Nociceptors are a subpopulation of dorsal root ganglia (DRG) neurons that detect noxious stimuli and signal pain. Veratridine (VTD) is a voltage-gated sodium channel (VGSC) modifier that is used as an “agonist” in functional screens for VGSC blockers. However, there is very little information on VTD response profiles in DRG neurons and how they relate to neuronal subtypes. Here we characterised VTD-induced calcium responses in cultured mouse DRG neurons. Our data shows that the heterogeneity of VTD responses reflects distinct subpopulations of sensory neurons. About 70% of DRG neurons respond to 30–100 μM VTD. We classified VTD responses into four profiles based upon their response shape. VTD response profiles differed in their frequency of occurrence and correlated with neuronal size. Furthermore, VTD response profiles correlated with responses to the algesic markers capsaicin, AITC and α, β-methylene ATP. Since VTD response profiles integrate the action of several classes of ion channels and exchangers, they could act as functional “reporters” for the constellation of ion channels/exchangers expressed in each sensory neuron. Therefore our findings are relevant to studies and screens using VTD to activate DRG neurons. Dorsal Root Ganglia (DRG) contain a heterogeneous population of sensory neurons that detect noxious and innocuous stimuli. DRG neurons that respond to noxious stimuli are known as nociceptors. Inflammation and nerve injury sensitise sensory neurons and result in hyperalgesia and allodynia112Drug screens for VGSC blockers use functional assays to test their sensitivity. These include FLIPR, calcium imaging and patch clamp electrophysiology; and employ Veratridine (VTD) as a VGSC “agonist” to test the pharmacological properties of candidate VGSC ligands4578VTD elicited heterogeneous calcium responses that differed in their profile and onset in sensory neurons, 7101314We classified VTD responses into four distinct profiles, DRG neurons are classified according to their soma size into small, medium and large diameter neurons161719We next investigated how the four VTD response profiles correlate with a commonly used functional marker of nociceptors. Capsaicin is a TRPV1 agonist used to activate peptidergic nociceptorsAs the SD profile occurred in a wide range of neuronal sizes, we examined the relationship between neuronal size and capsaicin sensitivity in the 111 neurons with the SD profile. Capsaicin-sensitive SD neurons had a smaller mean diameter than capsaicin-insensitive SD neurons, We next investigated how the four VTD response profiles correlate with sensitivity to another commonly used marker for nociceptors. α, β-methylene ATP is a specific agonist for P2×3 receptors that marks the non-peptidergic population of nociceptors23242523The SD subpopulation of neurons sensitive to α, β-methylene ATP had a smaller mean size (20 μm) compared to the subpopulation insensitive to α, β-methylene ATP (23 μm), Both the SD and RD response profiles were enriched in neurons insensitive to the two nociceptive markers; capsaicin and α, β-methylene ATP. This led us to hypothesise that these two response profiles could be functional markers for non-nociceptors, while the OS and ID response profiles could be functional markers for nociceptors. However, The OS and ID profiles did not occur preferentially in neurons sensitive to the nociceptive markers capsaicin  and α, β3031Neurons sensitive to any of the three agonists were three times more likely to respond to VTD  than not , The SD subpopulation of neurons sensitive to any of the three agonists have a smaller mean size (21 μm) compared to the subpopulation insensitive to any of the three agonists (26 μm), We also examined the distribution of sensitivity to the three agonists among neurons with the four VTD profiles, The present study examined VTD-elicited calcium responses in cultured adult mouse DRG neurons. VTD responses were heterogeneous. We categorised VTD responses into four distinct profiles based on the number of peaks and their decay rate. We report that VTD response profiles correlated with soma size and with commonly used pharmacological markers of nociceptor. To our knowledge this is the first detailed characterisation of VTD responses in DRG neurons.What do VTD response profiles represent? Most of the studies that looked at the effect of VTD on voltage-gated ion channels used patch-clamp electrophysiology to examine the effect of VTD on sodium8Our data shows that 30 μM VTD evoked robust responses in approximately 70% of sensory neurons. A higher dose of 100 μM VTD did not significantly increase the percentage of responding cells, Importantly, the inability of VTD to activate stable cells expressing Nav1.8 may explain the about 25–30% of DRG neurons that are unresponsive to up to 100 μM VTD, 36We categorised VTD calcium responses in cultured DRG neurons into four profiles which we named SD, ID, RD and OS. The four profiles were observed in similar relative frequencies from cultures prepared over many months, from different patches of mice and by different researchers. Furthermore, the frequency of occurrence of VTD profiles was not affected by the order of applications of agonists in our experiments, The most abundant response type is the OS (47–51% of VTD-responsive neurons) where VTD elicited oscillatory changes in [Ca]i levels. Interestingly, this is the only response profile VTD elicited in N2a and SH-SY5Y cells, 1238The other three types of VTD response profiles shared a profile characterised by a single peak but differed in their decay rate. The difference in the rate of signal decay could be due to a difference in the disassociation rates of VTD from the different VGSC subtypes. In addition, it has been shown that DRG neurons (particularly large-diameter neurons) produce calcium responses with slow decay rates (SD-like responses) when treated with VGKC blockerset al.A recent paper classified DRG neurons into 11 groups based on their mRNA profileThe data presented here raises several interesting questions. One question is what are the key molecular determinants of each of the four VTD response profiles? Answering this allows the use of VTD profiles as readout for these ion channels and exchangers in high throughput functional screens on primary DRG neurons. Considering that 25–30% of nociceptors do not respond to 30 μM VTD and that these are likely to be “silent” nociceptors, a second question is does the sensitization of DRG neurons by inflammation and nerve injury affect the number of VTD responsive neurons and their response profiles? Answering this question would allow the use of VTD response profiles as readout of drugs’ ability to cause or reverse sensitisation of DRG neurons in high throughput screens. Such screens have the advantage of showing the net effect of a drug on the various classes of ion channels that give rise to VTD response profiles.In conclusion, the present study shows that VTD induces calcium responses with heterogeneous profiles in TTX-S rich sensory neurons. VTD response profiles reflect distinct subpopulations of sensory neurons. These subpopulations overlap but are not identical to the subpopulations identified by classical functional nociceptive markers, 2. DRGs were then triturated with a P1000 pipette tip. The cell suspension was carefully layered on top of 15% Bovine Serum Albumin (Melford) in DMEM/F12 and centrifuged at 800 g, for 10 min at room temperature with the minimum deceleration speed. The cell pellet was then washed in culture medium composed of DMEM/F12 plus 10% Fetal Bovine Serum , 100 units/ml penicillin and 100 μg/ml streptomycin (Gibco). Cells were pelleted again, re-suspended in DRG culture medium and plated on glass coverslips coated with polyornithine . Cells were imaged 24 h after plating.Adult male C57BL/6 mice were sacrificed according to Schedule 1 of the Animal (Scientific procedure) Act 1986. DRG from all spinal levels were isolated and collected in PBS. PBS was then replaced with 1 mL Dulbecco’s Modified Eagle’s Medium/F12 (DMEM/F12) with Glutamax medium (Gibco) containing Dispase  and Collagenase Type XI  and left for 60 min at 37 °C and 5% CO2, 10 mM HEPES, 5 mM glucose, pH = 7.4 with NaOH) and incubated for 30 min at 37 °C. Coverslips were then washed with standard extracellular ringer solution and left for 15 min at 37 °C and 5% CO2 and then for another 15 min at room temperature. Cells were excited with 350 and 380 nm for ratiometric measurement of intracellular calcium using Cairn Dual OptoLED system. Cells were viewed using a 40X oil immersion objective. Images were acquired using a Hamamatsu C4742–95 camera. The cells were perfused with standard extracellular solution for at least 5 min to establish stable baseline. All recordings were performed at room temperature (23 ± 1 °C). Drugs were perfused at a flow rate of 3 mL/min. High potassium extracellular solution  was perfused at the end of the recordings, unless mentioned otherwise, as a depolarizing agent to identify viable neurons. Simple PCI 6 software was used for data acquisition, background subtraction and Fura-2,AM ratiometric measurement (F350/380 nm).DRG neurons were loaded with 2 μM Fura-2, AM (Molecular Probes) in standard extracellular solution , Capsaicin , α, β-methylene ATP , TTX , TNP-ATP triethylammonium salt , and allyl isothiocyanate .0) were calculated according to the following formula: F350/380 ratio in the presence of drug (F) during drug application – the mean of F350/380 ratio of the 2.5 min prior to drug application (F0). Statistical analysis was performed by calculating the mean percentage of responsive neurons (n) from the indicated number of mice for each figure (N). Sample mean of each set of experiments was calculated from multiple independent experiments (as specified in the result section) and compared to each other by one-way analysis of variance (ANOVA) with Sidaks’ post-test. Cell diameter measurements were performed using ImageJ software; cell area was obtained from a hand-drawn line delineating the soma. Soma diameter was then calculated using the following formula, Diameter = √(4.Area/π). For cell size comparison, two-tailed unpaired Student’s t-test was used for comparisons. All statistical analysis and comparisons were performed by GraphPad Prism software (version 7.00 for Windows). Area-proportional Venn diagrams were generated by BioVenn softwareCorelDRAW X8 software.Neurons were identified by their responsiveness to 40 mM KCl. We defined a response as an increase in (F350/380) ratio of > 6 SD above the baseline. Differences in fluorescence .Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


In the eighth TNM staging system proposal for NSCLC recently, classification of N stage is based on anatomical position of positive lymph nodes. We aimed to expand the sample volume to identify the value of positive lymph node number or ratio in prognosis and predictive effect for postoperative radiation. Clinicopathological characters of 109026 NSCLC patients were collected from the SEER Database. Kaplan-Meier curves and cox regression methods were used for survival analysis. Compared with positive lymph node number equal to 0, 1–3 and >3 groups were independent prognostic factors , so as the 0–50% and >50% positive lymph node ratio groups . And in the groups of N2&positive lymph node number ≥4 and N2&positive lymph node ratio >50%, postoperative radiation related to positive prognosis of NSCLC patients. In conclusion, positive lymph node number or ratio was associated with survival as an independent indicator in NSCLC. They also had predictive effects for postoperative radiation, while N nodal stage not. Non-small cell lung cancer (NSCLC) accounts for approximately 85% of all cases2, and the 5-year survival rate is still low despite great advance has been made in diagnosis and treatment.Lung cancer remains as the most common malignancy with high mortality worldwide3. Although the number of metastatic lymph nodes (MLNs) has been included in N staging system for breast, gastric and colorectal cancer. It has been reported number of positive lymph nodes had a better prognostic effect than anatomical position in these cancers4. Whereafter, due to the limited number of examined lymph node, the ratio of metastatic lymph nodes to total examined number was explored, and confirmed to have prognostic value for various tumors, even better than MLNs number10. The prognostic effects of positive lymph number and ratio for NSCLC have been reported in some studies13, and here, we used the Surveillance, Epidemiology, and End Results (SEER) Database aiming to expand the sample volume and confirm their values in prognosis. Besides, we also searched their effects for patients at N1, N2 stage to clarify if they could be influenced by N stage.Up to now, a series of prognostic indicators have been identified, but lymph node status still remains the important one currently for NSCLC patients14. As previously mentioned, positive lymph node number and ratio might have prognostic effects like N nodal stage, so we used a larger sample volume from 1988 to 2013 to further identify whether they have predictive values for postoperative radiation.Surgery is an effective way for NSCLC patients at early stage. However, among these operable patients, there are some harboring microscopic disease, and postoperative radiation might be performed aiming to reduce local recurrence and improve the outcome. Obviously, not all these patients could get benefits from it, some even could obtain detrimental results, so which group people should receive postoperative radiation was an important issue that have not been figured out. It is crucial to find predictive factors to determine the conduction of radiation after surgery. In 2006, Brian E. Lally and his colleagues searched SEER database and found postoperative radiation was related to an increase in survival in patients at N2 nodal stage but not in patients at N1 and N0 nodal stageThe information about patients was collected from the Surveillance, Epidemiology, and End Results (SEER) database, a population-based cancer surveillance program covering approximately 28% of the population of the United States. Patients would be included if they met the following criteria: (1) patients >20 years old; (2) diagnosed with NSCLC pathologically; (3) receiving tumor resection only or radiation after surgery; (4) survival month more than 4 months. The criteria of patients exclusion was as followed: (1) at M1 stage; (2) without complete information about N stage; (3) at N3 stage; (4) without definitive number of examined and positive lymph node; (5) with controversial information (e.g patients at N1 or N2 stages with 0 positive lymph node).In the process of selection, we also collected clinicopathological characteristics and follow-up information about patients, including age, gender, race, marital status, histological subtype, tumor size, N stage, differential degree, cancer position, treatment, year of diagnosis, number of examined and positive lymph node, survival status and survival months.For the optimal cutoff of positive lymph node number and ratio, we used χ2 scores which were calculated using the Cox proportional hazards regression model. We performed Kaplan-Meier (K-M) analysis to test if positive lymph node number and ratio were significant for prognosis or prediction for postoperative radiation in all patients and patients at different N stages. Hazard ratio with its 95% confidence interval for describing association of variables and survival was calculated by univariate and multivariate cox regression methods. All statistical calculations were performed by SPSS (version 20.0) software , and a two-sided p ≤ 0.05 was considered to be significant.The study was approved by ethic community of Shandong Provincial Hospital afflicted to Shandong University. All the experiments described here were performed in accordance with the approved guidelines.As presented in the flow chart of patients selection Fig. , 109026 2 scores shown in Table We used Cox proportional hazards regression model to determine the cutoff values for positive lymph node number and ratio. In accordance with the maximalχWhen we analyzed the prognostic effects of positive lymph node number and ratio in all included NSCLC patients by K-M curves, we found they were both associated with overall survival (OS) (p < 0.001 for both) significantly Fig. . CompareWhen stratified by N stage, positive lymph node number and ratio also related to prognosis of NSCLC patients at N1 stage or N2 stage . The same results were seen in N0, N1, N2, positive number = 0, positive number = 1–3, positive ratio = 0 and positive ratio = 0–50% groups ). But the results were not significant in positive number ≥4 and positive ratio >50% groups   N1&positive lymph node number =1–3; (2) N1&positive lymph node number ≥4; (3) N2&positive lymph node number = 1–3; (4) N2&positive lymph node number ≥4; (5) N1&positive lymph node ratio = 0–50%; (6) N1&positive lymph node ratio > 50%; (7) N2&positive lymph node ratio = 0–50%; (8) N2&positive lymph node ratio > 50%. And found that postoperative radiation would benefit patients in the subgroups of “N2&positive lymph node number ≥4” and “N2&positive lymph node ratio >50%”, patients in other ten groups obtained opposite results significantly. These suggested that the combination of N nodal stage and positive lymph node number or ratio was a good means to select potential NSCLC patients who could get benefit from postoperative radiation.Sometimes, postoperative radiation was performed for resected NSCLC patients to reduce local recurrence and improve survival. But results from a series of studies suggested that not all these patients would get benefits from radiation after surgery, some even would obtain detrimental result. In 1998, an article of meta-analysis containing nine studies with 2128 patients reported that whether postoperative had detrimental effect was determined by N nodal status. Significantly reduced survival was seen in patients at N0 and N1 stage, and patients at N2 stage seemed to advocate postoperative radiation but the result was not significantAlthough we had a large enough sample of NSCLC patients to conduct our analysis, there were some bias difficult to avoid due to the nature of respective study. And the SEER data is available data with heterogeneity and other limitations, we could not control the baseline of different groups to be the same. That is to say that those who used radiation might be quite different at the baseline from those without using radiation even among the NSCLC patients at the same N stage. So further exploration especially randomized clinical trials should be performed to confirm the result.In conclusion, our study analyzed 109026 patients with NSCLC between 1988 and 2013 from SEER database, and got the result that positive lymph node number or ratio was associated with survival as an independent indicator in NSCLC. And they also had predictive effects for postoperative radiation. For NSCLC patients in subgroups of “N2&positive lymph node number ≥4” and “N2&positive lymph node ratio >50%”, postoperative radiation use had a positive effect on survival significantly. And we could not get the result only according to the N nodal stage.


Supplemental Digital Content is available in the text Pmeta < 1 × 10−5). Of these, 2 SNPs at chromosome 17q21.31  exhibited cis effects on the expression of microtubule-associated protein tau  in 447 frontal-cortex samples obtained postmortem and profiled by RNA-seq (P < 1 × 10−15). Gene-based and pathway-enrichment analyses further implicated MAPT in regulating the sense of smell in older adults. Similar results were obtained after excluding participants who reported a physician-diagnosed PD or use of PD medications. In conclusion, we provide preliminary evidence that the MAPT locus may play a role in regulating the sense of smell in older adults and therefore offer a potential genetic link between poor sense of smell and major neurodegenerative diseases.Olfactory dysfunction is common among older adults and affects their safety, nutrition, quality of life, and mortality. More importantly, the decreased sense of smell is an early symptom of neurodegenerative diseases such as Parkinson disease (PD) and Alzheimer disease. However, the genetic determinants for the sense of smell have been poorly investigated. We here performed the first genome-wide meta-analysis on the sense of smell among 6252 US older adults of European descent from the Atherosclerosis Risk in Communities (ARIC) study, the Health, Aging, and Body Composition  study, and the Religious Orders Study and the Rush Memory and Aging Project (ROS/MAP). Genome-wide association study analysis was performed first by individual cohorts and then meta-analyzed using fixed-effect models with inverse variance weights. Although no SNPs reached genome-wide statistical significance, we identified 13 loci with suggestive evidence for an association with the sense of smell ( Olfactory dysfunction may not only result in poorer nutritional status and adverse effects on safety and quality of life, but is associated with higher mortality.3 Previous research suggests that the loss of sense of smell is among the earliest symptoms of neurodegenerative diseases such as Parkinson disease (PD)5 and Alzheimer disease (AD).6 For PD, this observation is consistent with the Braak hypothesis that Lewy body deposition first affects the olfactory bulb and lower brain stem before spreading into substantia nigra and cortex.7 Moreover, as the nasal cavity directly interacts with the environment, a dual-hit hypothesis was proposed that environmental neuro-toxicants may initiate PD development in the olfactory structures decades before PD clinical diagnosis.8 Therefore, research on the sense of smell in older adults may lead to a better understanding of the natural history and etiology of PD and potentially for other neurodegenerative diseases.The sense of smell decreases with age. Poor sense of smell affects over 20% of US older adults.ApoE ε4 allele may contribute to variable olfactory sensitivity and perception.10 Two earlier studies also suggest potential links to regions on chromosome 4q.12However, the genetic basis for the loss of the sense of smell is largely unknown. Limited studies have suggested that genetic variations at human olfactory receptors or the MAPT), a major susceptibility gene for PD14 and possibly for AD,15 may also play a role in regulating the sense of smell in older adults.Here, we report the first ever genome-wide meta-analysis on the sense of smell among 6252 US older adults of European descent from 3 cohorts: the Atherosclerosis Risk in Communities (ARIC) study, the Health, Aging, and Body Composition  study, and the Religious Orders Study and the Rush Memory and Aging Project (ROS/MAP). We further examined suggestive loci with gene-based analyses, pathway-enrichment analyses, as well as expression quantitative trait loci (eQTL) analysis using 447 postmortem prefrontal-cortex tissues. Overall, our results suggest that microtubule-associated protein tau  in 2011 to 2013. Of the 6523 participants of ARIC NCS, 4066 were eligible after excluding non-Whites (n = 1563), and participants without valid data on the smell identification test (n = 279) or genome-wide association study (GWAS) genotyping (n = 615). The Health ABC is a prospective study established in 1997 to 1998 to investigate risk factors for disability and functional decline among older adults.17 The sense of smell was tested among 2537 participants in 1999 to 2000. After excluding self-reported non-Whites (n = 976), participants without valid data on the smell identification test (n = 106) or GWAS (n = 111), 1344 were eligible for analysis. The ROS and MAP studies are longitudinal cohorts established in mid-1990s to investigate aging and AD incidence and progression among adults 65 years or older.19 The sense of smell was measured as part of its clinical evaluation. Our analysis included 1065 Whites after excluding participants without data on genotyping (n = 661) or the sense of smell (n = 644).Details of participating cohorts were published elsewhere.20 in ARIC and the 12-item Brief Smell Identification Test 21 in the Health ABC and ROS/MAP. Both tests assess participant's sense of smell to correctly identify 12 daily odorants, although the exact odorants were somewhat different. Briefly, participants were instructed to smell each odorant and choose the correct odorant from 4 possible answers in a multiple-choice format. One point was given for each correct answer with a total score from 0 to 12. Both tests have been validated and widely used in clinical and epidemiological studies.21All 3 cohorts used validated smell identification tests to examine the sense of smell: the 12-item Sniffin’ Sticks screening test ApoE genotypes by genotyping 2 ApoE variants at codons 130 and 176 (formerly 112 and 158) separately.All cohorts assessed global cognitive function during the same study visit in which the sense of smell was evaluated. The ARIC and ROS/MAP studies used the Mini-Mental State Examination (MMSE) that has a maximal score of 30, whereas the Health ABC study used the Modified Mini-Mental Status Examination (MMMSE) with a maximal score of 100. Each cohort also determined GWAS genotyping was performed using the Affymetrix Genome-wide Human SNP array 6.0 in ARIC and ROS/MAP, and Illumina Human1M-Duo BeadChip in Health ABC. Imputation was performed in each cohort using MACH based on HapMap Phase II CEU build 36 reference panel. Prespecified criteria were used for quality control of each cohort.22 or PLINK,23 using a linear regression model with analysis of allele dosages. The outcome was the natural log of the sense of smell score plus 1 to account for the few participants who had a score of 0. Potential confounders were adjusted for in 2 separate models: first for age, gender, study sites, cognitive function, and the first 2 principal components (PCAs) in Model 1, and then additionally for ApoE ε4 allele in Model 2. We further adjusted for ApoE ε4 because it is the most important genetic risk factor for AD and predicts poor sense of smell in all participating cohorts.Each cohort performed its own GWAS analysis following a standardized procedure with ProbABEL24 after filtering out SNPs with MAF < 1% 27 or low imputation quality (r2 < 0.3). We applied genomic control for each dataset before meta-analysis (e-Methods). We considered P value < 5 × 10−8 as genome-wide significant and P value between 1 × 10−5 and 5 × 10−8 as suggestive evidence for an association.We performed meta-analysis using a fixed-effect model with inverse variance weights in METAL,28 (Versatile Gene-based Association Study) for gene-based analysis and calculated individual gene-wise P values for each gene in relation to the sense of smell, and used binomial test to identity enriched ingenuity canonical pathways  based on the results of meta-analysis in Model 2, the enrichment test P values were derived from 10,000 rounds of permutation tests (e-Methods).We used VEGAShttp://regulomedb.org/)29 (e-Methods). We then generated RNA expression data using frozen postmortem dorsolateral prefrontal cortex (DLPFC) brain tissue from 447 ROS/MAP participants to define the biological consequence of the risk variants of identified SNPs (e-Methods).To explore the functional relevance of top SNPs from the GWAS analysis, we applied the publicly available data from the RegulomeDB . Although these studies used different screening tests of smell identification, the distributions of the sense of smell scores were comparable across cohorts .Table http://links.lww.com/MD/A526). Although no SNP reached genome-wide significance, 35 SNPs had a suggestive association with the sense of smell  or were potentially functional as annotated in the RegulombDB . Among these, 3 SNPs were predicted as eQTLs in frontal-cortex and cerebellum tissues in the RegulomeDB: rs199443 , rs2075650 , and rs2732614 . The rs199443 and rs2732614 were cis-eQTLs for MAPT in both frontal-cortex and cerebellum tissues,30 while rs2075650 was cis-eQTL for TOMM40 in lymphoblastoid cells.31The Q–Q plots for Model 1 showed relatively low genomic inflation factors , suggesting minimal population stratification . As shown in Table Pmeta < 1 × 10−5, Figure S3, http://links.lww.com/MD/A526); another 47 SNPs were in LD (r2 ≥ 0.8) with these 13 SNPs . Ten of these 13 SNPs overlapped with those from Model 1, including the 2 annotated eQTLs at chromosome 17: rs199443 (Pmeta = 3.02 × 10−6) and rs2732614 (Pmeta = 6.65 × 10−6). In contrast, the signal for rs2075650 at TOMM40 was drastically diminished (Pmeta = 0.09) after adjustment for ApoE ε4. For these 13 SNPs, we also repeated the meta-analyses using random-effect model and observed similar results. For example, the Pmeta was 3.73 × 10−6 for rs199443 and 8.25 × 10−6 for rs2732614 under the random-effect model.In the meta-analysis of Model 2 that further adjusted for 28 to identify genes that harbor multiple association signals based on Pmeta values from the model that adjusted for ApoE. The SNPs were mapped to 17,693 genes. No genes were statistically significant after Bonferroni correction . Genes with P < 1.0 × 10−3 were listed in Table S5, http://links.lww.com/MD/A526. Interestingly, MAPT (Pgene = 9.4 × 10−5) was among the top genes identified in this analysis along with nearby LRRC37A (Pgene = 2.9 × 10−5) and NSF (Pgene = 1.4 × 10−5), all located on chromosome 17. LRRC37A and NSF each harbor 1 of the 2 annotated eQTLs  whereas the expression of MAPT is regulated by both SNPs in brain tissues.30We conducted gene-based analysis using VEGAShttp://links.lww.com/MD/A526). The first analysis was based on results from Model 2 and we observed 21 significantly enriched canonical pathways with the permutation P < 0.05. These include the CDK5 and p38 MAPK signaling pathways both of which are implicated in neurodegeneration and MAPT is a member of both pathways. The second analysis were based on the 164 genes with Pgene < 0.01 identified by VEGAS. This analysis identified 113 enriched canonical pathways with the permutation P < 0.05. The CDK5 signaling pathway was again identified as 1 of the top ranked pathways.We further conducted 2 pathway enrichment analyses  and higher expression level of MAPT .We further examined MAPT is 1 of the most important susceptibility genes for PD, we further performed a sensitivity analysis for the 13 SNPs identified in Model 2 by removing potential PD cases based on self-reported physician diagnosis or use of PD medications  and obtained similar results .As MAPT as a potential susceptibility locus for poor sense of smell. This is particular interesting because MAPT is 1 of the most important susceptibility genes for late-onset sporadic PD14 and possibly for AD.15To the best of our knowledge, this is the first genome-wide meta-analysis on the sense of smell among older adults. The study population includes 3 well-characterized aging cohorts with objective measurements for smell identification. Although no SNP reached genome-wide significance, the preponderance of the evidence suggests 5 and AD.6 For example, in the Honolulu Asia Aging Study, individuals in the lowest quartile of the sense of smell score were 5 times more likely to develop PD in the next 4 years than those in the highest quartile5; further poor sense of smell was associated with higher prevalence of incidental Lewy bodies in postmortem brains, a condition that may represent prodromal PD.32 Similar observations have also been made for both mild cognitive impairment and AD.33 Therefore, the declining sense of smell in older adults may indicate underlying neurodegenerative processes. Characterizing the genetic and environmental determinants for the sense of smell in the context of aging may provide targets for intervention with enormous potential translational impact.Smell impairment is common among older adults, particularly in patients with neurodegenerative diseases. More importantly, recent data suggest that decreased sense of smell may precede the clinical onset of both PDMAPT locus has long been associated with the risk for PD.14 A recent meta-analysis of GWAS data further implicated MAPT as a susceptibility locus for late onset sporadic AD.15 The human MAPT encodes the microtubule-associated protein tau. It is notable that tau-related pathology was found in the bulbar component of the anterior olfactory nucleus only in patients of neurodegenerative disorders with marked loss of the sense of smell, such as AD, PD, and Lewy body dementia.35 Experimental studies further suggest that overexpression of tau may contribute to olfactory dysfunction in vivo.37 These findings indicate that overexpression or abnormal aggregation of tau may represent a common mechanism between olfactory dysfunction and several neurodegenerative diseases.The human MAPT is highly complex. It covers ∼1.8 Mb block of LD encompassing not only MAPT but also several other genes that have been linked to neurodegenerative diseases, such as STH (Saitohin) and NSF . We, however, in the RNA-seq data of 447 postmortem frontal-cortex tissues, found that rs2732614 and rs199443 had statistically significant cis effects on the expression of MAPT (P < 10−15), but not on NSF or LRRC37A. Additional gene-based and pathway-enrichment analyses also showed the potential of MAPT as a candidate susceptibility gene for poor sense of smell. Although our study provides fairly consistent evidence that MAPT may be a candidate susceptibility locus for the sense of smell in older adults of European descent, fine-scale mapping of this LD block with next generation sequencing will be necessary to identify potential causal locus.The genomic architecture in the region spanning ApoE ε4 is the most important genetic risk factor for late-onset sporadic AD38 and has been linked to olfactory dysfunction in several studies.9 The present study confirms that the ApoE ε4 allele is a risk factor for poor smell identification even after accounting for cognitive function. In our initial analysis, we also identified an intronic SNP in TOMM40 (rs2075650) in association with the sense of smell; but this signal was extinguished after adjustment for the ApoE ε4 allele. Although the mechanisms are yet unknown, preliminary studies showed that ApoE was expressed at high levels in various types of cells in the olfactory epithelium and its underlying lamina propria, and that ApoE might play a critical role in olfactory nerve regeneration in mice.40 Furthermore, experimental studies suggest that in contrast to the ApoE ε2 and ε3 alleles, the ApoE ε4 fails to promote neurite outgrowth in olfactory epithelium cultures, which may lead to olfactory dysfunction in AD.4143 and our study was conducted among old White adults, therefore the results should be interpreted in this context. Further studies are needed to confirm our findings and to examine whether they could be generalized to other populations.The main limitation of the current study was the lack of independent confirmation of GWAS signals. GWAS analysis often requires very large sample size to obtain adequate power to detect modest effects of underlying variants in complex diseases. However, few large population-based studies of older adults have both the sense of smell and GWAS data. We were able to identify data from 3 well-established cohorts with similar population characteristics and data collection. We chose to combine our samples for SNP discovery rather than set up the conventional 2-stage design to maximize power to detect signals. We did, however, follow up suggestive signals with functional and expression analyses. Finally, the determinants for the sense of smell are likely complexMAPT locus may regulate the sense of smell in older adults. This finding warrants independent confirmation and further mechanistic investigations into how MAPT region contributes to the loss of the sense of smell and the development of related neurodegenerative diseases.In conclusion, we report the results of the first comprehensive GWAS analysis to understand the genetic basis for the sense of smell in older adults of European ancestry. We provide novel evidence that the


Leptospira interrogans sv Pomona in Meat Workers in New Zealand”  The data presented in this article are related to the research article entitled “Comparison between Generalized Linear Modelling and Additive Bayesian Network; Identification of Factors associated with the Incidence of Antibodies against Leptospira interrogans sv Pomona (Pomona) infection, one of the most common Leptospira serovars in humans in NZ. This article provides an extended analysis of the data, illustrating the different steps of a multivariable  and especially a multivariate tool based on additive Bayesian networks (ABN) modelling.A prospective cohort study was conducted in four sheep slaughtering abattoirs in New Zealand (NZ)   The outcome was “Pomona” infection in meat workers and the main exposure variables of interest were “work position”, the usage of “protective equipment” (PPE), “hunting”, “home slaughter” and “farming”. The total data set comprised of 17 variables with 15 binary and 2 continuous variables, listed in the descriptive 22.1Leptospira interrogans sv Pomona infection. New infection occurred where a worker sero-converted or had an anamnestic response A prospective cohort study amongst 384 voluntarily participating meat workers from four purposively selected sheep abattoirs in the North Island of NZ was conducted. Study methods were described in detail by Dreyfus et al. 2.2Data were analysed using the software R, version 3.1.2 2.3(1)The first step was to find an optimal model , [7], [8(2)In the second step, the model was adjusted by checking it for over-fitting (3)In the third step of ABN analysis, the marginal posterior log odds ratio and 95% credible intervals were estimated for each parameter from the posterior distribution , expressAll analyses were conducted using the R package “abn”


Chaetopterus marine worm. We use optically trapped probes (2–10 μm) to induce microscopic strains and measure the stress response as a function of oscillation amplitude. Our results show that viscoelastic properties are highly dependent on strain scale (l), indicating three distinct lengthscale-dependent regimes at l1 ≤4 μm, l2≈4–10 μm, and l3≥10 μm. While mucus response is similar to water for l1, suggesting that probes rarely contact the mucus mesh, the response for l2 is distinctly more viscous and independent of probe size, indicative of continuum mechanics. Only for l3 does the response match the macroscopic elasticity, likely due to additional stiffer constraints that strongly resist probe displacement. Our results suggest that, rather than a single lengthscale governing crossover from viscous to elastic, mucus responds as a hierarchical network with a loose biopolymer mesh coupled to a larger scaffold responsible for macroscopic gel-like mechanics.We characterize the lengthscale-dependent rheological properties of mucus from the ubiquitous  Chaetopterus marine worm, also use mucus to trap and filter food, deter prey and build housing tubes /[|xmax|6πR], loss modulus, G" = [|Fmax|sin(Δϕ)]/[|xmax|6πR], and complex viscosity, η = (G′2+ G"2)1/2/ω where |Fmax|, |xmax|, Δϕ and R, are the amplitude of measured force, amplitude of the stage position, phase difference between F and x, and probe radius respectively . Fo. Foxmax/d fibers ,45. Becad fibers ,45. ThusG′~ω2 and G"~ω. As elastic effects become more important, i.e. as the frequency of perturbations approach and surpass the relaxation or disentanglement rate of network, G′ approaches and surpasses G", and both G′ and G" initially approach frequency-independent plateaus. As shown in G′~ω2 and G"~ω while for 4.5 and 6 μm probes, the scaling of G′ and G" is slightly reduced from terminal regime scaling and the magnitudes of G′ and G" are once again probe size independent. For 10 μm beads, G′ and G" both approach frequency-independent plateaus with a further reduced gap between G′ and G".To further characterize the lengthscale-dependent material properties of the mucus we measure the frequency dependence of the viscoelastic moduli . For a nG′/G"> for all four bead sizes as well as for macrorheology. This quantity can also be understood as the inverse of the loss tangent, tan δ = sinΔϕ/cosΔϕ, which quantifies the relative dissipation in the system. G′/G" values for all probe sizes converge as oscillation amplitude increases, in line with our interpretation of a threshold lengthscale necessary for the mucus mesh to appreciably contribute to measured mechanics.We quantify the relative elasticity of the mucus as a function of probe size by comparing the average ratio of the elastic modulus to viscous modulus <G′ and G″ values nearly >50× smaller than macrorheology measurements, with G′/G″ also substantially reduced [l3) linking together entangled extended mucins (l2).The results reported here are consistent with similar previous experiments with porcine gastric mucus in which passive microrheology with 1 μm beads measured  reduced . We note reduced . While tFmax| values for data collected in different mucus samples as well as in different regions of the same sample. However, the data trends with respect to oscillation amplitude and frequency were similar across all trials. This 20% heterogeneity is well above the ~5% polydispersity in nominal probe sizes and the ~2.6% instrument error in force detection ; as a continuum with principally viscous response features, characteristic of a loosely entangled polymer mesh (l2); or elastically, mimicking the macroscopic mucus properties (l3). These collective results suggest that the mucus can be modeled as a coupled two-fluid system comprised of (i) a loosely entangled polymer mesh coupled to (ii) a larger more elastic scaffold  provides the macroscale elasticity of the mucus at l3.We have used active microrheology to characterize the lengthscale-dependent structural properties governing the mechanics of scaffold . While ml1 is necessary for the passage of nutrients while l2 is needed for trapping larger pathogens and parasites. Finally, the macroscopic gel-like properties necessary for coating organs is provided by l3.This model can explain how mucus is able to simultaneously perform a multitude of functions across varying lengthscales. For example, S1 FigMeasurement techniques and instrumentation were identical to those shown in (PDF)Click here for additional data file.S2 Figσ2 that, as described in Refs 56 and 57, is inversely proportional to the trap stiffness k via the equipartition theorem (i.e. ½kσ2 = ½kBT). Because the index of refraction of the fluid can alter the trap stiffness, this calibration method, which can be carried out in the fluid of interest (i.e. mucus) is used to determine how the trap stiffness in mucus compares to that in water. The variances displayed in each plot are in units of 10−3 mV2. As shown the variance of the PSD voltage for all bead sizes in water (left) and in mucus (right) are all quite similar, within a factor of 2 of each other. The symmetry and goodness of Gaussian fits for nearly all of the distributions demonstrate that our optical trap is trapping properly and optimized for quantitative force measurements.Each distribution is the cumulative laser deflection data for 10 different trapped stationary probes (color scheme as in (PDF)Click here for additional data file.S3 Figx and y directions. The resulting diffusion coefficients, Dmucus and Dwater, calculated via the Einstein relation <Δx2> = <Δy>2 = 2Dt, were determined by tracking ~100 different probes for ~20 sec each at a frame rate of 10 fps. Error bars were determined by bootstrapping over 1000 subensembles. Measurements were carried out for probes coated with BSA, 2 kDa PEG-diamine and streptavidin (ST). Coating of carboxylated microspheres with PEG-diamine and BSA was done following the protocols published in Refs 39 and 61 which were modified from those provided by the microsphere manufacturer (Polysciences). ST-coated probes were purchased from Polysciences. Probes coated with PEG, following the cited protocols, have been shown to be mucoinert (no binding interactions with mucus)  whereas ST is a relatively “sticky” protein that nonspecifically interacts with a range of molecules. BSA-coated probes, assumed to be mucoinert, were used in all active microrheology data shown in manuscript. As shown, the diffusion of PEG-coated and BSA-coated probes in mucus are nearly identical, suggesting similar passivation efficacy, whereas ST-coated probes show slower transport, likely due to binding interactions with the mucus. Further, the results for the BSA- and PEG-coated probes are in agreement with the measured viscosities shown in Dmucus/Dwater is ~0.59 which is consistent with the viscosity of the mucus as actively measured with 2 μm probes (~2ηw), whereas for 6 μm probes Dmucus/Dwater is ~0.23 as expected provided an average measured viscosity of ~5ηw.2 μm (green) and 6 μm (blue) probes embedded in mucus and in water were imaged and tracked over time to determine the mean-squared displacements in the (PDF)Click here for additional data file.


In Escherichia coli, two tyrosine-family recombinases, XerC and XerD, bind to dif and carry out two pairs of sequential strand exchange reactions. However, what makes the reaction unique among site-specific recombination reactions is that the first step, XerD-mediated strand exchange, relies on interaction with the very C-terminus of the FtsK DNA translocase. FtsK is a powerful molecular motor that functions in cell division, co-ordinating division with clearing chromosomal DNA from the site of septation and also acts to position the dif sites for recombination. This is a model system for unlinking, separating and segregating large DNA molecules. Here we describe the molecular detail of the interaction between XerD and FtsK that leads to activation of recombination as deduced from a co-crystal structure, biochemical and in vivo experiments. FtsKγ interacts with the C-terminal domain of XerD, above a cleft where XerC is thought to bind. We present a model for activation of recombination based on structural data.Bacterial chromosomes are most often circular DNA molecules. This can produce a topological problem; a genetic crossover from homologous recombination results in dimerization of the chromosome. A chromosome dimer is lethal unless resolved. A site-specific recombination system catalyses this dimer-resolution reaction at the chromosomal site  The majority of bacterial species have circular DNA genomes. Prior to cell division each circular chromosome must be entirely replicated, unlinked and segregated to ensure that each daughter cell inherits a full genome complement. During or following replication DNA repair processes involving homologous recombination can produce a chromosomal crossover, and any odd number of these events between the circular DNA molecules results in a chromosome dimer2dif located in the replication terminus region578Escherichia coli dif site consists of two 11 bp sites that are an imperfect inverted repeat, separated by a 6 bp spacer, or central regiondif sites bound by XerCD can then be synapsed by protein-protein interactions between the two dif-XerCD complexes forming a pseudo-tetrameric structure11131415dif)2 is not by itself catalytically competent; it is not until FtsK is present that cleavage by XerD occurs12Most bacteria encode two tyrosine family recombinases XerC and XerD which bind to the specific chromosomal site dif sitesThe FtsK protein is a powerful DNA translocase that is involved in co-ordinating cell division with DNA unlinking and segregation to ensure that the closing septum does not trap or guillotine chromosomal DNA182122232526dif sites; FtsKγ binds specifically to 8 bp sequences termed KOPS that are highly skewed on each chromosome arm to point towards the dif site2829dif siteThe FtsKγ domain also plays another role in orienting the FtsK motor to translocate toward dif synapse to promote recombination.Here we present the structure of the FtsKγ domain interacting with the XerD recombinase and provide biochemical evidence to support the involvement of amino acids at the interaction surfaces in the activation of recombination. A mutant in XerD that disrupts the interaction with FtsKγ is severely impaired for recombination activity on plasmid substrates and is also incapable of supporting chromosome dimer resolution in cells. Further, we demonstrate that a 9 amino acid stretch from FtsKγ that encompasses the interaction surface is sufficient for activation of recombination. It is of note that the same region of FtsKγ that binds KOPS-DNA (between helices 2 and 3) is also responsible for the protein-protein interaction with XerD to activate recombination. Finally, we also provide structure-based models to explain how the FtsKγ-XerD interaction can influence the XerCD-dif requires the interaction of XerD with the γ domain of FtsK. To understand how this interaction can lead to activation of recombination, a fusion protein between the C-terminal domain of XerD, which contains the catalytic residues required for DNA cleavage and strand exchange, and FtsKγ was produced (XerDC–γ) and crystallized. The full length XerD-FtsKγ fusion protein has previously been shown to be a functional, self-activating, recombinase protein at dif sitesC-FtsKγ fusion protein was solved by molecular replacement and refined to a resolution of 2.3 Å (E. coli FtsKγ domain (2VE8) as search models30C–γ complex, but the flexible linker joining the two protein domains was not visible in the density. Details of the linker sequence and all amino acids for which there is insufficient electron density are shown in The activation of recombination at of 2.3 Å , using tC domain and the FtsKγ domain are structurally very similar to the previously determined individual proteins, with no major conformational changes revealed in either protein domain; overall the XerDC domain only varied by an average RMSD of 0.93 Å on matched Cα atoms, while the RMSD between matched Cα atoms for the FtsKγ domain was 0.77 Å. The density observed for FtsKγ was weaker than that for XerDC and increasingly diminished for atoms that were distal from its interaction with XerD, resulting in the final refined model having chain breaks in this region. However, the density for the interface between the two binding partners was sufficient to facilitate an in depth examination of the key molecular contacts .Both the XerDC is a 563 Å2 interface that involves two helices on the surface of XerDC .in vitro recombination reactions in the presence of XerC and a model plasmid containing two dif sites. The relative levels of recombination were again compared to wild-type proteins  shows that these are interactions which contribute greatly to the required association of these two proteins.Since the mutants in XerD for both in vitro and in vivo recombination data supports the interaction surface seen in the crystal structure being vital for the activation of recombination.Taken together the dif as well as, or even more efficiently than, a XerD-FtsKγ fusion : of the 5 amino acids found to be important for activation of XerD there are 3 identical and 1 similar in the H. influenzae protein whereas the L. lactis XerS (there is only a single Xer recombinase in this species) has only 1of the 5 amino acids identical to E. coli . Again, this is mirrored in the cross-species activities seen; E. coli FtsK can activate H. influenzae XerDE. coli FtsKγ alone cannot activate XerS from L. lactisA number of studies have used inter-species recombination assays using Xer recombinases and FtsKs from different organisms both for dif complexes has been observed in the absence of the FtsKγ domain1112dif synapse to the D* state must require interaction with DNA or with XerC, or likely both, in addition to the FtsKγ interaction. Indeed, this makes sense mechanistically; catalysis is only activated within the properly assembled XerCD-dif synapse reducing the occurrence of inappropriate XerD-mediated cleavage of the DNA.Synapsis between two XerCD-C–γ active-site structure to that of activated Cre or λ Int, it is clear that two key amino-acids of the XerD active-site are inappropriately positioned for catalysis13in vitro studiesBy comparing the XerDdif synapses along with data that shows that the very C-termini of both XerC and XerD are important for this reciprocal control of partner activation3611Another important feature of the Cre and λ Int synapses is the cyclic interaction between monomers obtained by donation of their C-termini to the adjacent monomer13loxP synaptic structure . We can, therefore, be confident that this is an important site of interaction between XerC and XerD, and that the FtsKγ domain is positioned to modulate this interaction.By modelling the structure of XerD onto the active structures of Cre or λ Int the XerD M and N helices can be re-arranged to produce an active-site close to those seen for other tyrosine recombinasestructure . There itructure , and amiion site . The Pyrdif, and this mutation is synergistic with mutation that alters E184, which directly contacts FtsKγ  +2 amino acids (SR) from the linking XbaI restriction enzyme site followed by FtsK residues 1261 to 1329) was amplified from the full length XerD-FtsKγThe XerDxerD-ftsKγ mutants were made by a two-step overlap PCR process, using mutagenic DNA primers and Phusion DNA polymerase (sequences available on request), and cloned into pBAD24. Selected mutant sequences were subcloned into pBAD24-XerD by PCR from the relevant mutant fusion using XerD primers as described previouslyxerC–ftsKγ-peptide fusion sequence was cloned by cutting the pBad24 XerC-FtsKγ fusionC-FtsKγ fusion overproduction and purification using Ni2+ resin was as previously described2, 1 mM DTT, using a VivaSpin 6 centrifugal concentrator  to a final concentration of ~9 mg/ml.XerDin vitro recombination assays were overproduced and purified as previouslyAll other proteins and fusion proteins for 2. Data was collected on MX2 beamline at the Australian Synchrotron, using a wavelength of 0.9184 ÅInitial hits were identified using 200 nl protein drops, using commercial screens (Molecular Dimensions) mixed by Mosquito nano-litre robot (TTP Labtech). Following initial screening, trigonal bipyramidal crystals were obtained by 2μl + 2μl hanging drop vapour diffusion in a solution of 100 mM Bicine (pH9), 10% (w/v) polyethylene glycol (8000 g/mol) over 2–5 days. Crystals were washed in paratone-N and flash frozen in liquid NE. coli XerD (PDB: 1A0P) and E. coli FtsKγ (PDB: 2J5P) as search models . The program PHASERC-FtsKγ in the asymmetric unit. The structure was built using Arp/WarpThe structure was solved by molecular replacement using the known structures of All structural alignments and structure figures were produced using PyMol .In vivo recombination assays using XerD-γ fusion proteins were carried out as previously published17E. coli strain GR51 (AB1157 xerC ftsK)xerC-γ fusion gene along with plasmid resolution reporter, pRB10, a pSC101 derivative  carrying two directly repeated dif sites, flanking the KmR gene cassette. pRB10 is almost identical to the pFX142 reporter used previously except that pRB10 lacks the duplication of restriction enzyme sites surrounding the two dif sites found in pFX142. Transformants were grown in LB with selection; following 16 h incubations plasmid DNA was recovered from the cultured cells and examined by agarose gel electrophoresis followed by SYBR green staining. Levels of parental and recombinant sized plasmid were quantified using ImageQuant software  and percentage recombination calculated.In vitro recombination, using the 2x dif reporter plasmid pSI56, was as previously describedlac::tetO180 GmR) and WX31 ΔxerD containing the relevant expression vector was prepared and grown in LB at 37 °C to stationary phase (~20 generations)3Assays were carried out essentially as previously published1734Accession codes: Coordinates and structure factors for the XerDC-γ complex have been deposited in the Protein Data Bank under the accession code 5DCF.How to cite this article: Keller, A. N. et al. Activation of Xer-recombination at dif: structural basis of the FtsKγ–XerD interaction. Sci. Rep.6, 33357; doi: 10.1038/srep33357 (2016).


Temporomandibular joint degenerative disease (TMJ-DD) is a chronic form of TMJ disorder that specifically afflicts people over the age of 40 and targets women at a higher rate than men. Prevalence of TMJ-DD in this population suggests that estrogen loss plays a role in the disease pathogenesis. Thus, the goal of the present study was to determine the role of estrogen on chondrogenesis and homeostasis via estrogen receptor alpha (ERα) during growth and maturity of the joint. Young and mature WT and ERαKO female mice were subjected to ovariectomy procedures and then given placebo or estradiol treatment. The effect of estrogen via ERα on fibrocartilage morphology, matrix production, and protease activity was assessed. In the young mice, estrogen via ERα promoted mandibular condylar fibrocartilage chondrogenesis partly by inhibiting the canonical Wnt signaling pathway through upregulation of sclerostin (Sost). In the mature mice, protease activity was partly inhibited with estrogen treatment via the upregulation and activity of protease inhibitor 15 (Pi15) and alpha-2-macroglobulin (A2m). The results from this work provide a mechanistic understanding of estradiol on TMJ growth and homeostasis and can be utilized for development of therapeutic targets to promote regeneration and inhibit degeneration of the mandibular condylar fibrocartilage. These alarming statistics suggest that the loss of estrogen during menopause may potentiate TMJ-DD. However, little is known regarding the role of estrogen in mediating TMJ growth, homeostasis, and degeneration.Temporomandibular joint degenerative disease (TMJ-DD) is marked by degradation and premature calcification of the extracellular matrix (ECM) of the articular mandibular condylar fibrocartilage. Patients with TMJ-DD experience pain during jaw movement (e.g. mastication and speaking) and are at higher risk for complete degradation of the joint and replacement surgery. TMJ-DD is a chronic form of TMJ disorder that specifically afflicts older patients and targets women at a higher rate than men. Specifically, 70% of people with TMJ-DD are 40–70 years old3. The articular fibrocartilage is comprised of fibrochondrocytes that produce collagen type 1 and 2 (Col1 and Col2) and proteoglycans. Degenerating joints are marked by degradation of this collagenous and proteoglycan-rich fibrocartilage matrix. Thus, it is crucial to investigate estrogen’s signaling effects on the synthesis and maintenance of the articular fibrocartilage extracellular matrix in both skeletally immature and mature tissue to delineate its role throughout aging and determine potential therapeutic targets.The mandibular condylar fibrocartilage functions as a growth plate cartilage to allow for longitudinal growth of the condyle and transitions to an articular cartilage after skeletal maturation4. The receptor complex then typically binds to the estrogen response element (ERE) and acts as an enhancer, recruiting cofactors to promote gene transcription6. In the nonclassical pathway, ERs, either dependently or independently of ligand binding, interact with other transcriptional pathways through protein-protein interactions likely involving phosphorylation modifications8. In other musculoskeletal tissues such as bone and hyaline cartilage, ERα is required for estrogen’s anabolic effects during development and remodeling to maintain homeostasis11. On the other hand, ERβ acts as a dominant negative regulator that can replace some of ERα’s roles in its absence14. However, the role of ERα on the growth and remodeling of the mandibular condylar fibrocartilage in adults is unclear. ERα is expressed in all cells of the mandibular condylar fibrocartilage emphasizing the importance this receptor must play in estrogen signaling in the TMJ15. As such, it is necessary to determine the role estrogen via ERα plays on growing and mature TMJ tissue.Estrogen modulates transcription via both classical and nonclassical pathways. In the classical pathway, estrogen binds to estrogen receptor alpha (ERα) or beta (ERβ) which results in a conformational change of the receptors, receptor dimerization, and translocation into the nucleus19. Results from these studies show cells of the mandibular condylar fibrocartilage respond to estradiol treatment resulting in a decrease in fibrocartilage cell proliferation and an increase in chondrogenesis19. Previous studies indicated that ERβ mediated estrogen’s role on condylar fibrocartilage cell proliferation but not the chondrogenic matrix effects suggesting the role of ERα in estrogen-mediated chondrogenesis19. Further, recent studies from our laboratory illustrated ERα regulates mandibular condylar fibrocartilage maturation in young male mice but does significantly play a role in mediating growth or remodeling in old male mice20. To our knowledge, the only study conducted that investigates the effect of estrogen on skeletally mature, female rodents was completed by Talwar et al.21. In this study, estradiol treatment in ex vivo and in vitro experiments revealed a decrease in fibrocartilage thickness and cell proliferation similar to effects seen in the growing mandibular condylar fibrocartilage. However, to date, there have not been any in vivo studies investigating the role of ERα in skeletally mature female mice. These results warrant the need to understand estrogen’s role via ERα on homeostasis and remodeling of the mandibular condylar fibrocartilage in adult female mice.Most of the studies investigating estrogen’s effects on the TMJ have been conducted in young rodentsThus, the goal of the present study was to determine the role of estrogen on mandibular condylar chondrogenesis and homeostasis via ERα during growth and maturity of the joint. Young and mature WT and ERαKO female mice were subjected to ovariectomy procedures and then given placebo or estradiol treatment. An ovariectomy model was employed to specifically evaluate the role of estrogen through ERα on the female condylar fibrocartilage. This is vital as ERαKO mice have elevated estrogen levels due to the negative feedback loop. Thus, in this model, endogenous estrogen is no longer produced and controlled concentrations of estradiol are given to the mice. The effect of estrogen via ERα on cartilage morphology, cell proliferation, cartilaginous matrix production, protease activity, and extracellular matrix integrity was assessed. In-depth transcriptome analysis was completed to further reveal the mechanisms by which estradiol signals via ERα to modulate mandibular condylar fibrocartilage health. The results from this work provide mechanistic evidence for the role of estradiol on TMJ growth and homeostasis and can be utilized for development of therapeutic targets to inhibit degeneration and promote regeneration of the mandibular condylar fibrocartilage.−/− mice results in increased sex steroid levels because of disturbed negative feedback regulation10. To avoid confounding endogenous sex steroid effects, all mice were ovariectomized and treated with placebo or estradiol with the sham group serving as the control. Estradiol treatment had no significant effect on fibrocartilage thickness as seen in Fig. . These changes in cell numbers may be due to a decrease in cell proliferation with estradiol treatment as shown by the trend in WT samples seen in Fig. Figure ig. Fig. . These c.The ovariectomy study design for 17-week mice is shown in Fig. Fig. .. Further, estradiol treatment increased Col2 immunostaining and positive staining of the sulfated glycosaminoglycans on the proteoglycans by SafO staining at 7-weeks . The changes in Col2 and SafO staining were not as prevalent in the 17-week samples .The role of estradiol via ERα on mandibular condylar fibrocartilage maturation was investigated via extracellular matrix gene expression and protein localization evaluation. The schematic in Fig. Fig. . Furthereks Fig. . The chales Fig. .Figure 22 fold change greater than absolute value of 2 with estradiol treatment in the WT mice as seen in Fig. 2 fold change greater than 2 . Interestingly, only 2 of these genes (Nkg7 and Dbp) are common between the genotypes at 7-weeks indicating other receptors are involved in estradiol-induced changes in the skeletally immature fibrocartilage. Pathway analysis utilizing gene ontology and outputting cellular components resulted in the information shown in Fig. 2 fold change greater than absolute value of 2 with estradiol treatment in 17-week old WT mice whereas none of these same genes are significantly upregulated in ERαKO samples as seen in Fig. (Supplementary Table 5). Further, utilizing the same gene ontology pathway analysis, no pathways were significantly affected by estradiol treatment in 17-week mice of both genotypes. All the genes that were significantly regulated at p < 0.01 and a log2 fold change greater than absolute value of 2 for all the groups are displayed in Supplementary Tables 6).To further decipher the mechanism by which estradiol alters the growing and mature mandibular condylar fibrocartilage, RNA sequencing was conducted to provide a non-biased complete scan of the entire murine genome in response to estradiol treatment in WT and ERαKO mice of both age groups. Volcano plots in Fig. n 2 Fig. . Interes. However, estradiol treatment in ERαKO mice had no significant effect on the qPCR gene expression of the Wnt inhibitors or activators examined . These results were confirmed by immunohistochemical staining of Sost .Full transcriptome analysis results were utilized to postulate a mechanism by which estradiol may regulate chondrogenesis in the mandibular condylar fibrocartilage. In compiling genes that were significantly regulated at p < 0.05 by estradiol in 7-week old mice, factors from the Wnt signaling pathway were seen to be affected as shown in Fig. Fig. . Howeverned Fig. . These rost Fig. .Figure 4 indicating the role of ERα in estradiol-based regulation of protease inhibitors.In the RNA sequencing analysis, a number of genes that are involved in anabolic processes in the fibrocartilage were upregulated at p < 0.01 as illustrated in the heatmap in Fig. Fig.  indicati but not in ERαKO mice except for MMP3 . Immunostaining of collagen type 1 and 2 cleavage epitopes further illustrated the role the decrease in protease activity exhibits on matrix protein stability as seen in Fig. The role of estradiol on protease expression and collagen cleavage was assessed to determine downstream effects of increased protease inhibitor activity. Estradiol treatment decreased qPCR gene expression of proteases including MMP3, MMP13, MMP9, ADAMTS4, and ADAMTS5 in WT Fig.  but not MP3 Fig. . Immunos, A2m, at both 100 and 200 nM, decreased MMP9 activity via zymography analysis . Similarly, Pi15 treatment at both 50 and 100 nM decreased MMP9 activity  and qPCR gene expression .Immunostaining of A2m in WT and ERαKO sections from the OVX experiment illustrates a decrease in protein localization (brown staining) in the OVX group and a recovery with estradiol treatment solely in the WT mice Fig. . In the Fig. , A2m, atsis Fig.  and qPCRig. Fig. . Similarity Fig.  and qPCRion Fig. .Figure 7The overarching goal of this study was to determine the role of ERα on the growth and homeostasis of the female mandibular condylar fibrocartilage to provide evidence for the sex predilection of TMJ disease and determine potential mechanisms for healthy joint maintenance. The major findings from this study indicate that estrogen via ERα promotes chondrogenesis by potentially suppressing the canonical Wnt signaling pathway in the mandibular condylar fibrocartilage and maintains homeostasis by upregulating protease inhibitors, thereby decreasing protease activity in the condylar fibrocartilage.22. However, the mandibular condylar fibrocartilage does not experience growth plate closure and instead continues to function as an articular cartilage after puberty23. In growing mice, we found that estrogen replacement caused a decrease in cell numbers in ovariectomized WT but not ERαKO mice. Previously, we have shown that estrogen replacement resulted in similar effects in ERβKO female mice19. Together the results suggest that both ERα and ERβ signaling are critical for estrogen’s inhibition of mandibular condylar fibrocartilage growth. Similar results have been reported in the long bones, where both female ERαKO24 and ERβKO25 mice have increased longitudinal bone growth. However, recent studies have suggested that local cartilage-specific ERα signaling is involved in estrogen’s inhibition of longitudinal bone growth plate cartilage thickness26 whereas the effects of ERβ on mediating longitudinal bone growth is unclear27. Thus, local administration of ERα-specific agonists may be a novel treatment option in skeletally immature patients with severe mandibular prognathism. On the other hand, we did not observe a significant effect of estradiol on growth in mature mice. Previously, the role of estrogen in skeletally mature female rats utilizing an explant culture model illustrated a decrease in fibrocartilage thickness and cell proliferation similar to young rodents, effects we did not observe in this study21. This difference may be due to differences in cell signaling within the in vivo OVX model compared to the in vitro explant culture model. Our results potentially indicate that targeting ERα with an ERα-specific agonist such as propylpyrazole triol (PPT) in skeletally mature patients may not be effective in inhibiting growth compared to young patients.The condylar fibrocartilage of the TMJ is unique compared to other articular cartilages. Specifically, the TMJ fibrocartilage acts like a growth plate cartilage and undergoes endochondral ossification to promote condylar growth28. Further, this analysis indicates the multiple receptors in which the estradiol ligand can interact and promote transcription beyond strictly binding to ERα in the growing mandibular condylar fibrocartilage. On the other hand, in the mature mandibular condylar fibrocartilage, all the genes that were up-regulated with estradiol were modulated via ERα suggesting this receptor is the sole mediator of estrogen-induced changes to the mature mandibular condylar fibrocartilage. In young growing mice, the mandibular condylar cartilage contains a large number of fibrocartilage progenitor cells29 that decrease in number with age30. As such, in the 17-week mice, the role of estrogen in mediating the activity of these progenitor cells may be diminished and only requires ERα for regulation. Overall, our results provide evidence for a differential role that ERα plays in the skeletally immature versus mature mandibular condylar fibrocartilage of female mice.Also, it was determined that in the absence of ERα, estradiol promoted myogenesis and/or muscle homeostasis in the growing, female mandibular condylar fibrocartilage whereas estradiol via ERα regulated non-specific extracellular cell processes. Therefore, estrogen treatment in young mice may highlight a potential novel role of ERα signaling in directing the mandibular condylar cartilage progenitor cells down the chondrogenic pathway as opposed to the myogenic pathway29 whereas an increase in canonical Wnt signaling decreases mandibular condylar fibrocartilage chondrogenesis31. In this study, it was determined that estradiol significantly increased Sost mRNA and protein expression in WT but not in ERαKO mice. Further, we determined that phosphorylated (inactive) β-catenin staining was also enhanced with estradiol treatment in WT mice with no observable change in ERαKO mice  are the cells most heavily expressing and responding to Sost, especially in response to estradiol treatment. Thus, these results suggest estradiol via ERα may promote mandibular condylar chondrogenesis by suppressing the canonical Wnt signaling pathway via Sost. However, in addition to Sost, it is possible that estrogen via ERα directly regulates transcriptional activation of Col234. Therefore, multiple studies are planned with the Sost antibody and the Col2 promoter to delineate the signaling pathway mediating estrogen’s transcriptional regulation of Col2 in the mandibular condylar fibrocartilage.It is evident from this study that estrogen replacement treatment for four weeks promoted TMJ chondrogenesis. To investigate the mechanism, we assessed the gene expression of a range of the cell fate mediators and transcription factors known to regulate differentiation of the progenitor cells There was no significant change in gene expression of any factors of the TGFβ family, Sox9, Pthrp/Ihh, or Notch that were studied in response to estradiol treatment. Previously, it has been shown that inhibiting the canonical Wnt pathway via Sost promotes chondrogenesis in the mandibular condylar fibrocartilage36. In this study, it was observed that estradiol significantly upregulated anabolic genes that function in the extracellular matrix of the articular fibrocartilage. Further, it was seen that estradiol via ERα decreased protease gene expression and cleavage of Col1 and Col2 in the fibrocartilage matrix. Previous characterization of the aging mandibular condylar fibrocartilage has illustrated the necessity of protease activity in the joint to maintain articulation after growth is complete37. Our analysis indicates estradiol plays a role in this process. RNA sequencing differential analysis pointed to the upregulation of protease inhibitors as a possible mechanism by which estradiol inhibits protease activity via ERα. Specifically, the protease inhibitors Pi15 and A2m were the two most significantly regulated genes and both shown to reduce MMP9 activity in the mandibular condylar fibrocartilage explant model. Proteinase 15, or peptidase 15, is a gene that encodes a trypsin inhibitor. To our knowledge, there is very limited understanding of Pi15 activity in specific tissues and cells38. As such, this presents a unique target to further pursue in homeostasis and degeneration of the mandibular condylar fibrocartilage. Future studies utilizing a wider range of Pi15 peptide concentrations and the recombinant peptide instead of synthetic are necessary to further probe the effect of Pi15 in homeostasis of the mandibular condylar fibrocartilage.The existence of a link between estrogen deprivation in post-menopausal women and TMJ-DD highlights the role estrogen may play in homeostasis of the mandibular condylar fibrocartilage. Specifically, clinical data suggests women with a single nucleotide polymorphism in the ERα gene may be at increased risk of developing TMJ-DD41. Cleavage of a unique set of amino acids found near the center of the polypeptide chain known as the “bait” region triggers a conformational change and resulting irreversible entrapment of the protease42. Consequently, a receptor binding domain is then presented on the surface of the A2m-proteinase unit that signals for cell-mediated endocytosis followed by proteolytic degradation within the lysosomes45. Studies have investigated the role of estrogen or similar molecules on A2m expression. Lim et al. illustrated an increase in A2m gene expression with administration of diestylstilbestrol, a synthetic estrogen, in chicken ovaries46. Interestingly, treating bovine granulosa cells with A2m results in a dose-dependent increase in estradiol production47. Further, the exact mechanism by which estrogen promotes transcription of A2m is not clear. In these studies, we show estradiol treatment increases A2m gene expression in only WT not ERαKO mice at 17-weeks. However, other studies in bone have shown A2m upregulation via estradiol independently of either ERα or ERβ suggesting this is not the sole mechanism48. Current studies pursuing the exact receptor and membrane vs. nuclear signaling are underway to pursue the mechanism in more depth.A2m is a large, tetrameric glycoprotein found in plasma of vertebrates and functions as a nonspecific protease inhibitor that has the ability to block proteases from all four classes49. In analyzing synovial fluid and in vitro chondrocyte culture from human patients with OA, A2m levels were significantly lower in OA patient synovial fluid and A2m treatment in chondrocyte culture decreased MMP3, MMP9, and MMP13 expression. Currently, the mechanism and timeline for A2m’s effects on downstream gene transcription have not been determined. However, our data illustrating a potential effect of A2m on MMP transcription coupled with past results in chondrocytes provides new avenues of study. Excitingly, a Phase 1/II clinical trial is currently underway investigating A2m for the treatment of tibiofemoral joint pain due to cartilage degeneration, osteoarthritis, inflammation, and meniscal degeneration. These studies are based on autologous A2m obtained from either the patient’s own blood or plasma in a relatively quick procedure at the medical office50. Further, recent datum suggests that molecular variants of A2m that can be synthesized prior to surgery are effective at reducing knee osteoarthritis in a rat model and provide an additional option for clinical feasibility51. As such, there is potential for similar treatment to reduce TMJ degeneration utilizing A2m.A2m has been shown previously to be a potential inhibitor of posttraumatic osteoarthritis in the knee53. Serum levels of these other hormones were not assessed but should be conducted in the future to further ascertain potential effects. Also, the importance of the subchondral bone in the bone-cartilage unit of the TMJ regarding joint health and OA diagnosis has been discussed. Recent studies from Shi et al. illustrate the correlation of low condylar bone quality as determined by bone mineral density and bone volume/total volume with increased TMJ-OA and the model as use for diagnostics in the future54. However, in these studies, we did not assess changes to the subchondral bone or condyle in response to OVX and estradiol treatment in either WT or ERαKO mice. Future studies must focus on the potential role of estradiol via ERα on the bone of the TMJ. Also, recent evidence from Ucer et al. illustrate distinct pathways by which sex steroid deficiency and aging promote bone loss independently55. Thus, it is likely that aging-specific mechanisms including mitochondrial dysfunction, reactive oxygen species accumulation, and DNA damage also play a role in mandibular condylar fibrocartilage degeneration independent of estrogen signaling. Lastly, while the role of estrogen via ERα on the articular fibrocartilage of the TMJ was assessed globally, future studies on the role of the receptor in the specific cells using cell-specific conditional knock-outs will be beneficial.We recognize every study has limitations. In the studies presented here, only the role of estradiol signaling on the mandibular condylar fibrocartilage was assessed. Insulin growth factor, progesterone, and relaxin levels are altered after ovariectomy and have been shown to affect articular cartilage and subchondral boneIn conclusion, the studies presented here illustrate the probable roles of estrogen via ERα on the growth and homeostasis of the mandibular condylar fibrocartilage of the TMJ. By investigating changes in young mice with growing condyles and comparing to adult mice with mature tissue, the importance of estrogen signaling at different ages was determined. In young tissue, Sost levels may be crucial to maintain healthy fibrocartilage growth and inhibit excess turnover and hypertrophic chondrogenesis. In older tissue, inhibition of protease activity is vital in supporting healthy extracellular matrix tissue that functions to withstand the loads of the TMJ. Both processes are controlled by estrogen via ERα signaling. Thus, targeting ERα with an agonist or delivering a protease inhibitor such as A2m to the mandibular condylar fibrocartilage may be an effective treatment to reduce TMJ-DD in post-menopausal women and partially address one component of the sex dimorphism of TMJ disease.-/+ (Ex3αERKO56) heterozygous male and heterozygous females were donated from Dr. Kenneth Korach at the National Institute of Environmental Health Sciences at National Institutes of Health to breed female WT and ERαKO mice. Total ERα−/− mice have increased sex steroid levels because of disturbed negative feedback regulation10. Thus, to avoid confounding endogenous sex steroid effects, all mice were ovariectomized and treated with placebo or estradiol to determine the effect of estradiol via ERα. WT and ERαKO female mice were divided into three groups: sham, ovariectomy with placebo treatment, and ovariectomy with estradiol treatment . To determine effects in the growing mandibular condylar fibrocartilage, 3-week old mice were treated for 4 weeks with placebo or estradiol. For the effects on the mature fibrocartilage, 13-week old mice were treated for 4 weeks. Mice were administered placebo or 17β-estradiol  for 4 weeks at a daily dose of 11 ng/g body weight for 7-week old mice (assuming 15 g body weight) and 7 ng/g body weight for 17-week old mice (assuming 25 g body weight). Doses similar to that used in this study resulted in serum estradiol concentrations of roughly 600 pM48. Normal serum levels of estradiol are between 70 and 110 pM during diestrus and between 350 and 730 pM during estrus of the female mouse estrous cycle57. Thus, the estradiol treatment in this study likely resulted in levels comparable to what is experienced during estrus. Further, the dose of estradiol utilized in these study matches the dose found effective to restore MCC thickness in female, ovariectomized WT mice of the same age58. Following 4 weeks of treatment, mice were injected intraperitoneally with 0.1 mg bromodeoxyuridine (BrdU) per gram body weight at both nineteen and three hours prior to euthanasia to track proliferating cells. Body and uterus weights were measured post sacrifice (data not included). For RNA sequencing, WT and ERαKO mice at 6- and 16-weeks were ovariectomized and treated with placebo or 17β-estradiol  for 1 week at doses detailed above . Lastly, WT mice at 13-weeks were utilized for the explant culture study .All experiments were performed in accordance with animal welfare based on an approved Institutional Animal Care and Use Committee (IACUC) protocol (#AAAH9166) from Columbia University. Breeding pairs of C57BL/6 ERαKOHistomorphometry techniques were employed to determine the effect of estradiol treatment via ERα on mandibular condylar fibrocartilage morphology. Whole mouse heads were sectioned into halves, fixed in 10% formalin for 4 days at room temperature and decalcified in 14% ethylenediaminetetraacetic acid (EDTA) (pH 7.1)  for 28 days. Subsequently, the samples were processed through progressive concentrations of ethanol, cleared in xylene, and embedded in paraffin. Sagittal serial sections of 5 μm thickness were made of the TMJ utilizing a Microm HM 355 s microtome . Sections representing the mid-coronal portion of the mandibular head were stained with hematoxylin and eosin (H&E) and Safranin-O (SafO) and used as the representative central section for analysis.Histomorphometry measurements were made in a blinded, nonbiased manner using the BioQuant computerized image analysis system . Analysis was performed on H&E sagittal sections corresponding to the mid-coronal portion of the mandibular condylar head. Average thicknesses were determined for the total cartilage thickness  at 37 °C  or 0.01 M citrate buffer  for 5 minutes at 60 °C  for unmasking, washed with PBS, and treated with a 3 vol% hydrogen peroxide in methanol solution for 10 minutes at room temperature to inhibit endogenous peroxidase activity. All sections were blocked with 10% normal goat serum (Life Technologies) to reduce non-specific binding of the antigen with the primary antibody. Immunohistochemical staining was performed using the SuperPicture™ Polymer HRP Broad Spectrum Detection Kit (Life Technologies) following the procedure recommended by the manufacturer. Further details are in the Supplementary Materials and Methods.et al.19. Expression of each gene of interest was determined relative to the Gapdh housekeeping gene (Gapdh – MM99999915_g1) utilizing the ΔΔCT method. Gene expression was analyzed for the following markers: collagen type 2 (Col 2a1 – Mm00491889_m1), collagen type 10 (Col 10a1 – Mm00487041_m1), SRY-box containing gene 9 (Sox9 – MM00448840_m1), parathyroid hormone-related peptide (PTHrP – Mm00436057_m1), indian hedgehog (Ihh – Mm00439613_m1), runt-related transcription factor 2 (Runx2 – Mm00501578_m1), fibroblast growth factor 2 (FGF2 – Mm00433287_m1), bone morphogenetic protein 2 (BMP2 – Mm01340178_m1), transforming growth factor beta – 2 (TGFβ-2 – Mm00436955_m1), mothers against decapentaplegic homolog 4 (SMAD4 – Mm03023996_m1), translocation-associated notch homolog 1 (Notch1 – Mm00627185_m1), sclerostin (Sost – Mm00470479_m1), lymphoid enhancer-binding factor 1 (LEF1 – Mm00550265_m1), twist-related protein 1 (Twist1 – Mm00442036_m1), protease inhibitor 15 (Pi15 – Mm00499734_m1), alpha-2-macroglobulin (A2m – Mm00558642_m1), matrix metalloproteinase 3 (MMP3 – Mm00440295_m1), matrix metalloproteinase 13 (MMP13 – Mm00439491_m1), matrix metalloproteinase 9 (MMP9 – Mm00442991_m1), aggrecanase-1 (ADAMTS4 - Mm00556068_m1), and aggrecanase-2 (ADAMTS5 – Mm00478620_m1). All primers were purchased from Applied Biosystems.After duration of treatment, mRNA from the condylar cartilage of all groups was extracted to analyze the effects of estradiol on chondrogenic markers and proteases via ERα. For each mouse, the MCC (left and right) was carefully isolated from all other soft tissue and dissected under a dissecting microscope. mRNA was extracted with TRIzol Reagent (Ambion by Life Technologies) following the manufacturer’s protocol and treated with DNase treatment and removal kit  to remove any residual DNA contaminants. Reverse transcription was performed to convert mRNA to cDNA utilizing the High Capacity cDNA Reverse Transcription Kit  in RNase-free conditions following the manufacturer’s protocol. Real-time polymerase chain reaction (RT-PCR) was conducted to assess the relative levels of genes of interest using the ViiA™ 7 Real-Time PCR System  following the protocol detailed in Chen RNA was extracted and purified from mandibular condylar fibrocartilage cells utilizing PureLink™ RNA Mini Kit  and following the manufacturer’s protocol. Volcano plots illustrating gene significant values as a function of log2 fold change were generated using GraphPad Prism. Selected genes that were differentially regulated in response to estradiol treatment with an adjusted p < 0.01 were analyzed utilizing the PANTHER overrepresentation test (release 20170413) and the GO Ontology database (released 2017-04-24) to determine the biological processes affected by estradiol treatment. Heat maps illustrating the relative gene expression were fabricated utilizing Gene Pattern from the Broad Institute and represent z-score values calculated by subtracting the sample mean from the raw expression value and then dividing by the standard deviation of the sample. Additional details can be found in the Supplementary Materials and Methods.−8 M, or 10−6 M 17β-estradiol , 100 nM and 200 nM A2m , or 50 nM and 100 nM Pi15  was added and incubated for 48 hours. Estradiol concentrations were chosen based on similar studies21 and further substantiated by evidence of circulating estrogen levels in humans59. A2m concentrations were determined based on previous evidence in a knee osteoarthritis model49. After 48 hours of treatment, supernatant was collected for zymogram assays and collagen type I and II cleavage ELISAs. Messenger RNA from both left and right samples was pooled together and utilized for RTPCR. For gelatin zymography and ELISA analysis, supernatant from only the left specimen was utilized.Explant cultures were utilized to further investigate the role of both estradiol and A2m on condylar fibrocartilage degradation. Entire mandibles with condyle and condylar fibrocartilage intact were harvested from 13-week old WT female mice and cultured in a 24-well plate with 1.5 mL of FBS-free BGJb medium supplemented with 1% Penicillin-Streptomycin (ThermoFisher Scientific) overnight. Following overnight incubation, media was removed, samples were washed 2 × with sterile PBS, and fresh media with either 0 M, 10Matrix metalloprotease (MMP) activity in response to A2m treatment to mandible explant cultures was assessed using gelatin zymography. After 48 hours of treatment in explant culture, supernatant (1.5 mL) was aspirated from the samples, concentrated utilizing a centrivap benchtop vacuum concentration (Labconco), reconstituted in 150 µL BGjb medium, and stored in −80 C until used. Tris-glycine gels (10%) with 0.1% gelatin incorporated as a substrate (ThermoFisher Scientific) were utilized and zymography run according to manufacturer’s protocol.After 48 hours of treatment in explant culture, supernatant (1.5 mL) was aspirated from the samples, concentrated utilizing a centrivap benchtop vacuum concentration (Labconco), reconstituted in 150 µL BGjb medium, and stored in −80 C until used. Detection of collagen type 1 and 2 cleavage fragments in explant culture were measured using a C1,2 C ELISA kit  according to manufacturer’s instructions.p < 0.05 or ^p < 0.01 as indicated in the figure legends.Values are presented as the mean ± standard deviation. Statistical significance of differences among means was determined by one-way analysis of variance (ANOVA) for protease gene expression datum or two-way ANOVA for all other data to account for genotype effects (WT vs. ERαKO). Post hoc analysis by the Bonferonni method using GraphPad Prism 7.03 was conducted for all data sets. Statistical significance was defined as either *All data generated and analyzed during this study are either included in this publication and the Supplementary Information, accessible through the NCBI GEO database (GSE110304), or available from the corresponding author upon reasonable request.Supplementary Information