High-energy-density biofuels are typically derived from the fatty acid pathway, thus establishing free fatty acids (FFAs) as important fuel precursors. FFA production using photosynthetic microorganisms like cyanobacteria allows for direct conversion of carbon dioxide into fuel precursors. Recent studies investigating cyanobacterial FFA production have demonstrated the potential of this process, yet FFA production was also shown to have negative physiological effects on the cyanobacterial host, ultimately limiting high yields of FFAs.Synechococcus elongatus PCC 7942. Stress response, nitrogen metabolism, photosynthesis, and protein folding genes were up-regulated during FFA production while genes involved in carbon and hydrogen metabolisms were down-regulated. Select genes were targeted for mutagenesis to confirm their role in mitigating FFA toxicity. Gene knockout of two porins and the overexpression of ROS-degrading proteins and hypothetical proteins reduced the toxic effects of FFA production, allowing for improved growth, physiology, and FFA yields. Comparative transcriptomics, analyzing gene expression changes associated with FFA production and other stress conditions, identified additional key genes involved in cyanobacterial stress response.Cyanobacterial FFA production was shown to generate reactive oxygen species (ROS) and lead to increased cell membrane permeability. To identify genetic targets that may mitigate these toxic effects, RNA-seq analysis was used to investigate the host response of A total of 15 gene targets were identified to reduce the toxic effects of FFA production. While single-gene targeted mutagenesis led to minor increases in FFA production, the combination of these targeted mutations may yield additional improvement, advancing the development of high-energy-density fuels derived from cyanobacteria. Escherichia coli[Synechocystis sp. PCC 6803 and Synechococcus elongatus PCC 7942 [Free fatty acids (FFAs) have recently garnered attention as potential precursors for renewable fuel production. FFAs are ideal targets for biofuel production: the pathway for FFA biosynthesis is already present in microorganisms for membrane production ; FFAs archia coli and on tS. elongatus PCC 7942 had reduced photosynthetic yields, suggestive of electron transport disruption, and the phycobiliproteins of these strains were aggregated at the cell poles rather than evenly dispersed throughout the thylakoid membranes, indicating dissociation of the phycobiliosomes [Synechocystis sp. PCC 6803 also had increased membrane permeability [S. elongatus PCC 7942 by the exogenous addition of both saturated fatty acid (SFA) and UFA [Exogenous FFAs are toxic to many organisms, including Gram-negative and Gram-positive bacteria, microalgae, fungi, protozoans, and multi-cellular organisms . In factWhile the mechanism(s) involved in FFA toxicity remain unproven, two possible mechanisms have been proposed based on the available evidence: (1) UFAs may react with reactive oxygen species (ROS) to generate toxic products such as hydroperoxides, free radical species, aldehydes, and oxylipins, and (2) the amphipathic structure of FFAs may allow them to intercalate into both the cell and thylakoid membranes . The genS. elongatus PCC 7942, to FFA production was quantified using RNA-seq, and from this analysis, differentially expressed genes were identified for either gene knockout or gene overexpression. The physiological responses and FFA productivities of these targeted mutants were assessed to determine the gene’s role in cellular FFA toxicity response. Additionally, the transcriptional response to FFA production was compared to other transcriptomic studies of cyanobacterial stress response to identify the key genes involved in general cyanobacterial stress response. From the analyses presented in this study, genetic targets were determined for improving cyanobacterial physiology during FFA production.In order to surmount the physiological effects of FFA production, the underlying mechanisms of FFA toxicity must be investigated along with the specific host response. This study explored the two proposed mechanisms of FFA toxicity through analysis of ROS generation and cell membrane permeability. The global transcriptional response of the cyanobacterial host, S. elongatus PCC 7942 was engineered for FFA production by gene knockout of acyl-ACP synthetase (SE01 = S. elongatus PCC 7942 Δaas) and introduction of a truncated E. coli thioesterase (SE02 = S. elongatus PCC 7942 Δaas ‘tesA) [aas was necessary to prevent recycling of FFAs through the β-oxidation pathway, while expression of the thioesterase released FFAs from acyl-ACP. Synthesis and excretion of FFAs resulted in detrimental physiological effects, ultimately limiting FFA biosynthesis. The two potential mechanisms of FFA toxicity, described previously, were investigated to determine the underlying cause of the observed physiological effects in the FFA-producing strains of S. elongatus PCC 7942, SE01 and SE02.Previously, 2 = 0.46, p = 0.0367)   Figure  A and C.2 = 0.48, p = 2.10 x 10-4)   as a defense mechanism against grazers [S. elongatus PCC 7942; amplification of these natural responses may allow for increased FFA production.Based on this preliminary assessment, both proposed mechanisms of FFA toxicity are possible and may be contributing factors of the observed toxicity. So the question remains, how can these toxic effects be prevented or mitigated? Microorganisms have evolved a multitude of mechanisms for coping with stressful conditions, including both general and stress-specific response mechanisms . Such na grazers . Along wS. elongatus PCC 7942 to FFA production, both the wild type (7942) and FFA-producing (SE01 and SE02) cultures were sampled at 100 h and 240 h for RNA-seq analysis. Genes differentially expressed during high FFA production were identified through six different comparisons: A) SE02:SE01 at 100 h, B) SE02:7942 at 100 h, C) SE01:7942 at 240 h, D) SE02:7942 at 240 h, E) 240 h:100 h for SE01, and F) 240 h:100 h for SE02. It should be noted that comparisons A and B are only valid for increased extracellular FFA production on a cell weight basis (mg/gDCW), while comparison F is only valid for enhanced extracellular FFA concentration (mg/L). All other comparisons  apply to both measurements of FFA production  and heat shock proteins (hsp20 and hsp90) were significantly up-regulated with FFA production. As described in the previous section, FFA production was accompanied by elevated ROS levels. To minimize ROS-induced cellular damage, oxidative stress response genes were elevated during FFA production, including ROS-degrading enzymes such as superoxide dismutase (Synpcc7942_0801) and glutathione peroxidases (Synpcc7942_1214 and Synpcc7942_0437). High light-inducible proteins have also been shown to enhance the ability of cyanobacteria to cope with oxidative stress response under high light [Genes showing increased expression during elevated FFA production were predominantly associated with known cellular stress responses. General stress response genes such as high light-inducible proteins , had elevated expression; SigD is a light-inducible sigma factor which activates transcription of psbA, the D1 protein of photosystem II (PSII) [sigD transcript levels, a PSII D1 protein (Synpcc7942_0893) showed enhanced expression. Many other PSII proteins were up-regulated, including several PSII D2 proteins (Synpcc7942_1637 and Synpcc7942_0655) and the PSII reaction center protein N (psbN). In 3 out of the 6 comparisons, transcript levels of the PBS degradation protein, nblA, were significantly increased by an average of 4.37-fold. Phycobiliprotein levels, however, did not change significantly in the FFA-producing cultures [Transcripts associated with photosynthesis were significantly up-regulated during FFA production. The RNA polymerase sigma factor, I (PSII) . In agregD Synpcc942_0672,E. coli, particularly TolC-AcrAB, have been identified as potential FFA exporters, although increased expression of these genes did not improve FFA production in the E. coli host [tolC homolog in S. elongatus PCC 7942, a hydrophobe/amphiphile efflux protein (Synpcc7942_2369) was found to be homologous to several E. coli efflux proteins including acrA, acrB, acrD, acrF, mdtB, and mdtF. However, gene expression of Synpcc7942_2369 was not significantly changed during FFA production. Several other transport proteins and porin proteins were found to be up-regulated during FFA production  was also down-regulated in comparisons B, C, and D. As you may recall, this gene was targeted for gene knockout in both SE01 and SE02. Therefore, the reduced transcription of aas in only comparisons of the wild type with the engineered strains  validates the data processing protocol developed for the RNA-seq analysis. Repression of carbon metabolism in FFA-producing cultures, as suggested by RNA-seq analysis, may serve as yet another indicator of severe cellular stress.Another conserved response of transcriptional repression is associated with carbon metabolism. Specifically, phosphoenolpyruvate (PEP) synthase was significantly down-regulated an average of 3.87-fold in 5 of the 6 comparisons. PEP synthase catalyzes an energy-consuming reaction, and reduced expression of this enzyme may serve as a stress-induced mechanism for energy conservation. Repression of PEP synthase also increases carbon flux through the glycolysis pathway, which feeds into the fatty acid biosynthesis pathway. Thus, the lower level of fatty acids available for membrane biosynthesis, due to fatty acid cleavage by the recombinant thioesterase, may cause the cell to increase carbon flux through the fatty acid biosynthesis pathway via PEP synthase repression. A sodium-dependent bicarbonate transporter showed an average 9-fold decrease in transcript level for 3 comparisons, suggesting a decrease in photosynthetic carbon fixation, and subsequently, a lower overall carbon flux may accompany FFA production. Lastly, the long-chain-fatty-acid CoA ligase or acyl ACP synthetase , however, is a bidirectional hydrogenase with undetermined physiological function. Proposed functions for this hydrogenase includes the delivery of electrons into respiratory complex I and the regulation of photosynthetic electron flow [Genes associated with hydrogen metabolism had reduced transcript levels during FFA production. The genes encoding the hydrogenase accessory proteins, ron flow . RepressMany regulatory proteins were down-regulated across multiple comparisons as well as genes associated with the global signaling molecule, cyclic-di-guanosylmonophosphate (c-di-GMP). Cellular c-di-GMP levels have been linked to the regulation of a variety of cellular processes, including cellular motility, virulence, heavy metal resistance, phage resistance, cell-cell communication, photosynthesis, exopolysaccharide production, and biofilm formation . The redaas deletion is required to prevent FFA recycling and SE02 had increased physiological effects during FFA production. The gene targets included hypothetical proteins, ROS-degrading proteins, and possible FFA exporters .Significant changes in relative cell concentration occurred most frequently for days 7, 10, and 13, following induction of the recombinant proteins Figure  A, C, E.Changes in relative extracellular FFA concentration were found to correlate with the changes in relative cell concentration for the hypothetical protein and ROS-degrading protein overexpression mutants; the mutants showing increased relative cell concentrations also had increased relative extracellular FFA concentrations Figure  B and D.Photosynthetic yield measurements were analyzed to serve as an estimate of overall cell physiology. For the control strain SE02a, photosynthetic yield declined severely after induction at day 4, with photosynthetic yield measurements approaching zero at day 7 Figure . Hypothe, Synechococcus sp. PCC 7002 transcriptome studies of temperature, oxidative, salt, and nutrient limitation  stresses [Synechocystis sp. PCC 6803 studies which analyzed transcriptional responses to temperature, oxidative, salt, osmotic, and ethanol stresses [S. elongatus PCC 7942, Synechococcus sp. PCC 7002, and Synechocystis sp. PCC 6803 were identified, as described in the Materials and Methods section, to facilitate the comparison. Many differentially expressed genes associated with FFA toxicity in S. elongatus PCC 7942 corresponded to homologs of differentially expressed genes in these previous stress studies  and a hypothetical protein . Another 4 genes were up-regulated in 11 or 12 of the stress conditions: the heat shock protein Hsp20 , the NAD(P)H-quinone oxidoreductase subunit 4 , RNA polymerase sigma factor SigD , and Beta-Ig-H3/fasciclin . These 6 genes are candidate targets for boosting the native stress response mechanisms of S. elongatus PCC 7942. The hypothetical protein and porin genes identified from targeted mutagenesis in the previous section were also investigated in this comparative transcriptomics analysis. Out of the 3 hypothetical protein targets , Synpcc7942_1655 was the only gene without homologs in Synechococcus sp. PCC 7002 and Synechocystis sp. PCC 6803. Both Synpcc7942_0122 and Synpcc7942_0900 had homologs in the other cyanobacterial species that were differentially expressed under other stress conditions, with down-regulation in 8 and 5 of the other stress conditions, respectively. Therefore, Synpcc7942_1655 may be a unique stress response gene in S. elongatus PCC 7942 or perhaps a response specific to FFA production, while Synpcc7942_0122 and Synpcc7942_0900 are likely involved in general stress response. The two porin targets, Synpcc7942_1464 and Synpcc7942_1607, were also differential expressed in the other cyanobacterial strains, showing up-regulation in 8 of the other stress conditions. The 9 genes identified through targeted mutagenesis and the 6 additional stress genes identified through this comparative transcriptomics analysis will be candidate targets for future improvement in cyanobacterial FFA production and general stress response.Genetic targets for improving FFA production in S. elongatus PCC 7942 confirmed the presence of intracellular ROS, and based on the known reactive properties of UFAs and ROS [This study investigated two proposed mechanisms of FFA toxicity in cyanobacteria: UFA reactivity with ROS to produce toxic compounds and the intercalation of FFAs in cellular membranes. Elevated ROS levels in FFA-producing strains of  and ROS , UFA degS. elongatus PCC 7942 to FFA production, natural mechanisms for alleviating FFA toxicity were identified. These mechanisms included the activation of genes contributing to general stress response, nitrogen metabolism, PSII photosynthesis, and protein folding as well as repression of genes involved in PSI photosynthesis and carbon and hydrogen metabolisms. Many of these cellular responses were expected, as they agree with the ‘core transcriptional response’ (CTR) genes determined for stress response in Synechocystis sp. PCC 6803 [S. elongatus PCC 7942 during FFA production.Through examining the transcriptional response of PCC 6803 . The difS. elongatus PCC 7942 were identified from the RNA-seq analysis, targeted mutagenesis experiments, and comparative transcriptomics analysis. Differentially expressed hypothetical proteins, ROS-degrading proteins, and potential FFA transporters were targeted to generate 15 mutant strains. The overexpression of ROS-degrading proteins improved cell growth, physiology, and FFA production, presumably due to ROS degradation and the subsequent reduction in cellular damage. Gene knockout of the two porin proteins (Synpcc7942_1464 and Synpcc7942_1607) improved FFA production and cellular physiology without significantly affecting cell growth. These porin proteins may therefore participate in FFA uptake rather than export. The overexpression of 3 hypothetical proteins  also improved cell growth, physiology, and FFA production. In addition to these genetic targets specific to FFA production, comparative transcriptomics of cyanobacterial stress responses identified potential targets for improving cellular stress response in general. These conserved stress response targets include a high light-inducible protein (Synpcc7942_1997), a hypothetical protein (Synpcc7942_0551), heat shock protein Hsp20 (Synpcc7942_2401), NAD(P)H-quinone oxidoreductase subunit 4 (Synpcc7942_1439), RNA polymerase sigma factor SigD (Synpcc7942_0672), and Beta-Ig-H3/fasciclin (Synpcc7942_1606). By targeting these stress response genes along with the targets confirmed in the targeted mutagenesis investigation, the physiological stress associated with FFA toxicity may be alleviated in FFA-producing strains of S. elongatus PCC 7942. By overcoming the physiological effects of FFA production, higher FFA productivities may be attained, advancing the development of cyanobacterial based biofuels.Genetic targets for addressing FFA toxicity in FFA-producing 2DCFDA), MP Biomedicals , Acros Organics (sodium molybdate(VI) dihydrate and cobalt(II) nitrate hexahydrate), Gold Biotechnology , Invitrogen/Life Technologies (SYTOX) and Fisher Scientific . DNA purification kits used in strain construction include the Zyppy Plasmid Miniprep Kit and Zymoclean Gel DNA Recovery Kit (Zymo Research Corporation). Enzymes used in cloning include DNA polymerases , restriction enzymes, and T4 DNA ligase from New England Biolabs. Primers were synthesized by Integrated DNA Technologies. Kits for FFA quantification and RNA isolation are described below. All strains and plasmids used in this study are listed in Additional file Chemicals used in this study were purchased from Sigma-Aldrich  and 50 μg/mL kanamycin monosulfate (Km)) as appropriate, to test tubes containing 4 mL of liquid media. After 5 to 7 days of growth, these test tube cultures were used as inoculums for both the large-scale FFA production experiments and the small-scale mutant screening experiments.All strains were grown at 30 °C with shaking at 150 rpm and approximately 60 μmol photons m730) was approximately 0.15. No antibiotics were added to the final experimental cultures to reduce any effects of the antibiotics on the experimental results. The large-scale cultures were grown under the previously described conditions and bubbled with air and 1% CO2. Additional setup information can be found in [‘tesA) expression was induced by the addition of 0.1 M isopropyl β-D-1-thiogalactopyranoside (IPTG) to a final concentration of 1 mM after approximately 4 days of growth. Samples were taken every 2 days for analysis of growth (OD730), photosynthetic yield (Fv’/Fm’), and FFA concentration. Every 4 days, samples were taken for measurement of ROS levels and cell membrane permeability. Samples for RNA-seq analysis were taken before induction (day 4) and 8 days after induction (day 12). Three biological replicates were performed for each large-scale experiment.The large-scale FFA production experiments were used for obtaining samples for RNA-seq analysis. For these experiments, a second inoculum was prepared by adding 1 mL of the test tube inoculum to a 500 mL baffled Erlenmeyer flask containing 100 mL of BG-11 medium and antibiotics as appropriate. These larger inoculums were grown for approximately 4 days under the aforementioned growth conditions. These cultures were added to 1 L media bottles containing 400 mL of BG-11 media so that the optical density at 730 nm (OD730), photosynthetic yield (Fv’/Fm’), and FFA concentration. Three biological replicates were performed for each small-scale experiment.Small-scale experiments were conducted to screen the 7 knockout and 10 overexpression mutants. For each mutant strain, two transformed colonies were selected for screening to account for possible variation among the transformants. This was particularly important for the overexpression mutants, which may have tested positive for PCR amplification of the inserted gene but could have had a mutation effecting gene expression. For the small-scale screening experiments, 1 mL of the test tube inoculum was added to 25 mL of BG-11 medium in a 125 mL baffled Erlenmeyer flask. After 4 days of growth, cultures were induced by adding IPTG to a final concentration of 1 mM. Samples were taken every 3 days to measure growth (OD730) using a PerkinElmer Lambda Bio spectrophotometer. A linear calibration curve relating OD730 and gDCW was obtained previously [v’/Fm’ values are averages of 3 technical replicate measurements. To measure extracellular FFA concentration, 0.5 mL of culture was centrifuged at 5000 x g for 3 min. The supernatant was transferred to a new tube and frozen at −20 °C until analysis. FFA concentration was analyzed using the Free Fatty Acid Quantification Kit from Biovision, as described previously [Bulk spectroscopic measurements were taken to assess cell growth, photosynthetic yield, and extracellular FFA concentration. Cell growth was estimated by measuring the optical density at 730 nm  to permeabilize the cell membrane for dye uptake. The mixture was incubated under culture conditions for 1 hour to allow for dye diffusion and ROS-mediated activation. The culture was diluted and analyzed using an Accuri C6 flow cytometer. For each measurement, 20,000 cells were analyzed, and cells with an increased fluorescence emission at 533 nm were determined to be positive for ROS. To measure cell membrane permeability, SYTOX Green Nucleic Acid Stain (Invitrogen/Life Technologies) was added to 0.3 mL culture samples to a final concentration of 300 nM. After 20 min of incubation in the dark at room temperature, the dyed cultures were analyzed using flow cytometry. Approximately 20,000 cells were measured for fluorescence emission at 533 nm, and an increase in fluorescence intensity at this wavelength was indicative of positive SYTOX staining (i.e. membrane permeable cells). Both ROS detection and cell membrane permeability are reported as the percentage of the cell population with positive staining.Single-cell analysis was used to measure the levels of ROS and cell membrane permeability in each culture. For ROS analysis, 0.5 mL of culture was mixed with Hp-values are reported in the text along with the R2 value associated with a linear regression analysis. To confirm statistically significant variation between the control strain SE02a and the targeted mutants, two-factor ANOVA analyses with replicates were performed for relative OD730 measurements (days 7 – 19) and relative FFA concentration (days 10 – 19). For the photosynthetic yield data, single factor ANOVA analyses were applied to the measurements from day 7, comparing the control (SE02a#1 and SE02a#3) and mutant data. Mutant strains with statistically significant (p < 0.05) changes relative to the control are indicated in Figures p-values from the ANOVA analyses are listed in Table S7 , there was a significant reduction in the number of aligned reads, with more than 96% of the total reads being discarded in the most severely affected sample. As a result, the best alignment for each read was selected, and no reads were discarded due to multiple alignments to the reference genome. The SAM files constructed by Bowtie were submitted to HTSeq v. 0.5.3p3 to obtain raw counts for each predicted gene feature in the S. elongatus PCC 7942 genome. Raw counts for each sample are shown in Additional file A total of 524 million reads corresponding to 52.9 billion bases of sequence data were generated from the 17 RNA samples. After sequencing, the de-multiplexed data files for each sample were analyzed using FastQC v 0.10.1 . The aveS. elongatus PCC 7942, Synechococcus sp. PCC 7002, and Synechocystis sp. PCC 6803. Differential gene expression data were obtained for each reference using either supplemental material or tables provided in the publications [S. elongatus PCC 7942 during FFA toxicity (determined in this study) using the list of homologs generated from MBGD and a Perl script generated for this comparison.Differentially expressed genes associated only with comparison F were excluded from the comparative transcriptomics analysis. Comparison F (SE02 – 240 h:100 h) includes an increase in extracellular FFA concentration but no change in FFA concentration on a per cell weight basis. Furthermore, the differentially expressed genes identified in comparison F suggest a reduction in cellular stress levels, contrary to the other 5 comparisons. These reasons justify the exclusion of comparison F. The Microbial Genome Database (MBGD) for Comparative Analysis was used to identify homologs among the three cyanobacterial species: ications -33,43. TThe following additional data files are available with the online version of this paper.ACP: Acyl carrier protein; c-di-GMP: Cyclic-di-guanosylmonophsophate; CoA: Coenzyme A; FC: Fold change; FFA: Free fatty acid; gDCW: Grams of dry cell weight; IPTG: Isopropyl β-D-1-thiogalactopyranoside; Km: Kanamycin monosulfate; MBGD: Microbial genome database; NAD(P)H: Nicotinamide adenine dinucleotide phosphate; NSII: Neutral integration site II; PBS: Phycobilisome; PCR: Polymerase chain reaction; PEP: Phosphoenolpyruvate; PSI: Photosystem I; PSII: Photosystem II; PUFA: Polyunsaturated fatty acid; RNA: Ribonucleic acid; ROS: Reactive oxygen species; RuBisCO: Ribulose-1,5-bisphosphate carboxylase/oxygenase; SE: Standard error; SFA: Saturated fatty acid; Sp: Spectinomycin dihydrochloride pentahydrate; UFA: Unsaturated fatty acid.The author declares that she has no competing interests.AMR conceived of the study, carried out the physiological studies, conducted the experiments for RNA sampling and isolation, processed and analyzed the RNA-seq data, constructed and tested the mutant strains, performed the comparative transcriptomics, interpreted the data, and drafted the manuscript.Table S2, listing the genes down-regulated during FFA production. Listing the genes up-regulated during FFA production. Click here for fileTable S6, listing the primers used in this study. Table S7, listing the p-values for the ANOVA analyses. Listing the strains and plasmids used in this study. Click here for fileTable S4, listing the down-regulated genes from the comparative transcriptomics analysis. Listing the up-regulated genes from the comparative transcriptomics analysis. Click here for fileContains the raw counts for each biological sample analyzed using RNA-seq.Click here for file


At any time, about 1% of people with severe and enduring mental illness such as schizophrenia require in-patient psychiatric rehabilitation. In-patient rehabilitation enables individuals with the most challenging difficulties to be discharged to successful and stable community living. However, the length of rehabilitation admission that is required is highly variable and the reasons for this are poorly understood. There are very few case-control studies of predictors of outcome following hospitalisation. None have been carried out for in-patient rehabilitation. We aimed to identify the factors that are associated with achieving discharge from in-patient rehabilitation by carrying out a case-control study.We compared two groups: 34 people who were admitted to the Rehabilitation Service at the Royal Edinburgh Hospital and discharged within a six year study period, and 31 people who were admitted in the same period, but not discharged. We compared the groups on demographic, illness, treatment and risk variables that were present at the point of their admission to rehabilitation. We used independent t tests and Pearson Chi-Square tests to compare the two groups.We found that serious self harm and suicide attempts, treatment with high dose antipsychotics, antipsychotic polypharmacy and previous care in forensic psychiatric services were all significantly associated with non-discharge. The non-discharged group were admitted significantly later in the six year study period and had already spent significantly longer in hospital. People who were admitted to rehabilitation within the first ten years of developing psychosis were more likely to have achieved discharge.People admitted later in the study period required longer rehabilitation admissions and had higher rates of serious self harm and treatment resistant illness. They were also more likely to have had previous contact with forensic services. This change over time is likely to be due to the drive in Scotland to manage mentally disordered offenders in conditions of lower security. There is a growing need for secure longer-term in-patient rehabilitation, particularly for people previously treated in forensic services. Admission to rehabilitation earlier in a person's illness may improve their outcome. At any time, about 1% of people with severe and enduring mental health problems such as schizophrenia require in-patient psychiatric rehabilitation . DischarInternational studies of schizophrenia and other psychotic disorders have found only limited evidence that demographic, illness and treatment variables predict outcome, either in terms of remission or disability -11. The NHS Lothian provides services for 800,000 people in Edinburgh and the Lothians and is the second largest NHS board in Scotland. The Rehabilitation Service at the Royal Edinburgh Hospital consists of an in-patient service with four wards and a total of 74 beds and a Community Rehabilitation Team. Two wards with 25 beds and 15 beds respectively offer high-dependency rehabilitation. This is for people with a high level of symptoms as well as significant risk histories and challenging behaviours. The two other wards provide longer-term complex care. This offers longer term admission, often for several years. This is for people with a high level of disability from a complex mix of conditions who also present a risk to themselves or others. One is a 19 bedded male only ward and the other a 15 bedded ward that provides a service for people with mental illness as well as serious physical health problems. Wolfson, Holloway and Killaspy have written a full description of the types of in-patient rehabilitation elsewhere . There wst April 2004 and ending 1st April 2010. The first group were admitted and discharged within the same period (n = 34). Many more people were discharged from the Service during this period, but only those admitted on or after 1st April 2004 were included in the study. The second group were admitted during the same time period, but had not achieved discharge by the end of the six years we studied (n = 31). This group of non-discharged patients was selected on April 1st 2010 and matched to the discharged group by ward of admission. There were no exclusion criteria.The sample consisted of two groups. Both of the groups were admitted to the Rehabilitation Service wards at the Royal Edinburgh Hospital in a six year period beginning 1In April 2004 a new set of baseline assessments was introduced by the Rehabilitation Service. These 20 page documents record the person's psychiatric, personal and past medical history as well as a risk assessment and information about their medication and physical health. The documents are produced by the multi-disciplinary team within the first two months of admission and are stored electronically. We designed data collection sheets for the study to gather data from these documents. The data gathering sheets are available from the first author on request. The date of discharge was gathered from a computerised patient management system.We used a case control study design to compare the two groups described above on demographic, illness, social, treatment and risk variables that were present at the point of their admission to in-patient rehabilitation. We chose these variables based on the literature around long-stay. Only variables that were reliably recorded were chosen.Independent t tests were used to compare the groups on continuous, normally distributed variables including age, date of admission and length of admission to rehabilitation. Pearson Chi-Square tests were used for categorical data and compared the groups on all other variables. The data was collected and analysed by the lead author. We consulted a statistician before analysing the data and carried out the analysis using Minitab for Windows.The South East Scotland Research Ethics Service confirmed that ethical approval was not required under NHS research governance arrangements.There were no significant differences between the two groups in terms of age, sex or diagnosis. This can be seen in table 2 = 4.7, P = 0.03) and previous care in forensic psychiatric services  were both significantly associated with non-discharge. Aggression, absconding and sexual offences or incidents  were also more common in the non-discharged group.Table Harmful or dependent substance use was very common and the rates were similar in the two groups. Alcohol dependence was present in 9% and opiate dependence in 11% of the total sample and harmful use of either was present in 46%.2 = 6.6, P = 0.01). Unfortunately in 15 of the discharged group and 11 of the non-discharged group (40% of the whole sample) it was not clear whether or not high dose antipsychotics had been prescribed in the past. These cases were excluded from the comparison. Antipsychotic polypharmacy was defined as the prescription of more than one regular antipsychotic. Polypharmacy in the past was significantly associated with non-discharge . There was no association between compulsory treatment under the Mental Health Act (Care & Treatment) (Scotland) Act 2003 and outcome.Table 2 = 2.4, P = 0.12).There were no statistically significant differences between the groups in the illness and social factors we studied . Three of the four university degrees conferred were to this group.We found that a history of self harm or suicide attempts, treatment with high dose antipsychotics and antipsychotic polypharmacy were all significantly associated with non-discharge. Previous treatment in forensic psychiatric services was also associated with non-discharge. The non-discharged group were admitted significantly later in the six year study period and had already spent significantly longer in hospital.A history of self harm or suicide attempts was significantly associated with non-discharge. Aggression and sexual offences or incidents were also more common in the non-discharged group. Self harm and suicide attempts before and after admission to hospital have been shown to increase the risk of suicide in people with schizophrenia . ChallenHarmful or dependent use of substances was more common in the sample than in the general population. In 2006 in Scotland 1.6% of people aged between 15 and 64 had dependent use of opiates or benzodiazepines . In our The prescription of clozapine, either at the point of admission to rehabilitation or ever in the past, was not associated with discharge. This is an important finding as it does not support the idea that improvement in rehabilitation is largely due to clozapine being prescribed. We found that both antipsychotic polypharmacy and the use of high dose antipsychotics in the past were significantly associated with non-discharge. It is likely that these are related to poorer outcome because they reflect a higher level of treatment resistance in the non-discharged group. Treatment resistance is defined by NICE as the "presence of poor psychosocial and community functioning that persists despite trials of medication that have been adequate in terms of dose, duration and adherence" . An assoThe social factors we examine were not significantly associated with outcome. This is in keeping with other studies, which have shown that social and demographic factors contribute little to predicting outcome in people with psychosis ,8.We noted that admission to rehabilitation within the first ten years of onset of psychosis was more common in the discharged group, although this did not reach statistical significance. This raises the question of whether earlier engagement in rehabilitation might improve outcomes, perhaps for the 10% of people who fail to achieve remission after their first episode of psychosis . This reThe non-discharged group were admitted later in time and had significantly higher rates of self harm or suicide attempts and higher rates of aggression and violence. They were significantly more likely to have previously been cared for in forensic psychiatric services. The characteristics of the people admitted to the Rehabilitation Service over time have changed, with a trend towards increased levels of challenging behaviour and more highly treatment resistant illness. This is likely to be because of the drive in Scotland to managing this challenging population in conditions of lower security. Scottish government policy recognised that people were admitted to the High Secure State Hospital for longer than was necessary due to a lack of effective local arrangements for mentally disordered offenders . As wellWe did not measure and correct for symptom severity. It may be that some of the associations of non-discharge are a result of more severe illness. However, we minimised this effect by matching the cases and controls by ward environment and demographically the two groups were similar. We didn't look at the process of rehabilitation and the interventions - for example skills training, family interventions and psychological therapies - that took place. These interventions would be expected to have an impact on outcome. The use of discharge as the primary outcome measure does have limitations as well as the benefits described above. It may not accurately reflect the level of disability  and doesIn a sample of rehabilitation service in-patients, we found that self harm, suicide attempts and previous care in forensic psychiatric services were all significantly associated with not having achieved discharge during the six year period we studied. Non-discharge was also associated with previous treatment with high dose antipsychotics and antipsychotic polypharmacy. This is likely to reflect higher levels of treatment resistance and possibly poorer engagement in the non-discharged group. There was a change over time in the characteristics of the in-patient rehabilitation population and this has important implications for service design. There is a growing need for secure, longer-term in-patient rehabilitation with high staff to patient ratios and access to a wide variety of therapeutic interventions, particularly for people transferred from forensic services. Secure longer-term rehabilitation could be provided as part of either forensic or rehabilitation services. Different areas will have to meet the challenge of this growing need in different ways depending on the design of their local services.The authors declare that they have no competing interests.JB collected the data, performed the statistical analysis and drafted the manuscript. All authors conceived of the study, participated in the design of the study and read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-244X/11/149/prepub


Retraction, atrophy and fatty infiltration are signs subsequent to chronic rotator cuff tendon tears. They are associated with an increased pennation angle and a shortening of the muscle fibers in series. These deleterious changes of the muscular architecture are not reversible with current repair techniques and are the main factors for failed rotator cuff tendon repair. Whereas fast stretching of the retracted musculotendinous unit results in proliferation of non-contractile fibrous tissue, slow stretching may lead to muscle regeneration in terms of sarcomerogenesis. To slowly stretch the retracted musculotendinous unit in a sheep model, two here described tensioning devices have been developed and mounted on the scapular spine of the sheep using an expandable threaded rod, which has been interposed between the retracted tendon end and the original insertion site at the humeral head. Traction is transmitted in line with the musculotendinous unit by sutures knotted on the expandable threaded rod. The threaded rod of the tensioner is driven within the body through a rotating axis, which enters the body on the opposite side. The tendon end, which was previously released (16 weeks prior) from its insertion site with a bone chip, was elongated with a velocity of 1 mm/day.After several steps of technical improvements, the tensioner proved to be capable of actively stretching the retracted and degenerated muscle back to the original length and to withstand the external forces acting on it.This technical report describes the experimental technique for continuous elongation of the musculotendinous unit and reversion of the length of chronically shortened muscle. Chronic rotator cuff tears lead to retraction of the musculotendinous unit, atrophy and hitherto irreversible fatty infiltration of the rotator cuff muscles -6. In thMuscle homeostasis is regulated by different mechanical signals ,14, primTo test our hypothesis that continuous traction might allow restoration of structure and function of chronically retracted muscle, an in-vivo technique had to be developed to apply continuous traction to a retracted musculotendinous unit. It is the purpose of this paper to present a new experimental tool and corresponding technique, which allows applying tension to a chronically retracted musculotendinous unit in the sheep model.An apparatus called “tensioner” allowing application of tension to the infraspinatus muscle-tendon-unit without necessitating limb immobilization was designed:Tensioner Figure :The tensioning device (“tensioner”) comprised a base plate (A) for fixation, which was welded to a longitudinal body (B). The body of the tensioner housed a threaded rod , which was driven out of the body (B) by turning a rotating axis (D) which entered the body (B) on the opposite side of the rod. The rotating axis was made of Nitinol® and had a length of 155 mm. At the end of the rotating axis, a steel wheel of 20 mm diameter was fixed to allow lengthening of the threaded rod. Turning of the rotating axis by 360° results in an extrusion of the rod by 1 mm. A clicking mechanism gives a small resistance after every full turn  of the axis and inhibits unwanted passive re-rotation of the rod. The rod displaces rotating clockwise and that the click mechanism prevents counter clockwise rotations. This axis (D) is connected to the body axis (B) with a universal (or Cardan) joint to allow angulations of axis (D) relative to the body axis (B). The steel wheel (E) mounted on rotating axis D is external to the sheep’s body. The Cardan joint is protected from spontaneous scarring and tangling with adjacent soft-tissue with a silicon tube. The apparatus is fixed on the scapular spine of the sheep with locking screws, which prevent angular shifting of the baseplate.Two different tensioners were manufactured and implanted during experiments. Tensioner No.1 was developed in collaboration with the AO, Development Center . For fixation of the tensioner in-vivo experiments, device No. 1 consisted of a base plate welded to the body of the tensioner. The frame was then fixed to the scapular spine using a maximum of four screws in the fixation plate  to secure the sutures holding the tendon to it during the “in-vivo-experiments”. Because of cut outs of four baseplates out of the scapular spine with tensioner No. 1, it was changed to the technically improved tensioner No. 2 over the course of the experiment. This tensioner No. 2 was performed in collaboration with Synthes . The second generation tensioner consisted of a longer base plate with eight screw holes for fixation of the base plate to the scapular spine . To reach more stability the multiaxial joint was made of stainless steel. Both, the Nitinol®-rod and the steel wheel had a higher diameter of 2 mm, and 32 mm respectively.In vitro experiments:Axial forces developed actively with both tensioners by extruding the threaded rod from the body, were measured using an Instron Universal Testing Machine 4202  with a 5kN load cell, mounted to the cross-bar. The sample was fixed to the lower table of the machine, using a vice holding the body of the scapula. The mean maximal axial force that can be developed by hand with each tensioner was tested by three different examiners .Three tensioner units of versions No. 1 and No. 2 were tested with cycling loading experiments. The tensioners were fixed on the scapular spine with 4 screws in tensioner No. 1 and six screws in tensioner No. 2, respectively. They were mounted on an Instron Universal Testing Machine 4202  with the adjustable fixation device, which allowed adjustment of direction of pull. The direction of load application corresponded to the axis of the infraspinatus muscle in vivo .The surgical approach was performed using a curved incision over the lateral aspect of the acromion  under geBy turning the steel wheel 360° once a day the tensioner pulled the tendon axially by a distance of 1 mm/day towards its original insertion.To document the elongation of the musculotendinous unit, computer tomograms were performed after implantation and every 14 days under general anesthesia. The position of the tensioner and the greater tuberosity were measured in relation to the midglenoidal plane.Subsequent to the elongation protocol, the tensioner was removed and the greater tuberosity was reattached as near as possible to its original insertion using 3.5 mm cortical bone screws and the previously prepared sutures. According to a previous study , the fixBoxplots and unpaired t-tests were performed to evaluate possible differences between the applied forces, displacements and maximal failure load of the two tensioners with SPSS version 15.0 for Windows . The level of significance was set at p < 0.05.Expansion force generation Figure The maximal possible load applied by the three examiners was 133 ± 19.03 N with tensioner No.1 and 183.3 ± 28.71 N with tensioner No. 2. The difference between the maximal possible expansion forces of the two tensioners was significant (p = 0.017). Boxplots of expansion forces applied to both tensioners are displayed in Figure Stability in cyclic testing Figure According to a previous study , the meaThe specific results are thoroughly reported and published separately in a paper describing the specific biological findings . ElongatHowever, in the remaining four sheep the elongation failed with tensioner No. 1: Of these, three sheep had an infection developed with abscess formation around the tensioner and the tendon-bone interface and the tendon disintegrated from the bone chip. In one sheep, the Fiber-Wire sutures were cut out of the bone chip and the tendon was again retracted. There were no revisions or reoperations prior to their failure in these four sheep.As a result of previous studies -9,12, itBecause progressive musculotendinous lengthening of a chronically retracted, musculotendinous unit has not previously been performed, a new method was needed. The present concept was selected for various reasons: First, we preferred to perform lengthening by a given distance per day (one mm) rather than by pulling with a specific constant load, as this method has been successful in lengthening muscle in the Ilizarov technique of bone lengthening ,26. MuscThe mechanical in-vitro analyses documented, that the apparatus is capable of delivering adequate tension to the musculotendinous unit during the extrusion of the rod and should be capable of resisting loads, which were exerted by in-vivo contraction of the muscles. Previous in vivo force measurements of the sheep’s infraspinatus muscle have shown that the contractile strength affected by fatty infiltration was constantly between 50 and 200 N, with a maximum force of 310 N ,24,27. FBoth tensioners showed in vitro enough fixation stability to counteract against the pullout strength of the muscle force. However, subsequent to cyclic loading the maximal resistance force was significantly lower in tensioner No. 1, which could be related to the increased fixation points of tensioner No. 2 with 6 screws. This may be a reason of failure by cut out of the tensioners’ body out of the scapular spine in four sheep.Nevertheless, in vivo lengthening of a musculotendinous unit up to its’ original length was feasible in two thirds of the sheep. Despite the fact, that reversion of the original length of a chronic retracted rotator cuff tear could be achieved, the in-vivo experiments, however, proved to be difficult and several problems occurred which were specific to the in-vivo situation: Although the four sheep with cut outs were revised using tensioner No. 2, the intra experiment tension of the musculotendinous unit remained at the same level before and after revision. None of the tensioners No. 2 showed any complication with fixation on the scapular bone or breakage of the Nitinol® rotating axis. To avoid material failure in future, the tensioners’ body should be fixed using at least six fixed angle screws and a diameter of at least 2 mm of the Nitinol® rotating axis is needed.Furthermore, infection occurred along the transcutaneous rotating axis extending to the body of the tensioner in three cases. In terms of animal protection in this demanding animal model, it is of tremendous importance to avoid complications with infections in future studies. Although we were able to standardize this model we think, a significant improvement of the method may be achieved if the force transmission to the tensioner would be performed through a short, subcutaneous mechanism, which is handled through the intact sheep’s skin. As study setup, surgeries and especially postoperative care were shown to be very demanding with a considerable rate of intraoperative and postoperative challenges, it is important that this device and animal model is used only by experienced surgeons and groups with intimate knowledge in handling sheep with postoperative care.This is the first series of experiments in which a mechanical device has been introduced for elongation and restoration of the original length of the musculotendinous unit. In future experiments, the functional and physiological properties of the muscle will be assessed during the phase of retraction and after lengthening, to evaluate if reversion of the muscle architecture does indeed result in an according restoration of the muscle’s physiological contractile properties, the final goal that this ongoing project and tendon repair in general are pursuing.MAZ and EF and DCM carried out the experiments, participated in the design of the study, performed the statistical analysis and drafted the manuscript. BvR carried out the surgical interventions. RF designed and manufactured the tensioner, CG and DCM conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.


This study aimed to evaluate the effectiveness of a rehabilitation intervention on physical dysfunction (PDF) and post-traumatic stress disorder (PTSD) in fracture victims 50 months after the Sichuan earthquake of 2008 and to identify risk factors for PTSD.This is a retrospective cohort study. Four hundred and fifty-nine earthquake-related fracture victims from Mianzhu city, Sichuan Province who did not qualify for disability pension participated. Two hundred and forty-five subjects received regular rehabilitation and 214 did not. Muscle strength, joint range of motion (ROM),sensory function, and sit-to-stand balance capacity were evaluated to assess PDF. The PTSD Checklist-Civilian Version (PCL-C) was administered to screen for PTSD. An ordinary least square regression was used to predict PTSD, and a logistic regression was used to predict PDF. In addition a Least Angle Regression (LARS) was carried out for PTSD to study the effects of rehabilitation and PDF at the same time.p<0.01) and PTSD prevalence (p<0.05) were significant in favor of the rehabilitation group. In addition, being female, average or above family income, having witnessed death and fearfulness were found risk factors for PTSD symptoms 50 months after the earthquake. Both PDF and rehabilitation were selected predictors by LARS demonstrating opposite effects.Unadjusted and adjusted group differences in physical dysfunction (PDF and PTSD were significantly reduced by the rehabilitation intervention. Future medical intervention strategies should consider rehabilitation in order to assist survivors in dealing with both physical and psychological effects of natural disaster. On May 12, 2008, an earthquake of magnitude 8.0 on the Richter Scale struck the densely populated Sichuan Province, in southwestern China. The Sichuan earthquake affected 46 million people resulting in 87,476 deaths, over 350,000 persons injured, leaving many physical and mental health problems in the affected area Past research has demonstrated a clear relationship between natural disasters and mental health problems Comprehensive rehabilitation efforts in the Sichuan earthquake zone, including therapeutic interventions, training and education, as well as vocational and social rehabilitation, were undertaken immediately and concurrently with clinical treatment We therefore aimed to examine the effect of rehabilitation on physical dysfunction and PTSD in fracture victims from the 2008 Sichuan earthquake, at 50 months after the disaster.A cross-sectional survey was conducted in July, 2012, 50 months after the disaster. We employed a retrospective cohort design comparing a group of survivors who underwent regular rehabilitation with a group who did not receive rehabilitation.We recruited participants from Mianzhu country, an area seriously hit by the earthquake. A total of 459 fracture victims were identified and enrolled. Bodily injury is an important risk factor for PTSD This research was approved by the Ethics Committee of the Nanjing Medical University and Nantong University. Written informed consent to participate in clinical examinations and surveys was obtained from all participants.The research team, which had undertaken three previous large-scale studies, was exclusively comprised of rehabilitation professionals including physiatrists, therapists , and nurses, all of whom had degrees in rehabilitative medicine and more than 2 years of clinical experience. Members of the team provided both comprehensive evaluation and management of the enrolled subjects. The team performed a critical role in integrating efforts of rehabilitation medical teams from more-developed areas of China and the local healthcare providers Physical examinations were undertaken by rehabilitation physicians and therapists using muscle strength, joint range of motion (ROM), sensory function and sit-to-stand balance capacity tests. If one or more diagnostic criteria presented a pathology that would require a rehabilitative intervention, we defined it as a physical dysfunction. To avoid interference and bias during assessments, patients and staff performing the examination were blinded, i.e. not advised about the purpose of the examinations. The results were reviewed by other rehabilitation professionals to ensure that no errors were made during data collection.Four questions were used to evaluate respondents’ earthquake exposures: 1) Was a close family member of you seriously injured or killed? 2) Was your house damaged? 3) Did you feel fear during the earthquake? and 4) Did you directly witness other people die? Four dummy variables were then generated based upon responses to the questions: casualties of family members, house damage, fearfulness during earthquake, and directly witnessing death.Diagnostic and Statistical Manual of Mental Disorders-IV , to identify PSTD-positive survivors. Scores for each question ranged from 1  to 5 (extremely). Summing the scores for each of the 17 items provided a total symptom severity score (range = 17–85). Scores between 20–39 are considered mild, 40–59 moderate, 60–79 severe, and scores above 80 extreme PTSD PTSD was assessed with the PTSD Checklist Civilian Version (PCL-C). The PCL-C is a valid and reliable standardized self-reported rating scale for screening PTSD symptoms Demographic data included information on gender, age, education, income and employment status.Data on earthquake exposures, PTSD, and demographics were collected in face-to-face interviews. Data were entered by two trained coders into Epidata 3.1. Inconsistencies between the independent coders resulted in verification of the raw data.Data were analyzed with SPSS 17.0 and Stata 12.Descriptive analysis of the data was performed for all variables investigated in the study. Chi-square and t-tests were performed to examine differences between rehabilitation and control group regarding demographics and exposures. A logistic regression model was used to predict the effect of rehabilitation on physical dysfunction. An ordinary least squares (OLS) regression was employed to predict PTSD symptoms. A log transformation of the PTSD scale was applied to meet the assumption of a normal distribution. Both regression models included the following independent variables: gender , being married (reference: not married), age, low education (reference: high school education or more), being the main earner of the family (reference: not), low income (reference: income at population average or above), casualties of family members (reference: none), house damage (reference: none), fearfulness during the earthquake (reference: none), directly witnessing death (reference: not), and belonging to the rehabilitation or control group (reference: control). As physical dysfunction and rehabilitation were highly negatively correlated an inclusion of both rehabilitation and dysfunction as predictors into the OLS regression of PTSD seemed inappropriate due to multicollinearity. Therefore, we performed an additional analysis using Least Angle Regression (LARS), i.e. a penalized variable selection algorithm that considers both parsimony and prediction accuracy of the model Four hundred and fifty-nine patients without disability were evaluated, 245 had received rehabilitation and 214 did not participate in rehabilitation. Persons in the rehabilitation group had significantly witnessed death more often. Other exposures did not differ between groups .2 = 57.65, p<0.001) as well as PTSD , both times in favour of the rehabilitation group  Many studies suggest that disasters and other traumatic events have short and long term health consequences,especially as regards the occurrence of functional disabilities as well as psychiatric disorders including PTSD, major depression, and anxiety In our study, apart from not having received physical rehabilitation and physical dysfunction, main risk factors for PTSD appeared to be female gender, having witnessed people die, fearfulness, and average or above income, while bereavement and home damage did not play a major role.Our finding that females are at higher risk for PTSD is consistent with previous research Differential earthquake exposures have been identified as important psychological risk factors before. Similar to our findings, Salcioguo and colleagues showed that PTSD was strongly related to fear during an earthquake in a sample of 769 Turkish survivors who had been relocated to a permanent housing site built in the epicentre region three years after the disaster Our result on witnessing death as a contributory factor for PTSD symptoms confirms findings from previous studies conducted 15 months In contrast, to previous research we did not find a significant influence of having lost a family member in our study population. As our study was conducted more than 4 years after the disaster, it is likely that our participants had already accepted their losses and were no longer in grief.Our finding on house damage not being a risk factor is difficult to interpret as solely 14 persons (3%) reported no damage of their residence and we do not have information on the severity of the damage. Moreover, at the time of the study most survivors had moved to new buildings that were better equipped and constructed than those previously damaged.Eventually, our result regarding higher income being a risk factor is surprising and not consistent with previous studies Some possible limitations of our study need to be addressed. There was no baseline data on PTSD of the interviewed participants shortly after the earthquake so that we could not study change or adjust for potential baseline differences between the study groups. However, since demographics and most exposures were similar (even a higher percentage of participants from the rehabilitation group witnessed death) baseline differences in favour of the rehabilitation group seem unlikely. Secondly, we did not address other mental health problems, such as anxiety and depression so that we do not have a complete picture of the mental health of the study population at the moment; an issue to be addressed by future research. Thirdly, we used a screening tool for PTSD and did not confirm these results with more intensive psychiatric examination, particularly due to resource constraints. Finally, several predictors in our models such as income and education were correlated which makes it difficult to demonstrate separate effects. However, additional analysis using penalized regression (LARS) showed that inclusion of all selected predictors resulted in optimal model fit.In conclusion, this is the first study to examine the association of rehabilitation and PTSD symptoms in Chinese fracture victims after the 2008 Sichuan earthquake. Our study suggests a positive impact of medical rehabilitation on PTSD symptoms. This finding may have important implications for prevention and intervention in post- traumatic mental health problems in survivors of natural disasters.


Most studies concerning the foraging ecology of marine vertebrates are limited to breeding adults, although other life history stages might comprise half the total population. For penguins, little is known about juvenile dispersal, a period when individuals may be susceptible to increased mortality given their naïve foraging behaviour. Therefore, we used satellite telemetry to study king penguin fledglings (n = 18) from two sites in the Southwest Atlantic in December 2007. The two sites differed with respect to climate and proximity to the Antarctic Polar Front (APF), a key oceanographic feature generally thought to be important for king penguin foraging success. Accordingly, birds from both sites foraged predominantly in the vicinity of the APF. Eight king penguins were tracked for periods greater than 120 days; seven of these  migrated into the Pacific. Only one bird from the Falkland Islands moved into the Indian Ocean, visiting the northern limit of the winter pack-ice. Three others from the Falkland Islands migrated to the eastern coast of Tierra del Fuego before travelling south. Derived tracking parameters describing their migratory behaviour showed no significant differences between sites. Nevertheless, generalized linear habitat modelling revealed that juveniles from the Falkland Islands spent more time in comparatively shallow waters with low sea surface temperature, sea surface height and chlorophyll variability. Birds from South Georgia spent more time in deeper waters with low sea surface temperature and sea surface height, but high concentrations of chlorophyll. Our results indicate that inexperienced king penguins, irrespective of the location of their natal site in relation to the position of the APF, develop their foraging skills progressively over time, including specific adaptations to the environment around their prospective breeding site. Integrated knowledge on the spatial distribution of long-lived animals, including all life history stages, are of paramount importance to fully understand their movements and distribution in view of potential population trends, threats and the subsequent implementation of appropriate conservation measures. Almost all information currently available for meso-predators such as sea turtles Thalassarche cauta) Diomedea amsterdamensis) D. exulans) albatrosses Aptenodytes forsteri) Pygoscelis papua) P. adeliae) penguins A.patagonicus).After fledging, young animals generally forage and disperse on their own using a portfolio of learned and/or genetically pre-determined behaviours King penguins have a circumpolar distribution Of the global population of over 1.5 million king penguin breeding pairs The aim of this study was to examine the post-fledging dispersal of juvenile king penguins in the South Atlantic using satellite telemetry. Juvenile king penguins were tracked simultaneously from South Georgia and the Falkland Islands to address the following questions: 1) How do juvenile king penguins disperse during their first year at sea with regard to the position of the APF? 2) Do they disperse potentially following a genetically pre-determined behaviour, or do they disperse randomly over the open ocean? 3) Do they use a different habitat from that of adults? 4) Do juveniles from the two breeding sites differ in their foraging habitats? 5) What are the key environmental parameters influencing their distribution?This study was approved by the Falkland Islands Government (R 09/2007) and the Government of South Georgia and the South Sandwich Islands and complied with the legal requirements in the United Kingdom. The king penguin colony in the Falkland Islands is located on private land (Johnson’s Harbour Farm); permission was given by the owners Osmund and Olive Smith . King penguins are not endangered (classified as ‘Least concern’ in the current IUCN red list) but are protected under the ‘Conservation of Wildlife and Nature Bill 1999’ in the Falkland Islands and under the South Georgia Environmental Management Plan in South Georgia Fieldwork was conducted in the king penguin breeding colonies located at Volunteer Beach , Falkland Islands, and at St. Andrews Bay , South Georgia, on 11 December 2007 and 13/14 December 2007, respectively. King penguin fledglings were randomly selected at each site  After their capture, penguins were weighed  before satellite transmitters were attached using black waterproof tape  and glue, adapted from the methods described by The KiwiSat 202 satellite transmitters used had maximum dimensions of 80×35×27 mm and weighed approximately 60 g, equivalent to about 0.6% of the mean penguin body mass. The flexible antenna of each device was 185 mm long and had a diameter of 2 mm: it originated with an angle of 60° at the rear of the device to further reduce drag Positional data obtained from Argos  were classified according to the quality of the positional fix, with location classes 0, 1, 2 and 3 representing accuracies of >1 km, <1 km, <350 m and <150 m, respectively 2–840×2400 cells) was chosen according to the available oceanographic data  of the receiver operating characteristic (ROC) plot was computed for each of the 100 replicate models with each set of validation data to estimate its predictive power through cross-validation We used the most parsimonious model to build a predictive map of current juvenile king penguin distribution in the Southern Ocean. To produce this map, we used the option in IDRISI Taiga Apart from some individual variability described below, no general differences in the migratory behaviour of birds from either site were apparent. This applied also to the various migratory parameters calculated . OverallGus, Iona and Leo) remained over the Falklands Plateau  and magellanic (Spheniscus magellanicus) penguins A tracking period of up to 261 days and a horizontal displacement of up to c. 12,000 km represents the longest tracking duration for king penguins yet recorded in the literature and potentially even one of the longest when compared with studies on other non-flying, air-breathing marine vertebrates. While it is well accepted that the attachment of external devices, especially those having an antenna, can greatly influence the foraging performance and ultimately survival of marine predators The foraging range of adult king penguins during the breeding season is one of the most extensively studied among marine air-breathing vertebrates  Remarkably, with the exception of While the foraging areas in general were, apart from the initial phase, quite similar for juveniles from both study sites, some differences were apparent when applying the GLM model. Of course it cannot be ruled out that genetically predetermined adaptations in their foraging behaviour exist, but our results indicate that behavioural adaptations linked to their breeding site are developed over time, presumably supported by foraging in association with congeners and other species, which has been shown to be the case in juvenile brown boobies


Purpose. To investigate the clinical value of 99Tcm-MDP SPECT for the diagnosis of unilateral condylar hyperplasia (UCH).  Methods. One hundred forty-nine patients who underwent mandibular 99Tcm-MDP SPECT between January 2009 and December 2012 were studied, including 105 cases that were clinically suspected of UCH and 44 comparable cases without UCH as a control group.  Results. Increased bone activity was observed in the affected condyles for all UCH patients. In the UCH group, the relative percentage uptake on the affected side was 59% (SD ± 4.3%), significantly higher than the 41% (SD ± 4.1%) uptake on the contralateral side (P<0.001). Similarly, the condyle/skull ratio was significantly higher for the affected side (1.66 ± 0.63) than for the contralateral side , specificity (61%), positive (84.4%) and negative (84.6%) predictive values, and accuracy (84.5%) for 99Tcm-MDP SPECT in the diagnosis of UCH were calculated. However, for the hyperplastic condyle, no correlation was observed between the thickness of each cartilage layer and the relative uptake in the SPECT image.  Conclusion. 99Tcm-MDP SPECT is accurate for diagnosing UCH and can provide a reference for treatment options. Unilateral condylar hyperplasia (UCH) is a rare disease that manifests as nonneoplastic overgrowth of the unilateral mandibular condyle, without analogous pathologic change in any other joint. This disorder often results in facial asymmetry, deformity, and malocclusion and is sometimes associated with obvious pain and dysfunction. It seems that UCH is common and frequently occurs in groups during the growth phase, especially in adolescence . FemalesThe asymmetry associated with UCH can be classified into three categories: hemimandibular hyperplasia, hemimandibular elongation, and a hybrid form. The first type is the result of unilateral growth in the vertical plane, leading to a sloping rima oris, with little or no deviation of the chin. Hemimandibular elongation is characterized by asymmetry in the transverse plane, resulting in deviation of the chin towards the contralateral side . ApproprAlthough condylar hyperplasia is a self-limiting condition, it is characterized by progressive and independent growth that always results in the bone volume of one condyle being greater than that of the other. According to the growth state, condylar hyperplasia is divided into an active phase and stationary phase. The activity level of the condyle is considered to be highly correlated with mandibular asymmetry , 8. When99Tcm-MDP (technetium methylene diphosphonate) SPECT in UCH.Because clinical and radiographic presentations in patients with condylar hyperplasia are highly diverse, the assessment of condylar growth activity has a vital role in managing these patients . Radionu99Tcm-MDP SPECT of the temporomandibular joint (TMJ) between January 2009 and December 2012, and 58 of the patients accepted high condylectomies and pathological examinations. The demographic and clinical characteristics of these patients are shown in 99Tcm-MDP SPECT because of other diseases such as malignancy, and specific tomography of the TMJ was performed after informed consent. To minimize the influence of unnecessary interfering factors on 99Tcm-MDP uptake in the TMJ, the control group was limited to patients with complete dentition and without skeletal metastasis, malocclusion, facial tumor, facial trauma, or TMJ disorder.In total, 105 patients with suspected UCH were observed in this study, including 42 males and 63 females aged 13–33 years, with an average of 21.5 years. As previously described , all sus99Tcm-MDP via elbow veins, SPECT bone images were obtained with an E.CAM SPECT scanner . By using high-resolution, low-energy collimators, the SPECT scanner collected data over 360° in a spherical field of rotation, using a 128 × 128 matrix at 15 seconds per frame. The data from each case were reconstructed and reformatted into coronal, sagittal, and axial planes.Three to six hours after the injection of 740–925 MBq 99Tcm-MDP uptake. To ensure that the left- and right-sided ROIs were of comparable size, the ROI was copied to the contralateral condylar region. The ratio of the unilateral count to the gross count of both sides was calculated for relative uptake [The SPECT images were analyzed by 2 experienced nuclear medicine physicians; the bone metabolism of each TMJ was routinely elevated by visual inspection, and a region of interest (ROI) method was applied. ROIs were drawn around each TMJ that represented the area of prominent e uptake ; 55% or The surgically removed condylar tissues were fixed by 10% paraformaldehyde, then embedded in paraffin, and serially cut for hematoxylin-eosin (HE) staining. Two experienced pathologists performed the pathological analysis independently from the results of the bone scan. The thickness of perichondrium, proliferation layer, and hypertrophic cartilage layer were measured under light microscope. Condyles in active growth were characterized by obvious cell hyperplasia, a thickened proliferative zone and hypertrophic layer, which were not observed in condyles in a stationary phase.t-test. The Pearson correlation test was used to determine the correlation between the relative uptake of affected condyles and the thickness of each cartilage layer. A P value of less than 0.05 was considered significant.The statistical analysis software SPSS version 16.0  was used, and values were represented as the mean ± standard deviation (SD). Comparisons of ROI counts were performed using Student's The control group had uniform and symmetrical radioactivity distribution on both sides of the TMJ based on visual inspection. However, for patients with UCH, the activity in the affected condylar region was obviously higher than that in contralateral condylar region, supported by a relatively concentrated area of radioactivity on the affected side . In antet = 6.590, P < 0.001). As for the ratio between the condyle and the skull, the values of the affected and unaffected condyle were also significantly different at 1.66 (SD ± 0.63) and 1.34 (SD ± 0.34) .Quantitative analysis of ROIs revealed differences in condylar activity . The meat = 0.762, P > 0.05). The condyle/skull ratios were 1.12 (SD ± 0.07) and 1.10 (SD ± 0.06), also not significantly different .However, no significant difference was found between the left and right condyles in the control group. The uptake percentages of the left and right condyles were similar at 50% (SD ± 1.7%) and 50% (SD ± 6.2%) . Similarly, the condyle/skull ratio was significantly higher for the affected condyles of the suspected UCH patients than for right condyles of the controls .The UCH group and the control group were also compared. The mean relative percentage uptake was significantly higher in the affected condyles of the suspected UCH patients than in the left condyles of controls ; 45 of them underwent condylectomy, and active growth was pathologically confirmed in 38 cases. To prevent deformity recurrence, 13 of the remaining 32 cases that presented no activity in the SPECT images also underwent condylectomy; pathologic analyses confirmed that 11 cases were in the stationary phases of growth , specificity of 61.1% (11/18), positive predictive value of 84.4% (38/45), negative predictive value of 84.6% (11/13), and accuracy of 84.5% (49/58).Visual and quantitative analysis of f growth . The effP < 0.01). The values for subjects in the stationary phase did not exceed 0.55. Condylar hyperplasia was defined as excessive growth of condylar cartilage, with thickening of the perichondrium, proliferative zone, and hypertrophic layer. No correlation was found between the relative percentage uptake and mean thickness of these layers in the 45 patients of the UCH group who underwent condylectomy and histological analysis .The histopathologic evaluation of the resected condyles showed that the condylar cartilage was divided into four layers: the perichondrium, proliferative zone, hypertrophic layer, and calcified cartilage layer. Condylar cartilage with active growth showed an obviously thicker proliferative zone and hypertrophic layer, accompanied by remarkable hyperplasia of undifferentiated mesenchymal cells from the proliferation layer and hyaline chondrocytes from the hypertrophic layer; periosteal thickening was observed in some cases . The per99Tcm-MDP SPECT in diagnosing UCH was evaluated. Our results found SPECT to be an accurate method for recognizing hyperactivity in a condyle, through analysis of the relative percentage uptake of 99Tcm-MDP, with a cutoff value of 55%. Increased radionuclide uptake in the hyperplastic condylar region can constitute evidence of continued abnormal growth.In the present study, the role of Currently, it is impossible to define an absolute age of presentation for UCH , 2, 16. 99Tcm-MDP SPECT of the TMJ can clearly show whether the bone metabolism is overactive or not. A relative uptake of at least 55% was regarded as an indication of hyperactive condyles, as this value distinguishes actively growing condyles from stationary ones [Radionuclide bone imaging has a unique advantage in evaluating the ongoing activity of condyle. ary ones , 15. Usiary ones –20, whicCurrently, surgery followed by orthodontic operation is the main treatment for condylar hyperplasia. Optimal treatment planning always depends on precise assessment of the condylar growth state. An active mandibular condyle with a related, progressing facial deformity often requires high partial condylectomy to limit the growth of the condyle. Additionally, to prevent recurrence, surgery to correct any residual deformity should be performed when the excessive growth of condyle has ceased . Convers99Tcm-MDP. Therefore, when the relative uptake exceeds 55%, dynamic observation combined with evaluation of clinical characteristics is recommended. Orthognathic surgery can be performed directly in cases without any progressing facial deformities. In cases with a progressing facial deformity, a longer follow-up of 6 to 12 months is necessary, and it is suggested that a SPECT examination be performed again after 6 months. If the relative uptake is less than 55%, only orthognathic surgery is required. Conversely, if the relative uptake remains above 55%, a partial condylectomy should be performed. If the result of the initial SPECT scan shows that the relative uptake is less than 55%, direct orthognathic treatment can be provided.A typical SPECT image of the hyperactive condyle in this study is characterized by an abnormally high uptake of Condylar cartilage can be divided into 4 layers according to surface depth: the perichondrium, proliferative zone, hypertrophic layer, and calcified cartilage layer. The pathological changes of condylar hyperplasia are mainly characterized by obvious thickening of the cartilage layer , 23. Var99Tcm-MDP SPECT is important for the assessment of condylar activity. It is an accurate method for identifying UCH patients with ongoing bone growth, and it is helpful to not only for the diagnosis of condylar hyperplasia but also for choosing an appropriate therapy.Our results suggest that


Next, lentiviral-mediated overexpression of GRβ in C2C12 was performed, and these cells were characterized for cell fusion and myotube formation, as well as sensitivity to Dex via the expression of ubiquitin ligases. GRβ overexpression increased mRNA levels of muscle regulatory factors and enhanced proliferation in myoblasts. GRβ overexpressing myotubes had an increased fusion index. Myotubes overexpressing GRβ had lower forkhead box O3 (Foxo3a) mRNA levels and a blunted muscle atrophy F-box/Atrogen-1 (MAFbx) and muscle ring finger 1 (MuRF1) response to Dex. We showed that GRβ may serve as a pharmacological target for skeletal muscle growth and protection from glucocorticoid-induced catabolic signaling. Increasing GRβ levels in skeletal muscle may cause a state of glucocorticoid resistance, stabilizing muscle mass during exposure to high doses of glucocorticoids.Unlike the glucocorticoid receptor α (GRα), GR β (GRβ) has a truncated ligand-binding domain that prevents glucocorticoid binding, implicating GRα as the mediator of glucocorticoid-induced skeletal muscle loss. Because GRβ causes glucocorticoid resistance, targeting GRβ may be beneficial in impairing muscle loss as a result of GRα activity. The purpose of this study was to determine how the overexpression of GRβ affects myotube formation and dexamethasone (Dex) responsiveness. We measured GR isoform expression in C Chronic glucocorticoid (GC) treatment and prolonged elevations of endogenous GC production cause skeletal muscle atrophy and reduce the adaptive response of skeletal muscle to injurious and atrophic events ,2,3,4. FWhile the physiological roles of GRβ are not completely understood, increased GRβ expression has been linked to GC resistance in patients suffering from severe asthma ,13,14,15Proliferation and differentiation of skeletal muscle myocytes are necessary for the cellular and molecular events that orchestrate skeletal muscle repair, adaptations to inactivity or exercise, as well as the basal maintenance of skeletal muscle size ,24. Furt2C12 muscle cells alters myotube formation and sensitivity to exogenous GC.It is well known that GCs induce muscle atrophy. The binding of GCs to the ligand-binding domain of GRα causes translocation to the nucleus and binding to glucocorticoid response elements (GREs) in the promoter region of genes. Specifically, GRα binds to GREs in the promoter of forkhead box O (Foxo) transcription factors and enhances expression . This re2C12 expresses both GRα and GRβ [2C12 myoblasts respond to the GC dexamethasone (Dex), causing a significant (p < 0.01) suppression of GRα and no change in GRβ expression. We have previously reported in mouse embryonic fibroblast (MEF) that Dex decreased GRα as part of a negative feedback loop [p < 0.001) increased GRβ protein expression  increase beginning one day into the differentiation process  by lentivirus and determined how elevated GRβ expression affected GC responsiveness and mRNA levels of MyoD and myogenin. GRβOE myoblasts had approximately 12.5-fold higher GRβ expression compared to vector cells  higher in GRβOE myotubes  A. A myotmyotubes B. Moreovmyotubes C, the numyotubes D, and thmyotubes E were alp = 0.025) reduced in GRβOE myotubes (p = 0.0091) blunted compared to vector myotubes (p = 0.1443) (p = 0.3678). Taken together, Dex-induced expression of atrogenes, particularly that of MAFbx, is reduced with overexpression of GRβ, providing a modest level of protection against the deleterious effects of GC exposure.GRα-mediated activation of the Foxo-atrogene (MAFbx and MuRF1) pathway is a well established mediator of muscle atrophy. Due to the ability of GRβ to inhibit GRα activity, we tested whether Dex-induced MAFbx and MuRF1 mRNA expression were abrogated in GRβOE myotubes. First, we measured mRNA levels of Foxo3a, a transcription factor known to regulate atrogene expression . Foxo3a myotubes A. As expmyotubes B,C. MAFbmyotubes B. Althou 0.1443) C. While 2C12 mouse muscle cells. We demonstrated that GRβ protein levels in C2C12 mouse muscle cells do not change following GC exposure, consistent with findings in human skeletal muscle cells [The detection of GRβ in mouse  has provle cells . Furtherle cells ,41,42,432C12 myotubes [in vivo is required before drawing significant conclusions. However, these data do allude to a mitogen-sensitive pathway for GRβ in skeletal muscle.Rats exposed to cortisone for five days had reduced insulin receptor phosphorylation and reduced insulin receptor substrate-1 (IRS1) content . Dex-indmyotubes . Overexpmyotubes . Importamyotubes . Our datMyoD and myogenin, and enhanced myotube formation. Collectively, these data support pro-myogenic and insulin-responsive properties of GRβ that may help preserve muscle mass in response to the negative affects of GC on skeletal muscle metabolism.Cell proliferation is a tightly regulated process interconnected with mitogen-sensitive pathways, particular kinases that are insulin-responsive. PTEN is a known regulator of cellular growth, and in our prior work we have shown that overexpression of GRβ enhances proliferation of 3T3 cells via inhibition of PTEN and increased phosphorylation of Akt , corroboIt is well documented that GC-induced GR activity induces muscle wasting by stimulating the proteolytic activity of the ubiquitin-proteasome pathway. The working paradigm suggests that in response to elevated endogenous or exogenous GCs, Foxo transcription factors increase MAFbx and MuRF1 expression to stimulate ubiquitin activity in skeletal muscle ,29,34. Iet al. [in vivo [2C12 myotubes [2C12 myotubes with no effect of protein synthesis rates [in vivo, MAFbx expression was regulated by a suppression of the canonical insulin signaling pathway (IRS1/PI3K/AKT) and an increase in IRS2/MEK/ERK signaling [Waddell et al.  determin[in vivo , muscle [in vivo . Interes[in vivo  due to emyotubes . Here, wis rates . Lastly,is rates . Throughignaling . These pin vivo approach to evaluate.The degradation of muscle regulatory factors is one mechanism by which MAFbx and MuRF1 induce destabilization of skeletal muscle and loss of muscle mass. Here, we show that overexpression of GRβ increased MyoD and myogenin gene expression, two muscle regulatory factors necessary for skeletal muscle development and regeneration . Through2, 21% O2, and 37 °C.All cells proliferated in HyClone DMEM  containing 10% FBS  supplemented with 1% penicillin/streptomycin. . Differentiation into myotubes was induced in cultures that reached ~90% confluence by switching to DMEM containing 2% horse serum (ATCC) and 1% penicillin/streptomycin. Dexamethasone  and insulin  treatments were for durations specified in the figure legends, at a concentration of 100nM (dexamethasone) and 100uM (insulin). For all experiments, cells were maintained in 5% CO2C12 mouse myoblasts were passaged in house. Cells passaged four times were used for experiments. To establish a C2C12 cell line with mouse GRβ stably overexpressed, mouse GRβ cDNA was ligated into the PacI/NotI sites of the pQXCIN vector that has an independent neomycin selection marker and transformed in DH5-α cells . The construct was co-transfected together with vectors expressing gag-pol, REV, and VSV-G into 293FT cells (Invitrogen) to generate a third generation lentiviral construct. Transfection was achieved using GeneFect  using 100 ng of total DNA per cm2 of the growth plate or well. The supernatants were harvested, and the cell debris was removed by centrifugation at 2000× g. The supernatant was used to infect C2C12 cells after the addition of polybrene  to establish cell lines with stable overexpression of GRβ mRNA (GRβOE) or those expressing an empty vector. After 72 h, the cells were selected using 500 mg/mL G418 [C4 cells per well). The growth rate was determined as a function of time for 0–4 days of proliferation. Cell proliferation was determined by a calorimetric assay using MTT -2,5-dipheyltetrazoline bromide) as previously described [Vector and GRβOE cells were plated in 12-well plates in DMEM containing  and quantified using the NanoDrop 2000 spectrophotometer . cDNA was synthesized using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). Real-time PCR amplification of the cDNA was performed using TrueAmp SYBR Green qPCR SuperMix  with a Step One Plus real-time PCR system . Changes in gene expression were determined using the quantitative ΔΔCt method and normalized to GAPDH. A list of primer sequences is in Cells were harvested and lysed in a RIPA lysis buffer containing Holt protease and phosphatase inhibitors . Protein content was determined using the BCA method (Pierce).Western blot analysis was performed as previously described . BrieflyFollowing four days of differentiation, plates were washed with PBS and fixed with cold 70% methanol/30% acetone for 10 min at room temperature. Myosin heavy chain staining was performed as previously described . Cells wBlinded investigators took all pictures and performed quantification after demonstrating proficiency using practice images. MHC positive (MHC+) cells were viewed at 10× magnification. A myotube was considered a MHC+ cell with two or more nuclei. Five to seven fields were viewed per well in a predetermined manner, starting from the center of the well; the stage was moved two complete fields to the right (field 1), two fields up (field 2), two fields to the left (field 3), two fields to the left (field 4), two fields to the down (field 5), two fields down (field 6) and two fields to the right (field 7). For each field, one picture of MHC+ cells and one picture of Hoechst-labeled nuclei were taken and merged. Quantification was performed on printed merged images of each field. To determine how many total nuclei were within myotubes, all of the nuclei within MHC+ cells that contained two or more nuclei were counted. The number of nuclei per myotube was determined by dividing the number of myotubes in each image by the total number of nuclei counted within myotubes. The fusion index was determined using the following formula: . A one-way analysis of variance (ANOVA) combined with Tukey’s post-test was used to determine changes in GR protein expression in response to insulin and dexamethasone and changes in MyoD, myogenin, GRα and GRβ mRNA levels through four days of differentiation and the responsiveness of MAFbx and MuRF1 to dexamethasone in GRβOE cells. A two-way ANOVA with a Tukey’s post-test was used to determine difference in proliferation rates from the MTT assay. A student’s in vivo, and testing whether its overexpression is protective. As new evidence begins to emerge on the role of GRβ in skeletal muscle, we believe that this GR isoform may lead to the ability to target GC resistance to skeletal muscle and minimize proteolytic activity during treatment. Importantly, future studies should employ techniques that preserve the important therapeutic effects of GCs in other tissues, while preventing skeletal muscle catabolism, such as tissue-specific transgenic mouse models. Lastly, the role of GC resistance in metabolic disease should also be considered when designing future models [The present study is the first to characterize the effects of GRβ overexpression on the myogenic program in C2C12 muscle cells. While these data are informative, they prompt several pertinent questions open for future investigation. For example, GC-mediated MuRF1 expression causes the degradation of structural proteins in skeletal muscle ,51,52,53e models .