Despite global efforts, stunting remains a public health problem in several developing countries. The prevalence of stunting among 0- to 5-year-old children in Armenia has increased from 17% in 2000 to 19% in 2010. A baseline study was conducted among preschool children in Berd, a region near the northeastern border of Armenia that has experienced intermittent military tension for over 20 years.We conducted a cross-sectional study including 594 children aged 6-month- 6 years old and their caregivers in our analysis, to assess the prevalence and determinants of stunting. We calculated the anthropometric measurements and hemoglobin levels of children; analyzed children’s stool and conducted a survey with children’s caregivers. We employed the hierarchical logistic regression model to explore the predictors of stunting among 25–72 months old children and multivariable logistic regression models to investigate the predictors of stunting among 6–24 months old children. Individual and residence level variables were included in the models including anemia, minimum dietary diversity, mothers’ height, the overall duration of breastfeeding, birthweight, child’s history of diarrhea and mean socio-economic score.p < 0.05). Each kilogram increase in birthweight was associated with 76% lower odds of being stunted . Mother’s height significantly decreased the odds of stunting among the children 25–72- months old . BMI was also a significant predictor of stunting among both age-groups.The prevalence of stunting was significantly higher among the 6–24 months old children (13.3%) compared to the children aged 25–72 months old (7.8%). We did not find any differences in the prevalence of stunting by place of residence in either age group. The 6–24 months old children who consumed at least four food groups during the previous day (minimum dietary diversity) had 72% lower odds of being stunted (The study results highlight the significance of mother’s height, birthweight, and adequate complementary feeding to reduce stunting. Further studies are needed to determine the possible association of anemia and stunting with the ongoing conflict in the region, as well as socioeconomic conditions and food insecurity in the region. Several studies have found that protracted conflicts have an adverse impact on health –3. CurreAccording to the World Health Organization (WHO), 54% of all child deaths are associated with undernutrition . DespiteSeveral factors influence stunting, including the socioeconomic status of the household, parents’ education, anemia, soil-transmitted helminths (STH) infections, diarrheal infections, dietary intake and complementary feeding –22. ShorIn a study conducted in Sri Lanka, maternal height was inversely related to the prevalence of stunting . Low birThere are limited data regarding infant and child nutrition status in the Republic of Armenia. The Armenian Demographic and Health Survey (ADHS), conducted every 5 years, is the primary source of information regarding nutrition and child growth patterns. According to the 2010 ADHS, the prevalence of stunting among 0- to 5-year-old children increased to 19% from 17% in 2000 . A recenAccording to the ADHS, the prevalence of stunting in 2010 was 16.1% in the Tavush province –33, resuThe growing trend of stunting in Armenia necessitates a thorough investigation to understand its risk factors to inform policies and interventions to reverse the trend. Although the determinants of stunting have been well investigated in several studies, the specific precursors of stunting might vary among different countries and regions . This diThe main aim of this study was to determine the prevalence and determinants of stunting among preschool children in Berd city and its seven surrounding rural communities, a vulnerable border region in the Tavush province in the Republic of Armenia, which is itself a low-middle income country. We specifically explored if there are significant differences in the prevalence of stunting among the children 6–24 months and 25–72 months old. We also examined if the prevalence of stunting in each age group was different by place of residence. We further investigated the influence of anemia, soil-transmitted helminths, feeding practices, dietary intake, maternal and household characteristics and birth outcomes on stunting among children 6–24 months and 25–72 months old.This cross-sectional study included anthropometric  measurements, blood tests to measure hemoglobin (HgB) levels of 6-month to 6-year-old children, and stool analysis for intestinal STH in 12-months to 6-year-old children. The survey also included a questionnaire for caregivers (mainly mothers) to understand the determinants of malnutrition in the target communities. The study was designed and conducted from September 2013 to March 2014.The study population for the anemia analysis and anthropometric measurements included all 6-month to 6 year-old children living in the city of Berd and seven surrounding rural communities . The only exclusion criteria were if the child was diagnosed with a blood coagulation or neurological disease; however, no child in our sample met the exclusion criteria.n = 352). Study participants were chosen from a sample frame consisting of all children living in Berd city, as registered in the Tavush province’s administrative records. The children from Berd were selected based on a stratified random sampling strategy. We chose age groups (year) as the strata. The number of the samples within each age group (stratum) were calculated separately based on n = 491).A representative sample of children from Berd city aged 6 months to 6 years old was randomly selected to participate in the study  were trained in the study protocols and assigned as study coordinators in their regions. The adherence to study protocols by coordinators and staff was monitored through frequent spot-checks. The anthropometric measurements, blood and stool analysis, and surveys with the parents/caregivers took place in the primary healthcare facilities in Berd and the local rural communities. The study coordinators obtained a written informed consent from the caregivers of all participants. After collecting the child’s anthropometric measurements, the caregiver participated in a survey administered by the physician or nurse. The survey questionnaire included questions about maternal and child characteristics, infant feeding practices, birth outcomes and child’s dietary intake, measured by 24-h recall.The HgB levels of the children were measured using HemoCue® HB 301, a U.S. Food and Drug Administration (FDA)-approved device designed for quick analysis of HgB in capillary, venous, or arterial blood –41. As rAscaris lumbricoides, and Trichuris trichiura, in stool samples [The Kato-Katz method was used to perform the stool analyses . This te samples . We provWe adapted the WHO, and UNICEF Infant and Young Child Feeding (IYCF) practices questionnaire to assess feeding practices as well as maternal and child characteristics in the target region , 44. TheChildren’s weight was measured using electronic scales. The children’s height (length in recumbent status for children younger than 24 months) was measured with appropriate measuring boards or stadiometers by the trained primary health care providers in the study sites.The primary outcome variable was stunting, analyzed as a dichotomous outcome. Stunting was considered positive for all the children whose height-for-age was below -2SD compared to the median height-for-age of the WHO growth standards .Anemia, one of the explanatory variables, was considered positive for the children with blood HgB levels lower than 110 g/L. Altitude adjustments were made to the HgB values of the children living in areas more than 1000 m above sea level . The preThe survey questionnaire administered to caregivers asked about child’s birth weight and birth length; child’s age; any breastfeeding; exclusive breastfeeding; breastfeeding duration; residence; mother’s height; mother’s participation in community training about child nutrition; accessibility of printed materials about child nutrition; any history of prolonged diarrhea or STH reported by the caregiver; and a detailed dietary intake assessment of the child during the last 24 h.We used a standardized socio-economic score as a composite of (a)mothers’ education; (b) the financial status of the family compared to neighboring households; (c)food insecurity. Further, we calculated the mean socioeconomic status score based on the place of residence. The information about birth weight, birth length and birthdate were confirmed based on administrative records.To assess the effect of conflict on stunting, we created a variable based on the geographical distance from the border and front lines of the conflict zone. The two villages closest to the front line were considered positive for being exposed to violence.Minimum dietary diversity, another explanatory variable, was created based on the scoring system of the WHO and UNICEF indicators  for the The data were double entered using SPSS 21.0 and analyzed using SAS 9.4 statistical software. We calculated frequencies and proportions to describe categorical variables and computed means and standard deviations for continuous variables. We used Chi-Square and ANOVA to assess the intergroup comparison for children 6–24 months and 25–72 months old separately. We further examined whether the association between stunting and main predictor variables was modified by place of residence using Breslow-Day test.p < 0.25 as well as the clinically significant variables to fit the multivariable logistic regression and hierarchical models  and Akaike’s Information Criterion (AIC) we compared the hierarchical models to estimate the predictors of stunting among 25–72 months old children and to examine the improvement in model fit.Model A was the multivariable logistic regression model including all the individual level predictors adjusted by age and gender of the child. Model B was the multilevel logistic regression model assuming variance components (VC) G matrix for all the residence groups and including only the random intercept to estimate the effect of a typical residence place level effect. In model C, we included only the individual level variables to account for the crude effects of these variables in the presence of random intercept. Model D included anemia as another individual level predictor. Subsequently, we included the residence-level mean socio-economic composite score variable in model E to explain the residence level variation in stunting. However, we did not observe a significant association between stunting and the residence level mean socio-economic composite score, and the inclusion of the variable did not improve the model fit. Model F included all the variables in model D, adjusted for age and gender. Finally, model G included all the variables in model E, adjusted for age and gender. We refrained from including random slope since it did not significantly improve the model fit when comparing to model F using LRT test.2. The multivariable logistic regression analysis resulted in six consecutive models. The final model included all the statistically and clinically significant variables that had the highest Hosmer–Lemeshow test score and pseudo R2 to predict the prevalence of stunting. Model A includes crude unadjusted predictors of stunting including birthweight, child’s history of any diarrhea reported by the caregiver, and minimum dietary diversity. Model B included all the individual level variables in model A adjusting for age of the child by months and gender of the child. We included anemia, standardized socio-economic score, and presence of soil-transmitted helminth infection in each of the three consecutive models subsequently. However, including the variables did not contribute to the model fit.The multivariable logistic regression models to estimate the predictors of stunting among 6–24 months old children were tested and compared by the Hosmer–Lemeshow goodness-of-fit test, AIC and LRT and Nagelkerke’s Rn = 674). We did not include children who were older than 6 years or younger than 6 months in the analysis. The final analysis included 594 children and their caregivers after excluding missing observations. Of the total number of children included in the study, 46.52% were girls, and 53.48% were boys; 34.43% were from Berd city, and 65.57% were from the rural communities named in methods above. Most of the caregivers included in the study (61.2%) had at least a high school level of education (≥10 years of education). More than 66.22% considered the financial status of their household to be average or above average compared to their neighbors. The prevalence of low birth weight, stunting, and anemia was significantly higher among the children aged 6–24 months compared to the children 25–72 months old. The prevalence of minimum dietary diversity was significantly higher among 25–72 months old compared to the 6–24 months old children. There were no significant differences in the prevalence of STH among younger and older children . However, when this variable was adjusted for age and gender of the child in model B, the effect estimate significance was reduced (OR = 2.83 p < 0.10). In model B, the children who had consumed at least four food groups (minimum dietary diversity) during the previous day of the investigation had significantly lower odds of stunting .As previously described in data management and statistical analysis, we fitted a multivariable logistic regression model among children between 6 and 24 months to identify the determinants of stunting among this age group (Table p < 0.01). BMI also appeared to be a significant predictor of stunting in model B. The odds of stunting increased by 55% with each unit increase in BMI score (OR = 1.55 p < 001). Finally, with each increasing month of age, the children had higher odds of being stunted . Therefore, significant predictors of stunting in children age 6–24 months were minimum dietary diversity, birthweight, BMI, and age.Also in model B, each kilogram increase in birth weight was associated with 76% lower odds of being stunted  and maternal height appeared to be protective against stunting. The odds of stunting seemed to decrease by 13% with each centimeter increase in maternal height . Finally, with each unit increase in the child’s BMI the odds of stunting were increased by 26%  , the lower the odds of stunting among 6–24 months -old children, which is consistent with the literature , 52–54. The association between BMI and stunting among children was one of the novel findings of our study. We found that the odds of stunting increased significantly among children in both age-groups with each unit increase in BMI adjusting for other predictors in our models Tables  and 5. TWe found that, with each centimeter increase in the mother’s height, the odds of stunting among all children aged 25–72 months old decreased by 13%. This finding, which is consistent with the literature, indicates the possible role of genetics and the intergenerational effect of malnutrition on the development of stunting , 60.We also found that the odds of stunting among boys were 2.36 times the odds of stunting among girls in the 25–72 months old. Several studies conducted predominantly in Sub-Saharan Africa have found similar results –63. ThisThe overall prevalence of anemia was high in our study population. However, anemia was not significantly associated with stunting in univariable and multivariable models among any of the age groups. The prevalence of anemia was significantly higher among the 6–24 months old children compared to 25–72 months old children in both the rural and urban areas. A possible explanation for this finding could be the lower dietary diversity among 6–24 months old children compared to children aged 25–72 months Table .A history of long episodes of diarrhea was only associated with stunting among the 6–24 months old children in the multivariable logistic regression. However, the association faded away after adjusting for age and gender and in hierarchical models. In fact, diarrhea was found to be associated with stunting in similar studies –69. DuriThe positive effect of overall duration of breastfeeding (months) on the development of stunting among 25–72 months old children, although very small (OR = 1.05), may indicate inadequate complementary feeding, which is also supported by the finding of low minimum dietary diversity. It could also indicate the inadequate frequency of breastfeeding during the day; however, the frequency of breastfeeding was not measured in this study.This is the first study in this restive border region assessing the trends and determinants of stunting. The study included the entire community in the rural areas (census), which indicates that the statistical inferences were accurate for the rural regions and the representative random sample from the Berd region also increases the generalizability of the results. The authors pretested the questionnaire along with study coordinators in the region to minimize the possibility of measurement errors.Nevertheless, there were several limitations in this study. The questionnaire utilized for the survey with mothers lacked questions regarding the water and sanitation situation of the respondents’ households. Although our instrument assessed the diet of the children based on 24-h recall questions, we do not exclude the possibility of recall bias. Recall bias may also have affected responses to other subjective questions like those related to diarrhea and breastfeeding duration. The stool analysis was also only conducted once and not in a modern laboratory. However, this misclassification would be non-differential. We also did not include a separate question to assess the frequency and level of border conflict in each study site in our survey. We did not find a significant association between residence-level mean socioeconomic score and stunting in our hierarchical models. We acknowledge that our questionnaire did not capture accurate information to calculate the socio-economic score for individuals. Only three variables were measuring the socioeconomic status of the study participants’ households. Furthermore, our questionnaire did not assess the family income and father’s occupation accurately, which could help us to determine the family’s socio-economic position better.This study identified several risk factors that are associated with the development of stunting among children in the conflict-ridden border regions of Armenia. Factors such as minimum dietary diversity and history of previous episodes of diarrhea indicate the necessity of adequately feeding children as well as the management of the diarrheal disease. The findings of this study will contribute to the development of appropriate interventions targeting timely complementary feeding in vulnerable regions to reduce stunting. Although anemia was not associated with stunting, the prevalence of anemia was very high among the 6–24 months old children and should be a focus of dietary intake interventions to promote optimal growth patterns among children in the region. Further research should consider a more thorough questionnaire to capture the socioeconomic status of the study participants, to determine the association between anemia and stunting, and to further assess the effect of the ongoing conflict in the region. The findings of this study can inform public health programmers and policymakers to focus on promoting appropriate complementary feeding, dietary diversity, and management of diarrhea for optimal growth among young children in this region.


Insults to the axons in the optic nerve head are the primary cause of loss of retinal ganglion cells (RGCs) in traumatic, ischemic nerve injury or degenerative ocular diseases. The central nervous system–specific leucine‐rich repeat protein, LINGO‐1, negatively regulates axon regeneration and neuronal survival after injury. However, the upstream molecular mechanisms that regulate LINGO‐1 signaling and contribute to LINGO‐1–mediated death of RGCs are unclear.The expression of SP1 was profiled in optic nerve crush (ONC)–injured RGCs. LINGO‐1 level was examined after SP1 overexpression by qRT‐PCR. Luciferase assay was used to examine the binding of SP1 to the promoter regions of LINGO‐1. Primary RGCs from rat retina were isolated by immunopanning and RGCs apoptosis were determined by Tunnel. SP1 and LINGO‐1 expression was investigated using immunohistochemistry and Western bolting. Neuroprotection was assessed by RGC counts, RNFL thickness, and VEP tests after inhibition of SP1 shRNA.We demonstrate that SP1 was upregulated in ONC‐injured RGCs. SP1 was bound to the LINGO‐1 promoter, which led to increased expression of LINGO‐1. Treatment with recombinant Nogo‐66 or LINGO‐1 promoted apoptosis of RGCs cultured under serum‐deprivation conditions, while silencing of SP1 promoted the survival of RGCs. SP1 and LINGO‐1 colocalized and were upregulated in ONC‐injured retinas. Silencing of SP1 in vivo reduced LINGO‐1 expression and protected the structure of RGCs from ONC‐induced injury, but there was no sign of recovery in VEP.Our findings imply that SP1 regulates LINGO‐1 expression in RGCs in the injured retina and provide insight into mechanisms underlying LINGO‐1–mediated RGC death in optic nerve injury. Under many traumatic, ischemic nerve injury or degenerative ocular conditions, such as glaucoma, the dysfunction and/or loss of RGC is the primary determinant of visual field loss and are the measurable endpoints in current research into experimental therapies.The lack of cellular and axonal regeneration in the event of neuronal injuries is due to myelin‐associated inhibitory factors.Upon axonal injury, transcription factors in neurons are activated, resulting in a cascade of changes in the transcriptome and priming of the degeneration and regeneration pathways.22.1A total of 52 male Sprague Dawley rats  and 40 newborn rats  were maintained in the Ophthalmic Animal Laboratory of Zhongshan Ophthalmic Center. All procedures involving animals were conducted in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. All experimental procedures were approved by the institutional animal care and use committee of Zhongshan Ophthalmic Center (Permit SYXK 2018‐025). All manipulations were performed with rats under general anesthesia with 2%‐3% inhaled isoflurane, and the eyes of the rats were administered topical 0.5% Alcaine eye drops (Alcon) prior to surgery, experimentation, and electrophysiology examination.2.2Optic nerve crush injury was performed as described previously.2.3XhoI/HindIII restriction enzyme sites in the pGL3‐Basic plasmid. For dual‐luciferase reporter assays, cells were cotransfected with 1 µg firefly luciferase plasmid harboring the promoter fragments and 100 ng of the Renilla luciferase reporter plasmid pRL‐TK. The cells were harvested at 36 hours after transfection, and Firefly activity and Renilla luciferase activity were measured using the Dual‐Glo Luciferase Reporter Assay System (Promega). Firefly luciferase activity was normalized to that of Renilla luciferase and is presented as relative luciferase units.The LINGO‐1 promoter fragment, comprising nucleotides −2104 to +121 bp of the LINGO‐1 5′‐flanking region relative to the transcription start site, was amplified by PCR  and fused upstream of the luciferase reporter in the pGL3‐Basic vector to generate the LINGO‐luciferase (Luc) reporter. Deletion reporter constructs of the 5′‐flanking region were generated by PCR using LINGO‐1–Luc as the template and a common reverse primer. The forward primers were as follows: 5′‐GAAGGCGAACAAGGCACTG‐3′ for LINGO‐1 (−1268 to +121)–Luc, 5′‐AGCTGAGCCCAGACTAAG‐3′ for LINGO‐1 (−789 to +121)–Luc, 5′‐ATGGCAGTGTGCAGTGAC‐3′ for LINGO‐1 (−383 to +121)–Luc, 5′‐CTCCCTGGCTCGCTGCTC‐3′ for LINGO‐1 (−122 to +121)–Luc. All PCR products were subcloned into the 2.44/mm2 and cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS), BDNF , CNTF , and forskolin  in culture slides precoated with poly d‐lysine and laminin (Sigma‐Aldrich). For survival assays, SP1‐shRNA was transfected into cells using Lipofectamine RNAiMAX as recommended by the manufacturer. At 48 hours after transfection, the cells were treated with or without recombinant Nogo‐66  or LINGO‐1  in the presence of absence of SP1‐shRNA. After incubation for 12 hours, complete medium was replaced with DMEM without serum to induce apoptosis. Cell cultures with serum were performed in parallel as controls.Primary RGCs were isolated and purified by immunopanning.2.5Total RNA was extracted from retina tissues using TRIzol reagent (Invitrogen). RT‐qPCR was performed using PrimeScript RT Master Mix and SYBR Premix Ex Taq II  according to the manufacturer's instructions. The sequences of the primers used for qPCR were as follows: LINGO‐1, 5′‐CTTCCCCTTCGACATCAAGAC‐3′ and 5′‐AAGACGGACCACGACGAC‐3′; β‐actin, 5′‐TCACCCACACTGTGCCCAT‐3′ and 5′‐TCTTTAATGTCACGCACGATT‐3′; SP1, 5′‐TCCAGACCATTAACCTCAGTGC‐3′, and 5′‐ACCACCAGATCCATGAAGACC‐3′.2.6Retinas were homogenized, and total protein was extracted using a Protein Extraction Kit (Beyotime Biotechnology). The total protein samples were separated by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis, electro‐transferred to a nitrocellulose membrane, and exposed to anti‐SP1  and anti‐LINGO‐1  antibodies. Next, the membrane was incubated with a horseradish peroxidase–conjugated secondary anti‐rabbit antibody (CST); β‐actin served as the loading control. The protein bands were detected by enhanced chemiluminescence (Pierce).2.7After cardiac perfusion with 0.9% saline, the eyes of the rats were collected, fixed in 4% paraformaldehyde for 12 hours, and cryoprotected in 30% sucrose for 12 hours at 4°C. The eyes were next embedded in optimal cutting temperature medium and sectioned (10 μm thickness). The sections were permeabilized with 0.3% Triton X‐100 and blocked with 10% goat serum for 45 minutes. The slides were incubated with an anti‐Sp1  or anti‐LINGO‐1  primary antibody overnight at 4°C, washed three times, and incubated with the secondary antibodies  for 1 hours at room temperature. Nuclei were stained with 4′, 6‐diamidino‐2‐phenylindole, and the slides were mounted and visualized under a confocal microscope .2.8Terminal deoxynucleotidyl transferase dUTP nick‐end labeling (TUNEL) staining was performed using an in situ cell detection kit according to the manufacturer's instructions (Roche). Briefly, cultured RGCs were fixed in 4% PFA for 15 minutes at 4°C, blocked using 1% donkey serum, and permeabilized with 0.1% Triton at room temperature for 20 minutes. TUNEL detection solution was added, and the samples were incubated for 50 minutes at 37°C and costained with DAPI. TUNEL‐positive nuclei were quantified using ImageJ software. Finally, TUNEL‐positive cells were enumerated in high‐power fields of view of three wells per treatment, and the mean was calculated.2.912 GC/mL; GeneChem) was injected into the vitreous cavity of the rats in the experimental group at 14 days before ONC injury, avoiding damaging the lens and fundus hemorrhage and ensuring that the intraocular pressure did not increase markedly. The sequence of the SP1 shRNA was 5′‐GCAACAUGGGAAUUAUGAATT‐3′.After general anesthesia and ocular surface anesthesia, intravitreal injections were performed 2 mm behind the limbus using a Hamilton micro‐injector with a 30‐gauge needle. Five microliters of AAV2‐SP1 shRNA (1 × 102.102) were evaluated at 1.5, 2.5, and 3.5 mm from the optic nerve head across the five petals for each retina. For each petal, images of three regions representing the peripheral, medial, and central parts of the retina were acquired.Rats were sacrificed and eyes were collected, fixed with 4% PFA for 2 hours. The intact retinas were separated, and five radial incisions were made to create a petal shape. The retinas were permeabilized with 2% Triton X‐100, blocked with 5% goat serum for 4 hours, and incubated with an anti‐RBPMS antibody  at 4°C in a shaker overnight. After three times of washes, the retinas were incubated with a secondary antibody conjugated to Alexa Fluor 488  for 4 hours. The retinas were transferred to glass slides using an enlarged open‐pad tube, flattened, blotted dry, stained with DAPI, mounted, and RBPMS‐positive RGCs were visualized under a confocal microscope . RGCs were enumerated as described previously.2.112), the stimulation frequency was 1.0 Hz, the passband was 0.5‐50 Hz, and the stimulation frequency was 100. For quantitative analyses, the VEP system detection index was the N1 wave, P1 wave latency (ms), and N1‐P1 wave amplitude (μv). A visual stimulus of 1 Hz white light (9.49 c × s/m2) was generated by a full‐field Ganzfeld stimulator under dark‐adapted conditions. The amplitude of N1‐P1 and the latency of the N1 and P1 peaks were measured using Roland software (Roland Consult). The amplitude of N1‐P1 was determined as the interval from the trough of the first negative peak after light onset (N1) to the peak of the first positive wave (P1). The latency of the N1 and P1 waves was measured from light onset to the peak of N1 or P1.Before assessment of visual evoked potentials (VEPs), the rats were dark‐adapted for > 2 hours. The rats were anesthetized by intraperitoneal injection of 10% chloral hydrate. VEPs were evaluated using a Roland RETI‐scan system (Roland Consult) for a full‐field flash stimulator. The stimulus intensity was 5 dB , we performed volume scans using a noninvasive high‐resolution SD‐optical coherence tomography (OCT) instrument  at baseline and 2, 7, 14, and 21 days after surgery as described previously.2.13P value <.05 was considered indicative of significance. Kolmogorov‐Smirnov tests were used to assess data distribution for normality. The Student t test, one‐way analysis of variance (ANOVA), two‐way ANOVA, or repeated measure ANOVA was used to compare differences between groups.All experiments were performed in at least triplicate biological repeats. Data are presented as means ± standard deviations (SDs). Statistics was performed using the statistical package for the social sciences (SPSS) software. A 33.1To determine the transcriptional control responsible for increased LINGO‐1 expression in RGCs, we used a gene‐expression microarray to identify genes that mediated RGC death. The efficacy of RGCs isolation was verified by cell morphology and flow cytometry with CD90.1 and CD48 labeling Figure A,B. The Next, we determined the region responsible for transcriptional regulation of LINGO‐1 and the related transcriptional factors. A series of fragments of luciferase reporter constructs containing deletions of the LINGO‐1 5′‐flanking region were transfected into cells, and luciferase activity was assayed. One deletion fragment (−789 to +121 bp) showed dramatically higher promoter activity than pGL3‐Basic, similar to the activity of the full‐length fragment (−2104 to +121 bp). By contrast, luciferase activity was almost completely abolished by a different fragment (−383 to +121 bp). Thus, the region responsible for transcriptional control of LINGO‐1 was located at nucleotides −789 to −383 Figure . Computa3.2To investigate the role of SP1 in the regulation of LINGO‐1 expression, we transfected HEK293 cells with pCGN‐SP1 or control vector and quantified the LINGO‐1 mRNA level by qRT‐PCR. SP1 overexpression resulted in a significantly higher LINGO‐1 mRNA level compared to the control Figure . To detein trans,Because LINGO‐1 is implicated in potentiating neuronal apoptosis under serum‐deprivation conditions,3.3To examine the association between LINGO‐1 and SP1 in the ONC‐injured retina further, double immunofluorescence staining of LINGO‐1 and SP1 was performed. The control RGCs exhibited low LINGO‐1 and SP1 expression; in contrast, LINGO‐1 and SP1 expression was considerably higher in ONC‐injured RGCs. In addition, LINGO‐1 and SP1 were colocalized in the RGCs Figure . Western3.4Inhibition of LINGO‐1 promotes the survival of RGCs following ONC,3.5To examine the neuroprotective effects of inhibition of SP1, we monitored changes in RNFLT by OCT, a noninvasive method of assessing degenerative changes in converging axons of RGCs. The average RNFLT was 1.5 mm from the center of the optic nerve head Figure . The RNF2)  Figure  encompas) Figure . Therefo3.6The preservation of RGC structure motivated us to evaluate the functional recovery of the visual circuits after injury to the optic nerve by assessing the VEP at 1‐day pre‐ONC (baseline) and at 7, 14, and 28 days post‐ONC. Impairment of visual function after ONC was evidenced by reduced amplitude of N1‐P1 waves and prolonged latency of N1 waves Figure . However4Death of, and axonal injury to, RGCs is important in retinal neuropathy. Similar to CNS neurons in other neurodegenerative diseases, death of RGCs is irreversible and can directly disturb visual pathway signal transmission, resulting in impaired visual function. Glaucoma is a retinal neurodegenerative disease characterized by the loss of, and axonal injury to, RGCs. Therefore, protection of RGCs is important in the treatment of retinal neuropathies such as glaucoma. In this study, we used the ONC model to explore the mechanism(s) underlying the loss of RGCs that occurs during the development of glaucoma.During the course of nerve injury, the myelin‐associated inhibitory protein LINGO‐1 mediates neuronal survival and axonal regeneration, thus contributing to neurodegeneration.syt11,In view of the important pathophysiological role of LINGO‐1 in mediating the death of RGCs, we investigated the upstream regulatory factors. In microarray analyses, we found that SP1 was upregulated in the RGCs of ONC‐injured retinas, and LINGO‐1 promoter analyses revealed that SP1 increased the expression of LINGO‐1 by binding to its promoter region. Furthermore, SP1 knockdown antagonized LINGO‐1–induced apoptosis of RGCs under serum‐deprivation conditions. In addition, SP1 and LINGO‐1 were simultaneously upregulated in injured RGCs. These data imply that SP1 regulates LINGO‐1 expression at the transcriptional level and that upregulation of SP1 is implicated in LINGO‐1–mediated death of RGCs. Indeed, SP1 has been reported to play important pathophysiological roles in a variety of neurodegenerative diseases, including Alzheimer's, Huntington, and Parkinson's diseases, and to regulate disease‐related pathogenic genes such as APOE, LRRK2, and P16INK4A.We evaluated the role of SP1 in the LINGO‐1–mediated death of RGCs. Intravitreous injection of AAV2‐SP1 shRNA was performed at 2 weeks before ONC to knock down SP1 expression in RGCs; this resulted in reduced expression of LINGO‐1. To investigate the neuroprotective effects of inhibition of SP1, we used OCT to monitor RNFLT and assess the axons of RGCs. Inhibition of SP1 promoted survival of RGCs and increased RNFLT, consistent with our previous report of the neuroprotective effects of LINGO‐1 antagonism.We then evaluated the protective effects of inhibition of SP1 on visual function. Surprisingly, inhibition of SP1 did not significantly modulate N1‐P1 amplitude or N1 latency. The two major possible explanations for these conflicting findings are that inhibition of SP1 preserved the structure of RGCs but did not restore visual function. First, inhibition of SP1 may not protect the subcellular organelles of RGC axons from damage such as disintegration of the synaptic assembly or may fail to promote synapse repair after damage. BDNF, CTNF, and NT3, among other factors, promote neuronal survival and synaptic regeneration after injury.We report the upregulation of SP1 and LINGO‐1 expression in RGCs in the ONC‐injured retina. In vitro, SP1 regulated LINGO‐1 expression at the transcriptional level and promoted the LINGO‐1–mediated death of RGCs. Furthermore, SP1 knockdown had a neuroprotective effect in vivo. Our results provide important insight into the mechanism of LINGO‐1–mediated death of RGCs in patients with glaucoma. Further studies are required to increase our understanding of the mechanism(s) of RGC‐related synaptic damage and formulate a neuroprotective strategy for glaucoma involving stimulation of the visual system.The authors declare no conflict of interest.Fig S1Click here for additional data file.


Pink1, Parkin and Fbxo7, three autosomal recessive familial genes of Parkinson’s disease (PD), have been implicated in mitophagy pathways for quality control and clearance of damaged mitochondria, but the interplay of these three genes still remains unclear. Here we present that Fbxo7 and Pink1 play a reciprocal role in the regulation of their protein levels. Regardless of the genotypes of Fbxo7, the wild type and the PD familial mutants of Fbxo7 stabilize the processed form of Pink1, supporting the prior study that none of the PD familial mutations in Fbxo7 have an effect on the interaction with Pink1. On the other hand, the interaction of Fbxo7 with Bag2 further facilitates its capability to stabilize Pink1. Intriguingly, the stabilization of Fbxo7 by Pink1 is specifically observed in substantial nigra pars compacta but striatum and cerebral cortex. Taken together, our findings support the notion that Fbxo7 as a scaffold protein has a chaperon activity in the stabilization of proteins. Parkinson’s disease (PD) is the second most common progressive neurodegenerative disease after Alzheimer’s disease, characterized by tremor, rigidity and bradykinesia due to dopaminergic (DA) loss in substantial nigra pars compacta (SNc) , 2. It iPink1, a mitochondrially localized serine/threonine kinase gene, acts as an important upstream regulator of Parkin in control of mitophagy –18. As aPink1 is processed by multiple proteases in the inner mitochondrial membrane –21. The Although previous study demonstrated that Fbxo7 and its PD familial mutants directly interact with Pink1 , the effDue to the lack of specificity and efficiency of commercialized Pink1 antibodies to detect the endogenous Pink1, we decided to create a knockin (KI) HEK 293A cell line, in which a cassette containing 3XFlag, a P2A motif and a neomycin resistant gene was integrated into the Pink1 locus in chromosome 1 for the expression of Pink1 fused with 3XFlag at the c-terminus of Pink1and a cleaved form of neomycin driven by P2A. To do this, we constructed a donor plasmid carrying a cassette with 3XFlag, P2A, neomycin, and two arms homologous to target the regions of the Pink1 locus on Chromosome 1, and a plasmid carrying genes for CRSPR/Cas9, tracer RNA and sgRNA specific for creating a break near the stop codon of Pink1 . 24 hourFbxo7, as a subunit of SCF E3 ubiquitin ligase, is involved in the regulation of proteasomal activity by recruiting its substrates . To testWe have demonstrated that Bag2 is capable of inhibiting the ubiquitination of Pink1 and stabilizing the endogenous PF-Pink1. We then asked whether Bag2 directly interacts with Fbxo7 and the PD familial forms of Fbxo7. To this end, we carried out an in vitro binding assay using the recombinant proteins purified from E. coli. The in vitro binding assay revealed that Bag2 directly interacted with Fbxo7 and the PD familial forms of Fbxo7 . LikewisBurchell et. al. reported that the expression of Fbxo7 is unable to rescue the phenotype caused by Pink1 mutant in a Drosophila model of PD and pinpointed that Pink1 kinase activity is inevitable for functional regulation of Fbxo7 in Pink1-Parkin signaling cascade . We thenThe work of the past few years has documented that mutations in Fbxo7 are associated with a severe form of autosomal recessive PD , 9, 34. The growing lines of evidence demonstrate that Fbxo7 is a subunit of the SCF E3 ligase that promotes proteasomal activity via facilitating the recognition of substrates , 33, 36.Previously, it has been reported that Bag2 has a critical role in the stabilization of Pink1 through inhibiting the ubiquitination of Pink1 , 27. In In addition to the effect of Fbxo7 on Pink1, we found that the loss of Pink1 causes a decrease of Fbxo7 in SNc rather than other brain tissues such as striatum and cortex, raising a question as to whether the expression of Pink1 has a positive effect on the protein level of Fbxo7. Burchell et. al. reported that the expression of Fbxo7 fails to rescue the phenotype caused by Pink1 mutant in drosophila and suggested the importance of Pink1 kinase activity in Fbxo7 mediated mitophagy . To exteem1Smoc, NM-KO-191011) were purchased from Shanghai Model Organisms, China. All animal procedures were approved by the Institutional Animal Care and Use Committee of Fujian Medical University. Animals were maintained in strict accordance with the Guidelines for the Use and Treatment of Animals put forth by the NIH’s Guidelines for the care and use of Laboratory Animals.Pink1 KO mice , mouse anti-GAPDH , mouse anti-Flag , mouse anti-β-Actin , mouse anti-Myc , mouse anti-V5 , mouse anti-Ubiquitin , rabbit anti-Bag2 , mouse anti-HA , anti-mouse horseradish peroxidase-conjugated secondary antibody  and anti-rabbit horseradish peroxidase-conjugated secondary antibody  for IP Western blots, anti-mouse horse radish peroxidase-conjugated secondary antibodies  and anti-rabbit horse radish peroxidase-conjugated secondary antibodies  for regular Western blots. All primary antibodies were used at 1:5000 dilution, except anti- β-Actin, which was diluted 1:50,000 for Western blot analyses. The secondary antibodies for IP were diluted 1:5000. The secondary antibodies for Western blots were diluted 1:10,000.Pink1 sgRNA sequences: 5’-CACCGGTGATGTCCCTGCATGGAGCTGG-3’and 5’-AAACCCAGCTCCATGCAGGGACATCACC-3’; Pink1 Hom1 primers: 5’-TCCCCGACCTGCAGCCCAGCTCATCTCCTGAGAGCAGATCTG-3’ and 5’-CCGGAACCTCCTCCGCTCCCCAGGGCTGCCCTCCATGAGCA-3’; Pink1 Hom2 primers: 5’-AGTTCTTCTGATTCGAACATCGAACATGGCATCCTCTGTGTC-3’ and 5’-TGGAGAGGACTTTCCAAGCCTTTACTGCATGTTGACGCT-3’; siRNAs for human Fbxo7 were purchased from Santa Cruz Biotechnology (sc-75010).Myc-Bag2 construct was a gift from Dr. Suneil K. Kalia. Pink1-V5 was a gift from Dr. Mark Cookson. Pink1-Flag was inserted into the pAdTrack vector by using NotI and HindIII. pFETCh_donor (EMMM0021) was a gift from Eric Mendenhall and Richard M. Myers  [http://n2t.net/addgene:48138; RRID: Addgene_48138) [Fbxo7 was cloned into pCI-neo with 2xMyc at its C-terminus. The e_63934) . pSpCas9e_48138) .HEK293A, SH-SY5Y and HeLa cells were cultured with 10% fetal bovine serum  in Dulbecco's modified Eagle's medium .The assays were carried out as previously described .The KI HEK 293A cell were generated as previously described , 44. BriStatistical significance was determined via Prism 8.0. All data presented as mean ± SD.


CircRNAs recently have shown critical roles in tumor biology. However, their roles in prostate cancer (PCa) remains largely unclear.via qRT-PCR and FISH. Sanger sequencing, actinomycin D, gDNA, and cDNA, RNase R assays were used to assess the circular characteristics of circNOLC1. CCK-8, colony formation, transwell migration assays, and mice xenograft models were conducted to evaluate the functions of PCa cells after circNOLC1 knockdown and overexpression. RNA pulldown, luciferase reporter assay, FISH (fluorescence in situ hybridization), and CHIP were utilized to illustrate the further mechanisms of circNOLC1.CircRNA microarrays were performed in immortal prostate cell line RWPE1 and PCa cell lines as DU145, PC3, LNCaP, C4-2, and 22RV1. Combined with upregulated circRNAs in PCa tissues, circNOLC1 expression was validated in PCa cells and tissues in vitro and vivo. Additionally, we found that circNOLC1 could upregulate PAQR4 expression by sponging miR-647, leading to the activation of PI3K/Akt pathway. Moreover, NF-kappaB was identified to bind to the NOLC1 promoter sites and upregulated both NOLC1 and circNOLC1 expression.Our research indicated that circNOLC1 was overexpressed in PCa cells and tissues, and circNOLC1 was more stable than linear NOLC1 mRNA. CircNOLC1 promoted PCa cells proliferation and migration via a miR-647/PAQR4 axis, and circNOLC1 is a potential biomarker and target for PCa treatment.CircNOLC1, elevated by transcription factor NF-kappaB, promotes PCa progression  Prostate cancer (PCa) is currently the first and second leading cause of new diagnosis and mortality, accounting for 21% of all new cancer cases and 10% of all death cases in men . AlthougCircular RNA (CircRNA), a novel type of non-coding RNA, is formed by covalently closed loop structures without free terminal ends . Due to in vitro assays and in vivo mouse model. Finally, the underlying mechanism of circNOLC1 were also evaluated. In summary, our works revealed that circNOLC1 could act as a oncogene in the progression of PCa, and may provide a novel target for the diagnosis and treatment of PCa.In this study, circRNA microarray in five common PCa cells combined with the published microarray data for PCa tissues, we identified 22 different circRNAs. Subsequently, circNOLC1 (circBase ID: has_circ_0000257) were chosen for further study to explore its role in the PCa proliferation and progression by using a series of Eighty prostate tissues and sixteen adjacent tissues were obtained from patients who underwent radical prostatectomy (RP) at Zhujiang Hospital of Southern Medical University from 2016 to 2018, and then the paraffin specimens were constructed as a tissue microarray (TMA). The clinical details of patients were collected from the medical records. All experimental procedures were approved by the Ethics Committee of the Southern Medical University Zhujiang Hospital. The median age of the enrolled patients was 67.5 years and average age was 65.1 (range: 20–97 years). Clinical TNM staging and Gleason scores of patient specimens were based on the American Joint Committee on Cancer (AJCC) Eighth Edition (2017) and the 2016 World Health Organization (WHO) classification of genitourinary tumors. The detailed clinicopathological information of all samples was presented in t-test (P: 0.05). Quantile normalization and subsequent data processing was performed through the R 4.0.2 software limma package. Differentially expressed circRNAs were identified via Fold Change filtering. Hierarchical Clustering was used to perform the distinguishable circRNAs expression pattern among the samples.Arraystar Human circRNA Array v2  was applied to analysis circRNA microarray. Total RNA from each sample was quantified with the NanoDrop ND-1000. Sample preparation and microarray hybridization were performed as outlined in the standard protocols stipulated by Arraystar, as described previously . Normali1 and circinteractome2. Overlapping candidates were considered as putative miRNA targets. Two algorithms (TargetScan and miRpathDB) were used to predict the potential miRNAs targeting the 3’-UTR of PAQR4. Data from The Cancer Genome Atlas (TCGA) was analyzed by Starbase 2.03.The circRNA-miRNA interactome was drawn by circBank2. Small interfering RNAs (siRNAs) of circNOLC1 and NF-kappaB, and miR-647 inhibitor or mimics were purchased from RiboBio Company . Lentivirus vectors  were utilized to establish cell lines stably overexpressing circNOLC1, and the transfected cells were selected in puromycin (2 μg/ml) for 1 week. All the target sequences were shown in DU145, PC3, C4-2, LNCaP, 22RV1 (PCa cell lines), RWPE1 , and HEK-293T cells were obtained from Cell Bank of Chinese Academy of Sciences, grown with RPMI-1640 medium  supplemented with 10% FBS  and incubated at 37°C in 5% CONuclear and Cytoplasmic Extraction Reagents  were used to separate nuclear and cytoplasm of cultured cells following the manufacturer’s protocol. And RNA extraction was described as followed.Total RNA was obtained from cells with TRIzol , while the cDNA was reverse-transcribed via utilizing PrimeScript RT reagent Kit (TaKaRa) following the manufacturer’s instructions. The genome DNA was isolated by special extraction kit . SYBR Green PCR Master Mix (TaKaRa) and Applied Bio-systems 7500 Fast Real-Time RCR System  were used for RT-qPCR analysis. Data were acquired from three independent experiments and normalized to GAPDH. All the primers were shown in To compare the stability of linear RNA and circRNA, Actinomycin D  was added into medium to block RNA transcription and the solvent dimethyl sulfoxide  was applied as a negative control. Cells were treated with actinomycin D or DMSO in a final concentration of 1 μg/mL for 0, 4, 8, 12, and 24 h, and then the RNA was extracted for RT-qPCR detection, using 18S as an internal reference. For RNase R treatment, 2 mg total RNA was incubated for 15 min at 37°C with or without 3 U/mg RNase R , and followed by RT-qPCR analysis.in situ Hybridization Kit , according to the official guidelines. DAPI was utilized to stain the nuclei. Subsequently, circNOLC1 and miR-647 were observed through a confocal microscope .CircNOLC1 and miR-647 were captured by Cy3-labeled probes  and Alexa 488-labeled probes . FISH experiment was conducted using Fluorescent Cell proliferation was measured by CCK-8 kit . 2,000 PCa cells were planted into 96-well plates. Then medium with 10%CCK-8 was added into each well, and incubated for 2 h. Subsequently, the absorbance values at 450 nm were detected by a microplate reader .PCa cells were seeded into 6-well plates and cultured with complete medium for 2 weeks. The formed cells were fixed with 4% paraformaldehyde for 10 min and stained with Giemsa  for 5 min. Each experiment was performed with three replicates.4) were resuspended in 300 μl serum-free medium and seeded into the upper chamber of the insert. Subsequently, the lower chamber was filled with 500 μl complete medium. After 24 h, the cells were fixed with 4% paraformaldehyde for 10 min and stained with Giemsa . Five randomly selected fields were photographed using an invert microscope at 200× magnification. The cell numbers of each image were counted by Image J software. Experiments were performed in triplicate.Cell migration assay was performed using transwell inserts  in 24-well plates. PCa cells , following the manufacturer’s instructions. Equal amounts of proteins were separated in 10% SDS-PAGE gels and transferred to PVDF membranes . The membranes were then blocked for 1 h with 5% no fat milk and incubated overnight at 4°C with the following primary antibodies: EMT marker , anti-PAQR4 , anti-β-actin . Subsequently, the membranes were immersed in the anti-rabbit or anti-mouse secondary antibodies  for 1.5 h at room temperature. Enhanced chemiluminescence (ECL) kit  was used to detect and visualize the protein.TM Magnetic RNA-Protein Pull-Down Kit , all procedures were followed as manufacturer’s instructions. Then the final RNA was extracted by TRIzol  and analyzed by RT-qPCR.CircRNA pulldown assay was carried out using Pierce5) were seeded into each well of a 12-well plate for 24 h. Then, a mixture of luciferase reporter vectors and miRNA mimics was transfected into cells. The relative luciferase activity was measured utilizing Luc-PairTM Duo-Luciferase HS Assay Kit . Each group was confirmed in triplicate.The sequences of circNOLC1 and its mutant types without miR-647 binding sites were designed and packaged into pEZX-MT06 vector , termed as circNOLC1-WT and circNOLC1-MUT. The miRNA mimics were purchased from RiboBio Company . HEK-293T cells (5 × 106 du145 cells stably transfected with circNOCL1 or Vector were subcutaneously injected into the mice axillae (n = 6 per group). Tumor volume was measured every 4–5 days and volumes were calculated using the formula: length × width2 × 0.5.All experimental animal procedures were authorized by the Animal Care and Use Committee of Southern Medical University and Specific Pathogen Free (SPF) conditions were used to raise animals. BALB/c nude mice were obtained from the Animal Center of Southern Medical University, Guangzhou, China. A total of 2 × 10Detection of Ki-67 and CD31 was performed on 3 μm thick paraffin sections with the indicated antibodies. Briefly, sections were incubated with primary antibodies Ki-67  or CD31  antibodies at 4°C overnight. Subsequently, Horseradish peroxidase (HRP)-conjugated secondary antibodies  were further incubated at 37°C for 30 min. Finally, the sections were stained with 3,3’-diaminobenzidine  for 5 min and examined with a microscope.® Plus Enzymatic Chromatin IP Kit , according to the manufacturer’s protocols. Anti-NF-kappaB antibody for CHIP was obtained from Cell Signaling Technology (#8242). The detailed binding sites between the promoter sites of NOLC1 and NF-kappaB were predicted by Consite4, as: GGGAAGTCCC. The specific primers for binding sites were shown in CHIP assay was performed using SimpleChIPSD. Two-tailed Student’s t-test or ANOVA was conducted to analyze the significance between variables. The relation between circNOLC1 expression and clinicopathological properties was analyzed using a χ2 test. P < 0.05 was considered as statistically significant.All statistical analysis was performed using GraphPad Prism 8 . The data were presented as mean ± To investigate the differential expressed circRNAs in PCa, we performed a microarray of the PCa cell lines, DU145, PC3, LNCaP, C4-2, and 22RV1 cells . We founCircNOLC1 is generated by head-to-tail splicing of NOLC1 exon 2–5 and contains 487 nucleotides . Actinomin vitro.To discover the function of circNOL1 in PCa, we randomly chose DU145 and C4-2 cell lines for silencing, PC3 and C4-2 for overexpression. We constructed three siRNAs targeting circNOLC1 to silence its expression. Finally, si-circ#02 exhibited the highest silencing efficiency measured by qRT-PCR and was chosen for the following experiments , left. MPlenty of reports have revealed that circRNAs mainly function as miRNA sponges in various cancers . To figuDue to previous reports, miR-647 could retard tumor progression via inhibiting downstream oncogenes . We subsConsite, and found that NF-kappaB scores ranked the most . We discovered 110 upregulated circRNAs both in five PCa cells, and 22 were overlapped overexpressing in PCa tissues. Among them, top five upregulated circRNAs were chosen for further validation, and circNOLC1 was significantly elevated in PCa cells and tissues. Meanwhile, circNOLC1 exhibited stronger stability than its linear mRNA. Through Nucleolar and coiled-body phosphoprotein 1 (NOLC1), the host gene of circNOLC1, was firstly discovered as a nuclear localization signal binding protein, which functions as a chaperone that shuttles between the nucleolus and cytoplasm , 1992. Pvia knockdown and overexpression of miR-647, and PAQR4 was confirmed to be the most likely downstream target gene. Further immunoblot validated that PAQR4 was negatively regulated by miR-647. These findings supported that circNOLC1 sponges with miR-647 promoted PCa progression via upregulating PAQR4 expression.Previous studies indicated that circRNAs exert their functions by various biological processes, such as miRNA sponges, protein-binding, and transcriptional and translational regulation . The ceRvia a miR-647/PAQR4 axis.Progestin and adipoQ receptor family member 4 (PAQR4) was reported to be high expression in PCa cells and tissues, and significantly improve PCa malignant phenotype by activating PI3K/Akt pathway . BesidesThe mechanism of circRNAs biogenesis is a remarkably complicated process . PreviouIn summary, we discovered a novel circRNA (circNOLC1) remarkably upregulated in PCa cells and tissues. Our results indicated that circNOLC1 participate in the malignant progression in PCa, mainly by sponging miR-647 to upregulate PAQR4 expression, thus activating the PI3K/Akt pathway. Also, NOLC1 and circNOLC1 can be regulated by NF-kappaB via binding to NOLC1 promoter sites. Our studies suggested that circNOLC1/PAQR4 axis could serve as a novel biomarker and therapeutic target for PCa .The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the ethics committee of Zhujiang Hospital, Southern Medical University. The patients/participants provided their written informed consent to participate in this study. The animal study was reviewed and approved by the ethics committee of Zhujiang Hospital, Southern Medical University. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.XM, DL, and CL conceived and designed the study. WC and SC carried out the experiments. TY, KW, and LZ collected and analyzed the clinical data. JL and XZ analyzed and interpreted the data. WC and DL wrote the manuscript. XM and DL executed the funding acquisition. All authors have reviewed and approved the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Streptomysis felleus (S. felleus) strain. This isolate was submitted to gene bank NCBI with accession number MH553077. In addition, physiological studies such as utilization of carbon, nitrogen, amino acid sources of potential isolated were studied. Further, optimization, purification and characterization of the novel compound producing strain may be helpful for discovering the new therapeutic microbial agent.In the current study, twenty-eight soil samples were collected from coalmine sites of Telangana, India. The isolates were purified and identified based on their culture characterization on oatmeal agar, glycerol asparagine agar, yeast extract-malt extract agar, inorganic salt starch agar, and starch casein agar medium. Further, the supernatant of all the isolates were tested for antimicrobial and antifungal activities. The biochemical and microscopic studies of isolated strains results indicates the potential isolate strains belongs to Streptomyces genus. Among all the strains the biological activity of BHPL-KSKU5 showed higher anti-bacterial and anti-funagal activity. The molecular characterization of BHPL-KSKU5 16s rDNA gene sequence and phylogenetic tree showed that is mostly related to the  A broad spectrum of antibiotic products like macrolides, β-lactams, polyenes, aminoglycosides, tetracyclines, peptides, and polyethers are produced by most of the Streptomyces sp  19°02'44.3''N 79°30'46.2''E, Bhupalpally (BHPL) 18°27'05.0''N 79°51''14.0''E, Godavarikhani (GDK) 18°42'45.0''N 79°31'48.4''E, Kothagudem (KGDM) 17°28'51.3''N 80°40'19.4''E, Sathupally (SPL) 17°12'18.2''N 80°47'39.6''E, Madhamari-Kalyankhani (MM-KK) 19°00'20.7''N 79°28'08.1''E, and Ravindhrakhani (RK) 18°53'17.9''N 79°30'04.9''E,Telangana, India, during 2019 using an open-end soil borer (10 cm in depth and 25 cm in diameter), then air-dried for 24 hr . The soi2.2Samples were allowed to 10 fold serial dilution with one gram of soil dissolved in sterile water and were inoculated on starch casein agar. Streptomycin (30 µg/L) and amphotericin B (50 µg/L) were added to the medium to retard bacteria and fungi growth, respectively. Later all the plates were incubated at 28 ± 2 °C for 4 to 10 days. The suspected colonies structures isolates with brown and white colonies were purified. The pure cultures were streaked on to respective medium plates.2.3The pure isolates were prepared for actinomycetes plates and placed 3 to 4 sterile coverslips. The plates were incubated at 28 ± 2 °C for 4 to 7 days. The coverslips were removed at 3 days of interval and observed under the high power magnification. The arrangement of conidiospores on aerial and substrate mycelia was observed and compared with the Bergeys Manual of Determinative Bacteriology.Cultural characteristics were conducted by growing the organism on oatmeal agar, glycerol asparagine agar, yeast extract-malt extract agar, inorganic salt starch agar, and starch casein agar media after 14 days of culturing at 28 °C. Morphological characteristics of aerial hyphae, spore chain, spore mass, spore surface, the colour of aerial and substrate mycelia, and diffusible pigments production were conducted by growing the organism on ISP-4 medium for 7-days and accessed via light microscope.The culture plates containing actinomycetes were prepared, and coverslips were placed at an angle of 45 °C and allowed to incubate at 28 °C for 7 days, later coverslips were carefully removed and examined under a microscope. The aerial and substrate of conidiospores' mycelial structure have been attributed to the Bergeys Manual of Determinative Bacteriology , Baciilus cereus (B. cereus), Streptococcus pneumonia (S. pneumonia), Bacillus subtilis (B. subtilis) and Micrococcus luteus (M. luteus) and five gram-negative bacteria Enterobacter aerogenes (E. aerogenes), Klebseilla pneumonia (K. pneumonia), Escherichia coli (E. coli), Salmonella paratyphi (S. paratyphi) Pseudomonas aeruginosa (P. aeruginosa) and Candida albicans  were screened against antimicrobial compound produced by actinomycetes isolates. The tested bacteria were obtained from the department of Botany and Microbiology, King Saud University, Riyadh, Saudi Arabia and Microbiology laboratory, Kakatiya Medical College, Warangal, India.Five different gram-positive bacteria 2.8B. subtilis, B. cereus, S. aureus, S. pneumoniae, M. luteus, E. coli, E.aerogenes, K. pneumoniae, S. paratyphi and P.aeruginosa. Muller Hinton agar plates were prepared and lawn culture of bacteria was made. The nutrient broth was cultivated on bacterial sample organisms for 24hr. For the preparation of the bacterial lawn, a 100 mg cultivation of each bacterial species was used (1 × 10-5 CFU/mL). Agar wells of 6 mm diameter were prepared with the help of a sterile cork borer. The wells were loaded with crude extract 900 µg/mL, negative control (DMSO), along with 30 µg/mL of streptomycin as a positive control. The plates were incubated 24 hr at 37 °C and the zone of inhibition was calculated  was tested by employing agar well diffusion assay, the crude obtained from all the isolates was carried on against the test pathogens namely lculated .C. albican testing fungus was conducted in a saline solution (0.85%), NaCl medium, and customized to 0.5 Mc Farland and (10-8 CFU/mL) turbidity. A sterilized spreader was used to spread 1 mL of the sample culture into PDA plates. Agar wells of 6 mm diameter were prepared. The wells were loaded with crude extracr (900 µg/mL), negative and positive control after the plates were incubated for 48 hr. The inhibition zone diameter was measured in millimetre. Based on the size of the zone of inhibition, the potent antibacterial compound contains isolate were selected.Approximately 50 mL of potato dextrose agar was poured onto the sterilized petri plate utilizing the agar well diffusion assay to assay the anti-candidal activity . The C. 2.9The data were evaluated using (SPSS V.10.0) and Excel. All the data expressed as mean ± standard deviation (SD) for each test for triplicate.3A total 28 soil samples were obtained from various coalmine soil samples of Telangana, India. From these actinomycetes colonies  were purified by sub-cultured and stored at 4 °C and used for further studies. The isolates aerial spore mass colour was categorized into 3 groups grey, ash, and white series . Most of3.1The fermented broth containing antimicrobial compounds of selected potential Streptomyces was extracted by centrifugation. All the crude extracts were used for antimicrobial activity.3.2-KSKU5 isolate showed a broad range of antimicrobial activity, which showed higher zone of inhibition against S. paratyphi and C. albicans (18 ± 0.4 mm and 16. ± 0.4 mm) hence, the BHPL-KSKU5 was selected for further investigations.All the isolates exhibited antimicrobial and anti fungal activity  against 3.3-KSKU5 with broad-spectrum antimicrobial activities was selected for further characterization based on morphological, physiological, and molecular identification by 16S rDNA molecular analysis.The actinomycetes isolate BHPL-KSKU5 formed a flexuous spore chain on aerial mycelium  was performed by PCR amplification. The 16S rDNA gene sequence was used for BLAST analysis. Based on the maximum similarity score, the first 10 sequences were used and aligned with multiple alignment ClusalW software. Using the MEGA 7 the distance matrix phylogenetic tree was developed. It revealed that BHPL-KSKU5 found 98% similarity with the existing species of S. felleus based on nucleotide homology and phylogenetic analysis (c). The sequence was submitted to Genebank and obtained accession number MH553077.The molecular characterization a, b was analysis c. The se4Actinomycins produce the different types of bioactive compounds, which contain antibiotic, anticancer, and antimicrobial activities . ActinomActinomycetes have a significant property in digestion and production of certain components such as proteins in the form of keratin and certain vitamins . A largeS. felleus (BHPL-KSKU5) was found to develop a flexuous spore chain with a smooth surface, this is the main characteristic feature of Streptomyces, these study was supported by In the current study, actinomycetes isolate namely S. felleus (BHPL-KSKU5) showed positive results in carbon utilization to dextrose, L-arabinose, maltose, starch, D-arabinose, fructose, mannose, and lactose but it unable to utilize mannitol, sucrose, xylose, and D-galactose. The utilization of different nitrogen sources in strain BHPL-KSKU5 showed positive results to sodium nitrate and asparagine, whereas it did not utilize ammonium sulphate, ammonium nitrate but utilized potassium nitrate, and calcium nitrate. The utilization of amino acids was also tested. Among the tested amino acids, namely DL-2 amino-N-butric acid, L-tyrosine, DL-tryptophan, DL-ornithine, L-lysine, DL-leucine, L-arginine, DL-alanine and L-leucine were utilized. Whereas it did not utilize L-glutamic acid, L-cystine, and DL-aspartic acid, these results could be utilized as a taxonomic criterion at genus level identification.For the characterization of actinomycetes isolates metabolites, various physiological tests were carried out . In the -KSKU5) was studied, and it was found that Streptomyces sp, which is found 98% similarity with the existing species of Streptomyces felleus.In identifying actinomycetes 16S rDNA gene sequence played a vital role which is evident by many workers . In currS. felleus (BHPL-KSKU5) could produce antimicrobial compounds in starch casein broth. Antimicrobial compounds normally produced from plant ingrediants, but nowadays which also produced from soil microbes such as Actinomycetes; it was great intrest due to which acting as a potent alternative antibacterial agents . The preS. felleus was done. Hence, coalmine soil is the best suitable source for the isolation of diverse actinomycetes.Further, studies need to be done to purify, charecterize the antimicrobial compound and its optimization for large-scale production. Therefore, the present study suggests that the diversity of antimicrobial producing actinomycetes in coalmine soils of Telangana were predominantly more. The phenotypic, genotypic characterization of the antimicrobial compound from 5S. felleus, which contain more antibacterial and antifungal activity. Further, this study may be helpful to develop potential antibacterial and antifungal agents against different pathogens.Actinomycetes are the highest microorganisms with the potential to produce novel antibiotics as industrially important secondary metabolites. Actinomycetes could be found in different environments such as soil, husk, and other than the source and our preferred site of coalmine soils are more tolerant of their growing and novel antibiotics and significant secondary metabolites. In present study totally 28 coalmine soil samples were collected from different districts in Telangana. The novel strain BHPL-KSKU5 was identified and characterized as The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


SARS-CoV-2 (the causative agent of COVID-19) is a major public health threat and one of two related coronaviruses that have caused epidemics in modern history. A method of screening potential infectible hosts for preemergent and future emergent coronaviruses would aid in mounting rapid response and intervention strategies during future emergence events. in vitro validation, we identified 5 key amino acid differences between murine and human ACE2 that mediate SARS-CoV-2 infection, generating a chimeric humanized murine ACE2. Additionally, we examined the ability of the humanized murine ACE2 receptor to permit infection by an additional preemergent group 2B coronavirus, WIV-1, providing evidence for the potential pan-virus capabilities of this chimeric receptor. Finally, we predicted the ability of these determinants to inform host range identification of preemergent coronaviruses by evaluating hot spot contacts between SARS-CoV-2 and additional potential host receptors. Our results identify residue determinants that mediate coronavirus receptor usage and host range for application in SARS-CoV-2 and emerging coronavirus animal model development.The angiotensin-converting enzyme 2 (ACE2) receptor is a major severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) host range determinant, and understanding SARS-CoV-2-ACE2 interactions will provide important insights into COVID-19 pathogenesis and animal model development. SARS-CoV-2 cannot infect mice due to incompatibility between its receptor binding domain and the murine ACE2 receptor. Through molecular modeling and empirical  Since its emergence in December 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected tens of millions, leading to over 2 million deaths worldwide , and othIn addition to providing new insights into coronavirus receptor interactions and host range, characterizing determinants of ACE2 species restriction also has implications for SARS-CoV-2 animal model development. Mouse models have been essential for understanding the pathogenesis of coronaviruses and have been key resources for the preclinical development of vaccines and antiviral therapies –7. Howev–9–14–Based on previous SARS-CoV research and published structures , 18, we In order to evaluate the impact of mACE2 humanization at each of these interaction hot spots, we first modeled the interaction between SARS-CoV-2 RBD and mACE2 . From thTo experimentally test these models, a panel of hmACE2 receptors was generated to directly evaluate whether these predicted contact residues are essential for SARS-CoV-2 infection. The receptors were introduced into mouse delayed brain tumor astrocytoma (DBT-9) cells , 20, norGiven the importance of the three ACE2 interaction hot spots for SARS-CoV-2 infection, we asked whether these same determinants were important for infection by other preepidemic group 2B coronaviruses. We modeled interactions between hmACE2.3 and two related group 2B CoVs, preepidemic SARS-like bat CoV WIV-1  and pang10.1128/mBio.03149-20.3FIG S3FIG S3, TIF file, 1.6 MB.Structure-based multiple sequence alignment of the RBD from SARS-CoV, SARS-CoV-2, WIV-1, and P5L. Download Copyright © 2021 Adams et al.2021Adams et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the in vitro  as well as those from species reported to support little to no infection    25, 27), 27in vichicken) , providi, ferrets (Mustela putorius), mink (Mustela lutreola), domestic cats (Felis catus), pigs (Sus scrofa), guinea pigs (Cavia porcellus), dogs (Canis lupus), mice (Mus musculus), and chickens . (b to h) Predicted hotspot contacts between SARS-CoV-2 RBD and the ACE2 molecule from humans (b), macaques (c), ferrets (d), mink (e), cats (f), pig (g), guinea pig (h), dog (i), mouse (j), and chicken (k). (l and m) Predicted hotspot contacts between SARS-CoV-2 variant Y453F and mink (l) and human (m) ACE2. (n) Predicted hotspot contacts between SARS-CoV-2 variant N501Y and human ACE2. Download FIG S1, TIF file, 1.2 MB.ACE2 hotspot contacts are predictive of SARS-CoV-2 host range. (a) Aligned hotspot residues of the ACE2 molecule from humans, macaques , pig (b), guinea pig (c), dog (d), chicken (e), and mouse (f). Download Copyright © 2021 Adams et al.2021Adams et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the in vivo by gene editing  and SARS-CoV RBD hACE2 complex (PDB ID 2AJF). WIV-1 RBD was modeled using the reference sequence, GenPept accession no. AGZ48828.1, and P5L RBD was modeled using GenBank accession no. MT040335.1. Additional receptor interactions were modeled from the following translated reference sequences: chicken, GenBank accession no. XM_416822.5; guinea pig, GenBank accession no. XM_023562040.1; ferret, GenBank accession no. XM_004758886.2; pig, GenBank accession no. EU518378.1; dog, GenBank accession no. NM_001165260.1; macaque, GenBank accession no. NM_001135696.1; and mink, GenBank accession no. MT560518.1. Contacts were identified and visualized in the PyMOL molecular graphics system (Schrödinger LLC). Structure-based multiple sequence alignment was generated with UCSF Chimera.The ACE2 panel was aligned using Geneious Prime (version 2020.0.5). The hACE2 sequence is found under GenBank accession no. MT461669), icSARS-CoV-2-nLuc (GenBank accession no. MT461671) (KF367457.1). Mouse delayed brain tumor astrocytoma (DBT-9)  , and icW (DBT-9) , 20 cellEach mouse, human, and chimeric ACE2 was expressed in pCDNA3.1 vector that conjugates a C-terminal 6×-His tag to the end of the expressed protein. The hmACE2 panel was generated with a combination of site-directed mutagenesis and custom double-stranded gBlocks gene fragments (IDT). DBT-9 cells were transfected using Lipofectamine 2000 transfection reagent (Invitrogen). Expression of each receptor was verified via staining against a 6×-His epitope  and Western blotting.CT) method and fold change over viral RNA in empty vector-transfected cells. SARS-CoV-2 genome equivalents were calculated against a standard curve with a diagnostic genomic assay primer set against Nsp4  of 1 for 1 h. Inoculum was removed, cells were washed with Dulbecco's phosphate-buffered saline (DPBS), and medium was replaced. Receptor usage was analyzed 24 h postinfection. RNA was collected via TRIzol (Invitrogen) and extracted using Direct-zol RNA MiniPrep kit (Zymo Research). Viral RNA was quantified in duplicate via reverse transcriptase quantitative PCR (qRT-PCR) using TaqMan Fast Virus 1-Step Master Mix (Thermo Fisher Scientific) on a QuantStudio 3 (Applied Biosystems). Viral RNA was quantified with a subgenomic diagnostic assay primer set . Host 18nst Nsp4 . ReceptoAll relevant data sets generated or analyzed in this study have been included in the article. Additional information, including replicates, is available upon request.


Friendship plays a crucial role in maintaining social connectedness in late life. Volunteering helps older adults to stay socially engaged and often times provides the opportunity to meet and make new friends. A small literature suggests that volunteering may be associated with friendship, but many studies are limited by reliance on small, non-probability samples and simplistic analytic approaches. The literature is also unclear on how volunteering behaviors relate to specific characteristics of friendships and whether there are gender differences that condition these relationships. Using the 2014 and 2018 waves of the Health and Retirement Study , we investigate whether volunteer status and hours volunteered in 2014 are associated with friendship characteristics in 2018  among community-dwelling adults aged 50 years and above . We also examine whether gender moderated these relationships. Volunteer status and hours in 2014 were positively associated with the number of close friends and contact frequency in 2018. Only those who volunteered 200 hours or more in 2014 were positively associated with friendship quality in 2018. Regarding gender differences, men who volunteered 200 hours or more in 2014 had higher friendship quality in 2018 than women, while women who volunteered 100-199 hours in 2014 had greater contact frequency in 2018 than men. Hence, our results suggest volunteering is integral in shaping late-life friendships and volunteering might be more critical for understanding friendship characteristics among older men and women.


V‐raf murine sarcoma viral oncogene homologue B1 (BRAF) is a proto‐oncogene that regulates cell proliferation and survival. BRAF V600E‐mutated lung cancer has aggressive characteristics and is resistant to chemotherapies. Combination of BRAF‐specific inhibitor dabrafenib and mitogen‐activated protein kinase kinase (MEK) inhibitor trametinib is the standard treatment for BRAF V600E‐mutated lung cancer. We report a case of BRAF V600E‐mutated lung adenocarcinoma, which presented with respiratory distress due to deterioration of advanced cancer. The tumour responded rapidly and significantly to BRAF/MEK inhibitors, and the patient's symptoms improved within 2 weeks. BRAF/MEK inhibitors are effective treatment in BRAF‐mutated lung cancer even under critical conditions. Combination of BRAF ‐specific inhibitor dabrafenib and MEK inhibitor trametinib is the standard treatment for BRAF V600E‐mutated lung cancer. We report a case of BRAF V600E‐mutated lung adenocarcinoma, which presented with respiratory distress due to deterioration of advanced cancer. The tumour responded rapidly and significantly to BRAF/MEK inhibitors, and the patient's symptoms improved within 2 weeks. V‐raf murine sarcoma viral oncogene homologue B1 (BRAF) is a proto‐oncogene that encodes a serine/threonine kinase, which is a component of the mitogen‐activated protein (MAP) kinase pathway that regulates cell proliferation and survival. BRAF mutations are common in a wide variety of cancers. The incidence of BRAF mutations in non‐small cell lung cancer (NSCLC) is 1%–3%, including in Japan.In February 2020, a 70‐year‐old woman with no smoking history underwent a chest x‐ray at a yearly medical check‐up, which showed no abnormalities. In September 2020, she noticed a low degree of fever, fatigue and cough. In October 2020, she was diagnosed with advanced lung adenocarcinoma. The patient was referred to the Department of Medical Oncology for treatment. Due to the worsening of her general condition, she was not eligible for conventional chemotherapy. While waiting for the results of molecular testing and programmed death‐ligand 1 (PD‐L1) expression analysis of biopsy tissue, in November 2020, the patient presented at our emergency department with respiratory distress. Her oxygen saturation was 88% with supplemental oxygen at 5 L/min via a cannula. A simple chest x‐ray showed bilateral hazy opacities, and a computed tomography (CT) scan showed multiple lung metastases with severe lymphangitis carcinomatosa and multiple liver metastases , has been approved which examines BRAF V600E mutation, in addition to the most common EGFR mutations, and ALK and ROS1 rearrangements.The BRAF V600E mutation leads to the constitutive activation of MAP kinase, which increases its oncogenic properties. The BRAF V600E mutation in NSCLC leads to adenocarcinoma with micropapillary features, indicating the aggressive behaviour of the tumours. The BRAF V600E mutation is associated with poor prognosis and lower response rates to platinum‐based chemotherapies in NSCLC.Targeted therapies against the BRAF V600E mutant protein are designed to inactivate the catalytic activities of BRAF and have been approved for patients with BRAF‐mutated NSCLC. In a phase 2 clinical trial, combination therapy with dabrafenib and trametinib in V600E‐mutated NSCLC showed efficacy.In summary, we describe a case of BRAF‐mutated lung adenocarcinoma, which recovered quickly with BRAF/MEK inhibitors. The case illustrates it is worth attempting BRAF/MEK inhibitors in BRAF‐mutated lung adenocarcinoma even under critical conditions.None declared.Takayo Ota drafted the original manuscript. All authors were involved in patient management, and read, critically reviewed and approved the manuscript.Appropriate written informed consent was obtained for publication of this case report and accompanying images.


Science in 2005, where it was asked ‘Where and why does liquid end and glass begin?’. This question continues to attract considerable interest of scientists.The glass transition is the most striking dynamic feature of glass-forming liquids and rigid glass, manifest in the following two scenarios . First, 5 K/s) glasses including metallic [gT peak in HQ glass that has undergone a certain duration of annealing below gT. The reheating and the subsequent cooling are conducted at the standard rate of 20 K/min. The glass cooled at this rate is usually defined as the ‘standard’ glass. The cooling rate for a HQ glass is more than six orders of magnitude higher than the standard rate. The magnitude of the SGT peak is quantified by the difference in isobaric heat capacity (pC) between the annealed HQ glass and the standard glass in the temperature range of the SGT peak, and it is expressed as ΔpC@Tg,shadow. To distinguish from the real reversible glass transition peak, the endothermic pre-peak was expressed as the shadow glass transition (SGT) by Yue and Angell [As a crucial contribution to understanding of the calorimetric glass transition, several groups of scientists independently observed a striking pre-endothermic event, that is an endothermic pre-peak before the real glass transition starts. This phenomenon occurs in annealed hyperquenched (HQ)  using the chip-based fast scanning calorimetry (FSC) without performing annealing [40Ni40-xCuxP20 glass, a crystallization peak appears subsequent to the glass transition. As illustrated in Fig. pC@Tg,shadow and ΔpC@Tg, respectively. Figure gT. This implies a bridge between the dynamics and the thermodynamics of β relaxation. Interestingly, Yang et al. also discovered that the SGT temperature  first decreases with quenching rate, and then remains unchanged when the quenching rate exceeds 106 K/s. This indicates that a critical g,shadowT value exists, below which the glass structural domains with the lowest potential energy cannot be rejuvenated to the average energy level of the standard glass.As a significant step towards understanding of SGT, Yang nnealing . In doinet al. provides a great advancement in revealing the nature of the shadow glass transition. Their findings have implications for the connection between the shadow and real glass transitions, as well as for correlation of the shadow glass transition with both β relaxation and physical properties of hyperquenched glasses [In summary, the work by Yang  glasses . Their wConflict of interest statement. None declared.


We sought to evaluate androgen receptor (AR) and PI3K pathway activity in ovarian cancer cell lines and tissue and determine if either pathway was correlated with growth of ovarian cancers. AR expression and activity were quantified using immunohistochemistry (IHC) and RT-qPCR in six ovarian cancer cell lines and 51 tissue samples. Phospho-mTOR and AKT expression were quantified by IHC as well. Cell growth was assessed in the presence of AR modulating drugs and metformin. We found that despite robust AR expression and activity, no cell line was dependent on androgen for growth. However, metformin inhibited activity in five of the six cell lines. Patient tissues had large variation in AR expression and activity, as well as in expression of phospho-mTOR and AKT, but none of these variables correlated with progression-free survival (PFS). AR expression and activity did not predict the dependence of ovarian cancer cell lines on androgens for growth, and AR expression and activity did not correlate with PFS. This result suggests that AR expression as a criterion for patient selection for clinical trials evaluating molecules targeting AR may not predict response for ovarian cancer patients. NCT01974765) evaluating single agent enzalutamide, an AR antagonist, in AR-positive ovarian cancers (defined as ≥5 % positivity by IHC), regardless of histological subtype.Epithelial ovarian cancer (EOC) is the most lethal gynecologic malignancy with 22,530 new cases and 13,980 deaths expected in 2019 . These aSeveral observational studies have reported improved survival in cancer patients taking metformin in several malignancies including ovarian cancer . The antIn this study, we quantified the expression and activity of AR and PI3K/AKT pathways in ovarian cancer cell lines and tumor tissue samples and examined the sensitivity of the cell lines to enzalutamide and metformin. Our findings have important implications for ongoing trials with these agents.Cells were provided by collaborators but were originally obtained from the ATCC. SKOV3 cells were maintained in McCoy's 5a Medium Modified with 10% FBS. A2780 cells were maintained in RPMI-1640 with 10% FBS. OV-90 cells were maintained in a 1:1 mixture of MCDB 105 medium containing a final concentration of 1.5 g/L sodium bicarbonate and Medium 199 containing a final concentration of 2.2 g/L sodium bicarbonate, with 15% FBS. OVCAR3 cells were maintained in RPMI-1640 supplemented with 20% FBS and 0.01mg/ml insulin. OVCAR8 cells were maintained in DMEM with 10% FBS. COV362.4 cells were maintained in DMEM with L-glutamine (300mg/L) and 10% heat inactivated fetal bovine serum. For some studies, cells were transferred to media containing charcoal dextran treated (C/S) FBS. Luciferase assays: Cells were transfected using Lipofectamine LTX & Plus (Thermofisher) with PSA-luciferase  and pRL-AR and a 20-gene panel that has been previously used to define an “AR activity score;” [FKBP5, MED28, ELL2, KLK2, PMEPA1, CENPN, C1ORF116, NKX3.1, KLK3, EAF2, TMPRSS2, HERC3) were robustly expressed and androgen-induced in the six ovarian cancer cell lines. The transcript levels, relative to housekeeping genes, were compared to those from LNCaP cells, a prototypical prostate cancer cell line with robust AR activity, and the mean percent of LNCaP transcript levels was reported as the AR activity score.RNA was isolated from cultured cells or from cancerous and benign tissue sections, as marked by the study pathologist, of an unbaked FFPE slide using the FFPR RNA easy kit (Qiagen), as we have done previously . RNA was score;”  this 20  score;” ,16. We fFor growth curves, cells were transferred to C/S medium three days before they were divided and plated at a density of approximately 20,000 cells/well in 48 well plates, in quadruplicate. The following day, vehicle or drugs were added to the cells. Proliferation was determined by measuring the DNA content of the cells in each well. Cells were fixed in 2% PFA, followed by staining for 5min at RT with 0.2ng/mL 4',6-diamidino-2-phenylindole (DAPI) in PBS. The cells were washed with PBS, then read on a fluorescence plate reader (FPR) using 365/439 excitation/emission wavelengths. A Student’s t test was used to determine significant differences among populations.Cell lysates were resolved via SDS-PAGE and transferred to nitrocellulose membranes. Membranes were blocked in 5% milk and probed overnight with antibodies including AR , phospho-Ser473 AKT (GeneTex), phospho-Ser2448 mTOR (GeneTex), or controls: p84, actin, or GAPDH (GeneTex).Patient slides and clinical data were collected under City of Hope IRB16430. IHC was performed using the same antibodies as used for Western blotting. The “IHC score” was determined by taking the average percentage of positively staining cells x the staining intensity (0-3) across eight 40x-fields within the marked cancerous area. Example images were obtained using an Aperio Digital Pathology Slide Scanner.Data were analyzed using a Student’s t test. All analyses were performed at a significance level of *P<0.05. Experimental data are presented as the mean plus or minus standard error.The AR is widely expressed in the normal ovarian epithelium  and alsoAs metformin has shown activity in ovarian cancer patients, growth in the panel of ovarian cancer cell lines was examined in the presence of this drug. All cell lines examined, except OV90, were extremely sensitive to metformin . The OV9There is a well-documented inverse relationship between AR and PI3K activity in prostate cancer; when AR is inhibited, it increases PI3K activity and vice-versa . To inveWe performed IHC for AR in 51 primary ovarian cancer tissue samples representing 48 patients (some with mixed histologies), including low and high grade serous, endometroid, clear cell, and mucinous subtypes, the majority of which were high grade serous . Using aWe also performed IHC for phospho-mTOR and phospho-AKT, another key downstream marker of PI3K activation, on slides of high-grade serous ovarian cancer tissue and scored them using the same system as for AR IHC. Again, we found a wide range of expression among the high-grade serous samples for both phospho-mTOR  and phospho-AKT . To determine if an inverse relationship existed between the PI3K and AR pathways in the high-grade serous cancer tissues, we looked for correlations between AR IHC or AR activity scores and IHC scores for the phosphoproteins. There were no significant correlations. We next investigated the relationship between the IHC and qPCR scores and clinical variables in 38 high-grade serous patients. None of our markers correlated with PFS. However, the AR activity score was lower tumors of patients assessed after neoadjuvant therapy (n=7) than in tumors of patients assessed prior to receiving any chemotherapy (n=31)  . This suFurthermore, we found that AR expression was associated with BRCA1 and BRCA2 status. The mean AR expression in patients with peathogenic BRCA mutations (n=6) was higher compared to the mean AR expression in non-BRCA mutation patients (n=29) .The AR is known to be expressed in ovarian cancer cells, but little is known about the impact of AR expression and activity on the natural history of the disease. In this study, AR expression and degrees of increased signaling activity in response to an androgen agonist, DHT, varied among the six ovarian cancer cell lines. A more comprehensive assessment of AR signaling, the AR activity score, also was evaluated and correlated with AR expression. However, hormonal manipulation of the cell lines suggested that AR stimulation alone was not sufficient to promote growth of these cell lines.We also evaluated whether AR expression or AR activity of their tissue correlated with PFS in 38 patients. Neither AR expression nor the AR activity score was correlated with PFS. The patient samples examined were high-grade serous adenocarcinoma histology, which limits the application of our findings to other ovarian cancer histologic subtypes. Unlike the cell lines, AR expression did not correlate strongly with the AR activity score in tissue samples. This may simply reflect differences in the techniques, or it may imply the existence of cancers with incongruous AR expression and activity, analogous to “AR indifferent” prostate cancers that arise more frequently in highly-treated patients .It is possible that the genes used to define the AR activity score are less relevant in ovarian cancer than they are in prostate cancer. One or several of these genes may be more strongly regulated by transcription factors other than AR in ovarian cancer. This may explain why several samples that had little AR expression detected by IHC still had high AR activity scores. Our results from the cell line experiments suggest caution should be used when using AR expression or activity as eligibility criteria for patient selection on clinical trials that are evaluating molecules targeting AR in ovarian cancer. AR expression or activity may not reflect cancer cell dependency on the androgen pathway and therefore may not predict response to AR antagonists or AR modulators.BRCA1 and BRCA2-mutated high-grade serous ovarian tumors compared to non-mutated BRCA tumors. This is consistent with published data suggesting that BRCA1 status correlated with AR expression in TNBC. Decreased expression of AR has been measured in BRCA1-wild type TNBC cells [BRCA1-mutated malignant cells may have up-regulated AR.In breast cancer, AR expression is associated with improved overall survival regardless of subtype of breast cancer or co-expression of ER. In triple negative breast cancer (TNBC), AR expression is seen in 12-55% of cases and is being evaluated as a therapeutic target in this patient population; in a recent phase II trial of TNBC with AR expression, there was a clinical benefit rate of 35% at 16 weeks and a median PFS of 14.7 weeks with enzalutamide . The resBC cells  and suggNCT02122185). The beneficial effects of metformin extend to other tumors as well. In a colorectal cancer patient population, patients with diabetes treated with metformin had superior overall survival [Other evaluations using the ovarian cancer cell lines included assessing their sensitivity to metformin. All cell lines were sensitive to inhibition by metformin, except for OV90. The OV90 cell line has a BRAF mutation that causes increased PI3K activity, effectively bypassing metformin. These results are consistent with a growing body of literature on the anti-tumor effects of metformin. Metformin has been shown to inhibit ovarian cancer growth and increases sensitivity to paclitaxel in mouse models . In 341 survival .Finally, we investigated the interaction between the PI3K/AKT and AR pathways in ovarian cancer cell lines treated with metformin and enzalutamide. Increased activation of the PI3K/AKT pathway conferred cisplatin resistance in an ovarian cancer cell line, implicatWe evaluated 6 ovarian cancer cell lines and 51 tumor samples from patients with ovarian cancer of various histologies, predominantly high-grade serous adenocarcinoma which comprises more than 70% of ovarian cancers. Although the AR activity score increased to varying degrees in all cell lines exposed to metformin, the activity of the PI3K pathway, as measured by phospho-mTOR, was not increased by the addition of enzalutamide, an AR antagonist. Although there appeared to be some interaction between AR and PI3K signaling in the ovarian cancer cell lines, it was not reciprocal as it is in prostate cancer. Assessment of additional markers of AR and PI3K signaling, and use of PI3K inhibitors that are more selective than metformin, would be useful to clarify the relationship between these pathways in ovarian cancer.The limitations of our study include the small sample size and limited representation of other histologic subtypes except for high-grade serous adenocarcinoma in patient samples. While our study did not support robust reciprocal feedback between AR and PI3K/AKT pathway activity in ovarian cancer, the small sample may prevent this finding from being extrapolated and further studies are warranted in different ovarian cancer histologies. Other limitations include some inherent subjectivity in quantifying immunohistochemistry and technical difficulties in performing and interpreting phospho-proteins by IHC in FFPE tissue.In summary, in our evaluation of 6 ovarian cancer cell lines and 51 ovarian cancer patient tissue samples, AR expression and AR activity did not consistently correlate with each other, growth of cancer cells, or PFS. Caution must be exercised when using AR expression as a selection criterion for clinical trial participation as a way to predict response to AR antagonists. Our study corroborates growing literature supporting metformin as an anti-tumor agent in ovarian cancer. A reciprocal relationship between the AR and PI3K pathway in ovarian cancer is not yet confirmed as it is in prostate cancer.