Myostatin signals through miR-34a to regulate Fndc5 expression and browning of white adipocytes.

BACKGROUND/OBJECTIVES: Myostatin (Mstn) has a pivotal role in glucose and lipid metabolism. Mstn deficiency leads to the increased browning of white adipose tissue (WAT), which results in the increased energy expenditure and protection against diet-induced obesity and insulin resistance. In this study, we investigated the molecular mechanism(s) through which Mstn regulates browning of white adipocytes. METHODS: Quantitative molecular analyses were performed to assess Mstn regulation of miR-34a and Fndc5 expression. miR-34a was overexpressed and repressed to investigate miR-34a regulation of Fndc5. Luciferase reporter analysis verified direct binding between miR-34a and the Fndc5 3'-untranslated region (UTR). The browning phenotype of Mstn-/- adipocytes was assessed through the analysis of brown fat marker gene expression, mitochondrial function and infrared thermography. The role of miR-34a and Fndc5 in this browning phenotype was verified through antibody-mediated neutralization of FNDC5, knockdown of Fndc5 by small interfering RNA and through miR-34a gain-of-function and loss-of-function experiments. RESULTS: Mstn treatment of myoblasts inhibited Fndc5 expression, whereas the loss of Mstn increased Fndc5 levels in muscles and in circulation. Mstn inhibition of Fndc5 is miR-34a dependent. Mstn treatment of C2C12 myoblasts upregulated miR-34a expression, whereas reduced miR-34a expression was noted in Mstn-/- muscle and WAT. Subsequent overexpression of miR-34a inhibited Fndc5 expression, whereas blockade of miR-34a increased Fndc5 expression in myoblasts. Reporter analysis revealed that miR-34a directly suppresses Fndc5 expression through a miR-34a-specific binding site within the Fndc5 3'UTR. Importantly, Mstn-mediated inhibition of Fndc5 was blocked upon miR-34a inhibition. Mstn-/- adipocytes showed reduced miR-34a, enhanced Fndc5 expression and increased thermogenic gene expression, which was reversed upon either neutralization of Fndc5 or Fndc5 knockdown. In agreement, Mstn-/- adipocytes have increased mitochondria, improved mitochondrial function and increased heat production. CONCLUSIONS: Mstn regulates Fndc5/Irisin expression and secretion through a novel miR-34a-dependent post-transcriptional mechanism. Loss of Mstn in mice leads to the increased Fndc5/Irisin expression, which contributes to the browning of white adipocytes.

Survival following spinal cord infarction.

STUDY DESIGN: Retrospective open cohort. OBJECTIVES: To calculate the survival of patients with spinal cord infarction and to compare the cause of death in patients with different mechanisms of ischaemic injury. SETTING: Spinal Rehabilitation Unit, Melbourne, Victoria, Australia. METHODS: Consecutive admissions between 1 January 1995 and 31 December 2008 with recent onset of spinal cord infarction. Linkage to the Registry of Births, Deaths and Marriages (Victoria) was used to determine survival following discharge from in-patient rehabilitation and cause of death. RESULTS: A total of 44 patients were admitted (males=26, 59%), with a median age of 72 years (interquartile range (IQR) 62-79). One patient died during their in-patient rehabilitation programme. In all, 14 patients (n=14/44; 33%) died during the follow-up period. The median survival after diagnosis was 56 months (IQR 28-85) and after discharge from in-patient rehabilitation was 46 months (IQR 25-74). The 1- and 5-year mortality rates were 7.0% (n=3/43; 95% confidence interval (CI)=2.4-18.6%) and 20.9% (n=9/43; 95% CI=11.4-35.2%). There was no statistically significant difference in survival between patients with the different aetiologies of spinal cord infarction (other vs idiopathic: χ(2)=0.6, P=0.7; other vs vascular: χ(2)=1.9, P=0.3). There was no relationship between survival and gender (χ(2)=0.2, P=0.6), age (χ(2)=3.0, P=0.08), level of injury (χ(2)=0.0, P=1) or American Spinal Cord Society Impairment Scale grade of spinal cord injury (χ(2)=0.02, P=0.9). CONCLUSION: Patients with spinal cord infarction appear to have a fair survival after discharge from in-patient rehabilitation, not withstanding the occurrence of risk factors of vascular disease in many patients.

Retrospective case series of outcomes following spinal cord infarction.

BACKGROUND AND PURPOSE: There are very few studies of functional and rehabilitation outcomes in patients with spinal cord injury (SCI) owing to infarction. METHODS: Retrospective chart review of consecutive admissions to a tertiary medical unit specializing in SCI rehabilitation, Melbourne, Australia. All admissions between 1 January 1995 and 31 December 2008 with a recent onset of SCI owing to ischaemia were included. Outcome measures included the following: demographic characteristics, American Spinal Injury Association (ASIA) Impairment Scale (AIS), length of stay (LOS), medical complications, accommodation, support services, continence, mobility and Functional Independence Measure (FIM) motor scores. Outcome measures recorded at admission, discharge and at 12 months post discharge. RESULTS: Forty-four patients were admitted for rehabilitation (men = 26, 59%), with a median age of 72 years (interquartile range [IQR], 62-79). On admission, 41 (93%) patients had paraplegia. The majority of patients (n = 33, 75%) had an incomplete SCI. Aetiology was vascular in 19 (43%) patients, idiopathic in 11 (25%) and other in 14 (33%). The median LOS in rehabilitation was 85 days (IQR, 24-129). The most common complications were pain (n = 34, 77%), urinary tract infection (n = 25, 57%), spasticity (n = 12, 27%), cardiac failure (n = 11, 25%) and pneumonia (n = 9, 20%). At rehabilitation discharge most patients (n = 35, 80%) had no change in their AIS grade. Despite this, the FIM motor subscale on admission (median = 28; IQR, 21-34) had significantly improved (P < 0.0000) by discharge (median = 66; IQR 42-78). CONCLUSION: Despite their comorbidities and limited change in AIS, these patients had significant improvement in functional abilities during impatient rehabilitation.

Inhibition of myostatin protects against diet-induced obesity by enhancing fatty acid oxidation and promoting a brown adipose phenotype in mice.

AIMS/HYPOTHESIS: Although myostatin-null (Mstn (-/-)) mice fail to accumulate fat in adipose tissue when fed a high-fat diet (HFD), little is known about the molecular mechanism(s) behind this phenomenon. We therefore sought to identify the signalling pathways through which myostatin regulates accumulation and/or utilisation of fat. METHODS: Wild-type, Mstn (-/-) and wild-type mice treated with soluble activin type IIB receptor (sActRIIB) were fed a control chow diet or an HFD for 12 weeks. Changes in gene expression were measured by microarray and quantitative PCR. Histological changes in white adipose tissue were assessed together with peripheral tissue fatty acid oxidation and changes in circulating hormones following HFD feeding. RESULTS: Our results demonstrate that inactivation of myostatin results in reduced fat accumulation in mice on an HFD. Molecular analysis revealed that metabolic benefits, due to lack of myostatin, are mediated through at least two independent mechanisms. First, lack of myostatin increased fatty acid oxidation in peripheral tissues through induction of enzymes involved in lipolysis and in fatty acid oxidation in mitochondria. Second, inactivation of myostatin also enhanced brown adipose formation in white adipose tissue of Mstn (-/-) mice. Consistent with the above, treatment of HFD-fed wild-type mice with the myostatin antagonist, sActRIIB, reduced the obesity phenotype. CONCLUSIONS/INTERPRETATION: We conclude that absence of myostatin results in enhanced peripheral tissue fatty acid oxidation and increased thermogenesis, culminating in increased fat utilisation and reduced adipose tissue mass. Taken together, our data suggest that anti-myostatin therapeutics could be beneficial in alleviating obesity.

Characterization of 12 microsatellite loci for the Pacific lamprey, Entosphenus tridentatus (Petromyzontidae), and cross-amplification in five other lamprey species.

The Pacific lamprey (Entosphenus tridentatus) is an anadromous fish that is of conservation concern in North America and Asia. Data on Pacific lamprey population structure are scarce and conflicting, impeding conservation efforts. We optimized 12 polymorphic microsatellite loci for the Pacific lamprey. Three to 13 alleles per locus were observed in a sample of 51 fish collected from the West Fork Illinois River, Oregon. Observed heterozygosity ranged from 0.235 to 0.902 and expected heterozygosity ranged from 0.214 to 0.750. Cross-species amplification produced 8 to 12 polymorphic loci in four other Entosphenus species and in the western brook lamprey (Lampetra richardsoni). Two loci appear to be diagnostic for distinguishing Entosphenus from Lampetra. These markers will be valuable for evaluating population structure and making conservation decisions for E. tridentatus and other lamprey species.

Myostatin-deficient mice exhibit reduced insulin resistance through activating the AMP-activated protein kinase signalling pathway.

AIMS/HYPOTHESIS: Myostatin-null mice (Mstn(-/-)) have reduced body fat and increased tolerance to glucose. To date the molecular mechanisms through which myostatin regulates body fat content and insulin sensitivity are not known. Therefore, the aim of the current study was to identify signalling pathways through which myostatin regulates insulin sensitivity. METHODS: Wild-type (WT) mice and Mstn(-/-) mice were fed either a control chow diet or a high fat diet (HFD) for 12 weeks. Glucose tolerance testing and insulin stimulated glucose uptake by M. extensor digitorum longus (EDL) were used as variables to determine insulin sensitivity. Quantitative PCR, Western blotting and enzyme assays were used to monitor AMP-activated protein kinase (AMPK) levels and activity. RESULTS: Mstn(-/-) mice exhibited reduced fat accumulation and peripheral insulin resistance when compared with WT mice, even when they were fed an HFD. Furthermore, treatment with a myostatin antagonist also increased insulin sensitivity during HFD. Consistent with increased insulin sensitivity, we also detected elevated levels of GLUT4, AKT, p-AKT and insulin receptor substrate-1 in Mstn(-/-) muscles. Molecular analysis showed that there is increased expression and activity of AMPK in Mstn(-/-) muscles. Furthermore, we also observed an increase in the AMPK downstream target genes, Sirt1 and Pgc-1α (also known as Ppargc1a), in skeletal muscle of Mstn(-/-) mice. CONCLUSIONS/INTERPRETATION: We conclude that myostatin inactivation leads to increased AMPK levels and activity resulting in increased insulin sensitivity of skeletal muscle. We propose that, by regulating AMPK in skeletal muscle and adipose tissues, myostatin plays a major role in regulating insulin signalling.

Scottish Stroke Research Network: the first three years.

Research networks were introduced in the UK to facilitate and improve clinical research and stroke was seen as a priority topic for local research network development. The Scottish Stroke Research Network (SSRN) is one of 11 stroke research networks in the UK. In this article we review the progress of the Scottish Stroke Research Network in the three years since inception. Between 2006-2009 the number of active hospital research sites has increased from 10 to 22 expanding to involve 20 stroke research nurses. There was a corresponding 58% increase in recruitment of participants into stroke studies, from 376 in 2006/07 to 594 in 2008/09. The majority (17/20) of our current studies are interventional. Data from one of these, the CLOTs trial (Clots in Legs Or sTocking after Stroke), demonstrates that the annual recruitment in Scotland increased from a median of 94 (range 6-122) patients per year in the six years before the SSRN, to 140 (135-158) patients per year after SSRN involvement. We currently screen about 50% of Scottish stroke patients and approximately 5% of Scottish stroke patients are participating in research studies that we support. The SSRN has made good progress in the first three years. Increasing the recruitment of screened patients remains a challenge.

Listening to people with intellectual disabilities who misuse alcohol and drugs.

There is a dearth of research that has explored alcohol/drug use and misuse by people with intellectual disabilities. The aims of the present study were twofold: (1) to examine the insights of 10 people with intellectual disabilities into the reasons why they may misuse alcohol or drugs, and what impact this behaviour may have on them; and (2) to explore the services that they receive. Ten individuals with intellectual disabilities who were deemed to be misusing alcohol/drugs were purposively selected and interviewed. One overarching theme of the reasons for such misuse was labelled as 'self-medicating against life's negative experiences'. This was divided into two sub-themes: 'psychological trauma' and 'social distance from the community'. All the participants reported that their main source of support came from intellectual disability services, acting in both educational and liaison roles. Although seven of the individuals were referred to mainstream addiction services, they perceived this service as negative. In order to address these underlying problems, better access to a wider range of specialist services is required. Intellectual disability and mainstream addiction service providers also need to be more effective in the prevention and treatment of substance misuse by employing techniques such as motivational interviewing.

Oxidative modification of triacylglycerol-rich lipoproteins.

Lipoprotein oxidation is thought to play a pivotal role in the evolution of atherosclerosis. Low-density lipoprotein (LDL) is the main source of oxidized lipid in the arterial wall. Oxidation of LDL alters its properties in a number of ways, making it more atherogenic, but oxidation of other lipoprotein classes may also be important. Common mechanisms are likely to contribute to the oxidation of all lipoprotein classes, with enzyme-mediated oxidation likely to be most important. Antioxidant content, fatty acid composition, particle size and the presence of seeding hydroperoxides also influence oxidative reactions. Larger triglyceride-rich lipoproteins are less likely to enter the arterial wall than LDL, but when oxidized will deliver a greater oxidant load to the arterial wall.

Quality of care in family planning clinics in Jamaica. Do clients and providers agree?

This paper uses data from 199 providers and 20 simulated clients collected at 50 public sector and Non Governmental Organization (NGO) health facilities islandwide in 1995 to compare the two groups' views on quality of care of family planning services. Each of the five components of quality of care studied can be improved in Jamaica. Nearly two-thirds of the simulated clients felt able to freely choose a contraceptive method; however, more adequate and appropriate information needs to be imparted to clients through improved counselling, including promotion of dual method use (against STD/HIV/AIDS and conception). The requirement that a woman must be menstruating to receive services has inadvertently resulted in many clients going away empty-handed (without counselling or condoms) when they visit family planning clinics. While providers generally treat clients well, training and service delivery practices need to be revised to improve the technical competence of providers. All of the providers would recommend these clinics to others, compared to a little over half of the simulated clients. Both the providers and simulated clients said that privacy should be strengthened, particularly in small facilities in rural areas. Many of these aspects of quality of care are being improved in Jamaica's public sector health facilities. Managers can learn more about quality of care by seeking the knowledge, opinions and experiences of both providers and clients.

Scale-down model to simulate spatial pH variations in large-scale bioreactors.

For the first time a laboratory-scale two-compartment system was used to investigate the effects of pH fluctuations consequent to large scales of operation on microorganisms. pH fluctuations can develop in production-scale fermenters as a consequence of the combined effects of poor mixing and adding concentrated reagents at the liquid surface for control of the bulk pH. Bacillus subtilis was used as a model culture since in addition to its sensitivity to dissolved oxygen levels, the production of the metabolites, acetoin and 2,3-butanediol, is sensitive to pH values between 6.5 and 7.2. The scale-down model consisted of a stirred tank reactor (STR) and a recycle loop containing a plug flow reactor (PFR), with the pH in the stirred tank being maintained at 6.5 by addition of alkali in the loop. Different residence times in the loop simulated the exposure time of fluid elements to high values of pH in the vicinity of the addition point in large bioreactors and tracer experiments were performed to characterise the residence time distribution in it. Since the culture was sensitive to dissolved oxygen, for each experiment with pH control by adding base into the PFR, equivalent experiments were conducted with pH control by addition of base into the STR, thus ensuring that any dissolved oxygen effects were common to both types of experiments. The present study indicates that although biomass concentration remained unaffected by pH variations, product formation was influenced by residence times in the PFR of 60 sec or longer. These changes in metabolism are thought to be linked to both the sensitivity of the acetoin and 2,3-butanediol-forming enzymes to pH and to the inducing effects of dissociated acetate on the acetolactate synthase enzyme.

Physiological responses to mixing in large scale bioreactors.

Escherichia coli fed-batch cultivations at 22 m3 scale were compared to corresponding laboratory scale processes and cultivations using a scale-down reactor furnished with a high-glucose concentration zone to mimic the conditions in a feed zone of the large bioreactor. Formate accumulated in the large reactor, indicating the existence of oxygen limitation zones. It is suggested that the reduced biomass yield at large scale partly is due to repeated production/re-assimilation of acetate from overflow metabolism and mixed acid fermentation products due to local moving zones with oxygen limitation. The conditions that generated mixed-acid fermentation in the scale-down reactor also induced a number of stress responses, monitored by analysis of mRNA of selected stress induced genes. The stress responses were relaxed when the cells returned to the substrate limited and oxygen sufficient compartment of the reactor. Corresponding analysis in the large reactor showed that the concentration of mRNA of four stress induced genes was lowest at the sampling port most distant from the feed zone. It is assumed that repeated induction/relaxation of stress responses in a large bioreactor may contribute to altered physiological properties of the cells grown in large-scale bioreactor. Flow cytometric analysis revealed reduced damage with respect to cytoplasmic membrane potential and integrity in cells grown in the dynamic environments of the large scale reactor and the scale-down reactor.

Quality of care in family planning clinics in Jamaica. Do clients and providers agree?

This paper uses data from 199 providers and 20 simulated clients collected at 50 public sector and Non Governmental Organization (NGO) health facilities islandwide in 1995 to compare the two groups' views on quality of care of family planning services. Each of the five components of quality of care studied can be improved in Jamaica. Nearly two-thirds of the simulated clients felt able to freely choose a contraceptive method; however, more adequate and appropriate information needs to be imparted to clients through improved counselling, including promotion of dual method use (against STD/HIV/AIDS and conception). The requirement that a woman must be menstruating to receive services has inadvertently resulted in many clients going away empty-handed (without counselling or condoms) when they visit family planning clinics. While providers generally treat clients well, training and service delivery practices need to be revised to improve the technical competence of providers. All of the providers would recommend these clinics to others, compared to a little over half of the simulated clients. Both the providers and simulated clients said that privacy should be strengthened, particularly in small facilities in rural areas. Many of these aspects of quality of care are being improved in Jamaica's public sector health facilities. Managers can learn more about quality of care by seeking the knowledge, opinions and experiences of both providers and clients.

Using the Microcyte flow cytometer to monitor cell number, viability, and apoptosis in mammalian cell culture.

The Microcyte is a novel, portable flow cytometer based on diode laser technology whose use has been established for yeast and bacterial analysis. We present data that demonstrate its suitability for routine mammalian cell counting and viability determination. To extend its range of applications in the field of animal cell culture biotechnology, a test to determine the number of apoptotic cells present has been developed for use with the instrument. Apoptosis was induced in hybridoma cell cultures by treatment with camptothecin. Apoptotic cells were labeled with biotinylated Annexin V and then visualized using a streptavidin-allophycocyanin conjugate. Their numbers were counted, and the cell size of the apoptotic cell population was determined using the Microcyte.

Studies related to the scale-up of high-cell-density E. coli fed-batch fermentations using multiparameter flow cytometry: effect of a changing microenvironment with respect to glucose and dissolved oxygen concentration.

Multiparameter flow cytometric techniques developed in our laboratories have been used for the "at-line" study of fed-batch bacterial fermentations. These fermentations were done at two scales, production (20 m(3)) and bench (5 x 10(-3) m(3)). In addition, at the bench scale, experiments were undertaken where the difficulty of achieving good mixing (broth homogeneity), similar to that found at the production scale, was simulated by using a two-compartment model. Flow cytometric analysis of cells in broth samples, based on a dual-staining protocol, has revealed, for the first time, that a progressive change in cell physiological state generally occurs throughout the course of such fermentations. The technique has demonstrated that a changing microenvironment with respect to substrate concentration (glucose and dissolved oxygen tension [DOT]) has a profound effect on cell physiology and hence on viable biomass yield. The relatively poorly mixed conditions in the large-scale fermentor were found to lead to a low biomass yield, but, surprisingly, were associated with a high cell viability (with respect to cytoplasmic membrane permeability) throughout the fermentation. The small-scale fermentation that most clearly mimicked the large-scale heterogeneity (i.e., a region of high glucose concentration and low DOT analogous to a feed zone) gave similar results. On the other hand, the small-scale well-mixed fermentation gave the highest biomass yield, but again, surprisingly, the lowest cell viability. The scaled-down simulations with high DOT throughout and locally low or high glucose gave biomass and viabilities between. Reasons for these results are examined in terms of environmental stress associated with an ever-increasing glucose limitation in the well-mixed case. On the other hand, at the large scale, and to differing degrees in scale-down simulations, cells periodically encounter regions of relatively higher glucose concentration.

The procedural validity of retrospective case note diagnosis.

OBJECTIVE: Deriving diagnoses from retrospective case note examination is a common practice in psychiatric research. The Operational Criteria (OPCRIT) diagnostic checklist is essentially a checklist built up of operational criteria defined by a comprehensive glossary and is designed to assign reliable diagnoses from case notes. However, the validity of such a procedure compared with procedures involving prospective assessment has never been tested. We examined the procedural validity of the OPCRIT diagnostic system in relation to four other diagnostic procedures mostly employing prospectively gathered information. METHOD: Three experienced psychopathology raters rated the case notes and clinical abstracts, using the OPCRIT method of diagnostic assignment, of 50 subjects who had participated in an early procedural validity study as an adjunct to the DSM-IV Field Trial for psychotic disorders. The setting was the Early Psychosis Prevention and Intervention Centre (EPPIC), which focuses on first episode psychosis. RESULTS: The pairwise concordance with the other procedures for DSM-III-R diagnoses assigned by OPCRIT using ratings derived from either the clinical abstracts or the case notes was found to be only poor to moderate when compared with the pairwise concordance of the four other procedures. The per cent agreement between OPCRIT clinical abstracts diagnoses and the other procedures ranged from 49% to 60% with kappa values between 0.30 and 0.45, and for OPCRIT case note diagnoses and the other procedures the per cent agreement range was between 44% and 57% and the kappa values were between 0.35 and 0.49. CONCLUSIONS: The procedural validity of diagnoses assigned via the application of checklists of operational criteria to case notes and clinical abstracts alone is unacceptably poor. Such sources need to be buttressed by other data, particularly direct patient interview and informant material.

Transmission of viral disease in the operating room setting.

The transmission of viral disease in the operating room is an evolving problem. Advances in transfusion medicine have made blood supply safer than ever, whereas changes in transfusion practice have minimized the amount of blood transfused. The epidemiology of viral infection is changing in both the general population and among healthcare workers. Vaccination and prevention hold the key to future risk reduction for patients and providers.