Objective: To determine the symptomatic and disease-modifying capabilities of sEH and COX inhibitors during joint inflammation.Methods: Using a blinded, randomized, crossover experimental design, 6 adult healthy horses were injected with lipopolysaccharide  from E. coli in a radiocarpal joint and concurrently received the non-selective cyclooxygenase (COX) inhibitor phenylbutazone (2 mg/kg), the sEH inhibitor t-TUCB (1 mg/kg) or both  intravenously. There were at least 30 days washout between treatments. Joint pain , synovial fluid concentrations of prostanoids , cytokines  and biomarkers of collagen synthesis (CPII) and degradation (C2C) were measured at pre-determined intervals over a 48-h period. The anti-apoptotic effect of COX and sEH inhibitors was determined via ELISA technique in primary equine chondrocytes incubated with TNF-α (10 ng/ml) for 24 h. Apoptosis was also determined in chondrocytes incubated with sEH-generated metabolites.Results: Combined COX and sEH inhibition produced significantly better control of joint pain, prostanoid responses, and collagen synthesis-degradation balance compared to each compound separately. When administered separately, pain control was superior with COX vs. sEH inhibition. Cytokine responses were not different during COX and/or sEH inhibition. In cultured chondrocytes, sEH inhibition alone or combined with COX inhibition, but not COX inhibition alone had significant anti-apoptotic effects. However, sEH-generated metabolites caused concentration-dependent apoptosis.Conclusions: Combined COX and sEH inhibition optimize pain control, attenuate loss of articular cartilage matrix during joint inflammation and cytokine-induced chondrocyte apoptosis. Inflammation of the synovial lining  and cartilage damage are prominent features of osteoarthritis (OA), strongly correlating with pain sensitization and disease severity. Synovitis can be present in all stages of OA, tends to worsen with radiographic severity and may contribute to the progression of cartilage damage –5. In eqLameness and pain continue to be the primary complaint associated with OA in horses and humans. In horses and humans, cyclooxygenase (COX) inhibitors are first-line therapies to control OA pain yet this strategy is only partially effective and can cause serious adverse effects in both species , 15. In t-TUCB (trans-4-{4-[3-(4-Trifluoro-methoxy-phenyl)-ureido]-cyclohexyloxy}-benzoic acid) and several others developed for the human enzyme are equally potent against equine sEH  aged 8.5 ± 3 years (range 5–13 years old) and weighing 462 ± 50 kg (range 397–536 kg) were used. Horses were considered healthy and free of radiocarpal joint disease on the basis of complete veterinary work-up that included general physical and orthopedic examinations, complete blood cell counts and plasma chemistry profile. Horses were housed as a group on paddocks for the duration of the study with water available t-TUCB, was kindly provided by Dr. Bruce Hammock , synthesized and characterized according to established methodology  or from Sigma-Aldrich, Inc. , and the 8,9-, 11,12-, and 14,15-DiHETs were obtained from Cayman Chemical . The sEH inhibitor, hodology –44. EquiE. coli O55:B5  into one randomly assigned radiocarpal joint for the first injection, with subsequent injections alternating between joints, as in a previous study from our laboratory (t-TUCB (1 mg/kg) or a combination of both drugs . We intentionally chose a relatively moderate dose of phenylbutazone to avoid fully blocking pain or prostanoid production, reasoning that this would allow interactions with the varying doses of the sEH inhibitor to be revealed. t-TUCB was dissolved in dimethyl sulfoxide  to final concentrations of 100, 30, and 10 mg/ml and filter-sterilized with 0.2 μm pore size sterilizing-grade membranes prior to administration (0.01 ml/kg). Drugs were administered slowly (30–45 s) as a single intravenous injection using separate jugular vein catheters, at the same time that the joints were injected with LPS. In the phenylbutazone-only treatment, horses also received the vehicle diluent of t-TUCB  to control for possible DMSO anti-inflammatory effects . Treatment responses were determined by assessing lameness (pain on ambulation) in a straight-line trot on a hard, flat surface. Kinematic parameters were assessed by calculating the vector sum of the head height difference relative to the stride cycle using an inertial sensor system   and synovitis was induced by injecting 2 ml of a freshly prepared solution of lipopolysaccharide (LPS) from boratory . There wlowly 30– s as a s effects  Horses iMO, USA) , 48 and 2, thromboxane (Tx) B2 , interleukin (IL)-β, IL-6, and tumor necrosis factor (TNF)-α . Use of equine synovial fluid with type II collagen biomarkers have been previously validated  all synovial fluid samples for type II collagen synthesis (CPII) and degradation (C2C) biomarkers , prostaglandin (PG) Ealidated , as have2 at 37°C until reaching ~80–90% confluency and then passaged to 12-well culture dishes at a density of 10,000 cells/ml for 24 h. Next, the chondrocytes were changed to serum-free medium and incubated with TNF-α (10 ng/ml) along with the COX inhibitor PBZ, the sEH inhibitor t-TUCB or both at several concentrations for 24 h. The inhibitors were used at their approximate half-maximal inhibitory concentration or IC50 , 80% inhibitory concentration or IC80  and 10-fold > IC80  for the equine enzymes . At the end of the 24-h incubation period, cells were harvested and apoptosis was determined in duplicates using ELISA technique as per the manufacturer's instructions .Primary equine articular chondrocytes of second passage  were cultured in high-glucose Dulbecco's Modified Eagle Medium  supplemented with 10% heat-inactivated fetal calf serum and 1% penicillin/streptomycin (Sigma-Aldrich) in an atmosphere with 5% CO enzymes , 50. In in vivo studies were analyzed using linear mixed-effects model whereby treatment and time were included as fixed factors and horse as random factor if normally distributed, or the Friedman test if not. Normality was determined primarily by visual inspection of QQ plots and the Shapiro-Wilk normality test. Chondrocyte apoptosis data were considered normally distributed by visual inspection of QQ plots and the Shapiro-Wilk normality test, and were analyzed by One- or Two-way ANOVA. In all cases, the two-stage step-up method of Benjamini, Krieger, and Yekutieli was used as multiple comparison test while correcting for multiple comparisons by controlling the False Discovery Rate. The Geisser-Greenhouse correction was applied for data showing different scatter. Since there were missing data for TxB2, IL-1β, IL-6, and TNF-α, the number of analyzed data points is indicated in the text. Significance level was set at p < 0.05. Data are presented as mean±SEM.Data were analyzed using GraphPad Prism . Areas under the curves (AUC) were calculated using the trapezoidal method for some of the variables, as indicated in the Results section. Data from the Results of kinematic and kinetic parameters are shown in 2 were significantly lower during treatment with combined inhibition of COX and sEH compared to COX or sEH inhibition individually. All doses of sEH significantly potentiated the inhibition of PGE2 production by the COX inhibitor. We also determined the synovial fluid concentrations of TxB2 during treatment with the highest dose of the sEH inhibitor (1 mg/kg t-TUCB) alone or when combined with the COX inhibitor (n = 4/6 horses). Results showed that TxB2 levels were not significantly different between COX inhibition alone and the combined COX and sEH inhibition, but was significantly higher than either of these during sEH inhibition alone. Thus, sEH inhibition added a significant inhibitory effect on COX-2-generated PGE2 but not on COX-1-generated TxB2. We also assessed cytokine responses within the joint (n = 4/6 horses), IL-6 (n = 4–6/6 horses), and TNF-α (n = 5–6/6 horses), given their demonstrated association with joint pain and cartilage loss in OA  similar TxB2 levels between combined COX and sEH inhibition and COX inhibition alone, and (ii) significantly higher TxB2 during sEH inhibition alone. Taken together, these results demonstrate that sEH inhibition added a significant inhibitory effect on COX-2, but not on COX-1, during synovitis, which should mitigate the activation of PGE2-responsive signaling pathways in chondroncytes  vs. “classical” signaling via its membrane bound receptor (mIL-6R)  . Investi(mIL-6R) . A decre(mIL-6R) . Further(mIL-6R) .We also sought to determine whether or not COX and/or sEH inhibition could protect the articular cartilage matrix during synovitis because (i) type II collagen degradation by matrix metalloproteinases (MMP) correlates with symptomatic radiographic and pre-radiographic OA , (ii) cy2 and improved collagen synthesis-degradation balance. These are important findings because PGE2 is pro-nociceptive, activating PGE2-responsive signaling pathways in chondrocytes that sensitizes them to apoptosis (The medical treatment of OA focuses primarily on alleviating pain symptoms, and no currently available therapy also targets the joint pathology and progressive cartilage damage. In our study, targeting sEH in addition to COX during synovitis and joint inflammation resulted in significantly better pain control, lower synovial fluid concentration of PGEpoptosis , 65, 66,poptosis . Since Tpoptosis , 52 and poptosis . An incrpoptosis  might expoptosis . Taken tpoptosis , we suggOur study has several possible limitations worth considering. The majority of our horses were castrated males and only one was a sexually intact female, thus the study was not balanced by sex or gonadal status. While there has been no study examining gonadal status or sex predilection for OA in horses, the condition tends to be more prevalent in older women than in men and is attributed to changes in sex hormones associated with menopause . The repIn summary, our study provides multiple lines of evidence suggesting that combined COX and sEH inhibition provides an effective mode of therapy in this large animal model. Our findings indicate that this therapeutic strategy would enhance symptomatic pain control and attenuate loss of articular cartilage matrix and apoptotic death of chondrocytes, ultimately resulting in reduced cartilage damage and disease progression.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.The animal study was reviewed and approved by University of Minnesota Institutional Animal Care and Use Committee.AG and TT carried out the analysis and interpretation of the data. LT, DG, ED, CB, EW-H, and AG collected and assembled the data. TT and AG drafted the manuscript. LT, TT, DG, ED, CB, and EW-H revised the article for important intellectual content. TT and AG obtained funding for conceived and designed the experiments. All authors provided final approval of the article prior to submission.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


PtenPC−/− mice and patient-derived xenograft (PDX) models. Mechanistically, the SWI/SNF ATPase subunit Brg1 and the nuclear receptor AR (androgen receptor) were identified as essential substrates for OTUD6A in PCa cells by a mass spectrometry (MS) screening approach. Furthermore, OTUD6A stabilized these two proteins by erasing the K27-linked polyubiquitination of Brg1 and K11-linked polyubiquitination of AR. OTUD6A amplification exhibited strong mutual exclusivity with mutations in the tumor suppressors FBXW7 and SPOP. Collectively, our results indicate the therapeutic potential of targeting OTUD6A as a deubiquitinase of Brg1 and AR for PCa treatment.Ovarian tumor (OTU) subfamily deubiquitinases are involved in various cellular processes, such as inflammation, ferroptosis and tumorigenesis; however, their pathological roles in prostate cancer (PCa) remain largely unexplored. In this study, we observed that several OTU members displayed genomic amplification in PCa, among which ovarian tumor deubiquitinase 6A (OTUD6A) amplified in the top around 15–20%. Further clinical investigation showed that the OTUD6A protein was highly expressed in prostate tumors, and increased OTUD6A expression correlated with a higher biochemical recurrence risk after prostatectomy. Biologically, wild-type but not a catalytically inactive mutant form of OTUD6A was required for PCa cell progression. In vivo experiments demonstrated that OTUD6A oligonucleotides markedly suppressed prostate tumorigenesis in  OTUD6A, a deubiquitinase, is amplified in prostate cancer and correlates with poor survivability, increasing the growth of prostate cancer cell lines and PDX models. OTUD6A stabilizes the expression of Brg1 and AR through the removal of K27- and K11-linked polyubiquination. The treatment strategies for localized PCa mainly include androgen deprivation therapy or AR (androgen receptor) inhibitor treatment to antagonize AR signaling4. However, drug resistance and tumor recurrence often occur and lead to lethal castration-resistant prostate cancer (CRPC), which has a median survival time of approximately 14 months5. Thus, discovering novel therapeutic targets is necessary for the treatment of PCa, especially for CRPC.Prostate cancer (PCa) accounts for 7% of newly diagnosed cancers in men and is one of the leading causes of cancer-associated death in men worldwideSPOP has been reported to be the most common gene with single mutations in prostate cancer, and mutations of SPOP occur in up to 15% of PCa cases7. SPOP exerts its tumor suppressor roles mainly by promoting the degradation of its oncogenic substrates, such as AR, ERG, BRD4, and Nanog9. As such, targeting the specific substrates could optimize the treatment strategy for PCa patients with different genetic statuses of SPOP11. Another example is the HECT-type E3 ubiquitin enzyme NEDD4, which has been found to be amplified and overexpressed in prostate cancer. As a specific E3 ligase for the tumor suppressor PTEN, NEDD4 can either promote PTEN degradation through polyubiquitination or import PTEN into the nucleus through monoubiquitination13. In addition, a number of deubiquitinases (DUBs) have emerged as alternative and important therapeutic targets for prostate cancers. For example, DUB3 can promote BET inhibitor resistance and PCa progression by deubiquitinating BRD4 and is considered a viable therapeutic target to overcome BET inhibitor resistance in PCa14. To date, approximately 100 human DUBs have been identified and classified into six families: USPs, UCHs, OTUs, MJDs, JAMMs, and MCPIPs16. Here, we found that many OTU family members displayed high DNA amplification with unclear function in PCa.Recently, specific ubiquitination enzymes or deubiquitination enzymes have attracted much attention for their regulation of the stability and activity of key proteins in PCa. Among these, CUL318. For example, OTUB1 depletion affects DNA damage repair because OTUB1 binds to and inhibits UBC1319, OTULIN regulates cell death and inflammation by deubiquitinating LUBAC20, and OTUD7B binds and deubiquitinates TRAF3, thereby controlling NF-κB activation21. A20 regulates multiple immune cell functions and autoimmunity by regulating ubiquitin-dependent NF-κB and cell survival signals22. Here, by mining diverse public PCa datasets, we found that many OTU family members showed high DNA copy number amplification, among which OTUD6A was the most frequently amplified. OTUD6A was previously reported to deubiquitinate and stabilize Drp1 to regulate mitochondrial morphology in colon cancer23. In this study, we report that OTUD6A can stabilize Brg1 and AR by removing Brg1 K27-linked polyubiquitination and AR K11-linked polyubiquitination, respectively, to promote PCa progression in vitro and in vivo, suggesting that targeting OTUD6A is a reasonable strategy for PCa therapy.OTU family members, also called OTU domain-containing deubiquitinases, have emerged as regulators of important signaling cascades, the abnormal expression, and activation of which are thought to be vital pathogenic factors in diverse human diseases and pathological processes, such as cancer, infection, and inflammationOTUD6A and OTUD6B displayed high amplification rates (15–20%) in all three datasets , we observed that many OTU family members showed high genomic amplification in PCa, among which ets Fig. . This fiOTUD6A mRNA expression in human prostate cancer samples by analyzing the TCGA, Michigan, and GSE21032 prostate cancer datasets24. The results showed that OTUD6A mRNA levels were highly increased in tumors compared to normal tissues among these datasets   . We found that compared to that in adjacent normal tissues, the OTUD6A protein level in prostate tumor tissues was remarkably elevated Fig. . Moreove01) Fig. . TogetheOTUD6A in the PCa cell lines LNCaP, C4-2, VCaP and PC-3 by using two different shRNA constructs. Importantly, we found that the growth and colony formation of these PCa cells were markedly inhibited upon OTUD6A depletion  but not the catalytically inactivated form of OTUD6A (C152A) reversed the reduced cell proliferation mediated by OTUD6A knockdown in PCa cells  could partially rescue the decreased Myc-CaP tumorigenesis mediated by OTUD6A depletion in a syngeneic immunocompetent FVB mouse model  was injected into the ventral prostate (VP) tissues of 2-month-old PtenPC−/− mice. Three months later, the physiological phenotype and tumorigenesis were monitored, and the results demonstrated that the size of the ventral prostate glands in AAV-shOTUD6A-injected PtenPC−/− mice were remarkably reduced compared with that in AAV-GFP group mice, and Ptenfl/fl mice were used as the negative control  model with a surgical specimen of radically resected prostate tumor from a patient with a postoperative pathological diagnosis of acinar adenocarcinoma, Gleason score 5 + 4. Similarly, the xenograft growth in mice with oup Fig. . These r27, whereas PC-3 cells are deficient in AR -mass spectrometry (MS) approach in two CRPC cell lines VCaP and PC-3; VCaP cells are positive for AR expression and show a growth response in the presence of androgen29, and AR is commonly recognized as a key driver of prostate cancer, even CRPC3. Therefore, we hypothesized that Brg1 and AR are both essential substrates for OTUD6A to perform its oncogenic roles in PCa. To this end, we initially detected that OTUD6A could immunoprecipitate both Brg1 and AR endogenously in VCaP cells, while Brg1 only bound to OTUD6A but not AR, indicating that Brg1 and AR form complexes with OTUD6A individually  is an alternative catalytic ATPase subunit present in the SWI/SNF complex that shares 75% sequence homology with Brg1Collectively, our results indicate that OTUD6A binds to Brg1 and AR individually to assemble distinct complexes.32 thus, we determined whether OTUD6A could affect the stability of its substrates Brg1 and AR. We found that depletion of OTUD6A remarkably reduced Brg1 and AR protein levels; without affecting their mRNA levels in VCaP, LNCaP, and PC-3 cells . The results showed that, upon ectopic expression of OTUD6A, Brg1 polyubiquitination was diminished in 293 T cells transfected with wild-type or K27-only mutant ubiquitin, suggesting that OTUD6A mainly cleaves K27-linked polyubiquitin chains in Brg1 41, were found to be differentially expressed (34 upregulated and 16 downregulated) by at least 2.0-fold (P < 0.05) upon depletion of either OTUD6A or Brg1 in prostate cancer cells  is associated with aggressive PCa45. Regulation of AR and Brg1 can be mediated through genetic or epigenetic modifications or other mechanisms, among which ubiquitination is one of the most important. Specific ubiquitin ligases, such as SPOP, Siah2, and RNF6, and deubiquitinases, such as USP14, USP26, and USP12, have been reported to be involved in the regulation of AR via the ubiquitin-proteasome system (UPS)47. With regard to Brg1, SCFFBW7 has been reported to be the Cullin3 ubiquitin ligase mediating the polyubiquitination and degradation of Brg136. However, there are few reports about the deubiquitination enzymes of Brg1. Here, we demonstrate that Brg1 and AR can specifically bind to the deubiquitinase OTUD6A, which erases FBW7-mediated polyubiquitin chains of Brg1 and SPOP-mediated polyubiquitin chains of AR in PCa cells.In prostate cancer, AR plays a pivotal role in the regulation of tumorigenesis and tumor progression. In addition to AR, a number of transcriptional regulators, such as ERG, BRD4, and Brg1, have also been identified as crucial effectors for PCa tumorigenesis48 OTUD5/DUBA is involved in the regulation of interferon signaling;49 OTUD2/YOD1 and VCPIP are involved in the regulation of p97-mediated processes;50 and OTUB1 in the regulation of the DNA damage response17. In addition, the OTU family has been reported to be involved in the regulation of cancers. For example, OTUB2 promotes breast cancer metastasis by stabilizing the YAP/TAZ protein51, and Snail1-OTUD7A (Cezanne2) signaling participates in the regulation of hepatocellular carcinoma cell proliferation and metastasis52. OTUD3 facilitates PTEN-related tumor suppressive responses and inhibits breast tumorigenesis, while in lung cancer, OTUD3 promotes tumorigenic phenotypes by stabilizing GRP7854. OTUD6A was reported to deubiquitinate and stabilize Drp1 and to regulate mitochondrial morphology in colon cancer23. In our study, we found that many OTU family members exhibited DNA amplification in PCa, while OTUD6A showed a specific and high amplification rate (15–20%), which emphasizes the potential roles of OTUD6A in the regulation of PCa. In vivo and in vitro studies showed that wild-type OTUD6A but not its catalytically inactive mutant C152A promoted PCa progression, highlighting that the function of OTUD6A in PCa cells is dependent on its deubiquitination activity. In addition, the ubiquitination assay showed that the C152A mutation largely but not completely caused OTUD6A to lose its deubiquitinase activity, as reported23, suggesting that an additional catalytic site might be involved in the regulation of the deubiquitination activity of OTUD6A.OTUD6A belongs to the OTU family of DUBs. Sixteen OTU DUBs have been revealed to exist in humans, and most OTU family members regulate cell signaling cascades. For example, A20, OTUD7B/Cezanne, and OTULIN are involved in the regulation of NF-κB signaling;PtenPC−/− mice and a PDX model of PCa, suggesting therapeutic potential of targeting OTUD6A in PCa. Ding et al. reported that Pten loss stabilizes the Brg1 protein by regulating the AKT/GSK3β/FBXW7 axis28. In our study, we found that OTUD6A was upregulated in Pten-depleted prostate cancer tissues ; PC-3, VCaP, and LNCaP cells were kindly provided by Cell Bank, Chinese Academy of Sciences; Mouse prostate cancer cell line Myc-CaP was obtained from Dr. Xuanming Yang ; Human embryonic kidney 293 (HEK293T) cell line was obtained from Dr. Wenyi Wei . All the cell lines were cultured according to ATCC instructions. Cells were detected to be mycoplasma-free, and were characterized by Genesky Biopharma Technology using short tandem repeat (STR) markers. Reagents used in this study included MG132 (Meilun Biotech), Puromycin (Meilun Biotech), and Cycloheximide (CHX) (MedChemExpress).The pcDNA3.1-Flag-OTUD6A, pcDNA3.1-Flag-OTUD6A-C152A, pcDNA3.1-Flag-OTUD6B, and pCMV5-HA-Brg1 plasmids were obtained from Dr. Wenyi Wei . The pcDNA3.1-HA-AR and pcDNA3.1-HA-BRM were constructed by cloning the coding DNA sequence of AR gene and BRM gene into the pcDNA3.1-HA vector, respectively. The pCDH-OTUD6A-mouse and pCDH-OTUD6A-C153A-mouse were constructed by cloning mouse-derived OTUD6A or OTUD6A-C153A mutant sequence into the pCDH-vector. And different fragments of OTUD6A, Brg1, and AR were generated by subcloning the corresponding cDNAs into the pE.T-28a, pcDNA3.1-Flag or pcDNA3.1-HA vectors. The pRK5-HA-Ubiquitin-WT plasmids were purchased from Addgene (#17608) and the other mutants were generated by PCR assays as indicated.OTUD6A are: #1: sense, 5′-CTACGACGACTTCATGATCTA-3′; #2: sense, 5′-CATGATCTACTGCGACAACAT-3′. And mouse-derived OTUD6A are: #1: sense, 5′-GCAGTGTGGATTCGGTTACAG-3′; #2: sense, 5′-GCCTACTACATGCGGAAACAT-3′. Human-derived shBrg1#1: sense, 5′-CCGAGGTCTGATAGTGAAGAA-3′; shBrg1#2: sense, 5′-CGGCAGACACTGTGATCATTT-3′; shAR: sense, 5′-AAGCAGGGATGACTCTGGG-3′. The corresponding lentivirus were packaged and generated by transfecting each pLKO.1 lentiviral plasmids with psPAX2 and PMD2.G packaging plasmids into HEK293T packaging cells using a polyethyenimine  transfection protocol.The shRNA sequences of the pLKO.1 lentiviral vector to knockdown human-derived Different prostate cancer cell lines transfected with indicated plasmids or infected with indicated lentivirus were seeded into 96-well plates at an appropriate density of 1000–2000 cells per well with the recommended culture medium and cultured for the indicated amounts of time. Relative cell growth ratio curves were detected using an SRB assay and data were shown as mean ± SD of three independent experiments.OTUD6A at a density of 5 × 106 in 0.2 mL of PBS/matrigel matrix were inoculated subcutaneously on the right flank of NOD/SCID male mice (6 weeks old) purchased from Shanghai SIPPR BK Laboratory Animals (6 mice in each group). Mouse prostate cancer cells Myc-CaP-shScramble, Myc-CaP-shOTUD6A#1, Myc-CaP-shOTUD6A#2, Myc-CaP-shOTUD6A#1 + OTUD6A WT or Myc-CaP-shOTUD6A#1 + OTUD6A C153A (1 × 106 cells in 200 μL PBS) were injected subcutaneously on the flanks of 6-week-old male FVB mice  (6 mice in each group). The tumor volume of the mice was measured twice weekly and the tumor volume was estimated as (D × d2)/2 . Three weeks later, the mice were sacrificed by cervical dislocation after carbon dioxide inhalation and tumors were harvested.Study protocols involving mice were approved by the Institutional Animal Care and Use Committees (IACUC) of Shanghai Jiao Tong University. Human prostate cancer cells C4-2-shScramble or C4-2-sh3, mixed with matrigel hydrogel, and then transplanted subcutaneously on the dorsal side of male NOG/SCID mice . Each sample was inoculated with 3–5 NOG mice, and the generation after tumor growth was the P0 generation. A similar operation was performed to transfer the tumor piece to the next generation. After reaching generation 3 (P3), tumors were implanted subcutaneously on the right dorsal flank of 6-week-old NOG/SCID mice for subsequent experiment. When the tumor volume reached 100–150 mm3, mice were intratumoral injected with PBS, AAV-GFP or AAV-shOTUD6A in a volume of 20 μL/100 mm3 (2–3 sites per tumor) (titer: 1.6 × 1012 vg/ml) once a week. AAV-GFP and AAV-shOTUD6A were serotype 9 adeno-associated virus (AAV9), which clone a scramble sequence or a specific sequence targeting OTUD6A into pHBAAV-U6-MCS-CMV-EGFP vector, respectively . Tumor volume were recorded three times weekly. After treatment for approximately 30 days, all the mice were sacrificed by cervical dislocation after carbon dioxide inhalation and the tumors were extracted.For PDX (patient-derived xenograft) mice models, fresh tumor tissue fragments of prostate cancer patients were collected from Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University, School of Medicine after the informed consent was obtained from the donors. All experiments were performed in accordance with relevant guidelines and regulations. The treated tumor tissue were cut into several small pieces of 10–15 mmPten-floxed mice and Probasin-Cre mice were kindly provided by Dr. Jun Qin . AAV-GFP or AAV-shOTUD6A were injected into ventral prostate (VP) part of PtenPC−/− male mice at 2-month-old. For intraprostatic delivery, 2 μL viral suspension (titer: 1.6 × 1012 vg/ml) was injected into the VP part at a rate of 50 nL/min using a 10 μL syringe with a blunt 32G needle. Animals after the procedure were kept warm using a heat lamp for recovery. Three months later, the mice were sacrificed and the VP part of PtenPC−/− mice were extracted, weighed, embedded in paraffin and sectioned.Prostate cancer cells seeded into 6-well culture dishes at a density of 500–1000 cells per well. After two weeks culturing, cells were fixed in 0.4% paraformaldehyde and stained with 1% crystal violet, and then scanned by Epson v33 and counted in four independent fields in each sample. Data were shown as mean ± SD of three independent experiments.Treated cells were lysed in EBC buffer  supplemented with protease inhibitor cocktail  and phosphatase inhibitors  and were collected into 1.5 mL EP tube when they reached to 100% confluency in 6 or 10 cm dishes. For IP, 1 mg lysates were incubated with the appropriate indicated antibody (1–2 μg) and ProteinA Sepharose beads (Sigma) overnight at 4 °C. Immuno-complexes were then washed four times with NETN buffer . After final spin, the beads were boiled with 50 μl of 2 × SDS Laemmli buffer for 15 min, and the samples were subjected to IB. For IB analysis, cells were collected and total protein lysed with EBC buffer described above. After boiling with SDS laemmli buffer, equal amount of protein was separated by SDS-PAGE gel and immunoblotted with standard protocols. Antibodies used in this study are as follows: OTUD6A , Brg1 , AR , PTEN , HA , Flag , GFP , Myc , Ub , β-Tubulin .http://david.abcc.ncifcrf.gov). The results were shown in Supplementary Data http://proteomecentral.proteomexchange.org) via the iProX partner repository with the dataset identifier PXD031307.VCaP and PC-3 cells were introduced with Flag-OTUD6A and Flag-M2 IP detection was performed as described above. The affinity purified OTUD6A-associated proteins were analyzed by Easy-nLC 1200  and Orbitrap Exploris 480 . After the mass spectral data was extracted by Proteome Discovere software, the database was retrieved by Sequest search engine. DAVID bioinformatics functional annotation tool was used to identify enriched Gene Ontology (GO) pathway terms  and sonicated. The lysates were incubated with nickel-nitrilotriacetic acid (Ni-NTA) matrices (QIAGEN) for 4 h at room temperature. The his pull-down products were washed twice with buffer A, twice with buffer A/TI (1 volume buffer A and 3 volumes buffer TI), and one time with buffer TI . The pull-down protein samples were subjected to IB analysis.293T cells were transfected with His-Ub and the indicated plasmids for 48 h. Cells were treated with 10 μM MG132  for 12 h before harvest. Cells were lysed in buffer A  for indicated time points and then were lysed in EBC buffer for IB analysis.90 pairs of normal and neoplastic prostate tissues from Shanghai Changhai Hospital were taken for IHC staining. Sections were incubated with primary antibodies against OTUD6A  or Brg1 (CST#72182) antibody at a 1:100 dilution. The staining extent was categorized as follows: 0, <5%; 1, 5–25%; 2, 26–50%; 3, 51–75%; 4, >75%. The intensity was categorized as follows: 0, negative; 1, weak; 2, moderate; 3, strong. And IHC staining scores were calculated by multiplying staining extent and staining intensity. Images were taken using a Nikon ECLIPSE Ni microscope.OTUD6A or shBrg1, were collected and then lysed by TRIzol at room temperature. RNA-seq transcriptome library was prepared following TruSeqTM RNA sample preparation Kit form Illumina  using 1 μg of total RNA. The RNA-seq data were analyzed on the free online platform of Majorbio Cloud Platform (www.majorbio.com). GSEA was used for cellular pathway analysis. The results were shown in Supplementary Data https://ngdc.cncb.ac.cn/gsa.RNA-sequencing data was generated by Majorbio, Shanghai. PC-3 cells, which were stably infected with shScramble, shTotal RNA was isolated from cells using TRIzol (Vazyme) and transcribed into cDNA (Vazyme). Real-time PCR was performed using a LightCycler 96 System (Roche) and SYBR Green PCR Master Mix (Vazyme). Primers used in this study are as follows: GAPDH, 5′-GCACCGTCAAGGCTGAGAAC-3′ (forward) and 5′-TGGTGAAGACGCCAGTGGA-3′ (reverse); OTUD6A, 5′-TTCTTCAGCAACCCCGAGAC-3′ (forward) and 5′-CATAGCGCAGGTAGACCAGG-3′ (reverse); Brg1, 5′-GGATGCCGTGATCAAGTACAAG-3′ (forward) and 5′-TGAAGGTCTGTGCGTTCTGG-3′ (reverse) and AR, 5′-GAACAGCAACCTTCACAGCCG-3′ (forward) and 5′-TGCTGTTGCTGAAGGAGTTGC-3′ (reverse).The ChIP assay was performed using the EZ ChIP Kit (Millipore) according to the manufacturer’s protocol. Primers for detecting AR promoter are as follows: 5′-AGCAAAAGAGACCCAGGCAA-3′ (forward) and 5′-GAGGTCCCATAAGCCCTGTG-3′ (reverse).t-test, Wilcoxon’s signed-rank test or one-way ANOVA test. All analysis was conducted by SPSS program . Different cutoff values, P < 0.05 (*), P < 0.01 (**) and P < 0.001 (***) were considered significant.Statistical evaluation of in vitro and in vivo experiments was calculated using two-tailed Student’s Further information on research design is available in the Peer Review FileSupplementary InformationDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4Reporting Summary


Prokineticin 1 (PROK1) was reported as an angiogenic factor, which is associated with tumor progression, cell invasion, and metastasis in colorectal cancer. Although the association between PROK1 expression in primary cancer lesion and patient prognosis was reported, it is unclear whether plasma PROK1 concentration may be a predictive factor in colorectal cancer patients. This study investigated the association between PROK1 concentration in plasma and prognosis in colorectal cancer patients.We measured preoperative PROK1 plasma levels using ELISA method, while PROK1 expression in primary cancer lesion was evaluated using immunohistochemistry (IHC). The association between plasma PROK1 levels and cancer-related survival rate (CRS) was evaluated. Additionally, we examined whether simultaneous PROK1 expression in both primary cancer lesions and plasma was correlated with CRS. The cancer-related survival rate was calculated using the Kaplan-Meier method, and survival estimates were compared using the log-rank test.P = 0.006). Additionally, simultaneous PROK1 expression was associated with a more significant decrease of 5-year CRS than both negative groups in all stages  and stage III . Multivariate analysis showed simultaneous PROK1 expression was independently associated with worse CRS .We have gathered eligible 130 CRC patients retrospectively. Out of 130 patients, 61 (46.9%) were positive on IHC in primary cancer, and 69 (53.1%) were negative, while 43 (33.1%) had high-value PROK1 in plasma. Out of these 43, 30 (25.4%) also had concomitant higher IHC expression in primary cancer. The plasma PROK1 levels tended to increase with advancing stages. The plasma PROK1-positive group had a lower 5-year CRS than the negative group (63.6% vs. 88.2%; PROK1 expression in preoperative plasma reflects poor prognosis in patients undergoing curative resection for colorectal cancer. The plasma PROK1 level may be a potential predictive marker, especially in stage III colorectal cancer patients.The online version contains supplementary material available at 10.1186/s12957-021-02421-0. Colon cancer is the third most common cancer in the world. Further improvement of the treatment for that cancer still has been required . Some guAngiogenic growth factors have been considered to play an important role in the proliferation of colorectal cancer . Some drRecent studies reported that PROK1 expressed not only endocrine glands but also some malignant tumors, including prostate cancer , pancreaThe present study aimed to examine the association between preoperative PROK1 concentration in the plasma of patients with colorectal cancer and their prognosis.This was a single-center, cohort study. Eligible subjects were chemotherapy-naive colorectal cancer patients who underwent surgery with D2 or D3 lymph node dissection (stages I‑IV: UICC-TNM 8th ) and undThe concentration of PROK1 in plasma was measured using an enzyme-linked immunosorbent assay (ELISA) with the OmniKine (Human) EG-VEGF ELISA kit  according to the manufacturer’s instructions. The test kit showed less than 1% cross-reactivity with human Prokineticin-2 by the manufacturer. The cut-off points for PROK1 concentration were determined using the receiver operating characteristic (ROC) curve. We determined the concentration above the cut-off value as positive and below the value as negative.TM . After three washes with phosphate-buffered saline (PBS), we used the Envision system  for the application of a secondary antibody for 30 min at room temperature and washed out the solution with Tris-buffered saline (TBS). Diaminobenzidine was applied to the sections for 4 min to evaluate the expression of PROK1, and then immersed in a hematoxylin solution for counterstaining. We picked up 5 hot spots in cancer tissue and measured the area of cancer tissue using the Image J software. We categorized staining intensity into three grades: weak, moderate, and strong. While we considered moderate and strong staining areas as positive, we calculated the average of the percentage in PROK1 positive area among five hot spots. We then determined it positive if the average was more than 30% : 0.62, negative likelihood ratio: 0.56, positive likelihood ratio: 1.48, and diagnostic odds ratio: 2.45, sensitivity: 70.6%, specificity: 50.2%).


We showcase a methodology to discover topics via Latent Dirichlet Allocation, which can reliably produce human-interpretable topics over an open news corpus that continually grows with time. Our system generates topics, or distributions of keywords, for news articles on a monthly basis, to consistently detect key events and trends aligned with events in the real world. Findings from 2 years worth of news articles in mainstream English-language Canadian media indicate that certain topics feature either women or men more prominently and exhibit different types of language. Perhaps unsurprisingly, topics such as lifestyle, entertainment, and healthcare tend to be prominent in articles that quote more women than men. Topics such as sports, politics, and business are characteristic of articles that quote more men than women. The data shows a self-reinforcing gendered division of duties and representation in society. Quoting female sources more frequently in a caregiving role and quoting male sources more frequently in political and business roles enshrines women’s status as caregivers and men’s status as leaders and breadwinners. Our results can help journalists and policy makers better understand the unequal gender representation of those quoted in the news and facilitate news organizations’ efforts to achieve gender parity in their sources. The proposed methodology is robust, reproducible, and scalable to very large corpora, and can be used for similar studies involving unsupervised topic modelling and language analyses.We present a topic modelling and data visualization methodology to examine gender-based disparities in news articles by topic. Existing research in topic modelling is largely focused on the text mining of  Gender equality is one of the UN’s 17 Sustainable Development Goals . By all The underrepresentation of women in certain areas of the news such as politics, business, or sports is well documented . There iIn this paper, we tackle one measure of gender equality in the media: the number of times men and women are quoted in news articles. More specifically, we present a scalable topic modelling methodology to explore how many times men and women are quoted across news topics. Our unsupervised large-scale analyses are enhanced with corpus-based studies of the language used in male-prominent and female-prominent articles, thus revealing not only which topics are different, but exactly how they are different in their language.Our analyses rely on statistics provided by the Gender Gap Tracker,We apply topic modelling, an unsupervised machine learning technique , to thisOur results show that there are clear trends in the proportion of men and women quoted depending on the topic of the article they are quoted in. Women are consistently quoted more frequently than men in the topics “Lifestyle”, “Arts and Entertainment”, and “Healthcare”, whereas men are quoted more frequently than women in the topics “Business”, “Politics”, and “Sports”. This comes as no surprise, regrettably, as the frequency of quotes seems to reflect a gendered cultural division of duties, where women are placed into the role of caregiver and nurturer and are less represented in the spheres of politics and business. This gendered division of duties and representation in media is no doubt self-reinforcing; quoting female sources more frequently in a caregiving role and quoting male sources more frequently in political and business roles enshrines women’s status as caregivers and men’s status as leaders and breadwinners . Since tWe would like to acknowledge that the terms we use in this paper are simplifications of a complex reality. We use the terms “women”, “men”, “female sources”, and “male sources”, implying a binary opposition that we know is far from simple. We know, at the same time, that lack of gender representation in many aspects of society is a reality. The aim of this study is to quantify that gender gap by analyzing language and the traditional associations of names with men and women.Data was obtained for the Gender Gap Tracker and analyzed for this related project. The Gender Gap Tracker data is scraped daily from the websites of seven major Canadian news outlets in English . We builWe analyzed 24 months worth of data, a total of 612,343 articles. As we see in sources. For the latter, we first identify quotes  in the text and then associate a speaker with them, using coreference resolution. Finally, we look up the gender of the speakers  using a mix of sources: an in-house cache of commonly quoted public figures and an API that stores the gender of people based on their first or full name. Thanks to this NLP pipeline, we obtain gender statistics  for each article, whose aggregated form is shown in Using the Gender Gap Tracker’s existing Natural Language Processing pipeline , the datpeople mentioned refers to all unique individuals named in an article, regardless of whether or not they were quoted. These names are extracted and identified by our NLP pipeline via Named Entity Recognition. People quoted refers to the subset of people mentioned that were quoted one or more times in an article, identified via coreference resolution. The counts of people mentioned and people quoted are per article, as we do not keep track of the same individual across articles, that is, our unit of analysis is the article.At this point, we would like to elaborate on our nomenclature. In this work, To illustrate this more clearly, consider a “set” of two articles. If the politician Chrystia Freeland is mentioned in both articles and has three quotes in one article and none in the other, this means two women were mentioned (one woman in each article) and one woman was quoted (one woman quoted three times in one article) in the set of all articles. If, however, Chrystia Freeland was quoted once in one article and once in the other, and another politician, Christine Elliott, was mentioned in the same two articles (but not quoted), two women were mentioned and two women were quoted in that set, with Chrystia Freeland counting as one woman in each article that she was quoted.Numbers alone tell a compelling story about the lack of female voices in the media, as we show in more detailed analyses of the Gender Gap Tracker data . Our goatopics, or themes, in a collection of unstructured documents  is by far the most popular. Because of LDA’s inherently non-deterministic and statistical qualities, methodological decisions involving data preprocessing, choosing the number of topics, tuning the algorithm hyperparameters, and evaluating the model’s results become all the more important .Gibbs sampling, in which a Markov chain of random variables is constructed with each variable dependent on the previous ones—the limiting value of this distribution equals the true posterior. The algorithm is run on the Markov chain defined on the hidden variables for a particular corpus and a number of samples are drawn using a Markov Chain Monte Carlo algorithm, following which the approximate distribution is constructed from the collected samples. While sampling-based methods are guaranteed to be identical to the true posterior under limiting conditions and can produce less biased results overall, they are quite computationally expensive and do not scale as well as variational Bayes methods do, as the corpus grows in size .By contrast with verbs, we chose to retain a large portion of nouns during text preprocessing, due to the importance of nouns in topic interpretation. It has been shown that a combination of lemmatization and limiting the corpus to just nouns provides a coherence advantage and lower word intrusion in topic modelling . While wOur final stopword list, a sample of which is shown in Term lemmatization is performed using a lookup process during run time, where each token is checked for presence in a dictionary of lemmas provided by the user. To do this, we turned to the spaCy NLP library,In this section, we discuss the key LDA hyperparameters we tuned to achieve our best results. Some additional modelling experiments are discussed in the The online variational Bayes algorithm was observed to converge quite rapidly (within 100–150 iterations). By contrast, we found that expectation maximization can take upwards of 200 iterations to converge. This makes sense, since the online algorithm is known to speed up convergence by using stochastic optimization and is less prone to getting stuck in local optima. We did not notice much of a difference in terms of perplexity as well as topic interpretability when running the online algorithm for 150 iterations and above. Minor differences in topic keywords were noticed for specific months’ results when we used just 100 iterations. As a result, we fix the maximum number of iterations to 150 for all our training runs, regardless of the size of the data involved each month.Three relative pruning hyperparameters are used during data preprocessing: maximum document frequency, minimum document frequency, and minimum term frequency. Controlling these values prunes, i.e., removes, those terms that occur both very frequently and very rarely from the vocabulary. As a result, these hyperparameters must be chosen carefully, so as to not hinder topic interpretability or miss larger themes in the data.We found that a maximum document frequency of 80% The minimum document frequency hyperparameter , the minimum term frequency is specified as a positive integer. A minimum term frequency of 1 means that no terms are pruned from a document, whereas setting The minimum term frequency hyperparameter prunes terms that occur fewer than a specified number of times Setting a finite vocabulary size limits the dimensionality of the random matrices that are solved for during LDA , our scenario is well-suited to e press” . Corpus e press” .For the language analyses, we rely on two key concepts from corpus linguistics, keyness and dependency bigrams. Keyness aims to identify large differences between the frequency of word-forms in two or more corpora . KeynessThe playerkickedtheball, the main verb kicked is connected to the noun ball via a direct object relationship, wherein kicked is the head and ball is the dependent. The dependency bigram returned using this process is “ball_kick”.A relatively more advanced form of corpus analysis, dependency bigrams identify verb-direct object combinations and their frequencies within a corpus . To extrDependency bigrams reveal linguistic aspects of the male and female corpora, unlike keyness, which mainly highlights textual or lexical differences. We obtained much more useful results with dependency bigrams rather than the more standard corpus analysis methods such as collocation or n-gram frequencies . This ishttps://gendergaptracker.research.sfu.ca/apps/topicmodel), we obtain per-month, per-outlet mean topic gender prominence measures for each of these topics over the 2-year period. We define gender prominence as the difference in mean topic weights between the female and male corpus or dataset for a given topic. A topic is categorized as having male prominence if the mean topic weights from the male corpus are greater than those from the female corpus. See the last section in the We examined nine topics that feature regularly in most months between October 2018 and September 2020. The topics cover a broad range of domains typically seen in the news, as seen on the vertical axis of The values for prominence range from positive red in , indicatOther topics that display strong female prominence over time include “Lifestyle” , “Healthcare and medical research”, and “Legal and court cases”. The primary reason for the “Lifestyle” topic being female-prominent is that it regularly features mothers involved in childcare, women reliving their past experiences, and female experts offering personal care advice. The “Legal and court cases” topic tends to feature more women mainly due to cases that pertain to sexual assault or women’s rights, featuring both victims and experts who are women. The “Healthcare and medical research” topic is particularly interesting, because, in Canada, a sizable proportion of healthcare experts and Public Health Officers happen to be women . This leOn the other hand, the “Business and market events”, “Sports”, “Federal politics”, and “United States politics” topics are almost entirely male-prominent throughout the period studied. The “Business” topic typically includes keywords pertaining to investments, analysts’ predictions on the stock market, and in-depth discussions on company futures, interest rates, and profits. Historically, these areas tend to be dominated by men, partly explaining why the topic intensity for “Business” is always much lower for the female corpus. Similarly, the “Sports”, “Federal politics”, and “United States politics” topics feature discourse that generously quotes male athletes, coaches, and politicians, who are not only more in numbers in society, but are also quoted much more often than women.In general, the heat map in distribution over topics. Mathematically, this means that the topic modelling process returns a one-dimensional vector of length 15 (we only model a maximum of 15 topics each month) for each document, where each component of the vector, which we call a topic weight, represents how strongly or weakly that topic’s keywords are associated with that document. To begin the combined topic and language analyses, we identify a topic of interest that exhibits strongly male or female gender prominence for a particular month. We then query that month’s data from our database and sort in descending order of topic weights for that topic. Sorting the articles in this order places all articles that are strongly related to that topic’s keywords on top. The full corpus, in sorted order for a particular topic, is then split into two corpora, each with male-majority and female-majority sources . We chose 200 articles for empirical reasons—we observed that in most cases, the maximum topic weights for each article rapidly dropped to less than 0.5 after a few hundred samples. Following these steps, we perform keyness analysis and extract dependency bigrams on five topics: “Sports”, “Business and market events”, “Lifestyle”, “Healthcare and medical research”, and “Crimes and sexual assault”. Below we discuss our analyses of these five topics for certain months.The first topic whose language we study in detail is “Sports”, for the months of June, July, and August 2019. August 2019 was an outlier because it was the only month for which the “Sports” topic demonstrated an overall female prominence. August 2019 was also particularly interesting because “Sports” was the strongest topic in both the female and male corpora. To better understand these trends, we performed keyness and dependency bigram analyses for the top articles from each of these 3 months. The keyness results uncover lexical differences between the female and male corpora, whereas the dependency bigrams show syntactic  differences based on subject-direct object relationships, that is, events and relationships that the articles capture.Yankees, Red Sox) and teams from the Canadian Football League . The dependency bigram plots indicate that there are differences in language across the female and male corpora, based on frequency counts of the most common bigrams.  The male corpus contains much higher occurrences of general verbs describing events in baseball/football games, such as hit homer, score goal, allow run, and give lead. The female corpus seems much more focused on winning, celebrating, and breaking records .From the keyness index plot, we can see that the female corpus contains numerous terms pertaining to the FIFA women’s world cup held in France that month. The names of Canadian and American soccer players and those of their female coaches appear, as do other terms related to women’s soccer federations. The male corpus, on the other hand, contains terms largely related to baseball . The male corpus continues to highlight terms and player/coach names from baseball (Blue Jays) and the CFL (Redblacks). The dependency bigram plots also reveal comparable results, with the female corpus using more vocabulary related to winning cups/medals and setting/breaking records. On the other hand, the male corpus much more frequently highlights terms that describe specific aspects of games, such as giving the opponents the lead or allowing them a run.July 2019  follows Finally, in August 2019, shown in win cup and break record), whereas articles that feature men’s sports focus on the finer details of each game. Perhaps authors that write about women’s sport highlight women’s achievements and success stories because of the limited time and space they have, compared to the coverage given to men’s sports. After all, the baseball and football seasons in Canada are each several months long, whereas events like the FIFA world cup and the Wimbledon tournament occur in the span of just 1 month. It does seem that, when it comes to sports, men just need to play to feature in the news; women need to win.This analysis of the top “Sports” articles across three summer months in 2019 shows that articles that feature women’s voices tend to focus more heavily on achievements and records , we see considerable amounts of coverage in the female corpus during certain times of the year. Sustaining a high level of coverage, however, seems to require more women role models across a wider range of sports, as well as longer seasons over which women’s sports are regularly featured. Since the COVID-19 pandemic in early 2020, our topic model gender prominence results show that women’s sport has once again taken a back seat in the news.The underrepresentation of women in sports news has been amply documented, showing that coverage has not kept pace with the increase of women’s participation in sports . HoweverWe consider 2 months, October and December 2018, for which this topic exhibits a particularly strong male prominence. The top 200 articles with the strongest topic intensities in each month were chosen for comparison.milk, shave) and small/local businesses . The dependency analysis gives us expressions like have impact, cover cost, pay debt, and run business. The male corpus contains terms related to larger financial instruments, stocks, trading, and company performance metrics. The male corpus also frequently features verbs pertaining to high-level business policy and decision-making .In October 2018 , in the gender, workforce, child, and racialized. The male corpus, as always, contains terms very specific to financial and business operations . Inspecting the dependency bigrams, we can see that the female corpus highlights terms such as cut production and make decision—this was primarily due to then-Alberta Premier Rachel Notley’s numerous comments on the oil industry’s production cuts. Other terms such as close gap, spend money, and provide service clearly point to some level of gender disparity between the two corpora.December 2018, the month for which the “Business” topic exhibits the strongest male prominence in all our results, showcases a similar disparity in the kind of language used across both corpora, as seen in The Globe and Mail, which we know quotes, on average, the smallest proportion of women.We find that the “Business and market events” topic regularly exhibits the largest disparity between the male and female corpora, in terms of language used, for all months of data studied. It seems that women’s voices are rarely featured in articles that cover business, especially big business, finance, and the stock market. The kind of language used (as seen in the dependency bigrams) shows that the focus of articles that quote women tends to be primarily small/local businesses and the challenges of working at or running a  business. It is also important to note that a large portion of articles in this topic were published by The “Lifestyle” topic, which covers content related to families, personal experiences, holidays, and shopping, exhibits strong female prominence for all months in which it appears. tbsp, tsp) to clothing and fabrics. Interestingly, not as many women’s names are seen for this month’s keywords as for other topics in other months. The male corpus keywords include several names of people, including Suresh Doss, a Toronto-based food writer. The dependency bigrams for both the male and female corpora focus mainly on the festive season, as well as acts of gift-giving and donating to charities.Keywords from the female corpus for December 2019 highlight a range of lifestyle topics, from food recipes  emerge, mainly because multiple outlets tend to write about the same topic, typically quoting the same people.We find that the “Lifestyle” topic, unlike other topics, tends to name regular people who share their life experiences with the media—this can lead to the drowning out of other keywords such as common nouns or adjectives in the keyness results. As a result, keyness is slightly less useful in interpreting textual disparities across the corpora for this topic. On occasion, interesting terms , while the male corpus shows a similar focus toward terms pertaining to men’s health . Interestingly, the bigrams from the female corpus seem to point to an additional emphasis on the effects of women’s personal choices on fertility and pregnancy . The male corpus bigrams, in this case, do not seem to have a particular focus other than the availability and effects of various treatments in the healthcare domain .The female corpus for March 2019  containsOnce the COVID-19 pandemic began spreading in Canada in March 2020, there was a stark shift in healthcare coverage. Because of the seriousness of the pandemic, there were multiple COVID-related topics in March. We focus our attention on four topics that were clearly female dominant throughout this period . The text of the top 100 articles for each topic (ordered by topic intensity) was collected and separated into male and female corpora, just as before. This gave us 400 articles in either corpus to analyze for their language used during this month. Keywords and dependency bigram for this corpus are shown in confirm case is significantly higher (>200) in the female corpus than the male corpus. This makes sense, considering that, for the most part, the Provincial Health Officers confirming the numbers happened to be women.Some of the keywords in the female corpus are names of women who are medical experts (Dr. Vera Etches and Dr. Penny Sutcliffe) or place names related to the spread of COVID-19 in various regions of Canada and the world. Similarly, the male corpus also names prominent medical researchers and scientists (Dr. Srinivas Murthy and Dr. Brent Roussin). The dependency bigrams show a very similar distribution across both male and female corpora, with terms pertaining to symptoms, case counts, and public health guidelines. Notably, the frequency count of the bigram In general, we observe that Canadian media outlets tend to provide a good degree of coverage to women’s healthcare and health issues throughout the year. This is helped by the fact that a relatively large proportion of healthcare experts and medical officers in Canada are women, which explains the high female prominence of the topic in most months of the year. The most notable of these is Dr. Bonnie Henry, the Chief Provincial Health Officer of British Columbia, who is the top quoted woman by far throughout the spring/summer of 2020, due to the COVID-19 pandemic. This leads to the conclusion that having women in prominent positions increases their chances of being quoted by the media, though this is, naturally, beyond the control of news organizations and journalists.In exploring our results, we observed that topics pertaining to women’s rights and sexual assault emerged around February and March, in both 2019 and 2020. We explore these topics for these periods using corpus studies, as shown below.hijab, trans, hormone). The issue of the gender pay gap in sports was also a major one during this period, shown through keywords such as fifa and cup. The male corpus keywords primarily feature names, presumably of men involved in crimes and their victims. Some key dependency bigrams from the female corpus include protect athlete, involve victim, and take action, indicating that coverage was focused on the action taken toward women’s justice. The male corpus highlights bigrams related to the crimes themselves, convictions, and legal matters.In February 2020, terms pertaining to women’s and trans rights appear in the female corpus . The male corpus keywords once again consist of names of the perpetrators or victims involved. In general, the language used in the female corpus (as seen in the bigrams) tends to focus on the victim’s accusations and details of the crimes, and on the convictions, defense, and accounts of the victims in the male corpus.In February 2020, as seen in take stand, tell story, have right) are used quite frequently in the female corpora, highlighting the role the media plays in spreading awareness about women’s issues and rights in Canada.It is quite interesting that topics around women’s rights  tend to emerge at the same time each year, around February and March, which is the time when International Women’s Day is celebrated. Analyzing the language used, we also find it heartening that strong action verbs  is. Almost every sport has a men’s and women’s division. But it’s often the men’s that is more lucrative and/or prominent. In that case, setting the baseline to the number of prominent or professional men athletes, would, in our view, simply reinforce the existing bias. This is why we assume a 50% baseline, that is, the approximate proportion of women in the world, in order to highlight the existing gap in women’s representation in the media.α and β, reliably produces topics that align well with events from the real world, while also effectively scaling to large datasets. In contrast to prior work that recommends removing stopwords as a post-processing step . During feature transformation, a common approach is to tokenize the corpus into bigrams . Words tThe second potential limitation pertains to model evaluation. Although there exist clean, well-tested implementations of popular topic model quality metrics such as Pointwise Mutual Index (PMI) and UMass’ topic coherence , Spark’sFinally, with respect to reproducibility, a common practice to reproduce results in LDA is to fix the random seed across runs. This should in principle work well, since it ensures that the Dirichlet samples are drawn from the same random distribution across runs. However, because Spark is a distributed system, there is an additional layer of randomness in the methodology, i.e., that of the distributed system itself. Due to the way Spark distributes data, the order of samples passed to an executor cannot be guaranteed, nor can the same random number generator seed be guaranteed on each executor. As a result, this stage of the process is non-deterministic. This causes a very minor deviation in the topic keywords discovered across multiple runs, even with the same random seed on the exact same data. However, our numerous experiments over multiple months of news data confirm that this does not affect topic interpretability or the overall robustness of the process.A study of bias is not complete unless the full gender spectrum is considered, as well as other forms of diversity such as status as visible minority, disability, or age. Our language-based analyses rely on names to assign gender to individuals and are not well suited to study such forms of diversity. Additionally, potential harms from assigning specific labels to individuals need to be considered . NeverthOne area we are mindful about is the relative lack of work that studies topic modelling and gender disparities in languages other than English. The Gender Gap Tracker project has also been collecting news articles from Canadian French-language news outlets since October 2018, which we have not yet analyzed. Research on morphologically rich languages such as Russian or Swedish has shown that effective pre-processing techniques, such as lemmatization, can greatly improve topic interpretability . Studyinstated vs. claimed) or in the way that the speaker is introduced, as an expert or merely a source. Analyses of transitivity structure in clauses can yield insights about the type of roles women are portrayed in  is updated every month, providing numbers and visualizations that can help Canadian newsrooms and policy makers monitor whether progress is being made. We open-source our code and visualization framework, so that others can also apply this methodology.Our public dashboard (


In the last decade, molecular cytopathology has emerged as a rapidly evolving field of diagnostic and predictive pathology , with anPisapia et al. provides an overview of the role of the modern cytopathologist in the morpho-molecular diagnosis of advanced stage NSCLC, focusing on the adoption of currently available NGS assays in different cytopreparations .From a clinical standpoint, cytology is a first-line diagnostic procedure in many neoplastic and non-neoplastic settings. In advanced-stage cancer patients, cytological samples may be the only material available for both diagnosis and molecular biomarker testing to select patients for targeted therapies . InteresSchmitt et al., along with immunocytochemistry (ICC), fluorescent in-situ hybridization (FISH), reverse transcription-polymerase chain reaction (RT-PCR), as testing method for gene fusion detection; advantages and disadvantages of each assay and specific requirements for the application to routine cytological material are reviewed.RNA and DNA based NGS approaches are also discussed by Patel et al. confirmed that pleural effusion supernatants from patients with metastatic NSCLC represents a robust source of DNA for NGS. Moreover, analytical and pre-analytical challenges in the utilization of cell-free DNA (cfDNA) extracted from pleural effusion supernatant are detailed. In particular, optimization of specimen storage and collection, determination of specimen cellularity, and nucleic acid quantity and quality are identified as challenges in the preanalytic phase; on the other hand, challenges in the analytic phase are represented by selection of a sequencing methodology with appropriate depth, targets, and interpretation of results.Maximizing cytological material for molecular testing is definitely a relevant goal for modern cytopathologists; in this view, several studies have investigated the possibility of using supernatants for molecular analysis . Data frTejerina et al. summarizes the knowledge of the role of cytopathology in the analysis of PD-L1, addressing important issues such as pre-analytical phase, cyto-histological correlation, and inter-observer agreement.Besides targeted therapy, over the past decade immunotherapy has emerged as one of the most promising cancer treatments  and the Canberk et al. covers the main epigenetic categories—DNA methylation, histone modification, and RNA-silencing—and their translational potential on thyroid cytology specimens.In addition to the predictive response to tumor targeted treatments, molecular profiling of cytological samples is also currently useful in diagnostics to stratify atypical and undetermined cytology diagnoses into low and high-malignancy risk categories. Moreover, advances in the epigenetic field supporting the differentiation between benign, borderline, and malignant lesions, as well as their role as prognostic factors in malignant neoplasms, are increasingly acknowledged . RecentlLau et al. reviewed the current state of deep machine learning and AI, that have potential applications to cytology specimens, including exfoliative samples , fine needle aspiration (FNA), or liquid biopsy samples ; moreover, the authors provided an overview of recent multiplex systems, such as antibody barcoding with cleavable DNA (ABCD), single cell analysis for tumor phenotyping (SCANT), fast analytical screening technique-fine needle aspiration (FAST-FNA), and portable fluorescence-based image cytometry analyzer (CytoPAN), that have shown great promise in their ability to automatically analyze several biomarkers concurrently with high sensitivity, even in paucicellular samples, lending themselves well as tools in FNA.Finally, a new challenge for modern cytopathologists addressed in this Research Topic is represented by the application of digital imaging, artificial intelligence (AI), and multiplex modalities to cytological material . Lau et In conclusion, to this day, cytological samples are still underutilized to select oncological patients for targeted treatments and we argue that a continuous educational effort and a timely update in both clinical and methodological molecular cytopathology aspects are required to better exploit the role of this approach in molecular medicine. All authors contributing to this Research Topic provided an overview of the current state of the art in molecular cytopathology and allowed to convey to the practicing cytopathologist an update on novel testing methodologies and further clarify cytological sample requirements, improving patient management, and treatment.EV, ML, and SR-C equally contributed in conceptualization, writing—review, and editing. All authors contributed to the article and approved the submitted version.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Fibrin is a major component of thrombi formed on the surface of atherosclerotic plaques. Fibrin accumulation as a consequence of local blood coagulation activation takes place inside atherosclerotic lesions and contributes to their growth. The imbalance between thrombin-mediated fibrin formation and fibrin degradation might enhance atherosclerosis in relation to inflammatory states reflected by increased fibrinogen concentrations, the key determinant of fibrin characteristics. There are large interindividual differences in fibrin clot structure and function measured in plasma-based assays and in purified fibrinogen-based systems. Several observational studies have demonstrated that subjects who tend to generate denser fibrin networks displaying impaired clot lysis are at an increased risk of developing advanced atherosclerosis and arterial thromboembolic events. Moreover, the majority of cardiovascular risk factors are also associated with unfavorably altered fibrin clot properties, with their improvement following effective therapy, in particular with aspirin, statins, and anticoagulant agents. The prothrombotic fibrin clot phenotype has been reported to have a predictive value in terms of myocardial infarction, ischemic stroke, and acute limb ischemia. This review article summarizes available data on the association of fibrin clot characteristics with atherosclerotic vascular disease and its potential practical implications. Growing evidence indicates that the formation of denser fibrin networks, which are less susceptible to lysis, characterizes patients with atherosclerosis and arterial thromboembolic events. Several cardiovascular risk factors, such as hyperlipidemia, hypertension, smoking, or diabetes have also been shown to be associated with unfavorably altered fibrin clot properties in the general population. Low-dose aspirin, statins, better diabetes control, or smoking cessation have been shown to increase fibrin clot permeability and its susceptibility to lysis. Moreover, it has been shown that non-vitamin K antagonist oral anticoagulants (NOACs) are able to improve fibrin clot characteristics and contribute to the reduced risk of adverse clinical outcomes. The current review article summarizes available basic research and clinical papers deposited on PubMed over the last decade regarding associations between fibrin clot phenotype and atherosclerotic vascular disease, supported by the seminal papers from previous years. Moreover, data on novel therapeutic strategies, which can potentially influence fibrin clot characteristics, have been discussed.Atherosclerosis is a major cause of cardiovascular disease that encompasses coronary artery disease, cerebrovascular disease, peripheral arterial disease, and aortic atherosclerosis. The current concept of the pathogenesis of atherosclerosis is based on chronic inflammation associated with modified lipid deposition and dysregulated immunity within the arterial wall ,2,3. TheNeovascularization within the advanced plaque contributes to its gradual growth in part due to intraplaque hemorrhages. An increased density of microvessels has been found in ruptured atherosclerotic plaques , suggestThe role of blood coagulation in the development and progression of atherosclerotic vascular disease reaches beyond thromboembolic complications. Macrophages can produce tissue factor (TF) , expressAdventitial fibroblasts in normal arteries are able to express TF, while in atherosclerotic lesions, TF is also expressed by SMCs, foam cells, and macrophages, which can additionally release microparticle-derived TF . TF was Several studies have suggested that increased fibrinogen concentration, a key determinant of fibrin formation and its characteristics, is a risk factor for atherosclerotic vascular diseases, in particular coronary artery stenosis and myocardial infarction (MI) ,21,22. IThe localization of fibrin degradation products  within the human arterial wall suggests their potential atherogenic properties . Higher Fibrin acts as a scaffold for intravascular blood thrombi, enhancing platelet aggregation and thrombin generation, leading to a further increase in fibrin formation . Fibrin [s associated with typical changes in fibrin structure, such as lower fibrin fibers diameter, lower pore size area between particular fibers, and an increased number of branch points, along with faster fibrin formation and prolonged lysis time  can be described using clot permeability (Ked time) ,38,39, ted time) , or the ed time) . Fibrin ed time) ,42,43. Asis time .The majority of well-established cardiovascular risk factors which have been reported to be associated with prothrombotic fibrin clot properties are presented in s and impaired clot susceptibility to lysis, have been reported in patients with acute MI, at least in part associated with increased oxidative stress and the extent of inflammation [s and prolonged lysis time, related to a higher body mass index, higher blood pressure and higher C-reactive protein levels [Acute MI is a leading cause of mortality in high-income countries and is a major thrombotic manifestation of atherosclerotic lesions in coronary arteries . Dense fammation . Prolongammation . It has ammation . In a coammation . As expen levels . Platelen levels . Serum ln levels .s [It has been demonstrated that fibrin is the main constituent 60%) of intracoronary thrombi obtained during thrombectomy in acute STEMI (within 12 h since the symptom onset), with amounts increasing with time. Increased intracoronary fibrin content has also been found to be positively associated with denser plasma fibrin networks, reflected by reduced Ks , which i% of intrs , who havIt is unclear whether fibrin clot composition differs among patients with the same condition. Proteomics data has shown that clot-bound protein composition can influence fibrin properties . A preliFrom a practical point of view, the issue of prognostication based on plasma fibrin clot characteristics appears to be of vital importance. Growing evidence indicates that the prothrombotic fibrin phenotype can predict cardiovascular events. A PLATO substudy performed on 4354 patients following acute MI has shown that a validated turbidimetric assay employed to assess plasma clot lysis time and clot maximum turbidity at hospital discharge, while on dual antiplatelet therapy, is able to predict adverse clinical outcomes during a 12 month follow-up period . After as and prolonged clot lysis time in patients with a history of MI [s but not lysis time [Stable CAD, defined as angina pectoris, MI history, or presence of atherosclerotic plaques, has been associated with unfavorably modified fibrin clot properties . Reducedry of MI . Moreovesis time . Similarsis time . The rs4sis time . In a losis time . Altogets when compared to controls, and it was associated with 31% prolonged CLT; the two alterations in fibrin properties predicted PAD progression during long-term follow-up [s with no difference in CLT was found in patients with a history of acute lower limb ischemia compared to individuals without any history of such event [s and prolonged CLT at baseline, accompanied by elevated thrombin generation and von Willebrand factor antigen; however, the fibrin clot variables cannot predict re-intervention, amputation, and death during further 3-year follow-up [Prothrombotic fibrin clot properties were observed for the first time in 2009 in patients with intermittent claudication, a typical manifestation of peripheral arterial disease (PAD) ,75 whichollow-up . Similarch event . Interesch event . Moreoveollow-up .Scott et al.  have shos and shortened CLT, despite no impact on plasma fibrinogen levels [s and a shortened clot lysis time in patients with LDL cholesterol < 3.4 mmol/L free of clinically evident CAD, like in the JUPITER trial with rosuvastatin [Statins (3-hydroxy-methylglutaryl coenzyme A reductase inhibitors) may exert several cholesterol-independent antithrombotic effects, including the down-regulation of TF expression and enhanced protein C activation via increased endothelial thrombomodulin expression . Althougn levels . Favorabvastatin . This efNovel therapeutic strategies to lower LDL cholesterol based on fully humanized monoclonal antibodies that bind free plasma proprotein convertase subtilisin/kexin type 9 (PCSK9) , differes after one week and 41% reduced Ks after two weeks when compared to values observed during treatment [Aspirin was the first effective antiplatelet therapy for the prevention of ischemic events in patients with atherosclerotic vascular disease. Aspirin treatment has been shown to be associated with the formation of thicker fibrin fibers and improved clot susceptibility to lysis in stable CAD patients . The mecreatment . It is us increase by 13% and shortening of clot lysis time by 28% [Antihypertensive therapy with ACEI has been found to modulate fibrin clot properties in association with reduced complement component C3 levels ; howevere by 28% . It has e by 28% .NOACs, including rivaroxaban and apixaban, which are selective and direct FXa inhibitors, and dabigatran as a direct thrombin inhibitor, are used to prevent and treat thromboembolic events . Clots fs and CLT in clinical practice [The formation of dense fibrin networks which are relatively resistant to lysis has been observed in patients with atherosclerotic vascular disease, in particular CAD and PAD, along with those who experience arterial thromboembolism. The prothrombotic features of fibrin clots that are largely determined by environmental factors can be improved by the control of cardiovascular risk factors, in particular the normalization of glycemia and statin use. Growing evidence suggests that measures of clot characteristics, such as clot permeability and clot lysis time, may predict arterial thromboembolic events. Therefore, fibrin clot measures could serve as prognostic markers in patients at risk of arterial thromboembolism. However, there is a need for large studies to validate the available observations and standardization of the assays used to characterize the clot phenotype, though the first step initiated by the scientific subcommittee of the International Society on Thrombosis and Haemostasis has been made to implement the measurement of Kpractice ,103.Taken together, several studies demonstrated that the formation of more compact fibrin networks displaying lower susceptibility to lysis are implicated in the progression of atherosclerosis and the occurrence of thromboembolic manifestations, in particular MI see . The obs


C. elegans. Animals grown continuously in these conditions display reduced movement and body length. Loss of mechanical contact stimuli in microgravity elicits decreased endogenous dopamine and comt-4 (catechol-O-methyl transferase) expression levels. The application of exogenous dopamine reverses the movement and body length defects caused by simulated microgravity. In addition, increased physical contact made comt-4 and dopamine levels rise. It also increased muscular cytoplasmic Ca2+ firing. In dop-3 (D2-like receptor) mutants, neither decrease in movement nor in body length were observed during simulated microgravity growth. These results strongly suggest that targeting the dopamine system through manipulation of the external environment (contact stimuli) prevents muscular changes and is a realistic and viable treatment strategy to promote safe human deep-space travel.Progressive neuromuscular decline in microgravity is a prominent health concern preventing interplanetary human habitation. We establish functional dopamine-mediated impairments as a consistent feature across multiple spaceflight exposures and during simulated microgravity in   •C. elegans grown under microgravityDopamine levels, movement and body are reduced in •Loss of mechanical contact in microgravity activates a D2-like dopamine receptor•Reinstating physical contact stimulation improves these reductions Space medicine; Aerospace Engineering Over almost 60 years, hundreds of humans have flown into space, spending up to one year living on the International Space Station (ISS). Unlike living on Earth, spaceflight presents a multitude of environmental stressors such as microgravity (μG) and cosmic radiation exposure. The direct consequences of these unique features of spaceflight upon health are widespread, including declines in the skeletal, neuromuscular, sensory, vestibular, and cardiovascular systems . AchieviCaenorhabditis elegans grown in μG displays reduced muscle cytoskeletal proteins, mitochondrial metabolic enzymes  and experience buoyancy during liquid culture in μG, resulting in almost complete loss of contact stimuli. Conversely, in the 1G Earth environment, nematodes settle in liquid and experience contact stimuli with each swimming movement. Altered mechanosensory signaling might therefore contribute to impaired neuromuscular health in-flight.Given the significant costs and practical limitations of studying astronauts, model organisms have also been employed. They have been found to demonstrate highly comparable molecular and physiological maladaptations to spaceflight that are parallel to those observed in people. For example, we have shown the nematode  enzymes , and DAF enzymes . This mo enzymes , paralle enzymes . HoweverC. elegans  DOP-1 and the D2R DOP-3, expressed in ventral cord motor neurons which innervate the body wall muscles, control locomotion (C. elegans body length (The neurotransmitter dopamine (DA) is a well-established modulator of mechanosensory plasticity in  elegans . C. elegnner ear . These Cre often . In addire often . While tcomotion . DOP-1 acomotion  with DOPcomotion . DA alsoy length  and synay length .Furthermore, as with vertebrates, DA regulates learning, decision making, and neuromuscular gait transitions . This crC. elegans grown under μG onboard the ISS over three separate ISS flight experiments  consistently display decreased comt-4 gene expression, a catechol-O-methyltransferase DA degradation enzyme  also displayed decreased comt-4 compared with ground controls  or cell ablation caused worms to crawl faster than wildtype on seeded plates  and D2R dop-3 (vs106).While μG-induced reductions in bending angle and body length in D1R dop-1 (vs100) mutants (dop-3 (vs106) mutants  gene promoter  adult  adult  and 5. T4) adult , assessmcomt-4 expression, DA levels or moving behaviors as compared with adults grown in the absence of the beads ] V), we observed the Ca2+ levels during muscle contraction during the swimming behavior of D1 adults grown in simulated μG in the presence or absence of beads and also in the 1G environment Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Atsushi Higashitani , LX703 dop-3 (vs106), TG2435 (vtIs1 [Pdat-1::GFP + rol-6] and ATU2301 (goeIs3 [Pmyo-3::SL1::GCamP3.35::SL2::unc54 3'UTR + unc-119(+)]; aceIs1[Pmyo-3::mitochondrial LARGECO+Pmyo-2::RFP]). To synchronize growth, approximately 50 adult hermaphrodites were transferred to a fresh NGM plate containing E. coli OP50 and cultured overnight at 20°C. Eggs laid were collected by flushing the adult animals with M9 buffer. After overnight incubation, L1 larvae were collected from the plate by using M9 buffer. Approximately 500 L1 larvae were cultured into 5 ml (35 mm x 60 mm polyethylene bag) of S liquid media containing 2 x freeze dried E. coli OP50 (NemaMetrix Co. Ltd.) at 20°C. Each bag was sandwiched with plastic mesh seats (50 mm x 75 mm (with 3 mm x 3 mm hole)) and setting into polycarbonate container  with sandwiched with hole pushing contents (iP-TEC® secondary container private mesh cushion). The container was subjected to microgravity treatment using a 3D-clinostat  was supplemented into the culture bag starting from L1 larvae at a final concentration of 50 μM in 5 ml 2 x freeze dried OP50 medium. To increase contact stimuli, 0.5 g of polyethylene microsphere beads (WPMS-1.00 ɸ250-300 μm (1.00 g / cc), Cospheric LLC, Santa Barbara) was added into 5 ml medium.C. elegans samples derived from our “CERISE”,“EPIGENETICS” and “MME” spaceflight experiments . Realtime quantitative reverse transcription polymerase chain reaction analysis was performed with the following primer sets: comt-4: fw 5’-CGC TGC GAT TCA CGA GAT G, rv 5’-GAA GCG CCG AGT AGG TAC GAT, eef-2: fw 5’-GAC GCT ATC CAC AGA GGA GG, and eef-2: rv 5’-TTC CTG TGA CCT GAG ACT CC.Total RNA was isolated using a TRI Reagent  following the manufacturer’s protocol. The residual DNA was eliminated by treating with DNase I  during an isolation process of total RNA. The isolated total RNA was subjected to DNA microarray and real-time qPCR. The isolated RNA was analyzed on the Approximately 300 to 500 frozen adult hermaphrodites were washed twice with M9 buffer and homogenized in 100 μl of 0.01 N HCl in the presence of EDTA and sodium metabisulfite. Endogenous dopamine extraction, acylation, and measurement were performed by Dopamine ELISA kit - Research .2+ levels of adult hermaphrodites in the culture bags were captured by a stereomicroscope  and a digital camera  system. Image J software was used to measure the strongest cytoplasmic Ca2+ intensity during flexion of the body wall muscles of an individual , focused on time-lapse microscopic images with CellSens standard software (Olympus).Movement activities of adult hermaphrodites cultured in each condition was measured by thrashing assay (moving frequency: Hz) and maximal bending angle (degree) described, respectively, with slight modification .The freqPdat-1::GFP were observed by scanning confocal microscopy  immediately after fixation with 100 mM NaN3 solution. Thirty worms were randomly selected for each condition. Number of beads /bubbles in CEP and ADE dopamine neuronal dendrites were counted in D1 (day 1) adults and D4 (day 4) adults.Body length and dopamine neuron morphology with Statistical analyses were performed using unequal variance t-test or one-way ANOVA followed by Tukey post hoc tests in RStudio software. P values less than 0.05 were classed as statistically significant. The same alphabet letters in any two groups indicate no significant difference between these groups.


Tau is a highly soluble protein mainly localized at a cytoplasmic level in the neuronal cells, which plays a crucial role in the regulation of microtubule dynamic stability. Recent studies have demonstrated that several factors, such as hyperphosphorylation or alterations of Tau metabolism, may contribute to the pathological accumulation of protein aggregates, which can result in neuronal death and the onset of a number of neurological disorders called Tauopathies. At present, there are no available therapeutic remedies able to reduce Tau aggregation, nor are there any structural clues or guidelines for the rational identification of compounds preventing the accumulation of protein aggregates. To help identify the structural properties required for anti-Tau aggregation activity, we performed extensive chemoinformatics analyses on a dataset of Tau ligands reported in ChEMBL. The performed analyses allowed us to identify a set of molecular properties that are in common between known active ligands. Moreover, extensive analyses of the fragment composition of reported ligands led to the identification of chemical moieties and fragment combinations prevalent in the more active compounds. Interestingly, many of these fragments were arranged in recurring frameworks, some of which were clearly present in compounds currently under clinical investigation. This work represents the first in-depth chemoinformatics study of the molecular properties, constituting fragments and similarity profiles, of known Tau aggregation inhibitors. The datasets of compounds employed for the analyses, the identified molecular fragments and their combinations are made publicly available as supplementary material. In particular, similarity estimations were performed by means of different types of fingerprints, identifying a high degree of structural diversity. Comparison of the molecular properties of active and inactive compounds confirmed the presence of significant structural diversity and allowed us to outline a set of molecular properties that compounds should possess in order to display high potency. A comparative analysis of the molecular fragments of Tau ligands corroborated the results obtained with the molecular descriptors and brought attention to the need for aromatic/heteroaromatic rings and hydrogen bond acceptors, as opposed to hydrophilic or saturated chemical moieties. Notably, an analysis of fragment combinations showed that active compounds are often organized into molecular frameworks composed by two aromatic or heteroaromatic rings separated by different 2- to 7-atom linkers, the latter moiety being very often decorated with H-bond acceptors or possibly consisting of rigid linear unsaturated functional groups. Altogether, the results of this study showed the possibility to identify a set of molecular properties and fragment combinations that can be useful for