Prostate cancer is one of the most commonly diagnosed cancers in men, and androgen deprivation therapy still represents the primary treatment for prostate cancer patients. This approach, however, frequently fails and patients develop castration-resistant prostate cancer, which is almost untreatable.Cancer cells are characterized by a hierarchical organization, and stem/progenitor cells are endowed with tumor-initiating activity. Accumulating evidence indicates that prostate cancer stem cells lack the androgen receptor and are, indeed, resistant to androgen deprivation therapy. In contrast, these cells express classical (α and/or β) and novel (GPR30) estrogen receptors, which may represent new putative targets in prostate cancer treatment.In the present review, we discuss the still-debated mechanisms, both genomic and non-genomic, by which androgen and estradiol receptors  mediate the hormonal control of prostate cell stemness, transformation, and the continued growth of prostate cancer. Recent preclinical and clinical findings obtained using new androgen receptor antagonists, anti-estrogens, or compounds such as enhancers of androgen receptor degradation and peptides inhibiting non-genomic androgen functions are also presented. These new drugs will likely lead to significant advances in prostate cancer therapy. Prostate cancer (PC) represents the most common type of cancer among male individuals and is the second leading cause of cancer death in men. In most cases, PC has a slow and symptom-free growth, whereas in the remaining cases it is more aggressive. Current treatments for clinically localized or advanced PC include radical prostatectomy, androgen deprivation therapy (ADT), radiation therapy, brachytherapy and cryotherapy. However, the efficacy of these therapies still remains unsatisfactory . New theSince the first observation on the role of androgens in PC progression  and the Stem cells (SCs) are present in normal tissues and have the ability to self-renew and differentiate into tissue-specific progeny , 6. CancProstate SCs and CSCs exhibit a robust ability to self-renew in the absence of appreciable amounts of AR -13. TheiHere we discuss the putative role of these receptors in prostate SCs and CSCs. New therapeutic strategies based on the use of novel compounds  are also reviewed, since they might provide important advances in PC treatment.Prostate gland is made up of basal, luminal, and neuroendocrine cells embedded in a fibro-muscular stroma , 17. LumCell lineage is a key factor in PC development, since the majority of PCs (95-99%) arise from the luminal cell lineage, with only 1-5% deriving from the neuroendocrine cell lineage. Basal cell-derived PCs have not been reported to date .+ stem/progenitor or intermediate cells increase after ADT or in CRPC [PC may originate from SCs that become CSCs as a consequence of accumulating mutations. Thus, prostate CSCs might arise from SCs or progenitor cells or differentiated cancer cells undergoing aberrations as a consequence of genetic mutations or changes in tumor microenvironment . CSCs ex in CRPC , 20-23. The human AR gene encodes for a protein with a mass of 110 KDa that consists of an N-terminal domain, a DNA-binding domain and a ligand-binding domain. The ‘hinge region’, which separates the ligand-binding domain from the DNA-binding domain, is made up of a short amino acid sequence that contains part of a bipartite ligand-dependent nuclear localization signal responsible for AR nuclear translocation .via the direct interaction of AR with Src-SH3 domain [in vitro or xenografted in mouse [AR controls the growth of prostate gland and is a hallmark of PC, since it is expressed throughout the various stages of the disease . Classic3 domain  or filam3 domain , 30. And3 domain  -amide peptide from the AR 628-646 sequence responsible for its interaction with filamin A, in which Ala628 and Lys632 residues were each replaced with an olefinic amino acid that allows them to be cross-linked. The peptide is thus ‘locked’ into its bioactive alpha-helical fold through the insertion of hydrocarbon staples. This modification increases affinity for targets as well as cell permeability, and decreases degradation of the stapled peptide, as compared to its unstapled counterpart . The staversus non-transcriptional), AR is constantly expressed in PC and its decrease reduces both androgen-independent and CRPC types [Whatever the mechanism  or AR activation by alternative signaling effectors . Analysiin utero enhance susceptibility to PC [de novo estrogen source [Genetically engineered, xenograft and cell culture models currently available do not accurately represent the molecular and cellular events underlying PC development and progression to CRPC. While the AR status of the most commonly used cell lines is generally known, expression levels of ERα and ERβ vary across different cell lines, and conflicting data have been reported for each cell type , 43. Thuty to PC -62. Use ty to PC . Furtherty to PC . These fn source  and aginn source . Secondln source -72. The n source  should bn source . In concvia ligand-dependent binding of ER (α or β) to target gene promoters [ERs, α or β, mediate estrogen effects in target cells . Human promoters . Estradiromoters .ERs also induce rapid, non-genomic effects independently of RNA or protein synthesis. Thus, ERs localized in the extra-nuclear compartment of target cells activate signal transduction pathways involving Src, MAPKs, adenylyl cyclase and PI3-K, or mediate a rapid increase in intracellular calcium levels . We alsoin vivo [The conflicting evidence on the role of ERs (α or β) collected so far in different experimental conditions  suggests that further analysis of ERα or β action should be carried out to gain valuable findings in relation to PC. The scenario seems to be even more complex because of the putative role of the novel ER, GPR30 in PC progression and androgen resistance. GPR30 contributes to physiological effects elicited by estrogens in target cells. In addition to mediating some of the rapid signaling events on estradiol stimulation , GPR30 also regulates gene transcription . A role in vivo . Again, in vivo . Thus, Gin vivo -91. Overin vivo .Prostate SCs or progenitor cells prevalently reside in the basal, but not in the luminal layer of human prostate epithelium. Since basal cells are AR negative, while luminal cells express high levels of AR, it was previously hypothesized that prostate SCs express undetectable levels of AR . This hy+8− basal cells, containing SCs or progenitors, lack AR. In contrast, cytokeratin 5+8+ intermediate and cytokeratin5−8+ luminal cells express AR [+ cells lack AR. In contrast, subsequent studies reported that CD133+ tumor-initiating cells express AR, as assessed by dual variable flow-cytometry [A number of markers  have been used to identify or isolate prostate SCs or CSCs , 96, 97.press AR , 101. Itpress AR . Again, ytometry . Howeverytometry , and AR-ytometry . Depletiytometry . In sum,The finding that prostate SCs and CSCs do not express AR calls for further comments. The use of cell markers to isolate SCs and prostate CSCs may generate a mixed cell population, significantly complicating the identification of pure AR-negative or -positive cells. Additionally, several normal or transformed cell types express low levels of AR, making the receptor unable to dimerize and translocate into the cell nucleus. These cells have, in most cases, been considered androgen insensitive because of their very low AR expression level, and hence the absence of AR-mediated gene transcription. Nonetheless, they still respond to androgens with activation of signaling effectors . Moreovevia a transcriptional mechanism in nuclei. Rapid activation of PI3-K/Akt signaling by androgens impinges on proliferation and differentiation of various cell types [Various signaling pathways are involved in prostate CSC functions. PI3-K/Akt pathway activation sustains survival and proliferation of PC progenitor cells. Again, Ras/MAPK and Stat3 signaling both sustain self-renewal of prostate CSCs . Experimll types . A direcll types . Conversll types , 114. Thll types , 115-119in vitro and in vivo. Re-expression of AR in PC3 cells decreases invasiveness in bone lesion assay and in vivo [in vitro or in xenograft models suggest that the androgen/AR axis plays a suppressor role in PC invasiveness. In contrast, much evidence implicates AR signaling in PC metastatic spreading at early and late stages. AR might promote pro-metastatic events through derangement of chemokine/chemokine receptor functions [in vitro and in vivo. Depletion of FOXA1 fosters PC metastasis and increases patient mortality [in vitro and in vivo. Clinical findings from CRPC specimens revealed a significant positive correlation between cyclin D1b and Slug expression, indicating that the cyclin D1b/AR/Slug axis is preserved in human PC [Most PC-related deaths are due to metastatic spreading, and human metastasis-initiating cells (MICs) have been found within sub-populations of CSCs that often express the features of epithelial-mesenchymal transition (EMT). These cells migrate and are putative candidates for metastasis-initiating activities . However in vivo . By inhi in vivo . Again,  in vivo , and the in vivo . In concunctions . AR contunctions , 127. CXunctions . CXCR4-eortality . Deregulortality . By moduhuman PC . Fusion human PC . SOX9, ahuman PC . In conchuman PC , 135.Altered function of extra-nuclear AR-associated signaling effectors or scaffolds might also be associated with PC metastatic events. We recently observed that AR-driven non genomic events trigger invasiveness of PC cells when they transit from hormone-dependent to hormone-independent status (unpublished data), making the extra-nuclear AR the culprit for invasiveness-initiating activity and maintenance of metastatic phenotype. A role for non-genomic AR functions was observed with regard to motility and invasiveness of various AR-expressing cancer cell types  -139. Altin vitro, thereby enabling analysis of growth and differentiation. Using this assay, it was shown that Wnt/β-catenin signaling activation increases the prostasphere size and self-renewal ability of PC cells [As discussed above, the role of estrogens in the etiology and progression of PC still remains unclear. A model for isolating SCs from mixed epithelial cell cultures utilizes a 3-D cell culture system, wherein only SCs survive and proliferate, forming spheroid structures of SCs or progenitors, also named prostaspheres. This system, initially used to isolate neural stem cells, has been successively extended to SCs from different tissues, including prostate , 147-150PC cells . TherefoIn addition to exhibiting membrane-associated prostate SC markers, such as CD117, Trop2, CD49f and ABCG2, prostaspheres derived from primary cultures of normal human prostate epithelial cells express almost undetectable levels of AR mRNA and are AR negative at protein level. In contrast, these cells express ERs (α or β1 or GPR30), in combination with high levels of retinoic acid receptor and retinoid X receptor. ER mRNA expression levels are significantly higher in normal progenitor cells than in LNCaP cells, with 6-fold higher ERβ1, 15-fold higher GPR30, and 125-fold higher ERα expression. These findings identify prostaspheres as potential targets for estradiol and retinoids . Table INormal prostaspheres derived from primary cultures of human prostate epithelial cells proliferate and grow upon stimulation with 1 nM estradiol . Finding+ subpopulation of the BPH-1 cell line [Findings obtained in prostaspheres derived from primary cultures of human prostate epithelial cells raise another important question. Since ERα and β are both expressed in prostaspheres, it might be argued that each receptor isoform mediates specific cellular responses upon estradiol binding. ERα or β might be specifically involved in non-genomic or genomic effects elicited by estradiol, thus contributing in a different way to hormone action. Despite intense investigations, we still have limited possibilities for using ligands that specifically bind ERα or β or even synthetic ligands that specifically affect genomic or non-genomic actions of steroids. Findings obtained using the synthetic ligand 8β-VE2, a specific ERβ agonist, showed that it induces apoptosis in both castration-resistant prostate CSCs  and a stell line . Thus, sPC represents the major cause of cancer-related death among the male population of Western society, and ADT is still used as the first line in PC treatment. However, ADT frequently fails or even induces paradoxical effects. The androgen/AR axis in PC and prostate CSCs has been investigated for several years. Much evidence from preclinical and clinical studies has shown that multiple androgen/AR signaling pathways operate in prostate tissue. Derangement of these pathways contributes to aberrant growth, maintenance of malignancy and PC invasiveness. At this stage, the disease becomes resistant to castration and is almost untreatable. In recent years, many therapeutic tools  have been designed to specifically target the functions of epithelial or stromal AR, or even the extra-nuclear functions of AR. Many of these molecules have been successfully exploited in preclinical or clinical models. Nonetheless, additional events sustain the disease and its escape from therapy. The role of estradiol/ER axis in PC and prostate CSCs is undeniable. Estradiol action in PC is commonly considered AR independent, with the focus on putative opposite effects, adverse or beneficial, exerted by the two ER isoforms, α and β, respectively. The novel ER, GPR30, has recently emerged as a therapeutic target in CRPC. Since PC co-expresses classical ERs and GPR30, a high degree of synergism and antagonism between various ligands and antagonists likely occurs in PC, adding further level of complexity to an already difficult therapeutic approach.Despite major advances in our understanding of the molecular basis of PC and in the generation of highly sophisticated tools for biological research, the questions addressed here suggest that we have to profoundly re-examine the mechanism of sex steroid receptor action in PC to better exploit new emerging targets and drugs. The power of novel genetically-engineered animal models or high-throughput screening in PC cells and/or biopsies will likely improve the design of new compounds that inhibit sex steroid receptor functions, leaving other useful receptor activities unaltered.


As a new strain of virus emerged in 2013, avian influenza A (H7N9) virus is a threat to the public health, due to its high lethality and pathogenicity. Furthermore, H7N9 has already generated various mutations such as neuraminidase R294K mutation which could make the anti-influenza oseltamivir less effective or ineffective. In this regard, it is urgent to develop new effective anti-H7N9 drug. In this study, we used the general H7N9 neuraminidase and oseltamivir-resistant influenza virus neuraminidase as the acceptors and employed the small molecules including quercetin, chlorogenic acid, baicalein, and oleanolic acid as the donors to perform the molecular docking for exploring the binding abilities between these small molecules and neuraminidase. The results showed that quercetin, chlorogenic acid, oleanolic acid, and baicalein present oseltamivir-comparable high binding potentials with neuraminidase. Further analyses showed that R294K mutation in neuraminidase could remarkably decrease the binding energies for oseltamivir, while other small molecules showed stable binding abilities with mutated neuraminidase. Taken together, the molecular docking studies identified four potential inhibitors for neuraminidase of H7N9, which might be effective for the drug-resistant mutants. Most patients presented with severe pneumonia, acute respiratory distress syndrome (ARDS), and multiple organ failure [As the WHO reported on June 27, 2014, there were 450 laboratory-confirmed cases of avian influenza A H7N9) virus human infection including 165 deaths  [http://pymol.org) [http://zinc.docking.org) [To perform the molecular docking between neuraminidase and potential inhibitors, we employed the H7N9 neuraminidase structures of A/Anhui/1/2013 (PDB code: 4MWQ) and A/Shanghai/1/2013 (PDB code: 4MWW), which were solved by Wu et al. at the resolutions of 2.00 Å and 1.90 Å, respectively . The PDBcsb.org) . The watmol.org) . Four sming.org) , 25.http://autodock.scripps.edu) to perform molecular docking. The AutoDock is an automated docking program designed for prediction of the binding among small molecules such as substrates or drug candidates and the receptor with known 3D structure [http://autodock.scripps.edu), the graphical user interface (GUI) program for setting up AutoDock software, was used to prepare the procedures of docking [http://fafdrugs3.mti.univ-paris-diderot.fr) [In this study, we employed AutoDock software  .P < 3 and tPSA > 75 .The molecular docking was performed among the neuraminidases from H7N9 A/Anhui/1/2013 and A/Shanghai/1/2013. Oseltamivir carboxylate could serve as a control to evaluate the neuraminidase binding ability of other molecules. The conformation variations, binding energies, and inhibition constants for the molecular docking among neuraminidase and small molecules were presented in ectively , which aTo further dissect the interaction between neuraminidase and the small molecules, the structures in complex derived from the best dock results with neuraminidase for oseltamivir carboxylate , quercetThe Supplementary Material of Table S1 provide the detailed results of the top 3 scored poses for the complexes of neuraminidases from A/Anhui/1/2013 and A/Shanghai/1/2013 docking with each small molecule.


We report precision measurements by Fourier transform spectroscopy of the vacuum wavenumber, line width, and relative signal strength of 928 lines in the Ar I spectrum. Wavelength in air and classification of the transition are supplied for each line. A comparison of our results with other precision measurements illustrates the sensitivity of Ar I wavelengths to conditions in the light source. Spectra of the noble gases are frequently observed in laboratory discharges, industrial plasmas, lighting discharges, sources for analytical spectrochemistry, and astrophysical sources. In response to the wide interest in these elements the NIST Atomic Spectroscopy Data Center is currently compiling the spectra and energy levels for all ionization stages of Ne, Ar, Kr, and Xe. A critical review of the literature revealed that no comprehensive description of the infrared spectrum of neutral argon has ever been published.53, 1 (1995)]. Their measurements, made with the high-resolution Fourier Transform Spectrometer at the National Solar Observatory (Kitt Peak), have been widely used as a source of wavelength standards. Measurements of neutral argon from the same spectra have been circulated as an unpublished line list. This list has been used in a number of laboratories to identify lines of argon in spectral sources and as a source of calibration wavelengths.In 1995 observations of the spectrum of singly ionized argon covering the range 222 nm to 5865 nm were published by Whaling, Anderson, Carle, Brault, and Zarem 1/2.σL = 0.5(W)/(S/N), where W (the FWHM) and S/N  appear in S/N > 104) this expression may underestimate ΔσL because it neglects self-absorption that may, if asymmetric, shift the line center, and it neglects the local increase in the noise level under a strong line in the FTS spectrum .As standards we used as many as possible of the 28 Ar II lines recommended by Learner and Thorne . These wJ-value selection rules, using Ar I level energies from Moore  value  is given. The last column gives the wavelength in air as determined from the vacuum wavenumber by a formula from Edlén , are to be preferred to those in Our Ar I wavelengths agree with those of Minnhagen  within tf, 5g, and 6g levels in Ar I, using spectra recorded with the same FTS used in the present experiment. In their hollow cathode source they used various Ar pressures but all were lower than ours; their average pressure of about 130 Pa (1 Torr) was lower than ours by a factor of 1/3. For the 4f → 3d transitions (66 lines), our wavenumbers agree beautifully with theirs; the mean deviation between the two sets of wavenumbers is 0.4(1.1) × 10−3 cm−1. For the 5g → 4f and 6g → 4f transitions (33 lines), our wavenumbers are, on average, greater than theirs by 6 × 10−3 cm−1. We attribute this difference to a pressure effect that appears to be strongly dependent on the angular momentum of the levels involved. This difference did not show up in our comparison with Norlén’s wavenumbers, even though his source pressure was lower than ours by a factor of 1/10, because the shift depends on the angular momentum of the levels involved and Norlén measured only transitions between s, p, and d orbitals in Ar I.Palmeri and Biemont  have mea−1 from the 6g levels in Ar I have an average width ≈300 × 10−3 cm−1, while nearby lines from 4d′ and 5p′ upper levels have widths between (18 and 22) × 10−3 cm−1. The dependence on l appears to be greater than the dependence on n; nine lines from 7p configurations in the same (3600 to 4000) cm−1 range have an average width of 38 × 10−3 cm−1. In these examples, the lines are only broadened and shifted at the pressure (270 Pa to 530 Pa) of our source, but the lines may not be seen at all from a source at higher pressure. Palmeri and Biemont [−1 from 7g and 7h levels of Ar I that are broadened beyond recognition as lines on our spectra.Any interaction between an excited Ar atom and neighboring atoms that shifts the energy of an excited level will also broaden it, and this effect is clearly seen in  Biemont  report lincreased the wavenumber of some lines from levels of high l or n. The shifted lines display a width noticeably greater than that of nearby transitions between s, p, and d levels. The wavenumbers, line widths, and relative signal amplitudes in the table are those one can expect to see from a hollow cathode discharge in Ar at a pressure of a few hundred Pa.We conclude that the gas pressure in our hollow cathode source has


P < 0.05). Further, CSF FFA levels were evidently lower in those 73 patients with favorable outcome when compared to those with unfavorable outcomes . Multivariate analysis results after possible confounders adjustment indicated that there was an increased risk of unfavorable outcome associated with CSF FFA levels ≥0.29 mmol/L . Collectively, CSF level of FFA at admission was suggested to be a useful, independent short-term prognostic marker in Chinese patient with AIS.In this study, prognostic value of cerebrospinal fluid (CSF) free fatty acid (FFA) levels in patients confirmed with acute ischemic stroke (AIS) was evaluated in a Chinese population. A prospective cohort designed study was conducted at our hospital of the Emergency department from November, 2012 to September, 2014. The National Institutes of Health Stroke Scale (NIHSS) score on admission was applied to assess CSF levels of FFA and specific severity degree of stroke. Evaluation of the prognostic outcomes of those stroke patients used the modified Rankin scale scores at 90-days. Logistic regression analysis analyzed the prognostic value of FFA. NIHSS score results suggested a positive relationship between levels of CSF FFA levels and severity of stroke. There was an obviously higher trend of CSF FFA levels in patients with CE stroke than those of the non-CE stroke patients, with statistically difference ( Stroke is a leading cause of mortality and subsequent serious long-term disability among survivors . During In the post absorptive state, free fatty acids (FFAs) are the preferred fuel source for the heart, liver, and skeletal muscle, and yielding large quantities of adenosine triphosphate ATP; . FFA entPathological processes, including ischemia, and subarachnoid hemorrhage could apparently lead to the increased cerebrospinal fluid (CSF) concentrations of FFAs, as supported by previous animal designed experimental evidence . FFA accProspectively, this study was performed within the department of Emergency of Laiwu City People’s Hospital. From November, 2012 to September, 2014, all eligible patients with first-ever AIS were included in the present experiment. All patients were examined by MRI with diffusion-weighted imaging (DWI). And available data were carefully and completely recorded for further investigation. Baseline characteristics, including ages, genders, and history of conventional vascular risk factors such as hypertension, diabetes mellitus, hypercholesterolemia, coronary heart disease, atrial fibrillation (AF) were obtained; previous cigarette smoking habit was also recorded. Clinically, the use of stroke scale-the National Institutes of Health Stroke Scale (NIHSS) was applied for the assessment of stroke severity executed by a neurologist in hospital . For a rAdditionally, exclusion criteria of this study were predefined as follows: (1) patients who were previously diagnosed with malignancy, acute or chronic inflammatory diseases; (2) the patients who presented to the hospital beyond 24 h following stroke onset; (3) patients who without a brain imaging (MRI) test. All enrolled subjects were gave written informed consent, or by their family, and legal guardian. Institutional Review Board of Laiwu City People’s Hospital was contacted to accept the performance of this experiment.The measurements of included subjects infarct volume was according to the formula 0.5 × a × b × c . The detection upper and lower limits were 3.00 and 0.01 mmol/L, respectively. In order to test the relationship between FFA and inflammation, CSF levels of high sensitivity C-reactive protein (Hs-CRP) were also tested . And jus2 test (Chi-square test) was used for the comparison of proportions, whereas the comparison of continuous variables applied the Mann–Whitney test. Spearman’s rank correlation was calculated to assess the relationship between CSF FFA and others factors. Further, multivariate logistic regression models was conducted to assess the associations between FFA and NIHSS score, infarct volume, as well as the influence of FFA levels on functional outcome, after the adjustment with possible confounders such as age, gender, time from onset to admission, stroke syndrome, stroke etiology, vascular risk factors, and CSF levels of Hs-CRP. The association between CSF FFA levels and AIS prognosis were expressed as adjusted odds ratios (OR) and 95% confidence interval (95%CI). The receiver operating characteristic (ROC) analysis was used to determine the optimal cut-off values from different predictive models for the comparison of different prognostic risk scores, combined with the calculation of area under the curve (AUC), which was selected as a summary measure over criteria and cut-point choices. Data were analyzed with SPSS software version 19.0  and the ROCR package . The difference was statistically significant when P < 0.05.In this study, discrete variables are expressed as percentage or frequencies and continuous variables as medians with interquartile range (IQR). The application of the χn = 89), 6–12 (n = 95), and 12–24 (n = 54) hours from symptom onset]. The primary baseline characteristics were illustrated in Table 1.In our study, CSF was collected after admission for standardized measurement of FFA and Hs-CRP in 238 patients. Of the 238 patients, 145 (60.9%) were male with a median age of 64 years . The median NIHSS score of patients was eight points  on the admission. A total of 59 (24.8%) patients were confirmed as AF and 47 (19.7%) with a family history of stroke diseases. For the purpose of this study, CSF samples were drawn at admission . Statistical analysis revealed that there was no significant influence of gender, age, stroke syndrome, and other risk factors of stroke on FFA in AIS patients . Interesting, we found that the patients with AF had elevated CSF FFA levels than others .The increase in CSF FFA levels correlated with increasing disease severity of stroke based on the NIHSS score. The levels of FFA in CSF were positively linked with the NIHSS score . Based on the AUC of 0.825, FFA showed a markedly greater discriminatory ability when compared with the Hs-CRP , the NIHSS score , and infarct volume . Further, FFA may improve the patients’ NIHSS score .At 90-day follow-up, 73 out of 238 patients (30.7%) showed an unsatisfactory functional outcome and the median mRS score was 4 . Compared to patients with favorable outcomes, the patients with unfavorable outcomes had elevated CSF FFA levels [0.36  mmol/L vs. 0.21  mmol/L, P < 0.0001). Multivariate analysis indicated that elevated risk of unfavorable outcomes was related to CSF FFA levels ≥0.29 mmol/L . In addition, four other indicators, including NIHSS score, age, infarct volume, and Hs-CRP, remained to be significant predictors for functional outcome.The study results also showed that CSF FFA levels ≥0.29 mmol/L was closely connected with an elevated risk of unfavorable outcomes for AIS patients , after multivariate adjustment for the outcome predictors including age, NIHSS score, and Hs-CRP.Cerebrospinal fluid free fatty acid levels in 29 patients who died showed greater than twofold increase as compared to patients who survived [0.47  mmol/L vs. 0.23  mmol/L, Previous animal study has demonstrated that the up-regulated levels of certain CSF biomarkers may reflect the pathological activities in central nervous system (CNS), and may be in relation to ischemic diseases . HoweverP < 0.0001). In another study, P < 0.01). However, the small sample (N = 25) should be referred. In our study, we confirmed above conclusion in a larger sample.P = 0.002) was found in our study. Whether the high CSF FFA plays an independent role in contributing the prognosis or just an epiphenomenon to disease severity is uncertain. As we all know, a severe stroke is essentially implicated with a poor outcome. Nevertheless, several other reasons have been involved in the unfavorable outcome of AIS patients with higher FFA levels. First, FFA may generate arrhythmia. Numerous experimental studies have also suggested the arrhythmogenicity of FFA . PreviouSeveral limitations of the current observational study should be noted while interpreting our results. Firstly, in our study, we did not measured the circulating CSF FFA levels, so it yielded no exactly data about when and how long the FFA levels were elevated in AIS patients. CSF level of FFA at admission was a useful and independent short-term prognostic indicator in Chinese patient with AIS. Further studies should be conducted to investigate the underlying mechanism between CSF FFA levels and the poor outcome of AIS. In this regard, the prognosis of stroke patients in China may be improved after elucidate the underlying mechanisms, if possible.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Background. The rising public health concern regarding traumatic brain injury (TBI) implies a growing need for rehabilitation services for patients surviving TBI. Methods. To this end, this paper reviews the practices and research on TBI rehabilitation in Hong Kong so as to inform future developments in this area. This paper begins by introducing the general situation of TBI patients in Hong Kong and the need for rehabilitation. Next, the trauma system in Hong Kong is introduced. Following that is a detailed description of the rehabilitation services for TBI patients in Hong Kong, as exemplified by a rehabilitation hospital in Hong Kong. This paper will also review intervention studies on rehabilitating brain-injured populations in Hong Kong with respect to various rehabilitation goals. Lastly, the implications of culture-related issues will be discussed in relation to TBI. Results/Conclusions. The intervention studies conducted in Hong Kong are generally successful in achieving various rehabilitative outcomes. Additionally, certain cultural-related issues, such as the stigma associated with TBI, may impede the rehabilitative process and lead to various psychosocial problems. Traumatic brain injury (TBI) is a condition involving brain damage caused by external forces, such as acceleration and deceleration, impact, blast waves, or penetrating injury. Its pathophysiology is characterized by shearing of white matter tracts, intra- and extracerebral hematomas, focal contusions, and diffuse swelling . This coSimilar to findings from studies conducted in Western populations , survivoAccording to Yeung et al. , in HongPatients who sustain trauma-related injuries are served by the trauma system as managed by the Hospital Authority (HA). The current trauma system, comprising five regional trauma centers—namely, Prince of Wales Hospital, Princess Margaret Hospital, Queen Elizabeth Hospital, Queen Mary Hospital, and Tuen Mun Hospital—was set up gradually in 2003 to provide trauma care services to various hospital clusters. For instance, Queen Elizabeth Hospital, which is based in central Kowloon, serves one such cluster by extending its trauma services to three other hospitals within its close proximity . These fFor the purpose of describing the rehabilitation of TBI patients in Hong Kong, we shall examine a rehabilitation hospital in Hong Kong: the MacLehose Medical Rehabilitation Centre (MMRC) . This exPatients usually receive short-term rehabilitation services in hospital settings. They may however, with an appropriate referral, turn to various nongovernmental organizations (NGOs) in Hong Kong for long-term care and rehabilitation. These NGO rehabilitation centers provide various outpatient services for discharged neurological patients and also facilitate the patient's reintegration into the community.At the time of this writing, there have not been any published trials on the rehabilitation of, specifically, TBI patients in Hong Kong. Given the paucity of TBI research in Hong Kong, it might be worthwhile to broaden the scope to other brain-injured populations, such as that of stroke. Stroke and TBI are highly similar in terms of pathophysiology. Hence, rehabilitation strategies beneficial to patients with stroke are likely to be beneficial to those with TBI as well . For then = 49) were randomly assigned to the mental imagery group or functional retraining group for three weeks (5 h/wk). In the mental imagery group, patients were taught to use mental imagery to analyze and identify their difficulties and practice certain tasks. The functional retraining group practiced similar tasks; however, unlike the mental imagery group, the entire process was more instructional and didactic and did not involve mental imagery. At the end of the intervention, the mental imagery group had significantly higher levels of task performance on both trained and untrained tasks.These studies have achieved various significant rehabilitative outcomes among brain-injured patients in Hong Kong via different modalities and objectives. For instance, improving the daily functioning of these patients is one such objective; these patients are likely to be impaired in many aspects of their daily functioning , 37. To The other relearning intervention focused on the relearning of motor skills . A totalN = 108) were randomly distributed between the intervention and control groups. The intervention group was administered a holistic care package that was delivered via weekly home visits that involved motivational interviewing and follow-up telephone calls for four weeks. Subsequently, the intervention group reported better physical functioning relative to controls immediately after the intervention and at a follow-up assessment four weeks later.Stroke and TBI patients may both experience problems related to mobility , 40; henN = 21) took part in an eight-week intervention (1.5 h/wk) that consisted of stroke education and physical exercise components, both of which were delivered via video conferencing to all participants. At the end of eight weeks, there were significant increases, relative to baseline, in measures of physical balance ability, self-esteem, quality of life, and knowledge of stroke. It should be noted that these results should be interpreted conservatively since there were no comparisons to a control group.The second study  had videThe third study  examinedBrain injuries can also result in a variety of cognitive deficits as mentioned earlier. Memory impairments are one of these deficits that was of key interest in Tam and Man's  intervenAttention is another cognitive domain where intervention work could be targeted. In one such experiment , 10 patiThe results of these intervention studies in Hong Kong are generally consistent with previous research in showing that functional outcomes among the brain-injured, such as those within the physical, psychosocial, and cognitive domains, can be improved via various rehabilitative approaches . HoweverThe disabilities or dysfunctions associated with TBI may not be the only concerns of TBI patients. TBI patients may experience certain psychosocial problems, which have less to do with the severity of the injury but perhaps more so to do with societal perceptions . These sOne cannot easily identify a TBI patient just from his/her appearance because TBIs are usually not associated with any physically obvious abnormalities unlike patients with physical disabilities. Hence, individuals with TBI can choose whether to conceal their disabilities or disclose them to others. In either scenario, there will be undesirable consequences. If they disclose their condition to others, they might risk being stigmatized by the society as being mentally ill . This isAs a result, it is likely that TBI patients, like those diagnosed with mental illness, would choose to conceal their conditions from others to avoid the stigma or even hide their condition from their family members to avoid implicating them . In factTaken together, the stigma encountered by TBI patients can hinder their rehabilitation in more than one way. This is a major concern that has yet to be adequately addressed in the current practices and research on rehabilitating TBI patients in Hong Kong. Certainly, more effort should be devoted to tackling such stigma. One effective way in which this can be accomplished is to promote more interpersonal contact with and exposure to this group of people. This method was found to be more effective than education alone in reducing various aspects of the stigma and can be implemented widely via school-based programs .The current rehabilitation practices for TBI patients in Hong Kong have generally satisfied the guidelines for the rehabilitation following acquired brain injury as laid out by the Royal College of Physicians and British Society of Rehabilitation Medicine . TBI patAdditionally, given that the patients' main caregivers are their families, it is crucial to involve them in the rehabilitation process prior to the patient's hospital discharge. However, such family involvements remain somewhat low, and perhaps this explains their unpreparedness and lack of the know-how in caring for their brain-injured relatives , 53. TheThe few reported researches on interventions for brain-injured populations conducted in Hong Kong have generally been effective in achieving certain specific goals such as the remediation of skills compromised by the injury and community integration. Despite the lack of local research on specifically rehabilitating TBI patients, these researches on other brain-injured populations do suggest that there are adequate resources, both in the community and in the health care infrastructure, to carry out similar rehabilitative efforts on TBI patients.Certainly, future research on TBI rehabilitation in Hong Kong will be useful in informing and guiding the current practices and ultimately to better the lives of TBI patients. Intervention efforts utilizing cognitive-based rehabilitative approaches on brain-injured populations in Hong Kong have been scarce. In light of the possible impairments in various cognitive domains  and the The present paper has reviewed the practice and research of TBI rehabilitation in Hong Kong. TBI rehabilitation in Hong Kong is carried out by a multidisciplinary team of health care professionals primarily within hospitals, as consistent with international guidelines. Despite limited resources, these rehabilitation services have generally satisfied most of the TBI patients' needs. However, there is room for improvement in rehabilitative efforts aimed at enabling them to return to work. While stigma is a major concern for these patients and their rehabilitation, it is hardly addressed in the current practices. Finally, research on TBI rehabilitation in Hong Kong is lacking; there is a need for more local research in this area to inform the current practice of TBI rehabilitation and more importantly to benefit TBI patients in Hong Kong.


The investigation revealed that staff perceived many families to be facing food insecurity and isolation during the school holidays, which may be alleviated through holiday club provision. Holiday clubs were viewed as a valuable source of support for children and adults, providing food, activities, and learning experiences. Staff were keen to see them implemented on a wider scale in future but suggested some areas that require attention in any future development of such provision. Findings are discussed in relation to current research, policy, and practice surrounding the health and wellbeing of children and families.Access to an adequate supply of nutritious food has been recognized as a basic human right. However, many families across the UK face food insecurity, which is thought to be exacerbated during school holidays. To address this issue, some schools and community groups have chosen to roll out holiday clubs, though research into the effectiveness of such interventions is limited and no studies to date have evaluated holiday clubs being organized through schools. In an effort to address some of the limitations in the research literature, the current qualitative investigation utilized semi-structured interviews with staff involved in holiday clubs in school  Sufficient access to food has long been recognized as a basic human right, which is critical to adequate health and wellbeing, and central to human dignity , 2. HoweA lack of food security  The importance of adequate food intake for children is evident across UK school food policy. Throughout the school day, children are provided with a range of meals and snacks, including breakfast, lunch, fruit, and milk, all of which must adhere to comprehensive nutritional standards . HoweverN = 459) of teachers surveyed in the UK believed that their pupils do not get enough to eat during the school holidays. Furthermore, research drawing on the views of parents has shown that the school holidays are a time of considerable financial difficulty for families, with some parents relying on sporadic meal patterns and cheap foods of low nutritional value to ensure that their children are fed  Why is there a need for holiday clubs? (2) What are the benefits of holiday club participation? and (3) What factors need to be considered in the development of holiday club provision?A semi-structured interview approach was adopted for the purpose of the current study. This method was deemed appropriate as it allowed participants to talk openly about sensitive topics, such as food insecurity and to freely express their views about the running of their holiday clubs. Interviews were carried out via the telephone so that staff could participate at a time that was appropriate to them without interfering with their day to day tasks within the holiday club setting.Fourteen staff members  from six holiday clubs were purposively sampled to participate in the current study. Purposive sampling refers to the method of selecting participants based on a particular characteristic that is relevant to the investigation . Staff iThe holiday clubs took place in school and community-based premises across the South of England and Wales for approximately 4–6 weeks of the school summer break. A summary of the features of each holiday club is presented in Table A semi-structured interview schedule was developed for the purpose of the current investigation, consisting of a series of open questions that invited participants to talk freely about various aspects of their holiday clubs. A copy of the interview schedule is included in Table Following ethical approval from Northumbria University Ethics Board , information about the research project was posted out to each holiday club. Completed consent forms were then collected from each holiday club by a member of the research team during August 2015 and those who provided written consent to participate were subsequently contacted and interviewed via telephone in September 2015. The interviews, which were carried out by a research assistant from Northumbria University, followed a semi-structured format to allow the interviewer to gain further insight and clarification on points of interest where necessary. All interviews were audio recorded and transcribed verbatim for subsequent thematic analysis. On average, the interviews lasted 18 min. Transcription was carried out by the interviewer and the accuracy of the transcripts was subsequently checked against the recordings by a second researcher. Thematic analysis was carried out by the second researcher and second coded by the interviewer as detailed under the following section.It has been proposed that qualitative interviews should continue until a saturation point is reached. Saturation refers to the stage at which no new themes are identifiable within the data and the inclusion of further interviews does not serve to add new themes to the data set , 21. In Data were coded and analyzed in accordance with published guidelines on the process of thematic analysis . An induStaff reflected on their knowledge of their local area and experiences of working in holiday clubs to share their views on the need for holiday club provision; the benefits of holiday club participation for children and adults; and potential areas for further development of holiday clubs. A summary of the themes that were identified across staff interviews is subsequently presented with themes, initial codes, and example quotes presented in Table Holiday Hardship; Inactivity and Isolation; and Evidence of Demand.Three themes relating to staff views on the need for holiday clubs were established through interviews with holiday club staff: Staff believed that holiday clubs were a worthwhile provision to support struggling families during holidays from school and believed that they should be made available in future. They were aware of families from various income levels being heavily reliant on school meals during term time and expressed concern that a lack of school meal provision during the school holidays could lead to children skipping meals or being provided with a limited range of foods, which are low in nutritional value. Furthermore, financial hardship was thought to limit family engagement in activities during the school holidays, which led to children and parents becoming quite sedentary and isolated within their communities.Staff reflections on their experiences of holiday clubs suggested that uptake was relatively low compared to their expectations. However, staff reported that attendance increased across the duration of the holidays and those families who attended did so consistently, thus evidencing a demand for the provision.Access to Food; Access to Activities; Social Opportunities; Familial Support; Informal Learning Experience; and Positive Experience for Staff.Six key themes encompassed the benefits of holiday clubs discussed by staff: In addition to providing a reliable and essential source of food for some children and their families, staff felt that the food provided at holiday clubs gave children opportunities to try new and more nutritious foods than they would have access to at home. Staff believed that the children liked trying new foods and felt that these new food experiences could have a positive influence on children’s dietary habits at home.In addition to food, holiday clubs offered an array of enjoyable activities for children to partake in each day. The activities on offer were valued as they were deemed to be more accessible than other activities on offer in the local area; they were believed to alleviate boredom and reduce the likelihood that children would engage in anti-social behavior. Moreover, children and adults learnt new skills and knowledge through the holiday club activities, particularly in relation to food, nutrition, and sports skills. The informal nature of these activities meant that children often did not realize that they were engaging in learning activities.The provision of food and activities provided a platform for children and adults to interact informally, creating a strong social element to the holiday clubs. Children, parents, and staff were able to spend time and build relationships with others that they would be unlikely to encounter at any other time. Moreover, staff felt that holiday clubs helped to break down perceived barriers to parents engaging with school and community interventions.Holiday clubs were also viewed as valuable sources of support, providing an environment where parents and children could go for various kinds of help, beyond the provision of food. By offering an accessible package of food, activities, and child care, holiday clubs were suggested to alleviate financial constraints on parents during the school holidays, while providing parents with peace of mind that their children were in a safe and fun environment.Finally, the staff involved in the holiday clubs found their experiences to be very rewarding. They gained satisfaction from helping others in their communities, gained professional development experience, and some benefited from earning extra income during the school holidays.Staffing Considerations; Prior Planning; Widening Participation; and Holiday Club Setting.On the whole, staff were very positive about holiday club provision and, as evidenced through their views on the benefits of holiday club participation, staff felt that holiday clubs had a lot to offer to children, parents, and staff. However, reflections on their own experiences led staff to suggest some minor areas for consideration should holiday clubs be implemented longer term. Potential areas for development discussed by staff were summarized across four themes: First, holiday clubs tended to bring together a number of organizations, including local authority representatives, school staff, and volunteers. Though staff recognized value in being able to draw on the expertise of various agencies, staff suggested that there needs to be more open communication between organizations to ensure that everyone is aware of their individual roles and responsibilities, and to make sure that nothing is overlooked. Moreover, some staff believed that taking on the responsibility of working in a holiday club was a big commitment and clubs could benefit from additional staffing. This was particularly the case for those clubs that relied on volunteers to work in the clubs day to day as they sometimes faced difficulties with having fewer staff than anticipated. Though when staff were paid for their time, there were issues with staff being paid different hourly rates for doing the same job as holiday club rates of pay were based on their term time rates of pay. This led to concerns that such issues with pay might deter people from becoming involved in the delivery of holiday clubs in future.Second, some staff felt that they had very little planning time prior to the commencement of the holiday clubs and believed that this would need to be addressed in future. Staff hoped to have more autonomy in terms of the activities they offered in future but, for those clubs in their first year of operation, they believed that they now had a bank of resources and experiences that they could draw upon in future.Furthermore, in terms of planning, it emerged that there were mixed views on the appropriate duration for the holiday clubs to run. Some staff felt that only offering provision for part of the week was good as it allowed children and staff some time away, while others suggested that it would have been useful to run for more days throughout the holidays or for a longer time period each day. Interestingly, in those clubs where breakfast was served, the start time of the clubs was thought to have an impact on children’s perceptions of the breakfast meal, which consequently impacted how much food they consumed in the mornings as some children had breakfast at home then went on to have more breakfast when they arrived at the holiday club.A further consideration for future holiday provision related to attendance rates. Although staff saw an obvious need for holiday clubs, the number of people attending the clubs overall was relatively low, which prompted staff to propose that more work would be necessary in future to ensure that those families who could benefit most from holiday provision are reached. However, it was suggested that directly targeting children and families to attend reduced opportunities for advertising and could potentially lead to attendance being stigmatized.Finally, staff across school and community settings spoke favorably about the venues that their holiday clubs took place in. Schools were thought to be particularly well placed to host holiday clubs given children’s familiarity with them and the abundance of resources and outdoor space that they offered; though the use of community venues was not thought to limit families’ holiday club enjoyment or access. Clubs based in community venues were more limited than schools in terms of outdoor space. However, all the holiday club venues had ample kitchen space and amenities to be able to offer cooked meals and this was something that staff recognized as important.The aim of the current study was to investigate the need for holiday club provision, the benefits of holiday club participation, and potential areas for future development from the perspective of holiday club staff. The findings of the investigation showed that staff believe there is a need for holiday club provision to help to relieve financial strain on families and to encourage them to remain active and engaged within their communities throughout the school holidays. Staff believed that holiday club participation conferred a range of benefits for families by providing access to meals and activities and offering social, learning, and support opportunities. Additionally, staff felt that they had benefited from their involvement in holiday clubs. They gained enjoyment and satisfaction from helping people within their communities and some benefited from additional income. Staff hoped to see holiday clubs made available in the future but were keen to see more careful planning and deployment of staff in future provision.The views of staff presented in the current study lend support to practitioners and policymakers who have argued that there is a need for accessible food and activity provision during the school holidays for children and families in the UK . There iThrough food and activity provision, holiday clubs in the current study were also thought to be able to provide a mechanism of support for families. In addition to relieving financial pressures, holiday clubs offered children and parents some respite and somewhere to socialize regularly with people from their local area. This is important as previous research has shown that children and parents are at risk of becoming isolated during the school holidays and generally receive a lack of support from their families and communities . Such isAdditionally, by supporting families through the provision of accessible childcare, holiday clubs were suggested to help parents to remain in employment and keep children engaged in activities, thus ensuring maintenance of a consistent routine, income stability for parents and less boredom for children. Given that The Trussell Trust have reported income instability as a key reason for people having to rely on food banks (see text footnote 1), supporting parents to retain employment during the school holidays could lead to a reduction in the high prevalence of families accessing food aid outside of school term time. Moreover, the prevalence of anti-social behavior tends to increase during the school holidays  and someA final key benefit of holiday clubs outlined by staff in the current study related to the learning opportunities that children and families were afforded. In some cases, learning took place through sessions designed to impart knowledge and skills, such as nutrition lessons and sports coaching. However, some staff felt that families were learning and being inspired to try new things simply by participating in holiday clubs. Such direct and indirect learning opportunities are particularly important for children, given concerns that during the summer break from school, children are at risk from summer learning loss , 17. StaThe current findings offer a timely contribution to the research literature in this area given recent calls by UK policymakers and practitioners for more research to be carried out to investigate the issue of holiday hunger and the impact of holiday provision . The vieTo conclude, the current investigation provides a useful contribution to the limited academic work in this area and provides a point of reference for policymakers, stakeholders, and academics with an interest in holiday clubs.PG contributed to study design, recruitment, analysis, and manuscript preparation. EC contributed to data collection, analysis, and manuscript preparation. PS contributed to study design and manuscript preparation. ML contributed to study design and manuscript preparation. KP contributed to study design, recruitment, and manuscript preparation. ES contributed to study design, recruitment, and manuscript preparation. MD contributed to study design, recruitment, analysis, and manuscript preparation.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


We investigated whether β2-AR-mediated signalling could be modulated in skeletal muscle via gene delivery to the target tissue, thereby avoiding the risks of β2-AR agonists. In mice, intramuscular administration of a recombinant adeno-associated virus-based vector (rAAV vector) expressing the β2-AR increased muscle mass by >20% within 4 weeks. This hypertrophic response was comparable to that of 4 weeks’ treatment with the β2-AR agonist formoterol, and was not ablated by mTOR inhibition. Increasing expression of inhibitory (Gαi2) and stimulatory (GαsL) G-protein subunits produced minor atrophic and hypertrophic changes in muscle mass, respectively. Furthermore, Gαi2 over-expression prevented AAV:β2-AR mediated hypertrophy. Introduction of the non-muscle Gαs isoform, GαsXL elicited hypertrophy comparable to that achieved by AAV:β2-AR. Moreover, GαsXL gene delivery was found to be capable of inducing hypertrophy in the muscles of mice lacking functional β1- and β2-ARs. These findings demonstrate that gene therapy-based interventions targeting the β2-AR pathway can promote skeletal muscle hypertrophy independent of ligand administration, and highlight novel methods for potentially modulating muscle mass in settings of disease.Anabolic β Consequently, clinical application of these compounds for muscle wasting has remained limited. We investigated whether stimulation of β2-AR signalling that promotes skeletal muscle hypertrophy might be achievable by means that circumvent the potential off-target effects of β2-AR agonists. Specifically, we hypothesised that administering gene therapy-based interventions to alter the expression of β2-AR pathway components could promote skeletal muscle growth independent of β2-AR agonist administration. This rationale was based on the emerging development of recombinant adeno-associated virus-based vectors (rAAV vectors) as tools for therapeutic gene delivery, owing to their propensity for achieving efficacious and targeted delivery of transgenes to the skeletal muscles of mammals2123Because sustained stimulation of β2-AR gene delivery using rAAV vectors can promote skeletal muscle hypertrophy in mice without administration of synthetic β2-AR agonists. Additionally, we observed that increasing the expression of specific G-protein subunits could exert hypertrophic and atrophic effects in skeletal muscle independent of ligand administration. These studies introduce targeted gene delivery as a new strategy for manipulating the β2-AR signalling pathway without administering β2-AR agonists, to promote skeletal muscle hypertrophy.Our studies identified that β2-AR abundance in muscle fibres, we used adeno-associated virus-based vectors encoding the β2-AR (AAV:β2-AR) or a gene-less cassette (control) to transduce the tibialis anterior (TA) hind-limb muscles of male eight-week-old C57Bl/6 mice. Optimisation of vector doses established that injection of muscles with 1 × 1010 AAV:β2-AR vector genomes (vg) produced a 22% increase in muscle mass within 28 days of vector administration, which was maintained for at least 84 days after vector delivery (the longest time point examined)  activity and immunolabelled for prevalence of the myosin type IIa isoform. Muscles examined four weeks after administration of AAV:β2-AR or control vector did not exhibit a difference in the proportion of fibres expressing the type IIa myosin heavy chain isoform, or the activity of SDH (To determine the effects of increasing βxamined) . Cross-se fibres , and an e fibres . Musclesy of SDH .via administration of AAV:β2-AR was comparable to that achieved by treating muscles with anabolic β2-AR agonists, additional cohorts of mice were administered AAV:β2-AR, or daily injections of formoterol (100 μg/kg) for 28 days. We observed that a single administration of AAV:β2-AR and 28 consecutive days of formoterol administration produced comparable increases in muscle mass  and inhibitory (Gαi) G-proteins to propagate intracellular signalling, we investigated whether treating the TA muscles of mice with AAV vectors that increase expression of either GαsL or Gαi2 affected muscle mass. Injection of TA muscles with AAV:GαsL four weeks prior to examination increased mass by 8% 281/β2mut mice, treated TA muscles exhibited a 35% increased mass , while minimising the potential for incurring off-target effects in other tissues.Although synthetic β2-AR expression in mouse limb muscles promoted increases in myofibre size and augmented protein synthesis. The hypertrophic effects of AAV:β2-AR administration were comparable in magnitude to those achieved with repeated administration of the potent β2-AR agonist formoterol. We did not find evidence that activation of mTOR was required to support muscle hypertrophy induced by β2-AR gene delivery, which contrasts with reports of β2-AR agonist-induced skeletal muscle hypertrophy requiring mTOR signalling2-AR via the PKC/GSK3β signalling axis2-AR administration could utilise similar mechanisms. These observations point to other possible advantages of developing skeletal-muscle-directed gene delivery as an alternative method for manipulating β2-adrenergic signalling in vivo. Further research is warranted to more comprehensively examine the similarities and differences between drug- and gene-based interventions targeting this signalling system in striated muscle.Using rAAV vectors to enhance β2-AR gene delivery can stimulate skeletal muscle hypertrophy, we investigated whether β2-AR-mediated effects could be potentiated by increasing the abundance of specific Gα protein subunits operating as signalling substrates for the β2-AR. Increasing expression of GαsL promoted a modest hypertrophic effect compared with AAV:β2-AR administration. Broadly, this stimulatory role of Gαs is consistent with earlier work demonstrating that muscle mass is reduced in Gαs knockout mice2-AR completely prevented the hypertrophic effects of β2-AR gene delivery. These findings are consistent with the possibility that Gαi2 possesses comparatively greater (relative to Gαs) affinity for interaction with the β2-AR, or that increased abundance of Gαi2 can outcompete Gαs for interaction with the β2-AR.Having established that βQ205L mutant3735Q205L promotes cell proliferation and recruitment lie outside the scope of the present study, the differences in effects of Gαi2Q205L reported elsewhere versus the effects of wild-type Gαi2 reported here appear to be attributed at least in part to differential actions within myogenic progenitor cells versus muscle fibres. Additionally, it cannot yet be ruled out that the Gαi2Q205L mutant isoform does not exert different effects on downstream signalling targets or is affected differently by feedback mechanisms.Although Gαi2 is considered to operate in opposition to Gαs, the inhibitory effects of over-expressing wild-type Gαi2 in muscle fibres described herein contrast with studies that documented protein accretion and cell growth after transducing myogenic cells with a constitutively active Gαi22-AR or formoterol. Muscles treated with AAV:GαsXL exhibited an increased proportion of myofibres expressing the type IIa myosin heavy chain isoform, whereas no such effect was noted in muscles receiving AAV:β2-AR. These findings lend support to the idea that the two vector-based interventions have differing effects upon the physiological properties of treated skeletal muscles. The marked muscle hypertrophy with administration of AAV:GαsXL was recapitulated in mice lacking functional β1- and β2-ARs, which do not exhibit an anabolic response when administered anabolic β-agonists1- and β2-ARs. It is not clear whether the observed muscle hypertrophy is a product of GαsXL possessing constitutive activity, or whether GαsXL proteins may be activated by other GPCRs in muscle fibres. Other receptors in muscle that could function as activators of ectopically expressed GαsXL include Fzd7 . All experimental protocols were approved by the Alfred Medical and Education Precinct Animal Ethics Committee (AMREP AEC). All surgical procedures were performed under inhalation of isoflurane in medical oxygen with post-operative analgesia. Eight to 10 week old, male, C57Bl/6 and β1/β2 mutant (β1/β2mut) mice were used for all experiments. Animals were fed standard chow diets with access to drinking water ad libitum while housed under a 12-hour light dark cycle. β1/β2mut mice were sourced and bred as described previously2-AR, AAV:Gαi2, AAV:GαsL (1 × 1010 vg) and AAV:GαsXL (1 × 109 vg) vectors (identified from preliminary dose-optimisation experiments) were diluted in 30 μl of Hank’s buffered saline solution (HBSS) and directly injected into the TA muscle. Control injections consisted of the administration of a viral vector lacking a functional gene into the contralateral limb. For systemic β-agonist treatments, intraperitoneal injections of formoterol at 100 μg/kg or saline were administered daily for 28 days. Rapamycin (ApexBio) was dissolved in DMSO to a stock concentration of 10 mg/ml, and a working concentration of rapamycin was formulated in a solution of 0.1% carboxymethylcellulose and 0.125% polysorbate-80. Mice received 2 mg/kg of rapamycin one day before and at the time of AAV:β2-AR administration by intraperitoneal injection. Mice were treated daily until experimental endpoint. For puromycin administration, mice received 0.04 μmol/g of puromycin (Life Technologies) via intraperitoneal injection exactly 30 min before experimental endpoint. Experimental endpoints were 28 days post viral vector administration unless indicated otherwise. Mice were humanely killed via cervical dislocation and the muscles rapidly excised and weighed before subsequent processing.2-AR (MBL) 1:500 and anti-GAPDH (Santa Cruz Biotechnology) 1:10000.All antibodies were purchased from Cell Signaling Technologies and used at a dilution of 1:1000, except anti-puromycin and anti-laminin B2 (Millipore) which were used at 1:5000 and 1:250 respectively, anti-β2-AR), Gnai2 (Gαi2), GnasL (GαsL) and GnasXL (GαsXL) (synthesized by GenScript) which were cloned into an AAV expression plasmid consisting of a cytomegalovirus (CMV) promoter and SV40 poly-A region flanked by AAV2 terminal repeats. The Gαi2, GαsL and GαsXL cDNA construct also included a flag-tag coding region at the 5′ end of the coding sequences. Viral vector production was performed as described previously6 cells on a 10 cm culture dish, 8–16 hours before transfection with 10 μg of a vector genome-containing plasmid and 20 μg of the packaging/helper plasmid pDGM6 by calcium phosphate precipitation. At 72 hours post transfection, the medium and cells were collected and homogenized through a microfluidizer (Microfluidics) before 0.22 μm clarification (Millipore). Purification of viral particles from crude lysates was performed using affinity chromatography over a heparin affinity column , and ultracentrifugation overnight prior to re-suspension in sterile physiological Ringer’s solution. The purified vector preparations were titered with a customized sequence-specific quantitative PCR-based reaction (Life Technologies).Traditional cloning techniques were used to generate cDNA constructs encoding Adrb2  and samples denatured for 5 min at 95 °C. Protein fractions were resolved by SDS-PAGE using pre-cast 4–12% Bis-Tris gels (Life Technologies), blotted onto nitrocellulose membranes (BioRad) and incubated with the appropriate primary antibody and detected as described previouslyvia ImageJ software analysis. Myosin type IIa positive fibres were counted and expressed relative to total number of myofibres counted per section (>600 fibres samples per muscle).Harvested muscles were embedded in optimum cutting temperature (OCT) cryoprotectant (Sakura Finetek) and frozen in liquid nitrogen-cooled isopentane. Frozen samples were cryosectioned at 10 μm thickness using a Leica CM1950 cryostat. Cross-sections were fixed in room temperature methanol and stained with hematoxylin and eosin as described previously2-AR primary antibodies overnight at 4 °C. Alexa-Fluor-488 and -594 secondary goat antibodies (Life Technologies) were used to detect β2-AR and laminin B2 primary antibodies respectively, followed by 3 min incubation in DAPI nuclear stain (Life Technologies) and mounting in HardSet Vectashield (Vector Laboratories). Images were captured using a BX61 light microscope (Olympus).OCT-frozen TA samples were cryosectioned at 8 μm thickness, fixed in methanol, washed in potassium phosphate buffered saline (KPBS) containing gelatin and blocked in a solution consisting of Tween-20, BSA, gelatin and KPBS. The sections were incubated in anti-laminin B2 and anti-βpost hoc analysis for specific group comparisons. All significant differences are reported with p < 0.05.All data are represented as the mean ± SEM. A paired Student’s t-test was used for comparisons between two conditions. Two-way analysis of variance (ANOVA) was used to measure statistical differences between multiple conditions with Tukey’s How to cite this article: Hagg, A. et al. Using AAV vectors expressing the β2-adrenoceptor or associated Gα proteins to modulate skeletal muscle mass and muscle fibre size. Sci. Rep.6, 23042; doi: 10.1038/srep23042 (2016).


Drosophila neuromuscular junction (NMJ), the postsynaptic structure, known as the subsynaptic reticulum (SSR), consists of elaborate membrane folds that link the synaptic contacts to the muscle, but its role in synaptic physiology is poorly understood.Synaptic transmission requires both pre- and post-synaptic elements for neural communication. The postsynaptic structure contributes to the ability of synaptic currents to induce voltage changes in postsynaptic cells. At the In this study, we investigate the role of the SSR with simultaneous intra- and extra-cellular recordings that allow us to identify the origin of spontaneously occurring synaptic events. We compare data from Type 1b and 1s synaptic boutons, which have naturally occurring variations of the SSR, as well as from genetic mutants that up or down-regulate SSR complexity. We observed that some synaptic currents do not result in postsynaptic voltage changes, events we called ‘missing quanta’. The frequency of missing quanta is positively correlated with SSR complexity in both natural and genetically-induced variants. Rise-time and amplitude data suggest that passive membrane properties contribute to the observed differences in synaptic effectiveness.Drosophila NMJ.We conclude that electrotonic decay within the postsynaptic structure contributes to the phenomenon of missing quanta. Further studies directed at understanding the role of the SSR in synaptic transmission and the potential for regulating ‘missing quanta’ will yield important information about synaptic transmission at the  Drosophila NMJ, the neurotransmitter glutamate is secreted and binds to glutamate receptors found on the postsynaptic cell [Drosophila NMJ are dense postsynaptic muscle membrane folds called the subsynaptic reticulum (SSR) [Drosophila NMJ, the SSR appears to be densely packed convoluted postsynaptic “leaflets” of muscle membrane, forming compartments that underlie and surround the bulbous endings of the presynaptic nerve terminal [Synaptic transmission is the process used by neurons to communicate with other cells. Key to this process is the synapse, which is the very close apposition of cellular membranes of the presynaptic and postsynaptic cells. At the tic cell , 14. Theum (SSR) , 12, 28.terminal , 19.Studies of the SSR have given rise to suggestions about its role in synaptic development and physiology. Some of the functions include acting as a site where glutamate receptors (GluR) are translated , a hub wThe SSR bears some similarity to junctional folds of mice, frogs and other vertebrate neuromuscular junctions. In some ways, the SSR also closely resembles it to dendritic spines typical of CNS neurons , which aDrosophila SSR plays in the physiology of synaptic transmission, however some models have been proposed by previous studies, including the idea that the SSRs compartmentalization and postsynaptic micro-domains house signals that shape synaptic response [Drosophila larval NMJ system.Relatively little is understood regarding the role that the response . It is gThe primary observation of this report is that, in our model synaptic system, not all synaptic currents result in a change in membrane voltage. We were able to make this observation by combining focal extracellular recordings with simultaneous intracellular measurements  Fig. 1)1). The eOre-R was selected to serve as a wild-type reference, whereas w; MHC::mCD8-GFP-Sh was used for live imaging and bouton identification. Synaptic boutons can be visualized with transmitted light and Differential Interference Contrast microscopy and allow for the placement of electrodes. However, fluorescently labeled boutons are easier to identify. The w; MHC::mCD8-GFP-Sh strain expresses a chimeric protein consisting of the CD8 transmembrane domain, GFP, and the cytoplasmic tail-domain of the Shaker potassium channel, under the control of the Myosin heavy chain promoter [To measure the occurrence of missing quanta we first used two control strains: promoter . Thus, CWe performed simultaneous recordings from 1b or 1s boutons and collected 50 extracellular events from 20 larval samples for each bouton from each genotype and thus analyzed 1000 synaptic events for each data set. A missing event was tallied when an extracellular synaptic event was detected with an extracellular electrode, however no corresponding intracellular response is detected from the intracellular electrode. As shown in Table Drosophila neuromuscular synapse and that there are differences between Type 1b and 1s boutons, with a greater preponderance of missing events at Type 1b boutons, we considered what may give rise to missing quanta.Having shown that there are missing quanta at larval While there may be several molecular and/or biochemical signatures that distinguish the two bouton types, for the remainder of this study we will focus on the well-known structural differences of the subsynaptic reticulum (SSR). It has been previously shown that the closely apposed presynaptic and postsynaptic membranes are connected to the muscle by the highly convoluted and tortuous membrane system of the SSR and that Type 1b boutons are composed of a larger and more complex SSR than Type 1s boutons . Indeed,Drosophila that either increase or decrease SSR complexity. We predicted that, as with Type 1b and 1s boutons, the proportion of missing events should vary in the same direction as SSR morphometry in genetic variants.There are several possibilities that may link SSR complexity to a role in the phenomenon of missing quanta. Since the SSR is an electrical connection between the point of synaptic contact and the postsynaptic muscle cell, the SSR likely acts as an electrotonic pathway between synapse and muscle. Therefore, passive membrane properties likely play a role, akin to the dendritic connection between dendritic spines and cell body of CNS neurons. In order to investigate whether electrotonic properties influence the existence of missing quanta we employed genetic variants of RalG20V in muscle increases the amount of SSR, whereas the ralG0501 allele reduces Ral protein expression and causes a decrease in the width and complexity of the SSR [Mutations of the Ral gene have been recently shown to increase or decrease the complexity of the SSR. Expression of  the SSR . We inveRalG20V expression showed the largest SSR, while Type 1b boutons of ralG0501 mutants were smaller. The SSR width in the control strains, OreR and MHC::mCD8-GFP-Sh fell between these two extremes. Type 1s boutons of the OreR and MHC::mCD8-GFP-Sh genotype contains the smallest SSR widths of 0.6 μm ± 0.03 and 0.7 μm ± 0.02 respectively (mean ± SEM). All other genotypes OreR Type 1b (1.02 μm ± 0.05), MHC::mCD8-GFP-Sh Type 1b (1.1 μm ± 0.06), and genotypes reared in a control background Mef2-gal4/+ (1.1 μm ± 0.05), ralG0501/+ (1.1 μm ± 0.04), and RalG20V/+ (1.1 μm ± 0.05) had varying similar SSR widths. 15 Type 1b boutons for each genotype and 15 Type 1s boutons for OreR and MHC::mCD8-GFP-Sh larvae was measured (n = 15).Although previous studies have quantified the extent of the SSR in these alleles using electron microscopy , we sougen masse from the test and control genotypes in the usual manner, analyzing 50 mEJPs from 10 muscle cells per genotype (n = 10)  Fig. . Data wepes Fig. . Muscle OreR and MHC::mCD8-GFP-Sh larvae showed similar results, with a range of mean amplitudes of 0.9–1 mV. All other genotypes showed similar amplitudes, with no significant differences in a range of 0.6–0.9 mV .The amplitude of mEJPs from control UAS-RalG20V/Mef2-gal4 contains the highest mean proportion of missing intracellular events, showing 10.8 % missing events. In contrast, ralG0501, which had the smallest Type 1b SSR, also showed the fewest proportion of missing intracellular events, with a mean value of 2.7 %.As described above for the control strains, we next quantified the extent of missing quanta in the larvae with expanded or reduced SSR. Interestingly, we found that genotypes with expanded SSR showed a greater proportion of missing events than those with reduced SSR Fig. a. UAS-Ra2 value of 0.8. For example, the Type 1s boutons from control strains and Type 1b from ralG0501/y have the smallest mean SSR widths and also the fewest proportion of missing events, while UAS-RalG20V/Mef2-gal4 have the largest SSRs and also the greatest proportion of missing quanta.To further analyze these data, and help us determine if there is a general relationship between SSR and missing quanta, we plotted SSR width versus missing events, for all bouton types and genotypes Fig. b. This aUAS-RalG20V/Mef2-Gal4 larvae, with a rise-time of 7.55 ± 0.5 ms, whereas the mEJPs from OreR Type 1s boutons had the shortest rise times of 5.19 ± 0.36 ms. Other bouton types fell in between these two values as plotted in a regression versus SSR width . A frequency distribution revealed a greater proportion of smaller mEJPs recorded from the Type 1b boutons  by the average mEJP amplitude, which was measured from the ensemble of mEJPs recorded from the whole muscle. The present results show that the quantal release of a nerve terminal would be underestimated by such a method. This calculation would be particularly effected by genetic variants that impact SSR structure, since as SSR complexity increases we might expect a greater number of missing quanta and therefore a greater error in estimates of quantal content. Our corroboration that mEJPs arising from the different nerve terminals have different amplitudes suggest that quantal size also contributes to the variation of EJP size, with Type 1b boutons having smaller mEJPs on average. Thus any genetic variants that tend to alter the relative proportion of mEJPs arising from one or the other bouton type would have a differential impact on estimates of average mEJP amplitude and therefore quantal content.Altogether our data introduce a new physiological parameter that may be a contributing determinant of synaptic strength. Further studies aimed at understanding whether the proportion of missing quanta is a physiological parameter that can be dynamically regulated will yield further understanding of the way in which postsynaptic structure influences synaptic transmission.Drosophila larval neuromuscular junction. We call these events missing quanta and they occur at a rate of 3 % of Type 1s and 8 % of Type 1b of synaptic events in the wild-type OreR larvae. We observed a positive correlation between SSR width and the proportion of missing quanta, in both the natural variants and by using alleles of the Ral gene which can up- and down-regulate SSR complexity. In turn, we hypothesized that SSR membranes act as an electronic connection between the synapse and muscle and present evidence that mEJP rise-time and amplitude vary in a manner consistent with this hypothesis. Further studies aimed at understanding whether the phenomenon of missing quanta can be dynamically regulated will prove fruitful to understanding the role of postsynaptic structures in synaptic effectiveness.In summary, we have demonstrated that not all synaptic currents generate postsynaptic voltage changes at the OreR). The following stocks used were: MHC::mCD8-GFP-Sh, UAS-RalG20V, RalG0501, which were all gifts from the lab of Dr. Thomas Schwarz. OreR and MHC::mCD8-GFP-Sh larvae served as a control for this study. Control fly strains used were Mef2-gal4/+, ralG0501/+, and UAS-ralG20V/+. UAS-ralG20V were expressed in the muscle by crossing these stocks to Mef2-Gal4 in the UAS-Gal4 system [All flies were maintained at 23–25 °C on Bloomington standard fly food medium. In this study the wild-type strain used was Oregon-R . Sharp intracellular microelectrodes were pulled and backfilled with 3 M KCl; they had resistances of ~30 MΩ. Extracellular electrodes for focal recordings [2, and was used to place over the bouton of choice on the NMJ. The tip of the pipette was polished using the microforge to an internal diameter of 6–8 µm, large enough to cover Type 1b boutons.Intracellular and extracellular recordings were performed on dissected wandering third instar larvae in HL3 bath solution  with addcordings , 33, 42 n = 20).Both intracellular and extracellular currents are recorded using an AxoClamp 2B amplifier (Axon Instruments). The analog signals were converted to a digital signal using a Digidata 1322A (Axon Instruments), and further analyzed using pClamp 8.2 software. Only muscles possessing an initial resting membrane potential of anything more negative than −60 mV was recorded. Intracellular and extracellular recordings were recorded simultaneously for 2–6 min. A total of 20 animals per genotype of each bouton type (Type 1b and Type 1s) was used for the electrophysiological recordings  to 0. Cursors were then used to measure the amplitude of the average trace. All data was then transferred to GraphPad Prism 5 for graphical and statistical analysis.Dissected larvae are fixed with 4 % paraformaldehyde  in phosphate buffered saline (PBS) for 20 min. Larvae were then washed in PBS with 0.1 % Triton X-100 (PBT). Larvae are then transferred into a new microcentrifuge tube containing PBT and are then blocked in blocking solution  for 30 min. Primary antibody was then added and allowed to incubate over night at 4 °C.® 555 (Life Technologies) was added (1:500) and allowed to incubate for 1 h at room temperature. Larvae were then washed in PBT for a final 4 times at 15 min each wash. All stained larvae are then placed on glass slides containing Vectashield mounting medium for fluorescence (Vector Labs).Monoclonal mouse anti-Discs large 4F3  was usedImages were collected on a Zeiss LSM 510 confocal microscope. Snap shots and Z-stacks were taken from muscles 4, 6, and 7 using a 40×/1.4 oil immersion lens or a 63×/1.4 oil immersion lens.Images were analyzed on ImageJ 1.48 Software where a Z-projection was created allowing all Z-stacks to stack to a single image showing maximum intensity from all stacks, or the stack showing the optimum intensity for a specific bouton was selected for measurement. An octagonal overlay with 8 visible connected spokes is placed over the bouton to be measured. Using the ImageJ line tool, lines are drawn on the octagonal spokes only where fluorescence is present. A total of 8 lines with varying lengths are recorded for each bouton and then were averaged to obtain the average width (μm) of Dlg fluorescence for that bouton Fig. a. Mean Dt test, ANOVA group comparisons, and column statistics were used on the data presented. A significance level of p < 0.05 was used for all experiments.All statistical analysis was performed on GraphPad Prism 5 software. Student