Staphyloccocus aureus, which is the main microorganism responsible for acne vulgaris. The mixture design was applied to study the interactions and optimize the independent variables that could match the prerequisites of selected dependent responses. A formulation containing 0.25 g of rosehip oil, 0.45 g of surfactant (Lauroglycol-90), and 0.3 g of cosurfactant (propylene glycol) was chosen as an optimized desirable formulation. The optimized batch was loaded with QRS and evaluated for in vitro and ex vivo permeation. The in vivo hepatotoxicity was assessed through topical administration. Permeability studies confirmed the enhanced permeation percentage of ITT (52.11 ± 2.85%) and QRS (25.44 ± 3.18%) of the optimized formulation, with an enhanced steady-state flux (Jss). The in vivo studies conducted on experimental animals demonstrated superior hepatoprotective activity of the prepared optimized formulation compared with other formulations of drugs and commercially marketed products. We anticipate that this optimized ITT formulation, followed up with good clinical evaluations, can be a breakthrough in the safe treatment of acne vulgaris.Acne vulgaris is a common skin disease that affects everybody at least once in their lives. The treatment is challenging because the stratum corneum contains rigid corneocytes surrounded by intercellular lamellae that are difficult to bypass. In the present study, we intended to formulate an effective nanoemulsion that could deliver isotretinoin (ITT) with enhanced solubility, permeability, and bioavailability across the skin. ITT can have a serious hepatotoxic effect if given too frequently or erratically. Therefore, to overcome the aforesaid limitation, quercetin (QRS), a hepatoprotective agent, was incorporated into the formulation. Initially, the ITT solubility was determined in various surfactants and cosurfactants to select the essential ingredients to be used in the formulation and to optimize a nanoemulsion that could enhance the solubility and permeability of ITT and its antimicrobial activity against  Staphylococcus aureus adapts to evolving microenvironments of the skin and may become an opportunistic pathogen causing acne or atopic dermatitis [S. aureus is a gram-positive, anaerobic organism that usually causes superficial sebaceous unit infections [S. aureus to acne is not entirely clear [Acne is the common skin disease for which most patients attempt to get a dermatologic cure ,2. Recenrmatitis . S. aurefections . Howeverly clear . In chroly clear . Acne trly clear .The most commonly used drug for acne is isotretinoin (ITT), which is a synthetic form of vitamin A. It is very effective for mild nodular and extreme papulopustular acne . ITT effConsequently, it is prudent to enhance the solubility of ITT and minimize its adverse effects by using an appropriate carrier.Several plant-derived natural products, characterized by a huge structural diversity, including phenolic compounds, are cited as antimicrobial agents and resistance-modifying agents . One sucThe self-nanoemulsifying drug delivery system (SNEDDS) is a nanocarrier that forms spontaneously from the mixture of an oil, surfactant, cosurfactant, and active drug compound in a homogenous, fine, oil-in-water, nanoemulsion liquid form .In recent years, much attention has been paid to SNEDDS. The systems have had reasonable success in improving the bioavailability of poorly soluble drugs, especially topical drugs. Nanoemulsions have also been used for the decontamination of radionuclei and photodynamic therapies. Consequently, nanoemulsions have many applications in the field of dermatology . SNEDDS Melaleuca alternifolia has a unique application in the treatment of acne. Owing to its improved bacteriostatic activity and ability to minimize lesion formation, rosehip oil has been used to treat seborrheic dermatitis and acne vulgaris. There are several drawbacks related to its topical application, such as the potential production of irritant or allergic contact dermatitis, decreased penetration in the affected area, and potential alteration of cutaneous flora [Numerous scientific studies were done over the years on the application of essential oils in nanoemulsions. An essential oil can be a suitable natural alternative to a synthetic skin penetration enhancer due to its low cost and its ability to improve penetration . Rosehipus flora . It was us flora . In the us flora ,31.In contrast to several conventional methods, the topical route offers an alternative form of drug delivery and attracts innovative research, particularly in the treatment of several skin diseases. The potential nanotechnology strategy circumvented the physicochemical and pharmacokinetic constraints of QRS and ITT. Nonetheless, very few studies have discussed the crucial findings of the in vivo results of this advanced method. Therefore, this study used a mixture design to create an optimized formulation of quercetin and ITT to improve their bioavailability and antimicrobial activity and to reduce ITT toxicity.ITT was purchased from Wuhan Senwayer Century Chemical Co., Ltd. . Rosehip oil was obtained from Hayward, CA, USA. Quercetin was procured from Water Import and Export Co., Ltd. . Lauroglycol-90 was generously gifted by Gattefosse . Propylene glycol, Tween, Span, and Cremophor were purchased from Sigma . All other reagents and chemicals used were of analytical grades.The solubility of ITT in rosehip oil and different surfactants and cosurfactants was determined. An excess amount of ITT was dissolved in approximately 3 mL of each vehicle individually. The mixture was then shaken on a water bath  for 72 h, and the temperature was maintained at 37 ± 0.5 °C. Once equilibrium was attained, the resulting mixture was centrifuged  at 8 °C for 30 min at 15,000 rpm. The formed supernatant was diluted with a suitable quantity of methanol and analyzed quantitatively for the ITT concentration using a UV-Visible spectrophotometer at 362 nm. The experiment was repeated three times, and the values were noted as the mean ± the standard deviation.Several formulations were designed with selected proportions of rosehip oil, Lauroglycol-90 surfactant, and propylene glycol cosurfactant and measured for particle size using the Zetatrac particle size analyzer  in accordance with the study design. Initially, 10 formulations were prepared  using diStatistical optimization of the response surface methodology (RSM) has proven to be a proficient tool in optimizing the various parameters in a multifarious process to obtain the desired responses; thus, it has been applied to formulate ITT-QRS SNEDDS. Among several models, the I-optimal point-exchange mixture design was selected to study the interaction of independent variables. Three factors were selected, namely, the percentage of rosehip oil (X1), surfactant (Lauroglycol-90) (X2), and cosurfactant (propylene glycol) (X3) . Data frVarious formulations of ITT-QRS nanoemulsions were prepared, as portrayed in the design mixture . First, Prepared ITT-QRS SNEDDS were characterized by various parameters to study the effect of selected variables.For each formulation, 100 μL of the formulation was diluted with 900 μL of distilled water to form an aliquot of 1 mL, and the globule size of the prepared sample was determined at 25 °C using a dynamic light-scattering technique  .The ex vivo permeation test was done with a simple, low-head dissolution testing instrument . The receptor compartment was filled with 450 mL of phosphate buffer with continuous stirring at 50 rpm. The temperature of the buffer solution was maintained at 37 ± 0.5 °C. The required area of skin (2 cm × 2 cm) was collected from a Wistar rat’s abdominal segment (hairless). Animals were collected from the Animal House at the Beni-Suef clinical laboratory center after obtaining approval from the ethical committee . Obtained skin was soaked in a phosphate buffer at a pH of 7.2 for 1 day before the study began. Afterward, the skin segment was placed between the donor and receptor compartments so that the dermal side would be in close contact with the receptor side. At specific intervals, aliquots of 3-mL samples were withdrawn, and the value of the Jss for each formula was analyzed using a UV-Visible spectrophotometer . The QRS and ITT were quantitatively measured at 269 nm and 299 nm, respectively.S. aureus inoculum into a petri dish. When set, the agar was punched with a sterile cork-borer 12 mm in diameter, and 100 μL of each formulation was added to the bores. After incubation at 37 °C for 18 h, the inhibition zones were measured with a ruler (n = 3). Sterile water was used as a negative control.An antibacterial activity test assessing the inhibition zones produced by the test formulations was performed by plating 20 mL of agar (cooled) plus an The obtained results from the experimental runs were further analyzed using the ANOVA  with a multiple-response optimization. The optimized formulation was selected based on the constraints of the selected responses.S. aureus. In addition, the following tests were done on the optimized formula.After the optimization of the experimental runs, an optimized formulation was prepared according to the concentrations proposed by the software and evaluated for globule size, ex vivo permeation, and inhibition zone against 2  was utilized. First, the dialysis membrane was soaked in a phosphate buffer (pH 7.4) for 15 min at 25 ± 0.5 °C. The receptor compartment was filled with the phosphate buffer, and the paddle was placed in the medium with continuous rotations (75 rpm). The temperature was maintained at 37 ± 0.5 °C throughout the experiment. Samples were withdrawn at various time intervals , and the ITT and QRS concentrations were analyzed at 269 and 299 nm, respectively.In vitro drug release studies were performed to calculate the amount of ITT and QRS released in the optimized formulation. A low-head dissolution testing instrument, PT-DT70, with a dialysis cellulose membrane diffusion area of 4 cmForty-two male adult mice weighing 20.5 ± 1.5 g were selected and adapted for 7 days before the study. The animals were divided into seven groups consisting of six animals in each group. All groups and the tested formulations are shown in p-value of less than 0.05 was noted as statistically significant.The in vivo results obtained were expressed as the mean ± the standard deviation and analyzed using the Statistical Package for the Social Sciences version 26 . A one-way ANOVA was followed by a post-hoc Tukey test to determine the significant differences between the quantitative variables. A The solubility of ITT in assorted vehicles is depicted in The enhanced solubility of ITT has been recorded with propylene glycol (20.1 ± 3.11 µg/mL). Propylene glycol is known for its great miscibility in polar and nonpolar solvents, as well as its ability to increase absorption and the degree of permeability . Thus, iThe particle sizes of all the prepared formulations were evaluated. First, the concentration of ITT to be used for further formulations was optimized. Formulation 1, having higher concentrations of ITT , resulted in a particle size of 114.11 ± 13.28 nm, 186.22 ± 13.62 nm, and 350.44 ± 21.32 nm . Therefo2/h and 3 to 27 mm, respectively , maximum Jss (Y2), and larger inhibition zone (Y3). The design projected 16 runs, and the observed responses are given in ectively . All obtBased on the fit summary of the responses  and the sequential sum of squares (Type I), different models were selected . No modep < 0.01%. Polynomial equations were generated using multiple regression analysis. Both the p-values and polynomial equations were used to estimate the true effect of the variables. p-values that were less than 0.0001 were considered statistically significant. Values greater than 0.1000 indicated that the model terms were not significant. If there were many insignificant model terms (not counting those required to support the hierarchy), model reduction could have been required. The lack-of-fit F-value (0.52) was nonsignificant, with only a 13.5% chance that a lack-of-fit F-value this large could occur due to noise.Response I: The selected model was statistically significant, F = 32, Adequate precision measured the signal-to-noise ratio. In general, a ratio greater than 4 is desirable. The obtained ratio (194.91) indicated an adequate signal to navigate the design space . ANOVA rp < 0.0001) between rosehip oil and other factors, but the predominant effect was for rosehip oil [Based on the ANOVA results, Response I was affected significantly by the synergistic effect of a linear mixture  and lack-of-fit F-value (4.36) for Response II confirmed that the selected model was significant, and the lack of fit was not significant, relative to the pure error. Adequate precision (84.54) indicated an adequate signal to navigate the design space. Based on the ANOVA results . The three selected components are depicted in the corners of the triangle, and the three-dimensional contour plots were drawn to study the interaction effects. The fitted linear special regression equation was generated as shown below.Response III: The adequate precision for the linear model of Response III was 36.366, which is greater than 4, suggesting that the model could navigate the design space. The ANOVA results confirmed the significant effect of the linear mixture . Based on these criteria, the desirability plot was generated with a D value of 1 . In concIn the end, the experimental design was further validated to confirm its accuracy. The optimized formulation was prepared with the given conditions and evaluated for the globule size, Jss, and inhibition zone. The relative error was calculated by comparing the predicted and practical values, as shown in The percentage of permeability through the cellulose membrane for the QRS and ITT was found to be 33.35 ± 2.17% and 59.43 ± 4.23% after 12 h . The JssEx vivo studies were conducted using a simple, low-head dissolution apparatus to establish the effect of the ITT-QRS formulations. The permeation was studied using rat full-thickness skin, and the results are summarized in Despite several notable advancements, hepatic safety and hepatic cell regeneration products are inadequate for treating several liver disorders. Numerous herbal extract formulations were also used in this approach ,45. QRS Consistent with these results, a significant rise in the MDA levels and lipid peroxidation and a reduction in the antioxidant enzymes were supported by a significant decrease in the GSH-Px level.p > 0.05) emerged in Group 5, which was treated with the formula that lacked QRS and the commercially available ITT formulation, and Group 3, which was treated with topical ITT in an aqueous dispersion, for all tests.Pretreatment with a formulation containing ITT plus quercetin for 14 days significantly increased the antioxidant characteristics and reduced the amount of lipid peroxidation, and this reduced the overall oxidative stress. All changes obtained with the QRS formulation (Group 4) were more beneficial in ameliorating hepatotoxicity compared with the formulation without QRS (Group 5). Additionally, Group 4 differed significantly from the marketed product (Group 7). No significant differences . A lower concentration of ITT favored small vesicle formation. The optimum formulation consisted of 0.25 g of rosehip oil, 0.45 g of a surfactant (Lauroglycol-90), and 0.3 g of a cosurfactant (propylene glycol). The range of relative error was acceptable (<5%), confirming the preciseness of the design. In vitro and ex vivo permeation studies confirmed the enhanced percentage of permeation of ITT and QRS, and in vivo studies further supported these results. The QRS had commensurate hepatoprotective activity in the prepared formulation and was found to be safe in comparison with formulations containing individual drugs and commercially marketed products. However, further clinical investigations are required to explore the commercial viability of the product.


Introduction: Autonomic dysfunction is a common and disabling non-motor symptom of Parkinson's disease (PD). We aimed to understand autonomic dysfunction in PD motor subtypes, the pattern of sympathetic skin response (SSR) to motor asymmetry, and the association of SSR with autonomic and motor dysfunctions.Methods: A total of 101 PD patients of Han Chinese were included. Unified PD rating scale (UPDRS), scales for outcomes in PD-autonomic symptoms (SCOPA-AUT), orthostatic hypotension, and SSR were evaluated.Results: SCOPA-AUT and incidences of orthostatic hypotension and absent SSR were worse in the subtype of postural instability gait disorder (PIGD) than the subtypes of tremor dominant and intermediate. SSR latency and amplitude were asymmetrical corresponding to the accentuation of motor severity. Patients with absent SSR had worse UPDRS and SCOPA-AUT scores. SSR parameters of the severe side in patients with SSR showed no independent association with the scores.Conclusion: Our results support that autonomic dysfunction is more severe in the PIGD than other subtypes and demonstrate an asymmetry of SSR in PD patients. Absent SSR may indicate worse autonomic and motor symptoms, but SSR parameters are not sufficient to evaluate the severity of the dysfunctions. Parkinson's disease (PD) is a common neurodegenerative disease caused by the progressive loss of dopaminergic neurons in the substantia nigra pars compacta. Patients not only suffer from motor inconveniences and non-motor symptoms such as cognitive impairment, psychosis, and autonomic dysfunction but also impaired quality of life . SimilarAutonomic dysfunction is one of the most common non-motor symptoms in PD and is considered to be heterogeneous as well . All regSympathetic skin response (SSR) is a simple and non-invasive method used to evaluate skin sympathetic sudomotor function based on conductance changes in response to stimuli , 13. ThiA total of 101 PD patients of Han Chinese ethnicity consisting of 49 men and 52 women were recruited in this study. The mean age of the patients was 65.2 ± 8.3 years. All patients were diagnosed by two movement disorder neurologists according to the UK Parkinson's Disease Society Brain Bank Criteria. Excluded participants were those with a family history of PD, secondary and atypical parkinsonism, or confounding factors potentially involving autonomic symptoms , on medications potentially influencing autonomic symptoms , and being diagnosed with cognitive impairment which would otherwise compromise our assessment. All included patients signed written informed consents. This study was approved by the ethics committee of the Second Affiliated Hospital and Yuying Children's Hospital, Wenzhou Medical University.Clinical information including age at onset, disease duration, L-dopa equivalent daily dosage (LEDD), scale for outcomes in PD for autonomic symptoms (SCOPA-AUT), and non-motor symptom assessment scale (NMSS) was collected through face-to-face interviews. Unified PD rating scale (UPDRS), Hoehn–Yahr stage, orthostatic hypotension, and SSR were evaluated 12 h after stopping antiparkinsonian medication, a practically defined OFF medication state . The sevEach of the patients was classified into subtypes of tremor-dominant (TD), postural instability and gait disturbance (PIGD), or intermediate as described previously , 21. In P < 0.05 was considered statistically significant.Data were analyzed using the statistical package of Predictive Analytics Software 19.0  for Windows. Distribution normality was evaluated by the Kolmogorov–Smirnov test. Differences in gender, orthostatic hypotension frequency, and absent SSR frequency between PD subtypes were assessed using the Chi square test. Differences in age, age at onset, UPDRS-Total score, UPDRS-III score, and SCOPA-AUT score were analyzed by one-way analysis of variance. Differences in disease duration, Hoehn–Yahr stage, LEDD, and NMSS score were analyzed using the Kruskal–Wallis test or Mann–Whitney test. Multivariate analysis was performed by multiple linear regression model or binary logistic regression model using the stepwise forward method with subtypes, gender, age, age at onset, duration, LEDD and UPDRS-III score as covariates. Differences in SSR parameters between the severe and less-severe sides were analyzed by the Wilcoxon signed-rank test. Spearman rank correlation was used to test the association between SSR parameters and scores of SCOPA-AUT or UPDRS-III. A two-tailed P > 0.05) among the three subtype groups (P < 0.001), UPDRS-III (P = 0.013), Hoehn–Yahr stage (P < 0.001), NMSS (P < 0.001), SCOPA-AUT (P < 0.001), orthostatic hypotension frequency (P = 0.011), and absent SSR frequency (P = 0.001). Numeric results of all these measurements were higher in the PIGD subtype compared to the other two subtypes. However, for the ones with SSR latency and amplitude detected, SSRs of the severe side in both upper and lower limbs showed no significant difference among the three subtypes  and orthostatic hypotension , urinary (P = 0.002), cardiovascular (P = 0.001) and thermoregulatory regions  and lower limbs , but not associated with SSR latencies of both limbs , but not associated with other ones (The patients with absent SSR had worse UPDRS-Total, UPDRS-III, and SCOPA-AUT scores compared with those with SSR . The latth limbs . In contth limbs . Analysiher ones . We alsoher ones . To clarher ones .Autonomic dysfunction is a frequent and disabling complication of PD. In this study, we analyzed autonomic symptoms in a currently largest group of Han Chinese PD patients. The results disclose a heterogeneity of dysautonomia in PD subtypes, an asymmetry of SSR, and the role of SSR absence and parameters in autonomic and motor symptoms.PIGD and TD are two typical motor subtypes of PD. A number of studies have demonstrated that certain non-motor symptoms differ among these two subtypes as well as the subtype in between , 22–24. While motor manifestations often develop and persist asymmetrically in PD , 9, we oOrthostatic hypotension is a key manifestation of cardiovascular dysautonomia in PD and is believed to be caused by degeneration of postganglionic sympathetic neurons which resulted from alpha-synuclein deposition. Such incidence in PD is around 30–40% . HoweverIn conclusion, our results support that autonomic dysfunction is more severe in patients of the PIGD subtype and demonstrate an asymmetry of SSR in PD. As evidenced by the analyses with SCOPA-AUT and UPDRS-III scores, absent SSR may indicate worse autonomic and motor symptoms, but SSR parameters of the severe side are not sufficient to evaluate the progression of the dysfunctions.The original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by Ethics Committee of the Second Affiliated Hospital and Yuying Children's Hospital, Wenzhou Medical University. The patients/participants provided their written informed consent to participate in this study.J-YW, M-YW, R-PL, YL, and XZ examined the patient and acquired and analyzed all clinical data. J-YW, M-YW, W-YZ, and BO assessed the orthostatic hypotension and sympathetic skin response. J-HZ and J-YW reviewed literatures and drafted the manuscript. XZ and J-HZ supervised the study. All authors read, revised, and approved the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


ACL reconstruction (ACLR) is a common procedure requiring rehabilitation in public hospital physiotherapy departments. The rate of re-rupture and reduced rates of return to sport following ACLR are concerning. Current guidelines recommend a progressive approach to rehabilitation based on objective criteria. The aim of this study was to determine whether a new public hospital model of care incorporating a phase-based program increased physiotherapist utilisation of objective outcome measures, improved service metrics including attendance and rehabilitation completion rates, and increased patient-reported activity and knee function.N = 132) were included in a retrospective chart review to assess utilisation of objective measures such as quadricep and hamstring strength assessment, patient attendance and rehabilitation completion. Phone followup (minimum 1 year) was conducted to retrieve patient-reported measures of knee function (IKDC) and activity . Patients were categorised by rehabilitation model of care (contemporary - time based [N = 93] vs new - phase based [N = 39]) and logistic regression used to assess the influence of patient factors and model of care on outcomes.Records from patients attending outpatient physiotherapy after ACL reconstruction  were low (30–38%). The probability of a patient receiving objective strength assessment was associated with model of care, sex, BMI and number of sessions attended. The probability of a patient being recorded as discharged from the program was significantly associated with model of care, and duration and number of sessions.Introduction of an updated model of care including a phase-based rehabilitation program increased physiotherapist utilisation of objective outcome measures in line with current ACLR rehabilitation recommendations, increased total rehabilitation duration and increased total number of sessions attended. Despite this, rehabilitation completion rates remained low, and self-reported activity and knee function remained equivalent.III, retrospective cohort study.The online version contains supplementary material available at 10.1186/s40945-020-00093-9. Anterior cruciate ligament (ACL) rupture is a common injury that usually occurs during non-contact pivoting or twisting movements of the knee , 2. A ruIncreased physiotherapist utilisation of objective outcome measures to guide clinical decision makingImproved service metrics such as attendance and rehabilitation completion ratesIncreased self-reported knee function and activity levelsVariation in rehabilitation exists with respect to setting, progression, duration and return to functional activities and sport , 9, 10. An observational cohort study with historical control was used to review service metrics and utilisation of objective outcome measures before and after a change in model of care for ACLR rehabilitation. Ethical approval was granted by the Metro South human research ethics committee (HREC/16/QPAH/732) prior to patient screening and data retrieval.N = 262). Procedures coded as revision surgery (N = 3) and surgeries outside the specified date range (N = 35) were removed. During the chart review process, revision ACLR procedures, a primary ACLR with concurrent surgical treatment of other ligaments  and those referred to an external physiotherapy service provider on discharge from the ward were excluded (N = 92). The final list included 132 patients who had undergone ACLR and rehabilitation at the hospital outpatient physiotherapy department. The final 132 patients were split into two groups using a surgery date of 1st of November 2016 as the group cut off. This resulted in all patients in the NEW model of care undergoing all rehabilitation using the updated model of care, however there may have been some crossover in those in the CONTROL group. .A list of patients who underwent primary ACLR at a metropolitan public hospital, between November 2014 and December 2017 was exported from the operating theatre management system by hospital administration , which was adjusted following feedback from the pilot review. All time-related data was referenced from the date of surgery. After the data collection tool was finalised, the pilot charts were reviewed again using the finalised tool. Data collection for the remaining 82 patient records was then undertaken by a single physiotherapist to negate the potential for limited inter-observer reliability.Follow-up phone calls were made between one- and three-years post-surgery to all patients who had undergone an ACLR and had received some form of rehabilitation at the physiotherapy outpatient department. Patients who completed rehabilitation, were removed due to lack of attendance, or withdrew during the rehabilitation process were contacted by a single physiotherapist using phone numbers provided by hospital administration. Data was collected regarding patient’s recollection of their rehabilitation and return to activity and sport. Reasons for patients failing to complete rehabilitation was also explored. Patients were sent an SMS advising them the research team would be attempting to call and included a link to a web-based form (Survey Monkey) asking their preferred time to be contacted. After three unsuccessful attempts to contact a patient, a web-based form (Survey Monkey) was communicated via text message for patients to complete in their own time.‘Physiotherapist adherence to objective measures’ and ‘service metrics’ were collected via the chart audits to assess adoption of the new model of care.Quadriceps strength,  or hand-held dynamometry (HHD)) OR 1 repetition max (1RM) leg pressHamstring strength (MMT or HHD)Kinetic chain strength Hop tests (single leg hop for distance or triple crossover hop)Balance  or single leg balance)Knee range of motion (ROM)Time to return to running (weeks)The following outcome measures were recorded as assessed or not assessed (yes or no):Number of individual sessions attendedNumber of group sessions attendedNumber of failed attendancesTotal occasions of serviceRehabilitation completion statusLength of physiotherapy input (weeks)Number of cliniciansThe following service metrics were also collected to assess changes in service utilisation by patients:IKDC subjective scores, Tegner activity scores and ACL rerupture rates were collected via follow-up phone calls. The IKDC is an 18 question evaluation that measures symptoms (7 items), activities of daily living (9 items) and sport (1 item), and comparative knee function .X2 analysis with likelihood ratio for categorical variables with > 2 responses, or Fisher Exact test otherwise. Attendance ratios were calculated between the total number of sessions labelled failed to attend relative to the total number of sessions for each group and compared with Fisher’s exact test. Loss of knee extension was defined as 5° or greater fixed flexion angle and the proportion of positive (> 5o) measurements were compared between groups with Fisher’s exact test. Backward stepwise binary logistic regression models were constructed to compare service metrics (quadriceps and hamstrings assessment) and rehabilitation status . A similar model was constructed for ipsilateral rerupture incidence with a weighting vector included to compensate for unbalanced proportions between response categories. Alpha for univariate analyses was set at 0.05, while model alpha for variable inclusion was set at 0.15. Model fit was assessed with adjusted R2 (%) and effect sizes expressed with odds ratio with 95% confidence intervals.The completed chart review spreadsheet  and patient followup response sheet  were transferred into Matlab  and linked by patient unique identifier (person-level linkage) into one combined dataset for analysis in a statistical software package . Categorical data was recoded to standardize spelling variations or for reasons listed Table . Continu2]; 64.4% male; 42.4% Non-Australian country of origin) meeting inclusion criteria for analysis  and the new model of care . The two groups were equivalent for baseline characteristics, except for the proportion of females )  and the duration (weeks) of physiotherapy input . Patients discharged due to a failure to attend appointments was 59% NEW compared to 49.5% CONTROL, and 10.3% NEW compared to 12.9% CONTROL (p = 0.61) withdrew from physiotherapy (Table N = 53), with 39% stating they believed they had been discharged, while 32% found it too hard to attend  was above the recommended threshold score of 75.9  in 55.6%The probability of a quadriceps strength assessment during rehabilitation was associated with longer physiotherapy duration and the NEW model of care Table . Those wOur results suggest that a phase-based progressive rehabilitation program leads to increased utilisation of a number of objective measures by treating physiotherapists. A phase-based model also appears to influence certain service metrics, resulting in a longer duration of physiotherapy rehabilitation and a higher number of attended physiotherapy sessions. It does not appear to increase rehabilitation completion rates, or rates of return to sport and activity. Following the introduction of a phase-based model of care, the incidence of physiotherapists using objective outcome measures significantly increased for assessment of quadriceps and kinetic chain strength or endurance , and neuromuscular control with single leg squat performance. This finding suggests that following the NEW model of care, physiotherapists were more frequently performing more comprehensive objective assessment to guide rehabilitation. The importance of monitoring and increasing lower limb muscle strength, particularly quadriceps, in rehabilitation post ACLR is well accepted within current literature , 22–24.There is currently no ideal duration or dose of physiotherapy identified in the literature , 17, 18.‘Thought they were discharged’ and ‘happy with knee’. Both these reasons indicate a potential disconnect between patient and physiotherapist expectations. In some cases, physiotherapists may be striving to achieve rehabilitation goals based on existing literature in sporting populations; however, not all public system ACLR patients may share these goals. The public hospital ACLR cohort includes a number of patients who participate in social sports but do not perform regular strength and conditioning exercise. This can make it difficult to engage patients to perform regular resistance exercise to achieve adequate strength gains recommended in ACLR rehabilitation [Follow-up phone calls investigated reasons why patients may fail to attend appointments, and subsequently be discharged from the service. Two of the most common reasons identified were litation . This calitation  and motiRegardless of the model of care, poor attendance rates, low rehabilitation completion rates and reduced functional outcomes were observed. This finding is somewhat surprising as it was hypothesised that a clear rehabilitation pathway guided by objective outcome measures would have increased patient’s functional outcomes and subsequently completion rates. While beyond the aim of this study, evaluating the relationship between quantitative objective data throughout the rehabilitation process with the patient’s level of function on discharge and follow-up could provide additional insight into the impact of criteria-based rehabilitation. These findings also highlight the importance of both patient selection for surgery, and the need for standardised education from all clinicians (surgeons and physiotherapists) regarding the intense rehabilitation requirements post ACLR. Heightening patients’ expectations may contribute to increased motivation and improved attendance . Given tAlthough this study has provided new information regarding the implications of new models of care in an ACL rehabilitation setting, its limitations should also be recognised. The retrospective, observational design precludes the determination of causal relationships between our findings and the model of care change. Additionally, some participants engaged in rehabilitation across both models due to the use of a specific cut-off date in group allocation. It should also be recognised that data collection was not blinded, and the researcher was involved in the service development and data collection. Data were captured from clinical notes that were not specifically documented for research purposes, and documentation was occasionally unclear and required interpretation, contributing to potential misclassification bias. When collecting data pertaining to physiotherapist use of outcome measures, if a physiotherapist had assessed that outcome measure at least once in their rehabilitation it was considered compliant, potentially leading to an over-representation of outcome measure use. A low response rate to the phone call follow-ups also made it difficult to extrapolate statistical relationships between the model of care change and return to sport or activity rates. Lastly, this study collected data on physiotherapy utilisation of outcome measures, however it did not report on specific clinical patient outcomes. A prospective study investigating whether rehabilitation attendance and duration in a public hospital setting impacts the achievement of certain evidence-based outcomes  would help inform ALCR rehabilitation models of care.This study determined that in patients electing to undergo supervised rehabilitation in a physiotherapy department in a public hospital, a new model of care incorporating a phase-based rehabilitation program achieved the following; an increase in physiotherapist utilisation of objective physical measures, namely, quadriceps strength, kinetic chain muscle strength, and single leg squat performance; an increased total duration of rehabilitation; and an increased total number of physiotherapy sessions attended. Despite this, rehabilitation completion rates decreased with the new model of care. Further work is required to understand the relationships between rehabilitation models of care and key patient metrics, particularly functional outcome.Additional file 1: Phases, Outcome Measures and Discharge Criteria.Additional file 2:  Clinical practice: models of care.


Allergic rhinitis (AR) is a type of inflammatory condition that includes a group of symptoms, mainly affecting the nasal mucosa. Nasal obstruction, sneezing, stuffy or runny nose, in addition to swollen, itchy, red and watery eyes are the most common symptoms of the disease. These symptoms are triggered as a result of increased inflammatory mediators such as histamine and leukotrienes. Studies have recently shown the role of vitamin D (vit.D) in many allergic and immune conditions, where receptors for the active form of vit.D  have been discovered on the surface of almost all types of inflammatory cells. Therefore, the present study was conducted to explore the level of vit. D in AR patients and its correlation with the severity of the disease. Two groups participated in the study; the first group included 49 patients who were diagnosed in a private otolaryngology clinic by the first author as having allergic rhinitis (AR group). The second one served as a control group and included 50 apparently healthy volunteers with no history of AR. The mean level of IgE and vit. D was found to be 326.3 and 10.2 ng/ml in the AR group, respectively, and 30.8 and 23.3 ng/ml in the control group, respectively. Ninety-three percent of AR patients have shown a deficiency in vit. D level, where 56% of this group showed severe deficiency. On the other hand, 34% of the control group has shown an insufficient level of vit. D. Additionally, 64% of AR patients have shown serum levels of IgE at values ranging between 100-299 ng/ml. Higher serum levels of IgE at values ranging between 300-599 ng/ml and 600-1000 ng/ml were observed in 25% and 11% of AR patients, respectively. The prevalence of low levels of vit. D in the AR group was significantly higher than that in the control group (P < 0.001). Vit. D deficiency is significantly related to severe AR symptoms and measuring serum vit. D level is recommended in the management plan of this group of patients. Allergic rhinitis is an inflammatory condition of the nasal mucosa. Typical symptoms include nasal obstruction/ congestion, itching, watery nose, and sneezing . AlthougTraditionally, AR was classified as seasonal (symptoms appearing in a particular season) or perennial (symptoms throughout the year). This classification is no longer employed since some allergens may be seasonal in some regions and perennial in others, and many patients have multiple seasonal attacks throughout the year . A more Symptoms of AR are triggered by inflammatory mediators such as histamine and leukotrienes released as a result of increased immunoglobulin E (IgE) production from plasma cells. This increased production of IgE is mediated by cytokines released from inflammatory T cells invading the mucosa of the nasal cavity in response to the exposure of the mucosa to exogenous allergens . Vit. D Regarding the specific role of vit. D in AR, a literature search has shown a discrepancy in findings. For example, Wjst and Hyppönen found that the incidence of AR increases with serum levels of vit. D in Finland . SimilarAccordingly, this study aims at examining the correlation between vit. D levels and AR in a sample population of the city of Mosul, Nineveh province in the north of Iraq.This study was conducted in Mosul city from March to October 2018 and involved two groups; the first group comprised AR patients diagnosed in a private fully equipped otolaryngology clinic by the first author as having allergic rhinitis (AR group). Forty-nine patients were clinically diagnosed, and nasal endoscopy was performed on every patient to exclude other conditions such as sinusitis, nasal polyposis, and nasal septal deviation. The diagnosis was made according to the “AR and its Impact on Asthma” (ARIA) guidelines, a runny nose and nasal obstruction as the main complaints . The secInclusion criteria included patients with AR attending the private clinic of the first author with age ranging between 18-55 years.Exclusion criteria involved AR patients who have a body mass index (BMI) greater than 26 kg/m2, patients with inflammatory or immunological conditions such as asthma, nasal polyposis, and rheumatoid arthritis, patients with chronic illnesses such as diabetes mellitus, renal insufficiency, and abnormal vit. D metabolism, in addition to patients receiving chronic or recent therapy with steroids, antihistamines, vit. D supplements and chemotherapeutic agents.All values were expressed as the mean ± standard deviation (SD) or standard error of mean (SEM) where indicated. Student’s paired t-test for single data comparison was performed. Pearson’s correlation was used to analyze the relationship between the studied parameters. GraphPad Prism 8.0 software was utilized to assess the statistical significance (P < 0.05) of any difference between the mean values.The studied sample was divided into two groups: the AR group included AR patients of the same age, sex and weight as the control group, which included healthy volunteers. Characteristics of the studied population are summarized in Most patients in the AR group (71%) had moderate to severe persistent AR. At the same time, the mild intermittent class was shown to be the least presented when compared to the other investigated classes of the disease, as shown in The mean level of IgE and vit. D was found to be 30.8 and 23.3 ng/ml, respectively, in the control group and 326.3 and 10.2 ng/ml, respectively, in the AR group. Ninety-three percent of AR patients have shown a deficiency in vit. D levels, where 56% of this group showed severe deficiency. On the other hand, 34% of the control group has shown an insufficient level of vit. D. Sixty-four percent of AR patients have shown serum level of IgE at values ranging between 100-299 ng/ml, while 25% and 11% of AR patients have shown higher serum levels of IgE at values ranging between 300-599 ng/ml and 600-1000 ng/ml, respectively. The prevalence of low levels of vit. D in the AR group was significantly higher compared to the control group (P < 0.001), as shown in Vitamin D is an essential nutrient required for healthy bones and the immune system. It has two major forms: ; both forms can be obtained from foods. However, only vitamin D3 can be made by the human body . The parThe association between immune diseases and 1,25(OH)2D3 had been documented since 1984. A link between allergic disorders  and vit. D levels had been reported in many epidemiological studies . SeveralAge, weight, type of food, skin pigment, lifestyle, residence and sun exposure are factors that could affect vit. D levels in one way or another. Elderly, as well as overweight or obese individuals, usually have low vit. D levels. Also, diets low in fish and dairy are associated with vit. D deficiency. People with dark skin, persons frequently using sunscreen, wearing long sleeve shirts, head cover, traveling by car, having a sedentary lifestyle, limited availability of the sun in the living area, as well as variation in sun exposure due to season variation, time of the day exposure and atmospheric components, could all affect the vit. D status , 20. IraThis study was directed to explore the relationship between vit. D serum level and the prevalence of AR in both patients and healthy subjects. Total serum IgE is not helpful for the diagnosis of AR according to the ARIA guidelines. Despite this, IgE was investigated in this study. Our hypothesis to include IgE was that vit. D is an immunomodulator, and its deficiency can lead to an increased allergic response. Therefore, IgE may be associated with vit. D deficiency for the same reason.The study revealed that 93% of patients diagnosed with AR had a deficiency in vit. D levels. We also found an association between IgE levels and vit. D deficiency in these patients; this association was found to be stronger in patients with moderate to severe persistent AR.The present study showed a significant difference in the mean serum level of vit. D between the healthy group and the AR group (p < 0.001). The statistics revealed that most of the patients were diagnosed as having the severe class of the disease (91.8%); the low number of patients in our study with mild AR  may be explained by the fact that these patients commonly depend on self-prescribed medications due to mild symptoms.Our study showed that the prevalence of vit. D deficiency is obvious among the AR group, which matches the findings of Sudiro et al., who reported a correlation between the severity of vit. D deficiency and the severity of AR. In addition, Vatankhah V. et al. described the AR group as a vit. D deficient group in comparison to people with normal vit. D levels , 21. HowHypovitaminosis D was reported in many literature studies conducted in the Middle East, revealing that, despite the plentiful sunny climate in this part of the world, the region registers low vit. D levels among different age groups. Factors like insufficient vit. D intake during infancy, economic causes, inadequate sun exposure, traditional clothing style, and urban living may contribute to the low level of vit. D. The findings of our study are consistent with these global annotations .The serum concentrations of vit. D that are associated with deficiency, adequacy, and optimum overall health are still tremendously questionable. The Institute of Medicine (IOM) states that the serum concentrations of vit. D less than 12 ng/ml may predispose people to a risk of vit. D deficiency. Serum levels between 12–20 ng/ml predispose to a potential risk of inadequacy. Almost everyone is considered to have appropriate vit. D levels at serum values greater than 20 ng/ml. Additionally, the committee of IOM reported that 20 ng/ml is the amount of serum vit. D serving the needs of about 97.5% of the population. Additionally, serum levels greater than 50 ng/ml may associate with possible adverse effects .Our study suggests that vit. D level is one of the parameters that have to be checked in AR patients, in accordance with the findings of other similar studies which correlate vit. D deficiency to AR , 24. TheVit. D deficiency is linked with the severity of AR, and monitoring serum vit. D levels is advisable in this group of patients. In addition, adjustment of vit. D levels is sensible in apparently healthy people as the results of this study have found a subclinical level of vit. D in a significant number of healthy volunteers. Moreover, future studies are recommended to investigate the role of the administration of vit. D as an add-on therapy for AR patients that have a low level of vit. D.The authors declare that there is no conflict of interest.


An optimal osteotomy angle avoids shortening of the first metatarsal bone after hallux valgus surgery and therefore reduces the risk of transfer-metatarsalgia. The purpose of the present ex-vivo study was to investigate whether augmented reality (AR) would improve accuracy of the distal osteotomy during hallux valgus surgery.n = 15 × 2) or without  overlay of a hologram depicting an angle of osteotomy perpendicular to the second metatarsal. Subsequently, the deviation of the osteotomy angle in the transverse plane was analyzed.Distal osteotomies of the first metatarsals were performed on a foot model by two surgeons with different levels of surgical experience each with . However, while the inexperienced surgeon performed more accurate osteotomies with AR with a mean angle of 6.4 ± 3.5° compared to freehand 10.5 ± 5.5° (p = 0.02), no significant difference was noticed for the experienced surgeon with an osteotomy angle of around 3° in both cases.Overall, AR decreased the extent of deviation and the AR guided osteotomies were more accurate (4.9 ± 4.2°) compared to the freehand cuts (6.7 ± 6.1°) by tendency (This pilot-study suggests that AR guided osteotomies can potentially improve accuracy during hallux valgus correction, particularly for less experienced surgeons. Hallux valgus deformity is a one of the most common deformities of the foot . Once coThe purpose of the present ex-vivo study was to investigate whether overlaying a hologram to reality would improve accuracy of the distal osteotomy for hallux valgus surgery.We hypothesized that overlaying a hologram will increase the accuracy of distal MT I osteotomy, especially when carried out by inexperienced surgeons.The experiments were performed on a foot dummy consisting of a polyamide forefoot skeleton with an exchangable first metatarsal bone (MT I) with a soft tissue cover, made of medical silicone Fig. .Fig. 1FTo build the 3 dimensional forefoot skeleton, a CT scan  from a cadaveric foot with hallux valgus deformity was conducted under axial load of 350 N in a custom made clamping system. The data of the 3 dimensional model of the foot were segmented and implemented in the preoperative planning software CASPA . The foot showed a moderate hallux valgus deformity with an intermetatarsal angle (IMA) of 15°. In order to obtain three different hallux valgus manifestations , the MT I was rotated parallel to the plantar plate to an IMA of 18°  and 13°  through the center of the sphere defined by the proximal articular surface of the MT I. A pluggable connector was incorporated in the model between the MT I and the medial cuneiform bone to permit exchange of the MT I after completion of an osteotomy. The polyamide skeleton was printed with 60 replaceable MT I  using selective laser sintering .The soft tissue cover of the foot dummy model was generated by using a plaster cast mold of the forefoot and midfoot of the cadaver. The cast was lined with a dividing layer  and filled with medical silicon . The polyamide skeleton model was subsequently immerged in the silicone before the silicon had cured.A dorsomedial approach through the silicone cover to the MT I was performed (AFV and SMZ). Finally, the model was fixed to a wooden frame with 3 dimensional markers attached to it for facilitated alignment of the hologram.For the augmented reality (AR) experiment, a cutting plane perpendicular to the axis of the second metatarsal bone and perpendicular to the plantar plate was integrated in the model to serve as cutting guide in the experiments Fig. . The modThe distal osteotomy of the MT I was performed using an oscillating saw blade . The cuts were alternately carried out freehand or with an overlaid hologram wearing the HMD Fig. . They wep < 0.05), assuming normal distribution of data. Cronbach’s Alpha was used to measure internal consistency (inter observer correlation). A post hoc power analysis was performed with G*Power .A second skeletal model Fig.  was prinResOrtho Foundation, University Hospital Balgrist. Funding source did not play a role in this investigation.p = 0.2)  more accurate with an angle of 6.4 ± 3.5° (range 19) compared to the freehand osteotomies with an angle of 10.5 ± 5.5°  with help of AR, and 6.7 ± 6.1° in anterior direction for freehand osteotomies. The difference between the osteotomy angle between freehand performance and experiments with AR was not significant if the combined average performances of both surgeons were considered (.2) Fig. . However.2) Fig. . For theP = 0.051). There was no significant difference in the precision of osteotomy angles for the different simulated manifestation of the deformity Fig. Although the more experienced surgeon also achieved better results with AR-support than the less experienced surgeon, this finding was not significant enough in our series of experiments  of our cases.The analysis showed excellent inter-observer reliability with a Cronbach’s Alpha of 0.978.The post hoc power analysis stated a power 1-β of 0.92 for an effect size d of 0.8 and a power of 0.61 for an effect size of d = 0.5 (α = 0.05).The aim of the present study was to investigate whether AR improves accuracy of the osteotomy angle in distal hallux valgus surgery. We hypothesized that overlaying a hologram helps surgeons perform the osteotomy more accurately, especially when inexperienced. We present the first report on potential merits of a promising new technology (AR) in a randomized ex-vivo study, and report the accuracy of the distal osteotomy during hallux valgus surgery with and without the support of this new technology with different levels of surgical experience.p = 0.02) by 4°. Without AR, the osteotomy revealed a mean deviation of 10.5° from the plane perpendicular to the second metatarsal. In accordance, the range of accuracy was much larger without AR-support. This was also true if the results of both surgeons were considered. The here observed important differences outline the power of AR guided osteotomies to decrease the learning curve of less experienced surgeons. This finding is in line with the results of other types of navigated surgery [When viewing the results for the two surgeons individually, the experienced surgeon did not perform the osteotomies more accurately with help of AR. The inexperienced surgeon however obtained better results when using AR technology. The accuracy of the osteotomy angle improved significantly for the osteotomies with AR ( surgery . A corre surgery . For an The power analysis showed a sufficient power for an effect size of 0.8, but not for an effect size of 0.5. The observed tendency towards a higher accuracy for AR performed osteotomies for both surgeons might become statistically significant for higher sample sizes. We believe an effect size of 0.8 to be sufficient, as small deviations in the osteotomy angle are not clinically significant.Furthermore, a higher sample size in this experimental set up also risks that the freehand osteotomies are performed more accurately due to a learning curve resulting from the AR guided osteotomies.Although a lot of effort was made to create a realistic foot model, artificial bones with a soft tissue cover were used. It is likely that the alignment of the hologram, although simplified by the frame of the foot, is a source of error. Further sources of error include precision of the augmented reality equipment, and performing the osteotomy guided by the hologram. Finally, the present study cannot quantitively assess the influence of each source of error Advancements of the holographic technology already allow a better alignment of the hologram.Considering the experimental setup, we conclude that AR is a powerful technology with the potential to improve accuracy in hallux valgus correction, particularly for less experienced surgeons. Following the here presented first report of use of AR in foot surgery, clinical studies are necessary to verify whether this promising technology will improve the outcome following hallux valgus surgery.


The role of the Androgen Receptor (AR) expression and its activity in the prognosis of hepatocellular carcinoma (HCC) remains inconclusive. The aim of this study is to analyze the role of the AR expression and its activity as prognostic biomarkers in HCC. Three-hundred and thirty-seven patients from The Cancer Genome Atlas (TCGA)  were included. To infer AR activity, the expression-profile of previously validated androgen responsive genes (ARGs) was included. AR activity was shown by the AR-Score-21 (21 ARGs) and AR-Score-13 (13 ARGs) that were computed based on the expression of the selected ARGs. Those ARGs whose expression was significantly different between histological grades were used for computing two new AR-Scores. HCC patients with higher AR expression showed a higher median overall survival (OS). AR-Score 21 and AR-Score-13 did not show any association with prognosis. Six of the 21 ARGs of the AR-Score-21 and 7 of the 13 ARGs of the AR-Score-13 showed a significant different expression profile among histological grades. Based on these differences, another two AR-Scores were computed (AR-Score-6 and AR-Score-7). They showed the relative increase of upregulated to downregulated ARGs in high-grade HCC. Higher AR activity inferred by these AR-Scores was associated with worse outcomes. The expression of AR is associated with a better prognosis in HCC. However, the activity of the AR seems to be qualitatively different among histological grades. The AR activity inferred by the shifted ARGs is associated with a worse prognosis in HCC patients. HCC has a male predominance, with more than double the incidence in men than in women4. This male dominance has also been reported for other cancers like gastric cancer5, bladder cancer6 or primary brain tumors7. The rationale of this male dominance in these cancers is not completely understood, but it may be associated with sexual hormones or their receptors9. This association has not completely confirmed unlike other tumors (e.g. prostate cancer or breast cancer) where the role of sexual hormones and their receptors have been studied in depth11.Hepatocellular carcinoma (HCC) is the most common primary tumor of the liver, with 854,000 new cases/year and it is the second cause of cancer related death around the world 12. The AR regulates the expression of specific target genes, also known as androgen-responsive genes (ARGs) which may or may not have one or more AREs13. Many ARGs have been identified and experimentally validated, mainly in prostate cancer cell lines14. The expression signatures of some of these ARGs have been used to calculate a score that putatively reflects the AR activity and can be used to identify different genomic prostate cancer subtypes15. Thus, the use of the ARGs expression profile to infer AR activity seems to be an adequate tool to study the role of AR activity in cancer.The androgen receptor (AR) is a transcription factor that, once activated , binds to the DNA in specific regions called androgen response elements (AREs)16. In other words, the ARGs expression profile seems to be different in tumoral and in normal tissues17. Furthermore, the ARGs expression also seems to be related with the period when the AR is exposed to androgens18. Therefore, AR activity and its associated up- or downregulated ARGs seem to vary among different microenvironmental and cellular conditions.However, ARGs may alter their expression profile depending on the microenvironment. In this respect, Pomerantz et al. showed that the AR binds to specific AREs in tumoral cells and normal prostate cells, in different ways19. Generally speaking, it has been demonstrated that AR expression is variable, but a higher expression in tumoral tissue than in normal surrounding tissue has been described22. Furthermore, the AR has been associated with hepatocarcinogenesis25. However, published literature about the relationship of the AR with HCC pathogenesis and evolution is controversial. On the one hand, a relationship between protein and mRNA AR expression with a better prognosis has been reported26. On the other hand, other authors have described a positive correlation between the AR expression at nucleus with more advanced tumoral stages and, consequently, with a worse prognosis20. In this regard, the recurrence rate of HCC patients was higher in patients with AR expression in the tumoral surrounding tissue and the survival rate was better in AR negative patients8. Consequently, more research about the role of the AR and, mostly, about the role of the AR activity is required. This is especially important if one bears in mind that there are various specific therapeutic approaches to block the AR pathways that could be useful for HCC management.The role of AR and its activity in HCC has not been fully studied. The AR is present in HCC, but also in normal surrounding parenchyma and normal liverTherefore, the aim of the present work is to study the role of the AR expression and its putative activity as a prognostic biomarker in HCC in a cohort of patients from The Cancer Genome Atlas (TCGA) project.Three hundred and sixty-three  were included from the TCGA database (from a whole cohort of 377 patients). All patients presented a confirmed HCC (fibrolamellar carcinoma patients were previously excluded [n = 3]). Only patients whose tumor samples have at least RNAseq data available were included (n = 363). Furthermore, patients whose pathologic stage was not registered were also excluded (n = 25). Thus, a final cohort of three hundred and thirty-seven patients were analyzed. Sample features are shown in Supplementary Table http://firebrowse.org/) (TCGA data version 2016_01-28). As mentioned above, only patients with RNAseq data available were included in the study. Apart from RNAseq data, clinical, mutational, copy number variations (CNVs) and reversed phase protein array (RPPA) data from the selected patients were also downloaded and included in a new database. It should be stated that all clinical and molecular data were not available for all patients. Details of this data generation are described elsewhere27. The study has been approved by the Hospital Universitario de Canarias’ ethics committee. Informed consent was obtained from all subjects by TCGA researchers. All experiments were performed in accordance with relevant guidelines and regulations.Data from TCGA was downloaded from Firebrowse , thus a dichotomization was performed using the median (111.24 RPKM). This variable was used to compare those patients with low and high AR-RNA expression with one another. Furthermore, a survival analysis was performed to determine differences in median overall survival (OS) and median progression free survival (PFS) between the two AR-RNA expression groups.AR-protein expression data were extracted as described above. The determination of AR expression in the RPPA was performed using a specific antibody . RPPA data were already normalized and the AR expression had a normal distribution . Furthermore, AR-protein expression was also dichotomized  and this variable was used for clinical and molecular data comparisons, as well as Kaplan–Meier curves and Log-Rank test analysis.14. They were used to infer the activity of the AR. As other studies did, the activation of the AR pathway was indicated by the AR-Score26. These previous publications computed the AR-Score based on the 21 ARGs expression reported by Hieronymus et al. This AR-Score was also computed in the present work, but another AR-Score, based on the expression of 13 ARGs expression reported by Bolton et al. was also computed. To distinguish one score from the other, the first one has been called the AR-Score-21 and the second one the AR-Score-13 (the number represents the number of genes included in each AR-Score).Apart from AR expression data (at protein and RNA levels), other gene expression profiles were also extracted. All of these genes have been previously validated as androgen response transcriptional targets (ARGs)15, a Z-score was computed for the expression of each gene in each sample by subtracting the pooled mean from the RNAseq expression values and dividing the result by the pooled standard deviation. Both AR-Scores for each sample were computed as the sum of the Z-scores of each ARGs signature. None of the AR-Scores presented a normal distribution . Both AR-Scores were also dichotomized (using the median) and this variable was used to compare groups of a low (< = p50) or high (> p50) AR-Score for Kaplan–Meier curves and Log-Rank test analysis.As described in other works17 , one can hypothesize that this variation may also be present in different tumoral histological grades. To confirm this hypothesis, the expression of the genes included in each AR-Score was compared between histological grades. Those genes whose expression was significantly different between histological grades were used for computing two new AR-Scores (one for the AR-Score-21 significantly different expressed genes and another for those from the AR-Score-13).Finally, regarding the fact that the ARGs expressions is different in normal and in tumoral tissueIn these two new AR-Scores, the relative increase of genes that are upregulated to those genes that are downregulated in higher histological grades (HG) was considered. They have been computed as follows:The two new AR-Scores were also dichotomized using the median as cutoff and they were used for survival analysis in the same way as the other AR-Scores.27. After excluding those patients whose RNAseq data was not available, the ten-most-common cancer related genes that show copy number alteration (CNA) and/or mutation were studied. Those genes . Comparative analysis of the distribution of these genetic events between the two expression groups of AR (RNA and protein) and AR-Scores was performed.Copy number variation and mutation annotation files were downloaded as described above and they have been analyzed as described in previous reportsAs previously stated, the median was used to generate two comparable groups for each data to study the differences between low and high AR and AR-Scores expression. Nonparametric statistical tests were used . Statistical significance was considered when the p-value < 0.05. However, bearing in mind the high number of comparisons during molecular analysis, a corrected p value was used for these variables, using the False Discovery Rate (FDR) method. Differences were considered statistically significant when FDR < 0.1.Kaplan–Meier curves and the Log-Rank test were used to study the differences in overall survival (OS) and progression free survival (PFS) between different AR (RNA and protein) and AR-Scores expression groups. The statistical significance for survival analysis was considered when the p-value < 0.05.Finally, correlation analysis between AR-Score and AR expression (at RNA and protein levels) was performed using the Spearman Rho test .RNAseq data showed a mean AR expression of 257.9 RPKM (Reads Per Kilobase Million) (SD = 381.0) and Reversed Phase Protein Analysis (RPPA) data showed a mean AR abundance of − 0.01 (SD = 0.31) in the probes of HCC. As expected, a significant positive correlation between AR at the protein and mRNA levels was found .Analysis of copy number variations (CNVs) showed that no patient presents an amplification or deletion of the AR gene. AR mutations were identified in 3 patients (0.9%). Two of these were missense mutations and the other was a truncating mutation. In any case, none of them were considered driver mutations.A comparative analysis between patients with low (< = p50) and high AR-RNA expression (> p50) was performed using the median of AR-RNA expression . Patients in the high AR-RNA group were older than those in the low AR-RNA group . Gender distribution also shows differences between groups . Significant differences were also identified in the histologic grade distribution (p = 0.002) and vascular invasion (p = 0.014). Low AR-RNA patients presented higher histologic grades (G3 and G4) and a higher proportion of cases with micro- (31.7%) and macrovascular invasion (9.2%)  in low AR-RNA patients (FDR = 0.07)  than patients in the high AR-RNA group    Log-. No diffAs performed with AR-RNA, the median of AR-protein expression  was used to generate two groups of patients (i.e. low and high AR-protein expression groups). Clinical and molecular variables were compared between these two groups. These comparisons did not show any statistically significant difference  while the median OS in patients with low AR-protein was 21.3 (95% C.I. [3.8–38.9]) Fig. . This diAs explained in “As performed with AR-RNA and AR-protein, two groups of AR-Score values (2 for each AR-Score) were generated using the median as cutoff (p50 AR-Score-21 = − 0.72 and p50 AR-Score-13 = − 0.72). Comparisons between groups were made.Firstly, regarding the comparison between low and high AR-Score-21 groups, a higher fraction of genome altered (p = 0.001) and aneuploidy score (p = 0.001) were found in low AR-Score-21 patients  and 20.9 (95% C.I. [13.9–27.9]) in the high AR-Score-21 group, without statistical significance   anSecondly, differences between low and high AR-Score-13 were identified in the histological grade distribution ; and more genetic alterations were present in the low AR-Score-13 group   and 21.0 months (95% C.I. [9.3–32.7]) in high AR-Score-13 group, without statistical significance  Fig. . The medAs mentioned above, ARGs expression can vary between normal and tumoral tissue and this variation may also be present in different tumoral histological grades. This variation may have prognostic implications. Therefore, an analysis was conducted of the changes in the genetic expression profile between histological grades in each AR-Score. New AR-Scores were computed and tested in survival analysis with this information.On the one hand, no difference in the mean AR-Score-21 was identified among histological grades  Fig. A, but a Gene symbols represent the normalized (Z) RNA expression of each gene.The AR-Score-6 did not have a normal distribution , thus a dichotomization of the variable using the median (p50 = 0.12) as cutoff was performed. Patients with low AR-Score-6 were older than high AR-Score-6 patients   while those patients in the low AR-Score-6 group presented a median OS of 83.2 months (C.I. 95% [43.2–123.3]) . The main difference was identified between grades 1 and 3   RNA expression of each gene.The AR-Score-7 data did not have a normal distribution , thus a dichotomization of the variable using the median (p50 = 2.10) as cutoff was performed. Patients in the high AR-Score-7 group were younger  and with a larger female representation  than patients with low AR-Score-7  while those patients in the low AR-Score-7 group had a median OS of 80.7 months (C.I. 95% 56.0–105.5]) (Supplementary Table .0–105.5)Finally, a correlation analysis showed a weak negative correlation between both new AR-Scores (AR-Score-6 and AR-Score-7) with AR-RNA expression   was performed. In stage I patients (167), a significant weak positive relationship between AR at RNA level and AR-Score-21 and AR-Score-13 was identified . Furthermore, in this group of patients, a weak negative correlation between AR at RNA level and AR-Score-6 and AR-Score-7 was identified . Regarding patients with stage II (86), a significant weak positive relationship between AR at RNA level and AR-Score-21 and AR-Score-13 was identified . In this group of patients, a weak negative correlation between AR at RNA level and AR-Score-6 and AR-Score-7 was also identified . Finally, in stage III patients (84) no relationship between AR at RNA level and AR-Score-21 and AR-Score-13 was identified. However, a moderate negative correlation between AR at RNA level and AR-Score-6 and AR-Score-7 was identified . No relationship between AR-Scores and AR expression at protein level was identified in any tumoral stage group.The present study analyzes the expression profile of AR (at mRNA and protein levels) and different ARGs (synthesized in different AR-scores) in HCC and determines their prognostic value. On the one hand, a shift in the ARGs expression profile between low and high histological HCC grades was observed. On the other hand, the activity of the AR was associated with a worse prognosis. However, AR expression seems to be associated with better outcomes. Thus, the activity of the AR and its expression have opposite effects in HCC patients. All these findings will be discussed below.16. Sharma et al. reported the existence of specific AREs for each tumoral stage28. Furthermore, Wang et al. described a different expression profile when the activation of the AR in prostate cancer was dependent on or independent from androgens29.ARGs expression can vary between normal and tumoral tissues. This has previously been demonstrated in prostate cancer. Pomerantz et al. compared prostate cancer and normal prostate tissues and concluded that the AR had specific AREs in each tissue31 and, therefore, they are both downregulated in higher histological grades  and NDRG1 (N-myc downstream regulated 1) upregulation is associated with a higher histological grade  was made, taking into consideration the shift of the ARGs in higher histological grades as mentioned above. In this respect, the new AR-Scores (i.e. AR-Score 6 and AR-Score 7) seem to reflect the oncogenic pathways activated through the activation of the AR in HCC see “”.20. Thus, without a subcellular fractionation protocol, no considerations about how much AR is activated can be inferred by the global AR expression. In this sense, the AR-Score would better indicate the presence of AR activation.The relationship between the modified AR-Scores with a worse prognosis showed that the presence of the AR is not enough to associate a worse prognosis to HCC. In fact, the present study shows that higher expression of AR (at mRNA and protein levels) was associated with a better OS Fig. . These f39. Furthermore, other authors have described an increase of the AR expression (at mRNA and protein levels) in HCC cell cultures free of testosterone and a prevention of the AR increase when testosterone is added40. These results agree with the negative correlation identified in the present study between the AR-Score-6/7 with the AR expression at both mRNA and protein levels  and H3K4 demethylation through AR activation suppressed the expression of the ARSome limitations should be considered in the present work. On the one hand, ARGs that are included in the analysis to generate the AR-Scores and subsequently to infer the AR activity have previously been validated in prostate cancer, but not in HCC cell lines. In this regard, new studies using Chip-Seq approaches should be performed to identify specific ARGs in the context of HCC. This would allow a more precise quantification of the AR activity in HCC and its relationship with prognosis.41. However, inferring the AR activity with the AR-Scores seems to be a suitable tool to investigate the role of this receptor in the prognosis of HCC and the results obtained from its use seem to concur with those reported in the literature.On the other hand, as mentioned above, the RPPA data from the TCGA indicate global protein expression, without quantifying the amount of protein in each subcellular compartment. This point makes it difficult to fit the results of the AR protein expression with those previously reported in literature. Furthermore, this data did not include the phosphorylation state of the AR which seems to play a role in the AR function and subcellular locationFinally, there is no available information about the androgen blood levels in this cohort of patients. These data would be of great value to help identify the putative origin of the AR activation (i.e. whether it is ligand-dependent or non-ligand dependent).HCC patients with high AR expression (at RNA and protein levels) have a longer median overall survival. Nevertheless, AR activity has a different relationship with prognosis. This may be related to the shift in the expression profile of ARGs among histological grades. The AR activity inferred by the relative expression of upregulated to downregulated ARGs in higher histological grades is associated with a worse prognosis. Overall, the results here are supported by the literature and open the door to new research in this field to define the adequacy of the AR as a new druggable target in HCC.Supplementary Information.


To further understand cellulose-solvent interactions in aqueous hydroxide solutions, cellulose behavior in aqueous solutions of NaOH combined with tetramethylammonium hydroxide (TMAH) or benzyltrimethylammonium hydroxide (Triton B), as well as urea, was investigated. The rheological properties of the solutions were assessed through flow sweeps at different temperatures, and the intermolecular interactions were probed using solvatochromic dyes. The results showed that NaOH combined with TMAH had synergistic effects on cellulose dissolution and was a better solvent for cellulose than the combination of NaOH with Triton B, in spite of the superior dissolution ability of Triton B alone. This somewhat unexpected finding shows that the base pair needs to be selected with care. Interestingly, addition of urea had no significant effect on the solvatochromic parameters or dissolution capacity of solutions of Triton B but rendered improved stability of solutions containing NaOH and/or TMAH. It seems that both urea and Triton B weaken the hydrophobic assembly effect of these solutions, but urea is excluded from interacting with cellulose in the presence of Triton B. This study provides further insight into dissolution of cellulose and the possibility of utilizing combinations of hydroxide bases to achieve improved solution properties. There is growing interest in using cellulose as a renewable raw material for products such as hydro-/aerogels, films, and various filaments for textiles and non-woven applications, and the dissolution of cellulose is often required for these applications as well as for certain analytical methods. The dissolution of cellulose is, however, not trivial, and much research has been invested in understanding the mechanisms behind it. One solvent that has been extensively researched due to its sustainable character and viability in large-scale applications is aqueous solutions of sodium hydroxide, which can dissolve cellulose to a very limited degree at temperatures below zero and concentrations around 2 M . The useThe use of additives to NaOH (aq) has been explored in order to improve NaOH (aq) as a solvent for cellulose, and urea is the additive that has gained the most attention. It has been shown that adding urea significantly improves the dissolution capacity and stability of these solutions . The mecLindman, Karlström, and Stigsson have suggested that the behavior of cellulose in aqueous systems  is largely governed by its hydrophobic assembly effect. The axial direction of the pyranose ring is more hydrophobic than the equatorial direction since the hydroxyl groups are all in an equatorial position. This leads to the network of hydrogen bonds in water around the hydrophobic region of the pyranose ring being broken. Consequently, the water molecules situated there suffer from fewer hydrogen bonds and might also have more restricted mobility , lea−  to dissolve cellulose delayed the gelation of the solution compared to dissolving cellulose in only NaOH or TMAH . This juydroxyls ), these w of 29,160 g/mol) as measured with GPC-MALS  was used. Granulated sodium hydroxide (NaOH), known commercially as Emplura, benzyltrimethylammonium hydroxide  and used as received.Microcrystalline cellulose (MCC), Avicel PH-101 purchased from FMC BioPolymer , a purified partially depolymerized cellulose made through the acid hydrolysis of specialty wood pulp, with a degree of polymerization of 180  was added in solution according to the method for Dissolution of cellulose see , and the−1], and increasing temperatures in series on the same sample.Rotational rheology measurements were performed to investigate the flow behavior of the solutions at different cellulose concentrations and temperatures. A TA Discovery Hybrid Rheometer (HR-3), with a sandblasted 40-mm plate-plate geometry with a gap of 1 mm, was employed, and the temperature was controlled with a Peltier plate with circulating cooling liquid. Samples were measured directly after dissolution with a water-filled solvent trap and brought to the desired temperature in the rheometer without pre-shearing. Flow sweeps were conducted with shear rates from 1 to 100 [sThe shear stress of a power-law fluid can be described by Equation (3). The flow index, n, was obtained graphically from the slope of the trendline fitted to the data in a plot of log stress vs. log shear rate.N,N-diethyl-4-nitroaniline, 4-nitroaniline, and Reichardt’s dye , and measurements with temperature control were conducted on a Cary 4000 spectrophotometer from Agilent. Plastic  disposable cuvettes with a light path length of 12.5 mm were used throughout the measurements, and water was used as the background. The maximum absorption of the peaks of interest ranged from 0.1 to 1.1. The Kamlet-Taft parameters were calculated according to Equations (4)–(7) [The dyes 4-nitroanisole,  dye see  were use (4)–(7) ,22,23.(This study followed the methods described in the articles by Kamlet and Taft and used the dye pairs (2) and (3) to obtain β and the pair (1) and (4) to obtain α.Aqueous solutions of bases were prepared as described above .FTIR spectra of the aqueous solutions of bases were collected using a Perkin Elmer Frontier spectrophotometer  equipped with an Attenuated Total Reflectance (ATR) sampling accessory, PIKE Technologies GladiATR. Samples were placed on the top of the ATR crystal and analyzed with a resolution of 4 cmThe dissolution limit was estimated by separation of larger, undissolved particles from the dissolved cellulose through the centrifugation of cellulose solutions see . This liThe solvents had different molecular volumes see , but theThrough a series of flow sweeps at increasing temperatures, we compared the temperature stability of the solutions and received information on cellulose-solvent interactions. The NaOH (aq) solution a was nevIn our previous study of the effect of combining NaOH and TMAH to dissolve cellulose, we found that combining these bases delayed gelation over time at temperatures 15–35 °C . In thisThe Triton B (aq) solution c displayThe results for the solutions that also contained urea confirm what was found for the dissolution capacity: the addition of urea enhanced the Newtonian behavior of the solutions containing NaOH and/or TMAH but did not have the same effect on the solutions containing Triton B . This isWe also found that both urea and the more hydrophobic bases stabilized the solutions as the temperature increased. This could indicate that both urea and the more hydrophobic bases interacted with cellulose through less temperature-dependent interactions, possibly van der Waals interactions in the less polar region of the pyranose rings.The ratios of the bases were further varied in order to observe the extent to which replacing NaOH with another base could change the properties of the solutions. Interestingly, a significant amount of the other base seemed to be required. Neither TMAH nor Triton B had any significant effect on flow properties when NaOH was replaced to a minor extent. We found that the base combination of NaOH and TMAH appeared to be optimal for the ratio of 50/50 see . More TrTo further investigate possibilities for cellulose-solvent interactions in the studied solutions, the solvatochromic parameters π, β, and α—the so-called Kamlet-Taft parameters—were determined as commonly indicative of solvent polarity/polarizability and hydrogen bonding capacity. These parameters are based on the interaction between a dye and the solvent and can indicate possible interactions between cellulose and solvents. Many dyes can be used to determine these interactions, but the absolute values obtained from different dyes should not be compared, instead trends should be compared, as demonstrated by Rani et al. .The π parameter measures the polarity and polarizability of a solvent, increasing with increasing polarizability and polarity with π = 0 for cyclohexane and π = 1 for dimethyl sulfoxide. According to the original method by Kamlet, Abboud, and Taft, at least seven different probes were used to measure π in order to minimize any specific interaction that could occur between a probe and a solvent. The average value of these seven π-values was then considered reliable enough to be used . In mostIt has been shown that it is important for a good solvent to meet the amphiphilic nature of cellulose chains . It is, The addition of urea generally increased π for both π probes see , which cThe β parameter measures the hydrogen bond acceptor ability of a solvent, with the reference point being the value of hexamethylphosphoramide set to 1 . Both waThe addition of urea to water slightly increased β. This means that urea acted as a slightly better hydrogen bond acceptor than water. When both urea and the base were present in an aqueous solution, there was no effect on β owing to the fact that it could not compete with the hydroxide ion as a hydrogen bond acceptor.+ < TMA+ << Triton B+ at 4 mol% base. Water has a high hydrogen bond donor ability, and if the cation does not contribute to the α value, then increasing the concentration of the base should lower α. The results in The α parameter measures the hydrogen bond donor ability of a solvent, where the value for methanol is set to 1 . As prevThe addition of urea to the solutions decreased α, indicating that urea is a weaker hydrogen bond donor than water (the bases offered no competition as hydrogen bond donors). The solutions with Triton B were an exception, i.e., the addition of urea had no significant effect on α, which was also found for the π values. This could mean that owing to Triton B’s strong interactions attributed to lower hydration and more potential for vdWs interactions, urea was completely excluded from interacting with the probe. This correlates with the finding that the addition of urea barely increased the dissolution capacity of Triton B.This finding can be summarized into possible interactions between cellulose and the bases; in these very basic solutions (pH ca. 14), one can expect a portion of the cellulose hydroxyl groups to be deprotonated, which would certainly have a positive effect on dissolution. Considering the polyelectrolyte effect, however, most of the hydroxyl groups can be considered to remain in their protonated state. The electrostatic interaction between a cation and a hydroxyl group would not be as strong as that observed between a cation and the zwitterionic alfa probe. This indicates that the superior dissolution power that Triton B has over NaOH or TMAH might not only be due to the hydrophobicity of the cation but also due to electrostatic interactions between the cation and cellulose.A comparison of the combination of NaOH and TMAH to the individual base solutions showed small differences in the measured Kamlet-Taft parameters, but overall, they were on the same levels despite clear differences in dissolution properties. Based on this finding, it seems that the improved dissolution properties do not primarily stem from an impact on polarity/polarizability or hydrogen bonding. When NaOH was combined with Triton B the behavior was quite different, i.e., the solvatochromic parameters seemed to be dominated by the properties of Triton B. This was especially evident in the high alfa value obtained and shows that Triton B probably enriches in the shell around the dye, and therefore the dye does not reflect the properties of the bulk solvent. To confirm this, we varied the ratio of NaOH/Triton B see  and NaOHIn our previous study , we founCombining bases with different properties and different advantages can have the same effect on cellulose dissolution as an additive. Adding urea to solutions of NaOH and/or TMAH, however, was found to further improve dissolution and can successfully be used together with these bases. Adding urea to Triton B did not seem to have a significant effect on cellulose dissolution, and the solvatochromic probes indicated that urea might be excluded from interacting with cellulose in the presence of Triton B. Both urea and Triton B are believed to weaken the hydrophobic effect by replacing water around the pyranose ring. The combination of NaOH with TMAH improved dissolution more than combining NaOH with Triton B. A tentative finding is that in order for a combination of hydroxide bases to have synergistic effects on cellulose dissolution, the presence of another base must still allow for the other to form the hydrates relevant for cellulose dissolution. Both urea and the more hydrophobic bases increased the temperature stability of the solutions at elevated temperatures, which was due to a larger contribution of less temperature-sensitive van der Waals interactions.