No correlations with clinical parameters were observed for pAR94 or pAR650 in HNPC or CRPC tumours. In contrast to our previous observation with serine 213, high pAR308 is significantly associated with a longer time to disease specific death (p = 0.011) and high pAR791 expression significantly associated with a longer time to disease recurrence (p = 0.018) in HNPC tumours and longer time to death from disease recurrence (p = 0.040) in CRPC tumours. This observation in CRPC tumours was attenuated in high apoptotic tumours (p = 0.022) and low proliferating tumours (p = 0.004). These results demonstrate that understanding the differing roles of AR phosphorylation is necessary before this can be exploited as a target for castrate resistant prostate cancer.We previously reported that AR phosphorylation at serine 213 was associated with poor outcome and may contribute to prostate cancer development and progression. This study investigates if specific AR phosphorylation sites have differing roles in the progression of hormone naïve prostate cancer (HNPC) to castrate resistant disease (CRPC). A panel of phosphospecific antibodies were employed to study AR phosphorylation in 84 matched HNPC and CRPC tumours. Immunohistochemistry measured Androgen receptor expression phosphorylated at serine residues 94 (pAR Prostate cancer has the highest cancer incidence in males and is the second highest cause of male cancer related mortality . As a whOne of the most challenging problems in the management of prostate cancer is the treatment of patients who no longer respond to hormone therapy and have failed docetaxel chemotherapy. CRPC has evolved from the clinical observation that surgical or medical castration, by blocking the production of testicular androgens, is not curative and virtually all patients have prostate cancer disease recurrence.Current practice aims to inhibit androgen production and action, thereby reducing stimulation of the androgen receptor (AR). Inhibition of testicular androgen production is achieved surgically  or chemically, using gonadotropin-releasing hormone (GnRH) agonists. The action of androgen may be blocked at a peripheral level using anti androgens, which inhibit ligand binding to AR and subsequent activation. Current clinical data implies that the AR is expressed and continues to mediate androgen signalling after failure of this therapeutic approach. However as this does not completely eliminate circulating androgens, sufficient concentrations of dihydrotestosterone may accumulate in tumour cells to maintain AR signalling, especially in the context of up-regulated receptor levels or increased sensitivity of the AR for activation. In addition, ligands of non-testicular origin  or ligand-independent activation can contribute to continued AR signalling. To overcome this and to improve patient treatment options, the mechanisms underlying the development of castrate resistance must be fully understood.The AR not only functions as a transcription factor but also as a core that incorporates multiple extracellular signals. AR effects are influenced by their levels of expression and posttranslational modifications, including phosphorylation on serine –4 and tyClinical data recorded for each patient included age , prostate specific antigen (PSA) at diagnosis , PSA at disease recurrence  and Gleason grade at diagnosis (7 range 6–9) and at disease recurrence (9 range 8–9). All patients underwent disease recurrence . The median time to death from disease recurrence was 2.1 (IQR 1.02–3.51) years. The median survival time was 5.82 (IQR 3.44–7.35) years. At last follow-up, 9 patients were alive, 53 patients had died of prostate cancer and 21 patients had died of causes not attributed to prostate cancer. Patients in this cohort were diagnosed with locally advanced (51) or metastatic prostate cancer (30) and subsequently received chemical or surgical castration . 57 of the 84 patients also received anti androgen therapy, this included all those who received GnRH analogues. Following disease recurrence, 31 patients received radiotherapy; no patients received taxane therapy. Clinical parameters associated with time to disease recurrence, time to death from disease recurrence and disease specific survival are shown in 308 and pAR650 ) exhibited and increase in expression in the transition from hormone naïve to castrate resistant disease  and compared using the log rank test. Expression of pAR94, pAR308 and pAR650 was not associated with clinical parameters in our cohort. However patients with tumours that had high expression of pAR791 in the cytoplasm had a significantly longer time to disease recurrence  than those patients whose tumours expressed low levels of pAR791p = 0.018 (p = 0.02). High pAR 791 expression was demonstrated to be an independent prognostic marker by Cox regression analysis when compared with the significant clinical parameters PSA at diagnosis, Gleason grade at diagnosis, metastases at diagnosis and radiotherapy (p = 0.019).To determine if protein expression was linked to time to biochemical relapse, Kaplan-Meier graphs were plotted for the hormone naïve tumours expressing low levels of protein (<median)  = 0.018 . The medversus high levels of phosphorylated AR (>median) and compared using the log rank test. Expression of pAR94 and pAR650 was not associated with clinical parameters in our cohort. However patients whose tumours expressed high levels of nuclear pAR308 were observed to have a significantly longer disease specific survival time compared to those patients whose tumours expressed low levels of nuclear pAR308  791. Patients whose tumours expressed high levels of nuclear pAR791 were observed to have a significantly longer time to death from disease recurrence compared to those patients whose tumours expressed low levels of nuclear pAR791p = 0.04 (p = 0.04). High pAR791 expression was demonstrated to be an independent prognostic marker by Cox regression analysis when compared with significant clinical parameters (p = 0.017).This observation of good prognosis was also observed for pARp = 0.04 . These p791 is significantly associated with a longer time to disease recurrence and time to death from disease recurrence, it was investigated if this expression was linked with tumour apoptotic or proliferation index. Kaplan-Meier graphs were then plotted for the castrate resistant tumours expressing low levels of protein (<median) versus high levels of protein (>median), stratified by apoptotic or proliferation index and compared using the log rank test.As high expression of pAR791 with a longer time to death from disease recurrence was negated in tumours with low apoptotic index (p = 0.784) and attenuated in tumours with a high apoptotic index (p = 0.022). The median time to death from disease recurrence for those with low pAR791 expression/high apoptosis was 1.5 years (1–2.2) years compared to 3.6 years (2.4–4.9 years) for those with high pAR791expression/high apoptosis  and negated in tumours with a high proliferation (p = 0.489). The median time to death from disease recurrence for those with low pAR791 expression/low proliferation index was 1.1 years (0.5–1.6) years compared to 3.4 years (2.4–4.3 years) for those with high pAR791 expression/low proliferation   histoscore units compared to 39  histoscore units, for tumours that do not have AR amplification.Data for AR gene amplification from the same cohort of patients was available for analysis. This allowed us to determine if AR amplification was associated with the phosphorylation levels of AR expression in this study. By dividing protein expression into tumours with or without AR amplification, tumours with AR amplification had significantly lower nuclear pAR= 0.034) . Median in vitro evidence into the clinical scenario, this study established that phosphorylation at specific serine residues on the AR are involved in the development of castrate resistant disease.One of the many challenges in the effective management of prostate cancer is the identification of molecular markers capable of predicting disease progression. Castrate resistant disease represents the lethal phenotype of prostate cancer. The main aim of this study was to investigate the effect of androgen receptor phosphorylation in the development and progression of prostate cancer and to determine if it influenced patient outcome. Translating the 308 expression were observed to have a survival advantage over those patients with low expression. This observation is consistent with the previous observation that pAR308 is associated with repression of AR activity and combined suggests that phosphorylation at this site may play a role in cell cycle arrest. It was surprising that levels of pAR308 expression increased with the development of CRPC disease when levels were associated with better prognosis, we hypothesis that as the disease progresses AR phosphorylation levels increase in response to signaling cascades being up-regulated, however due to the promoter selectivity associated with different phosphorylation sites this does not always result in advantage to the tumour.Utilizing this cohort, we have previously reported that phosphorylation of AR at serine 213 is associated with poor prognosis in CRPC ,15 and iin vitro by protein kinase A, protein kinase C and by members of the MAPK family, including the stress-induced kinases p38α and JNK [et al. have demonstrated that serine 650 phosphorylation regulates nuclear export, with the non-phosphorylatable S650A mutant receptor retained in the nucleus and a concomitant increase in a target gene expression [650 expression was not associated with clinical outcome measures.Serine 650 is phosphorylated  and JNK . Althoug and JNK . Yet oth and JNK . Gioeli pression . In addipression . However791 in hormone naïve tumours was observed to be significantly associated with a longer time to disease recurrence. High nuclear expression of pAR791 was also significantly associated with a longer time to death from disease recurrence in the castrate resistant tumours. Taken together these observations suggest high levels of AR phosphorylation at serine 791 have a beneficiary effect as patients are living longer. Due to this observation we further investigated whether high expression of pAR791 coincided with high apoptotic tumours or low proliferating tumours. Interestingly high pAR791 expression was significantly associated with disease specific survival from disease recurrence in patients with highly apoptotic tumours but this relationship was negated in tumours with low levels of apoptosis. Moreover, high pAR791 expression was significantly associated with disease specific survival from disease recurrence in patients with low proliferating tumours. Furthermore using the AR gene amplification data from this cohort it was possible to determine whether AR amplification resulted in increased protein expression and it was observed that pAR791 expression was significantly lower in castrate resistant patients with AR gene amplification. This result is in agreement with previous findings by Lin et al. who have demonstrated that AR is a substrate for Akt which phosphorylates the AR at serine 213 and serine 791. Here the authors observed that only AR phosphorylation at serine 213 could inhibit AR transactivation thus inhibiting AR targets genes such as p21, which modulates androgen/AR-mediated apoptosis. This would provide a rationale along the observations from this study that following phosphorylation by Akt at serine 791, the AR induces cellular apoptosis which in turn leads to an overall survival advantage in a subset of prostate cancer patients. Moreover this study highlights a differential role of Akt phosphorylation on the AR. We have previously observed that pAR213 expression is associated with the development of CRPC and observed a strong correlation between pAkt473 and pAR213 [791  but no correlation was made in the castrate resistant tumours. In addition when patients were split into AR amplification status it was observed that pAR213 expression was significantly higher in AR amplified tumours compared to non amplified tumours (p = 0.025) data not shown, whereas pAR791 expression is significantly lower in AR amplified tumours compared to non amplified tumours (p = 0.034). This data suggests that in the clinical setting another kinase may be responsible for phosphorylating the AR at serine 791.Interestingly, phosphorylation of the AR at serine 791 was observed to be significantly associated with disease progression. High cytoplasmic protein expression (above the median) of pARd pAR213 . HoweverThis biological marker study was performed in accordance to the reporting of tumour marker studies criteria (REMARK).A total of 84 prostate cancer patients were included in this study, with hormone naïve and castrate resistant tumours from each patient available for analysis . All tumours had patient identification removed, and the clinical information database was anonymised. Ethical approval was obtained from the Multicentre Research Ethics Committee for Scotland (MREC/01/0/36) and Local Research and Ethical Committees. Patients were only selected for analysis if they initially responded to hormone treatment  but subsequently relapsed (2 consecutive rises in PSA greater than 10%) and had both a hormone naïve and castrate resistant tissue sample available for analysis. Hormone naive tissue was obtained from 18 patients by TRUS guided biopsy and the remaining by TURP and castrate resistant tumours were all obtained by TURP, which was carried out to relieve bladder outflow obstruction.2) as identified by a uropathologist were removed from representative areas of high Gleason grade and areas of low Gleason grade of formalin fixed paraffin embedded TURP blocks. These cores were then embedded into recipient array blocks that were constructed in triplicate. Therefore for each patient sample there is an area of high Gleason and low Gleason grade taken in triplicate that accounts for tumour heterogeneity within the sample. Control cores of normal prostate, colon, breast, pancreas, tonsil, kidney, liver and lung tissue were included in each TMA.Cores of prostate cancer tissue  rabbits, 3 boosts, 3 bleeds, ELISA titration of all sera and ELISA titration of purified serum. This antibody was made by immunising host animals with conjugated phosphorylated peptide, following the final bleed of the animal the phospho-specific antibodies were selected by affinity purification. Two affinity columns were used; the first column coupled with the non-phosphorylated peptide and the second column with the phosphorylated peptide. The serum then passed through the first column and the non-retaining elute kept and used for the second purification. The eluate was purified on the second column in order to remove antibodies which might recognise the un-phosphorylated peptide. ELISA tests were performed to ensure that the recovered antibody is phospho-specific. Antibody specificity for pAR94, pAR308 and pAR650. was previously confirmed using a peptide competing assay  for 5 min. Endogenous peroxidase activity was blocked using 3% hydrogen peroxide and non-specific background staining was blocked using 5% horse serum in TBS for 20 min. pAR94 , pAR308 , pAR650 , and pAR791 , antibodies were used at the following concentrations  diluted in Dako antibody diluent (Dako A/S). All antibodies were incubated overnight at 4 °C. Bound antibody complex was visualized using EnVision plus kit (Dako A/S) followed by 3,3-diaminobenzidine tetrahydrochloride (Vector Laboratories). Nuclei were counterstained with haematoxylin before mounting with DPX. A positive (tissue know to express the antigen) and a negative control  were included in each immunohistochemistry run to exclude the possibility of false-negative and false-positive staining. Examples of staining expression patterns for all markers in both HNPC and CRPC tumours are shown in All immunohistochemistry was performed on TMAs with the exception of the TRUS biopsies which were 5 μm, archival formalin fixed, paraffin embedded prostate tumour sections on separate slides. Immunohistochemistry for pARin situ DAB) for light microscopy.Ki67 immunohistochemistry was performed by established protocols in the Department of Pathology, Glasgow Royal Infirmary with appropriate positive and negative controls. Dako anti-Ki-67  was used at dilution 1:100 for 30 min for immunohistochemistry on a Bond Max automated slide stainer  according to the manufacturer’s instructions, with Leica Envision detection system. Slides were lightly counterstained with haematoxylin, dehydrated and mounted with DPX. Apoptotic scoring was performed as per manufacturers instructions using the R & D systems Tunel labeling kit  were calculated using Cox Regression analysis. Multivariate analysis combined any significant proteins of interest with significant clinical parameters to establish if it was independent of these known prognostic markers in influencing patient outcome.The findings presented in the current study expand upon our previous work describing the association of phosphorylated serine 213 in prostate cancer tumours and demonstrate that phosphorylation of AR can positively and negatively regulate AR actions. Taken together, this data indicates that phosphorylation of the AR has the potential to regulate AR function in the development and progression of CRPC and it is conceivable that the functional consequences of phosphorylation at individual serine residues maybe gene specific. Further work is necessary to distinguish these possibilities.


The increase and spread of antibiotic resistance (AR) over the past decade in human pathogens has become a worldwide health concern. Recent genomic and metagenomic studies in humans, animals, in food and in the environment have led to the discovery of a huge reservoir of AR genes called the resistome that could be mobilized and transferred from these sources to human pathogens. AR is a natural phenomenon developed by bacteria to protect antibiotic-producing bacteria from their own products and also to increase their survival in highly competitive microbial environments. Although antibiotics are used extensively in humans and animals, there is also considerable usage of antibiotics in agriculture, especially in animal feeds and aquaculture. The aim of this review is to give an overview of the sources of AR and the use of antibiotics in these reservoirs as selectors for emergence of AR bacteria in humans via the food chain. The recent increase and spread of antibiotic resistance (AR) in human pathogens over the past decade has become a global problem, and is mainly due to the overuse and/or misuse of antibiotics in humans. However, there are many other sources of selective pressure such as use of antibiotics in agriculture that are known to be drivers of AR. Moreover, recent genomic and metagenomic studies in humans, animals, and the environment have led to the discovery of a large reservoir of new and/or unknown AR genes in these reservoirs that represent the so-called “ intrinsic resistome” that represent a large subset of non-acquired AR genes whose functions are multiple and complex in nature . AntibioAntibiotics are used in food animals and aquaculture as therapeutic agents, prophylactics, and growth promoters . For morEscherichia coli colonization in the chickens, as well as the acquisition of such resistance in E. coli in the gut of the farm family (Enterococcus faecium (vanA genes in pigs was likely due to the use of tylosin (macrolide antibiotic) because the ermB and vanA genes were found to be encoded on the same mobile genetic element  or for therapy have been known to select for AR bacteria that could be transferred to human pathogens since 1975 with the first demonstration of the use of oxytetracycline as a growth promoter in chickens, which demonstrated an increased selection of tetracycline-resistant m family ,b. This m family . Bacterim family . Avoparc faecium . This de element . A shortod chain . The rolod chain . Moreove Denmark .E. coli from the chicken gut has revealed that even short-term exposure can lead to resistant strains (O-phosphotransferases), which demonstrated the potential collateral effects of indirect selection of resistance to other classes of antibiotics  and the development of AR in  strains . AR geneibiotics .E. coli (ExPEC) that is responsible for community-acquired urinary tract infections  have also been detected in 12% of 309 commensal intestinal strains of E. coli from 128 Swedish infants who did not receive tetracyclines during their first year of life , sulfamethoxazole , tetracycline (tetA or tetB), and ampicillin (blaTEM; NDM-1 human carriage cases have been reported in several countries in India, China, and Pakistan as well as in the environment (Thanks to the use of new technologies, such as high-throughput pyrosequencing, metagenomic studies and exhaustive culture techniques, a description of human microbiota has begun to revolutionize our current understanding of the relationship between gut microbiota composition, several diseases and obesity . It is nears ago , recent m adults . Howeverm adults . One of m adults . Anotherm adults . Antibiom adults . Thus ARm adults , and them adults . Tetracy of life . A recen samples . Infant  species . Zhang e blaTEM; . Similarironment .E. coli was transferable to a susceptible strain in the gut of a children treated with ampicillin for UTIs. It has been shown that the administration of antibiotics could alter the bacterial community structure of the gastrointestinal microbiota in mice and that these modifications are different according to the antibiotic classes administered (ermB, can persist for long periods (4 years; Bacteroides spp., which could persist for as long as 2 years following clindamycin exposure. Antibiotic use could also lead to a collateral effect through the selection of AR bacteria among the normal microbiota, as has been demonstrated with the long-term persistence of clarithromycin-resistant Enterococcus species harboring the ermB gene immediately after treatment to eradicate Helicobacter pylori, which may persist for 1–3 years after antibiotic therapy (ermB gene, which may persist in some cases for up to 4 years post-treatment, demonstrating that the AR gene may be stabilized in the human gut microbiota following selection. Thus, the increased prevalence of AR and possibility LGT between bacteria are important consequences of the long-term persistence of AR genes in gut microbiota (Although antibiotic treatment in humans directly affects the pathogen responsible for the infectious disease, antibiotic use has been shown to lead to a collateral effect in commensal bacteria in the gut microbiota. In fact when AR genes are selected in the commensal gut microbiota, they could be further transferred to human pathogen. nistered . Moreovenistered  in humannistered . It has nistered  and in enistered ,2005. Denistered . Interes4 years; . Broad-r4 years; ,b. Löfma therapy . Similarcrobiota .While the overuse and misuse of antibiotics in humans is undoubtedly associated with the emergence and spread of AR bacteria and AR genes, at least in human infections, recent studies have demonstrated that the use of antibiotics in animals, aquaculture and most importantly, food production, is also a major factor contributing to the emergence of AR bacteria and the spread of AR genes. Moreover, because the potential pool of AR genes in these environments remains largely unknown, with thousands of AR genes yet to be discovered, future observation of AR in these ecosystems is warranted from an ecological perspective. This phenomenon is compounded by the immense number of methods that bacteria have developed for the exchange of AR genes via LGT, recombination, and transposition and also for the purpose of creating new AR genes, as recently exemplified with the discovery of the new chimeric carbapenemase encoding gene NDM-1 . FurtherThe author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Advancements in surgical techniques and anaesthetic agents in recent decades have allowed for the rapid expansion of day surgical services internationally. The aim of this study is to evaluate the patient's satisfaction (PS) with the day surgical services in an Irish teaching hospital.The study design was a survey, utilizing a phone questionnaire. 70 consecutively presenting patients undergoing 15 day surgery procedures in an Irish teaching hospital were invited to participate. Data collection was completed between June and July 2011. The survey evaluated 57 aspects of the DS process. Patients were asked to rate their level of satisfaction using a 5-point LIKERT scale ranging from 1-very dissatisfied to 5-very satisfied.70 patients consented to study participation, of which 85.57% (n=60) responded when phoned. Of these; 40% (n=24) male, 60% (n=36) female, mean age 47 years (range 17-84 years), 50% (n=30) had local anaesthetic procedures (LA) and 50% (n=30) general anaesthetic procedures (GA). Pre-operatively, patients were least satisfied with the waiting time: 35.59% (n=21) very satisfied; while 66.66% (n=40) were very satisfied with the nursing staff. 81.66% (n=49) of the patients were very satisfied with the medical care provided. Post-operatively, 94.44% (n=51) were very satisfied with the follow-up arrangements. However, only 54.55% (n=24) were very satisfied with the time taken to obtain results for x-rays and other tests. Regarding the supporting structures to the day surgery unit (DSU), patients were most satisfied with the cleanliness of the unit, where 86.66% (n=52) were very satisfied, while only 39.13% (n=18) were very satisfied with parking arrangements. Additionally, 84.21% (n=57) were very satisfied with the information leaflet regarding the procedure.Overall, patients were very satisfied with the care provided and service provision in the DSU. As day surgery services are expanded into the future, this study provides valuable insights into the current areas for improvement and development. Although the methodology used was very labour-intensive, the response rate was very high. Higher recruitment through e-mail or text messaging could have proven to be more time saving and cost efficient. However this might be at the expense of lower response rates.


We believe that Dr. Albert King’s letter regarding the STAR system paperWhile studying our comments, we ask the reader to keep in mind that all of this debate comes down to a very simple question: do you want to buy a helmet that reduces head acceleration? The STAR system is a complex experimental methodology that combines exposure and injury risk based on current scientific knowledge. Simply stated, it provides independent data to consumers to illustrate which helmets lower head acceleration.In order to understand the severity of Dr. King’s apparent conflict of interest, it is important to understand the entire timeline of our STAR paper relative to his letter to the editor. In May 2011, we published the STAR paper, after a 3-month open response period, and after full peer-review in the Annals of Biomedical Engineering (ABME). During this process, and for the entire next year, Dr. King did not submit any letters to ABME, nor did he submit any questions or comments to us regarding this paper.Once he submitted his letter, we contacted Dr. King, but he refused to discuss his letter or any potential conflicts. In order to gain clarity, Virginia Tech was forced to file a Freedom of Information Act request with Wayne State University so that we could document the following timeline, contracts, and statements.In May 2012, at the request of lawyers working for Xenith (a helmet manufacturer), a consulting company emailed experts around the country looking to retain anyone who would critique the STAR paper. This email stated that the Xenith helmet “did not fare well in the ratings.” They sent this email to Dr. King on May 9, 2012, and he responded to them within 2 h saying “it was a pleasure talking with you,” and he forwarded his CV. On May 17, 2012, Dr. King signed a contract with them entitled “Xenith LLC Consulting” which summarized the agreement that he would be paid $400/h and all reasonable expenses for his critique of the STAR paper.Over the next several weeks, Dr. King filled out time sheets recording his consulting hours as he prepared his report for this company. On June 17, 2012, Dr. King produced a draft report on “Albert I. King Inc” letterhead entitled “Review of the STAR Report by Rowson and Duma.” A few days later on June 21, 2012, Dr. King submitted a letter to the Editor of ABME that was virtually identical to this draft report. Dr. King provided no disclosure of any conflicts of interest in this letter. The next day and while the letter was under review, Xenith began nationally distributing a report from Dr. King, also dated June 21, 2012, that was virtually identical to his letter to the Editor of ABME. This report also does not include any disclosure of any conflicts of interest.etc. from lawyers as well as companies.” Dr. King responded “I received no personal compensation from anyone for writing this letter to the editor.” For a second time, Dr. King did not disclose any relationship or potential conflict of interest, even when directly asked by the Editor.On June 25, 2012, based on concerns raised about potential conflict of interest issues, Dr. King was specifically asked by the Editor of ABME to “provide any disclosures related to this work, in terms of retainers On June 29, 2012, Dr. King was informed that ABME had a copy of the May 2012 email request from a company representing Xenith’s lawyers, as well as a copy of his June 21, 2012 report that Xenith was distributing and which was effectively identical to his letter. Because of potential copyright issues, Dr. King was asked to write a different letter. He was also asked to complete a much more detailed conflict of interest form. This form specifically stated that Dr. King must “report all sources of revenue paid (or promised to be paid)…from sources with relevance to the submitted work.” On July 19, 2012, Dr. King submitted his revised letter with a new story about his conflict of interest. In this disclosure Dr. King states that “I was originally asked by Xenith Corp to write a written review of the paper in question with remuneration.”As the consulting story unfolded, we contacted Dr. King directly and informed him that we were aware of this serious conflict, and we asked for his explanation. Dr. King refused to respond to our questions; however, immediately following our inquiry, there were a series of emails between Dr. King, the consulting company, and Xenith. At one point Dr. King wrote Vin Ferrara, the CEO of Xenith, and stated that “I think we should cool it for a while.” These emails showed how Dr. King’s report changed from an “Albert I. King Inc.” report to a Wayne State University report when Dr. King stated that “it is now a public document and is likely to receive a lot of media attention.” Only then did Dr. King state that he did not want to be paid, but he added that “Mr. Ferrara can make a donation to Wayne State University.”What is clear is that Dr. King agreed to write his report for payment from Xenith (or their representatives), and that Dr. King deceived the scientific process by intentionally withholding information that he had in fact been retained by Xenith (or their representatives) to write his report.We have no financial interest whatsoever with the HIT System or Riddell. We have never received any funding or royalties or promise of payment in any form from any helmet manufacturer. We submit that we are one of the only truly independent helmet research laboratories in the world. Our funding is unbiased and comes from the NIH, DOT, DOD, Toyota Central Research and Development Laboratories, and Virginia Tech, all of which have no interest in any helmet company.Dr. King’s assertions that the HIT System is prone to large errors are not supported by any scientific peer reviewed data. Most importantly, the data collected in the referenced master’s thesis do not support his conclusions. In contrast, there is significant literature documenting the validation of the HIT System, which includes both laboratory and on-field testing.We summarize here the large body of research and development surrounding the HIT System and its validation, which has been employed on the field for the past 9 years at colleges and high schools across the country.r2 = 0.90) between the HIT system and the Hybrid III  and helmet locations. These locations and velocities were selected following detailed video review of on-field impacts to simulate the range of head contact sustained by NFL football players. The data were peer reviewed and published in ABME.1.2 m/s aRegarding Dr. King’s comments on helmet fit methods and selection of helmet size for validation, in our research, and to our knowledge in all associated published research, the manufacturer guidelines for helmet fit were followed. A medium-sized Riddell Revolution football helmet was determined to best fit the Hybrid III head due to the unique geometry of the Hybrid III head. The 22.5″ measurement for head circumference is close to the borderline between medium and large sized helmets. Head circumference is merely a starting point for proper helmet fit, with size and shape of the head ultimately affecting best fit. While the Hybrid III circumference is slightly over the medium range, it’s other features  dictate that a medium helmet should be used to achieve the manufacturer’s recommended fitting guidelines: The helmet should be (1) be approximately 1″ above the player’s eyebrows and (2) the skin of the forehead should move with the front pad. There should be no room for twisting of the helmet relative of the head.Dr. King references an unpublished thesis (the Wayne State study) that claims large absolute errors when a large Riddell Revolution IQ helmet equipped with HIT System is tested. Without explanation, a proper fitting helmet on the Hybrid III head was not evaluated, but rather a loose fit and a tight fit with a large helmet. The rationale behind these loose and tight fit conditions are not well supported by the methods and data provided in the thesis, but more importantly, those data are not publicly available and have not been peer reviewed. Dr. King offers no valid data to support his contention that the “the contact pressures between the helmet and the head were way above the comfort level of human volunteer subjects who tested both the medium and large sized helmets.”The unpublished Wayne State study performs impact tests at only one impact speed (9.7 m/s). This impact speed does not represent over 95% of the impacts players see in a game,In one published study, the HIT System was used to measure head acceleration resulting from impact in 32 clinically diagnosed concussions on the field, with an average linear acceleration of 105 ± 27 g.Additionally, Virginia Tech has utilized a more advanced helmet impact system in parallel helmet studies for the past 5 years. This custom helmet system utilizes 12 accelerometers per helmet (compared to 6 in the HIT system). This system was also validated over a wide range of impact velocities and helmet locations.Dr. King’s assertion that the measurement of resultant linear head acceleration in the laboratory in the STAR system used to rate football helmet is “potentially harmful to players who may not be diagnosed with a concussion…” is not supported by any scientific data. His argument is not logical. Reducing head acceleration is an important objective in reducing injury risk and obviously will not have a negative effect on injury incidence. In fact, understanding and managing the energy transferred to the human body during impact loading is the basis of the injury biomechanics field.Dr. King claims that it is inadequate to use a test based entirely on linear acceleration rather than one including rotational acceleration. He fails to understand that for the vast majority of impacts, if linear acceleration is reduced, rotational acceleration will also be reduced.Linear acceleration has been shown to be very well correlated with concussion from head impacts in football.Importantly, it must be emphasized that concussions are diagnosed only by trained medical personnel based on the onset of symptoms. They are not diagnosed by head acceleration measurements as Dr. King contends. The HIT System and the STAR system are not designed to be diagnostic tools. Instead, they provide valuable additional information related to head impacts in football. The HIT System provides biomechanical data on head impacts experienced by players wearing instrumented helmets during play. The STAR system allows a biomechanical evaluation of the relative performance of adult football helmets in their ability to reduce the probability of concussion.et al.2 = 4.52, p = 0.03]. Our analysis includes 308 instrumented player-years with 153,486 head impacts and eliminates the previous criticisms of Collins et al.To further illustrate the validity of the STAR system, we compare STAR values against available clinical data. The STAR system predicts the Riddell Revolution (4 star helmet) will result in a 54% reduction in risk of concussion compared to the Riddell VSR4 (1 star helmet). There are two clinical studies to compare this prediction. First, Collins Another study by the NFL examined the impact response of modern helmets using a linear impactor.et al.et al.Dr. King fails to consider that an injury risk curve is not only dependent on the injury data, but also the full exposure data associated with no injury. Due to the challenging and time consuming testing methods used by Pellman r2 = 0.99) and used these quadratic regressions to interpolate the values.To help Dr. King understand the findings of the STAR paper, we have provided plots below of the data from the 132 drop tests performed on 3 Riddell VSR4 helmets, with quadratic regression lines for each impact location  helmets that better manage the impact energy by resulting in lower head accelerations will reduce the risk of concussion (2) each test condition is weighted so that they represent how often each impact scenario is experienced on the field by players.In summary, we return back to the question that we started with: do you want to buy a helmet that lowers head acceleration? The STAR system uses current scientific knowledge to illustrate which helmets are better at reducing head acceleration. We will continue to purchase the best helmets for the Virginia Tech football team based on the STAR system.


The androgen receptor (AR) is a ligand-inducible transcription factor that mediates androgen action in target tissues. Upon ligand binding, the AR binds to thousands of genomic loci and activates a cell-type specific gene program. Prostate cancer growth and progression depend on androgen-induced AR signaling. Treatment of advanced prostate cancer through medical or surgical castration leads to initial response and durable remission, but resistance inevitably develops. In castration-resistant prostate cancer (CRPC), AR activity remains critical for tumor growth despite androgen deprivation. Although previous studies have focused on ligand-dependent AR signaling, in this study we explore AR function under the androgen-deprived conditions characteristic of CRPC. Our data demonstrate that AR persistently occupies a distinct set of genomic loci after androgen deprivation in CRPC. These androgen-independent AR occupied regions have constitutively open chromatin structures that lack the canonical androgen response element and are independent of FoxA1, a transcription factor involved in ligand-dependent AR targeting. Many AR binding events occur at proximal promoters, which can act as enhancers to augment transcriptional activities of other promoters through DNA looping. We further show that androgen-independent AR binding directs a gene expression program in CRPC, which is necessary for the growth of CRPC after androgen withdrawal. One of the major challenges in the management of prostate cancer is the treatment of patients who no longer respond to androgen deprivation therapy. Available treatments for androgen deprivation therapy resistant patients have had modest success, with improvements in survival measured in months . How proWhile mounting evidence shows that AR signaling is critical in both androgen-dependent prostate cancer (ADPC) and castration-resistant prostate cancer (CRPC), important differences in AR-mediated transcription have been observed. Gene expression profiling has shown that the androgen-dependent AR expression program characteristic of ADPC is significantly attenuated in CRPC ,13. To uin vitro and in vivo  and RNA-sequencing (RNA-seq) to characterize AR binding events in both the presence and absence of androgen in the well-established LNCaP/C4-2B cell culture model. This model shares strong similarities with the clinical progression from androgen-dependence to castration-resistance ,37. We oSupplementary File S1.LNCaP and C4-2B cells were maintained in RPMI 1640 media with 5% fetal bovine serum (FBS) as previously described . AntibodSupplementary File S1.LNCaP or C4-2B cells were cultured in phenol red-free RPMI 1640 media supplemented with 5% charcoal-stripped FBS (CSS) for 3 days. After treatment with ethanol or DHT (10 nM) for additional 4 h (for LNCaP) or 16 h (for C4-2B), ChIP experiments were performed as described previously ,39. For www.illumina.com) with modifications. Briefly, 10 ng of ChIP DNA was end-repaired, ligated to barcoded adaptors, size-selected on agarose gel (300–500 bp) and PCR amplified for 16 cycles using Phusion polymerase (Finnzymes). The libraries were sequenced in the Illumina Genome Analyzer IIx or HiSeq2000 system according to the manufacturer’s instruction. A summary of ChIP-seq experiments is provided in Supplementary File S1.The ChIP-seq libraries were prepared according to the Illumina Protocol  observed in at least one experiment (ChIP-seq reads were mapped to the human genome (hg18) using Bowtie . Reads tperiment . Bindingperiment ] were diperiment  followinperiment . Tag-wisperiment . In ordeDe novo motif discovery using MEME was performed within ±125 bp relative to the ChIP-seq peak center using default MEME–ChIP settings  Transfection protocol (Invitrogen). After transfection, cells were grown in phenol red-free RPMI 1640 containing 5% CSS for 48 h and then treated with ethanol or DHT (10 nM) for additional 16 h. Total RNA extraction and protein extraction were conducted for further assessment by RNA-seq, qRT–PCR and western blot.Supplementary File S1.RNA-seq was performed as reported previously with modifications ,52. BrieRNA-seq reads were mapped to the human genome (hg18) using Tophat . AlignedGAPDH) mRNA expression was analyzed for each sample in parallel. The primers are listed in Supplementary File S1.After the indicated treatments, total RNA from cells was extracted using TRIzol Reagent (Invitrogen). cDNA was prepared through reverse transcription (RT) using the iScript cDNA Synthesis Kit (Bio-Rad), and qPCR was conducted using SYBR Green PCR Master Mix (Applied Biosystems). Triplicate PCR reactions were conducted. glyceraldehyde 3-phosphate dehydrogenase . Five out of 10 androgen-independent AR-occupied regions are located at the promoter regions, which were cloned in reverse direction to minimize the promoter activity in luciferase assays. Also, 10 random genomic regions were subcloned into pGL4.26 vector and used as controls. The plasmid sequences were confirmed by Sanger sequencing. The primers for cloning are listed in 4 cells/well) were plated in 48-well plates and grown in phenol red-free RPMI 1640 containing 5% CSS for 2 days. Cells were then transfected with luciferase reporter plasmids (100 ng/well) using Lipofectamine LTX Reagent (Invitrogen). pRL-TK renilla luciferase plasmid (10 ng/well) (Promega) was co-transfected as an internal control. For the luciferase assay after AR knockdown, cells were transfected with AR siRNA  using Lipofectamine RNAiMAX Transfection Reagent and Reverse Transfection Protocol (Invitrogen), and then grown in phenol red-free RPMI 1640 containing 5% CSS for 2 days prior to reporter plasmid transfection. After plasmid transfection, cells were treated with ethanol or DHT (10 nM) for 24 h. Firefly and renilla luciferase activities were measured using the Dual-Glo Luciferase Assay System (Promega). Results are represented as firefly/renilla ratio.LNCaP or C4-2B cells (4 × 104 cells/well) were plated in 96-well plates and transfected with gene-specific siRNA at a final concentration of 15 nM using Lipofectamine RNAiMAX Transfection Reagent and Reverse Transfection Protocol. For proliferation assay, cells were maintained in phenol red-free RPMI 1640 containing 5% CSS with ethanol or different concentrations of R1881 as indicated for 5 days. The synthetic androgen R1881 was used instead of DHT to minimize metabolic degradation during incubation. The number of viable cells was analyzed using the CCK-8 kit (Dojindo Molecular Technologies). For apoptosis assay, cells were grown in phenol red-free RPMI 1640 containing 5% CSS with ethanol or DHT (10 nM) for 3 days after siRNA transfection. The Caspase 3/7 activity was measured using the Caspase-Glo 3/7 Assay kit (Promega).C4-2B cells (1 × 107 cells/150 mm dish) were grown in phenol red-free RPMI 1640 containing 5% CSS for 3 days. Cells were fixed with 1% formaldehyde for 10 min at room temperature, and then lysed in cold lysis buffer . The nuclei were harvested and suspended in digestion buffer containing 0.3% SDS and 2% Triton X-100. The chromatin was digested with BamHI or EcoRI (New England Biolabs) overnight at 37°C while shaking at 900 rpm. The reaction was then diluted with ligation buffer containing 0.1% SDS and 1% Triton X-100 in a final volume of 7 ml. Ligation was incubated at 16°C overnight with 2000 U T4 DNA ligase (New England Biolabs), followed by overnight incubation at 65°C in the presence of 10 μg/ml proteinase K (Sigma) to reverse cross-linking. The DNA was isolated by phenol–chloroform extraction and ethanol precipitation. The purified DNA was quantified and used as a PCR template.Chromatin conformation capture (3C) assays were performed as previously reported with modifications . BrieflySupplementary File S1.To create a standard for normalization of different PCR efficiencies, 3C control template was generated by digesting an equimolar mix of the PCR fragments spanning all restriction sites of interest followed by ligation to produce all possible ligation products . To contSupplementary Figure S1A). C4-2B is a CRPC cell line derived from a LNCaP xenograft that relapsed and metastasized to bone after castration. C4-2B cells show similar growth rates in the presence or absence of androgen. In the presence of androgen, C4-2B cell growth is inhibited by the AR antagonist bicalutamide, indicating androgen-dependent AR signaling remains functional . In the absence of androgen, however, growth of the C4-2B cells is minimally affected by bicalutamide but strongly inhibited by siRNA against AR . These results suggest that C4-2B cells in androgen-deprived conditions exhibit androgen-independent but AR-dependent growth. To understand how AR promotes C4-2B cell growth under androgen-deprived conditions, we asked whether AR genomic binding events in the absence of androgen are present and comparable with classic androgen-dependent binding events. We mapped AR binding sites in LNCaP and C4-2B cells in the presence and absence of DHT (10 nM) using ChIP-seq. We identified a total of 15 709 AR binding events in at least one sample at a P-value threshold of 0.01 (Supplementary File S2). Selected AD and AI-ORs were validated by ChIP–qPCR and showed good agreement with ChIP-seq data . We hypothesized that AI-ORs are responsible for the castration-resistant, AR-dependent phenotype in C4-2B cells.The LNCaP cell line, which expresses a functional albeit mutant AR, has a robust transcriptional response to androgen  and depe of 0.01 A. In lin of 0.01 ,20,22. M of 0.01  was usedSupplementary Figure S2). Interestingly, we also observed weak occupancy at AI-ORs in parental LNCaP cells . The 22RV1 line was derived from a CWR22 xenograft that relapsed during androgen ablation . Although no known motifs were enriched at AI-ORs, we identified a novel motif overrepresented at promoter AI-ORs compared with unbound promoters with no known match in the JASPAR . Pre-existing FoxA1 binding at AD-ORs was substantially enhanced after DHT treatment in line with previous studies . AR-bound promoters had high CpG (HCG) content  and displayed increased levels of AcH3 and H3K4me3 relative to unbound HCG promoters. AI-ORs at tRNA genes  is associated with both promoters and enhancers and frequently marks active AR enhancers ,69. UponNA genes . ImportaNA genes B, right.nd genes C.FigureCollectively, AI-ORs occur at locations with open chromatin structures such as HCG promoters associated with highly expressed genes and other open chromatin regions. The chromatin structure of these regions does not change upon DHT treatment and is independent of FoxA1 binding. These data are consistent with a model where in C4-2B cells a subset of genomic loci with pre-existing accessible chromatin serve as anchoring sites for androgen-independent binding of activated AR.We next sought to determine whether AI-ORs exhibited enhancer activity. We examined 10 AD-ORs and 10 AI-ORs in the context of a minimal promoter upstream of the luciferase gene in a transient transfection system. Both AD-ORs and AI-ORs displayed weak basal enhancer activity in LNCaP cells under androgen-deprived conditions compared with randomly selected genomic regions A. We obsSupplementary Figure S7A). We identified 431 DHT-upregulated genes in C4-2B cells (Supplementary File S2). In agreement with previous studies (Supplementary File S2). AI-upregulated genes showed strong genome-wide correlation with AI-ORs but not AD-ORs. Because genome-wide analysis identified a significant number of AI-ORs localized to promoters, we also asked whether AI-OR binding at the proximal promoter correlated with expression of the bound gene. Surprisingly, genes with AI-ORs at the proximal promoter did not show statistically significant upregulation in C4-2B DHT− versus LNCaP DHT− cells . These results suggest that promoter bound AI-ORs do not regulate the proximal gene, but instead, regulate gene expression through long-range interactions. The constitutively high expression and open chromatin structure of AI-OR bound promoters likely explains the absence of regulation of the proximal gene.In order to identify potential targets of AI-OR-mediated gene expression, we next used RNA-seq to identify genes regulated by AR in the presence or absence of DHT and after AR RNA interference , providing further evidence that AR regulates the expression of these genes. Interestingly, AI-upregulated genes also have a significantly increased probability of downregulation after DHT treatment , in line with the reduced enhancer activity of AI-ORs observed in luciferase assays , is located ∼110 kb away from this SYS1 flanking AI-OR . AR-mediated regulation of gene expression through promoter–promoter interactions is consistent with the observation that promoters can exhibit enhancer function and augment the transcriptional activity of other promoters through DNA looping . These results provide direct evidence that AI-ORs interact with the promoters of nearby genes that exhibit increased expression in androgen-deprived CRPC cells. Androgen-independent AR binding likely directly contributes to the androgen-independent AR-regulated expression program found in CRPC.We next sought to confirm the physical interaction between AI-ORs and the distal AI-upregulated genes using the quantitative 3C assay. Our results suggest that AR promoter binding does not regulate the proximal gene, but rather exhibits distal enhancer function. Here, we examined three AI-ORs, two of which were located at promoters. For example, AR was strongly bound to the promoter of the nockdown A, suggesng AI-OR B and is  regions C. Intere looping . In addiWe next investigated whether AI-upregulated genes are necessary for the survival and proliferation of CRPC cells after androgen withdrawal. We selected 10 AI-upregulated genes for functional analyses, all of which have an androgen-independent AR binding site within 150 kb and are downregulated after AR knockdown. Significant inhibitory effects on C4-2B proliferation after gene-specific RNA interference was observed in the absence of or at low concentrations of androgen (0.1 nM R1881), accompanied by a corresponding increase in apoptosis as determined by caspase-3 and -7 activities . NotablySupplementary Figure S11). Notably, AI-upregulated genes involved in cell cycle showed a strong spatial correlation with AI-ORs   on DHT ah AI-ORs B. GSEA uh AI-ORs ] showed h AI-ORs C. In addh AI-ORs  revealedh AI-ORs  , suggesting that similar pathways are activated in response to androgen-deprivation in different models of CRPC. It is important to note, however, that upregulation of LNCaP-abl genes was attributed to DHT-induced AR occupancies, in contrast to the androgen-independent occupancies identified here. Whereas we observed considerable overlap of AD-ORs between C4-2B and LNCaP-abl cells, AI-ORs were largely unique to C4-2B cells . These results suggest that the growth of CRPC can be driven by similar gene expression programs that can be upregulated through different transcriptional mechanisms. These commonly upregulated genes and pathways provide potential therapeutic targets for CRPC treatments against both androgen-dependent and androgen-independent AR signaling.The enrichment of mitotic cell cycle genes is consistent with previously reported ontology analysis of genes upregulated in the LNCaP-abl model of CRPC . We findGiven the importance of AR signaling in CRPC, there has been a dedicated interest in dissecting the mechanisms of AR function after androgen deprivation. Many lines of evidence suggest that androgen-dependent AR signaling remains functional in CRPC. It is known that the serum in clinical CRPC is never totally androgen free, that residual androgens are present within the prostate at levels capable of activating the AR despite castration and that enhanced intratumoral androgen synthesis has been commonly observed in CRPC ,26,74. FProstate cancer is a molecularly heterogeneous disease even within a single patient, and multiple mechanisms may co-ordinately contribute to CRPC progression. While ligand-dependent AR signaling continues to play an important role in the early stages of CRPC when residual androgen-mediated AR signaling is active, ligand-independent activation of AR may occur in an environment where androgen levels are below castrate levels following severe ligand-depriving therapies. Such therapies have been associated with complete elimination of testosterone in the tumor microenvironment (such as bone) and in some cases a loss of CYP17 (a key enzyme in androgen biosynthesis) in prostate cancer cells . More imPrevious studies have identified AR binding events in the presence of androgen in CRPC cells ,20,82,83Recent studies show that promoter–promoter interactions are widespread in human cells ,84, withWe conclude that androgen-dependent and androgen-independent AR signaling can coexist in CRPC, with their relative importance dependent on AR activity and androgen levels in tumor microenvironment. Androgen deprivation results in a dramatic alteration of genome-wide AR occupancies and reprogramming of AR-mediated gene expression. The androgen-independent AR signaling described here may be an important therapeutic target when androgen-deprivation therapy and anti-androgen treatments fail. More importantly, these results suggest a general mechanism whereby, hormone deprivation reprograms genome-wide hormone receptor binding and gene regulation.www.ncbi.nlm.nih.gov/geo) under accession number GSE40050.RNA-seq and ChIP-seq data have been deposited in the Gene Expression Omnibus (GEO) database (Supplementary Data are available at NAR Online: Supplementary Figures 1–12, Supplementary Files 1–3 and Supplementary References [88–90].The Concern Foundation; Wendy Will Case Cancer Fund; the Siteman Cancer Center Developmental Research Award in Prostate Cancer Research. Funding for open access charge: The Internal Fund from the Department of Medicine, Washington University in St. Louis.Conflict of interest statement. None declared.


To test the validity and reliability of a tool specifically developed for the evaluation of appropriateness in rehabilitation facilities and to assess the prevalence of appropriateness of the days of stay.The tool underwent a process of cross-cultural translation, content validity, and test-retest validity. Two hospital-based rehabilitation wards providing intensive rehabilitation care located in the Region of Calabria, Southern Italy, were randomly selected. A review of medical records on a random sample of patients aged 18 or more was performed.k statistic for the assessment of the appropriateness of days of stay were 93.4% and 0.82, respectively. A total of 371 patient days was reviewed, and 22.9% of the days of stay in the sample were judged to be inappropriate. The most frequently selected appropriateness criterion was the evaluation of patients by rehabilitation professionals for at least 3 hours on the index day (40.8%); moreover, the most frequent primary reason accounting for the inappropriate days of stay was social and/or family environment issues (34.1%).The process of validation resulted in modifying some of the criteria used for the evaluation of appropriateness. Test-retest reliability showed that the agreement and the The findings showed that the tool used is reliable and have adequate validity to measure the extent of appropriateness of days of stay in rehabilitation facilities and that the prevalence of inappropriateness is contained in the investigated settings. Further research is needed to expand appropriateness evaluation to other rehabilitation settings, and to investigate more thoroughly internal and external causes of inappropriate use of rehabilitation services. In recent years, developed countries have faced a growing demand for rehabilitation services, attributable to the ageing population and the increasing prevalence of disability due to technical advances in acute healthcare.The changed healthcare needs of the population and the growing costs related to rehabilitation services utilization have stimulated a growing interest in research aimed at the investigation of effectiveness of post-acute care It should be pointed out, however, that there is substantial heterogeneity in rehabilitation interventions according to diagnoses and types of patients. Clinical factors, complications and comorbidities, and physical and cognitive functioning should influence the selection of the appropriate care path for rehabilitation. For instance, patients undergoing orthopedic surgery may need rehabilitation care that can be different from patients needing pulmonary rehabilitation. Indeed, psychiatric disorders necessitate of specific and distinctive rehabilitation interventions. In Italy, post-acute rehabilitation refers to the care provided after a disabling event has occurred. Depending on the type of care provided, post-acute rehabilitation is set up at three different levels of care: the intensive rehabilitation (three or more hours a day of therapy), the high-specialty intensive rehabilitation , and the extensive rehabilitation (at least one hour but less than three hours a day of therapy). It can be delivered in a variety of healthcare settings, ranging from dedicated wards in hospitals providing also acute care to long-term care facilities. Each of these facilities can provide one or more levels of rehabilitation, and the aim is to provide rehabilitation care that best suites patient needs in the most appropriate setting.In the past, well-designed research has investigated appropriateness of services utilization in various settings, such as adults acute hospital care The evaluation of appropriateness of post-acute rehabilitation still remains challenging and unexplored. Reasons are mainly due to the particular case-mix of patients accessing rehabilitation and to the lack of a common set of terms and definitions for this particular type of care. So far, these factors have hampered the development of a universal tool for the evaluation of appropriateness in rehabilitation. However, a group of French authors has recently developed the first tool aimed at evaluating the appropriateness of hospitalizations in rehabilitation facilities that involves the use of explicit, objective and standardized criteria.To our knowledge, this tool has never been used in the Italian context, and information on the appropriateness of rehabilitation services utilization is very scarce in our country, although, according to the Ministry of Health, these facilities are facing a dramatic increase in their use and in the related costs for the National Health Service Within the vast and heterogeneous demands of rehabilitation care, patients who undergo orthopedic interventions provide a unique opportunity to test a tool for the evaluation of appropriateness, since they are a homogeneous population and represent a substantial proportion of subjects seeking intensive rehabilitation care.Therefore, our study was restricted to orthopedic intensive rehabilitation in order to assess the rate of appropriateness of the days of stay and to test the validity and reliability of this tool in our context.Two hospital-based rehabilitation wards providing intensive rehabilitation care located in the Region of Calabria, Southern Italy, were randomly selected among all of the orthopedic rehabilitation facilities accredited by the Regional Health System.A review of medical records on a random sample of patients aged 18 or more who were admitted to the selected rehabilitation facilities during the period March 2010 through July 2011 was carried out. The analysis was performed on random index days, which were preselected on a monthly basis. Patients admitted or discharged within 24 hours from the index day and all the outpatients were excluded from the analysis.The following data were recorded for each patient: socio-demographic characteristics , information related to hospitalization  to Italian and to adapt the appropriateness criteria to the Italian context followed published guidelines Then, a pilot study was conducted in a convenience rehabilitation facility in order to test the final draft of the tool.Subsequently, the translated version of the tool was submitted to a group of experts who were senior researchers in public health and in physical medicine and rehabilitation. They reviewed the format and content of the items, as well as the content validity of the tool as a whole. Eventual disagreement between physicians in reviewing medical records was resolved by discussion with the researcher in physical medicine staying in the facility.Two physicians, who were not involved in patient care and acquainted with the user manual, concurrently and independently reviewed 85 medical records with the aim of evaluating the inter-rater reliability.k statistic were used to assess the inter-rater reliability regarding the appropriateness of days of stay. Univariate and multiple logistic regression analyses were performed to explore the association of patients’ characteristics with the inappropriateness of hospital days. Univariate analysis was carried out using the appropriate test (chi-square test or t-test). Explanatory variables included in the multiple logistic regression model were the following: gender , age (continuous in years), marital status , distance from patient’s home to the rehabilitation facility (continuous in kilometers), length of stay from admission to index day (continuous in days), patient’s autonomy in performing BADL , Charlson comorbidity index . Adjusted odds ratio (OR) and 95% confidence intervals (CI) were calculated. The significance level was set at a P-value ≤0.05. All the analyses were programmed in Stata software program release 11.0 The overall agreement and the The Ethics Committee of the Mater Domini Hospital of Catanzaro  approved the protocol of the study (Prot. E.C. No. 03/CE/2010). Written consent was requested during the hospitalization, and only the patients who had given written permission for their personal data to be stored in the hospital database and used for research were included in the study.k statistic for the assessment of the appropriateness of days of stay were 93.4% and 0.82, respectively.The process of establishing content validity resulted in several modifications. The items were reduced from 16 to 11 to adapt their use to the Italian intensive rehabilitation setting. For instance, palliative care and vital sign monitoring were considered to be best suitable in facilities that are classified as high-specialty intensive rehabilitation. The criterion considering the type of rehabilitation professional and the duration of rehabilitation care was modified because it was not considered consistent with the Italian guidelines, which consider days of stay in intensive rehabilitation as appropriate if rehabilitation professionals provide care for at least three hours per day. Thus, the taking over criterion focused on capturing the selected process of care according to the local guidelines. In terms of test-retest reliability of the tool, the overall inter-rater agreement was excellent between the two reviewers, since the agreement and the Since no significant differences in appropriateness of days of stay between the two rehabilitation facilities studied were found, the results are presented in a combined form. A total of 371 patient days was reviewed, and their main characteristics and distribution of inappropriateness of days of stay according to various explanatory variables are presented in 2 = 8.22, 1 df, p = 0.004)  Table 1.The results of multiple logistic regression analysis Table 3 Among all the potential causes of inappropriateness, the study results showed that the primary reasons accounting for the inappropriate days of stay were social and/or family environment issues (34.1%), health conditions of patient that prevents the provision of rehabilitation care (31.8%), over cautious physician’s attitude (17.6%), and rehabilitation care that could be delivered at a lower level of care (16.5%).The present study represents the first attempt to assess the appropriateness of days of stay in rehabilitation facilities in Italy by using a tool developed and validated for this specific purpose. The tool is not merely a modified version of the AEP adapted to rehabilitation facilities, and it is intended to be used in the same way as the AEP. It consists of explicit, objective and validated criteria that were validated using a formalized expert consensus method k-statistic of 0.82. According to the classification of Landis and Koch, the latter value denotes a high level of reliability. This result is close to that obtained by the authors who developed the tool A version of the tool that was suitable for the Italian intensive rehabilitation was adapted and tested. The tool seems to be feasible to use, reliable, and to have adequate validity. Cross-cultural translation allowed to adapt the local version of the tool to international standards. Measurement of content validity, defined as the degree to which the content of a measurement tool is an adequate reflection of the construct to be measured, was assessed qualitatively and was necessary because of two reasons. First, the appropriateness criteria that had been drawn from the Italian guidelines on rehabilitation activities were included into the final draft of the tool. Second, some of the items from the original tool were not considered to be suitable for the Italian intensive rehabilitation. Content validity was judged to be good by our expert group, and the tool seems to require no further revision. Moreover, the tool showed to be highly reproducible since the agreement between the reviewers was of 93.4% with a Comparison across countries should be made cautiously, given the dissimilarities in the provision of rehabilitation care, the different case-mix of patients, and the modification that were made to the tool. For instance, as previously described in Italy rehabilitation activities are organized in three level of care, depending on care needs of patients, namely intensive rehabilitation, high specialty intensive rehabilitation, and extensive rehabilitation The results of multivariate analysis showed that some patient characteristics were significantly related to inappropriate days of care. Indeed, younger age was a predictor of inappropriateness of health service use and a similar findings has been reported in previous studies Reasons accounting for inappropriate days of care were not available when the present study was designed. However, following on our past experience on appropriateness in acute hospitals, a list of potential causes of inappropriateness was arranged. In the meantime, the authors of the original tool have published their list of reasons accounting for inappropriateness The study results should be read in the light of some limitations. First, retrospective data collection may have distorted the actual rate of inappropriateness, since it is influenced by the quality of medical records. Nevertheless, retrospective data collection is a common and accepted method for the evaluation of appropriateness, since many studies based on the AEP were performed by analyzing medical records of discharged patients. Second, data were collected in one Italian region that might not represent all intensive rehabilitation in Italy, and concern about generalizability and comparability of the findings may arise. However, we are confident that the findings of the study may be representative of the area examined and may be referred to the Southern part of our country. Third, the process of adaptation enabled us to trim the tool for evaluating the appropriateness in intensive rehabilitation only. Although this is a limit of the present study, the tool could serve as a canvas to be adapted in other settings by introducing slight and adequate modifications.In conclusion, the study findings showed that the tool used is reliable and have adequate validity to measure the extent of appropriateness of days of stay in rehabilitation facilities and that the prevalence of inappropriateness is contained in the investigated settings. Further research is needed to expand appropriateness evaluation to other rehabilitation settings, and to investigate more thoroughly internal and external causes of inappropriate use of rehabilitation services.


The androgen receptor (AR) plays a critical role in the proliferation of prostate cancer cells. However, its mechanism of action in proliferation remains unknown. An understanding of the mechanism of AR action in proliferation may lead to the development of effective strategies for the treatment of prostate cancer.1 phase was sufficient to prevent cells from entering S phase. Since the assembly of pre-replication complex (pre-RC) in G1 is required for the progression of cells from G1 to S phase, the effect of Casodex during mid-G1 suggested that the role of AR in proliferation might be to regulate the assembly of pre-RC. To test this possibility, we investigated the interaction between AR and Cdc6, an essential component of pre-RC in LNCaP cells. AR co-localized and co-immunoprecipitated with Cdc6, and Casodex treatment disrupted this interaction. AR-immunoprecipitate (AR-IP) also contained cyclin E and cyclin A, which play a critical role in pre-RC assembly and cell cycle entry into S phase, and DNA polymerase-α, PCNA, and ribonucleotide reductase, which are essential for the initiation of DNA synthesis. In addition, in cells in S phase, AR co-sedimented with components of the DNA replication machinery of cells that entered S phase.In this study we report that pulse treatment of synchronized LNCaP cells with Casodex, an AR-antagonist, for 4 hours in mid-G1 to S phase through a mechanism that is independent of its role as a transcription factor.Together, these observations suggest a novel role of AR as a component of the pre-RC to exert control over progression of LNCaP cells from G Prostate cancer is the most frequently diagnosed non-skin cancer and second leading cause of cancer deaths in American men Castration-resistant growth of prostate cancer is often associated with increased expression of AR 1 to S phase is fundamental to proliferation. We reported previously that Casodex , a specific inhibitor of AR, blocks the ability of G1 phase AR-positive LNCaP prostate cancer cells to enter S phase 1 to S phase. Progression of cells from G1 to S phase requires a cascade of sequential events in G1 phase that contribute to the assembly of the DNA replication machinery in cells that enter S phase. These events include a) loading of Cdc6, replication licensing factor RLF-B/Cdt1, and mini-chromosome maintenance (Mcm) proteins onto the origin recognition complex (ORC) to form pre-replication complex (pre-RC), and b) unwinding of DNA by helicase (Cdc45) associated with pre-RC, and the assembly of enzymes of DNA synthesis to form mega-complexes required for initiation of DNA synthesis at the origins of DNA replication  containing 10% fetal calf serum (FCS), 2.5 mM glutamine, 100 µg/ml streptomycin, and 100 U/ml penicillin (complete medium) in a humidified incubator with 5% COt with 18S RNA as internal control.RNA was isolated from control and Casodex treated cells and subjected to RT-PCR to determine PSA and GAPDH expression as described previously Isoleucine deprivation was carried out by our previously published method 1 to S phase was monitored by determining the ability of cells to incorporate 3H-thymidine into DNA as described previously 3H-thymidine  for 30 minutes at 37°C in a humidified incubator. The radioactivity incorporated into acid-precipitable material was then determined as described Since entry of cells into S phase is marked by the onset of DNA synthesis, the progression of synchronized cells from G2, 50 mM NaF, and 0.1 mM Na3VO4) supplemented with protease inhibitor cocktail  at a density of approximately 1×107 cells/ml. The cell suspension was then subjected twice to 30 pulses of sonication using a Branson Sonifier 250 , set at an output control of 2 and a duty cycle of 20, with intermittent cooling on ice. The sonicated cell extract was cleared by centrifugation in an Eppendorf Centrifuge 5415R at 6,000 rpm for 5 minutes at 4°C. Protein concentration in cleared extracts was assessed using BioRad Protein Assay reagent .Exponentially growing LNCaP cells harvested by scraping into phosphate-buffered saline (PBS) were pelleted by centrifugation for 10 minutes at 4°C at 2,000 rpm in a Sorvall RT7 centrifuge and resuspended in immunoprecipitation (IP) buffer  or anti-Cdc6 antibodies (H-304) . Immune complexes were then adsorbed to Pierce Protein A/G Agarose beads  for 2 hours at 4°C with gentle agitation. The adsorbed complexes were washed three times with IP buffer by centrifugation in an Eppendorf Centrifuge 5415R at 6,000 rpm for 5 minutes at 4°C, and then eluted with PAGE loading buffer . Control immunoprecipitates were prepared by using 4 µg/ml purified mouse or rabbit IgG  in place of anti-AR or anti-Cdc6 antibodies.1 phase (1 hour after release from isoleucine-block) or in S phase (24 hours after release from isoleucine-block) were harvested and the nuclear lysate was prepared by a slight modification of the method of Subramanyam et al 2, 70 mM HEPES (pH 7.6), 0.025 mM CaCl2, 1 mM phenylmethylsulfonyl fluoride (PMSF), 2 mM DTT, 1 mM EDTA] at a density of 2×107 cells/ml. The cell suspension was then homogenized in a motor-driven Wheaton homogenizer  until ∼90% of the cells were stained with trypan blue dye . The cytosolic supernatant was then separated from the nuclear pellet by centrifugation in an Eppendorf Centrifuge 5415R at 6,000 rpm for 5 minutes at 4°C. The nuclear pellet was suspended in a volume of buffer A equal to that used for homogenization and subjected twice to 30 pulses of sonication with a Branson Sonifier 250  set at an output control of 2 and a duty cycle of 20, with intermittent cooling on ice. The sonicated nuclear extract was cleared by centrifugation in an Eppendorf Centrifuge 5415R at 6,000 rpm for 5 minutes at 4°C. The DNA replication complex fraction was then isolated by subjecting the nuclear lysate to sucrose density gradient centrifugation essentially as described by Reddy and Pardee 1 or S phase cells was then subjected to Western blot analysis to assess the presence of enzymes of DNA synthesis and cell cycle regulatory proteins.Synchronized LNCaP cells in GKip-1, cyclin B , caspase 3 , or GAPDH . Immunoreactive bands were developed using horseradish peroxidase-conjugated secondary antibodies and SuperSignal WestPico chemiluminescent substrate  and visualized using X-ray film.Samples were dissolved in PAGE loading buffer  and subjected to denaturing 10% polyacrylamide gel electrophoresis (SDS–PAGE) and then transferred to nitrocellulose membranes. Individual membranes were probed with antibodies against AR (AR-N20 or AR-441), Cdc6 (H-304), prostate specific antigen , Lamin B, DNA polymerase-α (STK1), PCNA (PC10), ribonucleotide reductase , cyclin A (H-432), cyclin E (C-19) , p27LNCaP cells grown on glass slides were washed once with PBS, followed by fixation in 3.7% (vol/vol) formaldehyde for 20 minutes at 22°C. Cells were permeabilized in 0.5% (vol/vol) Triton X-100 for 15 minutes and blocked for 1 hour in 2% (wt/vol) BSA at 22°C. Slides were then incubated for 1 hour at 22°C with antibodies against AR (AR-N20 or AR-441) and Cdc6 or DNA polymerase-α followed by both goat-anti-mouse-fluorescein isothiocyanate (FITC)- and goat-anti-rabbit-tetramethylrhodamine B isothiocyanate (TRITC)-labeled secondary antibodies . After four washes with PBS, slides were mounted with Aqua-Poly/Mount . Confocal laser scanning microscopy was performed with a Zeiss LSM 410 upright confocal microscope. Images were processed, and colocalization analysis (which is colored yellow) was performed with a Zeiss LSM 410 Meta Excitation system with collection at 488 and 543 nm. Intensity Correlation Quotient (ICQ) analysis to quantify AR colocalization with Cdc6 or DNA polymerase-α was performed by using ImageJ software .LNCaP cells grown on glass slides were labeled with 10 µM BrdU  for 30 minutes, washed once with PBS and fixed in 3.7% formaldehyde for 20 min at 22°C. Cells were permeabilized in 0.5% (v/v) Triton X-100 for 15 min and blocked for 1 hour in 2% (w/v) bovine serum albumin at 22°C. Cells were then stained with mouse monoclonal antibodies against BrdU , followed by goat-anti-mouse-FITC-labeled secondary antibody. Slides were mounted with Vectashield Mounting Medium containing DAPI  and photomicrography was performed with a Zeiss Axiofot microscope equipped for epifluorescence.1, S, or G2/M phases) AR is required for proliferation of LNCaP cells, as each phase of the cell cycle lasts no more than few hours. Therefore, we used the non-steroidal anti-androgen Casodex  to selectively and acutely inhibit AR in order to determine the role of AR in progression of synchronized LNCaP cells through G1 and S phases.AR knockdown strategies have been used to demonstrate that AR is required for proliferation of prostate cancer cells 50 to inhibit AR activity is ≈50 µM Casodex in either FCS or CSS -containing medium S or CSS . Cells t0/G1 phase 1 and enter S phase 12 hours after release, and reach a peak of S phase 10–12 hours thereafter 1 vs. S phase. Therefore, in order to distinguish between an inhibitory effect of Casodex in G1 vs. in S phase, we treated synchronized LNCaP cells with Casodex starting at 0, 4, 8, 12, or 16 hours after release from isoleucine-block, and determined the ability of cells to incorporate 3H-thymidine (3H-TdR) at 20 hours after release, a time when control cells are in S phase , indicating a role of AR in G1. By contrast, the Casodex effect was blunted if treatment was delayed until 12–16 hours after release from isoleucine-block, a time when the cells have already committed to enter S phase. Casodex treatment of cells that had already entered S phase (16 hours after release from isoleucine-block) had very little effect on 3H-TdR incorporation  was more effective at blocking entry into S phase than was treatment during early-G1 (0–4 hours after release from isoleucine-block) or late-G1 phase (8–12 hours after release from isoleucine-block). This suggests AR involvement in cell cycle regulatory events in mid-G1 phase that are critical for LNCaP cells to progress from G1 to S.We then determined when in G1 blocked the ability of synchronized LNCaP cells to enter S phase  and G2/M  phases in LNCaP cells. We observed that cyclin E and cyclin A, but not a mitotic cyclin, cyclin B, were associated with AR-IP prepared from exponentially growing LNCaP cells  (Progression of cells from G with AR . Importae block) . Thus, t1 phase. While a role of AR in the control of proliferation of prostate cancer cells is evident from a number of studies that knocked down AR in androgen-sensitive and castration-resistant prostate cancer cells 1 phase for synchronized LNCaP cells to progress from G1 to S phase and that AR interacts with cell cycle regulatory proteins and enzymes required for initiation of DNA synthesis. While these observations remain to be validated in other AR-positive prostate cancer cell lines, the data presented here suggest AR involvement in proliferation of LNCaP cells through its interaction with components of the pre-RC necessary for the initiation of DNA synthesis and, therefore, for the progression of cells from G1 to S phase.The ability of cells to enter S phase is determined by events in G1 phase but not in those that had already entered S phase  that interface with AR in pre-RC may lead to the development of new and more effective strategies to suppress AR-dependent growth of prostate cancer. In addition, altered interaction of AR with pre-RC and replication machinery could circumvent the requirement for androgen and account for the continued critical role of AR in proliferation of castration-resistant prostate cancer cells. Therefore, analysis of pre-RC and replication machinery constituents in androgen-sensitive vs. castration-resistant prostate cancer cells may yield valuable information about the mechanism of AR action in prostate cancer cell proliferation and may lead to the identification of potentially more effective therapeutic targets for prostate cancer treatment.


Total deficiency of these adrenoceptors (TKO) results in cardiac hypotrophy and negative inotropy. TKO represents a unique mouse model mimicking total unselective medical β-blocker therapy in men. Electrophysiological characteristics of TKO have not yet been investigated in an animal model.Beta-adrenoceptors (β-AR) play an important role in the neurohumoral regulation of cardiac function. Three β-AR subtypes  at the age of 15 weeks. Standard surface ECG, intracardiac and electrophysiological parameters, and arrhythmia inducibility were analyzed.The surface ECG of TKO mice revealed a reduced heart rate , prolonged P wave  and PQ time  compared to WT. Intracardiac ECG showed a significantly prolonged infra-Hisian conductance . Functional testing showed prolonged atrial and ventricular refractory periods in TKO . In TKO both the probability of induction of atrial fibrillation  and of ventricular tachycardias  were significantly reduced.TKO results in significant prolongations of cardiac conduction times and refractory periods. This was accompanied by a highly significant reduction of atrial and ventricular arrhythmias. Our finding confirms the importance of β-AR in arrhythmogenesis and the potential role of unspecific beta-receptor-blockade as therapeutic target. The downstream mechanism includes phosphorylation of phospholamban and sarcoplasmatic/endoplasmatic reticulum calcium ATPase (SERCA) that enhances intracellular calcium dynamics 2-AR is additionally associated with the regulation of cardiac growth and remodelling 3-adrenergic stimulation is connected to nitric oxide liberation by means of endothelial nitric-oxide-synthase (eNOS) activation The myocardial beta-adrenergic receptors (β-AR) play a pivotal role in regulating cardiac autonomic function. Three β-AR subtypes have been described and identified by molecular cloning so far: β1- and β2-AR but also are associated with effects related to the β3-AR 1-blocking power with additional β3-agonistic effects 1-blockers are commonly given with higher priority than non-selective beta-blockers since several side effects (e.g. bronchospasm) 2-AR and β1-blockers have been proven effective to treat heart failure in large clinical trials Chronic β-adrenergic stimulation, e.g. in patients with dilated cardiomyopathy and heart failure, results in a down-regulation of the β-AR 1/2/3-AR knockout (TKO  =  β1/2/3-KO  =  β1−/−β2−/−β3−/−). These mice show cardiac hypotrophy and negative inotropy accompanied by a decreased SERCA 2a activity 1- and β2-AR but also the β3-AR with its partly converse effects. Electrophysiological characteristics of TKO mice have not been investigated systematically before.Jiminez et al. 1/2/3-AR knockout. Isogenic SV129 wild type (WT) littermates (β1+/+β2+/+β3+/+) were used as controls. Generation of the TKO-mice has been described previously We performed the studies on 15-week-old mice with a ubiquitous βin vivo transvenous electrophysiological investigations were performed in all mice (10 TKO and 10 WT), using a single catheter technique as described before The The registration and recording of the intracardiac electrograms and transvenous atrial and ventricular stimulation maneuvers were carried out as previously described Functional electrophysiological parameters were determined using a modified multi-programmable simulator . Sinus node recovery time (SNRT) was calculated after fixed-rate atrial pacing and defined as the maximum return cycle length after 10 s fixed-rate pacing at S1S1 cycle length 120 ms. Wenckebach periodicity (WBP) and 2∶1-AV-nodal block (2∶1-block) were evaluated by fixed rate atrial pacing . WBP was defined as longest S1S1 cycle length with loss of 1∶1-AV-nodal conduction; 2∶1-block as longest S1S1 with 2∶1 AV-nodal conduction.Performing programmed stimulation maneuvers , atrial, ventricular and AV nodal refractory periods  were evaluated. ARP was defined as longest S1S2 with absent atrial response, AVNRP as longest S1S2 with loss of AV-nodal conduction, and determined after programmed atrial stimulation. VRP was measured analogously after ventricular extrastimulus pacing.The induction of atrial fibrillation (AF) was tested by atrial burst stimulation . Rapid and fragmented atrial electrograms with irregular AV-nodal conduction for ≥1 s were defined as AF Data were expressed as mean ± one standard deviation. Differences between the two groups were assessed using a two-tailed Student's t-test. Discrete variables were analyzed by 2-sided Fisher's exact test. A Spearman nonparametric test was performed to assess the correlation of refractory periods and episodes of arrythmias. A p-value <0.05 was regarded as statistically significant.During the whole observation time of the surface ECG all animals presented with sinus rhythm without spontaneous supraventricular or ventricular premature contractions. No atrio-ventricular conduction blocks could be registered. Representative ECG recordings are presented in In TKO mice we found a significant reduction in heart rate  and significantly prolonged parameters for P wave duration , PQ interval  and QT time  compared to WT . QRS intIntracardiac ECG recordings showed equal supra-Hisian  but prolonged infra-Hisian  conduction times in TKO compared to WT . FunctioThe probability of induction of AF  was signVTs were inducible in 77.8% of the WT animals with a frequency of 5.0 episodes per animal . In comp1/2/3-AR is conformable with a viable mouse model. This unique model allows the evaluation of a complete β-AR knockout mimicking a total β-AR blockade. To our knowledge there is no data available of cardiac electrophysiology in a setting of complete β-AR blockade. Since human medical therapy with β-blocking agents does not lead to a complete blockade, this mouse model provides an excellent opportunity to further analyse cardiac β-AR function.β-AR stimulation is a major neurohumoral mechanism in the regulation of the cardiac function 1-, β2-, β1/2- and β3-AR knockout mice have shown a certain cardiotoxic effect for the β1-AR and a cardioprotective effect for the β2- and β3-AR. Stimulation of the β1-AR mediates apoptosis 1-AR leads to a more severe cardiomyopathy than overexpression of β2-AR The multifarious modes of action of the beta receptors are not yet fully understood. Studies with β2-AR by activation of Gi, PI3K and protein kinase B 2-AR results in an elevated rate of isoproterenol-induced apoptosis 2-AR knockout but not a β1-AR knockout resulted in a rapid death rate due to elevated levels of MAPK An anti-apoptotic effect is suggested for the β3-AR is also associated with cardioprotective effects by downstream signalling through NO due to activation of eNOS, iNOS and nNOS 3-ARs are activated only with higher concentrations of catecholamines than β1- and β2-ARs attributing them a protective function during sympathetic overstimulation 3-AR knockout mice with pressure-overload by aortic constriction resulted in augmented myocardial fibrosis, hypertrophy, ventricular dilatation and mortality 3-AR did also lead to short term improvement in cardiac function in the setting of animal models with heart failure The recently characterised β1-AR or the combined loss of β1+β2-AR led to a decrease in resting heart rate. Moreover the parameter of heart rate variability was predominantly dependent on the β1-AR 2-AR did not lead to differences in resting heart rate 1-AR and β1+β2-AR knockout mice 1/2/3-deficiency resulted in a markedly reduced heart rate. Moreover we registered prolonged global conduction times on atrial and ventricular levels as well as in the specific conduction system.Addressing specific parameters of cardiac electrophysiology it could be demonstrated that the single loss of βAlthough TKO mice displayed a reduced ejection fraction in a recent study, no functional impairment in a voluntary running wheel test but rather an increased willingness to run was found 2-AR in the cardiomyocytes of the interventricular septum close to the specific cardiac conduction system compared to normal working myocardium In patients with heart disease, Hisian and infra-Hisian conduction delay are associated with increased mortality Accompanying the known negative inotropy and the now demonstrated conduction delays we found a profound suppression of atrial and ventricular arrhythmias. No signs of heart failure, occurrence of spontaneous AV-blocks, or sudden cardiac deaths were present in this mouse model.1-blocker) and not metoprolol (a β1/2-blocker) or carvedilol  had a positive effect on mortality as a result of a reduction in ventricular arrhythmias in a mouse model of dilated cardiomyopathy. They concluded, that the effect might have been due to a significant shortening of the QT time. Here we could verify that TKO leads to a drastic reduction of the inducibility of VTs even though no changes in QTc interval were present at all. Hence, other mechanisms seem to be responsible for the protective effect found in TKO.The QT interval as well as the atrial and ventricular refractory period plays an important role in arryhthmogenesis The assumption that the suppression of inducible arrhythmias in TKO is due to a prolonged ARP and VRP is supported by the fact that in chronic medical beta-blocker treatment in humans, atrial and ventricular refractory periods show significant prolongations 1-AR blockade. Though the question, which beta-blockade is most relevant cannot finally be answered and is certainly highly dependent on the underlying cardiac disease. Undoubtedly the blocking of β1-AR in cases of heart failure is cardioprotective and improves cardiovascular outcome 2-AR (but not β1) blockade has been found to be highly antiarrhythmic in heart failure due to a reduction of sarcoplasmatic reticulum calcium load 2-AR than to β1-AR 3–AR: in sepsis 3-AR can be determined, but whether this increase is a protective response or might even contribute to further myocardial dysfunction is still a matter of debate. The ‘physiological break-mechanism’ of the β3-AR to sympathetic overstimulation plays a secondary role in the TKO model as no stimuli of the β1- and β2-AR can emerge.Regarding antiarrhythmic treatment, a non-selective medical beta blockade might not be inferior to a selective β1/2-, β1/3-, and β2/3-AR knockout to further clarify the role of the antiarrythmic effect of the individual receptor blockade. Moreover, our data provide the basis to further test possible beta-blocker side-effects that might occur independently of the β-AR in a unique mouse model.Based on our present results, a general recommendation cannot be given to use an unselective complete medical beta blockade to treat arrythmias; however, the data show that TKO mice exhibit a highly antiarrythmogenic potential. Further studies should address the electrophysiological phenotypology of mice with a single β-AR knockout, as well as combined βTKO results in significant prolongations of cardiac conduction times and refractory periods. This was accompanied by a highly significant reduction of atrial and ventricular arrhythmias. Our findings confirm the importance of β-AR in arrhythmogenesis and their role as therapeutic target.