Hedgehog (Hh) signaling pathway is an evolutionarily conserved signal transduction pathway that plays an important regulatory role during embryonic development, cell proliferation, and differentiation of vertebrates, and it is often inhibited in adult tissues. Recent evidence has shown that Hh signaling also plays a key role in rheumatic diseases, as alterations in their number or function have been identified in rheumatoid arthritis, osteoarthritis, ankylosing spondylitis, systemic sclerosis, and Sjogren’s Syndrome. As a result, emerging studies have focused on the blockade of this pathogenic axis as a promising therapeutic target in several autoimmune disorders; nevertheless, a greater understanding of its contribution still requires further investigation. This review aims to elucidate the most recent studies and literature data on the pathogenetic role of Hh signaling in rheumatic diseases. The Hedgehog (Hh) gene was first discovered in Drosophila in 1980 by Nusslein-Volhard, C. and Wieschaus, E., and was named Hedgehog because its mutation developed Drosophila larvae into a hedgehog-like morphology, which was also confirmed in vertebrates lately ; Dhh is Activation of Hh signaling includes canonical and non-canonical pathways, the canonical pathway is the most extensively studied and clearly understood currently, which is also known as the ligand-dependent pathway . In the Numerous studies have shown that aberrant expression of Hh signaling may take part in a wide range of diseases, and current studies have confirmed the involvement of this pathway in a variety of tumors. Similarly, scholars have also explored the engagement of the Hh signaling pathway in the pathogenesis (Rheumatoid arthritis (RA) is mainly characterized by chronic inflammation of the synovium and progressive joint destruction. Fibroblast-like synoviocytes (FLSs) are the key effectors that mediate synovitis and joint destruction in RA . AbnormaCompared with systemic lupus erythematosus (SLE), ankylosing spondylitis (AS) and healthy controls, the expression of Shh in serum of RA patients was significantly increased; the level of Shh in RA patients was correlated with rheumatoid factor (RF) and anti-cyclic citrullinated peptide antibodies (anti-CCP Ab) positively, but it had no correlation with erythrocyte sedimentation rate (ESR) . SimilarIn addition to the involvement of FLSs, damage of chondrocytes is also an important pathophysiological process in RA pathogenesis. In particular, cartilage damage is associated with irreversible disability in RA. Therefore, special attention should be given to the treatment of cartilage damage. The expression of Shh, Ptch1, Smo, and Gli1 proteins increased in AIA rats cartilage tissue, and the level of these proteins were proportional to the degree of chondrocyte damage . MoreoveThere is also cross-linking between the Hh signaling and other pathways. Smo is associated with the proliferation of RA-FLSs and participates in the migration of RA-FLSs by activating the Rho GTPase signaling pathway . Shh sigOsteoarthritis (OA) is a degenerative disease characterized by pain and loss of joint function and is the most common cause of chronic disability in adults. Therefore, early diagnosis and effective treatment are crucial for OA. There is currently no effective treatment for OA. Therefore, a better understanding of the pathogenesis of OA will be beneficial to improve our knowledge of the disease . The patThe Hh signaling is activated in OA and thought to influence chondrocyte differentiation as well as osteoblastogenesis . Olex A Temporomandibular joint (TMJ) OA is another type of OA that exhibits degeneration of the articular cartilage and bone of the mandibular condyle and glenoid fossa/protrusion. In TMJOA rats, Ihh signal-related protein levels in cartilage were elevated and showed a time-dependent relationship with the severity of cartilage degeneration, suggesting that activation of Ihh signaling is associated with cartilage damage . Ihh is in vitro studies have shown that Ihh does not cause degradation of ECM in healthy chondrocytes even in the presence of IL-1β and that IL-1β downregulated the expression of Ihh. Thus, there may be other factors other than IL-1β involved in the degradation of OA ECM by Hh. Primary cilia are involved in the development and maintenance of articular cartilage and regulated Hh signaling (Interleukin-1β (IL-1β) is colocalized with MMP-13 to sites of cartilage degradation in OA joints and is a key mediator of OA pathogenesis . Inhibitignaling . Depletiignaling . Cholestignaling . Furtherignaling . Smo mutignaling .in vivo (Systemic sclerosis (SSc) affects the skin and various internal organs including the lungs, the gastrointestinal tract and the heart. The accumulation of extracellular matrix (ECM) components in SSc is caused by increased production of ECM by activated fibroblasts . The proin vivo . These fin vivo . Subsequin vivo . HOTAIR in vivo . These fAnkylosing spondylitis (AS) is characterized by inflammation and new bone formation (NBF) in the spine and joint . During in vitro (Sjogren’s Syndrome (SS) is mainly involved in salivary glands and lacrimal glands. Several studies have demonstrated that Hh signaling participates in the morphogenesis of salivary glands and it has the potential to promote salivary glands , 59. Shhn vitro . In the n vitro . The expn vitro . Hh signn vitro . Fiaschin vitro . Finallyn vitro . TherefoWith the intensive study of the Hh signaling in RA, the targeted therapy is also gradually increasing. After the administration of cyclopamine, a specific inhibitor of Shh pathway, the proliferation and expression of Shh, Smo, Gli1 mRNA were both reduced in RA-FLSs . Gli-antAs for chondrocytes, Cyclopamine effectively decreased the expression of Shh, Ptch1, Smo, and Gli1 genes in AIA rats , while rIn the endothelial cells of RA synovial tissues, Cyclopamine significantly reduced the expression of Shh pathway-related proteins in EA.hy926 endothelial cells, decreased cell viability and survival, and promoted apoptosis. Similarly, Smo-siRNA inhibited the proliferation of endothelial cells and promoted its apoptosis . Scube p+ osteoblastic progenitor cells reduces subchondral local immune inflammatory responses and attenuates articular cartilage degeneration in TMJOA mouse model, providing a potential method for TMJOA treatment  has been used in the treatment of RA  expression is elevated in fibroblasts from SSc patients. It is associated with increased activation of TGF-β . Investiin vitro experiments, and there is no relevant clinical research. Moreover, research on specific inhibitors of this pathway is still missing. Expression of target genes can be inhibited by si-RNA, thereby inhibiting the activation of this pathway, but this method is difficult to apply to patients. GANT61, GDC-0449 and LDE223 are specific small molecule inhibitors of this pathway, at the moment there is little/no knowledge on the pharmacokinetics  of these agents and its toxicity. Cyclopamine is the most studied inhibitor of the Hh pathway, and its clinical use is limited due to its insolubility in water and organic solvents. The inhibitory effect of the above drugs on this pathway is not specific, and there is a lack of research on its adverse reactions in rheumatism.It is not difficult to see that there have been many studies on the targeting treatment of the pathway. However, it is worth noting that these studies are all animal or With the deepening of the current research on the Hh signaling, its role in rheumatic diseases has also been verified. In conclusion, the aberrant activation of it is involved in the inflammatory proliferation of FLSs, the differentiation and proliferation of chondrocytes, osteoblasts, and fibroblasts, and can stimulate the proliferation of endothelial cells and salivary gland epithelial cells, providing a new perspective for the treatment of multiple rheumatic diseases (This article is mainly written by YS. HX wrote part of the manuscript and proofread the manuscript. JK and LB helped us collect literature information and draw pictures. LZ reviewed the manuscript and proposed final revisions. All authors contributed to the article and approved the submitted version.This work was supported by the National Natural Science Foundation of China [grant number 81771768] and by the basic research project of Shanxi Science and Technology Department [grant number 202103021224342].The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


RABL3 which is implicated cell proliferation and motility. Two genomic regions, located on BTA6 and BTA26, showed marked non-additive effects. These regions harbor genes, such as WDR19 and ADGRA1, that are directly involved in male fertility, including sperm motility, acrosome reaction, and embryonic development. The gene-set analysis revealed functional terms related to cell adhesion, cellular signaling, cellular transport, immune system, and embryonic development. Remarkably, a gene-set analysis also including Holstein and Jersey data, revealed significant processes that are common to the three dairy breeds, including cell migration, cell–cell interaction, GTPase activity, and the immune function. Overall, this comprehensive study contributes to a better understanding of the genetic basis of male fertility in cattle. In addition, our findings may guide the development of novel genomic strategies for improving service sire fertility in Brown Swiss cattle.Improving reproductive performance remains a major goal in dairy cattle worldwide. Service sire has been recognized as an important factor affecting herd fertility. The main objective of this study was to reveal the genetic basis of male fertility in Italian Brown Swiss dairy cattle. Dataset included 1102 Italian Brown Swiss bulls with sire conception rate records genotyped with 454k single nucleotide polymorphisms. The analysis included whole-genome scans and gene-set analyses to identify genomic regions, individual genes and genetic mechanisms affecting Brown Swiss bull fertility. One genomic region on BTA1 showed significant additive effects. This region harbors gene  Most research has focused on female fertility, while male fertility has received less attention. It is worth noting that the service sire has a direct influence not only in the fertilization process but also in early embryo development. Indeed, Ortega and collaborators have recently proved that the reduced ability of low fertility bulls to establish pregnancy is multifactorial and encompasses sperm fertilizing ability, preimplantation embryonic development, and placenta and embryo development after conceptus elongation and pregnancy recognition2. Therefore, current evidence suggests that bull fertility should not be overlooked in breeding schemes aimed at improving cow reproductive performance.Pregnancy establishment in cattle is a complex process that involves numerous consecutive events, including gametogenesis, fertilization, blastocyst formation, conceptus elongation, pregnancy recognition, and placenta development. All these consecutive events should be accomplished in a well-orchestrated manner to achieve a successful pregnancy. The relative importance of the maternal, paternal, and embryonic factors on pregnancy establishment is still largely unknown4. In addition, several transcriptomic, proteomic, and metabolomic studies have discovered numerous differences between the spermatozoa of high-fertility and low-fertility bulls7. Furthermore, multiple genomic approaches have been used to identify genetic variants associated with bull fertility. Both candidate gene studies and whole-genome scans have identified genomic regions and positional candidate genes associated with male fertility in both beef and dairy cattle9. Overall, current evidence suggests that bull fertility is influenced by genetic factors, and hence it could be improved by genetic means. Additionally, the identification of biomarkers for bull fertility could assist farmers, breeders, and AI companies make accurate management and selection decisions, such as using or marketing semen from high-fertility bulls or early culling of predicted subfertile bull calves10.There is increasing evidence that genetic factors explain part of the differences in conception rate among sires. Some semen production and quality traits, such as ejaculate volume, sperm concentration, sperm motility, and sperm viability, have moderate heritability11. We used almost 400k breeding records from 130k lactating cows and more than 1200 bulls. The evaluation model included factors related to the bull under evaluation, such as bull age, bull inbreeding, and AI organization, and factors associated with the cow that receives the dose of semen, including herd-year-season, cow’s age, parity, and milk yield. Notably, we revealed a substantial variation in conception rate among Brown Swiss bulls, with more than 20% conception rate difference between high-fertility and low-fertility bulls11.The Brown Swiss breed, one of the oldest dairy breeds in the world, has a great significance for the dairy industry. The breed is raised worldwide due to its adaptability to different climates, including Europe, both North and South America, and the Middle East. Europe has the largest Brown Swiss population, which is led by Switzerland, Germany, and Italy. Interestingly, we recently developed a bull fertility evaluation in the Italian Brown Swiss population using cow field dataThe main objective of this study was to reveal the genetic basis of male fertility in the Italian Brown Swiss dairy cattle population. We performed alternative whole-genome scans and gene-set analyses to identify genomic regions, individual genes and genetic mechanisms affecting Brown Swiss bull fertility. We also compared the results obtained in Brown Swiss with those previously obtained in Holstein and Jersey. The identification of genes and gene networks affecting bull fertility could have multiple benefits, including better understanding of this complex trait in cattle and promote the development of novel tools to make enhanced management and selection decisions on male fertility in the Brown Swiss breed.11. Briefly, we first evaluated cow pregnancy success  using factors related to both the bull under evaluation and the cow that receives the dose of semen. Factors related to the cow included lactation number, age, days in milk at breeding, and total milk yield as fixed effects, and herd-year-season and both cow additive genetic and permanent environmental as random effects. We then estimated sire conception rate using only the factors closely related to the bull. Factors related to the bull included age and inbreeding as fixed effects and AI company and service sire as random effects. Estimates of sire conception rate were calculated as deviation from the mean, which is set as 0, so each 1-point difference reflects 1% more conception rate. For bulls with multiple SCR records, the most reliable SCR record, i.e., the SCR record with most breedings, was used in this study. Sire conception rate values ranged from − 22.3 to 9.9%, with number of breeding ranged from 50 to 8110. Supplementary Table Phenotypic data consisted in sire conception rate (SCR) records from a total of 1102 Italian Brown Swiss bulls. These sire conception rate records were estimated using cow field dataGenotype data for 572,528 single nucleotide polymorphism (SNP) markers located in autosomal chromosomes were available for all the 1102 Italian Brown Swiss bulls with SCR records. This set of SNP markers is a subset of the markers available in the BovineHD Genotyping BeadChip (Illumina Inc.). The SNP information, including chromosome and position, was based on the bovine reference genome ARS-UCD-1.2. SNP markers that were monomorphic or with calling rate less than 95% were removed from the dataset. After quality control, a total of 454,556 SNP markers were retained for subsequent genomic analysis.13.The relevance of additive and non-additive effects, namely dominance, recessive and overdominance effects, on service sire fertility was evaluated on a genome-wide scale using a two-step mixed-model-based approachy is the vector of SCR records, b is the vector of fixed effects, u is the vector of random animal effects, and e is the vector of random residual effects. The incidence matrices X and Z relate phenotypic records to fixed and animal effects, respectively. The random effects were assumed to follow a multivariate normal distribution with G is the genomic relationship matrix, and I an identity matrix. The variance–covariance matrix for this animal model was estimated as In the first step, the following model was fitted:XSNP is the design matrix for the genetic marker under study and βSNP is the regression coefficient, also known as SNP effect. Every SNP genotype XSNP was recoded using single numeric variables as , ,  and  for testing potential additive, dominance, recessive, and overdominance effects, respectively. These models are also known as 1-degree-of-freedom models14. Note that these models assume that R package MixABEL15.In the second step, the following model was fitted for every SNP:16, a three-step SNP-based gene-set analysis was implemented:Following Han and PeñagaricanoR package biomaRt and the most recent bovine genome annotation (ARS-UCD-1.2) were used to map SNPs to genes. Specifically, SNPs were assigned to genes if they were located within the genomic sequence of an annotated gene or within 5 kb either upstream or downstream the gene. The distance of 5 kb was used to capture proximal regulatory regions and other functional sites that may lie outside  but close to each gene. If a SNP was found to be located within or close to more than one gene, all these genes were included in subsequent analyses. Finally, a gene was considered as associated with Brown Swiss bull fertility if that gene contained at least one SNP with a significant additive effect .The first step in the gene-set analysis consisted of the assignment of SNP markers to bovine genes. The Bioconductor Different functional databases, including Gene Ontology, KEGG pathways, Medical Subject Headings (MeSH) and InterPro, were used to define functional sets of genes. Genes assigned to the same functional term can be considered as members of a group of genes (gene-set) that share some properties, typically their involvement in the same biological process or molecular function.P-value of observing k significant genes in the term was calculated byS is the total number of significant genes associated with bull fertility, N is the total number of genes tested, and m is the total number of genes in the gene-set term. This overrepresentation analysis was performed using the R package EnrichKit, developed by Lihe Liu and Francisco Peñagaricano, available at https://github.com/liulihe954/EnrichKit.The significant association of a given functional term with bull fertility was assessed using the Fisher’s exact test, a test of proportions based on the cumulative hypergeometric distribution. This test was performed to investigate a potential overrepresentation of significant genes in each gene-set term. The 16. For Jersey, we reanalyzed a dataset containing SCR records and 88,499 SNP markers from 1487 bulls17. The goal was to identify functional gene-sets, biological processes and molecular mechanisms that explain part of variation observed in dairy bull fertility in the three dairy breeds.Gene-set analyses were also performed using bull fertility records extracted from Holstein and Jersey breeds. For Holstein, we reanalyzed SCR records from 11,280 US Holstein bulls with 290,071 SNP markers11. As such, the present study was specially performed to identify genetic factors that explain part of the variation observed in sire conception rate among Italian Brown Swiss bulls.Improving reproductive performance remains a major goal in dairy cattle worldwide. Several studies have demonstrated that paternal factors play a major role on pregnancy establishment. Recently, our group developed a bull fertility evaluation in the Italian Brown Swiss cattle population using confirmed pregnancy records. We evaluated more than 1200 Brown Swiss bulls based on cow field data, including 400k breeding records from 130k lactating cows. Interestingly, we found a substantial variation in conception rate among Brown Swiss bulls, with more than 20% conception rate difference between high-fertility and low-fertility bullsrs43239680, is located within an intron of gene RABL3. Interestingly, RABL3 plays an important role in cell proliferation and cell motility18. It has been also shown that RABL3 is vital for embryonic development, as the complete ablation of this gene in mice leads to embryonic lethality19. Overall, our results suggest that genetic variation in RABL3 may explain part of the variation observed in male fertility in the Brown Swiss breed.The importance of additive effects on service sire fertility was evaluated on a genome-wide scale using a two-step mixed-model-based approach. Figure 20. In addition, the 1-degree-of-freedom model facilitates the interpretation of the results because the βSNP directly estimates the expected phenotypic difference between genotypic classes.It is believed that non-additive effects are important for fitness-related traits such as fertility. Here, the potential role of non-additive effects on sire fertility in Brown Swiss cattle was evaluated using three different 1-degree-of-freedom tests corresponding to complete dominance, complete recessive, and pure overdominance effects. Alternatively, we could have used a genotypic model with two degree-of-freedom that fits simultaneously additive and non-additive effects. Note that if one is interested in identifying pure non-additive effects, namely complete dominance/recessive or pure overdominance, then the use of 1-degree-of-freedom tests is recommended because it maximizes the statistical powerrs133071278 and rs41601831, is shown in Fig. We identified two genomic regions, located on BTA6 (57.6 Mb) and BTA26 (50.7 Mb), with significant recessive effects on sire conception rate  and calcium ion transport (GO:0006816), are also involved in many processes related to spermatogenesis and early embryo development30. Gene-sets involved in embryonic development, including muscle cell differentiation (GO:0042692) and super elongation complex (GO:0032783), were also associated with variation in bull fertility. These findings provide further evidence that paternal factors contribute to early embryo development in cattle31. Finally, gene-sets directly related to the immune system showed a significant enrichment of genes associated with bull fertility. The immune system impacts pregnancy establishment in different ways, from the spermatogenesis in the male through the fertilization in the female reproductive tract32.Our enrichment analysis revealed several significant functional terms related to cell adhesion. Note that most of the events that occur before and during the fertilization process, including gametogenesis, gamete transport, and sperm-oocyte interaction, involve cell adhesion events. Therefore, the impaired function of genes involved in cell adhesion might result in early pregnancy failuresOverall, a successful pregnancy establishment requires a very well-orchestrated cascade of events. Our findings show that genetic variation underlying different processes, including cell adhesion, cell motility, and the immune response, explain part of differences observed in male fertility in Brown Swiss cattle.None of the genomic regions or individual genes identified in this study were previously reported as significantly associated with male fertility neither in Holstein nor in Jersey. This may be due to multiple causes, namely the major genetic variants affecting male fertility in Brown Swiss are not segregating in Holstein or Jersey, or these major genetic variants are segregating in these two dairy breeds but are not in high linkage disequilibrium with the markers in the SNP chips or simply false-positive/false-negative results. Notably, the gene-set analysis performed across the three dairy breeds identified a set of functional terms that are associated with male fertility in all the breeds Fig. . These gRABL3, WDR19 and ADGRA1, with known roles in sperm biology and fertilization. We also performed a gene-set analysis to gain additional insights into the genetics of male fertility in cattle. Our analysis using Brows Swiss data identified functional terms involved in cell adhesion, cellular signaling, cellular transport, embryonic development, and immune system. Remarkably, a gene-set analysis also including Holstein and Jersey data, revealed significant processes that are common to the three dairy breeds, including cell migration, cell–cell interaction, GTPase activity, and the immune function. Overall, this study sheds light on the genetic variants and mechanisms that explain part of the variation observed in dairy bull fertility. These findings may contribute to the development of novel strategies for improving service sire fertility, including the use of biologically informed genomic prediction models.We performed an integrative genomic analysis to better understand the genetic basis of male reproductive performance in Brown Swiss cattle. We identified genomic regions in BTA1, BTA6, and BTA26, associated with sire conception rate. These regions harbor genes, such as Supplementary Table 1.Supplementary Table 2.


Giardia duodenalis is a world-widely distribution intestinal protozoan parasite that can infect human and a broad range of mammals. It can cause a number of clinical symptoms including diarrhoea, abdominal pain, vomiting, severe dehydration and anemia etc. in animals and humans. Eight genetic assemblages (A to H) have been identified in G. duodenalis based on molecular analysis. In this study, the infection rates and genotypes of G. duodenalis in swine are investigated in Fujian province, southeastern China. G. duodenalis assemblages E was found in pigs in the present study. These results provide epidemiological data for giardiasis prevention, control and public health.Giardia duodenalis, an intestinal parasite, is widely distributed in humans and various animals, such as pigs, cattle and cats. The clinical symptoms of giardiasis are characterized as including abdominal pain, acute or chronic diarrhea, and bloating and weight loss in humans and animals, leading to public and veterinary health problems worldwide. However, the prevalence and genotypes of G. duodenalis in pigs in Fujian Province, southeastern China, have not been reported. In the present study, 725 fecal samples were collected from six cities  in Fujian Province and analyzed for G. duodenalis prevalence and genotypes using nested PCR targeting the beta-giardin (bg), glutamate dehydrogenase (gdh) and triosephosphate isomerase (tpi) genes. The results shown that total occurrence rate of G. duodenalis was 26.9% (195/725) in pigs, with significant differences in the prevalence among different regions  and groups . 195, 11 and 6 samples were detected at the bg, tpi and gdh loci, respectively. Each one belonged to a subtype of assemblage E and was analyzed using sequences obtained in this study. Based on phylogenetic analyses of sequences from the three genetic loci, only one MLG E1 was found. The results indicated that pigs may present a potential zoonotic risk of spreading G. duodenalis infection from animals to humans in this area. The findings of the present study also provide basic data for the prevention and control of G. duodenalis infection in pigs and humans in China. Giardia duodenalis  is an intestinal protozoan parasite distributed worldwide and transmitted through the fecal-oral route [G. duodenalis cyst-contaminated water and food or by direct touch with infected animals, which causes abdominal pain, acute or chronic diarrhea, and bloating and weight loss [G. duodenalis infection in animals, such as pigs, cattle, dogs, cats and wildlife [G. duodenalis has a significant health impact on both humans and animals.al route . Humans ght loss ,3. Acuteght loss ,4. Some wildlife ,6,7. ThuG. duodenalis based on genetic analysis. Among them, assemblages A and B are found in both humans and a variety of mammals, and the remaining six assemblages are strongly host-specific [G. duodenalis infection in pigs all over the world. In the published studies, assemblages A, B and E were identified in pigs, with assemblage E being the predominant genotype [To date, eight assemblages (A–H) have been identified in specific ,9. It isspecific ,11,12,13genotype ,15,16,17G. duodenalis using only a single gene, and even sometimes a limited sensitivity, genotyping of G. duodenalis is most commonly achieved by the amplification of several target genes, such as the small subunit of ribosomal RNA (SSU rRNA), beta-giardin (bg), triose phosphate isomerase (tpi) and glutamate dehydrogenase (gdh) [bg, tpi and gdh) was developed and has been applied to provide more genetic information and contribute to understanding possible zoonotic transmission linkages [G. duodenalis infection in pigs in China is available. Thus, the current study was intended to characterize G. duodenalis in pigs in Fujian Province and to assess the public health potential of G. duodenalis in pigs.Due to the inadequate genetic characterization of se (gdh) ,8. In relinkages ,19. HoweIn total, 725 fresh fecal samples were collected from six regions  in Fujian Province (Location: 24′ N to 28′ N and 116′ E to 121′ E), southeastern China . These sE.Z.N.A® Stool DNA kit  according to the manufacturer’s protocol. G. duodenalis in fecal specimens was determined using nested PCR analysis of the beta-giardin (bg) gene. The primers and amplification used in this study have been previously described [TM  and were visualized under UV light.Approximately 200 mg of feces per sample was placed in a sterile centrifuge tube, and the potassium dichromate solution was washed three times with distilled water. Genomic DNA was extracted using the commercial escribed ,7. The fG. duodenalis positive secondary PCR products from the bg, tpi and gdh genes were sequenced bidirectionally with secondary PCR primers using an ABI PRISMTM 3730 XL DNA Analyzer  at the Sune Biotech Co., Ltd. . Then, the obtained DNA sequences were aligned with references downloaded from the National Center for Biotechnology Information (NCBI) website (http://www.ncbi.nlm.nih.gov/BLAST (accessed on 16 August 2022)) using the software Clustal X v.2.1 ) to identify G. duodenalis assemblages/subtypes.http://www.megasoftware.net (accessed on 16 August 2022)) software was used to perform phylogenetic analyses using the neighbor-joining method with the Kimura 2-parameter model. Bootstrap analyses with 1000 replicates were used to assess the robustness of cluster formation.MEGA X version 10.1.7 (χ2) using the software SPSS 22.0 . Values were considered to be statistically significant when p < 0.05.Differences in infection rate between husbandry parameters, including groups and locations, were compared with the chi-square test  gene. The highest infection rate was seen in specimens from Sanming , followed by Nanping , Fuqing , Zhangzhou , Longyan  and Putian , and was statistically significant . Among the six groups of pigs, suckling pigs  had the highest infection rate, followed by weaned pigs , boars , sows , nursery pigs  and fattening pigs , and the difference was statistically significant   fecal samples tested positive for  < 0.05) .bg gene locus, all 195 G. duodenalis positive samples belonged to assemblage E, among which subtypes E5 and E15 were found, and also showed 100% nucleotide identity with a cattle-derived sequence (GenBank accession number: MH158458) and a Tibetan sheep-derived sequence (GenBank accession number: KY633473) respectively. In total, 11 and 6 Giardia-positive samples belonging to assemblage E15 were identified at the tpi and gdh gene loci, respectively, and showed 100% nucleotide identity with a pig-derived sequence (GenBank accession number: KJ668136) and another pig-derived sequence (GenBank accession number: KJ668145), respectively. Unfortunately, attempts to access data at the tpi and gdh gene loci failed in all assemblage E5 samples. Phylogenetic analyses show that all sequences generated in this study at the gdh, bg and tpi loci were grouped in well-defined clusters with suitable references and matched previously published sequences retrieved from GenBank , which was close to the infection rate detected using PCR based on the bg, tpi and gdh loci and using ELISA in Shanghai (26.88%) [bg loci was lower than the following that were reported: 31.1% in Australia in 2009 [bg, tpi and gdh loci was higher than most reports on G. duodenalis: 17.4% in Denmark in 2006 [G. duodenalis prevalence.26.88%)  and Nige(26.88%)  in 2019. in 2009 , 66.4% i in 2009  and 57.1 in 2009 . However.9% 195/7, which w.88%  and in 2006 , 8% in S in 2006 , 14.8% i in 2006 , 4.26% i in 2006  and 0.97 in 2006 . Many faχ2 = 86.508, p < 0.05), with the highest prevalence in suckling pigs and the lowest in fattening pigs, which were similar to those of Australia and Denmark [G. duodenalis in Shaanxi Province showed that the infection rate of sows was the highest (10.5%) [Infection rates were statistically significant among different groups  , which w (10.5%) . MultiplG. duodenalis in Shaanxi Province found that the main zoonotic genotype was assemblage A (20%) [G. duodenalis.The alignment of nucleotide sequences obtained in this investigation showed only one genotype of assemblage E in Fujian Province. The previous report shows that ungulates, including swine, are easily infected by assemblage E, which can also infect humans . This in A (20%) . Assembl A (20%) . The disbg, tpi and gdh. In addition, coinfection with assemblage A and assemblage E was identified in one fattening pig [G. duodenalis was different among regions. However, more data are needed on the specific mechanism due to few reports on the MLG typing of G. duodenalis in pigs.In total, six samples were amplified at three loci at the same time. Only one MLG E1 was formed. In Shaanxi Province, eight samples were amplified to form four MLGs at the three loci of ning pig . The resG. duodenalis induces a strongly adaptive immune response in both humans and animals. After infection, a large number of parasitic-specific IgA are produced, and the CD4+ T cell response is helpful for the production of IgA and the control of infection [G. duodenalis infection, there is, unfortunately, currently no protective vaccine available for human use, and drug treatment regimens have had varying effects [nfection . Pigletsnfection . This is effects . As an o effects . TherefoG. duodenalis was 26.9% (195/725) in pigs in Fujian Province, southeastern China, and only one MLG was formed. To the best of our knowledge, this is the first report of G. duodenalis infection in pigs in Fujian Province. Considering that pigs are necessary economic animals in China, a better understanding of G. duodenalis in pigs will help to develop more targeted prevention and control measures, which have essential implications for the control of G. duodenalis infection in pigs and humans.In conclusion, the total infection rate of


Aggregation of α-synuclein (α-syn) into amyloid fibrils is closely associated with Parkinson’s disease (PD). Familial mutations or posttranslational truncations in α-syn are known as risk factor for PD. Here, we examined the effects of the PD-related A30P or A53T point mutation and C-terminal 123–140 or 104–140 truncation on the aggregating property of α-syn based on the kinetic and thermodynamic analyses. Thioflavin T fluorescence measurements indicated that A53T, Δ123‒140, and Δ104–140 variants aggregated faster than WT α-syn, in which the A53T mutation markedly increases nucleation rate whereas the Δ123‒140 or Δ104‒140 truncation significantly increases both nucleation and fibril elongation rates. Ultracentrifugation and western blotting analyses demonstrated that these mutations or truncations promote the conversion of monomer to aggregated forms of α-syn. Analysis of the dependence of aggregation reaction of α-syn variants on the monomer concentration suggested that the A53T mutation enhances conversion of monomers to amyloid nuclei whereas the C-terminal truncations, especially the Δ104–140, enhance autocatalytic aggregation on existing fibrils. In addition, thermodynamic analysis of the kinetics of nucleation and fibril elongation of α-syn variants indicated that both nucleation and fibril elongation of WT α-syn are enthalpically and entropically unfavorable. Interestingly, the unfavorable activation enthalpy of nucleation greatly decreases for the A53T and becomes reversed in sign for the C-terminally truncated variants. Taken together, our results indicate that the A53T mutation and the C-terminal truncation enhance α-syn aggregation by reducing unfavorable activation enthalpy of nucleation, and the C-terminal truncation further triggers the autocatalytic fibril elongation on the fibril surfaces. A30P and A53T are frequent mutations firstly identified in Italian and German families, respectively, with autosomal dominant inheritance for PD27. Several mutations including the A30P and A53T are known to lead to early onset of PD, despite large variations in the age of disease onset have been observed33. The A53T mutation promotes aggregation of α-syn in vitro and exhibits PD-like pathology in cellular and animal models35. In contrast, the effect of the A30P mutation on aggregation of α-syn has been variously reported to slow down36, accelerate39, or not affect the aggregation rates40. The recent kinetic analysis of aggregation into amyloid fibrils for these α-syn variants suggested that the mutations enhance the lipid-induced primary and secondary nucleations without affecting fibril elongation33.Although most cases of PD are sporadic, there are point mutations in the N-terminal region of α-syn that cause familial PD. Currently, eight familial PD mutations  have been reported42. The C-terminally truncated fragments of α-syn are another characteristic species enriched in the intraneuronal deposits of PD patients44. The accumulation of aggregated α-syn in lysosome leads to incomplete degradation by cathepsins, resulting in the generation of C-terminally truncated fragments 43. These C-terminally truncated fragments have higher amyloid-forming propensity and amyloid-seeding potency than full-length α-syn45. In addition, it was reported that amyloid fibrils of the Δ104‒140 or Δ120‒140 fragments of α-syn are more twisted than wild-type (WT) fibrils, and the greater twisted morphologies are propagated onto WT fibrils upon cross-seeding46. Overexpression of C-terminally truncated forms of α-syn in Drosophila and in murine demonstrated that these fragments tend to form more pathologic inclusions than full-length protein, inducing dopaminergic cell death47.Familial PD mutations are also known to enhance the intracellular truncation of the C-terminal regions of α-syn49, which forms interface of two protofilaments in the fibril core52. Truncations at N122 or N103 residues remove 7 or 14 acidic residues from the C-terminal domain which contains 15 negatively charged residues. Aggregation kinetics of these α-syn variants were analyzed by the Finke–Watzky 2-step model, which has been applied to separate homogeneous nucleation from heterogeneous autocatalytic fibril growth55. We also estimated the contribution of secondary processes  to fibril growth based on the dependencies of fibril formation on the total protein concentration57. In addition, we explored the effect of temperature on the kinetics of the aggregation of α-syn variants into amyloid fibrils to uncover the thermodynamic mechanism of nucleation and fibril elongation of the α-syn variants.In the present study, we asked how the PD-related mutations or truncations modulate aggregation and fibril formation of α-syn using the point (A30P and A53T) and C-terminally truncated (Δ123‒140 and Δ104‒140) variants . The time courses of increase in ThT fluorescence intensity were fitted by the empirical sigmoidal curves composed of an initial lag phase, a rapid growth phase, and a final plateau phase59 Fig. A, underlk1) and autocatalytic fibril growth (k2)55. As shown in Figs. k1 and k2 values, we also calculated the induction period tinduction (piecewise) and the maximum rate of fibril growth62 which correspond to lag time for nucleation and apparent rate constant for fibril growth, respectively, in the empirical sigmoidal equation. Comparison of the induction periods and maximum rates further demonstrated that the C-terminal 123‒140 or 104‒140 truncations greatly decrease the induction period and increase the maximum rate for fibril growth, whereas the A53T mutation significantly decreases the induction period  and the lag time  and increase in maximum rate , the Δ104‒140 truncation enhances not only the saturated elongation process but also the autocatalytic secondary nucleation, leading to great acceleration of amyloid fibril formation by α-syn.To understand the aggregation mechanism of the α-syn variants, we investigated the dependencies of kinetic parameters of amyloid fibril formation on the monomer concentration of α-syn. Figure kapp values based on the fitting by the sigmoidal equation were shown in Fig. kapp for WT, A30P, and A53T variants. Such dependency of seeding efficiency on the relative concentration of seed fibrils to monomers agrees with the nucleation-polymerization model at equilibrium between monomer and nuclei72. In contrast, the lag time for the Δ123‒140 and Δ104‒140 variants did not change even in the presence of 6 μM of seed fibrils that corresponds to 30% of monomer concentration  and fibril growth (k2) obtained from the Finke–Watzky 2-step model analysis increased with temperature except for k1 values of the Δ123‒140 and Δ104‒140 variants, in which k1 values decreased with increasing temperature. This suggests that the thermodynamic pathway for nucleation of the Δ123‒140 and Δ104‒140 variants is different from that for other α-syn variants.To further gain thermodynamic characteristics of fibril formation by the α-syn variants, we explored the effects of temperature on the fibril formation kinetics. Figure k1 and k2 76. Regarding the fibril growth process, the unfavorable activation entropy of α-syn is thought to arise because resolvation of the fibril surface dominates over desolvation of the free monomer78. Large elimination of negatively charged residues by the C-terminal 104‒140 truncation may alter the balance of resolvation of the fibril surface and desolvation of the monomer, resulting in the favorable activation entropy observed for the Δ104‒140 variant   of monomeric α-syn with negatively charged C-terminal ends of fibrils43, and the resulted aggregates can spread between neighboring cells in a prion-like manner92. Thus, our findings will provide novel molecular basis to understand the relationships between the aggregation and propagation properties of α-syn variants and PD pathogenesis.In summary, the present study provides for the first time the thermodynamic basis for the enhanced aggregation and fibril formation of the PD-related α-syn variants as summarized in Fig. E. coli BL21 Star (DE3) with N-terminal thioredoxin and hexa-histidine tags, and isolated by Ni-affinity chromatography as previously reported93. The fused tags were cleaved with HRV-3C protease, and then removed using Ni-chelating resin column. The obtained α-syn proteins have two extra amino acids, Gly-Pro at the N-terminus. The purity of proteins was estimated as at least 95% by Coomassie-stained SDS-PAGE.Recombinant human α-syn and its PD-related variants  were expressed in 95. The solution was centrifuged at 10,000g for 30 min at 4 °C to remove insoluble aggregates, and then dialyzed against PBS containing 0.02% NaN3 overnight. After dialysis, the solution was again centrifuged at 10,000g for 30 min at 4 °C and the supernatant was used in the experiment. The protein concentration was determined by the Lowry method. The monomer solution supplemented with 10 μM ThT was pipetted into multiple wells of 96-well black plate  The plate was incubated at 37 °C with shaking at 500 rpm in the presence of a Teflon polybead  in each well. ThT fluorescence intensity at 485 nm with an excitation wavelength of 440 nm was measured using Infinite M200 microplate reader  at each time points.The lyophilized α-syn proteins were dissolved in 20 mM glycine buffer (pH 8.0) and solubilized by addition of 2 M NaOHFor preparing seed fibrils, 200 μM of monomeric α-syn solution was incubated in 96 well plate at 37 °C, with shaking at 500 rpm in the presence of Teflon polybeads. The formed fibrils were precipitated by ultracentrifugation in an Optima MAX-TL  with a TLA-120.2 rotor at 80,000 rpm for 1.5 h at 15 °C. After the centrifugation, the supernatant was carefully removed, and the pellet was washed twice with PBS. The resultant pellet of amyloid fibrils was resuspended in PBS by sonicating in the bath-type ultrasonicator  for 1 min. The collected amyloid fibrils were stored at 4 ºC, and re-sonicated for 1 min before use.96,F is the fluorescence intensity, F0 is the initial baseline at the lag phase, and Fmax is the final baseline at the plateau phase. kapp is the apparent rate constant for fibril growth. t1/2 is the half time for fibril formation at which the ThT fluorescence intensity reaches 50% of the intensity at plateau phase. The lag time for nucleation is calculated as t1/2 − 2/kapp.ThT fluorescence data for the fibril formation of α-syn were analyzed using the sigmoidal Eq.78,0 is initial concentration of monomeric protein, k1 and k2 are the rate constants for the nucleation and autocatalytic fibril growth, respectively. The kinetic parameters tinduction (piecewise) and maximum rate vmax were calculated using following Eqs.62,The ThT fluorescence data were also analyzed according to the Finke–Watzky 2-step nucleation-autocatalytic growth model78,kB is the Boltzmann constant and h is the Planck constant. The activation enthalpy (ΔH‡) and entropy (ΔS‡) were obtained from the slope and y-intercept of the linear fitting based on Eq.  at 10,000g for 30 min at 15 °C. The flow-through fraction in filtration was regarded to contain only monomeric α-syn. After overnight incubation with 4 M urea, the protein concentration of each fraction was determined by the Lowry method. The amount of oligomer (> 100 kDa) was determined by deducting the protein concentration in flow-through fraction in filtration from that of supernatant in ultracentrifugation. The amount of fibrillar forms of α-syn was calculated by deducting the protein concentration in the supernatant fraction of ultracentrifugation from that of pellet fraction.Separation and quantification of oligomers and fibrils were performed according to the previously reported protocolProteins were subjected to SDS-PAGE with 15% gels and were transferred to polyvinylidene difluoride membranes . After the blocking of membranes with 4% Block-Ace , the membranes were probed with the anti-α-syn polyclonal antibody  for WT, A30P, A53T, and Δ123‒140, or with anti-α-syn N103 fragment polyclonal antibody  for the Δ104‒140, followed by horseradish peroxidase (HRP)-conjugated AffiniPure goat anti-rabbit IgG antibody  and ECL Prime Western Blotting Detection Reagent .Far-UV CD spectra were recorded from 190 to 260 nm at 25 °C using a Jasco J-1500 spectropolarimeter . The 20 μM of α-syn solutions in PBS before and after fibrilization were subjected to the measurements in a 1 mm quartz cuvette, and the results were corrected by subtracting the buffer base line.TEM measurements were performed on a JEOL JEM-1200EX transmission microscope  with an acceleration voltage of 80 kV. The sample was negatively stained with a phosphomolybdic acid solution for TEM observation.78.TIRFM measurements were performed on a fluorescence microscopic system based on an inverted microscope . ThT was excited using an argon laser, and the fluorescent image was filtered with a bandpass filter and visualized using an image intensifier coupled with a SIT camerap < 0.05.Data were analyzed via one-way analysis of variance followed by Dunnett' multiple comparisons test by means of Prism 8 software . Results were regarded as significant for Supplementary Information.


Appropriate tooth root formation and tooth eruption are critical for achieving and maintaining good oral health and quality of life. Tooth eruption is the process through which teeth emerge from their intraosseous position to their functional position in the oral cavity. This temporospatial process occurs simultaneously with tooth root formation through a cascade of interactions between the epithelial and adjoining mesenchymal cells. Here, we will review the role of the Wnt system in postnatal tooth root development. This signaling pathway orchestrates the process of tooth root formation and tooth eruption in conjunction with several other major signaling pathways. The Wnt signaling pathway is comprised of the canonical, or Wnt/β-catenin, and the non-Canonical signaling pathway. The expression of multiple Wnt ligands and their downstream transcription factors including β-catenin is found in the cells in the epithelia and mesenchyme starting from the initiation stage of tooth development. The inhibition of canonical Wnt signaling in an early stage arrests odontogenesis. Wnt transcription factors continue to be present in dental follicle cells, the progenitor cells responsible for differentiation into cells constituting the tooth root and the periodontal tissue apparatus. This expression occurs concurrently with osteogenesis and cementogenesis. The conditional ablation of β-catenin in osteoblast and odontoblast causes the malformation of the root dentin and cementum. On the contrary, the overexpression of β-catenin led to shorter molar roots with thin and hypo-mineralized dentin, along with the failure of tooth eruption. Therefore, the proper expression of Wnt signaling during dental development is crucial for regulating the proliferation, differentiation, as well as epithelial-mesenchymal interaction essential for tooth root formation and tooth eruption. Proper tooth root formation and tooth eruption are crucial for achieving lifelong oral and overall health, as it directly impacts fundamental human functions, including mastication and speech. The dental follicle, a loose sac-like structure surrounding the tooth bud, contains mesenchymal progenitor cells that play a crucial part in these processes; it contains precursor cells for cementoblasts, periodontal ligament cells, and cryptal bone osteoblasts, therefore, it serves as a critical component of tooth root formation. Tooth root formation starts after the dental crown formation. In the late bell stage, the inner and outer enamel epithelial fuse together and form Hertwig’s epithelial root sheath (HERS). The HERS lies between the dental papilla and dental follicle . The intTooth eruption is driven by the coronal bone resorption induced by the dental follicle and the force generated by the elongation of the tooth root . Dental The Wnt signaling pathway is widely known to play an essential role in multiple stages of tooth development, starting from the initiation of the crown formation to the completion of the tooth root formation. Furthermore, Wnt signaling is also involved in the tooth-eruption process. This review aims to outline the current knowledge on the roles of Wnt signaling in postnatal tooth development.2+ signaling pathway  and the dental mesenchyme, including the cells in the dental papilla and the dental follicle adjacent to HERS , 41. Axielopment .Moreover, canonical Wnt signaling is found to crosstalk with other important signaling pathways to regulate normal tooth root formation. For example, the TGF-β signaling pathway, which is required for root odontogenesis. TGF-β is found to have a cooperative role with Wnt signaling. It is suggested that TGF-β might suppress canonical Wnt signaling to inhibit osteogenic potential and secures odontogenic potential in mesenchymal cells , 45.Shh-creER impairs the structural integrity of the HERS and prematurely disrupts the bilayer structure of HERS. This inactivation further interrupts odontogenesis with the reduced messenger RNA (mRNA) expression of Osx, Nfic, Msx1, and Msx2, and eventually, resulting in shorter molar roots. These findings suggest that β-catenin has an important role in modulating the structural integrity of HERS which is important for dentinogenesis and root development  displays taurodontism with shortened root molars. The inactivation of Wnt10a in K14 expressed cells hinders epithelial cell proliferation and induces the compensatory elevation of Wnt4a in the dental papilla. This results in the excessive proliferation of dental papilla and eventually disrupts the pulp chamber floor formation. Interestingly, the deletion of Wnt10a in the dental mesenchyme results in normal molar formation [fl/flOcn-cre; Ctnnb) during molar tooth root development, the differentiation and proliferation of odontoblasts are impeded, resulting in completely disrupted tooth root development. The phenotype manifests as a rootless molar with normal tooth eruption in the first and second molars , the conditional ablation of Wntless genes, which controls Wnt ligand trafficking and secretion, also hampers odontoblast differentiation and polarization, causing short molar roots with thin dentin and enlarged root canal  causes delayed tooth root formation with short and thick HERs formation, and the absence of furcation in mice molar at P12. At P17.5 the mutant mice exhibit shortened roots at the furcation region due to the mesenchymal hypo-proliferation and delayed odontogenesis during tooth root elongation. However, the deletion of Ror2 in the epithelial cells during tooth root formation does not affect the tooth root morphology nor cell differentiation, supporting the cell-type-specific function of Ror2 in dental mesenchymal cells ), displayed short and distorted molar roots. The cell-specific β-catenin stabilization accelerates odontoblast differentiation, causing a thickened dentin with a hypo mineralized matrix ) also showed hampered root development in mutant mice, resulting in a shorter tooth root with a thin and hypo-mineralized root dentin, associated with premature odontoblast differentiation , cementum, gingiva, and alveolar bone , 53. Thetoblasts . Ror2 extoblasts . It has toblasts .fl/fl, Ocn-cre; Wlsfl/flOcn-cre; Ctnnb1), disrupts the differentiation of cementoblast and reduces cementum secretion. This specific inactivation of canonical Wnt signaling also compromises collagen synthesis and fibrogenesis, resulting in reduced Col1a1 expression in the periodontium. The absence of Wnt secretion in osteocalcin-producing cells significantly widens the periodontal ligament space with the disorganized periodontal ligament and defective collagenous extracellular matrix , suggesting that Wnt signaling has a role in PDL mineralization (+ cells (PDL and cementum progenitor cells)  induces a sharply decreased thickness of the cellular and acellular cementum after 3 weeks of induction /+, Col1a1-cre; Ctnnblox(ex3)/+) or by homozygously deleting Axin2, a negative regulator of Wnt signaling, expedites cementoblast differentiation, induces excessive cellular cementum formation, and increases Col1a1 expression in cementoblasts and periodontal ligaments with narrow periodontal space. /+; R26RtdTomato/+) in the periodontal complex shows a similar cementum hyperplasia phenotype (Dmp1-cre (Dmp1(8kb)-cre; Ctnnblox(ex3)/lox(ex3)) amplifies Wnt signaling in the periodontium, causes an excessive cellular cementum, and an abnormally calcified periodontal ligament, eventually leading to dental ankylosis . The treated cells express more ALP and other osteogenic markers, including Osx, Runx2, and Msx2 in a dose-dependent manner  and osteocalcin (OCN), indicating the inactivation of cementoblast differentiation and reduction of mineralization. Moreover, Wnt3a stimulates cell proliferation when cells are incubated with a Wnt3a-containing conditioned medium /protein kinase A (PKA) mechanisms  the proper amount of bone resorption and bone formation, and (2) appropriate differentiation and development of cells derived from the dental follicle mesenchymal progenitor cells . Wnt/β-cchanisms . As a rechanisms .Wntless genes in osteoblasts  severely reduces bone mineral density and impacts the bone ultrastructure, by upregulating the RANKL expression and downregulating the OPG expression, causing elevated osteoclast differentiation and activity. Additionally, the deletion of β-catenin in osteoblasts also increases the osteoclast function and number without any effect on the bone formation, resulting in lower bone mass , is found to improve the pulp reparative response to injury by reducing cell apoptosis and promoting cell proliferation and differentiation (LacZ/LacZAxin2), as well as in liposomal Wnt protein-injected mice. The improved bone healing is likely due to the increased proliferation and differentiation of skeletal progenitor cells (The role of Wnt signaling in tooth development can also serve as a solid foundation for clinical implication and regenerative dentistry. Accumulating evidence suggests the potential role of this pathway in dental, periodontal, and bone regeneration. The amplification of Wnt signaling, using Axin2-deficient mice (ntiation . Moreoveor cells . Furtheror cells . Lastly,or cells , 79. TheAxin2 and Dkk1, provide negative feedback to fine-tune the signaling pathway. The inactivation of Wnt/β-catenin signaling results in the lack of or short tooth root with thin cementum and increased periodontal space, whereas constitutive stabilization of the signal leads to a short root with thick cementum and narrow periodontal space. Moreover, this conserved signaling pathway also has significant functions in controlling bone formation and resorption, which indirectly influences tooth eruption. Wnt signaling controls bone volume and architecture through the modulation of osteoblastogenesis and osteoclastogenesis. Osteoclasts play important role in creating the path of tooth eruption. Resultingly, the constitutive activation of Wnt signaling causes osteoclast dysfunction and eventually leads to the failure of tooth eruption (Wnt signaling has a crucial role in several stages of tooth development. Wnt signaling activities are observed in the dental epithelium and the dental mesenchyme starting from the embryonic stage. In prenatal tooth formation, this pathway governs tooth number, size, and position. The lack of canonical Wnt signaling impedes tooth development, while overexpression of the pathway causes ectopic tooth formation . Postnateruption . The inc


Follicle-stimulating hormone signaling is essential for the initiation and early stages of spermatogenesis. Follicle-stimulating hormone receptor is exclusively expressed in Sertoli cells. As the only type of somatic cell in the seminiferous tubule, Sertoli cells regulate spermatogenesis not only by controlling their own number and function but also through paracrine actions to nourish germ cells surrounded by Sertoli cells. After follicle-stimulating hormone binds to its receptor and activates the follicle-stimulating hormone signaling pathway, follicle-stimulating hormone signaling will establish a normal Sertoli cell number and promote their differentiation. Spermatogonia pool maintenance, spermatogonia differentiation and their entry into meiosis are also positively regulated by follicle-stimulating hormone signaling. In addition, follicle-stimulating hormone signaling regulates germ cell survival and limits their apoptosis. Our review summarizes the aforementioned functions of follicle-stimulating hormone signaling in Sertoli cells. We also describe the clinical potential of follicle-stimulating hormone treatment in male patients with infertility. Furthermore, our review may be helpful for developing better therapies for treating patients with dysfunctional follicle-stimulating hormone signaling in Sertoli cells.  Spermatogenesis is a process that is under complex regulation to achieve successive germ cell proliferation and differentiation . StartinAs the only type of somatic cell in seminiferous tubule, Sertoli cell (SC) functions as a ‘nurse’ to care for spermatogenesis via paracrine actions to provide necessary nutrition and factors as well as forming necessary structures such as the blood-testis barrier (BTB) and Sertoli cell-Germ cell adhesion complex –9. In hihpg) model  -thymidine assay, which indicates mitosis activation, found that low levels of endogenous FSH decrease [3H]-thymidine incorporation and the final SC number both in vivo and in vitro [hpg mice with recombinant FSH or expressing an FSH transgene in hpg mice also counteracted the negative effect of FSH deprivation on SC proliferation. FSHR mutation also decreased the SC number in mice, supporting these results [FSH regulates SC proliferation only during foetal and early postnatal life. Pioneering works using the [in vitro –73. Injein vitro –77. In a results , 22, 78.Molecular mechanism underlying this stimulatory effect has been elucidated Table . The maic-myc encodes the transcription factor c-Myc, which is important for promoting cell growth and maintaining vitality [c-myc mRNA was elevated by FSH stimulation via a cAMP-dependent pathway [Furthermore, genes regulated by FSH signaling that promote SC proliferation have been identified. Most genes are related to DNA replication, the cell cycle, cytoskeletal rearrangement and stem cell factors. Among them, cell-derived Myc (c-Myc) and type D1 cyclin (Cyclin D1) have been linked to the FSH signaling pathway. The proto-oncogene vitality . In prep pathway . Further pathway . Cyclin  pathway . By acti pathway . Hypoxia pathway –86. HIF1 pathway –132. Dur pathway . Other g pathway . Moreove pathway . Interes pathway . OCT4 is pathway . WhetherSC differentiation begins after SC proliferation cessation during puberty in all species . During The main pathway by which FSH regulates SC differentiation is the cAMP/PKA signaling pathway. Although debates exist regarding whether FSH promotes SC differentiation, some evidences support our hypothesis. Firstly, FSH deprivation or FSHR knockout in mature mouse SCs led to low sperm counts and the SC transition from differentiation to proliferation . SecondlGlut1) mRNA level to augment glucose uptake while HIF2 promotes the expression of tight junction protein ZO-1, ZO-2 and Occludin levels to establish the BTB [c-jun) and increases jun-B mRNA level to regulate the transcription factor activator protein-1 (AP-1). AP-1 participates in the transcription response to hormones and growth factors which are necessary for SC differentiation [FSH regulates SC differentiation directly and indirectly via targeting direct functional factors and transcription factors respectively Table . First c the BTB . Also, intiation .In addition to transcription factors, direct functional factors involved in structural establishment, biochemical reactions and cell morphology were identified. Asp synthetase (ASNS), which promotes Asp accumulation in SCs, is regulated by FSH to induce its transcription in rat SCs. FSH activates the cAMP/PKA signaling pathway to regulate ASNS expression and its function in SC metabolism . BesidesApoptosis is important for maintaining a healthy microenvironment and cell number. SC apoptosis is also regulated by FSH, through the cAMP/PKA signaling pathway. FSH activates the cAMP/PKA signaling pathway to stimulate the expression of N-arachidonoylethanolamine hydrolase (FAAH) and inhibits SC apoptosis that caused by N-arachidonoylethanolamine (AEA) in mice . AEA iniIn summary, FSH promotes SC proliferation via the cAMP/PKA/ERK and PI3K/Akt/mTORC1 pathways while regulating SC differentiation and apoptosis via the cAMP/PKA pathway. Through sequential and corporative regulation of these three processes, FSH provides a healthy and functional microenvironment for spermatogenesis. However, the pathways that are activated in different processes and molecule function vary between different experiments. One explanation is that no precise boundaries exist between different stages. At the time of SC differentiation, SC proliferation can also occur. Besides, different species have different developmental stages. The two stages may overlap in some species such as rats . Differe1, A2, A3, A4, intermediate and type B spermatogonia [pale and Adark spermatogonia. Type B spermatogonia are the differentiating cells [Residing in the basement area of the seminiferous tubule, spermatogonia stem cells function as the original sources for the final spermatozoa . In rodeatogonia , 151. Inng cells . DecadesMaintaining the spermatogonia pool ensures normal spermatogonia stem cell self-renewal and proliferation of undifferentiated progenitors. FSH has been shown to positively regulate spermatogonia pool maintenance in vivo and in vitro –155. ImpMolecular mechanism behind FSH regulation has been elucidated Table . Glial cUndifferentiated type A spermatogonia are under regulation of signaling network to differentiate into differentiated type B spermatogonia and then preleptotene spermatocytes . FSH seeMolecular mechanism behind FSH regulation has been elucidated Table . FSH actFSH has been shown to protect spermatogonia from apoptosis which is important for steadiness of spermatogonia pool. Bcl-2-related ovarian killer (Bok) is proapoptotic member of the Bcl-2 gene family. FSH downregulates Bok mRNA level in rat testes to inhibit apoptosis . SupportTo sum up, FSH signaling in SCs induces paracrine action to maximize the capacity of spermatogonia ecology by maintaining the undifferentiated spermatogonia pool, promoting spermatogonia differentiation and spermatogonia survival. How to achieve a balance between molecules promoting self-renewal and molecules promoting differentiation in response to FSH stimulation remains to be further investigated. The answer may reside in spermatogonia themselves because it was reported that germ cells can control the local balance of GDNF, BMP4 and kit ligand levels .Transition of type B spermatogonia into spermatocytes facilitates meiosis while surviving spermatocytes are essential for quantitative spermatogenesis. FSH is shown to be indispensable for entry into meiosis and positively regulates spermatocyte survival Fig. .hpg mice with exogeneous FSH or transgenic expression of FSH restores the number of spermatogonia and spermatocytes [In vitro study using coculture system containing SCs and spermatogonia showed that FSH initiates the differentiation of secondary spermatogonia into primary spermatocytes in newts . Injectiatocytes , 171. Thatocytes , 172. Fuatocytes , 173. ThDetailed mechanisms that FSH adopts to guarantee entry into meiosis are as follows Table . ActivinFSH is also pivotal for spermatocyte survival. In FSH-suppressed adult rats and gonadotropin-suppressed adult men, the spermatocyte apoptosis rate showed a significant increase , 177.WheAnother factor, Galectin-3  is reported to inhibit both the intrinsic and extrinsic apoptotic pathways by blocking cytochrome c release and Fas (death receptor)/Fas-ligand (member of the tumor necrosis factor family of death-inducing ligands)cross linking respectively . FSH indAdditionally, FSH can inhibit early meiotic spermatocyte apoptosis via inhibition of transcription factor AP-1 in human SCs. Activation of AP-1 occurs before the activation of effector caspase such as caspase 3 . CaspaseIn summary, FSH exerts its effect at the beginning of meiosis by promoting entry into meiosis and the survival of spermatocytes. This effect may be mainly due to the sufficient number of Sertoli cells and spermatogonia. Further studies are recommended to focus on the effect of FSH on the transition from spermatogonia to spermatocytes as well as whether FSH has effect on the transition from primary spermatocytes to the secondary spermatocytes.In humans, FSH induces SC proliferation and spermatogonia proliferation and differentiation, while FSH alone is not essential to complete meiosis and spermiogenesis , 185. TeCurrently, FSH treatment is mainly administrated to two types of patients: patients with hypogonadotropic hypogonadism (HH) and normogonadotropic patients with idiopathic impairment of spermatogenesis . The finIn patients with hypogonadotropic hypogonadism, the lack of gonadotropin FSH stimulation or defects in gonadotropin-releasing hormone synthesis and secretion lead to azoospermia or severe oligozoospermia , 192. OnFSH treatment appears to be beneficial for normogonadotropic patients with idiopathic impairment of spermatogenesis. Meta-analyses revealed that FSH therapy in these patients increases the rate of clinical pregnancies in female partners . HoweverLet’s return to the exogeneous gonadotropin administration method. How to expand the half-life of gonadotropin is important for treatment efficiency. One way is to conjugate the gonadotropin to polyethylene glycol (PEG). PEGylated FSH not only retains FSH activity but also results in improved bioavailability . AnotherRecently, the relationship between diabetes mellitus and male infertility attracts attention –215. As Fshr-knockout mice and men carrying Fshr mutations are similar, the Fshr-knockout mouse model still has great clinical potential [Hope still exists. Conversation in spermatogenic processes between human and mouse are revealed in previous studies . Since totential . The ideotential .FSH signaling in SCs establishes the appropriate microenvironment for spermatogenesis. FSH signaling plays a dominant role in determining the number and function of SCs. FSH signaling also maintains the spermatogonia pool and induces spermatogonia differentiation through paracrine actions. In addition, FSH signaling promotes entry into meiosis and the survival of germ cells. However, few molecules involved in these paracrine actions have been found. We could detect the changes within protein and mRNA expression level of receptors on the surface of gem cells that are associated with different spermatogenic processes, and then determine whether the levels of their ligands changed after the administration of FSH signaling in SCs. Mass spectrometry and single-cell transcriptomics will be helpful. Moreover, combining the transgenic mouse model with human infertility is necessary to develop therapies for diseases related to dysfunctional FSH signaling.


A 69‐year‐old man with left eye pain and visual impairment was diagnosed with syphilitic optic neuritis, who was successfully treated by penicillin. Although it is difficult to decide syphilis as the direct cause of optic neuritis, it is essential to diagnose syphilis in every patient with optic neuritis. It is difficult to decide whether syphilis is the cause of optic neuritis. All patients with optic neuritis should receive serological tests for syphilis, and patients with positive results should be administered with penicillin G prior to steroid. A 69‐year‐old man had left eye pain 5 days previously with subsequent deterioration of his visual acuity (VA), which became finger counting at 10 cm with impaired left direct light reflex and relative afferent pupillary defect on admission. Fundoscopy examination showed left optic disk edema Figure . Twenty‐SON is a rare type of neurosyphilis.The authors state that they have no Conflict of Interest (COI).SY involved in concept, literature search, and drafting of manuscript. MT and TMN involved in concept and literature search. SI‐Y involved in concept and revision of manuscript.This manuscript conforms to the provisions of the Declaration of Helsinki in 1995 (as revised in Brazil 2013).Written informed consent was obtained from the patient to publish this report in accordance with the journal's patient consent policy.


Antidepressants and antipsychotics have a wide range of cardiac side effects. Although the absolute risk is considered low, some are potentially life-threatening.We aim to review the main cardiological complications of antidepressants and antipsychotics and their management. We will consider 1) QTc prolongation and arrhythmia 2) heart rate 3) blood pressure 4) myocarditis.Review of cardiological complications of antidepressants and antipsychotics.Qtc prolongation is correlated with arrhythmia risk. QTc is obtained with Bazett’s formula, which has limitations. All inpatients and some outpatients starting antipsychotic should undergo ECG. Increased QTc can result in different approaches, depending on severity. Most antidepressants do not significantly affect QTc, except for escitalopram and tricyclics, mostly in overdose. Sinus tachycardia can occur with most antipsychotics. Tricyclics can also produce this effect. Other causes should be excluded, and management can be achieved with bisoprolol. Other antidepressants most commonly produce a slight decrease in heart rate or have a minimal to no effect. Antipsychotics can cause hypertension or hypotension depending on the degree of affinity to specific adrenergic receptors. Tricyclics can lead to postural hypotension. Antidepressants interfering with noradrenaline can cause hypertension. Myocarditis is mostly associated with clozapine. Patients should be screened for clinical signs and laboratory findings - especially in the presence of risk factors. Suspicion should prompt echocardiological examination and confirmation leads to cardiology referral.Weighing the risks and benefits of these medications is a continuous process. Management of cardiological complications is possible and may involve a multidisciplinary approach.No significant relationships.


Giardia duodenalis is the underlying cause of a significant number of outbreaks of gastrointestinal illness in humans and animals worldwide. The purpose of this study was to elucidate the prevalence and genetic diversity of G. duodenalis in captive alpine musk deer (Moschus chrysogaster) in China. A total of 202 fecal samples were collected from three farms in Gansu Province, China. Identification of G. duodenalis was conducted by nested PCR targeting the genes coding for SSU rRNA, β-giardin (bg), glutamate dehydrogenase (gdh) and triosephosphate isomerase (tpi). The overall prevalence of G. duodenalis in captive alpine musk deer in surveyed area was 19.3% (39/202). Two G. duodenalis genetic assemblages were identified, namely assemblage A and E. Mixed genotype infections (A+E) were found in 15.4% (6/39) of positive samples. Multilocus genotyping (MLG) analysis of G. duodenalis isolates revealed six novel assemblage A MLGs formed by two newly-described MLG-subtypes which belonged to sub-assemblage AI. To the best of our knowledge, this is the first report on MLG of G. duodenalis isolates in captive alpine musk deer in China. The presence of zoonotic assemblages and sub-assemblages of G. duodenalis in deer species suggests that these animals may potentially act as a reservoir of this protozoan for humans. Giardia duodenalis  is the most prevalent protozoan pathogen, commonly found in the intestinal tract of humans and animals worldwide  or indirectly   . Approxiountries . Giardiaountries . Nitroimountries .Giardia species have been recognized  considering different host distribution  in the world, which are mainly distributed in the Qinghai Tibet Plateau, Sichuan and Gansu Province . Farm owners’ consent was obtained prior to proceeding to fecal sample collection from selected animals.In September 2020, 202 fecal samples were collected from three farms in Gansu Province, China , according to manufacturer’s instructions. The extracted DNA was stored at -20°C until PCR assay. Four genes were used for G. duodenalis genotyping by nested PCR, namely SSU rRNA, β-giardin (bg), glutamate dehydrogenase (gdh), and triosephosphate isomerase (tpi) . Nucleotide sequences were aligned and edited with DNAstar Lasergene Editseq 7.1.0 (https://www.dnastar.com/software/lasergene/) and Chromas Pro 2.1.10 (http://technelysium.com.au/wp/chromaspro/). Genotypes and subtypes of G. duodenalis were determined by aligning reference sequences available in NCBI GenBank database using ClustalX 2.1 . To determine genetic diversity among the isolates, concatenated sequences (bg-tpi-gdh) from each isolate at the three analyzed loci were aligned with reference sequences. Neighbor-joining (NJ) analysis was performed using MEGA 7.0 (http://www.megasoftware.net/) to infer the phylogenetic relationships of concatenated sequences based on the Kimura-2 parameter model.All positive secondary PCR products from SSU rRNA, bg and tpi genes of G. duodenalis are available in the NCBI GenBank database under the accession numbers OM273018-OM273020, respectively.Representative nucleotide sequences of G. duodenalis by PCR at the SSU rRNA locus  and assemblage E . Mixed genotype infections (A+E) were found in 6 of 39 samples.Overall, a total of 39 samples  were confirmed to be bg gene was obtained from 18 of 39 G. duodenalis-positive isolates; among these, 13/18 (72.2%) of isolates were identified as belonging to genetic assemblage A and 5/18 (27.8%) of assemblage E  and A3 (n=9) sequences were identical to the sequences MK639171 and MK639172, respectively. In addition, A2 (n=2) sequences showed a SNP (C21T) compared to the sequence MK639173. At the gdh locus, seven and three isolates were successfully amplified and identified as assemblage A and E, respectively. The subtypes A1 (n=3) and A2 (n=4) were consistent with the sequences MN047217 and the MK645799, respectively. Moreover, the three assemblage E sequences were identical to the sequence MK645786.Sequence analysis of the bg, tpi and gdh loci, and formed six assemblage A MLGs after removal of sequences of mixed infection samples  have historically been bred in China, which include siberian musk deer (M. moschiferus), forest musk deer (M. berezovskii), black musk deer (M. fuscus), alpine musk deer (M. chrysogaster), himalayan musk deer (M. leucogaster), and anhui musk deer (M. anhuiensis) (Six species of musk deer (uiensis) . In prevuiensis) . In partuiensis) .Giardia spp. infects a broad range of hosts including humans, livestock, companion animals, wildlife and birds  in Croatia  in alpine musk deer, which is higher than that reported in sika deer (0.6% and 0.8%) and forest musk deer (2.2%) in China. The discrepancies in infection rates of G. duodenalis may be related to the differences in geographical location, sampling season, animal species, and sample size. To the best of our knowledge, this is the first study to isolate and characterize G. duodenalis from alpine musk deer in China using MLG.nd birds . However Croatia . In the gdh, bg and tpi genes have currently been widely used for genotyping G. duodenalis isolates from human and animal samples in order to obtain high-sequencing resolution  and D (mainly found in canines) have also been reported occasionally in these animal hosts  in the genomes of assemblages A and E  can be found in the article/supplementary material.The animal study was reviewed and approved by Research Ethics Committee of Tarim University. Written informed consent was obtained from the owners for the participation of their animals in this study.ZC, QW, XH, JB, BZ, BW, and XG performed the experiments. ZC drafted the manuscript. MQ and JL critically revised the manuscript. All authors contributed to the article and approved the submitted version.The research was funded by the National Natural Science Foundation of China (32102689), Science and Technology Planning Project of Henan Province (222102110240), the Program for Young and Middle-aged Leading Science, Technology, and Innovation of Xinjiang Production & Construction Group (2018CB034), and Scientific research project of Xuchang University (2022GJPY009 and 2022YB034).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Thoracic aortic disease affects people of all ages and the majority of those aged <60 years have an underlying genetic cause. There is presently no effective medical therapy for thoracic aneurysm and surgery remains the principal intervention. Unlike abdominal aortic aneurysm, for which the inflammatory/atherosclerotic pathogenesis is well established, the mechanism of thoracic aneurysm is less understood. This paper examines the key cell signaling systems responsible for the growth and development of the aorta, homeostasis of endothelial and vascular smooth muscle cells and interactions between pathways. The evidence supporting a role for individual signaling pathways in pathogenesis of thoracic aortic aneurysm is examined and potential novel therapeutic approaches are reviewed. Several key signaling pathways, notably TGF-β, WNT, NOTCH, PI3K/AKT and ANGII contribute to growth, proliferation, cell phenotype and survival for both vascular smooth muscle and endothelial cells. There is crosstalk between pathways, and between vascular smooth muscle and endothelial cells, with both synergistic and antagonistic interactions. A common feature of the activation of each is response to injury or abnormal cell stress. Considerable experimental evidence supports a contribution of each of these pathways to aneurysm formation. Although human information is less, there is sufficient data to implicate each pathway in the pathogenesis of human thoracic aneurysm. As some pathways i.e., WNT and NOTCH, play key roles in tissue growth and organogenesis in early life, it is possible that dysregulation of these pathways results in an abnormal aortic architecture even in infancy, thereby setting the stage for aneurysm development in later life. Given the fine tuning of these signaling systems, functional polymorphisms in key signaling elements may set up a future risk of thoracic aneurysm. Multiple novel therapeutic agents have been developed, targeting cell signaling pathways, predominantly in cancer medicine. Future investigations addressing cell specific targeting, reduced toxicity and also less intense treatment effects may hold promise for effective new medical treatments of thoracic aortic aneurysm. Thoracic aortic aneurysm (TAA) is characterized by progressive aortic dilation, often asymptomatic until dissection or rupture occurs. One in five TAA result from gene mutations affecting vascular smooth muscle cells (VSMC) or the extracellular matrix (ECM) . GeneticPathological features of TAA include ECM degradation, VSMC dedifferentiation, inflammation and endothelial cell (EC) dysregulation ,6,7. TheThe transforming growth factor β (TGF-β) signaling pathway has been the focus of investigation for genetic TAA ,12. BothDeciphering the contributions of intersecting signaling pathways to TAA is key to developing effective therapeutics. As a fundamental step towards supporting future studies, this review examines the evidence for the roles of different cell signaling pathways in TAA. The functions of each pathway in VSMC and EC, and interactions between pathways, are described. Evidence for the involvement of each pathway in the pathogenesis of TAA is reviewed and finally, potential therapeutic interventions are examined.The principal anatomical layers of the aorta are the intima, media and adventitia. The intima is a monocellular layer of ECs, separating the bloodstream from the inner aortic wall, and includes a basement membrane, subendothelium and internal elastic membrane. The intima has an essential role in vascular homeostasis, through ion and solute exchange and hemodynamic signaling. The media lies between the intima and outer adventitia, with elastin laminae, type I & III collagen, fibrillin-1 microfibrils and proteoglycans, forming the ECM that surrounds the VSMC, to form the lamellar units . The medPathological features of TAA include the degradation of the ECM, the loss of VSMC due to apoptosis, and the fragmentation of elastin laminae ,18,19,20Normal endothelium promotes vasodilation via nitric oxide (NO), contributes to the inhibition of VSMC proliferation and migration, and also inhibits platelet aggregation and adhesion . In contThe most recognized pathway associated with TAA is the TGF-β cascade ,32,33. TThere are multiple ligands in the TGF-β family, including TGF-β and bone morphogenic proteins (BMPs). Receptors are heterodimers of Type II (TGF-β and BMP subtypes) and Type I receptors, with the latter having several subtypes, notably Activin-like kinase 5 (ALK5) and Activin-like kinases 1/2/3/6 (ALK1/2/3/6). The ALK5 receptors transduce TGF-β signaling to activate SMAD2/3, whilst ALK1/2/3/6 transduce BMP signaling to activate SMAD1/5/8 . SignaliThere are several layers of regulation of TGF-β signaling . The firThe second level of regulation involves internalization and the recycling or destruction of activated receptors. Interestingly, some receptors are specifically associated with cholesterol-rich regions of the cell membrane and these receptors are particularly associated with activation of MAPK/ERK by TGF-β. Furthermore, the depletion of cholesterol in these regions appears to inhibit TGF-β induction of MAPK/ERK signaling . The degSKIL, the expression of which is increased by TGF-β -S-phenylglycine t-butyl ester (DAPT) attenuated aortic dilatation [In a murine model of MFS, Jespersen et al. have recently reported evidence of increased NOTCH3 expression and activation in ascending aortic tissue. The blockade of NOTCH signaling with the latation . There ilatation . In adulThere appears to be a role for NOTCH signaling in mediating VSMC apoptosis in TAA, with the actual outcomes likely dependent upon NOTCH subtype. It is less likely that alterations in NOTCH signaling are a primary cause of TAA, but rather that NOTCH functions as a modulator of the disease process.−/− Ldlr−/− mice, which show characteristic changes of medial necrosis in response to ANGII [−/− after ANGII exposure [The adverse remodeling of the ECM is associated with chronic ANGII/AT1R signaling, with increased collagen deposition and matrix glycosaminoglycan synthesis . In abdoto ANGII , as do texposure .FBN1 mutations), Loeys-Dietz syndrome  and familial TAA associated with ACTA2 mutations. Each of these syndromes is characterized by TAA at a young age and also by cerebral aneurysms (Loeys-Dietz and ACTA2). In a fibrillin-deficient Marfan mouse model, the loss of ANGII-AT1R signaling in EC is associated with reduced p-ERK1/2 activity and reduced TAA formation, suggesting a role for ANGII-stimulated ERK1/2 activity in pathogenesis of TAA [Fbn1C1039G/+ mouse model of MFS, through inhibition of ERK1/2 signaling mediated by AT2R signaling [Several murine models of genetic TAA have been studied, including Marfan syndrome , ACTA2, is also associated with TAA formation after ANGII. The mechanism appears to involve both the generation of ROS and the NF-κB-dependent induction of AT1R . The ANGutations . Mice deutations .Other signaling pathways, e.g., AKT, may protect from the development of TAA in response to ANGII. The stimulation of VSMC with ANGII results in increased MMP-9 synthesis, which may contribute to ECM remodeling. Although the TGF-β pathway shares the downstream elements of ERK1/2 with ANGII, the inhibition of TGF-β signaling in a murine model did not alter MMP-9 production, indicating that ANGII is the main stimulus for MMP9 upregulation in the VSMC . The expAT1R gene have been associated with increased aortic stiffness [Several lines of evidence suggest a role for ANGII/AT1R in pathogenesis of human TAA. Single nucleotide polymorphisms in tiffness . In pathtiffness . Thus, tFollowing experimental findings that the AT1R blocker may attenuate TAA formation in MFS mice , multiplThe evidence, experimental and clinical, supporting the role(s) of each of the signaling pathways in pathogenesis of TAA is summarized in Information on potential therapeutic interventions in TAA is limited, however evidence from other fields, particularly cancer treatment, points towards options for future investigation.An inhibitor of TGF-β and downstream signaling mechanisms, pirfenidone, was FDA approved in 2014 for the treatment of idiopathic pulmonary fibrosis, indicating the therapeutic potential of inhibiting TGF-β signaling in a clinical setting ,305.Several major classes of TGF-β inhibitors are currently being tested in clinical trials and include antisense oligonucleotides which cause degradation of TGF-β mRNA, TGF-β antibodies and ligand traps which prevent TGF-β binding to receptors, and small inhibitor molecules that inhibit TGF-β receptor kinases . AdditioTGF-β plays a major role in cancer progression and metastasis . IntriguTherapeutics targets of TGF-β in cancer are frequently being tested in combination with other cancer treatments to improve therapeutic potential. This need for TGF-β inhibitors in combination with other agents may also be relevant to the treatment of TAA, as the interactions between and redundancy of signaling pathways may mean the inhibition of TGF-β alone may not result in an effective outcome. This concept has been supported by the finding that TGF-β1 inhibitors combined with PI3K inhibitors are the most effective method for inhibiting the phenotype switch of human aortic VSMCs compared to the use of a single inhibitor .Several drugs targeting the WNT pathway are in the clinical trial phase of testing, predominately for the treatment of cancer. These include inhibitors of WNT protein secretion, competitive antagonists of WNT binding to Frizzled, monoclonal antibodies to Frizzled and antagonists of Dishevelled binding to Frizzled .Molecules targeting WNT secretion have shown promising results. For example, several inhibitors of porcupine, a protein that is essential for secretion of WNT, results in the inactivation of WNT ligands and the reduction of downstream signaling, arresting cell growth in preclinical models, with clinical trials now in progress for the treatment of solid tumors ,318,319.These molecules currently in development for cancer therapies also demonstrate clinical utility for the treatment of CVDs. The administration of a porcupine inhibitor after myocardial infarction in mice reduced adverse cardiac remodeling and fibrosis . AdditioWhile currently there are no FDA approved WNT pathway inhibitors, the repurposing of established agents has been an area of interest, with several FDA approved drugs shown to inhibit components of the WNT pathway . Thus, aThe PI3K/AKT pathway and the therapeutic potential of its inhibition has been extensively investigated in cancer. Numerous drugs inhibiting the PI3K/AKT pathway have been develop and progressed to human clinical trials. The inhibition of PI3K/AKT and/or mTOR strongly influence other pathways such as MAPK, downstream FOXO3a, WNT/beta-catenin and NOTCH , which mThere are two main classes of PI3K/AKT inhibitors. The first are pan-PI3K inhibitors which do not discriminate between the different PI3K isoforms and were the first generation of inhibitors to be developed. One of these drugs includes wortmannin, which reduced aneurysmal growth when administered in an experimental animal model of AAA . While tDisordered NOTCH signaling is a feature of many cancers and a variety of targeted therapies have been developed, including inhibitors of the cleavage of NOTCH receptor precursors and gamma-secretase inhibitors . The gamAs cardiovascular abnormalities appear to be a feature of impaired NOTCH signaling, NOTCH agonists may be a useful therapeutic tool. However, given the fundamental role of NOTCH in vascular homeostasis, the overall inhibition of the pathway by nonspecific agents are unlikely to be beneficial. Therefore, the more targeted inhibitors currently being investigated in the treatment of cancer may be more useful in the treatment of CVD.There have been some preclinical studies on abdominal aortic aneurysm using NOTCH inhibitors. In a murine model using ANGII infusion, aortic inflammation and elastin fragmentation were reduced by treatment with a NOTCH inhibitor ,334. WheThe ANGII pathway is a well-studied treatment target in CVD, including in TAA. Angiotensin receptor blockers (ARBs) and beta-blockers have each been proposed to reduce TAA growth rates in MFS and other genetic aortopathies ,335. TheThe magnitude of effect on slowing TAA growth is debated and available clinical studies do not all clearly differentiate individual effects of beta-blockers and ARB, as they may be used in combination. Data from experimental animal studies appear more robust than from clinical studies and the benefits of ARB use may be dependent upon the age of the treatment subject. In addition, other variables in human populations may impact the benefit of ARBs, including variability in underlying mutations causing MFS or LDS, epigenetic factors and drug dose and type . NotwithIn summary, the work done on investigating therapeutic targets of TGF-β, WNT, PI3K/AKT, NOTCH and ANGII signaling in the treatment of cancer highlight the potential for these pathways to be targeted in the treatment of TAA. However, there are a few factors that need to be considered when beginning to translate the work done in cancer to their use in the context of TAA.Firstly, several compounds which target these pathways show levels of toxicity when administrated for the treatment of cancer as the aggressive nature of the disease may require relatively high doses of a particular drug. However, this does not rule out their potential clinical utility in the treatment of TAA as it does not need to be approached in the same aggressive manner and therefore lower doses may be acceptable in TAA treatment, attenuating toxicity and potential side effects.Secondly, the aggressive nature of cancer may, on the other hand, mean certain drugs are not potent enough to show any advantageous therapeutic effect. However, a small effect on any of these pathways may be enough to influence TAA development and therefore may be beneficial in TAA treatment and are still worth pursuing.Finally, therapeutic targets of the five pathways discussed in cancer are frequently being tested in combination with other cancer treatments to improve therapeutic potential. This need for TGF-β inhibitors in combination with other agents may also be relevant to the treatment of TAA, as the interactions between and redundancy of signaling pathways may mean inhibition of TGF-β alone may not result in an effective outcome. This concept has been supported by the finding that TGF-β1 inhibitors combined with PI3K inhibitors are the most effective method for inhibiting the phenotype switch of human aortic VSMCs compared to the use of a single inhibitor .Multiple signaling pathways influence both EC and VSMC phenotype, often acting in synergy to either promote or inhibit cell proliferation and phenotype. Crosstalk between two or more pathways in determining cell fate appears to be common. Signaling outcomes consequent upon the activation of a specific pathway EC and VSMC may, however, differ and each cell type has the capacity to influence outcomes in the other.At this time a cognate integrative description of the mechanism of TAA formation remains elusive. There is evidence indicating potential for each of the major pathways described to contribute to TAA formation. Experimental and human studies support the roles of these pathways in TAA, however the evidence base remains fragmentary and further work is required, particularly in the field of signaling cross-talk.For many patients with TAA, the underlying cause remains unknown, including for many with non-syndromic h-TAA. It is quite likely that many of these patients have an underlying dysfunction/dysregulation of the signaling pathways described and an understanding of the key regulatory points in these pathways may illuminate potential new loci for the investigation of gene mutations in TAA.Despite great interest, there is very limited data on efficacy of novel treatments for prevention of TAA. The experience in cancer medicine illustrates potential new approaches, but at the same time also highlights the challenges of drug toxicity and off-target effects. The multiplicity of pathways influencing cell fate infer redundancy and, therefore, therapeutic attempts to influence a single pathway may be sub-optimal in altering TAA development. Although much work remains to be done, our improved understanding of the roles of multiple cell-signaling pathways upon cell fate in the vasculature can provide a foundation for future advances.