In the coronal plane this corresponds to a valgus orientation of the posteroinferior talar facet relative to the talar dome. In conclusion, a more plantarly and valgus orientated posteroinferior talar facet may be associated to chronic ankle instability.Since both the talocrural and subtalar joints can be involved in chronic ankle instability, the present study assessed the talar morphology as this bone is the key player between both joint levels. The 3D orientation and curvature of the superior and the posteroinferior facet between subjects with chronic ankle instability and healthy controls were compared. Hereto, the talus was segmented in the computed tomography images of a control group and a chronic ankle instability group, after which they were reconstructed to 3D surface models. A cylinder was fitted to the subchondral articulating surfaces. The axis of a cylinder represented the facet orientation, which was expressed by an inclination and deviation angle in a coordinate system based on the cylinder of the superior talar facet and the geometric principal axes of the subject's talus. The curvature of the surface was expressed as the radius of the cylinder. The results demonstrated no significant differences in the radius or deviation angle. However, the inclination angle of the posteroinferior talar facet was significantly more plantarly orientated (by 3.5°) in the chronic instability group (14.7 ± 3.1°) compared to the control group (11.2 ± 4.9°) ( p < 0.05). The posteroinferior talar facet stands in slight valgus relative to the superior talar facet. The talar morphology may be a risk factor for CAI.To investigate talar morphological differences between healthy controls and patients with chronic ankle instability (CAI) we modeled the superior and posteroinferior talar facets as cylinders. In relation to the superior facet the posteroinferior facet directed more plantarly in the CAI group (14.7 ± 3.1°) versus the controls (11.2 ± 4.9°) ( In particular, the orientation of the articulating surfaces as well as the deformation in the mid‐ and forefoot induced by foot loading play an important role in this alignment. Both the talocrural and subtalar joints can be involved in CAI.A three‐dimensional (3D) analysis of the orientation of the articulating facets in the mechanical chain of the hindfoot is currently missing in present literature. Previously, the determination of subtalar joint orientation was reported in a two‐dimensional (2D) manner e.g.,, 15, 16,As a result, the present study was designed to address the potentially present differences in the morphology of the talar facets between subjects with CAI and those without . A comparison was made between healthy controls and patients with CAI to assess the differences in orientation of the posteroinferior subtalar joint relative to the talar dome in 3D space, and of the curvature of these facets. The hypothesis is that this orientation and curvature is significantly different between patients with CAI and healthy controls.22.1Brilliance 64 CT scanner, Philips Healthcare, Amsterdam, The Netherlands). A total of 40 ankles from two groups of 20 healthy volunteers each with non‐symptomatic feet/ankles were included to form the control group for the present study.For this retrospective case‐controlled study, three datasets from three previous studies were used.2.2The talus was segmented from the 3D‐CT image and modeled as 3D polygons using custom made software,2.3The facets were modeled as a segment of a cylinder.X‐axis was set parallel with the cylinder's axis of TalusSF and was in a medial‐lateral direction. Next, the geometric principal axes of the subject's talus were determined. The Y‐axis was defined using the Gram‐Schmidt process with the determined X‐axis and the talar principal axis in antero‐posterior direction.Z‐axis was defined perpendicular to the X‐ and Y‐axis with the positive direction orientated superiorly. The orientation of a cylinder was defined by a direction vector, which was chosen in approximately the medial direction in this study. From this, it follows that the direction vector of TalusSF was parallel to the X‐axis, pointing in the negative direction.Another outcome of the present study was the interrelationship between the two facets of the talus. The orientation of TalusIF in respect to TalusSF describes (a part of) the talar morphology. To align all tali uniformly and reproducibly, a local orthogonal right‐handed coordinate system was defined with the following steps. All left tali were mirrored to right tali for further analyses in a corresponding orientation. The α) angle was determined between the direction vector and the XY plane, and the deviation (β) angle was determined between the negative X‐axis and the projection of the direction vector in the XY plane. Cylinder fittings and determinations of the angles were processed with custom made routines in Matlab .The orientation of TalusIF was defined by two angles Figure . The inc2.4The intraobserver reliability in cylinder orientation and radius, introduced by manual selection of the facet surfaces, was evaluated using three randomly chosen feet; one per study cohort. Surface selection was done three times with at least 1 week in between. Interobserver reliability was evaluated based on manual selection of the facet surfaces by two observers  of nine randomly selected hindfeet (three from the CAI group and six from the control group). The two observers were blinded to each other's selections.2.5t test in case of normally distributed data and with a Mann–Whitney U test for non‐normally distributed data. The Hegdes' g value for effect size was calculated. The Hedges' g value was interpreted as small effect (closest to 0.2); medium effect (closest to 0.5); or large effect (closest to 0.8).The orientation angles of TalusIF, the radii of the cylindrical fits, and the root mean square errors were analyzed for normal distribution with the Shapiro–Wilk test. For normally distributed data, outliers were identified as exceeding the value of three times the standard deviation from the mean. For non‐normally distributed data, outliers were identified as exceeding the value of 1.5 times the interquartile range from the median. An outlier in one of the orientation angles was removed from further analyses for both angles. An outlier in the root mean square error or radius of a cylinder facet was removed from further analyses for that facet. The differences between the control group and the CAI group were tested with an independent Student The intraclass correlation coefficient (ICC) was calculated as a measure of intraobserver and interobserver reliability. The ICC was interpreted as poor (≤0.40); moderate (0.40−0.75); substantial (0.75 – 0.90); or excellent reliability (>0.90).SD for normally distributed data and as median (interquartile range) for non‐normally distributed data.The statistical analyses were performed with SPSS . A significance level of 0.05 was used for all tests. Data are presented as mean ± 1 33.1n = 11) compared to the control group , with a large effect size, t(26) = −2.85, p = 0.008, g = 0.76, with a mean difference of 3.5° , that is, degrees of freedom were adjusted from 48 to 26. The deviation angle of TalusIF was not statistically significantly different between the CAI  and the control group , t(48) = 1.12, p = 0.263.The orientation angles were normally distributed. One outlier was identified in the inclination angle in the CAI group, and one outlier was identified in the deviation angle in the control group. The orientation of TalusIF was in an antero‐medial‐plantar direction. The inclination angle of TalusIF was statistically significantly more plantarly orientated in the CAI group  and the control group , t(48) = −1.06, p = 0.249  and the control group , t(26) = 1.40, p = 0.084. For TalusIF, Levene's test indicated unequal variances . Therefore, degrees of freedom were adjusted from 50 to 26.The cylinder radii were normally distributed. One outlier was identified for TalusSF in the control group, and one outlier in the CAI group for the root mean square error. The cylinder radii of TalusSF were not statistically significantly different between the CAI  and the control group , t and the control group , t(48) = 1.22, p = 0.228.The root mean square errors of the cylinder fit were normally distributed, except for TalusIF in the CAI group. After removal of this outlier, the remaining data were normally distributed. The root mean square errors of TalusSF were not statistically significantly different between the CAI (0.45 ± 0.09 mm, 3.4The intra‐ and interobserver reliability showed excellent reliability for each of the outcome measurements Table . The dif4The present study investigated the orientation and curvature of the superior and posteroinferior talar articulating surfaces of nonsymptomatic individuals and of patients with chronic ankle instability. Previous reports have identified a varus hindfoot deformity to be a risk factor in the development of CAI after a first ankle sprain.The most important finding was that the mean inclination angle of the cylinder's axis of the posteroinferior talar facet was statistically significantly more plantarly orientated, by 3.5 degrees, in the CAI group compared to the control group. In a coronal plane this corresponds to a talus with a slight valgus of the posteroinferior facet relative to the talar dome Figure .We expected to find a varus orientation of the subtalar joint in patients with CAI when comparing them to the healthy population. This would have been in accordance with earlier studies who found a correlation between varus malalignment and CAI.No statistically significant differences were demonstrated between the groups for the radii nor root mean square errors of the cylinder fits. The root mean square error indicates how well the facets fit to a cylinder. These results imply that the morphology of the two facets was comparable between the two study groups. Frigg et al.The present study focused on the bony morphology of the talus as a possible risk factor for CAI. Caution is required when interpreting the results of this one factor, namely the bony morphology, when in fact CAI is multi‐factorial.It is important to realize that the overall hindfoot alignment could still be varus, but that in our analysis of an isolated talus at the joint levels this could not be demonstrated. The patients could have an overall hindfoot varus alignment when the medial distal tibial angleThis study has several limitations other than not assessing the hindfoot alignment. The true articulating cartilaginous surfaces cannot be seen in CT images, and as such only the subchondral surfaces were analyzed. Nonhomogeneous thickness distribution of the cartilage may shift the cylinder. This could be addressed in a study that uses Magnetic Resonance Imaging. Further, only 12 CAI patients were included. After removal statistical outliers, this was further reduced. Future research could include more patients with CAI.Up to today, the mechanics of the subtalar joint are not fully elucidated. Different in vivo, ex vivo, and modeling studies have given more insight into the kinematics of the subtalar joints.The strength of this present study was that the interrelationship of the superior and posteroinferior talar facets could be determined in 3D without influence of the position of the foot in the CT scanner. The applied method for selecting the articular surface on 3D surface models proved to be very reliable based on the results of the intraobserver and interobserver analyses, but was laborious. A semi‐automatic procedure can make this step more efficient in the future.The present study provides a next step in identifying morphology of bony structures in the hindfoot being associated with CAI. It is the first study that analyzed the interrelationship of the talocrural and subtalar joints in 3D. Future studies should focus on a method to determine the orientation at each joint level in the chain from the knee to the ground in 3D with a golden standard for a frame of reference, preferably in a weight bearing condition.In conclusion, it was found that the morphology of the talus itself does not necessarily need to be in “varus” to contribute to CAI. It was demonstrated that CAI patients have a more valgus position of the posteroinferior talar facet that may be a risk factor for CAI by varus compensation. The relationship between the superior and posteroinferior facets of the talus in relation to the lower leg axis in varus and valgus deformities, the overall alignment, needs further research.All authors contributed to the study design. Roeland P. Kleipool, Jari Dahmen, Johannes GG. Dobbe, Geert J. Streekstra, and Leendert Blankevoort contributed to the data analysis, and Roeland P. Kleipool and Jari Dahmen contributed to the interobserver analysis. Roeland P. Kleipool, Sjoerd AS. Stufkens, Jari Dahmen, Gwendolyn Vuurberg, and Markus Knupp participated in interpretation of data and clinical significance. Roeland P. Kleipool drafted the manuscript, which was critically revised and approved by all authors.


Fibrotic signaling plays a pivotal role in the development and progression of solid cancers including renal cell carcinoma (RCC). Intratumoral fibrosis (ITF) and pseudo-capsule (PC) fibrosis are significantly correlated to the disease progression of renal cell carcinoma. Targeting classic fibrotic signaling processes such as TGF-β signaling and epithelial-to-mesenchymal transition (EMT) shows promising antitumor effects both preclinically and clinically. Therefore, a better understanding of the pathogenic mechanisms of fibrotic signaling in renal cell carcinoma at molecular resolution can facilitate the development of precision therapies against solid cancers. In this review, we systematically summarized the latest updates on fibrotic signaling, from clinical correlation and molecular mechanisms to its therapeutic strategies for renal cell carcinoma. Importantly, we examined the reported fibrotic signaling on the human renal cell carcinoma dataset at the transcriptome level with single-cell resolution to assess its translational potential in the clinic. Renal cell carcinoma accounts for approximately 3% of all adult malignant diseases and over 90% of kidney cancer . In 2020Cancer-associated fibrosis was found to play a critical role in tumorigenesis, immune evasion, metastasis, and drug resistance in various solid tumors . EpidemiIn this review, we summarized the pathogenic roles and underlying mechanisms of various RCC-associated fibrosis in the development and progression of RCC. In addition, we validated the reported findings in RCC patients datasets by using gene set enrichment analysis (GSEA) and single-cell RNA sequencing (scRNA-seq) data mining. Furthermore, the translational potentials of new therapeutic strategies targeting fibrotic signaling in RCC were also discussed.Fibrosis is a common feature frequently observed in RCC , charactvia cross-linked collagen matrix deposition. Such ITF is associated with decreased lymphocyte infiltration, poorer patient survival, and various carcinomas, including breast cancer , collagen 1 (COL1A1 and COL1A2), and collagen 6   . Also, s COL6A3) .Interestingly, in contrast to the previous findings, several studies show that tumor-related fibrosis might limit tumor growth and metastasis at the early stages of cancer . In termWith the emerging RNA sequencing technologies, from bulk RNA sequencing (bulk RNA-seq) to scRNA-seq, transcriptome profiling of RCC has been greatly utilized in biomarker discovery, cancer heterogeneity characterization, and studies regarding distant metastasis and therapy resistance. More importantly, understanding the role of fibrosis in RCC development enlightens further therapeutic target identification by data mining the transcriptomic database of RCC patients. Here, we performed gene set enrichment analysis, as previously described , of a puACTA2 (α-SMA), COL1A1, COL23A1, VEGFA, and TGFB1 as the top differentially expressed genes (DEGs) upregulated in ccRCC (2 fold change| ≥1 in collecting duct carcinoma (CDC), in comparison with the normal kidney tissue. Enrichment analysis targeting those DEGs further identified the pro-fibrosis collagen signaling pathway as the top-ranked enriched pathway activated in the CDC group. KRT17 (keratin 17), a wounded stratified epithelium-induced filament protein, was identified as the top DEG with the highest fold change of expression in CDC [KRT17 high-expressing basal-like cells were defined in fibrotic hypersensitivity pneumonitis patients with higher expression levels of COL1A1, FN1, and COL6A2 and upregulated activities in ECM organization by scRNA-seq analysis  plot by using the Normalized, ScaleData, and FindVariableFeatures functions, respectively, in the Seurat package to map the comprehensive cell landscape as shown in TGFB1 expression, together with three other key fibrosis regulators, FN1, VIM, and CXCR4, was also elevated under the RCC condition  to treat patients with advanced malignant melanoma and RCC . Howeverin vivo . MechaniFibrosis has long been recognized as a major contributor to cancer progression and invasion. In this review, we systematically summarized the clinical association between renal fibrosis and poorer RCC outcomes and how a fibrotic microenvironment interacts with RCC in the form of ITF, CAFs, and PC fibrosis. In addition, we further conducted transcriptomic analysis on an up-to-date scRNA-seq profile of human RCC to confirm the participation of diverse fibrotic signaling in RCC development at single-cell resolution. The crosstalk between common pro-fibrotic pathways, including TGF-β, WNT, mTOR, and NOTCH signaling pathways, and RCC has also been discussed. In conclusion, the discovery of the mechanisms through which fibrotic signaling promotes tumorigenesis and aggressiveness in RCC provides inspiration for the development of anti-fibrotic therapies, such as mTOR inhibitors or anti-TGF-β antibodies, as novel therapeutic strategies for renal cancer.


A standard bull breeding soundness evaluation (BBSE) identifies bulls with semen that is grossly abnormal. Nonetheless, semen samples classified as satisfactory based on these traditional approaches differ in fertility; perhaps there are submicroscopic differences in sperm characteristics affecting fertility. Therefore, a better understanding of molecular regulation of sperm function could promote development of novel, evidence-based approaches to predict male fertility. Recently the α4 isoform of Na/K-ATPase (ATP1A4) has received considerable attention, due to its testis- specific expression in post-meiotic germ cells and mature sperm, in addition to its regulation of sperm motility and capacitation. Using fresh bull sperm, we determined that ATP1A4 resided in specialized microdomains (raft and non-raft) of the sperm plasma membrane and activated specific signaling  molecules during sperm capacitation. Furthermore, ATP1A4 was the predominant isoform responsible for total Na/K-ATPase activity in capacitated sperm. Despite the widely accepted dogma of transcriptional/translational quiescence, bovine sperm translated ATP1A4 mRNA on mitochondrial or mitochondrial-type ribosomes, increasing their content and activity during capacitation. Proteomic analysis of raft and non-raft fractions revealed a significant interaction between ATP1A4 and plakoglobin, a member of the β-catenin family of proteins involved in cell adhesion, in the equatorial segment of capacitated sperm, suggesting a potential role in sperm-oolemma fusion. In frozen-thawed sperm, ATP1A4 content and activity was greater in high- versus low-fertility bulls. Additionally, ATP1A4-induced increases in ROS, calcium, actin polymerization and tyrosine phosphorylation were also involved in regulating post-thaw sperm function in these bulls. Overall, results demonstrated that ATP1A4 had unique roles in controlling several aspects of sperm physiology, acting through well-established enzyme activity and signaling functions. Consequently, isoforms of Na/K-ATPase are potential biomarkers for male fertility. Dairy and beef industries strive to achieve high pregnancy rates from genetically superior bulls. Therefore, fertility is more important than production traits. Based on the assessment from the American Gelbvieh Association’s Alliance marketing program on 110,000 feedlot cattle, estimated relative importance of reproductive traits to growth and carcass traits are in the ratio of 4:2:1, respectively . Bulls wHigh-throughput technologies such as mass spectrometry, along with appropriate bioinformatics tools, may provide better cues for annotation of proteins in the context of gamete biology. As proteins define a cell phenotype, changes at the proteome level could lead to differences in phenotypes influencing economically important traits and could enable identification of biomarkers of fertility. For example, content of P25b, a bovine sperm membrane antigen, was lower in semen from subfertile bulls than in high-fertility bulls . Compari+ and K+ gradients across the plasma membrane of most mammalian cells. In that regard, this enzyme is responsible for cell volume and pH, resting membrane potential, osmotic balance, and generation of a Na+ gradient essential for secondary transmembrane ion transport  has received considerable attention in recent years due to its sperm-specific expression . In addiet al. (2012) also reported that ATP1A4 activity was upregulated at the plasma membrane during sperm capacitation. ATP1A4 indirectly influences sperm motility by regulating intracellular pH, membrane potential and intracellular calcium release. ATP1A4 is linked to other K+ channels which are involved in depolarization and hyperpolarization. Consequently, ouabain inhibition of ATP1A4 caused sperm membrane depolarization  specifically expressed in sperm suggests that this protein has a specific role in sperm physiology. Consequently, it was no surprise that sperm from ATP1A4 KO mice demonstrated severe reduction in total motility due to a characteristic bend in the sperm tail and cell membrane depolarization  mediated activation of protein tyrosine kinase (PTK) induces tyrosine phosphorylation  in sperm  induces ERK1/2 activation via transactivation of EGFR and activation of PLC and PKC. In addition, Na/K-ATPase-ouabain interaction generates ROS from mitochondria through the Src/EGFR/Ras pathway  and non-raft  membrane fractions during capacitation. We also inferred that ATP1A4 interacted with Cav1 and EGFR in the raft fraction, whereas interactions of ATP1A4 with Src, EGFR, and ERK1/2 occurred in the non-raft fraction of ouabain-capacitated sperm. Therefore, we concluded that both raft and non-raft cohorts of ATP1A4 contributed to enzyme activity and phosphorylation of signaling molecules during bovine sperm capacitation.2), generating inositol 1,4,5-triphosphate (IP3) and diacyl glycerol (DAG), which in turn activate protein kinase C (PKC). Co-localization of ATP1A4 and PLC isoform (PLCζ) was performed to determine involvement of PLCζ in our raft signaling model. PLCζ was localized to the equatorial segment (ES) and ATP1A4 co-localised with PLCζ at the ES and postacrosome region in ouabain- capacitated sperm (3 binds to its cognate receptor (IP3R), leading to an increase in intracellular calcium, whereas PKC through other mediator proteins promotes polymerization of globular actin (G-actin) to filamentous-actin (F-actin). Within non-rafts, ATP1A4- ouabain interaction involves Src and EGFR leading to activation of ERK1/2 and protein tyrosine kinase (PTK)-mediated tyrosine phosphorylation of proteins. Increases in F-actin, intracellular calcium, and protein tyrosine phosphorylation contribute to capacitation- associated changes in sperm. The above-mentioned model assumes that activation/phosphorylation of signaling molecules occurs within the respective domains (raft or non-raft) of the plasma membrane. However, it is likely that a subset of the non-raft pool of ATP1A4 merges with the raft pool of ATP1A4 (or vice- versa), due to cholesterol efflux, either amplifying the existing signaling process or initiating a new downstream pathway during capacitation. All these possibilities open exciting areas for future investigation and could unravel the dynamic organization of signaling complexes mediated by ATP1A4 in sperm.We proposed a model regarding potential raft- and non-raft-mediated ATP1A4 signaling pathways  in the bed sperm . IP3 binIn the aforementioned study, we highlighted two distinct pools (raft and non-raft) of ATP1A4 that trigger specific downstream signaling pathways. However, during spermatogenesis, sperm acquire several unique proteins or their isoforms to meet its functional demands, including lactate dehydrogenase (LDH-C4), sperm adhesion molecule 1 (PH-20), and testis-specific isoform of Angiotensin Converting Enzyme (t-ACE). Therefore, it is likely that ATP1A4 interacts with other sperm proteins during capacitation. To test this, we used an immunoprecipitation-mass spectrometry approach to compare the interactome profile of ATP1A4 between raft- and non-raft membrane fractions from capacitated sperm.Proteomic analysis using a gel-based LC- MS/MS approach identified that the non-raft interactome was comprised of hexokinase, plakophilin- 1, 14-3-3 protein, cathepsin D, and heat shock protein. A disintegrin and metalloprotease (ADAM) and annexin A2 were exclusive to the raft fraction, whereas actin and plakoglobin were identified in both raft and non-raft fractions of ouabain-capacitated sperm. These differentially interacted proteins are putatively involved in sperm-oocyte interactions, metabolism, and protease activity and they also act as adaptor and cytoskeletal proteins, based on gene ontological information. We validated plakoglobin, among other proteins, due to its significant interaction with ATP1A4. Immunocytochemical staining demonstrated that plakoglobin was localized to the equatorial segment of uncapacitated and capacitated sperm. The ATP1A4 signal was predominantly localized to the anterior acrosome in uncapacitated sperm, became pronounced in the equatorial segment, and co-localized with plakoglobin in capacitated sperm .In epithelial cells, E-cadherin, catenins and the actin cytoskeleton mediate cell-cell adhesion , suggesting mitochondrial translation of ATP1A4 during capacitation.de novo protein translation from sperm mRNA is essential to supplement degraded proteins or to support functional changes during capacitation. This de novo protein production was also demonstrated in other mammals, suggesting widespread existence of this mechanism across species . The latter step is markedly enhanced in the presence of GTP. These initial steps recruit the large ribosome subunit (39 S) to join the small ribosome subunit (28 S) and thereby a fully functional mitochondrial ribsome is assembled. The initiation phase is completed, and translation can proceed with elongation and termination phases leading to production of a functional protein , band intensities at ∼110 and 37 kDa were partially inhibited, whereas bands at 75 and 50 kDa were completely abolished, suggesting that protein synthesis occurred during bovine sperm capacitation. Mitochondrial or mitochondrial-type ribosomes may perform a similar function in sperm, as cytoplasmic machinery is lost during spermatogenesis . Interes embryos , we hypoPerhaps post-translational modifications of ATP1A4 are carried out through hydrophobic and electrostatic interactions of membrane-associated molecular chaperones ultimately guiding them to appropriate folding and transportation pathways. Finally, insertion and anchoring of the protein to the plasma membrane could be facilitated by hydrophobic amino acids in the transmembrane domains of ATP1A4 (In the Canadian Dairy Network (CDN), bull fertility is assessed by 56-day non-return rates (NRR), based on a linear mixed model adjusted for several factors, such as month of insemination, age of the cow or heifer at insemination, breed of service sire, price of inseminating dose, AI technician, and an overall herd management effect, all combined into one index . The bovine artificial insemination (AI) industry in Canada uses high-fertility (HF) and low-fertility (LF) Holstein bulls for breeding purposes. Based on this FERTSOL assessment, bulls were classified as either high-fertile  or low-fertile .Since mature sperm DNA is generally transcriptionally quiescent, sperm functions are carried out by existing proteins without additional protein synthesis (with few exceptions). Therefore, sperm proteins may serve as molecular markers for variations in bull fertility , an intracellular calcium store receptor, is regulated by ouabain, thereby increasing calcium concentrations. The presence of IP3 ligand and its cognate receptor, IP3R has already been confirmed in sperm  may aid in development of improved laboratory assays for better prediction of fertility of bulls (Using fresh and frozen-thawed bull semen, we demonstrated the physiological relevance of ATP1A4 in regulation of sperm function and its potential as a fertility marker. In the raft and non-raft study, we determined that raft and non-raft pools of ATP1A4 stimulated independent signaling pathways, leading to capacitation. Deducing downstream pathways mediated by ATP1A4 could help understand the molecular basis by which this protein functions in normal sperm, which in turn could help develop diagnostic approaches to identify male infertility. Mass spectrometry revealed interesting candidate proteins such as plakoglobin that interacted with ATP1A4 during capacitation. The functional significance of PLCζ as a sperm oocyte activating factor and the role of plakoglobin has been well studied in sperm and testis, respectively. However, the relevance of both these proteins interacting with ATP1A4 during capacitation and fertilization remains to be investigated.Our results also indicated that there is ATP1A4 mRNA in capacitated sperm. Functionally intact and stable sperm mRNAs can be delivered to the oocyte during fertilization and are important during the critical window of activation of the embryonic genome and its epigenetic regulation (


Arthrometry has been introduced to evaluate the laxity of ankle joint in recent years. However, its role in the diagnosis of chronic ankle instability is still debatable. Therefore, we assessed the diagnostic accuracy of a digital arthrometer in terms of sensitivity and specificity.According to the inclusion and exclusion criteria proposed by the International Ankle Consortium, we recruited 160 uninjured ankles (control group) and 153 ankles with chronic ankle instability (CAI group). Ankle laxity was quantitively measured by a validated digital arthrometer. Data of loading force and joint displacement were recorded in a continuous manner. Differences between the control and CAI groups were compared using 2-tailed independent t test. A receiver operating characteristic curve analysis was used to calculate area under a curve, sensitivity and specificity.p < 0.001) with the largest effect size at 90 N. Statistical significance was found in the differences between the two groups in load–displacement ratio at 10–120 N, 10–40 N, 40–80 N and 80–120 N , with the largest effect size at 10–40 N. Load–displacement ratio at the interval of 10–40 N had the highest area under a curve (0.9226), with sensitivity and specificity of 0.804 and 0.863, respectively, when the cutoff point was 0.1582 mm/N.Load–displacement curves of the two groups were depicted. Differences of joint displacement between the control and CAI groups were compared at 30, 45, 60, 75, 90, 105 and 120 N, which were all of statistical significance (all The digital arthrometer measurement could quantitively analyze the ankle laxity with high diagnostic accuracy. The load–displacement ratio would be a reliable and promising approach for chronic ankle instability diagnosis.Level of evidence level II. Ankle sprain is one of the most frequently encountered traumatic injuries in clinical settings . It is cDespite the numerous studies of CAI, there is still no consensus about the gold standard of CAI diagnosis. The proper diagnosis of CAI is a difficult task because the association between inversion trauma history and ligament injury is still uncertain . Manual Recent literature showed that instrumented stress testing could quantify the laxity of talocrural joint; however, only few studies focused on the validity of arthrometers in terms of specificity and sensitivity. Croy et al.  found thThe purpose of this study was to design a cross-sectional study with standardized inclusion/exclusion criteria and an adequate sample size to quantitively assess the diagnostic accuracy of ankle arthrometers. In this study, we were to compare different diagnostic standards and calculate the diagnostic accuracy of each standard. Our hypothesis was that arthrometers would display an excellent diagnostic accuracy on CAI.A cross-sectional study was conducted to quantitatively investigate the difference in ankle joint laxity between the CAI and control groups from October 2020 to September 2021. This study was approved by the local ethics committee. All participants provided written informed consent.We made the inclusion and exclusion criteria according to the international Ankle Consortium . For theSample size was calculated by the formula designed for quantitative variables in cross-sectional studies:α/21− = Standard normal variate, which is 1.96 at 5% type 1 error (p < 0.05).ZSD = Standard deviation of variable. Value can be taken from previously done study.d = Absolute error or precision decided by researchers.SD was 5.66 mm according to a previous study , and d wA total of 338 subjects consented to the participation in the study and underwent the testing procedure. Among them, 25 were excluded: 12 had a history of ankle fracture or surgery, 6 experienced at least one episode of ankle sprain within 3 months prior to the enrollment, and 7 could not tolerate the stress applied by the arthrometer. At last, 313 subjects were included in the study. In total, there were 160 subjects assigned to the control group and 153 to the CAI group according to the inclusion and exclusion criteria  of a single measure was 0.897 , and the ICC2,2 was 0.963  when using the average of 3 measures, indicating an excellent test–retest reliability. Inter-tester reliability was evaluated by taking measurement of the same ankle made by 2 independent examiners, and the ICC2,1 was 0.949, indicating an excellent inter-tester reliability.The data were analyzed for only 1 ankle per individual. For individuals in the control group, the side to be included in the group was randomly selected. For individuals in the CAI group, the self-described “worse” side was chosen. On the tested side, the procedure was performed 3 times. Ankle arthrometer calculations were based on the average values of 3 consecutive measurements. Reliability of the arthrometer in our study was also validated. The intraclass correlation coefficient , medium (0.50) or large (0.80) . A receip = 0.047). This might be due to a larger proportion of male in the CAI group  than the control group . The CAI group’s CAIT scores were significantly lower than the control group (p < 0.001).Subjects’ demographic characteristics in comparisons between the control and CAI groups are shown in Table p < 0.001) with the largest effect size at the load of 90 N . with the largest effect size at 10–40 N (effect size = 1.85).By depicting each individual load–displacement curve, the overall load–displacement curves of the control and CAI groups were derived Fig. . For staA ROC analysis was used to calculate the diagnostic accuracy of each reference standard Table . DisplacIn this study, we demonstrated that arthrometers present with good to high diagnostic accuracy, with sensitivity and specificity of 0.804 and 0.863, respectively. Dynamic measurement may have an advantage over static measurement in diagnosing CAI (Table Researchers tended to apply a force to a high magnitude with arthrometers, ranging from 125 to 200 N , 18, 19.Nauck et al.  and LohrThe manual ADT is a physical examination routinely used to evaluate the laxity of the ankle joint complexes in patients with CAI because of its good practicability. However, it has been questioned for decades because of subjectivity. Vaseenon et al.  found thIt is been proposed to use arthrometers to perform ADT quantitatively and objectively . ArthromIn this study, the selection criteria of subjects were based on recommendations of International Ankle Consortium, which proposed selection criteria with the best available evidence . The incTypes of arthrometric devices also play a role in validating diagnostic accuracy. The Hollis ankle arthrometer and the LigMaster are two most frequently used arthrometers. The Hollis ankle arthrometer is reported to have a high to excellent reliability with ICC values between 0.82  and 0.99Differences in the selection of reference standards also influence diagnostic accuracy of arthrometers. A systemic review of in vivo arthrometer measurements showed that studies regarding diagnostic accuracy of arthrometers in an in vivo, clinical setting were still limited . In exisWe recognized several limitations of this study. First, we did not include copers in the study. Besides, there was a significant difference in BMI between the control and CAI groups, which might influence the results. The influence was supposed to be limited, however. Vuurberg et al.  already In conclusion, the digital arthrometer measurement could quantitively analyze the ankle laxity with high diagnostic accuracy. The load–displacement ratio would be a reliable and promising approach for chronic ankle instability diagnosis. The load–displacement ratio at 10–40 N had a high diagnostic accuracy with sensitivity and specificity of 0.804 and 0.863, respectively, which was suitable to diagnose patients with CAI in clinical settings.


Thymic stromal lymphopoietin (TSLP), long known to be involved in Th2 response, is also implicated in multiple inflammatory dermatoses and cancers. The purpose of this study was to improve our understanding of the expression of TSLP in the skin of those dermatoses. Lesional specimens of representative immune-related dermatoses, including lichen planus (LP), discoid lupus erythematosus (DLE), eczema, bullous pemphigoid (BP), psoriasis vulgaris (PsV), sarcoidosis, and mycosis fungoides (MF), were retrospectively collected and analyzed by immunohistochemistry. Morphologically, TSLP was extensively expressed in the epidermis of each dermatosis, but the expression was weak in specimens of DLE. In a semiquantitative analysis, TSLP was significantly expressed in the epidermis in LP, BP, eczema, PsV, sarcoidosis, and MF. TSLP expression was higher in the stratum spinosum in LP, eczema, BP, PsV, and MF and higher in the stratum basale in sarcoidosis and PsV. Moreover, we found positive TSLP staining in the dermal infiltrating inflammatory cells of BP, PsV, and sarcoidosis. Our observation of TSLP in different inflammatory dermatoses might provide a novel understanding of TSLP in the mechanism of diseases with distinctly different immune response patterns and suggest a potential novel therapeutic target of those diseases. S. aureus killing in the innate immune response [Thymic stromal lymphopoietin (TSLP), a type of epithelial-derived cytokine, was originally identified as a factor promoting B cell proliferation and development , 2. Subsresponse . Hematopresponse , 6.Initially, research about TSLP mainly focused on allergic disorders, such as allergic asthma, atopic dermatitis (AD), allergic rhinitis, eosinophilic esophagitis, and food allergies , 7, 8. I+ T cells attacking the keratinocytes. Previous research has reported that TSLP expression is elevated in the oral epithelium and sera of patients with oral LP [Combining clinical and histopathologic phenotypes with immunology and molecular genetics, noncommunicable inflammatory dermatoses could be classified into six immune response patterns . PsV is  oral LP , 17. Sar oral LP . Thus faTo better understand the exact location and pattern of TSLP expression in the skin of patients suffering from different dermatoses, we analyzed by immunohistochemistry retrospective samples of representative immune-related dermatoses, including LP, discoid lupus erythematosus (DLE), PsV, BP, eczema, sarcoidosis, and mycosis fungoides (MF).The dermatopathology database of the Department of Dermatology at Peking Union Medical College Hospital (Beijing) was queried to identify samples consistent with LP, LE, eczema, BP, PsV, sarcoidosis, and MF. Healthy controls (HC) for stains were recruited from normal surrounding skin in patients with nevi, cysts, or seborrheic keratosis. This study was performed in line with the principles of the Declaration of Helsinki (as revised in 2013). Approval was granted by the Ethical Review Committee of Peking Union Medical College Hospital (ZS-1735). Potential participants who had received systematic glucocorticoids, immunosuppressants, biologics, or long-term topical treatment were excluded from the study. The criteria for specimen selection are shown in Paraffin sections were deparaffinized and blocked with 3% hydrogen peroxide for 15 min, then incubated with goat serum solution for 1 h at room temperature. Next, samples were incubated with rabbit anti-human TSLP  overnight at 4°C, followed by incubation with HRP-conjugated AffiniPure goat anti-rabbit IgG . The specimens were then stained with DAB , followed by washing and counterstaining with hematoxylin .For quantitative image analysis, the slides were scanned using a NanoZoomer 2.0-RS , and five visual fields were selected randomly for each slide. Each section was graded semiquantitatively on a scale of 0–3  blindly assessed by two dermatologists in five randomly selected visual fields at high (400X) magnification of scanned sections. The section whose average grade is greater than one was considered a positive case. The gradient of the stratum basale and the stratum spinosum was mathematically calculated by subtracting the stratum basale score from that of the stratum spinosum.t-test was used to determine the significance of the gradient of the stratum basale and the stratum spinosum. A one-way ANOVA was used to assess general differences among each dermatosis group, and Bonferroni adjustment was used to assess differences between each dermatosis group pair. The grade correlation of TSLP expression between the stratum basale and the stratum spinosum was evaluated using the Pearson correlation test. Statistical significance was assigned at p < 0.05. SAS 9.4  was used for statistical analyses. The Prism 7 software  was used to generate statistical graphs.The grades of TSLP expression in the stratum basale and the stratum spinosum were presented as means ± standard deviations. A one-way ANOVA and Dunnett's test (the HC group was considered the control group) were used to determine the significance of grade. One-group Student's n = 10), DLE (n = 10), eczema (n = 10), BP (n = 10), PsV (n = 11), sarcoidosis (n = 10), MF (n = 10), and HC (n = 5), were collected for our investigation  and sarcoidosis (p =0.025) specimens and not significantly expressed in LP (p = 0.102), DLE (p = 0.360), eczema (p = 0.253), BP (p = 0.145), and MF (p = 0.436) (p = 0.002), BP (p = 0.031), PsV (p = 0.002), eczema (p = 0.041), and MF (p = 0.046) specimens and not significantly expressed in DLE (p = 0.217) and sarcoidosis (p = 0.309) (Positive rates of TSLP expression in the epidermis in each group are presented in = 0.436) . Similar= 0.309) . In the To further investigate the diversity of TSLP expression among these dermatoses, we compared the grade between every two groups. There were no significant differences in TSLP expression in both the stratum spinosum and the stratum basale grades among these dermatosis groups, which means TSLP expression did not significantly differ between each disease group overall.p = 0.022) specimens and lower than those in the stratum basale in sarcoidosis (p = 0.019) specimens. The differences of TSLP expression between the stratum basale and the stratum spinosum in other dermatoses were not significant .Morphologically, there were no apparent differences in TSLP stain intensity between the stratum basale and the stratum spinosum. However, mean semiquantitative grades in the stratum spinosum were significantly higher than those in the stratum basale in LP (p = 0.009), eczema (p = 0.030), BP (p = 0.027), and MF (p = 0.023), respectively. The differences of gradients between other dermatoses were not significant . Similar correlations could also be found in LP , DLE , eczema , BP , PsV , and sarcoidosis  specimens, whereas in MF  and HC  specimens, this correlation was not significant  . CorrelaThus far, no studies about the relationship between cutaneous sarcoidosis and TSLP have been reported yet, although one study about idiopathic pulmonary fibrosis showed that TSLP level in the bronchoalveolar lavage fluid of pulmonary sarcoidosis was not higher than that of normal controls . In our Regarding BP and PsV, the dermal infiltrating inflammatory cells were also positive for TSLP staining, which indicates that TSLP was expressed not only by keratinocytes but also by the infiltrating inflammatory cells in BP and PsV. This observation has not been reported previously, and the specific mechanism is still unknown. Nevertheless, TSLP was also expressed by mast cells, CD163+ macrophage cells, and endothelial cells in other immune-related dermatoses , 37, 38.+ T cells attacking the basal keratinocytes, but TSLP is expressed predominantly higher in the spinous keratinocytes. It may imply that the spinous cells play more important roles in pathogenesis of LP than previously thought, whereas in sarcoidosis, TSLP in the skin may come from the dermis or circulation. Moreover, if TSLP was produced from spinous keratinocytes in those dermatoses, it would be valuable to prevent TSLP generation topically. Recently, topical agents were found to reduce TSLP secretion in the epidermis [Although our study showed no differences of TSLP expression between the stratum basale and the stratum spinosum morphologically, TSLP expression is significantly more intense in the stratum spinosum in LP, which is not consistent with a previous report . By revipidermis , 40. TheFinally, this study has potential limitations. As mentioned above, the sample size of this research is small in some extent, whereas our study attempts to conduct a preliminary study and our intention is to raise a concern about TSLP and its effect in cutaneous diseases with different patterns of immune response. We would like to enlarge the sample size in the further study of specific dermatosis. Furthermore, we only detected the levels of TSLP in skin specimens of patients. Some immune-related dermatoses, such as PsV, BP and sarcoidosis, were not only skin diseases but also systemic disorders. It might be meaningful to detect the levels of TSLP in the sera or other samples and to compare the results with those in skin lesions. Moreover, TSLP interacts with IL-33 and other cytokines and mediates a Th2 immune response so that it is valuable to detect other cytokines and compare their expressions .As a whole, our study found that the expression of TSLP was elevated in different immune response dermatoses. This finding adds to the accumulating evidence of the importance of TSLP in each inflammatory disease. It remains to be elucidated if TSLP plays a role in some inflammatory diseases, such as DLE and sarcoidosis. Tezepelumab, the first-in-class anti-TSLP monoclonal antibody, was approved as a biological agent for the treatment of severe asthma . A rando


Malignant mesothelioma (MMe) is an aggressive neoplasm that occurs through the transformation of mesothelial cells. Asbestos exposure is the main risk factor for MMe carcinogenesis. Other important etiologies for MMe development include DNA damage, over-activation of survival signaling pathways, and failure of DNA damage response (DDR). In this review article, first, we will describe the most important signaling pathways that contribute to MMe development and their interaction with DDR. Then, the contribution of DDR failure in MMe progression will be discussed. Finally, we will review the latest MMe therapeutic strategies that target the DDR pathway. Malignant mesothelioma (MMe) is an aggressive high lethal cancer that mostly arises from mesothelial cells in the pleural and peritoneum . AlthougThe dysregulation of major signaling pathways has been the subject of a huge number of studies investigating their possible role in cancers development and tumorigenesis. On this basis, multiple studies have reported higher expression levels and activity of Hedgehog (HH), mTOR, MAPK, and Calcium signaling pathways that are associated with the worst outcome and survival in MMe patients Table . In the Hedgehog (HH) signaling is introduced as an embryonic/developmental signal transduction pathway that appears to play essential roles in embryonic development through the modulation of proliferation, differentiation, and body patterning as well as tissue homeostasis upon injuries in differentiated cells. Furthermore, this signaling pathway has been the subject of many studies investigating its possible influential function in the progression and maintenance of multiple types of cancers . There aTherefore, various studies have recently reported that HH target genes overexpression in MPM is associated with the worst clinical outcome. Higher gene expression of GLI1, SMO, YAP, and SHH are detected in MPM patients which are characterized by short overall survival , 15.Moreover, HH signaling activates in both tumor and tumor microenvironments. Thus, not only autocrine HH signaling but also HH signaling in the stroma of the tumor microenvironment plays a significant role in tumor growth . SolutioFurthermore, it has been suggested that there is a cross-talk between HH signaling and YAP signaling in MMe. YAP is a transcriptional regulator located in the nucleus in its active form and provokes cancer stem cell attributes, proliferation, chemoresistance, and metastasis in a variety of cancers including MMe. In general, above 70% of MPM is characterized by a high protein level of YAP . In vivoMoreover, HH signaling inhibition decreases cell proliferation by about 40–60%. Generally, the most effective strategy for HH suppression is implementing pharmacologic SMO antagonists (GDC-0449) or Gli1/2 antagonists (GANT61). ATO and ITRA are two FDA-approved drugs that can inhibit HH signaling in MPM cells . MoreoveAs a result, the significant effect of HH signaling in MMe cell proliferation has been proved in multiple studies. Thus, this signaling pathway can be a potential option for targeting and combinational therapy.mTOR is a serine/threonine kinase from the phosphoinositide-3-kinase-related kinase (PIKK) family. This protein kinase as a downstream component of the PI3K/AKT pathway has an imperative role in cell growth, proliferation, metabolism, migration, and overall tumorigenesis of multiple types of cancers . MoreoveAs we mentioned above, targeting the PI3K/AKT/mTOR signaling pathway as a therapeutic strategy improves MPeM tumor shrinkage. On this basis, a clinical trial study investigated the role of apitolisib as a PI3K-mTOR inhibitor in two young female MPeM patients. Both of them are still alive. Saoirse O Dolly et al. implemented combinational therapy for both patients. Platinum-pemetrexed chemotherapy was conductive to disease stabilization and apitolisib led to tumor shrinkage. Also, results showed that, in one of the patients, apitolisib alleviated both symptoms and CA-125 (tumor marker) and in another one, this drug led to a reduction of tumor volume. This data demonstrates that apitolisib has a considerable anti-tumor effect in both patients through the inhibition of PI3K-mTOR .Besides, p-mTOR expression is confirmed to influence MPM prognosis. P-mTOR is positively related to the early stage of mesothelioma and its positive rate experiences a significant decline in MPM late stage . Taken tTaken collectively, PI3K/AKT/mTOR inhibitors can increase the potency of MMe cell death and can be effective agents in improving MMe therapy in either single or combinational therapies.The mitogen-activated protein kinases (MAPKs) are serine-threonine protein kinases that are involved in different kinds of cellular processes like proliferation, differentiation, apoptosis or survival, and inflammation . MAPK paThe same as pI3K/AKT/mTOR pathway, RAF/MAPK pathway activation depends on RTKs  which upIt was observed that Pirfenidone alone or with cisplatin downregulates both MAPK/ERK and AKT/mTOR pathways which induce MPM cell proliferation. Both in vivo and in vitro results of this study show that Pirfenidone plays a significant role in improving the effectiveness of cisplatin through the modification of extracellular matrix-like changing the expression level of collagen genes, leading to accelerating the drug delivery to tumor tissue . In addiIn addition, Ras genes mutations are common in different types of human cancers . AlthougCalcium ions (Ca2 +) are practically involved in the progression of human cancers. New evidence shows that one of the characteristics of MMe cells is the alteration in the expression and activation of calcium signaling . The intMeanwhile, ion channels are responsible for Ca2 + homeostasis. On this basis, T-type Ca2 + channels as low voltage channels take part in Ca2 + transportation. The overexpression of T-type Ca2 + channels has been reported in MMe cell lines. Also, it has been proved that T-type Ca2 + channel blockers have general anti-cancer properties to alleviate cell growth and provoke cell death. It has been suggested that blocking these channels could be considered a therapeutic subject for MMe treatment , 48.2+ homeostasis or Ca2+ buffering [2+ handling impairment, leading to the reduction of Ca2+ uptake into the mitochondria and consequently the inhibition of mesothelial cells apoptosis. However, more investigations are needed to clarify this evidence [Moreover, Calretinin (CRT) is a calcium-binding protein controlling intracellular calcium signaling. There is some initial evidence that CRT links to mesothelioma transformation and mesotheliomagenesis through the regulation of intracellular Cauffering . CRT up-uffering . The higevidence . As mentevidence , 51  and reactive nitrogen species (RNS). Asbestos fibers, as the main cause of MMe, produce both ROS and RNS directly and indirectly . AlthougPARP1 is a DNA damage sensor that plays a considerable role in repair pathways, including BER, NHEJ, and HR, and also has functions in apoptosis and cell death induction . PARP exThe mismatch repair (MMR) system is one of the major DNA repair pathways mediated by MSH2, MSH6, MSH3, MLH1, and PMS2. In this pathway, MSH2 and MSH6 heterodimer or MSH2 and MSH3 heterodimer recognize and bind to small insertion/deletion loops and larger insertion/deletion loops, respectively. Moreover, a heterodimer of MLH1 and PMS2 is required for completing this complex and repairing the damages , 68. MLHFinally, NHEJ is the fifth DDR pathway in cells. KU70/80, DNA-PKcs, XRCC4-ligase4 complex, and certain DNA polymerases are introduced as the core components of NHEJ. KU70/80 is the first complex in the NHEJ pathway being responsible for break recognition . A studyThe average treatment for MPM patients most generally is surgery accompanying chemotherapy and radiotherapy. But only a few are eligible for these treatments, some show strong resistance to chemotherapy, and few have the proper conditions for surgery . Since DTrabectedin is another drug that is being tested in the laboratory for the treatment of MPM by targeting the DNA repair system . It has Studies have shown that HR is directly related to the response to trabectidine treatment. HR-defective cells are 100 times more sensitive to this drug. HR deficiency was associated with the persistence of unrepaired DSBs along with the S phase of the cell cycle pathway with apoptosis , 95. TheImmune checkpoint blockade ICI, which is an immune-regulating factor, that enhances the latent immune response kept in check by the tumor, is also under consideration against MPM. According to studies, the inactivation of the MMR genes such as mutL homolog 1 gene (MLH1), PMS1 homolog 2, mismatch repair system component gene (PMS2), mutS homolog 2 gene (MSH2), and mutS homolog 6 gene (MSH6) cause microsatellite instability of the MSI. The most common of these translated proteins form heterodimers fixing DNA lesion, are mutL homolog 1 (MLH1) / PMS1 homolog 2, mutation repair system M2 (PMS2) and mutS homolog 2 (MSH2) / mutS homolog 6 (MSH6). Thus, mutations in the genes of these proteins lead to immune mutations, and the MMR deficiency predicts the clinical advantage of ICI in many tumors; but because the MMR defects are rare in MPM patients and the MSI phenotype is absent, it is not yet related to the response of patients to ICI , 102. ReIn this paper, we have discussed the involvement and dysregulation of survival signaling pathways including, Hedgehog signaling, PI3K/AKT/mTOR signaling, MAPK signaling, and calcium signaling in MMe progression. Also, encouraging evidence showed the important role of DNA damage and DNA repair in MMe development. The importance of DNA repair failure is to the extent that 83% of MMe cases mutations were in charge of DDR. Since DNA repair mutations are confirmed among the most significant alterations in MMe development, DDR regulation can be a promising approach to stop cancer cells progression and provoke apoptosis. Therefore, much attention has been drawn to targeting DDR as a treatment strategy for MMe treatment. However, we believe that there is a long way to achieve this goal and further investigations are required to prove the efficacy of this strategy.


EMBO Reports, Carney and colleagues surprisingly find that the Slit‐Robo pathway, which is best known for its function in axon guidance, regulates the polarity of developing zebrafish fins . Intriguingly, they identify an intricate back and forth of signals between the mesenchyme and the AER. Slit ligands derived from mesenchyme act on Robo receptors in the AER to stimulate the production of sphingosine‐1‐phosphate, which then acts back on the mesenchyme to regulate cell polarity and orientation.Development of vertebrate limbs and fins requires that tissue growth is directed outwards, away from the body. How such directed growth is achieved is a fascinating biological problem. For limb/fin formation and outgrowth, signaling between mesenchymal cells and the overlying epithelium is essential. In particular, the epithelium at the distal margin of the growing limb/fin bud, termed the apical ectodermal ridge (AER), promotes directed outgrowth of the underlying mesenchyme, e.g., by providing polarization cues for mesenchymal cell migration. Several classical signaling pathways, such as fibroblast growth factor (Fgf), hedgehog, and Wnt signaling, are involved in the regulation of the cellular events that shape the limb/fin bud . In this issue of  A study in this issue shows that the Slit‐Robo pathway, best known for its function in axon guidance, regulates zebrafish fin polarity and morphogenesis. Slits are secreted glycoproteins that function as the main ligands of Roundabout (Robo) receptors. The Slit‐Robo signaling axis was first described as an essential regulator of axon path finding and neuronal migration. Since then, it was shown that Slit‐Robo signaling is involved in the regulation of various additional developmental processes such as migration of other cell types, cell proliferation, angiogenesis, and vascularization  is affected, suggesting a new link between Slit‐Robo and sphingosine‐1‐phosphate (S1P) signaling during fin development. S1P is a sphingolipid metabolite that acts through a family of receptors to regulate such divergent processes as cell migration, growth, and survival  results in a fine‐tuned progression of adhesiveness along the fin fold, which appears to regulate the directed migration of the mesenchymal cells.In this elegant work, the authors define a novel pathway regulating morphogenesis and directed growth of zebrafish fins. While experimental proof for an S1P gradient is still lacking, their findings open interesting new avenues for future research. It will for example be important to test to which extent this signaling axis is conserved in mammalian limb development. Furthermore, the downstream modulators between Robo receptors and S1P activation are so far not known. Robo receptors do not possess autocatalytic or enzymatic activity, and several signaling molecules mediate downstream signaling progression. The authors have tested some known mediators, which, however, seem not to be involved in establishing the Robo‐S1P axis. Importantly, their work provides a new paradigm for signals that can mediate Slit‐Robo signaling, and it will be intriguing to learn whether sphingosine‐1‐phosphate acts downstream of this important pathway also in other systems.


IJMS Special Issue “Perception, Transduction and Crosstalk of Auxin and Cytokinin Signals” (2021–2022).Auxins and cytokinins are considered the most important plant hormones, responsible for fundamental traits of the plant organism . These hReconstitution of the Cytokinin Signaling Pathway. Cytokinin signaling proteins are encoded by multigene families; therefore, determining the contribution of each component to a signaling pathway is challenging. This was addressed by researchers from the National Institute of Agricultural Sciences , who succeeded in reconstituting the cytokinin signaling pathway by the transient expression of genes encoding major players in the signal transduction chain in rice protoplasts [toplasts . Therebytoplasts . This netoplasts , fine-tuEvolution of the Cytokinin Perception Apparatus. Although the cytokinin signaling in typical model plants, such as Arabidopsis or rice, has been studied in-depth, the investigation of its evolution remains in its infancy. Hormone perception is based on sensing receptor proteins, so receptors are key components of signaling cascades. Lomin et al. (2021) [Physcomitrium patens, lycophyte Selaginella moellendorffii, and gymnosperm Picea abies. All the receptors or their individual sensory modules were analyzed in terms of their cytokinin-binding affinities, their ligand specificity, and the pH-dependencies of hormone binding. Through this integrative approach, new trends in the evolution of land-plant cytokinin receptors were identified. In particular, moss receptors were shown to strongly prefer isopentenyladenine (iP) over other forms of cytokinin nucleobases, whereas spruce receptors bound trans-zeatin (tZ) with much higher affinity than iP. These trends are consistent with the parallel evolution of active cytokinin forms which, in primitive plants, are mainly represented by iP, whereas in vascular plants starting from gymnosperms, specific enzymes (CYP735-like) for the direct synthesis of tZ from iP-type precursors appear.. (2021) , from thHow Do Cytokinins Get to Their Receptors? One of the recurring problems in cytokinin signaling research is the subcellular distribution of cytokinins between different cellular compartments. It is fundamentally important for any hormone, such as a cytokinin, to have access to a cognate receptor. The main cellular targets of cytokinins are currently thought to be the lumens of the endoplasmic reticulum (ER), where the sensory modules of the receptors are exposed [ exposed ,7. For o exposed , presentEnvironmental Cues Modulate Cytokinin Signaling. Plants’ survival in different climatic zones requires effective adaptation. The restructuring of the cytokinin regulatory system can play an important role in this [cis-zeatin signaling in apices. Since the publication of the article [ in this . A team  in this . They fo article , this scSynthetic Drugs with Unique Cytokinin or Anticytokinin Properties. The development of specialized methods for the analysis of cytokinin–receptor interactions [N6-benzyladenine with individual Arabidopsis receptors was studied. Three synthetic cytokinin S-enantiomers specific for AHK3 receptors were discovered: N6-((S)-α-methylbenzyl)adenine (S-MBA), 2-fluoro-S-MBA (S-FMBA) and 2-chloro-S-MBA (S-CMBA). Two other S-enantiomers were found to exhibit receptor-specific and chirality-dependent anticytokinin properties. These anticytokinins appear to act as allosteric regulators of the receptor activity. ractions ,12 creatractions , the intCytokinins + Auxins: Synergistic Morphogenic Effects. One of the most striking effects of cytokinins, observed almost immediately after the discovery of these hormones in 1955, is the ability, together with auxins, to induce organogenesis in undifferentiated callus tissue [s tissue . A high s tissue . The uniCytokinins + Auxins: Antagonistic Growth Effects. Despite the true synergistic effects in stimulating cell proliferation, auxin and cytokinin most often act as an antagonistic hormone pair [one pair . The crione pair . As a tyCytokinins + Auxins: Adaptation to Photoperiod Stress. Another example of a stress effect under the influence of the auxin–cytokinin duo was described in an article by a joint German–Czech group headed by Prof. Schmülling from the Free University of Berlin (Germany) [ahk2,3 mutants with loss of cytokinin receptor function, or gain-of-function YUC1 auxin synthesis mutants exhibited increased stress responses, while mutations causing the loss of the function of the auxin receptors TIR1, AFB2 and AFB3, in contrast, reduced stress levels. Thus, cytokinin signaling has been shown to play a protective role in this particular type of light stress, whereas auxin signaling enhanced the stress effect. This finding was in good agreement with earlier data on transgenic PHYB potatoes, which showed that cytokinin (kinetin) could reduce plant sensitivity to photoperiod [Germany) . In thistoperiod . Cytokinins + Auxins: Signaling Crosstalk at the Molecular Level. In the aforementioned research on the concerted action of cytokinins and auxins, the main topics were somehow indirectly related to the interplay between auxin and cytokinin signaling. Unlike these studies, the main purpose of the work by Kolachevskaya et al. [a et al.  from thea et al.  point toMicroRNA Fine-Tunes Auxin Signaling. miRNAs are short, single-stranded nucleic acid molecules that repress target gene expression, regulating plant growth and stress responses [TIR1/AFBs, ARF and AUX/IAA; (ii) the modulation of the free auxin content via the indirect regulation of auxin biosynthesis, metabolism and transport. The authors consider the latest studies on the miRNA-mediated regulation of the auxin pathway in the dicot Arabidopsis and the monocot rice, as well as new findings in other species, in order to provide a comprehensive picture of the important roles that miRNAs play in auxin signaling. The combination of certain miRNAs with the genes involved in auxin signaling creates specific regulatory modules that fine-tune development and stress responses in the plant kingdom. Some of these modules function as potent regulatory nodes to integrate auxin, cytokinin and other signaling pathways. Such regulation appears in a wide range of developmental processes in plants. The challenges remaining to be addressed are highlighted. esponses . The genesponses , the miRConcluding remarks. This Special Issue, “Perception, Transduction and Crosstalk of Auxin and Cytokinin Signals”, was aimed at bringing together researchers studying various aspects of the molecular basis of auxin and/or cytokinin perception and signal transduction (sduction . Indeed,


SARS-CoV-2 has been a pandemic threat to human health and the worldwide economy, but efficient treatments are still lacking. Type I and III interferons are essential for controlling viral infection, indicating that antiviral innate immune signaling is critical for defense against viral infection. Phase separation, one of the basic molecular processes, governs multiple cellular activities, such as cancer progression, microbial infection, and signaling transduction. Notably, recent studies suggest that phase separation regulates antiviral signaling such as the RLR and cGAS–STING pathways. Moreover, proper phase separation of viral proteins is essential for viral replication and pathogenesis. These observations indicate that phase separation is a critical checkpoint for virus and host interaction. In this study, we summarize the recent advances concerning the regulation of antiviral innate immune signaling and SARS-CoV-2 infection by phase separation. Our review highlights the emerging notion that phase separation is the robust modulator of innate antiviral signaling and viral infection. Sensing of pathogen-associated molecular patterns (PAMPs) by the corresponding pathogen recognition receptors (PRRs) is the first step in innate immunity signaling . Three kSARS-CoV-2, the causative agent of COVID-19, has led to a worldwide pandemic since 2019 . As a hiPhase separation, either liquid–liquid phase separation (LLPS) or liquid–gel phase separation, is considered the driving force for biomolecular condensates. Biomolecular condensates are critical for the formation of membrane-less organelles (MLOs) such as nucleoli, Cajal bodies, promyelocytic leukemia nuclear bodies (PML-NBs), and stress granules ,13,14,15In this review, we summarize the recent progress concerning the regulation of antiviral innate immune signaling and SARS-CoV-2 infection by phase separation. We especially emphasize the phase separation of the essential signaling molecules such as cGAS, STING, and IRF3 as well as the nucleocapsid protein (N) and non-structural 8 (NSP8) of SARS-CoV-2. The phase separation of viral proteins involved in modulating antiviral response is also discussed. Finally, we list the possible small molecules targeting the viral proteins, which are the potential drugs for the treatment of viral infections.cGAS, with 522 amino acids and 62 kDa, is the major receptor for dsDNA and microbial DNA . cGAS seThe condensate formation of cGAS is regulated by multiple cellular and viral proteins . GTPase 336/E337 are the two essential residues for phase separation. Moreover, the transmembrane domains of STING are also essential for its droplet formation since STING forms jigsaw puzzle-like condensates within the ER membrane [The STING protein, with 379 amino acids and 42 kDa, is the critical adaptor protein in cGAS–STING signaling. STING resides on the ER membrane in the resting state. The second messenger cGAMP, which is produced by cGAS upon dsDNA stimulation, binds to STING, stimulating its ER-Golgi translocation, aggregation, and TBK1 activation to induce the downstream type I interferon signaling ,37,38 F. STING imembrane . Functiomembrane . Anothermembrane . Distincmembrane . Consistmembrane ,40,41.RIG-I and MDA5 are the major RLR receptors (RLRs) for recognizing viral RNAs. The interaction of viral RNA with RLRs leads to its oligomerization and interaction with the downstream adaptor protein MAVS. The interaction of caspase activation and recruitment domains (CARDs) of RLRs with CARD of MAVS further results in MAVS aggregation, fibril formation, and downstream signaling activation ,43,44 F. With thIRF3, with 427 aa and 50 kDa, is the critical transcriptional factor in RLR and cGAS–STING signaling. It is phosphorylated by the upstream kinase TBK1/IKKε in the cytoplasm, which promotes its dimerization and translocation. After translocation into the nucleus, dimerized IRF3 binds to the regulatory elements of IFN gene promoters to stimulate the expression of type I interferons . IRF3 coIn vitro assays indicate that IRF3 forms liquid-like droplets . Domain Neurofibromatosis protein 2 (NF2) is a classic tumor suppressor. Another study indicates that missense mutations of the tumor suppressor neurofibromin 2 (NF2m) form condensates in the cytoplasm upon activation of the innate immune signaling . InteresIDR), the N-terminal domain (NTD), the linker region (LinkerIDR), the C-terminal domain (CTD), and the C tail (CIDR) -rich linker region, and the phosphorylation of this region affects the viral transcription and replication, which will be discussed later. NTD and CTD domains are both responsible for binding with viral or cellular RNA [Coronavirus virions encode four structural proteins that form the virion: nucleocapsid (N), envelope (E), membrane (M), and spike (S). The N protein of the coronavirus is the most abundant protein in infected cells and is strongly immunogenic . The N pl (CIDR) D. The Liular RNA . A recenular RNA . This suular RNA ,67. Due ular RNA . The NTDular RNA .Previous studies suggest that IDR is crucial for driving the robust phase separation of proteins . Given tIDR of the N protein and the ubiquitin-like (Ubl) domain of NSP3 [IDR, which is catalyzed by the host kinases shortly post-infection [After infection, the RNA genome of the SARS-CoV-2 virus is released into the cytosol, translated, and cleaved into individual nonstructural proteins (NSPs) by viral proteases . The NSP of NSP3 . The N p of NSP3 . More in of NSP3 . Therefo of NSP3 . In anot of NSP3 ,79, sugg of NSP3 ,81,82. Anfection . Studiesnfection ,70. A renfection . Moreovenfection ,81. ThesDuring the replication of the viral RNA, the SARS-CoV-2 N protein accumulates in cytoplasmic complexes along the membrane of the Golgi and other vesicles to initiate the process of virion assembly in cooperation with the membrane (M) protein . The DMVAs aforementioned, NSP8 and NSP7 form the RdRp complex with the core enzyme NSP12 . NSP8 prA characteristic feature of COVID-19, the disease caused by SARS-CoV-2 infection, is the dysregulated immune response with impaired type I and III interferons (IFN) expression and an overwhelming inflammatory cytokine storm . CurrentA proteomic study indicates that the N protein interacts with stress granule proteins . SG is aThe severity and mortality of COVID-19 are closely associated with virus-induced over-activated inflammatory responses and cytokine storms. However, the exact molecular mechanism governing the inflammation with viral infection is not understood. Recently, the N protein is shown to enhance the activation of NF-κB signaling in the context of viral infection . MechaniPhase separation of the innate immune signaling molecules plays positive and negative roles in the signaling cascade. Therefore, targeting the phase separation of innate immune signaling molecules needs to be specifically designed. Even though one study suggests that STING condensate formation plays a negative role in interferon signaling , anotherThe phase separation of the N protein is required for viral genome packaging, RTC formation, and counteracting host cell immune response. Multiple small molecules or peptides to disturb or facilitate the N condensate have been discovered ,63,79. GEven though phase separation is a critical modulator of gene expression, the function and molecular mechanism of phase separation in regulating innate immune signaling and emerging viral infection are still in their infant stage. Recently, a series of essential signaling molecules with phase separation properties was identified. More importantly, their LLPS or phase separation is critical for modulating the signaling transduction. The emergence of SARS-CoV-2 is a pandemic threat to the worldwide population. Targeting the viral RdRp or N protein is a promising strategy for combating the viral infection based on the low mutation of these proteins. Since N and NSP8 possess the phase separation property, small molecules targeting the condensates of N and NSP8 proteins are likely effective in inhibiting the viral infection. A couple of molecules based on the phase separation of N have been developed and found to be effective in vitro ,63,79. HProper innate immunity is beneficial for combating viral infection and cancer immunity, but overactivation of innate immunity may lead to autoimmune disease and even neuronal diseases. Phase separation targeting innate immunity may have a potential for treating the disorders listed above. As aforementioned, the NF2m leading to the condensate formation abrogates the TBK1–IRF3 signaling, which contributes to cancer immune evasion. On the other hand, autoimmune diseases related to self-DNA recognition rely on the cGAS–STING-IRF3 pathway to induce type I interferon production ,102. The


Caenorhabditis elegans, cellular stress stimuli and responses are communicated between tissues to promote organismal health- and lifespan. The nervous system is the predominant regulator of cell nonautonomous proteostasis that orchestrates systemic stress responses to integrate both internal and external stimuli. This review highlights the role of the intestine in mediating cell nonautonomous stress responses and explores recent findings that suggest a central role for the intestine to regulate organismal proteostasis. As a tissue that receives and further transduces signals from the nervous system in response to dietary restriction, heat- and oxidative stress, and hypoxia, we explore evidence suggesting the intestine is a key regulatory organ itself. From the perspective of naturally occurring stressors such as dietary restriction and pathogen infection we highlight how the intestine can function as a key regulator of organismal proteostasis by integrating insulin/IGF-like signaling, miRNA-, neuropeptide- and metabolic signaling to alter distal tissue functions in promoting survival, health- and lifespan.In multicellular organisms such as  Efficient protein folding is essential for the vitality of all cells within an organism throughout the different challenges that occur in a lifetime. Maintaining a functional proteome is therefore crucial for cellular health in all living organisms. This is achieved by the cellular proteostasis network, and predominantly enacted by molecular chaperones as well as degradation machineries such as the proteasome and autophagy  (FOXO-to-FOXO (-) signaling] (FOXO-to-FOXO (+) signaling, intestinal DAF-16 induces an upregulation of the DAF-16-dependent gene sod-3 in the epidermis and muscle, correlating with an increased lifespan (FOXO-to-FOXO (+) signaling was found to be dependent on the positive and negative regulation of the insulin-like genes ins-18 and ins-7, respectively. In particular, ins-7 knockdown in the intestine induced sod-3 expression in head muscles and enhanced lifespan whereas intestinal ins-18 knockdown prevented sod-3 expression in head muscles and caused a shortened lifespan creating a positive feedback loop that coordinates system-wide aging  signaling can be employed from the intestine to the neurons to regulate lifespan.The IlS pathway is the predominant pathway integrating different nutritional cues resulting in system-wide impacts through several aspects of healthspan including fertility ; reviewegnaling]  or througnaling] . In FOXOlifespan . This inde aging   trans-tissue communication  signaling (FOXO-to-FOXO (-) signals were found to be dependent on the lipid signal mdt-15 a subunit of a mediator complex that regulates the expression of genes involved in lipid metabolism, suggesting lipids could be enactors of this pathway. In age-associated protein misfolding disease models, the intestine was able to utilize this mdt-15-dependent FOXO-to-FOXO (-) signaling to improve muscular proteostasis and alleviated the age-dependent paralysis caused by muscular Aβ expression  is a risk factor for developing neurodegenerative disease  pathway [reviewed in l, cup-5 . This suof PolyQ . mir-83 lifespan . Despitevia the miRNA mir-60 by maintaining cellular homeostasis to promote survival and lifespan  and serotonin signaling (tph-1) in ASI and ADF neurons, respectively    and the pathway)   . Moreovepathway)  (Sakai epathway) . Thus, tSimilarly, behavioral cues initiated in the intestine can influence the responses to thermal stress by migrating away from dangerously high and low temperatures. For example, intestinal activation of the TORC2 substrate PKC-2, promotes cold-directed migration .Together these findings indicate the intestine is able to modulate risk-taking behaviors as well as pathogen avoidance to promote organismal proteostasis.via cross-seeding of amyloid species and priming certain responses of innate immune cells in the brain, such as glial cells , that is, often preceded by changes in gut microbiota. For example, a lack or decrease of certain bacterial species in the gut, can have a negative impact on PD or ALS pathogenesis in patients at an early disease stage .via the passive release of hormones and other macromolecules from the gut. Epithelial sensor cells in the gut, the enteroendocrine cell or neuropod cells, can also directly connect with vagal neurons to transduce mechanical, chemical, or bacterially derived sensory signals from the gut lumen to the brain, using glutamate as a neurotransmitter [reviewed in Interestingly, the mammalian brain senses gut stimuli As highlighted in this review, the intestine differentially affects different tissues, therefore further understanding of intercellular signaling events occurring between the gut and distal tissues, and in particular gut-to-neuron communication, could open exciting possibilities for future therapeutic interventions to improve brain homeostasis during aging.


The effect of the transforming growth factor beta (TGFβ) signaling pathway on joint homeostasis is tissue-specific, non-linear, and context-dependent, representing a unique complexity in targeting TGFβ signaling in joint disease. Here we discuss the variety of mechanisms that TGFβ signaling employs in the synovial joint to maintain healthy joint crosstalk and the ways in which aberrant TGFβ signaling can result in joint degeneration.Osteoarthritis (OA) epitomizes a condition of disordered joint crosstalk in which multiple joint tissues degenerate leading to overall joint deterioration. Synovial joint tissues, such as subchondral bone, articular cartilage, and synovium, as well as mesenchymal stem cells, each demonstrate aberrant TGFβ signaling during joint disease, whether by excessive or suppressed signaling, imbalance of canonical and non-canonical signaling, a perturbed mechanical microenvironment, or a distorted response to TGFβ signaling during aging.The synovial joint relies upon a sophisticated alliance among each joint tissue to maintain joint homeostasis. The TGFβ signaling pathway is a key regulator of the health of individual joint tissues, and the subsequent interaction among these different joint tissues, also known as joint crosstalk. Dissecting the sophisticated function of TGFβ signaling in the synovial joint is key to therapeutically interrogating the pathway to optimize overall joint health. The synovial joint consists of multiple tissues, including articular cartilage, subchondral bone, meniscus, and synovium, that interact biologically and mechanically to maintain joint health . During The transforming growth factor beta (TGFβ) signaling pathway plays an intricate role in maintaining healthy joint crosstalk by carefully regulating homeostasis of each tissue. The delicate balance of TGFβ signaling throughout the joint is non-linear and relies upon control of effector selection, mechanical cues, and spatial localization. Here, we discuss the multifaceted function of TGFβ signaling in joint homeostasis and disease.The TGFβ signaling pathway controls cell behavior through hierarchical, context-dependent, and coordinated regulation –4. The eThe TGFβ ligands are secreted as inactive ligands, consisting of the latency-associated peptide and the mature peptide. This complex is then secured to the extracellular matrix (ECM) through latent TGFβ binding proteins (LTBP) and other proteins, such as biglycan, fibrillin, and thrombospondin, many of which have also been implicated in joint homeostasis or disease . Not onlOnce latent TGFβ ligand becomes activated, it binds to two type II serine/threonine kinase receptors (TβRII), which then recruit and phosphorylate two type I serine/threonine kinase receptors (TβRI) to form a heterotetrameric transmembrane receptor complex . The typSMAD6 and SMAD7 are induced by TGFβ, activin, and BMP signaling and regulate signaling by competitively interfering with the receptor-Smad complex to prevent Smad activation . Ce. Ce25]. Joint loading presents a tissue-level mechanical cue that regulates the function of TGFβ ligand to modulate metabolic activity of chondrocytes. Both the superficial zone chondrocytes and the synoviocytes secrete latent TGFβ into the synovial fluid, which becomes activated with joint motion, and accumulates in the superficial zone of articular cartilage . MechaniSerpine1 and Smad7, and production of Tgfb1 mRNA through an ALK5-dependent mechanism, demonstrating that TGFβ signaling and TGFβ production respond to cartilage loading [Serpine1, Smad7, and Alk5, that is restored upon re-loading [Col10a1 mRNA, a marker of chondrocyte hypertrophy, is observed, suggesting a TGFβ-dependent mechanism that couples joint loading to cartilage homeostasis [Both physiologic and excessive loading of articular cartilage results in rapid induction of the canonical TGFβ signaling pathway . In resp loading . The los-loading . As a coeostasis .Alk5 expression increases, but the consequences of this change for joint homeostasis depend upon the cell compartment. In cartilage, loading reduces Alk1 expression and increases Alk5 expression to support canonical TGFβ signaling, unveiling one mechanism by which the balance between canonical and non-canonical TGFβ signaling in cartilage is preserved [In response to load in either cartilage or bone, relative reserved , 35. In reserved , 37. In reserved . Thus, treserved . The extSubchondral bone, likewise, is dynamically loaded during joint motion and provides mechanical support to the overlying cartilage. The load-induced response of bone is largely mediated by osteocytes, the primary mechanosensors of bone . OsteocyTGFβ plays a tissue-specific role in joint homeostasis and joint disease, contributing to the sophisticated function of TGFβ in the joint Table . For exaThe association of altered bone mass and mineralization with OA progression , coupledTGFB1 and SMAD3 gene expression, relative to non-arthritic joints, even in bone specimens taken from sites distant from the OA joint [SMAD3 [Studies of TGFβ signaling in whole-bone samples from OA human joints have found increased TGFβ ligand production and OA joint –14. Micrt [SMAD3 . These dt [SMAD3 , 14. TakTGFB1 mRNA, TGFβ1 ligand, and SMAD3 mRNA [COL1A1/COL1A2 mRNA of approximately 2:1 to levels 2–threefold higher than that in normal osteoblasts, which blunts the ability of osteoblasts to generate a mineralized ECM in vitro [COL1A1/COL1A2 ratio at the mRNA level and mineralize normally [SMAD3 in human OA osteoblasts is associated with the calcium:phosphorous ratio in bone mineral [The evaluation of primary osteoblasts isolated from human OA specimens supports an osteoblast-intrinsic role of TGFβ in OA. Similar to findings in whole bone extracts, primary osteoblasts obtained from OA bone express increased levels of AD3 mRNA –49. OA oin vitro , 47. Whenormally , a key r mineral , likely DKK2), which diminishes osteoblastic mineralization in response to BMP-2 [HGF) mRNA and HGF protein in OA osteoblasts increase TGFB1 expression, which subsequently inhibits the Wnt signaling pathway and blunts BMP-2-dependent mineralization [TGFβ signaling in OA osteoblasts exhibits an epistatic relationship with the Wnt signaling pathway, an important regulatory pathway in bone formation , 48, 49.Col1A1 promotor drives expression of Tgfb1 with the point mutation H222D, results in higher levels of active TGFβ1 secreted by osteoblasts [Camurati-Engelmann disease (CED) is a disease of poor bone quality due to excessive levels of TGFβ1. A mouse model of CED, in which the eoblasts . Coupledeoblasts . The subeoblasts . This MSOn the other hand, inadequate subchondral bone TGFβ signaling also has negative consequences for joint homeostasis. TGFβ signaling in osteocytes is essential for function of these cells, regulating their ability to maintain the lacunocanalicular network through osteocytic perilacunar/canalicular remodeling (PLR) , 42. In Rankl expression by osteocytes and interfere with osteoblast-osteoclast coupling during bone remodeling [As mediators of bone homeostasis, osteoclasts are inevitably involved in joint health. The altered mass and mineralization in OA subchondral bone suggest an imbalance in bone remodeling by osteoblasts and osteoclasts, for which TGFβ is a critical coupling factor . Osteoclmodeling , 57. TheSMAD3 mRNA levels are significantly higher in OA cartilage, in a manner that is unexplained by DNA methylation in the SMAD3 promoter region [ALK1 in human OA cartilage is correlated with chondrocytic MMP13 mRNA expression, whereas ALK5 mRNA correlates with aggrecan and type II collagen mRNA expression. Since ALK1 preferentially activates SMAD1/5/8, and ALK5 preferentially activates SMAD2/3, this suggests a role for TGFβ receptor and effector selection in expression of genes that are protective or deleterious for cartilage [Human osteoarthritic cartilage shows a complex disruption of homeostatic TGFβ signaling at various levels involving ligand, receptor, effector, and negative regulators, underscoring the importance of studying the hierarchy of altered TGFβ signaling in OA. There are at least three ways that altered TGFβ signaling in human cartilage is implicated in OA. First, excessive activity of the pathway is observed in human OA cartilage: the expression of genes encoding TGFβ1 and β2 ligands is positively correlated with cartilage degeneration . Likewisr region . Second,artilage . Third, artilage . SMURF2 In addition to direct regulation of the signaling pathway, TGFβ signaling can regulate, and be regulated by, microRNAs (miRNAs) to affect downstream signaling and OA gene expression. The expression of miR-140, for instance, is repressed by TGFβ signaling and significantly reduced in human OA cartilage –62, wherTgfb1, b2, and b3 mRNAs in the synovium and cartilage, relative to vehicle-injected controls [Findings in animal models reinforce the importance of an optimal level and type of TGFβ signaling in the joint. Increased TGFβ in the synovial fluid, whether by intra-articular administration of TGFβ or overexpression of TGFβ by the synovium, induces cartilage degeneration and osteophyte formation –66. Elevcontrols –66. Convcontrols . At the controls . In cartcontrols •. TogethTransgenic mouse models with disrupted TGFβ signaling in multiple tissues have likewise demonstrated the requirement of TGFβ signaling in cartilage health. Mice with a dominant-negative mutation of the gene encoding TβRII in the articular cartilage, synovium, periosteum, and perichondrium exhibit a severe OA phenotype with substantial cartilage loss and osteophyte formation . This muCol2a1 promoter, isolates the relative chondroprotective nature of TGFβ. As mentioned previously, SMURF2 levels are elevated in human OA cartilage, disrupting homeostatic TGFβ signaling [Smad3 develop OA and demonstrate decreased expression of genes encoding aggrecan and collagen II associated with increased MMP13 protein levels and enzyme activity in the articular cartilage, thus indicating that chondrocyte-intrinsic SMAD3 is essential for maintenance of healthy cartilage [Tgfbr2 in chondrocytes exacerbates OA and induces Runx2, Mmp13, and Adamts5 gene expression in articular cartilage [Tgfbr2 ablation were mediated by activities of proteinases encoded by Mmp13 and Adamts5, since deletion of either gene or treatment with an MMP13 inhibitor ameliorated the OA phenotype in this model [Mmp13 and Col10a1 gene expression through a RUNX2-dependent mechanism [Alk5 resulted in a spontaneous OA phenotype with articular cartilage degradation, synovial hyperplasia, osteophyte formation, and subchondral bone sclerosis, with reduced SMAD3 phosphorylation and increased Mmp13, Adamts5, and Col10a1 gene expression and elevated protein levels of MMP13 and ADAMTS5 [Because OA involves the coordinated disruption of multiple joint tissues, mouse models employing chondrocyte-intrinsic mutations in the TGFβ signaling pathway can diminish the apparently confounding effects of TGFβ signaling in joint crosstalk. Toggling this pathway experimentally by targeting the receptors, effectors, and negative regulators in chondrocyte-intrinsic models, for example, by driving transgenic expression of Cre recombinase or other genes using a ignaling . Mice wiignaling , demonstartilage . At the artilage  or ALK5 artilage  promotesartilage . Interesis model . Likewisechanism . Further ADAMTS5 .COL2AI gene expression and subsequently produce increased protein levels of collagen II [COL2A1 [SOX9 and COL2A1 to hypertrophic genes such as COL10A1 [Some of the effects of TGFβ signaling in cartilage are explained by its ability to control chondrocyte differentiation. Prior to chondrogenesis, MSCs require both ALK5-dependent SMAD2/3 activity and ALK1-dependent SMAD1/5/8 activity, in concert with SMAD4, to induce lagen II –76. As clagen II . On the lagen II –79. In M [COL2A1 . Through [COL2A1 , 78 and  [COL2A1  and by n [COL2A1 . Decreas COL10A1 . In addi COL10A1 , as mentSMAD3 variants identified in GWAS [Mmp13 and Col10 gene expression and tempering BMP signaling [Mmp13 mRNA and MMP13 protein requiring SMAD3 and a later induction of Mmp13 mRNA and MMP13 protein requiring p38 [Smad1, Smad5, BMP2, and BMP6 mRNA levels, resulting in increased phosphorylated SMAD1/5/8 and Col10 mRNA expression and accelerating chondrocyte maturation [Smad2 and Smad3 or inhibiting BMP signaling [Among the constituents of the TGFβ pathway that control chondrocyte homeostasis, the most well studied is SMAD3, motivated by the association of human OA and  in GWAS –21. In cignaling , 72, 81,ring p38 . Importaring p38 . Primaryignaling , 83, illAs in cartilage, a balanced level of TGFβ signaling in the synovium and synovial fluid is required for healthy joint homeostasis, with insufficient or excessive TGFβ levels compromising joint health. The ability of TGFβ to induce a suppressive or inflammatory immune response within the synovium is context-dependent . The synIn addition to the role of subchondral bone osteoblasts, osteoclasts, and osteocytes in synovial joint homeostasis, there is evidence of an influx of bone marrow MSCs into the subchondral bone during OA progression, which induce angiogenesis and differentiate into osteoblasts to further perturb the subchondral bone environment . Joint iWith increased levels of TGFβ in circulation after injury, targeting the TGFβ signaling pathway at the level of the ligand, rather than receptor, could likewise normalize the aberrant environment after injury. Local treatment to the subchondral bone or systemic treatment with an inhibitor of TGFβ1, β2, and β3 reduces the number and mobilization of nestin-positive MSCs in the subchondral bone and diminishes angiogenesis after ACL transection , 89. ThiAging can have broad effects on TGFβ signaling in multiple joint tissues. Because the prevalence of OA increases with age, considering the shift in function of TGFβ with age could identify a distinct age-related pathophysiology of cartilage degeneration. Furthermore, studying spontaneous age-related OA allows for the evaluation of joint degeneration in the absence of the substantial mechanical and inflammatory changes that occur with joint injury in post-traumatic OA models.Among the many roles of TGFβ in the joint is its interaction with IL-1, a pro-inflammatory cartilage destructive cytokine that is upregulated with age and following injury . TGFβ anAging cartilage exhibits decreased levels of TGFβ1, β2, and β3 ligands, diminished TGFβ-induced proteoglycan synthesis, and reduced the number of cells positive for TβRI and TβRII protein , 91. ThiAcross species, articular cartilage exhibits a shift from canonical to non-canonical TGFβ signaling during aging , 37, 93.Aging also impacts the TGFβ-induced collagen production in ligamental fibroblasts. With aging, the ability of medial collateral ligament (MCL)-derived fibroblasts to synthesize collagen in response to TGFβ is diminished relative to young controls . While oOne area of interest in the intersection of TGFβ and joint homeostasis is the extent of crosstalk with the nervous system. Nerve growth factor (NGF) is a key driver of the musculoskeletal pain response. Despite the lack of apparent sequence similarities, TGFβ and NGF demonstrate similarities in topological structure that place them in a common growth factor superfamily . Anti-NGCareful consideration must be taken when developing TGFβ-targeting treatments for OA due to the complex role of TGFβ in joint health and disease. Treatment strategies need exquisite control of multiple factors, including optimal dose, tissue-specific effects, selection of downstream TGFβ receptors and effectors, and local mechanical cues. For instance, injury can induce excessive levels of TGFβ ligand within the joint , suggestTherapeutics have been developed that target different levels of the TGFβ signaling pathway in the clinical setting, primarily in the context of cancer or fibrotic disease , 98. RecIn conclusion, TGFβ signaling plays a sophisticated function in maintaining healthy joint crosstalk that is non-linear and depends upon effector selection, physical and mechanical cues, and tissue-specific function to support joint health. Synovial joints facilitate smooth motion and load transfer through integrated function of multiple tissues, including articular cartilage, bone, ligaments, tendons, synovium, and menisci. Under normal conditions, the biological and mechanical activities of these tissues are exquisitely coordinated, yet in the setting of aberrant TGFβ signaling, one or more of these joint tissues can deteriorate leading to overall joint destruction. Regulation of the TGFβ signaling pathway can occur at different levels, and disrupting the homeostatic TGFβ signaling at any level of the pathway can have broad effects through crosstalk among multiple joint tissues.