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Background The skeletal muscle plays a central role in glucose homeostasis through the uptake of glucose from the extracellular medium in response to insulin. A number of factors are known to disrupt the normal response to insulin leading to the emergence of insulin resistance (IR). Advanced age and a high-fat diet are factors that increase the susceptibility to IR, with lipid accumulation in the skeletal muscle being a key driver of this phenomenon. It is debated, however, whether lipid accumulation arises due to dietary lipid overload or from a decline of mitochondrial function. To gain insights into the interplay of diet and age in the flexibility of muscle lipid and glucose handling, we combined lipidomics, proteomics, mitochondrial function analysis and computational modelling to investigate young and aged mice on a low- or high-fat diet (HFD). Results As expected, aged mice were more susceptible to IR when given a HFD than young mice. The HFD induced intramuscular lipid accumulation specifically in aged mice, including C18:0-containing ceramides and diacylglycerols. This was reflected by the mitochondrial β-oxidation capacity, which was upregulated by the HFD in young, but not in old mice. Conspicuously, most β-oxidation proteins were upregulated by the HFD in both groups, but carnitine palmitoyltransferase 1B (CPT1B) declined in aged animals. Computational modelling traced the flux control mostly to CPT1B, suggesting a CPT1B-driven loss of flexibility to the HFD with age. Finally, in old animals, glycolytic protein levels were reduced and less flexible to the diet. Conclusion We conclude that intramuscular lipid accumulation and decreased insulin sensitivity are not due to age-related mitochondrial dysfunction or nutritional overload alone, but rather to their combined effects. Moreover, we identify CPT1B as a potential target to counteract age-dependent intramuscular lipid accumulation and thereby IR.

International audience; Background and aims: Glycogen storage disease (GSD) type 1a is an inborn error of metabolism caused by defective glucose-6-phosphatase catalytic subunit (G6PC) activity. Patients with GSD 1a exhibit severe hepatomegaly due to glycogen and triglyceride (TG) accumulation in the liver. We have shown that the activity of carbohydrate response element binding protein (ChREBP), a key regulator of glycolysis and de novo lipogenesis, is increased in GSD 1a. In the current study, we assessed the contribution of ChREBP to nonalcoholic fatty liver disease (NAFLD) development in a mouse model for hepatic GSD 1a.Approach and results: Liver-specific G6pc-knockout (L-G6pc-/- ) mice were treated with adeno-associated viruses (AAVs) 2 or 8 directed against short hairpin ChREBP to normalize hepatic ChREBP activity to levels observed in wild-type mice receiving AAV8-scrambled short hairpin RNA (shSCR). Hepatic ChREBP knockdown markedly increased liver weight and hepatocyte size in L-G6pc-/- mice. This was associated with hepatic accumulation of G6P, glycogen, and lipids, whereas the expression of glycolytic and lipogenic genes was reduced. Enzyme activities, flux measurements, hepatic metabolite analysis and very low density lipoprotein (VLDL)-TG secretion assays revealed that hepatic ChREBP knockdown reduced downstream glycolysis and de novo lipogenesis but also strongly suppressed hepatic VLDL lipidation, hence promoting the storage of "old fat." Interestingly, enhanced VLDL-TG secretion in shSCR-treated L-G6pc-/- mice associated with a ChREBP-dependent induction of the VLDL lipidation proteins microsomal TG transfer protein and transmembrane 6 superfamily member 2 (TM6SF2), the latter being confirmed by ChIP-qPCR.Conclusions: Attenuation of hepatic ChREBP induction in GSD 1a liver aggravates hepatomegaly because of further accumulation of glycogen and lipids as a result of reduced glycolysis and suppressed VLDL-TG secretion. TM6SF2, critical for VLDL formation, was identified as a ChREBP target in mouse liver. Altogether, our data show that enhanced ChREBP activity limits NAFLD development in GSD 1a by balancing hepatic TG production and secretion.

Objective To compare Sonicision cordless ultrasonic dissector (SCUD) to LigaSure vessel sealing device (LVSD) for laparoscopic ovariectomy (Lap OVE) in dogs. Study design Randomized, paired prospective clinical trial. Animals Client-owned dogs (n = 22) presented for elective Lap OVE. Methods Dogs were randomly assigned to one of two protocols: protocol 1 required the left ovary resected using SCUD and the right ovary using LVSD; protocol 2 required the left ovary resected using LVSD and the right ovary using SCUD. Duration of ovary excision, complications, surgical smoke production, and collateral thermal damage were compared between SCUD and LVSD. Total surgery duration, postoperative convalescence, obesity, mesovarial fat score, and technique-associated costs were also recorded. Results Ovary excision was significantly faster with LVSD than SCUD. Surgical smoke production was significantly greater for SCUD than LVSD. Minor pedicle hemorrhage occurred 3 times with SCUD and one time with LVSD (not significantly different) and was easily corrected intraoperative. Presence of hemorrhage significantly increased ovary excision time. Technique-associated costs were lower for SCUD than LVSD. No significant differences were found in collateral thermal damage between SCUD and LVSD. Total surgery duration and convalescence time were similar to previous reports of Lap OVE in dogs at the authors' institution. Conclusions SCUD is a cost-effective alternative for Lap OVE, taking into account differences in technique and user preference.

Gastric carcinoma (GC) is the most common neoplasm in the stomach of dogs. Although incidence in the general population is reported to be low, breed-specific GC has a high incidence. Median age at presentation ranges from 8 to approximately 10 years. The disease is mostly located in the lesser curvature and antropyloric region of the stomach. Unfortunately, diagnosis is usually made when the disease is at an advanced stage and, therefore, prognosis is poor. Due to similarities in clinical presentation, diagnosis, histology and prognosis, canine GC may serve as a valuable model for human GC. Extensive pedigrees of canine gastric carcinoma cases could reveal insights for human gastric carcinoma. Putative species differences include the role of Helicobacter in pathogenesis, the wide array of genetic data and screening available for humans, and treatment protocols that are available for human GC.

Atypical E2F transcription factors (E2F7 and E2F8) function as key regulators of cell cycle progression and their inactivation leads to spontaneous cancer formation in mice. However, the mechanism of the tumor suppressor functions of E2F7/8 remain obscure. In this study we discovered that atypical E2Fs control tumor angiogenesis, one of the hallmarks of cancer. We genetically inactivated atypical E2Fs in epithelial and mesenchymal neoplasm and analyzed blood vessel formation in three different animal models of cancer. Tumor formation was either induced by application of 7,12-Dimethylbenz(a)anthracene/12-O-Tetradecanoylphorbol-13-acetate or by Myc/Ras overexpression. To our surprise, atypical E2Fs suppressed tumor angiogenesis in all three cancer models, which is in a sharp contrast to previous findings showing that atypical E2Fs promote angiogenesis during fetal development in mice and zebrafish. Real-time imaging in zebrafish displayed that fluorescent-labeled blood vessels showed enhanced intratumoral branching in xenografted E2f7/8-deficient neoplasms compared with E2f7/8-proficient neoplasms. DLL4 expression, a key negative inhibitor of vascular branching, was decreased in E2f7/8-deficient neoplastic cells, indicating that E2F7/8 might inhibit intratumoral vessel branching via induction of DLL4.Oncogene advance online publication, 18 September 2017; doi:10.1038/onc.2017.336.

In a previous study of phyllodes tumours, it has been shown that both the stroma and the epithelium can exhibit distinct molecular changes, suggesting that both are part of the neoplastic process. In view of this finding, it was decided to study stromal-epithelial interactions in these tumours by examining the Wnt-APC-beta-catenin pathway. beta-catenin and cyclin D1 immunohistochemistry was performed on 119 phyllodes tumours. Eighty-six (72%) showed stromal nuclear beta-catenin localization and in 57%, the staining was moderate or strong; however, of the eight malignant tumours in the series, seven showed absent or weak nuclear staining (p < 0.025). In no tumour was nuclear beta-catenin staining seen in the epithelial component. Moderate or strong stromal cyclin D I staining correlated with nuclear stromal beta-catenin staining (p

To compare Sonicision cordless ultrasonic dissector (SCUD) to LigaSure vessel sealing device (LVSD) for laparoscopic ovariectomy (Lap OVE) in dogs.Randomized, paired prospective clinical trial.Client-owned dogs (n = 22) presented for elective Lap OVE.Dogs were randomly assigned to one of two protocols: protocol 1 required the left ovary resected using SCUD and the right ovary using LVSD; protocol 2 required the left ovary resected using LVSD and the right ovary using SCUD. Duration of ovary excision, complications, surgical smoke production, and collateral thermal damage were compared between SCUD and LVSD. Total surgery duration, postoperative convalescence, obesity, mesovarial fat score, and technique-associated costs were also recorded.Ovary excision was significantly faster with LVSD than SCUD. Surgical smoke production was significantly greater for SCUD than LVSD. Minor pedicle hemorrhage occurred 3 times with SCUD and one time with LVSD (not significantly different) and was easily corrected intraoperative. Presence of hemorrhage significantly increased ovary excision time. Technique-associated costs were lower for SCUD than LVSD. No significant differences were found in collateral thermal damage between SCUD and LVSD. Total surgery duration and convalescence time were similar to previous reports of Lap OVE in dogs at the authors' institution.SCUD is a cost-effective alternative for Lap OVE, taking into account differences in technique and user preference.

This study aimed to evaluate efficacy of group (GRP) versus individual (IND) physiotherapy rehabilitation following lower limb intramuscular injections of Botulinum Toxin-Type A (BoNT-A) for ambulant children with cerebral palsy (CP). Following lower limb BoNT-A injections, 34 children were randomly allocated to GRP (n=17; mean age 7y8m SD 2.0; 13 males; Gross Motor Function Classification System (GMFCS) I=5, II=8, III=4) or IND physiotherapy (n=17; mean age 8y7m SD 2.0; 11 males; GMFCS I=9, II=5, III=3). Primary outcomes were the Canadian Occupational Performance Measure (COPM) and Edinburgh Visual Gait Score (EVGS) assessed at baseline, 10 and 26 weeks post intervention. There were no baseline differences between groups. GRP intervention had greater, but not clinically meaningful, improvement in COPM satisfaction (estimated mean difference EMD 1.7, 95% CI 0.4-3.1; p

Background Cerebral palsy is the most common cause of physical disability in childhood. Spasticity is a significant contributor to the secondary impairments impacting functional performance and participation. The most common lower limb spasticity management is focal intramuscular injections of Botulinum Toxin-Type A accompanied by individually-delivered (one on one) physiotherapy rehabilitation. With increasing emphasis on improving goal-directed functional activity and participation within a family-centred framework, it is timely to explore whether physiotherapy provided in a group could achieve comparable outcomes, encouraging providers to offer flexible models of physiotherapy delivery. This study aims to compare individual to group-based physiotherapy following intramuscular Botulinum Toxin-A injections to the lower limbs for ambulant children with cerebral palsy aged four to fourteen years. Methods/Design An assessor-masked, block randomised comparison trial will be conducted with random allocation to either group-based or individual physiotherapy. A sample size of 30 (15 in each study arm) will be recruited. Both groups will receive six hours of direct therapy following Botulinum Toxin-A injections in either an individual or group format with additional home programme activities (three exercises to be performed three times a week). Study groups will be compared at baseline (T1), then at 10 weeks (T2, efficacy) and 26 weeks (T3, retention) post Botulinum Toxin-A injections. Primary outcomes will be caregiver/s perception of and satisfaction with their child’s occupational performance goals (Canadian Occupational Performance Measure) and quality of gait (Edinburgh Visual Gait Score) with a range of secondary outcomes across domains of the International Classification of Disability, Functioning and Health. Discussion This paper outlines the study protocol including theoretical basis, study hypotheses and outcome measures for this assessor-masked, randomised comparison trial comparing group versus individual models of physiotherapy following intramuscular injections of Botulinum Toxin-A to the lower limbs for ambulant children with cerebral palsy. Trial registration ACTRN12611000454976

The orientations, geometries, and lipid interactions of designed transmembrane (TM) peptides have attracted significant experimental and theoretical interest. Because the amino acid proline will introduce a known discontinuity into an alpha helix, we have sought to measure the extent of helix kinking caused by a single proline within the isolated TM helical domain of WALP19. For this purpose, we synthesized acetyl-GWWLALALAP(10)ALALALWWA-ethanolamide and included pairs of deuterated alanines by using 60-100% Fmoc-l-Ala-d(4) at selected sequence positions. Solid-state deuterium ((2)H) magnetic resonance spectra from oriented, hydrated samples (1/40, peptide/lipid; using several lipids) reveal signals from many of the alanine backbone C(alpha) deuterons as well as the alanine side-chain C(beta) methyl groups, whereas signals from C(alpha) deuterons generally have not been observed for similar peptides without proline. It is conceivable that altered peptide dynamics may be responsible for the apparent "unmasking" of the backbone resonances in the presence of the proline. Data analysis using the geometric analysis of labeled alanines (GALA) method reveals that the peptide helix is distorted due to the presence of the proline. To provide additional data points for evaluating the segmental tilt angles of the two halves of the peptide, we substituted selected leucines with l-Ala-d(4). Using this approach, we were able to deduce that the apparent average tilt of the C-terminal increases from approximately 4 degrees to approximately 12 degrees when Pro(10) is introduced. The segment N-terminal to proline is more complex and possibly is more dynamically flexible; Leu to Ala mutations within the N-terminal segment alter the average orientations of alanines in both segments. Nevertheless, in DOPC, we could estimate an apparent kink angle of approximately 19 degrees . Together, the results suggest that the central proline influences not only the geometry but also the dynamics of the membrane-spanning peptide. The results make up an important basis for understanding the functional role of proline in several families of membrane proteins.

We have described a previously unreported entity of an intussuscepted neuroendocrine carcinoma of the appendix. Our patient was a 70-year-old man whose only complaint was insipient weight loss. Colonoscopy showed a malignant cecal “polyp”, and an extended right hemicolectomy was performed. We have reviewed the literature on the causes of appendiceal intussusception and their appropriate treatment options, and clarified the classification of neuroendocrine tumors of the gastrointestinal tract.

The orientations, geometries and lipid interactions of designed, specifically anchored, transmembrane (TM) peptides have attracted significant experimental and theoretical interest. Because Pro will introduce a known discontinuity into an alpha-helix, we have sought to measure the extent of helix kinking caused by a single proline within an isolated TM helical domain, and to address the question: To what extent do the individual N-terminal and C-terminal segments adjust their tilts with respect to the bilayer normal in response to the proline? For this purpose, we synthesized WALP19-P10, acetyl-GWWLALALAP10ALALALWWA-ethanolamide, and included pairs of deuterated alanines by using 60-100% fmoc-d4-Ala at selected sequence positions. Remarkably, solid-state 2H NMR spectra from oriented, hydrated samples (1/40, peptide/lipid; using DOPC, DMPC or DLPC) reveal signals from many of the Ala Cα deuterons as well as the Ala Cβ methyls; whereas signals from backbone Cα deuterons had not been observed for WALP19 without Pro. For example the magnitudes of the 2H quadrupolar splittings are 70 and 10.7 kHz for the Ala11 Cα-D and side-chain methyl groups, respectively. We are considering possible reasons for the apparent “unmasking” of the backbone resonances in the presence of the proline. At the same time, the observed backbone resonances provide valuable additional data points for evaluating the segmental tilt angles of the N- and C-terminal segments. In order to make available still more data points for the Geometric Analysis of Labeled Alanines (GALA), we also are substituting selected leucines with d4-Ala. Together the results suggest that the central proline influences not only the geometry but also the dynamics of WALP19.