To compare the efficacy and safety of self‐ versus physician‐managed titration of insulin glargine 300 U/mL (Gla‐300) in people with inadequately controlled type 2 diabetes.Take Control (EudraCT number: 2015‐001626‐42) was a 24‐week, multi‐national, open‐label, controlled, two‐arm, parallel‐group study in insulin‐naïve and pre‐treated participants, randomized 1:1 to a self‐ or physician‐managed titration of Gla‐300. The fasting self‐monitored plasma glucose (SMPG) target was 4.4 to 7.2 mmol/L. The primary outcome was non‐inferiority of glycated haemoglobin (HbA1c) change from baseline to week 24. Secondary outcomes included SMPG target achievement without hypoglycaemia, hypoglycaemia incidence, adverse events and participant‐reported outcomes (PROs).P < 0.0001) and superiority (P = 0.0247) of self‐ versus physician‐managed titration. Significantly more of the self‐ than physician‐managed group achieved SMPG target without hypoglycaemia . Overall, hypoglycaemia incidence was similar in each group. No safety concerns were reported. In both groups, similar PRO improvements were observed for distress related to diabetes disease burden and for confidence in diabetes self‐management, with even more individuals achieving a clinically relevant reduction in emotional burden and fewer individuals with high emotional burden in the self‐managed group.At week 24, the least squares (LS) mean HbA1c reduction was 0.97% (10.6 mmol/mol) and 0.84% (9.2 mmol/mol) in the self‐ and physician‐managed groups, respectively, with an LS mean difference of −0.13% [95% confidence interval −0.2619 to −0.0004] (–1.4 mmol/mol [–2.863 to –0.004]), demonstrating non‐inferiority (Self‐managed titration of Gla‐300 was superior to physician‐managed titration in terms of HbA1c reduction, accompanied by similar total PRO scores, with a clinically relevant reduction in emotional burden, and similar hypoglycaemia frequency. Inclusion criteria comprised age ≥18 years, T2DM diagnosis for at least 1 year before screening and at least 6 months on treatment with at least one non‐insulin antihyperglycaemic drug, with or without a BI. Insulin‐pretreated participants were required to have been on a stable dose (type of insulin and time/frequency of injection) within 3 months prior to screening. The main exclusion criteria included HbA1c <7.0% or >10.0% (<53 mmol/mol or >86 mmol/mol) for participants taking BI, or <7.5% or >11.0% (<58 mmol/mol or >97 mmol/mol) for insulin‐naïve participants, and unwillingness to self‐manage the titration algorithm. The full inclusion and exclusion criteria are presented in Background non‐insulin antihyperglycaemic drug(s), including sulphonylureas (SUs), administered at a stable dose for ≥12 weeks prior to screening were permitted according to local labelling guidelines. The type and dose remained unchanged during the study, unless safety concerns necessitated a dose reduction or discontinuation. Non‐study drug BIs were stopped by the time of randomization.All participants provided informed, written consent. The clinical trial protocol was approved by the relevant health authorities and the appropriate local institutional review board/independent ethics committees. The study was conducted in accordance with the Declaration of Helsinki and the International Conference on Harmonization guidelines for good clinical practice.2.2Participants were randomized 1:1 to self‐ or physician‐managed titration, using an interactive response system, stratified by HbA1c at screening (<8.5% vs ≥8.5% [<69 mmol/mol vs ≥69 mmol/mol]); previous use of insulin (yes vs no); and SU use at screening (yes vs no).2.3After randomization, Gla‐300 was self‐administered subcutaneously once daily, with doses according to the European Union label, taking into account history of insulin use . The timPaper diaries were dispensed to participants at each on‐site visit, and collected for review at each subsequent on‐site visit. Diaries contained instructions for managing glycaemic values according to each participant's allocated titration arm. Participants were trained at visits 1 and 2 (and re‐trained as necessary during the study) in how to complete these diaries to record information related to hypoglycaemic events, self‐monitored plasma glucose (SMPG) and insulin doses. Insulin dose was to be adjusted to a fasting SMPG target of 4.4 to 7.2 mmol/L as per 2015 ADA recommendations, while avoiding hypoglycaemia.The study consisted of a prespecified schedule of six on‐site and 10 telephone call visits. During visits, physician‐managed participants were instructed to self‐inject at a dose prescribed by the investigator. Self‐managed participants were instructed to continue the same titration approach, unless participant safety was at risk. Additional contacts  between scheduled visits were available at the discretion of the investigator.2.4Addition of a new anti‐hyperglycaemic drug or a dose increase of an existing anti‐hyperglycaemic non‐study drug, was performed based on the investigator's decision and local requirements. It was expected that the initiation of any rescue medication would be deferred until week 12 of treatment. Short‐term use  of short‐acting insulin  was not considered as rescue therapy.2.5The primary outcome was non‐inferiority of glycaemic control, as assessed by the change from baseline to week 24 in HbA1c, achieved using the self‐ versus physician‐managed titration algorithm for Gla‐300. Key secondary outcomes included the percentage of participants reaching fasting SMPG target without experiencing hypoglycaemia that was severe and/or confirmed by a glucose measurement of <3.0 mmol/L (see below for a full definition); safety and tolerability, including hypoglycaemic events; and change in PROs according to total and subscale scores using the Diabetes Distress Scale (DDS)2.6A sample size of 592 was required to ensure that the two‐sided 95% confidence interval (CI) for the mean difference between the two titration approaches would not exceed 0.3% HbA1c, with 80% power assuming the SD is 1.3%, with a one‐sided test at the 2.5% significance level and that the true difference between the two dosing regimens of Gla‐300 is zero.The primary efficacy analysis was performed in the intention‐to‐treat (ITT) population, comprising all randomized participants, irrespective of the titration arm at the time of the analysis. PROs were also analysed in the ITT population. The safety population was defined as all randomized participants who received at least one dose of Gla‐300, regardless of the amount of treatment administered.The primary endpoint was analysed using a mixed‐effect model with a repeated measures approach, including fixed categorical effects of titration approach, visit, titration approach‐by‐visit interaction, randomization strata as well as continuous fixed covariates of baseline HbA1c and baseline HbA1c value‐by‐visit interaction. A stepwise closed testing approach was used for the primary efficacy variable to assess non‐inferiority and superiority. Non‐inferiority was demonstrated if the upper bound of the two‐sided 95% CI for the difference in the mean HbA1c change from baseline to week 24 between titration approaches was <0.3% HbA1c. If non‐inferiority was demonstrated, superiority was tested and demonstrated if the upper bound of the two‐sided 95% CI for the difference in the mean HbA1c change from baseline to week 24 between the two titration approaches was <0. Further details on statistical methods can be found in the Supporting Information.The proportions of participants reaching fasting SMPG target and reaching fasting SMPG target without experiencing hypoglycaemia were analysed according to the mean of a log binomial regression model, adjusted on randomization strata of screening HbA1c, SU use, and previous use of insulin therapy. Safety endpoints were analysed descriptively.33.1Overall, 631 participants were randomized (ITT population) to self‐managed  or physician‐managed  titration Figure . A similDemographic and baseline characteristics were similar in the self‐ versus physician‐managed groups Table . Across Overall, 62% of participants had received previous BI treatment, with a mean (SD) duration of 3.5 (4.2) years. In the self‐ and physician‐managed groups, 37.9% and 38.5% of participants, respectively, were insulin‐naïve.3.2P < 0.0001) and superiority (P = 0.0247) were demonstrated for self‐ versus physician‐managed titration. Mean HbA1c reduction from baseline to week 24 was greater in insulin‐naïve versus pre‐treated participants  mean reduction in HbA1c from baseline to week 24 was 0.97% (10.6 mmol/mol) and 0.84% (9.2 mmol/mol) for self‐ and physician‐managed titration respectively, with an LS mean difference of −0.13% [95% confidence interval −0.2619 to −0.0004] (−1.4 mmol/mol [−2.863 to −0.004])  achieved an HbA1c target <7.0% (<53 mmol/mol)  in the self‐ and physician‐managed groups  LS mean change from baseline to week 24 was −1.72 mmol/L in the self‐managed titration group and −1.64 mmol/L in the physician‐managed titration group, resulting in an LS mean difference of −0.08 mmol/L  of any type was reported by 8.0% of participants in the self‐managed titration group and 11.4% in the physician‐managed titration group (relative risk 0.70 [95% CI 0.43 to 1.13]). Severe hypoglycaemia was reported in two of 312 participants [0.6%] in the self‐managed titration group, and one of 316 participants [0.3%] in the physician‐managed titration group.During the 24‐week on‐treatment period, 36.2% of participants in the self‐managed titration group and 37.0% in the physician‐managed titration group experienced at least one episode of hypoglycaemia, of any definition  in the documented symptomatic (23.1% and 22.8%), asymptomatic (17.0% and 20.9%), and confirmed and/or severe (33.3% and 34.2%) categories.The incidence of confirmed (≤3.9 mmol/L and <3.0 mmol/L) and/or severe hypoglycaemia was similar between self‐ and physician‐managed titration, regardless of prior insulin use .3.4Treatment‐emergent adverse events (TEAEs) were reported by 33.7% of participants in the self‐managed titration group, and 34.5% in the physician‐managed titration group during the 24‐week on‐treatment period . Rates o3.5There was a numerically higher mean daily insulin dose increase in the overall self‐managed versus physician‐managed group as well as in the insulin‐naïve and insulin pre‐treated subgroups of these titration approaches Table . During The LS mean (SE) change in body weight from baseline to week 24, was +0.84 (0.17) kg in the self‐managed titration group and +0.50 (0.17) kg in the physician‐managed titration group (LS mean difference 0.34 [95% CI –0.14 to 0.82]).3.6The mean (SD) total DDS was 2.04 (0.89) and 2.06 (0.99) at baseline (indicating “moderate distress”) in the self‐ and physician‐managed groups. The mean change in the DDS total score from baseline to week 12 was −0.23 (0.70) and −0.23 (0.79) for the self‐ and physician‐managed groups, respectively. This reduction in DDS score from baseline was maintained at week 24 in both groups (−0.23 [0.76] and −0.17 [0.90]). The decrease in DDS total scores indicated small improvements in both groups, with an LS mean difference from baseline to week 24 of −0.07 (95% CI −0.19 to 0.04), for self‐ versus physician‐managed titration . The proThe mean (SD) DES total scores at baseline were 4.00 (0.50) and 4.02 (0.51) in the self‐ and physician‐managed groups, indicating that both groups were reasonably confident in their diabetes self‐management. There was a mean increase in total DES scores from baseline to week 12 by 0.14 (0.43) and 0.06 (0.47) in the self‐ and physician‐managed groups, respectively. These increases in DES total score were maintained at week 24, with mean increases of 0.19 (0.45) and 0.12 (0.49) from baseline, and an LS mean difference (95% CI) of 0.07 (0.00 to 0.14) for self‐ versus physician‐managed titration, indicating that participants in both groups felt more confident in managing their diabetes . Similar4In the present study, self‐managed titration of Gla‐300 was both non‐inferior and superior to physician‐managed titration in terms of mean reduction in HbA1c from baseline to week 24. Slightly greater HbA1c reductions were observed in self‐ versus physician‐managed titration, regardless of previous insulin use, with more participants in the self‐managed group achieving the HbA1c target of <7.0% (<53 mmol/mol). Furthermore, a significantly higher proportion of participants in the self‐ versus physician‐managed groups achieved the fasting SMPG target without experiencing confirmed (<3.0 mmol/L) or severe hypoglycaemia.The results from the present study are generally consistent with those from two previous studies. ATLASIn AT.LANTUS and ATLAS, there was a higher risk of symptomatic hypoglycaemia with self ‐ vs physician‐managed titration.At week 24, Gla‐300 doses were numerically higher with self‐ versus physician‐managed titration, consistent with the results with Gla‐100 in ATLAS and AT.LANTUS.The PRO scores for both titration groups at baseline indicated moderate distress and reasonable confidence in self‐management. Total DDS and DES results improved similarly in both groups, while more individuals in the self‐managed group achieved a clinically meaningful improvement in the DDS emotional burden subscale score, and fewer individuals reported a high emotional burden compared to the physician‐managed group. These results indicate that, by taking responsibility for titrating insulin, patients did not experience more distress related to diabetes, had a tendency towards less emotional burden, and had similar confidence in self‐management of their diabetes versus physician management. In the ATLAS study, there was no difference in the treatment satisfaction or quality‐of‐life outcomes between self‐ or physician‐managed titration of Gla‐100. It remains to be evaluated whether the PRO improvements observed with Gla‐300 in the present study translate to benefits in treatment satisfaction, overall quality of life, and treatment adherence and persistence in real‐life settings.The study benefitted from the randomized trial design with a sufficiently large sample size to detect differences in the primary endpoint. Furthermore, the interactions between participants and research staff mimicked real‐life practice, with the same number of predefined visits in both groups. Participants were recruited from 79 centres in 10 European countries, with characteristics that were representative of the European T2DM population . There was good retention of participants in both titration groups, with ~98% completing the study. The study also benefitted from the simple titration algorithm and close follow‐up during the first 8 weeks. Given that barriers to effective insulin therapy include patient frustration at the time taken to achieve goals, empowering patients to titrate frequently using this simple titration algorithm is an important consideration for applying this approach in real‐life clinical practice. Other care approaches such as the Stepping Up model, whereby patients and practice nurses have an enhanced role in therapeutic decisions, have also shown improvements in insulin initiation rates and glycaemic control compared with standard practiceThe study was limited by the lack of ability to blind participants to their treatment; however, if anything, this may be more likely to bias perception in the self‐managed titration group. The weekly contacts during the study may not reflect the level of interaction that patients would receive in routine practice. Furthermore, since the self‐managed algorithm allowed more frequent titration than the physician‐managed algorithm, one cannot directly conclude whether the results were driven by the patient or the titration regimen. However, it does indicate that the more frequent dose adjustments in the self‐managed group translated to better glycaemic control, with more participants achieving glycaemic targets without hypoglycaemia, and comparable or lower levels of distress than in the physician‐managed group, with a low overall risk of hypoglycaemia. It is also important to note that although participant characteristics at baseline were similar in the two titration groups, the results may not be generalizable to all patients with diabetes because other factors such as education level may impact the success of self‐titration. The results do show, however, that self‐titration is effective if performed appropriately.The results of the Take Control study show that Gla‐300, a second‐generation BI analogue, with its prolonged, more stable and reproducible pharmacokinetic/pharmacodynamic profile,D. R.‐J. has received honoraria, teaching and research grants from AstraZeneca, Boehringer Ingelheim, Eli Lilly, GlaxoSmithKline, Novartis, Novo Nordisk, Pfizer and Sanofi‐Aventis. A.D., L.P. M.B. and A.R.C. are employees/shareholders of Sanofi. E.D. has received unrestricted research support from AstraZeneca, Novo Nordisk, Sanofi, Pfizer and Roche and has received consulting fees and/or honoraria for membership on advisory boards from AstraZeneca, Novo Nordisk, Lilly, Sanofi, GlaxoSmithKline, Pfizer, Almirall, Novartis, Abbott Laboratories, Esteve and Merck Sharp & Dohme. G.D. has received honoraria for lectures/advisory boards and research support (through the University of Athens) from Abbott, AstraZeneca, Lilly, ELPEN, MSD, Novartis, Novo Nordisk, Sanofi and Vianex. H.A.F. has received honoraria for advisory boards with Novo Nordisk, Sanofi‐Aventis, Lilly and Abbott. B.S. has acted as consultant, advisory board member or speaker for Sanofi‐Aventis, Novo Nordisk, Lilly, Merck Sharp & Dohme, Boehringer Ingelheim, AstraZeneca, DEXCOM and Ypsomed. K.S. has received honoraria from Sanofi Aventis, Novo Nordisk, Servier, AstraZeneca, Eli Lilly, MSD, Boehringer Ingelheim, Krka and research support from AstraZeneca, Pfizer, Sanofi Aventis, Novo Nordisk and Amgen. M.D. has acted as consultant, advisory board member and speaker for Novo Nordisk, Sanofi‐Aventis, Lilly, Merck Sharp & Dohme, Boehringer Ingelheim, AstraZeneca and Janssen, an advisory board member for Servier and as a speaker for Mitsubishi Tanabe Pharma Corporation and Takeda Pharmaceuticals International Inc. She has received grants in support of investigator and investigator‐initiated trials from Novo Nordisk, Sanofi‐Aventis, Lilly, Boehringer Ingelheim and Janssen.D.R.‐J. contributed to the original design of the trial, participated as an investigator, provided comments and input to all drafts of the manuscript and interpretation of the results. A.D. provided comments and input to all drafts of the manuscript and interpretation of the results. E.D. participated as an investigator, provided comments and input to all drafts of the manuscript and interpretation of the results. G.D. participated as an investigator, provided comments and input to all drafts of the manuscript and interpretation of the results. H.A.F. participated as an investigator, provided comments and input to all drafts of the manuscript and interpretation of the results. L.P. was esponsible for medical oversight during the trial, provided comments and input to all drafts of the manuscript and interpretation of the results. B.S. participated as an investigator, provided comments and input to all drafts of the manuscript and interpretation of the results. K.S. participated as an investigator, provided comments and input to all drafts of the manuscript and interpretation of the results. M.B. wrote the study outline, provided comments and input to all drafts of the manuscript and interpretation of the results. A.R.C. provided the analysis and critical interpretation of the PRO data. M.D. contributed to the original design of the trial, participated as an investigator, provided comments and input to all drafts of the manuscript and interpretation of the results.https://www.clinicalstudydatarequest.com.Qualified researchers may request access to patient‐level data and related study documents including the clinical study report, study protocol with any amendments, blank case report form, statistical analysis plan, and dataset specifications. Patient‐level data will be anonymized and study documents will be redacted to protect the privacy of trial participants. Further details on Sanofi's data sharing criteria, eligible studies, and process for requesting access can be found at: Table S1 Inclusion and exclusion criteriaTable S2: Gla‐300 starting dosesTable S3: Titration algorithmTable S4. Study objectivesTable S5. HbA1c reduction and hypoglycaemia incidence according to prior insulin useTable S6. Adverse events during 24‐week on‐treatment periodClick here for additional data file.Figure S1 Take Control study designClick here for additional data file.Figure S2 Participant flow diagramClick here for additional data file.Figure S3 Change in patient reported outcomes A) Diabetes Distress Scale (DDS) total scores B) Diabetes Empowerment Scale (DES) total scoresClick here for additional data file.


Bisphenol A (BPA) is a monomer primarily used in the production of polycarbonate plastic and epoxy resins. Bisphenol F (BPF) is apparently the main BPA replacement that is used increasingly. BPF has been detected in canned food, thermal paper receipts, and soft drinks. In the present experiment, we did both in vitro and in vivo studies to evaluate the effect of low and high-dose BPF exposures on testosterone concentration, oxidative stress, and antioxidants activity in reproductive tissues of male rats.n = 36) obtained from the rodent colony of Animal Sciences Department of Quaid-i-Azam University. The direct effects of BPF on the antioxidant enzymes and testosterone secretion were measured in vitro and in vivo studies. In an in vivo experiment, adult male Sprague Dawley rats (n = 42) were exposed to different concentrations of bisphenol F  for 28 days. Various biochemical parameters were analyzed including the level of catalase (CAT), superoxide dismutase (SOD), peroxidase (POD), reactive oxygen species (ROS), and lipid peroxidation (LPO). Moreover, sperm motility, daily sperm production (DSP), comet assay, and histological analysis were performed.Adult (80–90 days old) male Sprague Dawley rats (p < 0.05) induced oxidative stress biomarkers, i.e., ROS and LPO, while it did not change antioxidant enzyme and testicular testosterone concentration. Whereas, an in vivo study revealed that BPF induced dose-dependent effect and high-dose (100 mg/kg) exposure of BPF significantly reduced tissue protein (p < 0.05) content, CAT (p < 0.001), SOD (p < 0.05), and POD (p < 0.05) levels while significantly (p < 0.05) augmented ROS and lipid peroxidation. Furthermore, BPF reduces testosterone, LH, and FSH secretion in a dose-dependent manner. Significant (p < 0.001) reduction in plasma and intra-testicular testosterone, LH, and FSH was noticed at 100 mg/kg BFP dose. High-dose exposure reduces spermatogenesis.In vitro study showed that BPF exposure significantly (BPF showed an antagonistic effect on male reproductive hormones and induce alterations in testicular morphology. Increased oxidative stress and decreased testicular antioxidant status might be the underlying mechanism of BFP-induced testicular toxicity. Bisphenol A (BPA) is a monomer primarily used in the production of polycarbonate plastics and epoxy resins , 2. BPA BPF has a wide spectrum use in the plastic industry and it has been detected in 55 of the 100 tested urine samples with a concentration of 0.08 μg/L . Similar2O2, Ca2+, Mg2+, Hank’s balanced salt solution (HBSS) were bought from Sigma-Aldrich .Bisphenol F (BPF) with 99% purity was purchased from Santa Cruz Biotechnologies, USA. For the in vitro experiment, different materials as fetal bovine serum, penicillin/streptomycin, and Dulbecco’s modified Eagle’s medium (DMEM) were obtained from Thermos Fisher Scientific . CAT, N-acetyl-L-cysteine (NAC) and HSprague Dawley adult male rats (age 80–90 days) were obtained from the animal facility of Quaid-i-Azam University, Islamabad. Prior to the start of the experiment standard laboratory conditions were maintained. Animals were fed with laboratory feed and tap water was available freely for the animals. Protocols of handling of the animals were approved by the animal sciences department ethical committee.For bisphenol F (BPF) exposure on male rats, different experiments were conducted. Firstly, we conducted an in vitro experiment in which the direct effects of BPF on the levels of antioxidant enzymes and different concentrations of testosterone in the testis of rats were tested. While on the results of the in vitro study, an in vivo study was conducted in which the effects of different concentrations of BPF on the reproductive system of male rats were evaluated through subchronic study.n = 36 and n = 6 animals per group) Sprague Dawley male adult rats were used. In order to investigate the direct effects of BPF on the antioxidant enzymes and testosterone production, an in vitro study was conducted. In this study, different doses of BPF  were prepared in ethanol which was in accordance with [2) incubator for 2 hours. After the incubation period, all the incubated tissues were washed with saline and homogenized in 30 ml of phosphate buffer saline (PBS) and centrifuged at 30,000 for 30 min. Then the supernatant was collected and stored at − 80 °C for further investigation.In the in vitro study, a total of  were divided into six groups (n = 7/group) by randomization procedures explained elsewhere [Adult male Sprague Dawley rats , animals were euthanized and different organs were dissected and stored at − 80 °C for different tests. Blood was collected and centrifuged at 3000 rpm for 10 mins and plasma was separated and stored at − 20 °C for hormonal and different biochemical analysis by the researcher blind to the treatment groups. The reproductive organs as testicular tissues (left testis and left epididymis) were weighed and processed for antioxidant enzymes while right testis (transverse sections) and right epididymis were fixed in 10% formalin for histological analysis as explained by .Tissues collected from both in vitro and in vivo studies were further processed for the antioxidant enzymes and oxidative stress markers. Tissues were homogenized with an automatic homogenizer in phosphate buffer saline (PBS) and centrifuged at 30,000 rpm for 30 mins. After the centrifugation, the supernatant was removed and used for the hormonal analysis, protein estimation, and antioxidant enzymes , 19.Afsar et al.’s method was used to determine the catalase (CAT) activity , and theAfsar and colleagues method was used to determine the superoxide dismutase (SOD) activity . In this2O2, and 2.5 ml of phosphate buffer and the absorbance was read at 470 nm. Change in the absorbance of 0.01 as unit per minute was defined as one unit of POD.Peroxidase (POD) activity in the homogenate was determined by the spectrophotometric method of Carlberg and Mannervik, . In thisThe activity of lipid peroxidation by T-BARS was determined in the homogenate by the method used by Iqbal and coworkers  and the 2O2 standards and the homogenate was mixed with 140 ml of sodium acetate buffer with pH 4.8 in 96-well plates and incubated at 37 °C for 5 min. After the incubation, 100 ml of DEPPD and ferrous sulphate mix samples were added in each well with a ratio of 1:25 and were incubated at 37 °C for 1 min. With an interval of 15 s for 3 min, the absorbance was read at 505 nm at microplate reader.The assay of reactive oxygen species (ROS) was done according to the method of Hayashi et al. . In thisDetermination of total protein content in tissues was done following a commercial diagnostic kit  protocol. The results of protein were measured by plotting absorbance of the standard against samples. These values were expressed as milligram per gram of tissue.Quantitative EIA kits were used for the measurement of testosterone , luteinizing hormone (LH) , and follicle-stimulating hormone (FSH)  concentrations in the tissues and the assays were performed by the instructions with the kits. All the above assays were repeated with both inter- and intra-assay variations for more and precise results.Testicular tissues (testis and epididymis) were fixed in formalin for 48 h, dehydrated with different grades of alcohol, and cleared with the help of xylene. The paraffin sections (5 μm) were cut and stained with hematoxylin and eosin for histology and morphometry. Transverse sections (10–20/group) of testicular tissues were examined under a Leica Microscope (New York Microscope Company) equipped with a digital camera .For the morphometry, the images were taken at × 20 and × 40, and the results were done with Image J software. Area of different sections was calculated with the method of Jensen et al. . From × P < 0.05. One way ANOVA followed by Dunnet’s multiple comparison tests was used for the comparison of different groups with control using Graph Pad Prism software.All parameters of data points showed normal distribution and hence were reported as mean ± SEM and difference was considered significant at Antioxidant enzymes, i.e., CAT, POD and SOD, oxidative stress markers, i.e., reactive oxygen species (ROS) and TBARS, were determined in the testicular tissues after 2 hours incubation with different concentrations of BPF Table . There wP < 0.05) increases in LPO were observed as compared to the control. However, there was no significant increase observed in the low-dose-treated groups as compared to the control. Similarly, there is a dose-dependent augmentation in ROS levels in different treatment groups. In BPF 25 ng/ml and 50 ng/ml, significant (P < 0.05) increase in ROS was noticed, whereas in BPF 100 ng/ml, marked (P < 0.01) increase in ROS was examined as compared to the control group. Low doses of BPF did not induce any change in ROS level compared to the control group.Reactive oxygen species and LPO are considered important oxidative stress markers. In BPF 50 ng/ml and 100 ng/ml treated groups, significant , BPF 25 mg/kg (P < 0.01), BPF 50 mg/kg (P < 0.05), and BPF 100 mg/kg (P < 0.05) when compared to the control.Antioxidant enzymes in the testicular tissues after 28 days of different concentrations of subchronic exposure to BPF and control are presented in Table P < 0.05) reduction in CAT activity at doses of 5, 25, and 50 mg/kg treated groups as compared to control. Similarly, BPF treatment caused significant (P < 0.01) decline in CAT activity at dose levels of 100 mg/kg treated groups.BPF treatment caused significant (P < 0.05) increase in the LPO content was observed in BPF 50 mg/kg and BPF 100 mg/kg treated groups when compared to control. However, the other doses of BPF did not show a significant effect as compared to control.ROS and LPO level in different treatment groups are presented in Table P < 0.05) when compared to control. Total ROS was increased significantly (P < 0.001) in BPF 100 mg/kg as compared to control. However, total ROS was not altered by BPF 1, 5, and 25 mg/kg groups when compared to the control.Similarly, a significant increase in ROS level was observed in BPF 50 mg/kg (P < 0.05), BPF 25 mg/kg (P < 0.05), and BPF 50 mg/kg (P < 0.05) as compared to the control. On the other hand, BPF 100 mg/kg treatment group showed a significant reduction (P < 0.01) in protein levels as compared to control.Total protein in the testis showed a significant reduction in BPF 5 mg/kg (P < 0.05) in BPF 25 mg/kg and 50 mg/kg treated groups. Similarly, BPF treatment caused a significant reduction (P < 0.01) at a dose level of 100 mg/kg. However, BPF in 1 and 5 mg/kg treated groups did not affect testosterone concentrations significantly.Plasma testosterone, LH, FSH, and intra-testicular testosterone in the BPF different treated groups and control is presented in Table P < 0.05) as compared to the control. A significant reduction (P < 0.01) was also observed in BPF 100 mg/kg when compared to the control. On the other hand, BPF 1 and 5 mg/kg doses did not reduce plasma LH concentrations as compared to the control.Plasma LH concentrations reduced significantly in BPF 25 and 50 mg/kg (P < 0.01) as compared to the control. A significant reduction (P < 0.001) was also observed in BPF 50 and 100 mg/kg when compared to the control. On the other hand, BPF 1 and 5 mg/kg treatment did not reduce plasma FSH concentrations as compared to the control.FSH reduced significantly in BPF 25 mg/kg  as compared to the control. Intra-testicular testosterone was not different in BPF 1 and 5 mg/kg treated groups than control  reduction in epithelial height in BPF 50 mg/kg and 100 mg/kg groups when compared to the control. On the other hand, epithelial height was not different in BPF 1, 5, and 25 mg/kg treated groups than the control.Effect of BPF exposure on the seminiferous tubule area, interstitium area, seminiferous tubules diameter, and epithelial height in testicular tissue are presented in Table Transverse sections of testicular tissues of the control group were observed with thick epithelium, sperm-filled lumen, and seminiferous tubules Fig. . SeminifMorphometry of different parameters of caput and cauda epididymis region after different BPA exposures did not show any significant difference in any of the parameter  as compared to the control presented in Table The number of different cell types in the seminiferous tubules presented in Fig. Although numerous studies have been published on the effects of BPA on the reproductive functions of male rats, the underlying mechanisms remain unclear. BPA displays non-monotonic dose-response functions . CurrentIn the in vitro study, we incubated testicular tissues with different concentrations of BPF for 2 hours. The incubated tissues did not show any significant change on the antioxidant enzymes as CAT, SOD, and POD. BPA and its analogs have a toxic influence on the formation of ROS inside the body, and studies have also shown that these phenols not only increase in the levels of ROS but also lead into oxidative stress inside many cellular networks . SimilarExposure to BPF caused induction of ROS which lead into an increase in the levels of LPO and activation of antioxidant enzymes which are in line with the earlier studies where BPA exposure degraded protein and altered antioxidant enzymes . In the We observed the substantial change in both plasma and intra-testicular testosterone in the in vivo study. Our results are in accordance with previous researches which indicated altered levels of different hormones after exposure to BPA and some of its analogs , 34.The process of Spermatogenesis is controlled by different reproductive hormones and cellular interactions inside the testes. ROS and disturbed antioxidant enzymes lead to disturbed spermatogenesis . In the The results of our present study showed that BPF at higher dose exposures may possibly have outcomes in oxidative stress and disturbed reproductive hormones. Thus, the use of BPA analogs should be carried out with caution, especially until the effective risk assessment is conducted. Further studies need to analyze the molecular basis of these alterations both in vivo and in vitro studies which will let us understand how BPF can still have an effect on the physiology of different tissues inside the body.


SGCA). Due to lack of specificity, it is impossible to identify LGMD2D only by clinical symptoms and conventional immunohistochemical staining. The loss of any protein  that represent sarcoglycanopathy may cause reduction or absence of the other three proteins. Here, we report a patient with a complete loss of all the four proteins. Next generation sequencing (NGS) results showed a missense mutation (C.218 C > T) and a partial heterozygous deletion containing exons 7 and 8 of SGCA, which led to the final diagnosis of the patient. The discovery of this new mutation could broaden the spectrum of SGCA mutations, which may be associated with putative LGMD2D, especially when all the four proteins are completely missing.Limb‐girdle muscular dystrophy 2D (LGMD2D) is caused by mutations in the α‐sarcoglycan gene ( SGCA). Nowadays more than 50 different SGCA missense mutations have been reported.SCGA, which is located on chromosome 17q21 and encompasses 10 exons,SGCA variants have been recognized in humans according to the Human Genome Mutation Database. The most frequently reported mutation, in some populations up to one‐third of SGCA mutations, is a missense mutation at position 229 CGC ≥ TGC in exon 3 of SGCA, substituting arginine for cysteine in codon 77 (R77C). This is the most frequently reported.Limb‐girdle muscular dystrophy 2D (LGMD2D) is a rare autosomal‐recessive myopathy, caused by mutations in the α‐sarcoglycan (SGCA) gene  were normal. Thereafter, the boy was admitted to our hospital for further treatment. Neurological examination showed weakened bilateral knee tendon reflexes and hypertrophic bilateral gastrocnemius muscle. Limb strength was assessed at grade 4 and the Gower's sign was positive. The autoantibodies were normal. Biochemical items displayed myocardial enzymes as follows: myoblobulin 448.6 ng/mL, creatine kinase (CK) 14450 U/L, creatine kinase MB fraction 276 U/L, lactic dehydrogenase 961 U/L and normal electrolytes. The family members denied any relevant family history.A fresh muscle sample was taken from the patient's left quadriceps and immediately frozen in isopentane cooled by liquid nitrogen. Then the specimens were analyzed using the standard panel of histochemical stains including hematoxylin and eosin (HE), modified Gomori trichrome (MGT), nicotinamide adenine dinucleotide‐tetrazolium reductase (NADH‐TR) and acid phosphatase, adenosine triphosphatase (ATPase) stains, periodic acid‐Schiff (PAS) and oil red O (ORO).DNA (5 μg) extracted from the patient's muscle was interrupted and amplified. Four hundred and thirty genes associated with muscular dystrophy were captured by using liquid catch kit  and sequenced by a high‐throughput sequencing instrument Hisep2000 , which generated a set of data with mean depth of 414.1‐fold and had a 99.0% coverage of the target sequence at 200× or greater. Biological information of single nucleotide polymorphisms, insertions and deletions were analyzed. The mutant genes of his parents were verified by Sanger sequencing or qPCR according to the results of exon sequencing. The information of the sequenced target region was sorted and analyzed to find the frequency of the mutation in the 1000‐person genome and dbSNP132 database. The pathogenicity was analyzed using Polyphen‐2 software, GATK.An 8‐year‐old boy with clinical manifestations of progressive lower extremity weakness and post‐exercise myalgia without relevant family history was admitted to our hospital. Neurological examination showed bilateral gastrocnemius hypertrophy and proximal lower extremity weakness. Laboratory tests showed significantly increased levels of serum CK. Electromyography showed myogenic damage. Therefore, the patient was diagnosed initially as having muscular dystrophy of uncertain type. However, metabolic myopathy could not be excluded.HE Fig. A staininSGCA mutations were revealed. One was missense mutation c.218C > T (cytosine to thymine) leading to amino acid changes p.P73L (proline to leucine), which was further confirmed from the patient's father by Sanger sequencing. Another partial heterozygous deletion contained exons 7–8 and was confirmed by qPCR. This mutation was predicted to be a pathogenic mutation by Polyphen‐2 software , thus DMD/BMD was excluded. Further, LGMD2D was further confirmed by NGS. In LGMD2D patients, the defect of α‐sarcoglycan protein could cause the secondary reduction or complete absence of β‐, γ‐, δ‐sarcoglycan proteins. Any one reduction or absence of any single of sarcoglycan protein complex will result in a change in overall function, leading to pathogenic changes.SGCA. Throughout the mutations in the SGCA, a high relative frequency of mutations was reported in exon 3 .et al.In this report, we discovered a previously unknown point mutation (c.218 C > T (p.P73L)) in exon 3, adding to the growing spectrum of mutations in the Yanpeng Lu, study concept and design, acquisition of data, analysis and interpretation of data; Xueqin Song, study concept and design, critical revision of manuscript for intellectual content; Guang Ji, analysis and interpretation of data; Hongran Wu, performed a muscle biopsy, acquisition and analysis of pathology data; Duan Li, interpretation of data; Shuyan Sun, acquisition of data.This research did not receive any specific grant from funding agencies in the public, commercial, or not‐for‐profit sectors.


HIV‐1 transmission of CRF01_AE. Worldwide, men who have sex with men (MSM) are at elevated risk for HIV‐1 infection. Hence, understanding HIV‐1 diversity in a primarily Thai MSM cohort with acute infection, and its connections to the broader HIV‐1 transmission network in Asia is crucial for research and development of HIV‐1 vaccines, treatment and cure.Thailand plays a substantial role in global RV254/SEARCH 010 cohort were assessed by multiregion hybridization assay (MHAbce), multiregion subtype‐specific PCR assay (MSSPbce) and full‐length single‐genome sequencing (SGS). Phylogenetic analysis was performed by maximum likelihood. Pairwise genetic distances of envelope gp160 sequences obtained from the cohort and from Asia  were calculated to identify potential transmission networks.Subtypes and diversity of infecting viruses from individuals sampled from 2009 to 2015 within the MHAbce/MSSPbce results identified 81.6% CRF01_AE infecting strains in RV254. CRF01_AE/B recombinants and subtype B were found at 7.3% and 2.8% respectively. Western subtype B strains outnumbered Thai B′ strains. Phylogenetic analysis revealed one C, one CRF01_AE/CRF02_AG recombinant and one CRF01_AE/B/C recombinant. Asian network analysis identified one hundred and twenty‐three clusters, including five clusters of RV254 participants. None of the RV254 sequences clustered with non‐RV254 sequences. The largest international cluster involved 15 CRF01_AE strains from China and Vietnam. The remaining clusters were mostly intracountry connections, of which 31.7% included Thai nodes and 43.1% included Chinese nodes.CRF01_AE and subtype B, emergence of unique recombinant forms (URFs) are found in a moderate fraction of new HIV‐1 infections. Approaches to vaccine design and immunotherapeutics will need to monitor and consider the expanding proportion of recombinants and the increasing genetic diversity in the region. Identified HIV‐1 transmission networks indicate ongoing spread of HIV‐1 among MSM. As HIV‐1 epidemics continue to expand in other Asian countries, transmission network analyses can inform strategies for prevention, intervention, treatment and cure.While the majority of strains in Thailand are  As such, the country has served as a long‐standing base for research on HIV‐1 epidemiology, treatment, prevention, vaccines and cure strategies. Analyses for correlates of protection in the landmark HIV vaccine efficacy trial RV144 are still ongoing Despite numerous efforts to curtail the epidemic, HIV‐1 transmission persists in Thailand, largely affecting MSM and transgender women (TGW) env sequences (~2.5 kb) with a stringent genetic distance threshold to provide more resolution in identifying transmission clusters within RV254 and across the Asia region.In this study, using HIV‐1 full‐genome sequencing, we report molecular epidemiological data primarily from MSM in Bangkok diagnosed with acute HIV infection (AHI) from 2009 to 2015. These participants were enrolled in the RV254 cohort conducted to support vaccine and treatment research 22.1Plasma was collected as part of the study protocol RV254 , an ongoing prospective, longitudinal cohort study of participants with AHI in Bangkok, Thailand. The protocol was reviewed and approved by the institutional review boards of Chulalongkorn University  and the Walter Reed Army Institute of Research . All study participants provided informed consent. Samples used in this study were from the first available time‐point following HIV diagnosis but prior to ART and were collected a median of two days (IQR two to three days) following first documented positive HIV RNA test. Fiebig stages categorized upon enrolment ranged from Fiebig I to V 2.2Viral RNA extracted from plasma samples was screened for subtype reactivity through a multiregion hybridization assay (MHAbce) or multiregion subtype‐specific PCR assay (MSSPbce) as previously described HIV‐1 sequences were obtained either as a near full‐length genome, or as two half genomes overlapping by approximately 1.5 kb, by single‐genome sequencing (SGS) as previously described 2.3https://www.hiv.lanl.gov/content/sequence/VIRALIGN/viralign.html), and manually edited using Geneious Pro 5.6.7  https://www.phylo.org) using maximum likelihood trees reconstructed with RAxML by implementing the general time reversible (GTR+I+G) substitution model with gamma‐distributed rate heterogeneity and a proportion of invariable sites, which was the best model of evolution for the data set as evaluated by ModelFinder  or jModelTest 2 (CIPRES) http://jphmm.gobics.de/), visual inspection, confirmed by subgenomic phylogenetic analysis and plotted using RecDraw Full‐genome subtype and CRF reference sequences were downloaded from the Los Alamos National Laboratory (LANL) HIV Database Table . Alignme2.4https://www.hiv.lanl.gov/content/sequence/HelpDocs/geo_regions.html, shown in Table https://github.com/saraquigley/) and based on the D3.js JavaScript library (https://d3js.org) by Mike Bostock.Envelope gene (gp160) sequences downloaded from the LANL HIV Database were filtered to include one sequence per individual from the Asia geographic region  is comprised primarily of males (96.0%), MSM (92.1%), of various occupations from Bangkok. Their socio‐demographic characteristics are presented in Table 3.1A total of 288 out of 303 samples were successfully screened by MHAbce (n = 63) or MSSPbce (n = 225) for subtype analysis. Viruses were predominantly CRF01_AE , followed by CRF01_AE/B recombinants , subtype B , subtype C  and non‐typeable .Subtypes of viruses obtained by SGS from one hundred and thirty‐eight individuals were predominantly CRF01_AE , seven of which were classified based on half‐genome sequences only. Nearly a quarter of infections were CRF01_AE/B recombinant . There were eight subtype B infections (5.8%), and one each of subtype C, CRF01_AE/B/C recombinant and CRF01_AE/CRF02_AG recombinant (0.7% each). A full list of RV254 sequences included in the present analysis is shown in Table Phylogenetic trees with reference sequences and RV254 half genomes are shown in Figure By phylogenetic analysis, only one of the eight subtype B infections (2544072) was a Thai B′ variant, although it contained the GPGR motif in V3 that is more commonly associated with Western B strains Figure . The ded3.2env gp160 sequences of all subtypes from RV254 (n = 135) and Asia (n = 1794). Upon visual inspection of the alignment, two Asia sequences containing either an unusual 5′ insertion or deletion were identified and excluded from analysis (GenBank accession numbers EF036536 and KJ140245 respectively). The alignment used for the present analysis is contained in File To determine whether RV254 sequences were closely related to other sequences within the cohort and in Asia, phylogenetic analysis was performed on A network diagram with identified potential transmission clusters is shown in Figure Overall, 123 clusters were identified containing a total of 348 nodes and 444 edges as shown in Table The median cluster size was a dyad, and most clusters were dyads  or triads ; however, larger clusters were observed: 4‐node , 5‐ and 6‐node , and 7‐, 10‐, 12‐, 14‐ and 15‐node .The largest cluster (15 nodes) was comprised of CRF01_AE sequences from Vietnam (11) and China (4) sampled between 1997 and 1999, and included PWID (2), nosocomial (1) and unknown (12) risk factors. Another international cluster included one 14‐node cluster with sequences from Thailand (10), Japan (2), Indonesia (1) and Vietnam (1). One dyad included sequences from Malaysia and Singapore. Each of the other clusters was limited to nodes from the same country.For a large proportion of clusters, risk factor information was unknown for at least one node  or all nodes . Risk factors for at least one node in a cluster included PWID , MSM , heterosexuals , nosocomial exposures or homosexuals , and haemophiliac, blood transfusion or mother to child transmission . Six edges were pairs of one MSM and one heterosexual , and one edge was a homosexual and haemophiliac pair (subtype B KR‐KR).A vast majority of clusters were entirely Chinese  or entirely Thai . Ten (8.1%) clusters were Japanese, seven (5.7%) were South Korean, three (2.4%) each was either Myanmar or Singaporean, two (1.6%) were Pakistani and one (0.8%) cluster each were Indian, Malaysian or Taiwanese.Of the 54 clusters with individuals from China, 25 included at least one PWID node, 15 included at least one heterosexual node and 14 included at least one MSM node. Of the forty‐one Thai clusters, eight included at least one PWID node, six included at least one MSM node and two included at least one heterosexual node.44.1Strains of CRF01_AE and subtype B, and their recombinants still represent the majority of circulating subtypes of HIV‐1 in Thailand. The subtype distribution of strains harboured by this cohort are similar to that of the Bangkok MSM Cohort Study, another primarily MSM and TGW cohort conducted between 2006 and 2014 In Thailand, the major proportion of subtypes is represented by a predominance of CRF01_AE and a minority of subtype B strains, facilitating the sustained emergence of recombinants between the two. By way of comparison, in the Nigerian epidemic in which two major subtypes (G and CRF02_AG) co‐circulate, the relative proportion of recombinants is similar to that of the parental subtypes env V2 may confer protection against multiple circulating strains Despite the theoretical challenges, immune correlates analyses conducted on the RV144 vaccine trial have found that vaccine‐induced antibodies targeting 4.2env) mediates viral entry into host cells, and though it is partially shielded by glycans from the immune response, it possesses the highest genetic diversity and evolutionary rate of any gene in the HIV‐1 genome env‐based networks may yield insight into viral pathogenesis, for a strain that has a high degree of connectivity may possess some advantage with respect to infectivity or fitness env‐based transmission networks could provide more contemporaneous data to show that current vaccine candidates may still cover the majority of circulating subtypes despite increasing diversity and recombination, and could also inform selection for vaccines in the pipeline.The envelope glycoprotein  of the sequences we retrieved from the LANL HIV Database for the present analysis do not contain risk data. Risk reporting may be limited , due to stigma or criminalization of that risk, or to protect privacy in ongoing studies, among other reasons. Within a cluster though, risk for nodes with incomplete annotation may, to some extent, be deduced from association with nodes with known risk.pol gene sequences (approximately 1 kb) which are readily available from routine drug resistance screening but may possess limited resolution for transmission network analysis The method of identifying potential transmission networks is dependent on pairwise sequence comparisons, and therefore, the quality and quantity of input sequences supplies the foundation and boundaries of the proposed network. Selecting sequences of differential lengths and locations in the viral genome will undoubtedly affect the shape of the network. Many previous studies, including one with RV254 participants, have used partial The majority of individuals living with HIV, including those who may transit between countries, are never enrolled in a study cohort, and thus, the comprehensive depth of any analysis of a given transmission network may be limited. However, the identified transmission network remains a reflection of the at‐risk population that could be accessed for other studies in prevention, treatment, cure or interventions such as pre‐exposure prophylaxis. Timely application of pre‐exposure prophylaxis may be key in controlling the HIV‐1 epidemic among MSM, and indeed is expanding rapidly in Thailand 5The HIV‐1 epidemic in Asia is increasingly more complex at the molecular level and among transmission networks. The persistence of circulating viral subtypes coupled with repeatedly exposed and/or migrating hosts combine to promote the emergence of more diverse and unique recombinant forms of HIV‐1. Lessons learned from the relatively more mature Thai epidemic may be useful to consider while addressing and monitoring the expanding epidemics in other areas of Asia.The authors declare no competing interests.EK, DJC, NC, MDS, JLE, PPhanuphak, NP, RJO, JHK, MLR, NLM and JA conceived, designed, implemented and managed the RV254/SEARCH 010 study protocol. DC and ST conceived and designed this analysis. DC, ESB, MB, AMO, RS, WR and ST were responsible for data collection. PPham and SP provided bioinformatics support. DC, ESB, PPham, EB, GK and ST contributed to data analysis and interpretation. DC conducted the literature search, wrote the manuscript and generated figures. All authors critically reviewed and approved the final version of the manuscript. Click here for additional data file.Figure S1. Phylogenetic tree of subtype B strains from RV254 participants with reference Western subtype B (right tree branches) and Thai B′ strains (left tree branch). Pure subtype B strains from RV254 participants were primarily Western B  versus Thai B′ . Subtype J (open triangles) is plotted as an outsider group.Figure S2. Phylogenetic tree of env gp160 gene sequences from RV254 participants. Coloured circles depict RV254 subtyping by phylogenetic analysis: subtype CRF01_AE (red), CRF01_AE/B recombinant (purple), B (light blue), B′ (dark blue), C (yellow), CRF01_AE/B/C recombinant (green) and CRF01_AE/CRF02_AG recombinant (grey).Table S1. Summary and details of RV254 and subtype reference sequences included in the present analysis. a) Summary of sequences from RV254 participants obtained by single‐genome sequencing, b) details of sequences listed by participant ID; FL = full genome, LH = 5′‐half genome, RH = 3′‐half genome, LH + RH = 5′‐ and 3′‐half genomes, ENV = envelope gene only, c) details of subtype reference sequences (full genome), d) details of subtype B, B′, and J reference sequences (env gp160).Table S2. Details of Asia env gp160 sequences downloaded from the Los Alamos National Laboratory (LANL) HIV Database and included in the present analysis.Table S3. Socio‐demographic characteristics of RV254 study participants.Table S4. RV254 subtype B V3 loop tip motifs and their global frequencies.Table S5. Clusters identified in Asian HIV‐1 transmission network.Table S6. Description of nodes identified in Asian HIV‐1 Transmission Network. a) Summary of nodes, b) detailed list of nodes. Country codes: China (CN), India (IN), Indonesia (ID), Japan (JP), Malaysia (MY), Myanmar (MM), Pakistan (PK), Singapore (SG), South Korea (KR), Taiwan (TW), Thailand (TH), Vietnam (VN). Risk factors: blood transfusion (PB), haemophiliac (PH), heterosexual (SH), homosexual (SG), people who inject drugs (PWID), men who have sex with men (MSM), mother to child (MB), nosocomial (NO), unknown (N/A).File S1. Alignment of RV254 and Asia env gp160 sequences used in the present analysis.Click here for additional data file.


Cannabis is the most widely used illicit substance by Australian young people, including those engaged with youth alcohol and other drug (AOD) systems. While recreational cannabis use in young people may be a developmental activity for some, for others, this usage becomes regular and be associated with poorer long term outcomes. This study reports on the rates of cannabis use and co-existing psychosocial complexity factors in the Youth Needs Census (2013 and 2016) where workers report on all clients in the youth AOD system, a cohort considered highly vulnerable.Data was examined for two rounds of data collection for the Youth Needs Census, including 823 youth AOD service engaged young people in 2016 and 1000 AOD service engaged young people in 2013, to identify usage rates, psychosocial outcomes, and changes over time.Daily use of cannabis alone significantly exceeded daily usage rates for methamphetamines, alcohol, and cannabis used alongside other substances. Daily cannabis use was significantly associated with mental health problems, employment problems, education problems, family problems, and housing problems. Daily cannabis use was associated with most psychosocial complexity factors to the same extent as daily methamphetamine use and daily alcohol use, with daily cannabis users only showing lower incidence of the drug-related harm measure. Notably, daily cannabis use also increased from 2013 (47.5%) to 2016 (54.2%).It is imperative that the number of individuals using cannabis is considered alongside the severity of harm when assessing the social impact of this substance. Within cannabis users engaged with the youth AOD system, who often have high levels of psychosocial complexity, cannabis is used daily by a large proportion of these youths and may play a role in negatively impacting their lives. In the Australian Institute of Health and Welfare (AIHW) National Drug Strategy Household Survey (NDSHS) 2013 [Evidence indicates that a complex range of social, family, peer, and personal elements influence the substance use patterns of adolescents and young adults . CumulatAge of first use of cannabis is proposed to be an important factor in determining progression to heavy or problematic use –15. AdolWhilst many occasional users have no long term consequences of cannabis use, evidence indicates poorer outcomes in heavy users. The associated functional impacts of regular cannabis use may be significant , 28. ResThe aim of the present study was to examine the prevalence of cannabis use in young people engaged with youth (AOD) services, and to investigate the psychosocial complexity factors present in this cohort, including how they have changed over time. Methamphetamine use and alcohol use were analysed for comparison with cannabis use. Five hypotheses were formed based on AIHW usage data from the general population and empirical findings: (1) Cannabis would be more commonly and frequently used than other substances; (2) Daily cannabis use would be associated with mental health problems, drug-related harm, suicidality, employment problems, education problems, criminality, family problems, and housing problems; (3) These psychosocial complexity factors would be associated with daily cannabis use to a similar or greater extent than daily methamphetamine or alcohol use; (4) Daily cannabis use would have increased in frequency between the 2013 and 2016 census periods; and (5) The psychosocial complexity factors associated with daily cannabis use would have increased in frequency between 2013 and 2016.SD = 2.82 years), with ages ranging between 8 and 27 years. Only 3 individuals were over 25 years of age. The sample included 339 females, 665 males and 6 transsexual young people. The 2016 sample was characterized by an average age of 18.84 years (SD = 2.74 years), with ages ranging between 10 and 27 years. Notably, only 5 individuals were over 25 years of age. The sample included 294 females, 522 males, 5 transsexual young people and 2 individuals who did not nominate a gender identity (an option available in the latter census).The Youth Needs Census (ThYNC) was collected in 2013 and 2016 across Victorian youth AOD services. The 2013 sample collected surveys on 1000 young people and the 2016 collection reported on a further 823 young people. In 2013, the average age of the sample was 18.93 years . This was followed by an assessment of service utilization . The survey then went on to review demographic and psychosocial complexity issues experienced by the young person. These complexity factors in these young people included: (i) mental health problems ; (ii) drug-related harm ; (iii) engagement in employment or education; (iv) criminal and forensic issues ; (v) family problems ; and (vi) housing instability. These psychosocial complexity factors were further investigated using associated sub-questions, including items querying the experiences of verbal, physical, and sexual abuse, as well as neglect; and experiences of criminal violence and family violence. ThYNC also included Section 2  of the Australian Treatment Outcome Profile (ATOP), a collection of questions designed to measure an individual’s level of psychological wellbeing, physical wellbeing, and quality of life on a standardized 10-point scale . The ATOThe Youth Needs Census is a three-yearly activity in which all participating Victorian youth AOD sector workers complete a census survey on each client they are working with (most recently conducted in 2016 in Victoria). One survey was conducted on each young person enrolled in the youth AOD system on the census date. The ThYNC project received ethics approval from the Eastern Health Human Research Ethics Committee in 2013 (E28–1213) and 2016 (LR89/2016). All participants provided informed consent by reading a participant information and consent form before completing each survey online. Workers that did not provide informed consent were taken to an automatic thank you page and exited from the survey.To complete ThYNC, all workers were informed of the upcoming census date and provided a link to an online survey offered using the Qualtrics software package . Managers were asked to monitor completion at their sites but records were not obtained on the number of surveys completed in each service to ensure the voluntary nature of the study for youth workers and services. The Youth Needs Census (2013 and 2016) was a 56-item clinician rated multiple choice audit tool . ThYNC wFour variables were available as potential identifiers of problematic substance use: (i) daily or almost daily use; (ii) use within the past month; (iii) worker identification of young person as substance dependent; and (iv) worker identification of the substance of primary concern . Daily use, use in the past month, and dependence were coded yes or no, and were not mutually exclusive with other substances . Substance of primary concern was rated by substance, forming independent groups .Daily or almost daily cannabis use was selected as the primary focus of hypothesis-testing, due to this variable most objectively capturing high frequency of use. Past month usage was judged to be less indicative of high frequency use, whilst dependence was not used as a measure as it was not determined using structured clinical tools and was not included in the 2013 ThYNC. The substance of primary concern variable was not included as it did not allow for acknowledgement of poly-substance use and was determined solely on clinician opinion. Past month usage, dependence, and substance of primary concern were however retained as informative in describing the sample.Methamphetamines and alcohol were chosen for cross-substance comparison of psychosocial complexity factors. This was because cannabis, methamphetamines, and alcohol were the three most frequent substances of daily use in both the 2016  and 2013 samples , excluding tobacco. Tobacco products were very commonly used , but were not investigated in the present study as they were seldom identified as a primary concern  in youth services clients. No other substance exceeded 5% of the sample in terms of daily use, dependence, or substance of primary concern.V was calculated to estimate effect size [p < .05. Bonferroni correction was not applied due to concerns that it would be overly conservative given the categorical nature of the data [n = 295); (ii) daily use of cannabis only (n = 309); (iii) daily use of cannabis as well as methamphetamines, alcohol, or all three substances (n = 137); (iv) daily use of methamphetamines only (n = 35); and (v) daily use of alcohol only (n = 41). Cases of daily use of both methamphetamines and alcohol (n = 6) were excluded as this substance combination was not a focus of the study and the group size was minuscule.The majority of ThYNC data was categorical in nature. For this reason, non-parametric testing was the most appropriate form of inferential statistics. Hypothesis-testing was conducted by generating contingency tables and deriving chi-square as the omnibus test, with responses of “other” or “unsure” excluded from analysis . Cramer’ect size . The sigthe data . Initial2(2) = 2.50, p = .287, Cramer’s V = .06, sexual orientation, Χ2(1) = 0.22, p = .639, Cramer’s V = .02, Aboriginal and Torres Strait Islander, Χ2(1) = 1.03, p = .310, Cramer’s V = .04, or asylum-seeker demographics, Χ2(1) = 0.48, p = .489, Cramer’s V = .02.Figure n = 823) = .05, p = .162, whilst 20.4% also used alcohol, Spearman’s ρ(n = 823) = .11, p = .002. In the past month, 33.0% of young people engaging in daily cannabis use had used methamphetamines, Spearman’s ρ(n = 823) = .08, p = .019, whilst 59.6% had also used alcohol, Spearman’s ρ(n = 823) = .14, p < .001. With regards to dependence, 84.8% of young people engaging in daily cannabis use were rated as dependent on cannabis, Spearman’s ρ(n = 823) = .80, p > .001, whilst 13.0% were dependent on methamphetamines, Spearman’s ρ(n = 823) < .01, p = .998, and 12.3% were dependent on alcohol, Spearman’s ρ(n = 823) = .05, p = .163. Cannabis was the primary substance of concern for 65.2% of young people engaging in daily cannabis use, with methamphetamines and alcohol the primary substance of concern for 19.1 and 7.2% respectively, Χ2(1) = 148.59, p > .001, Cramer’s V = .46.Overlap in daily or almost daily substance use was present, with 14.1% of young people engaging in daily cannabis also using methamphetamines daily, Spearman’s ρ(2(4) = 432.58, p > .001, supporting the first hypothesis. Figure 2(1) = 7.32, p = .007, Cramer’s V = .12, employment problems, Χ2(1) = 4.63, p = .032, Cramer’s V = .09, education problems, Χ2(1) = 12.09, p = .001, Cramer’s V = .15, family problems, Χ2(1) = 10.04, p = .002, Cramer’s V = .13, and housing problems, Χ2(1) = 4.83, p = .028, Cramer’s V = .09, supporting the second hypothesis.A visual comparison of group numbers is displayed in Fig. n = 823) = −.17, p < .001, physical wellbeing, Spearman’s ρ(n = 823) = −.20, p < .001, and overall quality of life, Spearman’s ρ(n = 823) -.19, p < .001. Elaborating on the first hypothesis test, these data further indicate an association between cannabis use and poorer mental and physical health.Notably, the rate of mental health problems in daily cannabis users included a 37.9% rate of formal diagnosis of a mental health condition. In terms of the past experiences of young people using cannabis daily, the data indicated that these young people had high lifetime histories of neglect (39.9%), emotional abuse (51.1%), physical abuse (39.5%), sexual abuse (15.5%) and being victims of violent crime (24.4%). In relation to family violence more specifically, young people using cannabis were reported to have witnessed family violence in 35.2% of cases, experienced as the victim of family violence in 34.3% and the instigator of family violence in 18.2% of cases. Daily cannabis use was weakly negatively associated with psychological wellbeing, Spearman’s ρ(2(2) = 15.10, p = .001, Cramer’s V = .21. These results support the third hypothesis.To test the third hypothesis, the proportion of each psychosocial complexity factor was compared across cases of daily cannabis use only, daily methamphetamine use only, and daily alcohol use only. No significant differences between the three substances were present for mental health problems, suicidality, employment problems, education problems, criminality, family problems, or housing problems, although young people engaging in daily use of cannabis only were significantly less likely to experience drug-related harm, Χ2(1) = 23.89, p < .001, Cramer’s V = .25, suicidality, Χ2(1) = 4.00, p < .045, Cramer’s V = .11, employment problems, Χ2(1) = 5.96, p < .015, Cramer’s V = .12, and education problems, Χ2(1) = 4.89, p < .027, Cramer’s V = .11. No significant differences were present for mental health problems, criminality, family problems, or housing problems.To acknowledge the occurrence of poly-substance use, where an individual may engage in the use of multiple substances on a daily basis, as well as the low number of young people using only methamphetamines or alcohol daily, cases of daily use of cannabis only were compared to cases of daily use of cannabis plus methamphetamines and/or alcohol in terms of psychosocial risk factors. Young people combining daily use of cannabis with daily use of methamphetamines and/or alcohol were significantly more likely to experience drug-related harm, ΧThe above hypothesis tests measured strength of association by analysing the proportion of psychosocial complexity factors relative to group size. This approach allowed inferences to be drawn regarding the likelihood of individual risk based on substance use category, but does not consider the greater prevalence of cannabis use. Figure 2(1) = 8.09, p = .004, Cramer’s V = .07. There were no significant changesin daily methamphetamine use or daily alcohol use between 2016 and 2013. Cannabis use in the past month remained stable between 2016 and 2013, however there were significantly less cases of use in the past month for both methampetamines, 29.5% versus 34.9% respectively, Χ2(1) = 5.95, p = .015, Cramer’s V = .06, and alcohol, 53.1% versus 63.1% respectively, Χ2(2) = 18.61, p < .001, Cramer’s V = .10. Dependence was not assessed in the 2013 sample. Cannabis was identified as the primary substance of concern in a greater proportion of cases in 2016, up to 52.3% in 2016 from 44.4% in 2013. Methamphetamines as primary concern increased only slightly between cohorts, 31.2% in 2016 versus 30.0% in 2013. The increase in young people for whom cannabis was a concern was a result of displacing individuals for whom alcohol was the primary concern, with alcohol as primary concern decreasing to 16.5% in 2016 from 25.6% in 2013. These frequency changes in primary substance of concern were significant, Χ2(2) = 19.56, p < .001, Cramer’s V = .11.The 2016 and 2013 cohorts did not significantly differ in age or gender. The proportion of young people using cannabis daily significantly increased to 54.2% in 2016 from 47.5% in 2013, Χ2(1) = 29.44, p < .001, Cramer’s V = .13, and disengagement from education, Χ2(1) = 4.86, p = .028, Cramer’s V = .05, but a significantly lower rate of current criminal behaviour, Χ2(1) = 13.04, p < .001, Cramer’s V = .09. Rates of drug-related harm, suicide attempt presence, disengagement from employment, family problems, and housing problems remained stable between 2013 and 2016 in young people who were using cannabis daily.Investigating the fourth hypothesis, Fig. This study examined cannabis use and psychosocial complexity factors in young people engaged with youth AOD services in 2016 and 2013. The results provide a picture of the impact that daily cannabis use has on young people accessing youth AOD services. As predicted in the first hypothesis, cannabis was found to be the most common substance of daily use, both on its own and in combination with other substances. Supporting the second hypothesis, cannabis use in young people was associated with elevated rates of mental health problems, disengagement from employment, disengagement from education, family problems, and housing problems. Mental health problems, suicidality, employment problems, education problems, criminality, family problems, and housing problems were present with daily cannabis use at a similar rate to their co-occurrence with daily methamphetamine and alcohol use, supporting the third hypothesis, although drug-related harm, suicidality, employment problems, and education problems were exacerbated when daily cannabis use was accompanied by daily use of another substance. In accord with the fourth hypothesis, the proportion of young people using cannabis daily had significantly increased between 2013 and 2016, although only mental health problems and disengagement from education worsened in frequency between 2013 and 2016.The major finding of this study was that cannabis was the most frequent substance of daily use, monthly use, dependence, and primary concern in young people accessing youth AOD services. Rates of cannabis use were more than double the rates of methamphetamine and alcohol use in this sample, and this overwhelmingly higher prevalence of daily cannabis use was maintained even when daily use of cannabis and other substances was considered separately. These findings indicate that cannabis prevalence in individuals accessing youth AOD treatment has now overtaken that of alcohol, a legally accessible substance that was more commonly used than cannabis in the previous ThYNC census, and also show that despite widespread media coverage of methamphetamine problems e.g., , 36, canDaily cannabis use was associated with a range of psychosocial complexity factors, in a similar fashion that methamphetamines and alcohol were problematic for these young people. Only drug-related harm was proportionately more likely for young people using methamphetamines or alcohol daily as opposed to cannabis. Drug-related harm, suicidality, employment problems, and education problems were exacerbated when cannabis was used daily alongside other substances. Notably, these findings indicate that cannabis use in youths engaged with the youth AOD system in Victoria have significant negative personal, family and societal impact on young people. With half of youths engaged with youth AOD services using cannabis daily with the majority of these young people experiencing negative consequences, this highlights the complex social issue around effectively supporting young people with cannabis as the substance of concern. Indeed, when examined in terms of the number of people affected, individuals using cannabis daily constitute a significantly larger amount of young people experiencing psychosocial complexity factors than do those using alcohol or methamphetamines. Supporting young people presenting with daily cannabis use is therefore responsible for greater consumption of services.Importantly, cannabis use is also an increasing concern, with daily use increasing by 6.7% between the 2013 and 2016 censuses, with the data suggesting that increasing cannabis use was displacing alcohol use in young people accessing youth services. The degree to which daily cannabis use was linked to the eight psychosocial risk factors examined by the present study largely remained stable since 2013. This means that for the past 3 years, most psychosocial concerns have not increased in prevalence in disadvantaged youth who use cannabis daily. However, both mental health problems and disengagement from education have worsened since 2013, with criminality lessening. It is timely when considering cannabis use and psychosocial complexity that ThYNC is an association study so there is no implication that increasing rates and level of cannabis use predict psychosocial complexity, nor that this complexity is predictive of cannabis use. Rather the study highlights to clinicians and researchers that there is often high rates of psychosocial complexity in young people engaged with AOD services who use cannabis on a daily basis . This inThe findings should be qualified in the context of limitations to the present research. Firstly, the categorical nature of the present data hindered parametric analysis. As a large survey that is sub-population-scale in scope, ThYNC questions are typically rated in dichotomous (yes-or-no) format, sometimes including an “other” or “unsure” option, with a focus on being as clearly defined and objective as possible for workers. This method of data collection meant that potentially useful quantitative metrics allowing for more rigorous statistical analysis  were not available. Similarly, the large-scale nature of data collection for this study precluded the use of more involved standardised measures with established reliability and validity.A second limitation was that the chi-square tests used to analyse the data identify an association, but do not assess cause. The eight psychosocial risk factors that were examined feasibly have a bidirectional relationship with substance use. Accordingly, these factors may have led to individuals seeking treatment from youth AOD services , or mighThirdly, while not a limitation as such, it is important to qualify the scope of the present research. This study made use of two large-scale data sets recorded from youth AOD workers about the young people that they were working with. This means that the population being studied here are those accessing youth AOD services, with the large sample size and pattern of results indicating that cannabis use is an issue that should not be ignored in this population. However, these results may not generalise to the general population that is of interest when further quantifying the social impact of cannabis use. These data specifically relate to the issues surrounding youths who are presenting and engaging with youth AOD services, not a general measure of the impacts of cannabis youth in the general population. Whilst this limits generalizability to the wider youth community of Australia, this is deliberate focus. The results of both ThYNC surveys indicate that these young people are often disengaged with school and work, in unstable housing and experiencing other factors that make them less likely to be captured in large scale data collection efforts e.g., , 2. ThYNGlobally, mental, neurological and substance use disorders contribute to a significant proportion of disease burden . HoweverThe association between regular cannabis use and psychosocial complexity has important clinical ramifications. For clients presenting with regular cannabis use, clinicians may benefit form exploring their experience of psychosocial complexity. Likewise, in people presenting with clear psychosocial complexity, screening for cannabis use may prove important and require consideration in the case formulation. Overall this research again highlights that systematic assessment of substance use in AOD help-seeking young people is important in any debate around cannabis, particularly those considering the issues from developmental and harm minimization perspectives.


The authors would like to draw the reader's attention to the errors in the following article.J Diabetes Investig 2019; 10: 163–170.Kijmanawat A, Panburana P, Reutrakul S, Tangshewinsirikul C. Effects of probiotic supplements on insulin resistance in gestational diabetes mellitus: A double‐blind randomized controlled trial. On pages 163  and 164 (under ‘METHODS’), the period that a double‐blind, placebo‐controlled, randomized clinical trial was carried out should have been between July 2016 and February 2017.In Figure 1 on page 166, the period that GDM patients at GA 24 – 28 weeks were in Ramathibodi Hospital should have been between July 2016 and February 2017.The authors apologize for these errors and any confusion they may have caused.


Correlations between their expression and calcification levels (Alizarin Red S staining) were determined. In addition, the effect of PTHrP and hedgehog proteins on canine and human chondrocyte‐like cells (CLCs) was determined in vitro focusing on the propensity to induce calcification. The expression of PTHrP, its receptor (PTHR1) and hedgehog receptors decreased during loss of the NC phenotype. N‐terminal (active) hedgehog (Indian hedgehog/Sonic hedgehog) protein expression did not change during maturation or degeneration, whereas expression of PTHrP, PTHR1 and hedgehog receptors increased during IVD degeneration. Hedgehog and PTHR1 immunopositivity were increased in nucleus pulposus tissue with abundant vs no/low calcification. In vitro, hedgehog proteins facilitated calcification in CLCs, whereas PTHrP did not affect calcification levels. In conclusion, hedgehog and PTHrP expression is present in healthy and degenerated IVDs. Hedgehog proteins had the propensity to induce calcification in CLCs from degenerated IVDs, indicating that in the future, inhibiting hedgehog signaling could be an approach to inhibit calcification during IVD degeneration.Parathyroid hormone‐related protein (PTHrP) and hedgehog signaling play an important role in chondrocyte development, (hypertrophic) differentiation, and/or calcification, but their role in intervertebral disc (IVD) degeneration is unknown. Better understanding their involvement may provide therapeutic clues for low back pain due to IVD degeneration. Therefore, this study aimed to explore the role of PTHrP and hedgehog proteins in postnatal canine and human IVDs during the aging/degenerative process. The expression of PTHrP, hedgehog proteins and related receptors was studied during the natural loss of the notochordal cell (NC) phenotype during IVD maturation using tissue samples and de‐differentiation  Hedgehog and PTHrP expression decreases duringmaturation to increase back again in advanced stages of IVD degeneration; herein hedgehogs facilitate calcification A positive correlation between IHH or SHH expression and osteoarthritis (OA) has been described.Hedgehogs have been studied to some extent in joint cartilage degeneration.Previous work indicated that IVD degeneration resembles OA, for example, hypertrophic differentiation and calcification are phenomena that can occur in both tissues during degeneration.22.1In this study, hedgehog, PTHrP, and related receptor expression and function were determined in two different phases: during the loss of the NC phenotype (maturation phase) and in maturated until severely degenerated IVDs (degeneration phase) Figure . Canine ie, IHH and SHH) and PTHrP during loss of the NC phenotype, hedgehog‐ and PTHrP‐related gene and protein expression was studied in healthy‐maturated canine IVDs and NCs from healthy NCD canine IVDs. To determine the role of hedgehog and PTHrP in IVD degeneration, hedgehog‐ and PTHrP‐related protein expression was studied in maturated until severely degenerated canine and human IVDs. Additionally, the correlation between hedgehog, PTHrP and related receptor expression and calcification was determined in surgically removed human NP tissue across the range of histological IVD degeneration. Lastly, the effect of IHH, SHH, and PTHrP was determined on calcification of canine and human CLCs from degenerated IVDs in vitro.To determine the expression of possible relevant hedgehogs , 10% fetal bovine serum  and 1% penicillin/streptomycin . Samples for RT‐qPCR were collected at day 0, 2, and 4. RNA was isolated and cDNA was generated from the cultured NCs (n = 8) and native NPs (n = 6) as described previously.IHH, SHH, patched (PTCH1), smoothened (SMO), parathyroid hormone‐related protein (PTHrP), parathyroid hormone receptor‐1 (PTHR1), and transcription factors GLI1, GLI2, and GLI3 was performed and analyzed as described previouslyEΔΔCq) method was used. For each target gene, the Cq‐value of the test sample was normalized to the mean Cq‐value of four stably expressed reference genes : ΔCq = Cq mean ref − Cq target. Secondly, the ECqΔ‐value for the test and calibrator sample was calculated. In this formula, E indicates the amplification efficiency of the target/reference gene and the mean Cq‐value of all tested samples was used as calibrator. ECqΔΔ was calculated by normalizing the ECqΔ‐value of the test sample to one of the calibrators: ECqΔΔ = ECq testΔ/ECq calibratorΔ. For each target gene, the relative expression and standard deviations in gene expression were calculated.To study hedgehog and PTHrP expression during loss of the NC phenotype, hedgehog, and PTHrP related gene expression was studied in NCs from healthy NCD canine IVDs that lost their typical vacuolated phenotype and marker  expression during monolayer culture.2.3n = 8‐7‐8‐7‐7 for grades I‐V, respectively). Tissue was obtained <24 hours after death, and IVD slices were decalcified in 0.5 M EDTA (pH 7.0) for 3 months as previously described.Hedgehog‐ and PTHrP‐related immunopositivity was determined in canine and human NPs from healthy until severely degenerated (Thompson scores I‐V) IVDs. All dogs had been euthanized in unrelated experiments, approved by the Utrecht University Animal Ethics Committee, or were client‐owned dogs that were submitted for necropsy to the Faculty of Veterinary Medicine, Utrecht University. Thirty‐seven thoracolumbar or lumbosacral IVDs from dogs of various breeds , age (1‐16 years) and gender  were studied . The samn = 5‐4‐4‐5‐4 for grades I‐V, respectively, Human IVDs were obtained during standard postmortem diagnostics. The L2‐L5 part of the spine was collected (<48 hours after death), as approved by the scientific committee of the Pathology department . Anonymous use of redundant tissue for research purposes is a standard treatment agreement with UMCU patients . IVDs were used in line with the code “Proper Secondary Use of Human Tissue,” installed by the Federation of Biomedical Scientific Societies. Also, the human IVDs were chosen based on approximately equal representation of all Thompson scores  and immunohistochemically stained for N‐terminal hedgehog (IHH/SHH), PTCH1, SMO, PTHrP, and PTHR1 Table . NegativFor all staining, raw images were captured with a Leica DFC420C digital camera (Leica Microsystems) mounted to a BX60 microscope (Olympus) and Leica Application Suite (V4.2) software package. (Positively stained) cell numbers in each NP were manually counted. Adobe Photoshop CS6 was used to manually count (positively stained) cell numbers in four (canine) or six (human) randomly selected NP areas per IVD section as described previously.2.4Surgically removed NP tissue  was fixed in 10% v/v neutral buffered formalin and processed to paraffin wax. Following embedding, 4‐μm sections were cut and histologically graded using the previously published criteria.2.5−9 M dexamethasone  and 1 ng/mL bFGF ) at 21% O2, 5% CO2, 37°C. All canine and human IVDs were mildly degenerated (Thompson grade II/III). After 1 week, expansion medium was replaced by hypertrophic induction medium −7 M PTHrP , 1 μg/mL N‐terminal IHH  or 1 μg/mL N‐terminal SHH . Concentrations for PTHrP,−7 M dexamethasone and 10 mM β‐glycerolphosphate)−7 M PTHrP. After 14 days, monolayers were fixed in 4% neutral buffered formaldehyde for 2 hours. Alizarin Red S stainingPassage 2 CLCs from four canine donors  and four human donors  were plated at a density of 10 000 cells/well in 12‐wells plates  in expansion medium  were determined. Benjamini & Hochberg False Discovery Rate post‐hoc corrections for multiple comparisons were performed. A P‐value <.05 was considered significant. A χ² test was performed to determine whether calcium deposition (≤1 and > 1 intensity) increased per histologic degeneration grade .Statistical analysis was performed using IBM SPSS (version 22). All data were examined for normal distribution (Shapiro‐Wilks test). Kruskal‐Wallis and Mann‐Whitney 33.1To study the IVD maturation phase (defined by loss of the NC phenotype), hedgehog and PTHrP signaling‐related expression was studied in vitro in NCD canine NC‐rich NP tissue and 0 to 4 day cultured NC clusters described to de‐differentiate and lose their specific phenotype and marker  expression by gene expression analysis3.1.1IHH messenger RNA (mRNA) expression was significantly higher in NCD canine NP tissue than in 0‐ to 4‐day cultured NCs (P < 0.05), where it was hardly detectable  and decreased rapidly during culture , where it was hardly detectable  and their transcription factors GLI1 and GLI2 was hardly detectable  in canine NCs that lost their typical vacuolated morphology during culture.e Figure A. SHH mRPTHrP mRNA expression was significantly higher in freshly isolated NCs than in native tissue (P < .05) and decreased rapidly during NC culture .3.1.2In healthy, Thompson score I canine NPs, only NC clusters were detected Figure E. In mat3.1.3P < .05, Figure The N‐terminal hedgehog antibody demonstrating active hedgehog  signaling did not render specific staining in the canine samples and therefore, this antibody was only studied in human tissues. Immunopositivity (ratio of positive cells) for PTCH1 and SMO was significantly higher in canine NP tissues from healthy, Thompson score I NC‐rich IVDs than in NP tissue from maturated, Thompson score II CLC‐rich IVDs , whereas only CD dogs showed a positive correlation between SMO immunopositivity and macroscopic IVD degeneration grade . PTHrP immunopositivity significantly increased during canine IVD degeneration . Immunopositivity for PTHR1 increased from NPs of maturated, Thompson scores II to III IVDs to severely degenerated, Thompson score V IVDs . Although the current study demonstrates some differences in hedgehog‐PTHrP (related) expression between CD and NCD dogs, which may be related with their differential genetic background, the direction of the correlation between protein expression and IVD degeneration grade was similar.Considering the differential clinical representation of IVD disease among dog breeds,3.2.2postmortem , correlation are given in relation to the histological IVD degeneration grade. A significant positive correlation between PTCH1, SMO, and PTHrP immunopositivity and histologic IVD degeneration grade was encountered  demonstrated that PTHrP did not induce calcifications under the conditions studied . Moreover, the number of cells with PTHR1 and N‐terminal hedgehog immunopositivity was significantly higher in the NP tissues associated with high calcium deposition  and PTHrP expression increased during the IVD degeneration process in the studied species. This result is in line with the positive correlation between IHH expression and OA,4.3Based on their well‐described role in chondrocytes,4.4in vitro. Interestingly, SHH was even more potent in inducing calcification in human and canine CLCs than IHH. Together with the increased hedgehog expression in IVDs with high levels of calcium, this may suggest a possible role for SHH and/or IHH in calcification during IVD degeneration. In line with this thought, disrupted IHH signaling prevented hypertrophic chondrocyte differentiation and osteophyte formation in OA cartilage.In contrast to PTHrP, IHH is known to promote chondrocyte hypertrophy and calcification in OA.5in vitro. Taken together with the increased N‐terminal hedgehog immunopositivity in IVDs with high levels of calcium deposition, this may indicate that inhibition of hedgehog signaling could be a therapeutic approach to inhibit CLC calcification in IVD degeneration.Expression of hedgehogs, PTHrP and their receptors were high in NCs from young, healthy IVDs, decreased together with the loss of the NC phenotype, and increased again in the advanced stages of IVD degeneration. While PTHrP did not affect calcification, IHH and mainly SHH had the propensity to facilitate calcification in human and canine CLCs The authors declare no potential conflict of interest.The manuscript has been read and approved by all authors. There are no other persons who satisfied the criteria for authorship. Each author has contributed to a minimum of two of the four major parts of the submitted work and there are no “Gift Authorships.” Given the extent of the study, nine authors have been listed. They contributed as follows: F.C.B. collected material, performed most of the experiments, mined, and analyzed the data, participated in the design of the study, and drafted the manuscript. K.M.R., F.M.R., J.W.S., C.L.M., W.A.M.J., and Y.Z. assisted in performance of experiments and mining the data. L.B.C. and C.L.M. provided human IVDs. L.B.C., C.L.M., and D.C. contributed to the study design and data analysis. M.A.T. conceived the study, mined and analyzed data, assisted in drafting the manuscript and coordinated the process. All authors read and approved the final manuscript.Supporting information 1 Primers used for quantitative PCR of canine samplesClick here for additional data file.Supporting information 2 Canine and human IVD donors for immunohistochemistryClick here for additional data file.Supporting information 3 PTHrP immunopositivity in young canine and human NP tissueClick here for additional data file.Supporting information 4 Indian and Sonic hedgehog facilitate calcification in human and canine chondrocyte‐like cells in vitro.Click here for additional data file.Supporting information 5 PTHrP does not facilitate calcification in canine chondrocyte‐like cells in vitro.Click here for additional data file.