Many patients with Alzheimer's dementia (AD) also exhibit noncognitive symptoms such as sensorimotor deficits, which can precede the hallmark cognitive deficits and significantly impact daily activities and an individual's ability to live independently. However, the mechanisms underlying sensorimotor dysfunction in AD and their relationship with cognitive decline remains poorly understood, due in part to a lack of translationally relevant animal models. To address this, we recently developed a novel model of genetic diversity in Alzheimer's disease, the AD‐BXD genetic reference panel. In this study, we investigated sensorimotor deficits in the AD‐BXDs and the relationship to cognitive decline in these mice. We found that age‐ and AD‐related declines in coordination, balance and vestibular function vary significantly across the panel, indicating genetic background strongly influences the expressivity of the familial AD mutations used in the AD‐BXD panel and their impact on motor function. Although young males and females perform comparably regardless of genotype on narrow beam and inclined screen tasks, there were significant sex differences in aging‐ and AD‐related decline, with females exhibiting worse decline than males of the same age and transgene status. Finally, we found that AD motor decline is not correlated with cognitive decline, suggesting that sensorimotor deficits in AD may occur through distinct mechanisms. Overall, our results suggest that AD‐related sensorimotor decline is strongly dependent on background genetics and is independent of dementia and cognitive deficits, suggesting that effective therapeutics for the entire spectrum of AD symptoms will likely require interventions targeting each distinct domain involved in the disease. Sensorimotor deficits in Alzheimer's disease are distinct from AD‐related cognitive decline or normal age‐related decline. Notably, several of these models have been reported to also exhibit sensorimotor deficits, suggesting that motor impairments are an inherent part of the disease process.Significant progress in understanding the pathophysiology of AD has been made using mouse models incorporating familial mutations in amyloid precursor protein , while the other ~50% are nontransgenic littermate controls (termed Ntg‐BXDs). Mice from a total of 27 Ntg‐ and AD‐BXD  strains were used in this study; mice were genotyped for the 5XFAD transgene by either the Transgenic Genotyping Service at The Jackson Laboratory or Transnetyx, Inc. All mice were fed a standard laboratory mouse chow (Teklad 8604) and both food and water were available ad libitum. All mice were kept on a 12:12 light cycle and were phenotyped as previously described.Generation of the AD‐BXD panel was described in detail in References.All experiments involving mice were performed at the University of Tennessee Health Science Center and were approved by the Institutional Care and Use Committee at that institution and carried out in accordance with the standards of the Association for the Assessment and Accreditation of Laboratory Animal Care (AAALAC) and the NIH Guide for the Care and Use of Laboratory Animals.2.2Narrow beam: Briefly, the narrow beam task was used to assess motor and balance coordination by placing each mouse in the center of a 1 m long beam 12 mm wide that was elevated 50 cm above a table surface . The time taken in seconds for each mouse to cross the beam to a safe platform on either side was recorded. A maximum time limit of 180 seconds was imposed; if a mouse fell from the beam, the maximum time of 180 seconds was given. The average of three trials for the task was used to assess the performance for each mouse and the average used for statistical analysis.Sensorimotor function was assessed at 6 and 14 months of age in the Ntg‐BXD and AD‐BXD panels. A total of 894 mice  across a total of 27 AD‐ and Ntg‐BXD strains were included in this study. The sensorimotor tasks used were narrow beam, negative geotaxis (incline screen) and grip strength.Incline screen: The incline screen task was used to assess vestibular and/or proprioceptive function. Each mouse was placed nose‐down in the center of a wire mesh grid (1 × 1 cm) positioned at a 45° angle (Harvard Apparatus). The time taken for a mouse to reorient itself with its nose facing upwards (negative geotaxis) was recorded; the average of three trials for each mouse was used for analysis. As with narrow beam, a 180 seconds maximum time limit for righting was used.Grip strength: Muscle strength was measured using a standard grip strength meter. Each mouse was placed horizontally on the wire grid of the apparatus (Columbus Instruments) with all four paws grasping the grid and then gently pulled away from the grid by the base of its tail until its grip released to measure the force exerted. The average of three trials for each mouse was recorded and used for analysis.2.3https://www.synapse.org/#!Synapse:syn17016211). Strain averaged behavioral data has also been deposited at http://genenetwork.org. Heritability estimates for the sensorimotor phenotypic traits in the AD‐ and Ntg‐BXDs were determined by calculating the ratio of between‐strain variance to total sample variance  according to.All data and statistical analysis were performed using R version 3.5.3). A univariate ANOVA was used for each sensorimotor task using genotype, age, sex and strain as fixed factors. Data are reported as mean ± SE of the mean in both the main text and figure legends. A two‐sided Pearson's correlation test was used to assess the relationship of decline between the Ntg‐BXD and AD‐BXD on these tasks. CFA and CFM measures used for correlations in Figures .5.3. A u33.1Since balance and coordination skills are often impaired in human AD patients,1,886 = 24.5, P < .0001). There was a main effect of age on narrow beam performance  and presence of the AD transgene significantly exacerbated age‐related decline  . Furthermore, although there was a trend for 6 m AD‐BXD mice to perform worse on this task than their age‐matched Ntg‐BXD controls, this difference was not significant (adj. P > .1). Lastly, although there was no significant main effect of sex on narrow beam performance across the panel  < 1, n.s.), there were significant interactions between both age and sex  = 9.16, P < .005) and sex and genotype  = 7.9, P < .005), with post hoc comparisons suggesting that impairment due to age and AD status was greater in females than males  = 3.35, P < .0001; StrainAD: F27,474) = 2.7, P < .0001) and a high degree of heritability for this task (Table left panels), there is no effect of age, with most Ntg‐BXD strains performing as well or better at 14 m compared with 6 m , suggesting that age related decline is exacerbated in most  AD‐BXD strains. Notably, although there are significant main effects of both strain and genotype as described above, there is no significant correlation between the Ntg‐ and AD‐BXD strains on narrow beam performance in either males  or females   estimates comparing the between‐strain variance (an estimate of variation due to genetic factors) to total sample variance  indicate that genetic factors significantly influence phenotypic variation . There was also a significant effect of age on inclined screen performance  = 59.8, P < .0001). For this task, there was a significant interaction between age and genotype  = 20.33, P < .0001). As with the narrow beam task, post hoc multiple comparisons indicated that Ntg‐BXD mice do not exhibit significant age‐related decline (adj. P > .1), while inclined screen performance is significantly worse with age in the AD‐BXD mice (adj. P < .000001), suggesting a strong interaction between age and genotype on vestibular function and proprioception. Again, there was no main effect of sex on inclined screen performance  = 1.5, P > .1), nor was there a significant interaction between sex and age  = 1.14, P > .1). There was an interaction between sex and genotype  = 3.87, P < .05), which post hoc comparisons indicate is due to worse performance by AD mice relative to their Ntg counterparts, with females affected more than males  = 2.04, P < .005; StrainAD: F = 2.2, P < .0005), once again suggesting a strong influence of naturally occurring genetic variation on vestibular function and proprioception  < 1, n.s.; Age*StrainAD: F = 1.2, P > .1). We found a significant interaction between strain, sex and genotype, indicating that female mice from AD strains are impaired relative to male AD strains, which may account for the slight difference in decline between 6 and 14 m of age shown in Figure r = .21, P > .1, df = 21; males: r = −.13, P > .1, df = 15) ; however, it should be noted that there is very little phenotypic variation in the inclined screen task compared with CFM, with all female Ntg‐BXD strains performing very well on this task.Similar to the narrow beam task, we found no correlation between performance on the inclined screen test and long‐term CFM in male Ntg‐ and AD‐BXD strains or female AD‐BXD strains Figure B,C, sugg3.3 < 1, n.s.). As expected, there was a significant effect of age on grip strength, with both Ntg and AD mice exhibiting significant decline with age from 6 to 14 m  = 1444.70, P < .00001)  = 40.97, P < .00001)  = 3.02, P < .00001; StrainAD: F = 4.2, P < .00001) and a significant interaction between age and strain  = 2.6, P< .00001; Age*StrainAD: F = 2.34, P < .0005). Consistent with our finding that there was no influence of genotype on grip strength, we found no significant interaction of genotype with strain  < 1, n.s.). Taken together, these results suggest that age and genetic background profoundly impact muscle strength and decline and that this is not impacted by the presence of the AD transgene. As with the other sensorimotor traits assessed in this study, the correlation between grip strength in the Ntg‐BXD and AD‐BXD is not significant, although it is stronger than observed for narrow beam and inclined screen , suggesting that individual strains may perform more similarly at this task.We determined the influence of genetic background on grip strength by assessing average grip strength for each strain at 6 and 14 m Figure A. As sho44.1Although dementia and cognitive decline are the hallmark diagnostic symptoms of AD in humans, many AD patients also experience impairments in noncognitive domains, including phenotypes associated with sensorimotor function such as coordination, balance, muscle strength and proprioception.4.2We recently developed the first mouse model of genetic diversity in AD‐ the AD‐BXDs ‐ and showed that the inclusion of naturally occurring genetic variation introduces significant phenotypic variation in cognitive traits, including long‐term memory. Specifically, despite all strains carrying the same high‐risk 5XFAD transgene, some strains exhibit resilience to cognitive impairment while others show increased susceptibility.h2) estimates for all three sensorimotor domains evaluated in this study range from 0.6 to 0.9 , we do see a trend for worse narrow beam performance in the AD‐BXDs compared with Ntg‐BXDs at 6 m of age  suggests they are amenable to QTL mapping to identify candidate genes and loci associated with sensorimotor performance. However, the number of strains used in the present study is insufficient for this approach and additional strains will be needed to ensure we are adequately powered for such analysis. That said, we are still able to investigate the relative contributions of the two parental genomes of the BXD family (C57Bl/6J and DBA/2J) and how their influence may differ between cognitive and noncognitive phenotypes. For example, we recently reported that the B6 genetic background confers resilience to the cognitive effects of the 5XFAD transgene.Genetic reference panels such as the BXD family used here represent powerful tools for the analysis and genetic mapping of quantitative traits such as the sensorimotor traits investigated here. As such, they are ideal for discovering new genetic loci associated with complex traits.4.6The results presented here provide further evidence for the translational utility of our novel AD‐BXD panel, which we now show also exhibits sensorimotor decline similar to that reported in human AD patients. Moreover, this decline is strongly modified by genetic background and exhibits a high degree of heritability, consistent with the human disease. Thus, the AD‐BXD panel represents a novel tool to investigate sensorimotor deficits in normal aging and AD and facilitate the discovery of novel targets to address these deficits. Our finding that there is no correlation between motor and cognitive phenotypes in either the AD strains or the normal aging controls indicates that motor decline likely occurs via a mechanism distinct from cognitive decline in both AD and normal aging individuals. Future work will incorporate additional strains for genome‐wide mapping to identify potential modifier genes and omics analysis of CNS regions associated with sensorimotor tasks.K.M.S.O., S.M.N. and C.C.K. conceived of the experiments. S.M.N. conducted the behavioral experiments. K.M.S.O., A.R.O and C.C.K. designed the analyses. A.R.O. and K.M.S.O. performed the data analysis. A.R.O., C.C.K., S.M.N., A.R.D and K.M.S.O. contributed to the interpretation of results. K.M.S.O. wrote the manuscript. All authors approved of the final manuscript.


We sought to determine the proportion of bronchiolitis episodes attributable to respiratory syncytial virus (RSV) among ICD‐9 coded infant bronchiolitis episodes which were tested for RSV.Bronchiolitis healthcare encounters were extracted from Kaiser Permanente Northern California databases for years 2006 to 2009. We used ICD‐9 codes for bronchiolitis to capture bronchiolitis‐related healthcare encounters including hospital admissions , emergency department visits (EDV), and outpatient visits (OPV). We reported the monthly proportion of RSV‐positive bronchiolitis episodes among tested bronchiolitis episodes. We used logistic regression to assess association between bronchiolitis episodes and patient demographic and health care characteristics. We also used logistic regression to assess association between decision to test and patient demographics and health care characteristics.Among 10,411 ICD‐9 coded infant bronchiolitis episodes, 29% were RSV tested. Fifty one percent of those tested were RSV positive. Between December and February, and in infants ≤6 months, the proportion of bronchiolitis episodes that were attributable to RSV was 77.2% among hospitalized episodes, 78.3% among EDV episodes, and 60.9% among OPV episodes, respectively. The proportion of RSV‐positive bronchiolitis episodes varied based upon infant age at diagnosis, level of health care service used, and time of the year of the episode.Estimation of the proportion of ICD‐9 coded bronchiolitis episodes attributable to RSV is more specific when restricting to bronchiolitis episodes during peak months, younger infant age, and those requiring higher level of healthcare. Is it RSV?Perspective on this article:  AAPAmerican Academy of PediatricsCIConfidence IntervalEDVEmergency Department VisitICD‐9International classification of diseasesKPNCKaiser Permanente Northern CaliforniaOPVOutpatient VisitPCRPolymerase Chain ReactionPRIMAPrevention of RSV: Impact on Morbidity and AsthmaRSVRespiratory syncytial virus1Bronchiolitis is a lower respiratory tract infection characterized by increased respiratory effort and wheezing.The purpose of this study was to calculate the proportion of RSV‐positive bronchiolitis episodes among infant bronchiolitis episodes identified by ICD‐9 codes, utilizing a population of infants in whom both ICD‐9 codes as well as laboratory RSV detection methods, were available in electronic health records. Improving the specificity of ICD‐coded RSV‐bronchiolitis episodes may help surveillance and prevention strategies by identifying RSV peak season and target age groups.2This study was approved by both the Kaiser Permanente Northern California (KPNC) and Vanderbilt University Institutional Review Boards. Informed consent process was waived in accordance with 45 CFR 46.116 (d) by Vanderbilt University Institutional Review Board. Use of birth certificate data was approved by the State of California Committee for the Protection of Human Subjects.Infant (defined as age ≤ 12 months) bronchiolitis healthcare encounters were extracted from KPNC databases for the years 2006 to 2009, a subset of the “Prevention of RSV: Impact on Morbidity and Asthma (PRIMA)” cohort study.Encounters that occurred within 14 days of each other were considered as one episode and were classified based on the highest level of care visit type the infant experienced , while the bronchiolitis encounters that were 14 days or more apart were treated as independent episodes. More than one test and/or one detection method  could be performed during one bronchiolitis episode. The episode was classified as RSV positive if any of the tests were positive. If multiple tests per episode were negative, only the PCR result was retained. Since 2006, KPNC started using primarily PCR to test for RSV (78.3%), with antigen and culture methods constituting only 13.2% and 8.5% of the tests, respectively. In this study, we included ICD‐9We reported the monthly proportion of RSV‐positive bronchiolitis episodes among tested bronchiolitis episodes. The proportion of RSV‐positive bronchiolitis episodes was further calculated among the subset of tested bronchiolitis episodes: by infant age at the time of bronchiolitis diagnosis (≤6 months and ≤ 3 months), by both infant age at the time of bronchiolitis diagnosis (≤6 months) and the severity of the bronchiolitis episodes , and by gestational age . We conducted post hoc analysis using multivariable logistic regression to assess the association of infant age at bronchiolitis diagnosis, sex, season of the episode (peak RSV season vs. non‐peak season), severity of the bronchiolitis episodes , RSV‐immunoprophylaxis eligibility based on American Academy of Pediatrics (AAP) guidelines,3n = 608) ICD‐9 code 466.11 (RSV‐bronchiolitis), 0.2% (n = 25) ICD‐9 code 480.1 (RSV‐pneumonia), and 93.9%  ICD‐9 code 466.19 (non‐RSV bronchiolitis). Among the 10,411 ICD‐9 coded bronchiolitis episodes, the median infant age at the first bronchiolitis episode was 184 days . Sixty one percent of episodes  were in male infants, and 13.8%  were among preterm infants. Of total bronchiolitis episodes, 89.0% , 4.1% (n = 425), and 6.9% (n = 717) were OPV, EDV, and hospitalization, respectively.A total of 10,411 ICD‐9 coded bronchiolitis episodes among 6,655 infants were identified over the 4‐year period, 2006 to 2009. These included: 5.8%  of all ICD‐9 identified bronchiolitis episodes during infancy were laboratory tested for RSV. The proportion of bronchiolitis episodes that were tested within each of the three ICD‐9 codes was as follows: among 608 RSV‐bronchiolitis coded episodes, 59.4% were tested; among 25 RSV‐pneumonia coded episodes, 44.0% were tested; and among 9,778 non‐RSV coded bronchiolitis episodes, 27.3% were tested .In total, 29.2% (ed Table . Comparen = 7), and the RSV positive episodes occurred only among hospitalized infants in a single year (2007). The monthly ratio of percentage RSV positive bronchiolitis episodes to the percentage of RSV tested bronchiolitis episodes exceeded one between November and March, and identifies the RSV season in each of the study years , 54.4% were RSV positive. When we limited to bronchiolitis episodes occurring among infants ≤6 months and ≤ 3 months, 56.9% and 66.3% were RSV positive, respectively. When we stratified bronchiolitis episodes in infants ≤6 months by type of healthcare encounter, RSV positive bronchiolitis episodes were highest among hospitalized episodes (68.5%), compared with EDV (66.7%) and OPV (52.6%) Table . Further months, We next stratified RSV tested bronchiolitis episodes by established risk factors for RSV. A stratified analysis was conducted on bronchiolitis episodes occurring during the first 6‐months by gestational age, a known risk factor for RSV infectionWe conducted a multivariable logistic regression for outcome of RSV positive bronchiolitis episodes among tested episodes Table . YoungerIn addition to evaluating factors that were associated with testing for RSV, we conducted multivariable logistic regression including infant age at diagnosis, meeting AAP defined eligibility for RSV‐immunoprophylaxis guidelines, level of care, calendar month during which illness occurred, infant sex, and parent reported infant race as covariates Table . The rel4In a large population‐based study of infants who were continuously enrolled in an integrated healthcare system, we calculated the proportion of ICD‐9 defined and laboratory tested bronchiolitis episodes that were attributable to RSV. ICD‐9 coded bronchiolitis episodes occurring during the peak months of winter viral season, among younger infants, and among those with higher levels of care, were more likely to be attributable to RSV.In agreement with other studies,In a multi‐year surveillance study of 31 US ED sites, Makari and colleagues reported that 62% of ICD coded bronchiolitis EDV encounters were RSV positive by PCR during the peak period (January 15 to end of February).The majority of the infants in our study were deemed to be low‐risk (97%), born at term or later, and without underlying conditions. Three percent of episodes were among those born preterm, and RSV‐immunoprophylaxis eligible (high‐risk) infants who may or may not have received RSV‐immunoprophylaxis. In our multivariable regression analysis, there were decreased relative odds of having at least one RSV positive bronchiolitis episode for RSV immunoprophylaxis eligible vs. non‐eligible and for subjects receiving RSV‐immunoprophylaxis. Eighteen percent of those eligible to receive RSV immunoprophylaxis did not receive any RSV‐immunoprophylaxis and 1% of those ineligible, received at least one dose of immunoprophylaxis. It may be that the routine care of high‐risk infants is different from the care for term or later birth infants. Parents may be less likely to send high‐risk infants to daycare, may take extra caution with regard to ill contacts, and healthcare providers may do more testing to include a larger proportion of infants with a broader spectrum of respiratory illness, as evidenced by the higher proportion tested (34.6% vs. 28.3%).Our study has several strengths, including a large sample of bronchiolitis episodes which were tested for RSV. In addition, the majority of tests (78.3%) were done using the most sensitive clinical testing modality for RSV detection, PCR. Antigen and culture RSV detection results were used only when PCR was not performed. This approach overcomes the limitation of most surveillance studies, which are based on low sensitivity antigen RSV detection methods.Despite the strengths mentioned above, this study has important limitations to consider. First, the criteria for ordering of RSV laboratory tests was not standardized and varied by level of healthcare episode . Moreover, we do not know the proportion of RSV positive episodes among those 71% of non‐tested episodes. It is likely that the prevalence of RSV positive episodes would be lower if all ICD‐9 coded bronchiolitis episodes were tested. However, as shown in our regression results, tests were more commonly ordered for bronchiolitis episodes in younger infants, during peak months of winter viral season, and for infants with higher severity healthcare encounters. AAP guidelines5Estimates of RSV‐bronchiolitis burden and seasonal distribution are often based on ICD‐9 codes for bronchiolitis. We provide estimates of ICD‐9 codes for bronchiolitis being attributable to RSV based on results confirmed using RSV laboratory testing. The proportion of RSV positive bronchiolitis episodes varies by infant age at the illness, level of health care service used, and time of the year of the episode. Estimation of RSV positive bronchiolitis based on ICD‐9 codes can be improved by restricting to episodes occurring during peak months of winter viral season, among infants ≤6‐months age at time of illness, and among those requiring higher levels of healthcare. Strategies to improve the specificity of bronchiolitis‐coded episodes have implications for public health surveillance, the design of prevention strategies, and for research. As an example, identifying RSV peak season and target age group may help to efficiently channel scarce public health resources, as well as to implement prevention measures such as daycare closures or the testing of targeted prevention measures, to prevent disease spread. In addition, this information may inform the start and end of use of RSV immunoprophylaxis in high risk populations, improving prevention during periods of highest risk of infection, and potentially shortening duration, and, thus, cost of RSV immunoprophylaxis. Lastly, among those tested, the majority of RSV positive bronchiolitis episodes were among infants born at term or later gestational age, reinforcing that the greatest disease burden is among term infants.This study was approved by both the Kaiser Permanente Northern California (KPNC) and Vanderbilt University Institutional Review Boards.This work was supported in part by National Institute of Health (NIH) grants: K24 AI 77930 (Hartert), R01 HS018454 (Hartert), 5T32HL087738 (Turi).Dr. Kedir N Turi, Dr. Pingsheng Wu, Dr. Kecia N Carroll, Ms. Tan Ding, and Ms. Tebeb Gebretsadik have no conflicts of interest. Dr. Tina V. Hartert served as a consultant to Medimmune through a Vanderbilt University School of Medicine contract to define respiratory outcomes for clinical and vaccine trials. Consultant fees were paid to Vanderbilt. She has also served as a consultant to Novavax and Regeneron. Dr. Gabriel Escobar, Ms. Eileen Walsh, and Ms. Sherian Xu Li have received grant support from Medimmune, LLC and Astra Zeneca to conduct research on RSV infections in infancy.Conceptualization: Pingsheng Wu, Tina V. Hartert, Gabriel J. EscobarFunding Acquisition: Tina V. HartertInvestigation: Pingsheng Wu, Tina V. Hartert, Kedir N. TuriMethodology: Pingsheng Wu, Tina V. Hartert, Tebeb Gebretsadik, Tan Ding, Kedir N. TuriResources: Tina V. HartertSupervision: Tina V. HartertWriting – Original Draft Preparation: Kedir N. TuriWriting‐Review & Editing: Pingsheng Wu, Tina V. Hartert, Tebeb Gebretsadik, Gabriel J. EscobarFigure S1:Panel A. Total ICD‐9 coded bronchiolitis episodes and RSV positive episodes by month. Panel B. Acute RSV bronchiolitis (ICD‐9 code = 466.11) episodes and RSV positive episodes by month. Panel C. Viral pneumonia (ICD‐9 code = 480.1) episodes and RSV positive episodes by month. Panel D. Acute bronchiolitis due to other infectious organisms (ICD‐9 code = 466.19) episodes and RSV positive episodes by month.Click here for additional data file.Table S1. ICD‐9‐CM Diagnosis Code relevant to the diagnosis of bronchiolitis as captured from KPNC electronic records.Table S2. Distribution of RSV positive bronchiolitis episodes among all infants (≤12 months), those ≤6‐months, and those ≤3‐months of age, and by type of healthcare encounter at age ≤ 6‐months in the PRIMA‐KPNC study cohort, 2006–2009Click here for additional data file.


To identify baseline factors relevant to functional outcomes and health‐related quality of life in the elderly with intertrochanteric fractures.t‐test, ANOVA, Pearson's correlation, χ2‐test, and multivariate linear regression.For the present study, 168 patients with intertrochanteric fracture who were assigned to different treatments between January 2016 and December 2017 were retrospectively selected. Hip function was assessed by Harris hip score (HHS), and health‐related quality of life was evaluated by Barthel index (BI) of activities of daily living (ADL) and EuroQol 5‐dimensions (EQ‐5D) score, respectively. Data were analyzed by CI) −0.73 to −0.18, P < 0.01), serum albumin , and ADL at discharge . The Barthel index at final follow up in this cohort was 80.2 ± 18.1, and multivariable linear regression analysis showed that age , ADL score at discharge  and internal fixation  were associated with ADL at final follow‐up. EQ‐5D at final follow‐up was 0.74 ± 0.2, with which HHS  was positively associated.A total of 164 (97.6%) patients completed the follow‐up, with an average follow‐up time of 15.7 ± 6.9 months; 39 (23.8%) patients died during the follow‐up period and 125 (76.2%) patients were eligible for the functional analysis. HHS at final follow‐up of 125 patients was 71.8 ± 13.1, and the following were associated with hip functional recovery: age (−0.45, 95% confidence interval (We identify several baseline factors associated with hip functional outcome, health utility, and ADL in the elderly after an intertrochanteric fracture, of which we could modify mutable factors to achieve better outcomes. These findings could help to inform treatment and functional prognosis. Hip fracture is common and consists of one‐fifth of the operative work of an orthopaedic trauma center. It is a devastating health problem, associated with substantial mortality, morbidity, and costs. Millions of people are disabled as a result of hip fractures every year worldwide, which places a heavy burden on the healthcare systemFractures around the hip joint are associated with functional impairment and major disabilities. It is reported that over half of patients could not return to premorbid mobility statusRecently, the Fixation using Alternative Implants for the Treatment of Hip fractures (FAITH) investigators conducted an international, multicenter, allocation concealed, randomized controlled trial to assess the effects of sliding hip screws versus cancellous screws on reoperations over a 24‐month follow‐upDespite long‐term regular follow up over 24 months, the FAITH trial exclusively included patients with femoral neck fractures and excluded intertrochanteric fractures. Fractures around the trochanteric region were located outside of the hip joint capsule and were different from femoral neck fractures in relation to biomechanics and treatment options. Prognostic predictors for functional outcomes of patients with intertrochanteric fractures were still unknown. It was of great significance to identify additional factors associated with long‐term functional recovery, ADL, and health utility after an intertrochanteric fracture to optimize the care of these patients.Therefore, the purpose of this retrospective study was to: (i) evaluate overall long‐term hip functional recovery and quality of life of patients after an intertrochanteric fracture; (ii) identify risk factors associated with hip functional recovery, ADL and health utility; and (iii) help to assess rehabilitation potential and modify mutable risk factors to reach satisfactory outcomes.Inclusion criteria: (i): patients who were at 65 years of age and above with closed intertrochanteric fracture diagnosed by anterio–posterior and lateral plain film, CT or MRI; (ii): patients who had undergone intramedullary nail fixation ; (iii): patients who had undergone hemi‐arthroplasty  or conservative therapy; (iv): the main outcome measures included Harris hip score (HHS), Barthel index of activity of daily living, European Quality‐five dimension; and (v): it is a retrospective case control study.Patients were excluded if they met one of the following conditions: (i) patients with known metastatic cancer and pathology‐proven pathological fractures; (ii) fractures associated with poly‐trauma; (iii) younger than 65 years of age; (iv) patients were lost to follow‐up or with missing data.The medical and nursing record of patients with intertrochanteric fractures were retrospectively retrieved from the Department of Medical Records between January 2016 and December 2017. The data included demographic information, perioperative images and laboratory tests, and treatments. Fractures were classified according to Arbeitsgemeinschaft fur Osteosynthesefragen/Association for the Study of Internal Fixation (AO/OTA) classification. Patients enrolled in the cohort were followed up with telephone interviews or clinical visits. The hip joint function was evaluated according to HHS, and quality of life was measured by EuroQol 5‐dimensions (EQ‐5D) and Barthel index (BI) of ADL.Written informed consent was obtained from all participants. All clinical investigations are conducted according to the principles expressed in the Declaration of Helsinki.Patients were given general or spinal anesthesia.Patients were placed in a supine position on a traction table. Satisfactory fracture reduction was achieved by limb traction with a slight abduction and pronation with the assistance of fluoroscopy. An incision was made 3 cm above the apex of the greater trochanter, and the gluteus medius was incised to expose it.The nail was implanted percutaneously. The guide needle was inserted into the medullary cavity under fluoroscopy. Then proximal part of the femoral shaft was reamed with the reamer, and the main nail was inserted into the medullary cavity. The guidewire was inserted into the femoral neck with the help of an aiming arm. An anti‐rotation spiral blade  was then inserted. The distal locking nail was achieved with the guidance of an aiming arm.All patients were given prophylactic antibiotics  within 24 h postoperation and low molecular weight heparin for thromboembolism prophylaxis. Patients were mobilized to bear full weight, as tolerated, for 6 weeks postoperatively Fig. .Patients were given general or spinal anesthesia.A lateral approach was used. Part of the gluteus medius and most of the anterior and lateral joint capsules were removed, then the femoral intertrochanteric fracture was exposed.The femur was cut obliquely 1.5 cm above the small trochanter. Then we took out the femoral head and exposed the medullary cavity of the femur, and tested the mold as the joint tightness was good. After injection of bone cement, the femoral prosthesis stem was inserted and an appropriate femoral head  was installed to maintain anatomical ante‐version and equal length of the contralateral limb. We placed additional wires around the intertrochanteric region to restore the fractured greater trochanter.All patients received the same prophylactic antibiotics and thromboembolism prophylaxis as patients in the intramedullary nail group. Patients were mobilized to bear full weight, as tolerated, for 1 week postoperatively Fig. .Patients who refused surgical treatment and those with contraindications for surgery underwent conservative treatment. An anteroposterior radiograph showed an unstable intertrochanteric fracture of the left hip in a 43‐year‐old woman who fell at home 7 days previously  index comprises five dimensions: mobility, self‐care, usual activities, pain or discomfort, and anxiety or depressionThe Barthel index (BI) questionnaire contains 10 items: feeding, grooming, transfer, toilet use, mobility, dressing, going up and down stairs, bathing, bowel control, and bladder control. Scores for each activity reflect its relevance to the person's life. Items are summed with a total score range from 0 to 100. Higher scores reflect higher independence with ADL.2‐test was used for comparison of categorical variables and the independent t‐test was used for continuous variables. Multivariate linear regression analysis with a 95% confidence interval was used to evaluate factors influencing hip function, ADL, and health utility. The significance level was set to 0.05 and all P‐values were two‐tailed. All analyses were performed using the SPSS software .We used descriptive statistics to summarize all factors . The Pearson χA total of 168 patients aged 65 years and above with intertrochanteric fractures were included in the cohort between January 2016 and December 2017; 112 (66.7%) were women and 56 (33.3%) were men, with patients aged from 65 to 93 years (80.1 ± 8.7). According to AO classification, 49 (29.2%) cases were classified as 31A1, 105 (62.5%) as 31A2 and 14 (8.3%) as 31A3. A total of 20 (11.9%) patients had recurrent contralateral hip fractures.Of these 168 patients, 144 (85.7%) were treated with internal fixation, 10 (6.0%) underwent hip replacement, and 14 (8.3%) received conservative treatment. In the end, 164 (97.6%) patients completed follow‐up, with mean duration of 15.7 ± 6.9 months (Table P = 0.79), gender (P = 0.82), BMI (P = 0.43), fracture types (P = 0.06), ASA grade (P = 0.31), and recurrence of hip fracture (P = 0.57). Clinical outcomes in different surgical treatment groups are summarized in Table Between groups of different treatments , there were no significant differences in terms of mean age . Patients in three different age groups  scored 80.0 ± 10.7, 72.4 ± 15.0, and 68.7 ± 11.2, respectively, and the difference was statistically significant (P < 0.01). Patients in the ASA 1/2 group scored 73.7 ± 13.9 and in the ASA 3/4 group scored 68.4 ± 11.2, with significant difference (P = 0.03). Patients with hypoalbuminemia (<35g/L) scored 73.7 ± 12.3, and with normal albumin scored 68.0 ± 13.7; the difference between groups was significant (P = 0.02). No statistically significant interrelations were observed between HHS and gender, as well as fracture type, treatment options, recurrence, Charlson comorbidity index (CCI), body mass index (BMI), and hemoglobin levels . Higher albumin levels  and ADL score at discharge  also emerged as independent factors predicting higher HHS at final follow‐up. None of the other variables that we tested, including hemoglobin levels, were predictive of HHS . Patients in three different age groups  scored 90.0 ± 9.2, 82.2 ± 17.5, and 76.1 ± 19.2, respectively, and the difference was statistically significant (P = 0.01). Besides, there was a significant correlation between ADL at discharge and ADL at final follow up (P < 0.01). We observed no statistically significant interrelations between ADL at final follow up and gender, fracture type, recurrence, CCI, ASA grade, BMI, and albumin levels  of ADL at final follow up of 125 patients was 80.2 ± 18.1. Patients in internal fixation, hip replacement, and non‐operative treatment groups scored 81.6 ± 17.7, 65.7 ± 19.7, and 66.3 ± 13.2, respectively, with significant difference . A higher ADL score at discharge  and internal fixation  also emerged as factors independently predicting higher ADL at final follow‐up. None of the other variables that we tested were predictive of ADL at final follow‐up . Patients in three different age groups  scored 0.83 ± 0.13, 0.77 ± 0.18, and 0.70 ± 0.19, respectively, and the difference was statistically significant (P = 0.02). Patients in the ASA 1/2 group scored 0.78 ± 0.16, and in the ASA 3/4 group scored 0.68 ± 0.21, with significant difference (P < 0.01). No statistically significant interrelations were observed between EQ‐5D at final follow up and gender, fracture type, recurrence, BMI, hemoglobin values, and albumin levels (Table EuroQol 5‐dimensions (EQ‐5D) in patients who survived at final follow up was 0.74 ± 0.2. Patients in internal fixation, hip replacement, and conservative treatment groups scored 0.75 ± 0.18, 0.59 ± 0.24, and 0.61 ± 0.11, respectively, with significant difference (P < 0.01). No other factors were found to be significantly associated with EQ‐5D at final follow up  were related to higher ADL at final follow up as measured by the Barthel index. Finally, we revealed that post‐fracture EQ‐5D scores indicated that overall health utility was positively associated with hip joint function.In our study, hemoglobin levels did not emerge as an independent risk factor for impaired hip function both in simple and multivariable regression analysis. A recent meta‐analysis reviewed studies on variables associated with hip joint function and concluded that anemia on admission was associated with poor functional outcomes with a weak level of evidence. It suggested that patients with low hemoglobin on admission were more likely to be frail and had less muscle strength than those without anemiaet al. found that albumin levels could not independently predict better functional outcomesIn a study including 499 elderly patients with hip fractures, Mizrahi Garden classification of femoral neck fractures was based on the degree of displacement, which was judged by AP radiograph by determining the relationship of the trabecular in the femoral head to those in the acetabulum. Garden III and IV type indicated more displacement, and in that case, vessels that supplied to the femoral head along the neck would be damaged. The results of the FAITH trial strongly supported that displacement was associated with poor hip function. Femoral head necrosis was proposed as the underlying mechanism for the association between Garden classification and functional outcomeet al. found that HHS decreased in the elderly patients due to aging. Patients below 70 years of age had a mean HHS of 86.7 years, patients between 85 and 90 years old scored 79.3, and those older than 90 years only scored 77.1 on averageIn a study of 303 patients with intertrochanteric fractures, Tang We found that activities of daily living measured by Barthel index (BI) at final follow up was inversely related to older age. Hip joint function declined with aging, and compromised function limited mobility, which resulted in impaired ability to perform daily tasks. ADL at final follow up was associated with ADL at hospital discharge. Ishidou (2017) found that an increased risk of BI deterioration was associated with worse BI at discharge, which suggested that early rehabilitation could improve functional prognosisOur study suggested that health utility, measured by EQ‐5D, was strongly associated with hip function. A systematic review of studies investigating health status or health‐related quality of life following hip fractures found that mental status, pre‐fracture function, comorbidity, female gender, nutritional status, postoperative pain, length of hospital stay, and complications were associated with health‐related quality of life with strong evidenceThis study had several strengths. First, to the best of our knowledge, it is the first to focus on the baseline factors associated with hip function, ADL, and health‐related quality of life in a retrospective cohort of patients after an intertrochanteric fracture. In addition, inclusion of three different outcome measures contributed to the validity of the results. The study was strengthened by the more than 12‐month follow‐up period, which provided a long‐term assessment of the patients.Despite these strengths, the study was limited in its retrospective nature. Patients were not assigned to the groups randomly, which could have biased the results to some extent. It is possible that for larger groups of patients with various treatments, HHS differences would be significant and have sufficient statistical power. Finally, BI of ADL scales was the most widely used index in the previous literature, which has been validated for use in elderly patients. Nevertheless, it was not specifically used in the hip fracture population and could be affected by comorbidities. In comparison to generic scales, disease‐specific scales, including HHS and WOMAC, were more specialized in disease state. However, disease‐specific measures used in musculoskeletal research have focused on patients with osteoarthritis, and not specifically on hip fractures. In fact, functional outcomes also depended on the death rate as patients may have recovered if they remained alive.In this retrospective analysis, to begin with, we reviewed the functional outcomes and health‐related quality of life of patients after rehabilitation following an intertrochanteric fracture. Second, we identified age, albumin, and ADL at discharge as relevant factors for functional recovery. Age, ADL at discharge, and fixation type emerged as predictors for ADL at final follow up. In the end, we found that health utility was strongly associated with hip function.


Attentional biases have been observed in populations with psychological disorders, but have been under-investigated in populations with physical illnesses. This study investigated potential attentional biases in asthma as a function of mood. Asthma (N = 45), and healthy (N = 39) participants were randomly allocated to a depressed or a neutral mood state induction. They completed a visual probe task that measured participants’ reaction times to health-threat and neutral pictures and words. Compared to the healthy controls, the asthma group showed attentional bias towards health-threat pictures in depressed mood, and avoidance of health-threat pictures in neutral mood. Attentional biases were found in a group with a physical illness as a function of induced mood. It is suggested that attentional processes in people with physical illness may be important in relation to symptom perception and illness management. Current asthma treatments aim to control and manage the symptoms in order to prevent lung function deterioration and asthma attacks (GINA 2015). In spite of effective medication that allows individuals to function close to full capacity, there are high rates of uncontrolled or partly controlled asthma  then it slows down the colour naming. If participants are slower to colour-name threat words as opposed to neutral or positive words, then the biased attention towards threat is inferred  is presented in two different locations, typically one word on the top half of a computer screen and the other on the bottom. One stimulus in a pair represents a threat , the other stimulus in a pair is neutral, for example, it can be a word pair “death—table”. Then a visual probe  appears in the same place as one of the stimuli presented previously. The trials may be congruent, where the dot replaces a threat stimulus, or incongruent, where the dot replaces a neutral stimulus. This is one of the advantages of the task, because it allows calculation of congruence and incongruence indices corresponding to, respectively, attentional engagement and difficulty in disengaging attention from threat , and present conflicting evidence. Lowther et al.  found atIn contrast, Dudeney et al.  did not Another potential explanation for mixed findings of De Peuter et al.  and LowtImportantly, and in line with theories with regard to the impact of symptom perception processes in asthma, Dudeney et al.  did findThus, empirical observations so far point towards potential attentional bias in asthma, primarily focused on asthma-related stimuli as opposed to more general physical threat, or negative emotional stimuli. Current evidence, however, does not allow a confident prediction as to whether the bias will be observed at earlier or later attentional stages due to hypervigilance or difficulty in disengagement. The duration of stimuli presentation in the current study was set at 500 ms to replicate the methodology employed by De Peuter et al. , the onlEven though the past findings with regard to attentional bias in asthma are mixed, there is one observation that appears to be quite consistent, and that is the correlation between greater attentional bias and negative affect. The study by De Peuter et al.  points tJanssens et al.  proposedTherefore, negative mood may play a role in directing attention towards internal sensations or external cues. It may be responsible for activating an illness schema and increasing attentional bias, thus leading to symptom overperception and undermining asthma control. Consequently, the current study employed a mood induction technique in order to investigate a direct influence that depressed mood may have on attentional bias in a sample of adults with asthma with no current comorbid mental health disorders (such as depression or anxiety). Factoring mood in would also allow a more concrete prediction regarding where in the attentional process a bias may occur. In depression, attentional bias tends to manifest at a later voluntary elaborative stage, as in difficulty in disengaging from negative stimuli  reported a primary asthma diagnosis by a GP or referred specialist, b) had no other current acute or chronic medical or mental health diagnosis, and c) were prescribed a reliever inhaler. Participants reported illness duration (months/years), severity, and daily asthma interference with functioning on a scale from 0  to 100 (extremely interfering) , a 14 item scale for populations with non-psychiatric medical conditions, so that the scores are not affected by the physical symptoms of the medical condition  assessed momentary mood  appeared either above or below the fixation point  and stayed on the screen either until participants responded or for 2500 ms. The inter-trial interval was 1000 ms. Each threat cue could appear in one of two positions (above or below the fixation point) and an arrow replaced the threat cue or the neutral cue. Figure 5 mm appez key) or to the right (m key). Participants were instructed to concentrate on detecting the arrows and not the words or pictures, which might have preceded them. They were asked to be as fast and as accurate as possible and maintain their gaze at the centre of the screen.The following instructions were given to the participants: to look at the fixation cross on the monitor, and when an arrow appeared on the top or bottom half of the screen, press a key on the computer keyboard to indicate whether the arrow pointed to the left , after which they had the opportunity to ask any questions or rerun the practice. Each experimental word or picture pair was presented four times, so that each threat cue was presented in two positions (above and below the fixation point) and the arrow replaced the threat cue  or the neutral word . Therefore, the presentation of the 48 pairs  produced 192 experimental word trials and 192 pictures trials; in total, 384 experimental trials. Of those trials, half had the arrow pointing left, and half had the arrow pointing right. Trials were presented in a random order, where the word and picture trials were intermingled. The task took on average 14 min to complete.p > .99. The 48 monochrome picture pairs matched the words . The neutral halves of the picture-pairs matched the threat pictures in content and complexity (e.g. doctors performing surgery—people playing a board game).The 48 word-pairs, 16 health-threat and neutral (e.g. cancer-feline), 16 activity and neutral (e.g. jogging-chrome), 16 neutral and neutral (e.g. flute-flint), were selected from previous research on attention  for words and 6.1 (SD = 1.7) for pictures. The ratings for activity/exercise were 6.1 (SD = 0.6) for words and 6.3 (SD = 0.5) for pictures.Six people with asthma and 7 healthy individuals who did not take part in the visual probe task, rated the relevance of the health-threat stimuli to illness in general on a scale from 0—not at all relevant to 10—extremely relevant. The mean ratings were 6.9  (in milliseconds) data were filtered by removing RTs for incorrect answers, for responses below 200 ms and above 2000 ms, and those more than 2 Attention bias scores were calculated following the formula ((TUDL – TLDL) + (TLDU − TUDU))/2, where T—threat, N—neutral stimulus, U-upper and L-lower denoted the position above or below the fixation point, and D was the target arrow  − ((NUDU + NUDL + NLDU + NLDL)/4)). Positive scores suggest difficulties disengaging from threat, reflecting slower RTs on incongruent threat-neutral trials than on neutral–neutral trials, indicating a difficulty in switching attention from a threat towards the arrow appearing in place of a neutral stimulus.t-tests. To check if the induction affected each group differently a three-way 2 × 2 × 2 ANOVA factorial design was conducted with group and induction as between-subjects and time as within-subjects variable.The effect of mood manipulation was checked by conducting two 2 × 2 ANOVAs with induction  as the between subjects and time  as the within-subjects variable. Positive and negative affect scores (PANAS) were the dependent variables. Significant interactions were clarified by conducting paired-samples t tests. Correlational analysis was conducted to test the association between self-report measures and attentional bias scores. Independent correlation coefficients were compared using z-scores and two-tailed tests of significance  and induction  as between-subjects independent variables. Stimulus  and threat type  were repeated measures. Significant interactions were clarified using independent samples The computer tasks were run on a desktop computer  and a Toshiba Satellite A100 laptop (15.4-inch screen). The presentation of the task and data collection was conducted using E-Prime software version 1.1 (SP3) (1.1.4.4). Participants sat in a comfortable position in front of the computer screen. The distance between the participant and the screen was approximately 50 cm. Previous research suggested that any variation in distance between the participant and computer screen had no significant influence on the effect  participants were randomly allocated to undergo either depressed or neutral mood state induction. A random numbers list was used to allocate participants, where odd numbers were assigned to depressed mood and even numbers to neutral mood. Thus, there were 22 asthma participants and 21 controls allocated to the depressed mood induction, and 23 asthma and 18 control participants allocated to the neutral mood induction.Participants were tested alone in a single session lasting approximately 1 h. The investigator explained the procedure before obtaining written consent. Participants completed the mood induction, then the visual probe task, and self-report measures at the end. Participants were debriefed and compensated for their time (£10).f = .25, for the ANOVA of bias scores 4-way interaction with between measures of group  x induction , and within measures of stimulus  and threat type   Cohen, .F = 13.05, p = .001, ηp2 = .137. Negative affect increased significantly following the depressed mood induction t(42) = 3.19, p = .003 the mean difference was 2.5 (SD = 5.2).The analysis of negative affect scores demonstrated a significant induction*time interaction F  = 7.10, p = .009, ηp2 = .080. Positive affect decreased significantly after the depressed mood induction; the mean difference was 6.1 (SD = 5.9), t (42) = 6.77, p < .001. Positive affect also decreased significantly after the neutral mood induction, but to a lesser degree; the mean difference was 3.0 (SD = 4.6), t (40) = 4.16, p < .001.Positive affect scores also showed a significant induction*time interaction p > .98 for negative, and p > .78 for positive affect, indicating the induction had similar effect on both groups.The ANOVA with the added group  factor did not show a significant three-way interaction group*induction*time, with F  = 4.69, p = 0.010, ηp2 = .055, 90% CI  and a significant group*induction interaction F  = 9.46, p = .003, ηp2 = .106, 90% CI  but there were no significant main effects or lower level interactions, all p > .250.The ANOVA revealed a significant four-way interaction of group*induction*stimulus*threat p > .250. In contrast, for the pictorial stimuli there was a significant three-way interaction of group*induction*threat F  = 4.69, p = .010, ηp2 = .055, 90% CI  and similarly, there was a significant two-way group*induction interaction F  = 8.32, p = .005, ηp2 = .094, 90% CI .To clarify the four-way interaction two 3-way ANOVAs were conducted separately for linguistic and pictorial stimuli. No significant main effects or interactions were detected for linguistic stimuli, all p > .250. In contrast, ANOVA for health-threat pictures showed a significant group*induction interaction, F  = 11.08, p = .001, ηp2 = .122, 90% CI . Most importantly, independent samples t-tests revealed that in a neutral mood, the asthma group showed significant attentional avoidance of health-threat pictures  compared to the control group , t (39) = 2.72, p = .010, d = .883, 95% CI . However, the asthma group in a depressed mood showed a significant attentional bias towards health-threat pictures  compared to the control group , t41 = 2.16, p = .037, d = .676, 95% CI   = 3.58, p = .001, d = 1.093 95% CI . That was not the case for the control group, p > .250.Analysis within each group revealed that people with asthma allocated to the neutral mood induction showed attentional avoidance compared to those allocated to the depressed mood induction who demonstrated attentional bias towards health-threat pictures, t-tests were conducted to compare each mean against zero, where zero signifies an absence of the bias either towards or away from Health-Threat pictures or words  = 3.37, p = .003, 95% CI , d = 0.72, 95% CI . This suggests that potentially the attentional bias is underpinned by the difficulty in disengaging attention from Health-Threat pictures experienced by the asthma group induced into depressed mood.For the congruence scores, one sample t(21) = 2.54, p = .019, 95% CI , Hedges g = 0.78, 95% CI .The paired-samples comparison between congruence and incongruence scores for the Health-Threat pictures within the asthma depressed group similarly showed a significant difference, r45 = .32, p = .033 and for Health-Threat pictures r45 = .31, p = .041. These results indicate that greater asthma severity is related to greater difficulty in disengaging attention from health threatening stimuli. The correlations between illness duration or asthma interference scores and attentional indices were not significant, all p > .10.Correlational analysis of attentional bias scores and asthma duration and severity showed that there was a significant positive association between asthma severity level and attentional incongruence scores for Health-Threat words r22 = − .42, p = .052. In contrast for the control group in neutral mood greater attentional bias was associated with higher anxiety r18 = .43, p = .074. Comparison of these two correlations showed a significant difference z = 2.63, p = .008. The equivalent correlations were not significant in the induced depressed mood group.Correlational analysis of anxiety scores (HADS) and attentional bias scores was conducted to determine whether biased attention in asthma might have been due to elevated anxiety. In the neutral mood, two correlations showed a trend towards significance, but more importantly showed diametrically opposing patterns. For the asthma group in neutral mood, greater attentional bias towards health-threat pictures was associated with lower anxiety, The current study investigated attentional processing in people with asthma in an induced depressed or neutral mood. In support of the first hypothesis, the asthma group in an induced depressed mood showed an attentional bias towards health-threat pictures, compared to the asthma group in a neutral mood and to the healthy controls in either a depressed or a neutral mood. The results also indicated that this attentional bias may be due to difficulty disengaging from health threat, as opposed to hypervigilance towards health threat.The significant association between depressed mood and attentional bias in asthma is in line with previous findings  and thus potentially the conferral of exaggerated importance upon minor symptoms and a consequent diminution in levels of fine control over asthma , treatment , disability , illness consequences (e.g. dying), covering a range of potential health-threat. Any of these concepts could be key in biasing attention in negative mood, and different concepts may lead to different consequent cognitive, emotional and behavioural processes. Anxiety and fear may lead to an increase in vigilance and catastrophizing in relation to minor physical sensations. Anger and irritability may lead to increased rumination and brooding, hostility, and undermine treatment compliance. Sadness and hopelessness may lead to increased rumination, difficulties in shifting attention away from symptoms or health-threat, and undermine beliefs in treatment effectiveness. Distinguishing maladaptive attentional bias towards more general health-threat from beneficial amplified attention towards asthma-specific information may be aided by further investigating the exact nature of the information that consumes attentional resources and the nature of its interaction with specific types of emotional states.In contrast to the hypothesis that attentional bias, albeit of a smaller magnitude, would be observed in the neutral mood, participants with asthma demonstrated attentional avoidance of health-threat pictures following neutral mood induction. The result, though not predicted, echoes similar findings from other studies in respiratory disorders. Dudeney et al.  detectedOne explanation of the avoidance of health-threat demonstrated in the neutral mood asthma group may have to do with the nature of the mood induction. The neutral mood induction consisted of Velten statements, for example, participants were instructed to think about a statement “Boeing’s main plant in Seattle employs 35,000 people”. The induction appeared to be successful, because the positive mood was reduced afterwards, as would be expected when we are trying to induce a neutral mood state. However, Velten neutral mood induction is similar to a distraction induction developed for the purposes of altering a cognitive state . The current proposed attentional training paradigm may be similarly effective in addressing inaccurate perception of asthma symptoms  or varying the exposure durations in order to explore the time course of attention .The mood manipulation in the study was successful. Depressed mood induction led to increased negative affect and reduced positive affect. However, negative affect  is not the same as depressed mood, and depressed mood is not depression. Therefore, it is too early to make conclusions about the effect of clinical levels of depression or of naturally occurring depressed mood on attentional bias in asthma. The study also did not include other moods for comparison, such as, a positive mood or anxious mood, again making it difficult to make firm conclusions about the role of depressed mood specifically rather than negative affect in general. It may be that it is the general elevation in emotional level, greater emotional arousal, that effected the attentional bias, compared to a dampening of emotional level resulting from the neutral mood induction. A future study could compare a group of asthma patients with comorbid depression with a group of asthma patients without comorbid depression, or measure naturally occurring depressed mood among people with asthma.The current findings indicate a presence of potentially maladaptive attentional patterns in asthma, characterised by attentional bias towards health-threat in depressed mood and by avoidance of health-threat in neutral mood. This distorted attentional pattern may be the mechanism behind inaccurate symptom perception in asthma. Attentional training could be utilized to improve the accuracy of symptom perception. This in turn may have a wider impact, for example in decreasing stress and anxiety, improving adherence to prescribed medication, reducing the burden on health services, and increasing patients’ well-being and quality of life.


Context:Equisetum giganteum L. (Equisetaceae) is an endemic plant of Central and South America used in traditional medicine. Natural drugs have been frequently used in the treatment of a myriad of diseases, proving to be an alternative to synthetic chemicals, and have been intensively studied in the prevention of sicknesses, including oral diseases.Objective: This study evaluated the in vitro antiadherent activity of E. giganteum extract against Candida albicans biofilms.Materials and methods: Crystal violet and colony-forming units assays were used to quantify the total biofilm biomass and biofilm living cells on a denture base acrylic resin pretreated with hydroethanolic extract of E. giganteum in different concentrations , after 24 h of biofilm development.Results:Equisetum giganteum affected biofilms by reduction of biomass and living cells per area of acrylic specimens. The results revealed reduction of 15–44% of the biofilm mass and reduction of numbers of colony-forming units (CFUs) present in biofilms (79%) compared to the untreated control (CTRL/PBS). At all concentrations, it demonstrated important antiadherent activity on Candida albicans biofilms, the main microbe in denture stomatitis.Discussion and conclusion: The present findings show that E. giganteum antimicrobial effects may qualify the extract as a promising natural alternative for topical treatment or prevention of denture stomatitis. The usage of drugs made of natural products shows advantages in relation to synthetic drugs on the market, such as lower cost, lower toxicity, and in relation to the occurrence of microbial resistance. Candida albicans is a microorganism that colonizes the mucosa in a commensal way, yet it may become pathogenic as an opportunist microorganism, being responsible for serious infections in immunosuppressed patients  , inert polymers, or proteins are called adhesins , commonly known as cavalinha, cola de caballo, horsetail, or giant horsetail, is an endemic to Central and South America and is used in traditional medicine. For example, all over Brazil and Argentina, it is commercialized as raw material for herbal medicines and as a food supplement  is greater than in initial biofilms. It has been reported that the higher the amount of EPS, the greater the microbial resistance /cm2) in agar, and the total biomass using crystal violet assay (CV).This new study evaluated the antimicrobial/antiadherent action of E. giganteum aerial parts were collected in July 2012 at Jardim Botânico Municipal de Bauru, SP, Brazil (22°20′30′S and 49°00′30′W). The plant was identified at the Herbarium of UNESP, São Paulo State University "Júlio de Mesquita Filho", UNBA , where a voucher specimen was preserved under reference number 5795. The hydroethanolic extract (70% EtOH) of E. giganteum was prepared as previously described by Alavarce et al.  initially obtained using silicone patterns from an acrylic matrix were fabricated, with rectangular shape, measuring 10 × 10 × 4 mm. The specimens were polymerized in digital water bath at 73 °C for 90 min followed by heating for 30 min at 100 °C. To obtain a roughened surface, one surface of each acrylic resin specimen was chosen and manually abraded with high-grade sandpaper   was obtained by immersion of specimens into sterile phosphate-buffered saline, containing 1.05 mM monobasic potassium phosphate, 155.17 mM sodium chloride and 2.966 mM dibasic sodium phosphate ; and CTRL/NaOCl (positive control) was obtained by immersion of specimens into 1% sodium hypochlorite solution  for 10 min  frozen culture stocks at 80 °C were incubated in tryptic soy broth (TSB)  for 24 h, at 37 °C, under 180 rpm for reactivation. Afterwards, cells were harvested, washed with PBS, and standardized to 1 × 107 cells/mL in PBS  containing 1 mL of standardized cell suspension. The acrylic specimens were incubated for 90 min, at 37 °C, under 75 rpm (adhesion period). After that, non-adherent organisms were removed by washing with 1 mL PBS and acrylic resin specimens were incubated in new wells for 24 h, at 37 °C, under 75 rpm to allow biofilm development, submersed in 1 mL RPMI-1640  , and 50 μL of 10−4 dilution were plated in duplicate on Sabouraud Dextrose agar . The plates were incubated at 37 °C under aerobic conditions for 24 h. After this period, the colony-forming units were counted, transformed into actual counts based on known dilution factors, and the total number of CFUs per unit area (Log10 CFU/cm2) of acrylic specimens was counted. Each group included three specimens, and the experiment was repeated four times.After 24 h of biofilm development, specimens were placed into a polypropylene tube containing 1 mL of sterilized PBS. Adherent microorganisms were removed from the specimens by ultrasonication  for 20 min . All steps were carried out at room temperature. Each group included two specimens, and the experiment was repeated six times.The CV staining assay was performed according to the protocol used by Peeters et al. , with miData obtained from CFU and CV assays were presented as means and standard deviations and statistically analyzed by Kruskal–Wallis test or ANOVA, complemented by Miller test or Tukey test, respectively, at a significance level of 5%, on the statistical software Prisma 6.0 . E. giganteum extract  showed fewer colonies compared to the negative control (CTRL/PBS) (Log10 CFU/cm2). These results showed cell density reduction of up to 79% in relation to CTRL/PBS  against C. albicans, and this result was statistically similar to 1% NaOCl. No growth inhibition was observed in the negative control (CRTL/PBS). Only the 8 mg/mL concentration was not statistically similar to CTRL/NaOCl, presenting a slightly higher number of colonies in comparison to the other extract concentrations. Yet, it presented a 50% reduction in relation to CTRL/PBS.The resin pretreatment with CTRL/PBS . As showE. giganteum allowed formation of C. albicans biofilm regardless of extract concentration. However, differences occurred depending on the concentration . This was most evident from the concentration of 16 mg/mL. In the highest concentration, the reduction in biofilm cell viability was 79%.Our results showed that the resin surface previously treated by brushing with Results obtained from both methods of biofilm analysis were compared and correlated, because in general the lower number of viable cells counted in the biofilm (CFU assay) matched a minor amount of extracellular matrices remaining on the acrylic resin surface (CV assay). Therefore, we suggest that, for this reason, the highest CFU values (extract concentrations 8 and 4 mg/mL) in CV assay showed no statistical difference with the negative control (PBS). From these two methods, it was possible to verify that the extract brushed on the surface of specimens was able to reduce not only the number of remaining viable cells, but also the amount of EPS.E. giganteum on C. albicans biofilms, since this extract is able to act on different targets using different mechanisms for biofilm development  results in better outcomes from lower concentrations of the herbal extracts  we can obtain a more effective antifungal/antimicrobial activity in lower concentrations than with the extract (Wolfender et al. E. giganteum antimicrobial potential and its ability to eliminate or prevent the adhesion of C. albicans to resin surface may qualify the extract as a promising natural alternative for topical treatment or prevention of denture stomatitis. After further testing, it will be able to be clinically applied in microorganisms-related infectious diseases, such as DS.These results suggest that


As academic, governmental, and commercial use of space advances, data availability and validated processing methods will enable better planning, execution, and analysis of parabolic flight experiments, and thus facilitate future space activities.Parabolic flights provide cost-effective, time-limited access to “weightless” or reduced gravity conditions, facilitating research and validation activities that complement infrequent and costly access to space. Although parabolic flights have been conducted for decades, reference acceleration profiles and processing methods are not widely available. Here we present a solution for collecting, analyzing, and classifying the altered gravity environments experienced during parabolic flights, which we validated during a Boeing 727-200F flight with 20 parabolas. All data and analysis code are freely available. Our solution can be integrated with diverse experimental designs, does not depend upon accelerometer orientation, and allows unsupervised classification of all phases of flight, providing a consistent and open-source approach to quantifying gravito-inertial accelerations (GIA), or  Parabolic flights serve as valuable proving grounds for experimental efforts to maximize the research potential of the International Space Station4 and to accommodate increasing interest in commercial space flight.6Parabolic flights are cost-effective, ground-based analogs that achieve variable ase Fig. . Modifieg levels for all flight phases. We demonstrate this approach using a small (65 g) battery powered commercially available accelerometer and vibration measurement system. Together, these tools and products reflect a comprehensive solution for experiment planning, execution, and analysis of g level and vibrations during parabolic flight.Here we address the limited availability of open access acceleration datasets containing parabolic flight profiles and enable unsupervised and precise characterization of timing and g, Mars g, Lunar g, 0 g, and 0 g. All other parabolas targeted 0 g. Data were collected for 1.77 h during all phases of flight from a Slam Stick X™ (Mide Technology Corp.) mounted in the rear of the research section . Four sets of parabolas were performed with 5, 6, 4, and 5 parabolas, respectively. The first set targeted, in order, Mars g level or g (Methods).In any given experiment, one accelerometer orientation may be more appropriate than another. Thus, our phase of flight identification is based on a metric that is independent of accelerometer orientation: the Euclidean norm of the accelerometer  axes, which we refer to as 8 Conceptually, this process finds the point for which a statistical property , has minimum total residual error summed across two groups, e.g., before and after the change point. Here residual error is the difference between an observed value and the statistical property for the group.To facilitate our analysis Fig. e using ag level, gfilt, to identify differences in mean g levels in an unsupervised manner  from these flights, neither the acquisition hardware, the raw data, nor the code implemented for analysis, are currently available to the public. Indeed, when reviewing the literature, we found no such easily accessible parabolic flight acceleration data nor published analysis methodology.Parabolic flights provide the opportunity to perform simulated space research in a cost-effective manner. Recently, Lambot and Ord (2016) evaluated data from over 400 parabolic flights and assessed the quality of reduced g levels and durations achieved for 20 parabolas. In addition, the code implementing our methodology to categorize all phases of flight and characterize g levels and durations of parabolas is publicly available in order to facilitate future parabolic flight research. In addition, our methods could be adapted to analysis of data from suborbital flights, drop towers, or studies involving launch and landing accelerations.Here, we provide the following: (1) a commercially available hardware solution for data acquisition; (2) raw and calibrated data for all phases of flight; (3) data analysis methodology that is independent of accelerometer orientation, and (4) characterization of Our hardware solution, the Slam Stick X™, offers a compact, flexible, low-power, high resolution solution for acceleration and vibration monitoring with favorable comparison to alternatives (Methods). The small size facilitates integration into experiments and measurement of the local GIA environment, which is not constant across the aircraft. In addition, mounting with double-sided tape is simple, robust, and does not impact the frequency response (Methods).9 identified a “sweet zone” in the middle of the parabola with low acceleration deviation. Our data are consistent with this conclusion , although Lambot and Ord9 used, after unspecified low-pass filtering, a much more stringent tolerance (± 0.01 g) that was not met by our filtered data. We encourage future parabolic flight experimenters to release raw data as well as to provide data processing details  to facilitate validation and improvement of processing algorithms, as well as refine expected g levels and support planning and analysis that can be tailored to specific experimenter needs.Lambot and OrdDue to the limited availability and high cost of actual space environments, it is imperative that we continue to utilize parabolic flights as a means to simulate space – and to understand the accuracy and limitations of this modality. By making our data and methods available we hope to enable others to better plan, execute, and analyze parabolic flight experiments, and thus to help facilitate future space activities.www.mide.com) was selected based on its size (76 mm × 30 mm × 15 mm), low mass (65 g), integrated battery, manual and USB interfaces, and combination DC  and piezoelectric (TE 832M1) accelerometers to enable accuracy at both low  and high frequencies (up to 20 kHz sample frequency). The aluminum body was selected to provide improved high frequency response. Additional integrated sensors included temperature and pressure (NXP MPL3115) and control pad temperature and pressure (TE MS8607).The Slam Stick X™ , higher sampling frequencies, a wider operating temperature range (− 40 °C to + 80˚C) and much lower ( > 10 × ) mass and volume. Another COTS option is the Lansmont 3X90  , although the Slam Stick X™ specifications provide benefits in several areas .Alternative data acquisition systems include many commercial off the shelf (COTS) accelerometers, as well as the NASA Suborbital Flight Environment Monitor (SFEM)One consideration for parabolic flight experiments is that the GIA environment is not constant across the aircraft. In some cases, it may be adequate to have a single reference flight profile to be used by multiple experiment teams. However, some applications may be better served through measurement of the local GIA environment of a given experimental apparatus. Here, the small size of our solution facilitates direct incorporation into a payload, as well as placement in the desired location or orientation.When selecting a data acquisition solution, it is also important to consider how the mounting of the accelerometer itself may impact the frequency response; in our case, use of double-sided sticky tape represents both an extremely practical and low bias option, enabled by the low device mass. Because no additional materials separate the accelerometer from the aircraft, there is no need to correct for the frequency response of the mounting interface.Zero Gravity Corporation (ZGC) utilizes a standard system of mounting hardware to the aircraft structure consisting of a baseplate  bolted to the aircraft structure using four clearance holes at the corners of a square with 50.8 cm (20 in) sides, centered on the baseplate. Washers (McMaster Carr 92503A230) were used for mounting in combination with AN-6 steel bolts (3/8 inch) provided by ZGC. The Slam Stick X™ was mounted to a standard baseplate with double-sided sticky tape (3 M 950), which is the preferred mounting method due to its vibration frequency response (near unity) and robustness: this method has previously been validated during vibration testing at over 75 g at 1 kHzThe raw IDE file generated by the Slam Stick X™ was converted to calibrated MAT  files using the ide2csv.exe command line utility (Mide Technology Corp.) using the factory calibration. Note that data calibration and export functions can also be performed directly using Slam Stick Lab. Here we focus on data from the DC accelerometer. We note that temperature varied less than 1 °C and pressure showed a typical regulated profile and was highly stable during parabolas .z axes, which we hereafter refer to as the g level or g. As this variable is a positive scalar, it does not capture directional fluctuations in the gravity vector. Thus, for characterization of phases of flight, vector-based statistics should be used. For example, we estimated the mean g level during a 0 g parabola by averaging the x, y, and z components, and then computing the norm.In any given experiment, one accelerometer orientation may be more appropriate than another. Thus, we based our phase of flight identification method (below) on a measure that is independent of the accelerometer orientation: the Euclidean norm of the x, y, and g level is unity on Earth under non-accelerated conditions; as a verification of our accelerometer calibration, we found the norm under lab bench conditions (14.2 s recording) to be 0.9840 (rms) and 0.9840 ± 0.0055 (mean ± SD), consistent with < 2% error. This is a lower bound when vibration or specific force other than that caused by gravity is present, consistent with the rms value (1.07) observed during flight. Specific force was concentrated in the z axis as measured by root mean square (rms) values , consistent with the accelerometer orientation  as described below. Next, we utilized change point detection8 as implemented by the MATLAB FindChangePts function.For parabola identification, we first filtered the raw data using a zero-phase 12th order Butterworth filter using the g level gfilt to identify differences in mean g levels. A known number of change points was specified based on the parabola number within each set, e.g., two times the number of parabolas, plus two additional transitions  for each set of parabolas. In our case sets of 5, 6, 4, and 5  parabolas become 12, 14, 10, and 12 change points. This total number of change points (48) was specified and FindChangePts identified all g level change points in an unsupervised manner . Despite its simplicity, this classifier achieved good separation between classes  was then performed, first by categorizing any periods with duration > 100 s as “other”, then by segmenting data according to ses Fig. .g, N = 17, range 17 to 24 s), 23.7 s , and 28.9 ± 0.7 s . The g levels achieved were 0.041 ± 0.005 g (0 g) and 0.159 g (lunar g). Both Mars parabolas achieved 0.356 g, indicating high consistency between parabolas targeting similar g levels. Higher g levels were significantly associated with longer-duration parabolas , although not when lunar and Mars data were excluded .Parabola durations (mean ± s.d.) were 19.5 ± 1.4 s (0 k-means).Some limitations are inherent in our study, which focused solely on one flight and 20 parabolas. If analyzing multiple flights, with parabolas performed under more varied conditions, it is possible a slightly more complex classification strategy might be required; however, based on the wide separation between “parabola,” “hypergravity,” and “other” classes, this is not expected to present a significant challenge to standard unsupervised classification approaches . The PSD was computed via the MATLAB pwelch function with default parameters. To select the HPF, we examined the cumulative sum of the PSD , which revealed a sharp increase in power above 0.01 Hz. We chose this value (HPF = 0.01 Hz) to maximize the low frequency content of the filtered data while rejecting as much spectral power from higher frequencies as possible. As an example, filtering at HPF = 0.01 Hz preserves parabola dynamics, whereas filtering at HPF = 0.001 Hz does not . Our selected value provides the smoothest data for identifying parabolas, while still accurately representing g level transitions. A manual procedure identified similar values, e.g., adjusting the HPF toward DC until the rapid transitions between g levels showed systematic bias, then setting the HPF to 10 × this value, also gave HPF = 0.01 Hz.To optimize the filter, we selected a HPF based on the x, y, z, respectively), consistent with low frequency aircraft accelerations mainly due to pitch maneuvers. The g level was near unity during periods of relative calm  direction but little in other directions (0.0161, 0.0681, 1.0623 for g level on duration was performed using the MATLAB fitlm function. Confidence intervals were determined using the MATLAB coefCI function.Regression of parabola https://github.com/CarrCE/zerog.The MATLAB scripts implementing our analysis are available at: https://osf.io/nk2w4/.Raw and calibrated data are available via the Open Science Framework at: Supplementary Information: Acceleration Profiles and Processing Methods for Parabolic Flight


IN vitro, primary cultured human ovary granulosa cells (GCs) were isolated from the follicular fluid (FF) extracting from PCOS patients and controls. FF, especially which extracted from PCOS patients with a high expression of serum testosterone, significantly induced cell apoptosis and inflammation in human GCs. To examine the communication between PCOS and kidney injury, a human proximal tubular epithelial cell line (HKC‐8) was cultured and administered FF. Interestingly, FF from PCOS patients with a higher level of serum testosterone induced fibrotic lesions in HKC‐8 cells. These data suggest serum testosterone plays a critical role in PCOS and PCOS‐associated kidney injury. Serum testosterone may serve as a promising indicator for kidney fibrotic injury outcomes in PCOS patients.Polycystic ovary syndrome (PCOS) is closely related with the onset and development of metabolic abnormalities. However, the correlation between PCOS and kidney injury has not been clarified, and the underlying mechanism remains unknown. Herein, we performed a prospective survey in 55 PCOS and 69 healthy participants. Furthermore, the correlation analyses between serum testosterone and renal functional manifestations of patients and healthy subjects, including urinary albumin to creatinine ratio (UACR), urinary κ‐light chains (KapU), urinary λ‐light chains (LamU), urinary α1‐microglobulin (α1‐MU), and urinary β2‐microglobulin (β2‐MU), were analyzed. Compared with that in normal subjects, the levels of serum testosterone and UACR were significantly higher in PCOS patients. Serum testosterone is significantly correlated with the disease severity of PCOS. Although urinary excretions of KapU, LamU, α1‐MU, and β2‐MU did not increase in PCOS patients, they had a significantly positive correlation with the extent of serum testosterone in PCOS patients.  Polycystic ovary syndrome (PCOS) is the most common type of endocrine disorders in women of reproductive age. Up to 20% in women of childbearing age worldwide are suffering PCOS , dyslipidemia, hypertension, anxiety, depression, obstructive sleep apnea, endometrial cancer, fatty liver, and cardiovascular diseases  is a gradual loss of kidney function over months to years. Without early preventing and treatment, CKD could eventually progress into the disease state of uremia, an end‐stage kidney disease with significantly increased mortality , λ‐light chains (LamU), α1‐microglobulin (α1‐MU), and β2‐microglobulin (β2‐MU), the markers of tubular proteinuria  were obtained from additional five PCOS patients with hyperandrogenism, five PCOS patients with normal expression of testosterone and five control participants. FF was collected from the PCOS patients with hyperandrogenism and controls.PCOS was diagnosed according to the 2003 Rotterdam Criteria, with at least two of the following symptoms: (1) oligomenorrhea/amenorrhea; (2) polycystic ovaries (PCO) on ultrasonography; (3) clinical and/or biochemical signs of hyperandrogenism (hirsutism or acne) and exclusion of other etiologies . Clinical features including menstrual cycle, the number of antral follicles (AFC), body mass index (BMI), mean arterial pressure (MAP) as well as endocrine and biochemical parameters  were recorded. The demographic and clinical data of the patients with PCOS are presented in Tables g for 10 min, and supernatants were obtained and stored at −80°C until further analysis. The urinary albumin to creatinine ratio (UACR), KapU, LamU, α1‐MU, β2‐MU, and urinary creatinine (UCr) were measured by automatic‐specific protein analyzers  in National Clinical Research Center of Kidney Disease in Nanfang Hospital, Southern Medical University. The kits were purchased from SIMENS9. The value of the UACR as well as KapU, LamU, α1‐MU, and β2‐MU were normalized to UCr.One hundred and twenty‐four urine samples from 55 PCOS patients and 69 control subjects were collected before participants were treated with assistant reproductive technology. The urine samples were centrifuged at 3000in vitro fertilization and embryo transfer. In brief, ovarian hyperstimulation was achieved with the use of recombinant follicle‐stimulating hormone (FSH) from the third day of menstruation and adjusted as needed according to the ovarian response. Gonadotropin‐releasing hormone antagonist (GnRHant) was started in patients until they were triggered after the fifth day of COH. Transvaginal ultrasound‐guided oocyte retrieval was performed 35 h after the injection of human chorionic gonadotropin (hCG). The follicular fluid (FF) samples were obtained from the first‐punctured follicle (18−25 mm in diameter) during the oocyte aspiration. The samples were then centrifuged at 800g for 10 min, and the supernatants were stored at −80°C until further analysis. The testosterone expression in FF was analyzed. The GCs were isolated from FF obtained during oocyte retrieval for IVF from each subject. Briefly, FF was first centrifuged at 400g for 10 min. The pellet was resuspended with PBS, and then layered on 50% Percoll and centrifuged at 400g for 20 min. The volume ratio between PBS and 50% Percoll was 3:2. Three layers could then be distinguished, and the middle white layer was collected and washed with PBS at 600 g for 8 min at least twice. One milliliter of TRIzol reagent was added to the collected GCs  until RNA analysis.An antagonist protocol for controlled ovarian hyperstimulation (COH) was used in the cycle of HKC‐8 cells, a human kidney proximal tubular cell line, were cultured in DMEM–Ham’s F12 medium containing 10% fetal bovine serum. HKC‐8 cells were treated with DMEM medium containing 10% FF for 24 h. The cells were then collected for various analyses.−ΔΔCT). Relative levels of mRNA were reported after normalization with β‐actin. The composition of the primers was as follows: caspase3: forward 5’‐GAAATTGTGGAATTGATGCGTGA‐3’, reverse 5’‐CTACAACGATCCCCTCTGAAAAA‐3’; NLRP3: forward 5’‐CGTGAGTCCCATTAAGATGGAGT‐3’, reverse 5’‐CGACCCTGTCCCTCAAATCC‐3’; actin: forward 5’‐CTCACCATGGATGATGATATCGC‐3’, 5’‐AGGAATCCTTCTGACCCATGC‐3’.The mRNA expression levels of caspase 3 and NLRP3 in GCs were analyzed by qRT‐PCR. Total RNA was extracted with TRIzol reagent  and cDNA was synthesized with the Reverse Transcription System kit . qRT‐PCR was performed using SYBR Green PCR MasterMix (Applied Biosystems), according to the kit instructions by an ABI PRISM 7000 Sequence Detection System . The PCR reaction system contained 12.5µL SYBR Green PCR MasterMix (Applied Biosystems), 5µL diluted RT product (1:10), and 0.5 mmol/L sense and antisense primer sets. The expression levels of various genes were determined by the comparative CT method . After separation on 10% SDS‐PAGE gels, the protein was transferred to membranes, which were later incubated with primary antibodies: anti‐fibronectin , anti‐α‐SMA , and α‐tubulin  overnight at 4°C. The signals were visualized using the enhanced chemiluminescence system . Relative protein abundance was calculated after normalizing with α‐tubulin.HKC‐8 cells were cultured on coverslips and fixed with 4% paraformaldehyde for 15 min at room temperature and immersed in 0.2% Triton X‐100 for 10 min. After blocking with 10% donkey serum for 30 min, the cells were immunostained with primary antibodies against fibronectin  and a Cy3‐conjugated secondary antibody . Photos were taken with fluorescence microscopy .n (%). Student’s t test and the Mann–Whitney U test were used to compare normally and non‐normally distributed variables, respectively. Correlations were conducted using Spearman’s correlation coefficient for non‐normally distributed data. Continuous variables among groups were compared using one‐way analysis of variance, followed by the LSD test. Statistical analyses were conducted with SPSS 22.0 software. A value of P < 0.05 was considered statistically significant.Continuous variables are shown as the means ± SD, and categorical variables are expressed as A total of 124 participants aged between 23 and 40 were enrolled. Among 55 patients with PCOS, hyperandrogenism  was observed in 24 (43.64%) patients.2), and testosterone (T) in the PCOS group were significantly higher compared to controls. There were no significant differences in GLU, FSH, and prolactin (PRL) between two groups (Table Two PCOS patients (3.64%) showed a slight increase in blood pressure  and higher BMI index . While the blood pressure in each of 69 controls was normal. One control subject showed slight increases in fasting glucose (GLU) (6.42 mmol/L) and BMI index 26). None was diagnosed hyperglycemia among 55 PCOS patients. Due to the small proportion of participants with the abnormal rate of BP and GLU, hierarchical analysis was not performed. The groups were matched in terms of age. Whereas, the BMI, length of the menstrual cycle, number of total antral follicles , basal levels of luteinizing hormone (LH), estradiol (EP < 0.05) in PCOS patients compared with controls. However, SCr and BUN showed no significant difference. Although no significant differences in the levels of KapU, LamU, α1‐MU, and β2‐MU, there was a rising trend for these tubular proteinuria markers in PCOS patients. Additionally, the proportion of patients with UACR higher than 7 mg/g was 45.45% in PCOS patients and 44.93% in controls.Previous studies have shown that PCOS links to abnormalities including obesity, DM, and cardiovascular disease. Notably, all these abnormalities are major risk factors for the progression of CKD  , and metabolic dysfunction. Notably, PCOS patients often complicate with hyperandrogenism. To further clarify the responsibility of testosterone for the disease severity of PCOS, we analyzed the correlation between serum testosterone and the length of the menstrual cycle or number of total antral follicles . We also assessed the association of testosterone with BMI, MAP, and GLU, the three metabolic symptoms that commonly display in PCOS. As shown in Table 1) Table . HoweverTo identify the role of serum testosterone in PCOS‐associated kidney injury, we performed Spearman’s correlation analysis in two groups. First, the correlation between serum testosterone and UACR in two groups was analyzed. There was no correlation between them in two groups (data not shown). We next assessed the urinary levels of KapU, LamU, α1‐MU, and β2‐MU, which represent the proteinuria originated from renal tubular epithelia cell injury. All of them were performed correlation test with serum testosterone. As shown in Table P < 0.01), suggesting the important role of testosterone in GCs injury.To further confirm the effects of testosterone on PCOS injury, we cultured primary GCs. The cells were collected from the follicular fluid (FF) samples of five PCOS patients with hyperandrogenism (with high testosterone), five PCOS patients with non‐hyperandrogenism  and five healthy controls. First, the levels of testosterone in FF were analyzed Fig. A. We nexTo further identity the correlation between PCOS and kidney injury, we cultured HKC‐8 cells and treated cells with FF from PCOS patients with hyperandrogenism and healthy controls. The fibrotic injury markers of fibronectin and α‐SMA were assessed by western blot analyses. As shown in Figure PCOS has a close association with the progression of metabolic abnormalities such as obesity, diabetes and hypertension, which are the major causes of kidney disease  in our study. We then explored the association between serum testosterone and the disease severity of PCOS. Abnormality of the menstrual cycle is one of the critical characteristics in PCOS patients. The prolongation of the menstrual cycle could result in the high frequency of anovulation, which would lead to amenorrhea, infertility, endometrial hyperproliferation and even carcinogenesis (Barry et al., .64% in oOther studies have revealed testosterone in the FF in PCOS patients is higher than that in controls (Qu et al., We further investigated the role of testosterone in PCOS‐associated kidney injury. We performed correlation analyses of serum testosterone with the urinary excretion levels of KapU, LamU, α1‐MU, and β2‐MU, the markers of kidney tubular injury (Yu et al., To the best of our knowledge, this is the first study that demonstrates a definitive relationship between PCOS and kidney injury. First, our clinical analysis showed that UACR is higher in PCOS patients than controls, suggesting that PCOS is closely associated with kidney injury. Second, we found that serum testosterone in PCOS patients are positively correlated with the urinary protein excretion levels of which reflect the injury in kidney tubules. These results demonstrate that testosterone plays an important role in PCOS‐associated kidney injury. Third, we analyzed the effects of FF from PCOS patients with hyperandrogenism on HKC‐8 cells. We found the upregulated fibrotic lesions in PCOS FF‐treated HKC‐8 cells, suggesting that testosterone mediates PCOS‐related kidney injury through promoting the fibrosis in tubular epithelial cells.In this study, because mass spectrometry has not yet been established in our clinics, we tested the total testosterone in serum by the chemiluminescence immunoassay but not the “gold standard” testing method of testosterone status such as liquid chromatography‐tandem mass spectrometry. Future studies should be designed to analyze testosterone by multiple methods. Furthermore, a larger sample size including various ethnic backgrounds from different regions should be recruited to verify the conclusion and deeply explore the underlying mechanisms. Specially, studying a prospective cohort of women with PCOS and BMI more than 30 would be more meaningful to assess the association between PCOS and renal injury.Although more studies are needed, these results provide proof of principle that serum testosterone plays a pivotal role in the onset and development of PCOS and PCOS‐associated kidney disease. Continued monitoring and early intervention of serum testosterone would be of great value to prevent the high risk of kidney injury for PCOS patients. Overall, serum testosterone may be regarded as a novel indicative marker for clinical prevention and treatment of PCOS and the related kidney diseases.The authors declare that there are no competing interests associated with the manuscript.This study was approved by the Ethical Committee Faculty of Nanfang Hospital, Southern Medical University, and informed consent was obtained from all of the participants recruited into this study and was carried out in accordance with the World Medical Association Declaration of Helsinki. All methods were performed in accordance with the relevant guidelines and regulations. The number of ethical approvals is NFEC‐2018‐154.Table S1. Demographic characteristics of the participants in the study.Table S2. Parameter values of control subjects and PCOS patients.Table S3. Parameter values in isolated human granulosa cells.Click here for additional data file.