The Rho guanine dissociation inhibitor α inhibits skeletal muscle Rac1 activity and insulin action.

The molecular events governing skeletal muscle glucose uptake have pharmacological potential for managing insulin resistance in conditions such as obesity, diabetes, and cancer. With no current pharmacological treatments to target skeletal muscle insulin sensitivity, there is an unmet need to identify the molecular mechanisms that control insulin sensitivity in skeletal muscle. Here, the Rho guanine dissociation inhibitor α (RhoGDIα) is identified as a point of control in the regulation of insulin sensitivity. In skeletal muscle cells, RhoGDIα interacted with, and thereby inhibited, the Rho GTPase Rac1. In response to insulin, RhoGDIα was phosphorylated at S101 and Rac1 dissociated from RhoGDIα to facilitate skeletal muscle GLUT4 translocation. Accordingly, siRNA-mediated RhoGDIα depletion increased Rac1 activity and elevated GLUT4 translocation. Consistent with RhoGDIα's inhibitory effect, rAAV-mediated RhoGDIα overexpression in mouse muscle decreased insulin-stimulated glucose uptake and was detrimental to whole-body glucose tolerance. Aligning with RhoGDIα's negative role in insulin sensitivity, RhoGDIα protein content was elevated in skeletal muscle from insulin-resistant patients with type 2 diabetes. These data identify RhoGDIα as a clinically relevant controller of skeletal muscle insulin sensitivity and whole-body glucose homeostasis, mechanistically by modulating Rac1 activity.

Dynamic Changes to the Skeletal Muscle Proteome and Ubiquitinome Induced by the E3 Ligase, ASB2ß.

Ubiquitination is a posttranslational protein modification that has been shown to have a range of effects, including regulation of protein function, interaction, localization, and degradation. We have previously shown that the muscle-specific ubiquitin E3 ligase, ASB2ß, is downregulated in models of muscle growth and that overexpression ASB2ß is sufficient to induce muscle atrophy. To gain insight into the effects of increased ASB2ß expression on skeletal muscle mass and function, we used liquid chromatography coupled to tandem mass spectrometry to investigate ASB2ß-mediated changes to the skeletal muscle proteome and ubiquitinome, via a parallel analysis of remnant diGly-modified peptides. The results show that viral vector-mediated ASB2ß overexpression in murine muscles causes progressive muscle atrophy and impairment of force-producing capacity, while ASB2ß knockdown induces mild muscle hypertrophy. ASB2ß-induced muscle atrophy and dysfunction were associated with the early downregulation of mitochondrial and contractile protein abundance and the upregulation of proteins involved in proteasome-mediated protein degradation (including other E3 ligases), protein synthesis, and the cytoskeleton/sarcomere. The overexpression ASB2ß also resulted in marked changes in protein ubiquitination; however, there was no simple relationship between changes in ubiquitination status and protein abundance. To investigate proteins that interact with ASB2ß and, therefore, potential ASB2ß targets, Flag-tagged wild-type ASB2ß, and a mutant ASB2ß lacking the C-terminal SOCS box domain (dSOCS) were immunoprecipitated from C2C12 myotubes and subjected to label-free proteomic analysis to determine the ASB2ß interactome. ASB2ß was found to interact with a range of cytoskeletal and nuclear proteins. When combined with the in vivo ubiquitinomic data, our studies have identified novel putative ASB2ß target substrates that warrant further investigation. These findings provide novel insight into the complexity of proteome and ubiquitinome changes that occur during E3 ligase-mediated skeletal muscle atrophy and dysfunction.

Morphologically high-grade trichoblastic carcinoma: a clinicopathological study with long-term clinical follow-up.

AIMS: Trichoblastic carcinomas (malignant trichoblastomas) are rare and poorly documented neoplasms characterised by malignant transformation of a pre-existing benign trichoblastoma, and are subdivided histologically into low-grade and high-grade tumours. Whereas morphologically low-grade trichoblastic carcinomas show indolent behaviour, morphologically high-grade trichoblastic carcinomas have been associated with a poor prognosis, but little is known about their true biological potential. The aim of this study was to better define the clinicopathological features and outcomes of morphologically high-grade trichoblastic carcinomas. METHODS AND RESULTS: Four high-grade trichoblastic carcinomas were retrieved from departmental files, and the clinical and histopathological features and follow-up were recorded. The tumours presented as nodules on the scalps, necks and shoulders of adults (median age, 40 years; range, 30-55 years) with a female predominance of 3:1. Three patients had a longstanding history with recent change. Histologically, three tumours were characterised by an expansile cellular nodule composed of sheets of pleomorphic epithelioid cells with brisk mitotic activity and necrosis arising in a background of a benign trichoblastoma. One tumour showed a more gradual transition from a benign trichoepithelioma to an undifferentiated carcinoma with infiltrative growth and perineural infiltration. All patients were alive with no evidence of recurrence or metastasis following complete excision after a median follow-up of 96 months (range, 30-180 months). CONCLUSIONS: The correct diagnosis of high-grade trichoblastic carcinoma relies on adequate sampling and recognition of the benign trichoblastic precursor lesion, i.e. trichoblastoma or trichoepithelioma. Despite the concerning histological features of the malignant component, the tumours appear to be less aggressive than previously thought.

Intravascular Follistatin gene delivery improves glycemic control in a mouse model of type 2 diabetes.

Type 2 diabetes (T2D) manifests from inadequate glucose control due to insulin resistance, hypoinsulinemia, and deteriorating pancreatic ß-cell function. The pro-inflammatory factor Activin has been implicated as a positive correlate of severity in T2D patients, and as a negative regulator of glucose uptake by skeletal muscle, and of pancreatic ß-cell phenotype in mice. Accordingly, we sought to determine whether intervention with the Activin antagonist Follistatin can ameliorate the diabetic pathology. Here, we report that an intravenous Follistatin gene delivery intervention with tropism for striated muscle reduced the serum concentrations of Activin B and improved glycemic control in the db/db mouse model of T2D. Treatment reversed the hyperglycemic progression with a corresponding reduction in the percentage of glycated-hemoglobin to levels similar to lean, healthy mice. Follistatin gene delivery promoted insulinemia and abundance of insulin-positive pancreatic ß-cells, even when treatment was administered to mice with advanced diabetes, supporting a mechanism for improved glycemic control associated with maintenance of functional ß-cells. Our data demonstrate that single-dose intravascular Follistatin gene delivery can ameliorate the diabetic progression and improve prognostic markers of disease. These findings are consistent with other observations of Activin-mediated mechanisms exerting deleterious effects in models of obesity and diabetes, and suggest that interventions that attenuate Activin signaling could help further understanding of T2D and the development of novel T2D therapeutics.

TBC1D13 is a RAB35 specific GAP that plays an important role in GLUT4 trafficking in adipocytes.

Insulin stimulates glucose transport in adipocytes by triggering translocation of GLUT4 glucose transporters to the plasma membrane (PM) and several Rabs including Rab10 have been implicated in this process. To delineate the molecular regulation of this pathway, we conducted a TBC/RabGAP overexpression screen in adipocytes. This identified TBC1D13 as a potent inhibitor of insulin-stimulated GLUT4 translocation without affecting other trafficking pathways. To determine the potential Rab substrate for TBC1D13 we conducted a yeast two-hybrid screen and found that the GTP bound forms of Rabs 1 and 10 specifically interacted with TBC1D13 but not with eight other TBC proteins. Surprisingly, a comprehensive in vitro screen for TBC1D13 GAP activity revealed Rab35 but not Rab10 as a specific substrate. TBC1D13 also displayed in vivo GAP activity towards Rab35. Overexpression of constitutively active Rab35 but not constitutively active Rab10 reversed the block in insulin-stimulated GLUT4 translocation observed with TBC1D13 overexpression. These studies implicate an important role for Rab35 in insulin-stimulated GLUT4 translocation in adipocytes.

Follicular induction in a case of molluscum contagiosum: possible link with secondary anetoderma-like changes?

Molluscum contagiosum (MC) can rarely show follicular neogenesis as a result of proliferation of virus-infected basal cells. We report a case of molluscum contagiosum showing multifocal areas of primitive follicular induction involving the adjacent surface epidermis. The underlying dermis showed histological features suggestive of secondary anetoderma. The pathogenetic mechanisms behind development of these microscopic features and the putative link between follicular induction and secondary anetoderma are explored. Awareness of this unusual phenomenon will be helpful in avoiding a misdiagnosis of a superficial basal cell (trichoblastic) carcinoma in such cases.

A new approach for measuring the work and quality of histopathology reporting.

AIMS: Cancer datasets drive report quality, but require more work to inform compliant reports. The aim of this study was to correlate the number of words with measures of quality, to examine the impact of the drive for improved quality on the workload of histopathology reporting over time. METHODS AND RESULTS: We examined the first 10 reports of colon, breast, renal, lung and ovarian carcinoma, melanoma resection, nodal lymphoma appendicitis and seborrhoeic keratosis (SK) issued in 1991, 2001 and 2011. Correlations were analysed using Pearson's partial correlation coefficients. Word count increased significantly over time for most specimen types examined. Word count almost always correlated with units of information, indicating that the word count was a good measure of the amount of information contained within the reports; this correlation was preserved following correction for the effect of time. A good correlation with compliance with cancer datasets was also observed, but was weakened or lost following correction for the increase in word count and units of information that occurred between time points. CONCLUSIONS: These data indicate that word count could potentially be used as a measure of information content if its integrity and usefulness are continuously validated. Further prospective studies are required to assess and validate this approach.

Low-grade Hidradenocarcinomas: A Clinicopathologic Study of an Unusual Carcinoma That Can Mimic its Benign Counterpart.

Hidradenocarcinomas are rare cutaneous adnexal malignancies with sweat gland differentiation that can show a broad spectrum of histomorphologic appearances, ranging from low to high grade. The diagnosis of low-grade hidradenocarcinoma can be challenging and may be mistaken for benign hidradenomas, especially on superficial and partial samples. We performed a retrospective analysis of 16 low-grade hidradenocarcinomas, obtained from 4 large academic institutions. All neoplasms presented clinically as nodular lesions that ranged in size from 1.5 to 6.0 cm. All patients were adults and their age ranged from 33 to 74 years of age. All cases shared features similar to hidradenomas in the surface and mid portion of the tumors and all tumors had 1 or more histomorphologic clues to malignancy, including the presence of an asymmetric and infiltrative growth pattern (especially at the base of the tumors), perineurial invasion, and a desmoplastic stromal reaction. In the tumors evaluated for immunohistochemistry, the tumor cells were positive for p63, EMA, AE1/AE3, MNF116, and CK7. Three patients underwent sentinel lymph node biopsy, and 2 cases showed metastatic disease to regional lymph nodes. All cases (including the 2 cases that had regional lymph node metastasis), showed no local recurrence or distant metastasis observed after a complete re-excision of the tumors (follow-up range from 6 to 72 mo). Our study highlights the salient clinical and histopathologic features of low-grade hidradenocarcinomas and emphasizes the potential diagnostic pitfalls in distinguishing this entity from other neoplasms. Our results indicate that a combination of thorough histopathologic inspection is necessary to support the diagnosis of this rare neoplasm. These tumors can be exceedingly difficult to diagnose and awareness of the subtle features of low-grade hidradenocarcinoma is of importance are as it remains a diagnostic challenge for practicing pathologists.

Cutaneous Myoepithelial Neoplasms on Acral Sites Show Distinctive and Reproducible Histopathologic and Immunohistochemical Features.

Cutaneous myoepithelial neoplasms are a heterogenous group of neoplasms with mixed tumors typically affecting the head and myoepitheliomas showing a predilection for the extremities. Their malignant counterparts, myoepithelial carcinoma, and malignant mixed tumor are exceptionally rare in the skin, and the morphologic criteria for malignancy are only poorly defined. The aim of the present study was to characterize the clinicopathologic features of myoepithelial neoplasms presenting on acral skin. The clinical and histopathologic features of 11 tumors were recorded, and follow-up was obtained. Immunohistochemistry was performed for S100, SOX10, glial fibrillary acidic protein, keratins, epithelial membrane antigen, p63, p40, smooth muscle actin, desmin, and PLAG1. The tumors mainly affected the feet of adults (range: 26 to 78 y; median: 47 y) with a predilection for the great toe and a male predominance of 1.8:1. Most tumors (91%) displayed a lobular architecture composed of solid and nested growth of epithelioid cells with plasmacytoid features in a myxoid or angiomatous stroma. Scattered cytologic atypia and rare duct differentiation were frequently noted. Three tumors with confluent cytologic atypia, infiltrative growth, and lymphovascular invasion were classified as malignant. By immunohistochemistry, the tumors were positive for S100, SOX10, keratins AE1/AE3, CK5/6 and CK7, and PLAG1. Local recurrence and bilateral pulmonary metastasis were observed in a patient presenting with a histopathologically benign-appearing tumor. Two patients with malignant tumors experienced local recurrences, and 1 developed metastasis to soft tissue, lung, and mediastinal lymph nodes. All patients are currently alive, all but 1 with no evidence of disease after a median follow-up interval of 96 months (range: 2 to 360 mo). In conclusion, acral myoepithelial neoplasms show distinctive and reproducible histopathologic and immunohistochemical features. They are best regarded as a distinctive subset of mixed tumors with features reminiscent of their salivary gland counterparts. While most tumors pursue a benign disease course, histopathologic features appear to be a poor indicator of prognosis.

Fine-tuning the cardiac O-GlcNAcylation regulatory enzymes governs the functional and structural phenotype of the diabetic heart.

AIMS: The glucose-driven enzymatic modification of myocardial proteins by the sugar moiety, ß-N-acetylglucosamine (O-GlcNAc), is increased in pre-clinical models of diabetes, implicating protein O-GlcNAc modification in diabetes-induced heart failure. Our aim was to specifically examine cardiac manipulation of the two regulatory enzymes of this process on the cardiac phenotype, in the presence and absence of diabetes, utilising cardiac-targeted recombinant-adeno-associated viral-vector-6 (rAAV6)-mediated gene delivery. METHODS AND RESULTS: In human myocardium, total protein O-GlcNAc modification was elevated in diabetic relative to non-diabetic patients, and correlated with left ventricular (LV) dysfunction. The impact of rAAV6-delivered O-GlcNAc transferase (rAAV6-OGT, facilitating protein O-GlcNAcylation), O-GlcNAcase (rAAV6-OGA, facilitating de-O-GlcNAcylation), and empty vector (null) were determined in non-diabetic and diabetic mice. In non-diabetic mice, rAAV6-OGT was sufficient to impair LV diastolic function and induce maladaptive cardiac remodelling, including cardiac fibrosis and increased Myh-7 and Nppa pro-hypertrophic gene expression, recapitulating characteristics of diabetic cardiomyopathy. In contrast, rAAV6-OGA (but not rAAV6-OGT) rescued LV diastolic function and adverse cardiac remodelling in diabetic mice. Molecular insights implicated impaired cardiac PI3K(p110α)-Akt signalling as a potential contributing mechanism to the detrimental consequences of rAAV6-OGT in vivo. In contrast, rAAV6-OGA preserved PI3K(p110α)-Akt signalling in diabetic mouse myocardium in vivo and prevented high glucose-induced impairments in mitochondrial respiration in human cardiomyocytes in vitro. CONCLUSION: Maladaptive protein O-GlcNAc modification is evident in human diabetic myocardium, and is a critical regulator of the diabetic heart phenotype. Selective targeting of cardiac protein O-GlcNAcylation to restore physiological O-GlcNAc balance may represent a novel therapeutic approach for diabetes-induced heart failure.

Characteristics of meta-analyses and their component studies in the Cochrane Database of Systematic Reviews: a cross-sectional, descriptive analysis.

BACKGROUND: Cochrane systematic reviews collate and summarise studies of the effects of healthcare interventions. The characteristics of these reviews and the meta-analyses and individual studies they contain provide insights into the nature of healthcare research and important context for the development of relevant statistical and other methods. METHODS: We classified every meta-analysis with at least two studies in every review in the January 2008 issue of the Cochrane Database of Systematic Reviews (CDSR) according to the medical specialty, the types of interventions being compared and the type of outcome. We provide descriptive statistics for numbers of meta-analyses, numbers of component studies and sample sizes of component studies, broken down by these categories. RESULTS: We included 2321 reviews containing 22,453 meta-analyses, which themselves consist of data from 112,600 individual studies (which may appear in more than one meta-analysis). Meta-analyses in the areas of gynaecology, pregnancy and childbirth (21%), mental health (13%) and respiratory diseases (13%) are well represented in the CDSR. Most meta-analyses address drugs, either with a control or placebo group (37%) or in a comparison with another drug (25%). The median number of meta-analyses per review is six (inter-quartile range 3 to 12). The median number of studies included in the meta-analyses with at least two studies is three (inter-quartile range 2 to 6). Sample sizes of individual studies range from 2 to 1,242,071, with a median of 91 participants. DISCUSSION: It is clear that the numbers of studies eligible for meta-analyses are typically very small for all medical areas, outcomes and interventions covered by Cochrane reviews. This highlights the particular importance of suitable methods for the meta-analysis of small data sets. There was little variation in number of studies per meta-analysis across medical areas, across outcome data types or across types of interventions being compared.

Regulation of glucose transporter 4 translocation by the Rab guanosine triphosphatase-activating protein AS160/TBC1D4: role of phosphorylation and membrane association.

Insulin-stimulated translocation of the glucose transporter GLUT4 to the plasma membrane in muscle and fat cells depends on the phosphatidylinositide 3-kinase/Akt pathway. The downstream target AS160/TBC1D4 is phosphorylated upon insulin stimulation and contains a TBC domain (Tre-2/Bub2/Cdc16) that is present in most Rab guanosine triphosphatase-activating proteins. TBC1D4 associates with GLUT4-containing membranes under basal conditions and dissociates from membranes with insulin. Here we show that the association of TBC1D4 with membranes is required for its inhibitory action on GLUT4 translocation under basal conditions. Whereas the insulin-dependent dissociation of TBC1D4 from membranes was not required for GLUT4 translocation, its phosphorylation was essential. Many agonists that stimulate GLUT4 translocation failed to trigger TBC1D4 translocation to the cytosol, but in most cases these agonists stimulated TBC1D4 phosphorylation at T642, and their effects on GLUT4 translocation were inhibited by overexpression of the TBC1D4 phosphorylation mutant (TBC1D4-4P). We postulate that TBC1D4 acts to impede GLUT4 translocation by disarming a Rab protein found on GLUT4-containing-membranes and that phosphorylation of TBC1D4 per se is sufficient to overcome this effect, allowing GLUT4 translocation to the cell surface to proceed.

Mechanisms involved in follistatin-induced hypertrophy and increased insulin action in skeletal muscle.

BACKGROUND: Skeletal muscle wasting is often associated with insulin resistance. A major regulator of muscle mass is the transforming growth factor ß (TGF-ß) superfamily, including activin A, which causes atrophy. TGF-ß superfamily ligands also negatively regulate insulin-sensitive proteins, but whether this pathway contributes to insulin action remains to be determined. METHODS: To elucidate if TGF-ß superfamily ligands regulate insulin action, we used an adeno-associated virus gene editing approach to overexpress an activin A inhibitor, follistatin (Fst288), in mouse muscle of lean and diet-induced obese mice. We determined basal and insulin-stimulated 2-deoxy-glucose uptake using isotopic tracers in vivo. Furthermore, to evaluate whether circulating Fst and activin A concentrations are associated with obesity, insulin resistance, and weight loss in humans, we analysed serum from morbidly obese subjects before, 1 week, and 1 year after Roux-en-Y gastric bypass (RYGB). RESULTS: Fst288 muscle overexpression markedly increased in vivo insulin-stimulated (but not basal) glucose uptake (+75%, P < 0.05) and increased protein expression and intracellular insulin signalling of AKT, TBC1D4, PAK1, pyruvate dehydrogenase-E1α, and p70S6K, while decreasing TBC1D1 signaling (P < 0.05). Fst288 increased both basal and insulin-stimulated protein synthesis, but no correlation was observed between the Fst288-driven hypertrophy and the increase in insulin-stimulated glucose uptake. Importantly, Fst288 completely normalized muscle glucose uptake in insulin-resistant diet-induced obese mice. RYGB surgery doubled circulating Fst and reduced activin A (-24%, P < 0.05) concentration 1 week after surgery before any significant weight loss in morbidly obese normoglycemic patients, while major weight loss after 1 year did not further change the concentrations. CONCLUSIONS: We here present evidence that Fst is a potent regulator of insulin action in muscle, and in addition to AKT and p70S6K, we identify TBC1D1, TBC1D4, pyruvate dehydrogenase-E1α, and PAK1 as Fst targets. Circulating Fst more than doubled post-RYGB surgery, a treatment that markedly improved insulin sensitivity, suggesting a role for Fst in regulating glycaemic control. These findings demonstrate the therapeutic potential of inhibiting TGF-ß superfamily ligands to improve insulin action and Fst's relevance to muscle wasting-associated insulin-resistant conditions in mice and humans.

Acral Spitz Nevi: A Clinicopathologic Study of 50 Cases With Immunohistochemical Analysis of P16 and P21 Expression.

Spitz nevi on acral sites are rare and poorly documented. The combination of Spitzoid cytomorphology and atypical architectural features of the junctional component may lead to an erroneous diagnosis of melanoma. To study the clinicopthologic and immunohistochemical features, 50 Spitz nevi localized on the distal extremities were retrieved from departmental files. Clinical data and follow-up were obtained and the histologic features were analyzed. P16 and P21 immunohistochemical staining of the dermal component was compared with that of 10 acral lentiginous melanomas and 10 acral nevi. Acral Spitz nevi affected young adults (median: 24.5 y; range: 4 to 61 y) with a female predominence and a predilection for the feet. They were pigmented measuring 1 to 15 mm (median: 4 mm). Follow-up, available for 45 patients (median: 48 mo; range: 4 to 228 mo), revealed no local recurrence, metastasis or mortality. Histologically, acral Spitz nevi were composed of large epithelioid and/or spindled melanocytes. They were well circumscribed with occasional asymmetry. The junctional component was broad with a lentiginous and nested growth and shoulder formation. Marked pagetoid spread and transepidermal elimination of junctional nests were common features. Focal active regression of the dermal component was frequently noted but there was no nuclear pleomorphism or dermal mitotic activity. Acral Spitz nevi were characterized by strong and diffuse P16 and P21 expression, which differs from acral nevi and acral lentiginous melanoma. Acral Spitz nevi are a distinctive subgroup of Spitz nevi with benign behavior. Awareness of the subtle histologic differentiating features and the distinctive P16/P21 expression pattern allows separation from melanoma.

Acute bidirectional manipulation of muscle glucose uptake by in vivo electrotransfer of constructs targeting glucose transporter genes.

Analysis of conventional germ-line or tissue-specific gene manipulation in vivo is potentially confounded by developmental adaptation of animal physiology. We aimed to adapt the technique of in vivo electrotransfer (IVE) to alter local gene expression in skeletal muscle of rodents as a means of investigating the role of specific proteins in glucose metabolism in vivo. We utilized a square-wave electroporator to induce intracellular electrotransfer of DNA constructs injected into rat or mouse muscles and investigated the downstream effects. In initial studies, expression of green fluorescent protein reporter was induced in 53 +/- 10% of muscle fibers peaking at 7 days, and importantly, the electrotransfer procedure itself did not impact upon the expression of stress proteins or our ability to detect a reduction in 2-deoxyglucose tracer uptake by electroporated muscle of high-fat-fed rats during hyperinsulinemic-euglycemic clamp. To demonstrate functional effects of electrotransfer of constructs targeting glucose transporters, we administered vectors encoding GLUT-1 cDNA and GLUT-4 short hairpin RNAs (shRNAs) to rodent muscles. IVE of the GLUT-1 gene resulted in a 57% increase in GLUT-1 protein, accompanied by a proportionate increase in basal 2-deoxyglucose tracer uptake into muscles of starved rats. IVE of vectors expressing two shRNAs for GLUT-4 demonstrated to reduce specific protein expression and 2-deoxyglucose tracer uptake in 3T3-L1 adipocytes into mouse muscle caused a 51% reduction in GLUT-4 protein, associated with attenuated clearance of tracer to muscle after a glucose load. These results confirm that glucose transporter expression is largely rate limiting for glucose uptake in vivo and highlight the utility of IVE for the acute manipulation of muscle gene expression in the study of the role of specific proteins in glucose metabolism.

Integrated expression analysis of muscle hypertrophy identifies Asb2 as a negative regulator of muscle mass.

The transforming growth factor-ß (TGF-ß) signaling network is a critical regulator of skeletal muscle mass and function and, thus, is an attractive therapeutic target for combating muscle disease, but the underlying mechanisms of action remain undetermined. We report that follistatin-based interventions (which modulate TGF-ß network activity) can promote muscle hypertrophy that ameliorates aging-associated muscle wasting. However, the muscles of old sarcopenic mice demonstrate reduced response to follistatin compared with healthy young-adult musculature. Quantitative proteomic and transcriptomic analyses of young-adult muscles identified a transcription/translation signature elicited by follistatin exposure, which included repression of ankyrin repeat and SOCS box protein 2 (Asb2). Increasing expression of ASB2 reduced muscle mass, thereby demonstrating that Asb2 is a TGF-ß network-responsive negative regulator of muscle mass. In contrast to young-adult muscles, sarcopenic muscles do not exhibit reduced ASB2 abundance with follistatin exposure. Moreover, preventing repression of ASB2 in young-adult muscles diminished follistatin-induced muscle hypertrophy. These findings provide insight into the program of transcription and translation events governing follistatin-mediated adaptation of skeletal muscle attributes and identify Asb2 as a regulator of muscle mass implicated in the potential mechanistic dysfunction between follistatin-mediated muscle growth in young and old muscles.

Simple, robust strategies for generating DNA-directed RNA interference constructs.

We describe two complementary strategies for preparing DNA-directed RNA interference (ddRNAi) constructs designed to express hpRNA. The first, oligonucleotide assembly (OA), uses a very simple annealing protocol to combine up to 20 short nucleotides. These are then cloned into appropriately designed restriction sites in expression vectors. OA can be used to prepare simple hairpin (hp)-expressing constructs, but we prefer to use the approach to generate longer constructs. The second strategy, long-range cloning (LRC), uses a novel adaptation of long-range PCR protocols. For LRC, entire vectors are amplified with primers that serve to introduce short sequences into plasmids at defined anchor sites during PCR. The LCR strategy has proven highly reliable in our hands for generating simple ddRNAi constructs. Moreover, LCR is likely to prove useful in many situations in which conventional cloning strategies might prove problematic. In combination, OA and LRC can greatly simplify the design and generation of many expression constructs, including constructs for ddRNAi.

Child deaths due to injury in the four UK countries: a time trends study from 1980 to 2010.

BACKGROUND: Injuries are an increasingly important cause of death in children worldwide, yet injury mortality is highly preventable. Determining patterns and trends in child injury mortality can identify groups at particularly high risk. We compare trends in child deaths due to injury in four UK countries, between 1980 and 2010. METHODS: We obtained information from death certificates on all deaths occurring between 1980 and 2010 in children aged 28 days to 18 years and resident in England, Scotland, Wales or Northern Ireland. Injury deaths were defined by an external cause code recorded as the underlying cause of death. Injury mortality rates were analysed by type of injury, country of residence, age group, sex and time period. RESULTS: Child mortality due to injury has declined in all countries of the UK. England consistently experienced the lowest mortality rate throughout the study period. For children aged 10 to 18 years, differences between countries in mortality rates increased during the study period. Inter-country differences were largest for boys aged 10 to 18 years with mortality rate ratios of 1.38 (95% confidence interval 1.16, 1.64) for Wales, 1.68 (1.48, 1.91) for Scotland and 1.81 (1.50, 2.18) for Northern Ireland compared with England (the baseline) in 2006-10. The decline in mortality due to injury was accounted for by a decline in unintentional injuries. For older children, no declines were observed for deaths caused by self-harm, by assault or from undetermined intent in any UK country. CONCLUSION: Whilst child deaths from injury have declined in all four UK countries, substantial differences in mortality rates remain between countries, particularly for older boys. This group stands to gain most from policy interventions to reduce deaths from injury in children.

Predicting the extent of heterogeneity in meta-analysis, using empirical data from the Cochrane Database of Systematic Reviews.

BACKGROUND: Many meta-analyses contain only a small number of studies, which makes it difficult to estimate the extent of between-study heterogeneity. Bayesian meta-analysis allows incorporation of external evidence on heterogeneity, and offers advantages over conventional random-effects meta-analysis. To assist in this, we provide empirical evidence on the likely extent of heterogeneity in particular areas of health care. METHODS: Our analyses included 14 886 meta-analyses from the Cochrane Database of Systematic Reviews. We classified each meta-analysis according to the type of outcome, type of intervention comparison and medical specialty. By modelling the study data from all meta-analyses simultaneously, using the log odds ratio scale, we investigated the impact of meta-analysis characteristics on the underlying between-study heterogeneity variance. Predictive distributions were obtained for the heterogeneity expected in future meta-analyses. RESULTS: Between-study heterogeneity variances for meta-analyses in which the outcome was all-cause mortality were found to be on average 17% (95% CI 10-26) of variances for other outcomes. In meta-analyses comparing two active pharmacological interventions, heterogeneity was on average 75% (95% CI 58-95) of variances for non-pharmacological interventions. Meta-analysis size was found to have only a small effect on heterogeneity. Predictive distributions are presented for nine different settings, defined by type of outcome and type of intervention comparison. For example, for a planned meta-analysis comparing a pharmacological intervention against placebo or control with a subjectively measured outcome, the predictive distribution for heterogeneity is a log-normal (-2.13, 1.58(2)) distribution, which has a median value of 0.12. In an example of meta-analysis of six studies, incorporating external evidence led to a smaller heterogeneity estimate and a narrower confidence interval for the combined intervention effect. CONCLUSIONS: Meta-analysis characteristics were strongly associated with the degree of between-study heterogeneity, and predictive distributions for heterogeneity differed substantially across settings. The informative priors provided will be very beneficial in future meta-analyses including few studies.