience a health shock or disability onset after age 50. In this article, we highlight key information collected from HRS respondents that benefits disability policy research and the body of knowledge that has resulted from this information. Our main goal is to identify from this research experience potential improvements in data collection and documentation that would further strengthen the HRS as a data source for disability policy researchers.

ß-subunit myristoylation functions as an energy sensor by modulating the dynamics of AMP-activated Protein Kinase.

The heterotrimeric AMP-activated protein kinase (AMPK), consisting of α, ß and γ subunits, is a stress-sensing enzyme that is activated by phosphorylation of its activation loop in response to increases in cellular AMP. N-terminal myristoylation of the ß-subunit has been shown to suppress Thr172 phosphorylation, keeping AMPK in an inactive state. Here we use amide hydrogen-deuterium exchange mass spectrometry (HDX-MS) to investigate the structural and dynamic properties of the mammalian myristoylated and non-myristoylated inactivated AMPK (D139A) in the presence and absence of nucleotides. HDX MS data suggests that the myristoyl group binds near the first helix of the C-terminal lobe of the kinase domain similar to other kinases. Our data, however, also shows that ATP.Mg2+ results in a global stabilization of myristoylated, but not non-myristoylated AMPK, and most notably for peptides of the activation loop of the α-kinase domain, the autoinhibitory sequence (AIS) and the ßCBM. AMP does not have that effect and HDX measurements for myristoylated and non-myristoylated AMPK in the presence of AMP are similar. These differences in dynamics may account for a reduced basal rate of phosphorylation of Thr172 in myristoylated AMPK in skeletal muscle where endogenous ATP concentrations are very high.

Muscle-specific deletion of SOCS3 increases the early inflammatory response but does not affect regeneration after myotoxic injury.

BACKGROUND: Muscles of old animals are injured more easily and regenerate poorly, attributed in part to increased levels of circulating pro-inflammatory cytokines. The Janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling cascade is a key mediator of inflammatory cytokine action, and signaling via this pathway is increased in muscles with aging. As a negative regulator of JAK/STAT signaling, a key mediator of myogenic proliferation and differentiation, altered expression of suppressor of cytokine signaling (SOCS3) is likely to have important consequences for muscle regeneration. To model this scenario, we investigated the effect of SOCS3 deletion within mature muscle fibers on injury and repair. We tested the hypothesis that reduced SOCS3 function would alter the inflammatory response and impair muscle regeneration after myotoxic injury. METHODS: Mice with a specific deletion of SOCS3 within mature skeletal muscle fibers were used to assess the effect of SOCS3 deletion on muscle injury and repair. Twelve-week-old or 24-month-old SOCS3 muscle-specific knockout (SOCS3 MKO) mice and littermate controls were either left uninjured or injured with a single injection of notexin (10 µg/ml) into the right tibialis anterior (TA) muscle. At 1, 2, 3, 5, 7, or 14 days post-injury, the right TA muscle was excised and subjected to histological, western immunoblotting, and gene expression analyses. Force production and fatigue were assessed in uninjured muscles and at 7 days post-notexin injury. RESULTS: In uninjured muscles, SOCS3 deletion decreased force production during fatigue but had no effect on the gross or histological appearance of the TA muscles. After notexin injury, deletion of SOCS3 increased STAT3 phosphorylation at day 1 and increased the mRNA expression of the inflammatory cytokine TNF-α, and the inflammatory cell markers F4/80 and CD68 at day 2. Gene expression analysis of the regeneration markers Pax7, MyoD, and Myogenin indicated SOCS3 deletion had no effect on the progression of muscle repair after notexin injury. Inflammation and regeneration were also unchanged in the muscles of 24-month-old SOCS3 MKO mice compared with control. CONCLUSIONS: Loss of SOCS3 expression in mature muscle fibers increased the inflammatory response to myotoxic injury but did not impair muscle regeneration in either adult or old mice. Therefore, reduced SOCS3 expression in muscle fibers is unlikely to underlie impaired muscle regeneration. Further investigation into the role of SOCS3 in other cell types involved in muscle repair is warranted.

When phosphorylated at Thr148, the ß2-subunit of AMP-activated kinase does not associate with glycogen in skeletal muscle.

The 5'-AMP-activated protein kinase (AMPK), a heterotrimeric complex that functions as an intracellular fuel sensor that affects metabolism, is activated in skeletal muscle in response to exercise and utilization of stored energy. The diffusibility properties of α- and ß-AMPK were examined in isolated skeletal muscle fiber segments dissected from rat fast-twitch extensor digitorum longus and oxidative soleus muscles from which the surface membranes were removed by mechanical dissection. After the muscle segments were washed for 1 and 10 min, ∼60% and 75%, respectively, of the total AMPK pools were found in the diffusible fraction. After in vitro stimulation of the muscle, which resulted in an ∼80% decline in maximal force, 20% of the diffusible pool became bound in the fiber. This bound pool was not associated with glycogen, as determined by addition of a wash step containing amylase. Stimulation of extensor digitorum longus muscles resulted in 28% glycogen utilization and a 40% increase in phosphorylation of the downstream AMPK target acetyl carboxylase-CoA. This, however, had no effect on the proportion of total ß2-AMPK that was phosphorylated in whole muscle homogenates measured by immunoprecipitation. These findings suggest that, in rat skeletal muscle, ß2-AMPK is not associated with glycogen and that activation of AMPK by muscle contraction does not dephosphorylate ß2-AMPK. These findings question the physiological relevance of the carbohydrate-binding function of ß2-AMPK in skeletal muscle.

Trends in disability and program participation among U.S. veterans.

BACKGROUND: Disability is increasingly part of the lives of veterans and more research is needed to understand its impact on veterans' participation in disability benefit programs. OBJECTIVE/HYPOTHESIS: We examine how recent trends in receipt of service-connected disability compensation from the Department of Veterans Affairs (VA) compare to trends in self-reported disability and participation in Social Security Disability Insurance (DI) and Supplemental Security Income (SSI) among veterans. METHODS: We use 2002-2013 data from the Current Population Survey to describe trends in receipt of VA disability compensation and to compare between trends in self-reported disability and DI/SSI participation for veterans versus nonveterans. RESULTS: The percentage of veterans reporting they receive VA disability compensation increased substantially from 2002 to 2013 and was especially notable among younger (ages 18-39) and older (ages 50-64) veterans. From 2009 to 2013, self-reported disability increased among the younger and older veterans but not among middle-age veterans and nonveterans, and self-reported cognitive disability increased substantially among young veterans. DI/SSI participation among older veterans increased more than for nonveterans over the period examined. CONCLUSIONS: Effective policies are needed to incentivize work among young veterans and to help them obtain both the skills they need to succeed in the labor force and the supports (such as psychiatric health services) they need to do so. Older veterans are facing increasing challenges in the labor market, and further research is needed to determine whether these challenges are primarily related to health, a growing skills gap, or poorly-aligned incentives.

Predicting Disability among Community-Dwelling Medicare Beneficiaries Using Claims-Based Indicators.

OBJECTIVES: To assess the feasibility of using existing claims-based algorithms to identify community-dwelling Medicare beneficiaries with disability based solely on the conditions for which they are being treated, and improving on these algorithms by combining them in predictive models. DATA SOURCE: Data on 12,415 community-dwelling fee-for-service Medicare beneficiaries who first responded to the Medicare Current Beneficiary Survey (MCBS) in 2003-2006. STUDY DESIGN: Logistic regression models in which six claims-based disability indicators are used to predict self-reported disability. Receiver operating characteristic (ROC) curves were used to assess the performance of the predictive models. PRINCIPAL FINDINGS: The predictive performance of the regression-based models is better than that of the individual claims-based indicators. At a predicted probability threshold chosen to maximize the sum of sensitivity and specificity, sensitivity is 0.72 for beneficiaries age 65 or older and specificity is 0.65. For those under 65, sensitivity is 0.54 and specificity is 0.67. The findings also suggest ways to improve predictive performance for specific disability populations of interest to researchers. CONCLUSIONS: Predictive models that incorporate multiple claims-based indicators provide an improved tool for researchers seeking to identify people with disabilities in claims data.

Molecular Insights into the Enigmatic Metabolic Regulator, SnRK1.

Sucrose non-fermenting-1 (SNF1)-related kinase 1 (SnRK1) lies at the heart of metabolic homeostasis in plants and is crucial for normal development and response to stress. Evolutionarily related to SNF1 in yeast and AMP-activated kinase (AMPK) in mammals, SnRK1 acts protectively to maintain homeostasis in the face of fluctuations in energy status. Despite a conserved function, the structure and regulation of the plant kinase differ considerably from its relatively well-understood opisthokont orthologues. In this review, we highlight the known plant-specific modes of regulation involving SnRK1 together with new insights based on a 3D molecular model of the kinase. We also summarise how these differences from other orthologues may be specific adaptations to plant metabolism, and offer insights into possible avenues of future inquiry into this enigmatic enzyme.

Regulation of Starch Stores by a Ca(2+)-Dependent Protein Kinase Is Essential for Viable Cyst Development in Toxoplasma gondii.

Transmissible stages of Toxoplasma gondii store energy in the form of the carbohydrate amylopectin. Here, we show that the Ca(2+)-dependent protein kinase CDPK2 is a critical regulator of amylopectin metabolism. Increased synthesis and loss of degradation of amylopectin in CDPK2 deficient parasites results in the hyperaccumulation of this sugar polymer. A carbohydrate-binding module 20 (CBM20) targets CDPK2 to amylopectin stores, while the EF-hands regulate CDPK2 kinase activity in response to Ca(2+) to modulate amylopectin levels. We identify enzymes involved in amylopectin turnover whose phosphorylation is dependent on CDPK2 activity. Strikingly, accumulation of massive amylopectin granules in CDPK2-deficient bradyzoite stages leads to gross morphological defects and complete ablation of cyst formation in a mouse model. Together these data show that Ca(2+) signaling regulates carbohydrate metabolism in Toxoplasma and that the post-translational control of this pathway is required for normal cyst development.

FT011, a Novel Cardiorenal Protective Drug, Reduces Inflammation, Gliosis and Vascular Injury in Rats with Diabetic Retinopathy.

Diabetic retinopathy features inflammation as well as injury to glial cells and the microvasculature, which are influenced by hypertension and overactivity of the renin-angiotensin system. FT011 is an anti-inflammatory and anti-fibrotic agent that has been reported to attenuate organ damage in diabetic rats with cardiomyopathy and nephropathy. However, the potential therapeutic utility of FT011 for diabetic retinopathy has not been evaluated. We hypothesized that FT011 would attenuate retinopathy in diabetic Ren-2 rats, which exhibit hypertension due to an overactive extra-renal renin-angiotensin system. Diabetic rats were studied for 8 and 32 weeks and received intravitreal injections of FT011 (50 µM) or vehicle (0.9% NaCl). Comparisons were to age-matched controls. In the 8-week study, retinal inflammation was examined by quantitating vascular leukocyte adherence, microglial/macrophage density and the expression of inflammatory mediators. Macroglial Müller cells, which exhibit a pro-inflammatory and pro-angiogenic phenotype in diabetes, were evaluated in the 8-week study as well as in culture following exposure to hyperglycaemia and FT011 (10, 30, 100 µM) for 72 hours. In the 32-week study, severe retinal vasculopathy was examined by quantitating acellular capillaries and extracellular matrix proteins. In diabetic rats, FT011 reduced retinal leukostasis, microglial density and mRNA levels of intercellular adhesion molecule-1 (ICAM-1). In Müller cells, FT011 reduced diabetes-induced gliosis and vascular endothelial growth factor (VEGF) immunolabeling and the hyperglycaemic-induced increase in ICAM-1, monocyte chemoattractant protein-1, CCL20, cytokine-induced neutrophil chemoattractant-1, VEGF and IL-6. Late intervention with FT011 reduced acellular capillaries and the elevated mRNA levels of collagen IV and fibronectin in diabetic rats. In conclusion, the protective effects of FT011 in cardiorenal disease extend to key elements of diabetic retinopathy and highlight its potential as a treatment approach.

Rat skeletal muscle glycogen degradation pathways reveal differential association of glycogen-related proteins with glycogen granules

Glycogenin, glycogen-debranching enzyme (GDE) and glycogen phosphorylase (GP) are important enzymes that contribute to glycogen particle metabolism. In Long-Evans Hooded rat whole muscle homogenates prepared from extensor digitorum longus (EDL, fast-twitch) and soleus (SOL, oxidative, predominantly slow twitch), it was necessary to include alfa-amylase, which releases glucosyl units from glycogen, to detect glycogenin but not GDE or GP. Up to ∼12 % of intramuscular glycogen pool was broken down using either in vitro electrical stimulation or leaving muscle at room temperature >3 h (delayed, post-mortem). Electrical stimulation did not reveal glycogenin unless alfa-amylase was added, although in post-mortem muscle ∼50 and ∼30 % of glycogenin in EDL and SOL muscles, respectively, was detected compared to the amount detected with alfa-amylase treatment. Single muscle fibres were dissected from fresh or post-mortem EDL muscles, mechanically skinned to remove surface membrane and the presence of glycogenin, GDE and GP as freely diffusible proteins (i.e. cytoplasmic localization) compared by Western blotting. Diffusibility of glycogenin (∼20 %) and GP (∼60 %) was not different between muscles, although GDE increased from ∼15 % diffusible in fresh muscle to ∼60 % in post-mortem muscle. Under physiologically relevant circumstances, in rat muscle and within detection limits: (1) The total cellular pool of glycogenin is always associated with glycogen granules, (2) GDE is associated with glycogen granules with over half the total pool associated with the outer tiers of glycogen, (3) GP is only ever weakly associated with glycogen granules and (4) addition of alfa-amylase is necessary in order to detect glycogenin, but not GDE or GP