Migratory flows play a significant role in the spread of human T-lymphotropic virus 1/2 (HTLV-1/2). In the last decade, a substantial migration of individuals occurred from Haiti and Venezuela to Brazil. However, data on the prevalence of HTLV-1/2 infection among these international migrants in Brazil are scarce. This study describes the prevalence of this infection among immigrants and refugees in Central Brazil.A cross-sectional study was conducted with 537 international migrants in the State of Goiás, Central Brazil. Participants were interviewed, and blood samples were collected. Serological screening for anti-HTLV-1/2 was performed using an enzyme-linked immunosorbent assay , and seropositive samples were submitted for confirmation by a line immunoassay .n = 5) were refugees from Venezuela, resulting in a rate of 2.26% for anti-HTLV-1/2, HTLV-1 (0.45%) and HTLV-2 (1.81%) among Venezuelan refugees. Of the demographic and behavioral characteristics evaluated, unprotected sexual intercourse and having more than one sexual partner (≥2) in the previous 12 months were associated with HTLV-1/2 seropositivity among Venezuelans.The majority of participants were males (54.4%), between 18 and 50 years old , self-declared black (55.1%), reported 1 to 12 years of formal education (70.9%), and were either Venezuelans (47.9%) or Haitians (39.7%). Additionally, 50.1% were immigrants, 49% were refugees, and five were Brazilian children (0.9%) born to Haitian immigrant parents. The overall prevalence of anti-HTLV-1/2 was 0.95% (95% CI: 0.31–2.28), with HTLV-1 at 0.19% and HTLV-2 at 0.76%. All seropositive individuals (This study revealed, despite the low seroprevalence of HTLV-1/2 among international migrants in Central Brazil, evidence of HTLV-1 and HTLV-2 infections in Venezuelan refugees. In addition, their characteristics highlight that specific social and health programs should be implemented for these emergent and socially vulnerable migrant groups. The human T-lymphotropic virus type 1 (HTLV-1) is a retrovirus associated with severe diseases worldwide, including adult T-cell leukemia/lymphoma (ATL) and progressive tropical spastic paraparesis/HTLV-1-associated myelopathy (TSP/HAM). Additionally, other inflammatory diseases are associated with this virus .Despite estimates that at least 5–10 million people worldwide are infected with HTLV-1, its epidemiology is poorly understood . These rCurrently, it is estimated that 281 million people are international migrants worldwide , and appMigratory flows contribute to the emergence of infectious agents in new geographic areas; and have indeed played a significant role in the introduction and spread of HTLV-1 and HTLV-2 in Brazil . High HTA cross-sectional study was conducted among international migrants (immigrants and refugees) residing in the State of Goiás, Central Brazil. Thus, the study was performed in three municipalities in Goiás, which had received most international migrants during the study, including Goiânia , Aparecida de Goiânia, and Anápolis, between July 2019 and May 2021 .For this study, the term international migrant is an umbrella term used to refer to any person living in a country other than where they were born , includiα < 0.05), 80% statistical power (β = 20%), a precision of 1% and an effect design of 1.2%. However, a convenience sample of 537 individuals was obtained (80% of the target sample) due to the lack of knowledge of the exact number of immigrants and refugees in Goiás, the difficulty of accessing them, and linguistic and cultural challenges in addition to the COVID-19 pandemic. The inclusion criteria for this study were: being an international migrant in Goiás and having lived in Brazil for up to 10 years. In addition, children aged less than 2 years were excluded due to the difficulty of drawing their blood samples.During the data collection period, there were no data on HTLV-1/2 prevalence among international migrants from Latin America in Brazil for up to 10 years. Therefore, the sample size was estimated to be 681 individuals, considering a hypothetical prevalence of 1.5% for HTLV-1/2 infection, a significance level of 95% . All subjects who voluntarily agreed to participate in the study signed an informed consent form.Data and blood samples were collected at private locations, such as churches and non-governmental organization facilities. Data collection instruments were prepared in Portuguese, English, and the native languages of the participants in the study . In addition, interviewers fluent in these languages were trained for this data collection by the project team. Participants were interviewed face-to-face by trained interviewers in their language after they agreed to participate in the study and signed the Free and Informed Consent Form. Those under 18 years of age were consented using the Terms of Informed Consent of Children and Adolescents and Free and Informed Assent.A total of 537 international migrants agreed to participate in the study and were interviewed using a structured script containing questions about sociodemographic data, risk characteristics associated with HTLV infection, and also about their medical history. Then, a blood sample (10 ml) was collected from all participants.Of the 537 international migrants who consented to participate, a total of 528 (98.3%) serum samples were tested for anti-HTLV-1/2  using an enzyme-linked immunosorbent assay (ELISA). Reactive samples were confirmed using a line immunoassay . Samples that tested positive by LIA were considered positive for HTLV-1 or 2 infections.n = 254). All Venezuelans in the study reported being breastfed during childhood, which precluded further statistical analysis on this variable. For this study, p-values <0.05 were considered statistically significant.The collected data were analyzed using the Statistical Package for the Social Sciences (SPSS) version 20.0. Descriptive analyses were performed using frequency distributions, mean values, and standard deviations. Prevalence of anti-HTLV-1/2 was calculated using a 95% confidence interval (95% CI). Fisher’s exact test was used to evaluate the association of demographic and behavioral characteristics with the presence/absence of HTLV-1 or HTLV-2 among Venezuelan migrants who were tested for anti-HTLV-1/2 (n = 24), Dominicans (n = 18), Ecuadorians (n = 7), Cubans (n = 5), Equatorial Guineans (n = 3), Colombians (n = 2), Peruvian (n = 1), French Guianese (n = 1), and Bahamian (n = 1) ( (n = 1) .Among the 528 international migrants in Central Brazil tested for anti-HTLV-1/2 antibodies, five  were found to be anti-HTLV-1/2 positive by ELISA. After confirmatory testing (LIA), one was positive for HTLV-1 (0.19%), and four were positive for HTLV-2 (0.76%). All HTLV-1/2 seropositive individuals did not report any symptoms. They were all refugees from Venezuela, resulting in a rate of 2.26% (5/221) for anti-HTLV-1/2, HTLV-1 (0.45%) and HTLV-2 (1.81%) among Venezuelan refugees.As shown in p < 0.001). Additionally, seropositivity was higher among those who reported ≥2 sexual partners in the last 12 months than those with only one sexual partner .In this study, most participants were Venezuelans 47.8%) and Haitians (39.7%), reflecting the shift in international migration patterns in the last decade in Brazil .8% and H. Since 2This study found a prevalence of anti-HTLV-1/2 antibodies of 0.95% (95% CI: 0.31–2.28) among immigrants and refugees in Central Brazil, HTLV-1 (0.19%) and HTLV-2 (0.76%). Notably, all seropositive individuals were refugees from Venezuela, yielding a prevalence of 2.26% for anti-HTLV-1/2, HTLV-1 (0.45%), and HTLV-2 (1.81%) among Venezuelan refugees. These results align with those reported by Abreu et al. , which fDespite limited data on HTLV-1/2 infection in Venezuela using new screening and confirmatory assays, previous studies detected anti-HTLV-1 and HTLV-2 antibodies among Venezuelans, specifically in blood donors at the Municipal Blood Bank of Caracas (0.11%) . AdditioIn line with other studies , which rSince HTLV-1 and 2 infections are often asymptomatic, these viruses are silently spread from mother to child, mainly by breastfeeding, and through horizontal transmission . In factSistema Único de Saúde - SUS) may be limited.Notably, most of the anti-HTLV-1/2 seropositive individuals had a lower educational level. This characteristic was also observed in the study population, which in addition to the cultural and language barriers , may contribute to the high unemployment (35.9%) observed. This cultural and linguistic divide can exacerbate poverty, social discrimination, and marginalization in the group. Consequently, the group’s access to public health services through the free Unified Health System (In this study, although the individuals who were HTLV-1/2 seropositive were clinically asymptomatic at the time of sampling, they were referred for clinical follow-up, as this infection can take many years to develop into severe related diseases. It should be noted that HTLV-1 is associated with ATL and TSP/HAM, as well as other inflammatory diseases .This study has some limitations that should be considered. Initially, the convenience sampling used can compromise the external validity of the results, though the sociodemographic and migration characteristics of the studied population are consistent with those reported elsewhere , 27. AlsThe findings of this study revealed not only a low seroprevalence of HTLV-1/2 among international migrants in Central Brazil but also the circulation of HTLV-1 and HTLV-2 within the Venezuelan refugee population, an emerging and socially vulnerable group. Therefore, specific health programs should be implemented for immigrants and refugees, mitigating barriers and promoting the implementation of appropriate interventions aimed at the well-being of all international migrants. Considering language and cultural barriers, promoting health, and facilitating early diagnosis of symptoms associated with HTLV-1/2 are some appropriate first steps toward controlling/preventing this infection.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.The studies involving humans were approved by Committee on Ethics in Research of the Federal University of Goiás. The studies were conducted in accordance with the local legislation and institutional requirements. Written informed consent for participation in this study was provided by the participants' legal guardians/next of kin.TAM: Conceptualization, Methodology, Writing – review & editing. LM: Methodology, Writing – review & editing. KS: Methodology, Writing – review & editing. TLM: Methodology, Writing – review & editing. GS: Methodology, Writing – review & editing. ÁS: Methodology, Writing – review & editing. MC: Conceptualization, Methodology, Writing – review & editing. KC: Conceptualization, Methodology, Writing – review & editing. ST: Conceptualization, Formal analysis, Methodology, Writing – original draft. RM: Conceptualization, Supervision, Writing – original draft, Writing – review & editing.


The perception of human T-cell leukemia virus type 1 (HTlV-1) infection as a “silent disease” has recently given way to concern that its presence may be having a variety of effects. HTLV-1 is known to cause adult T-cell leukemia (ATL), an aggressive cancer of peripheral CD4 T cells; however, it is also responsible for HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Most patients develop ATL as a result of HTLV-1 mother-to-child transmission. The primary route of mother-to-child transmission is through the mother’s milk. In the absence of effective drug therapy, total artificial nutrition such as exclusive formula feeding is a reliable means of preventing mother-to-child transmission after birth, except for a small percentage of prenatal infections. A recent study found that the rate of mother-to-child transmission with short-term breastfeeding (within 90 days) did not exceed that of total artificial nutrition. Because these preventive measures are in exchange for the benefits of breastfeeding, clinical applications of antiretroviral drugs and immunotherapy with vaccines and neutralizing antibodies are urgently needed. There are seven known carcinogenic viruses: Epstein–Barr virus ), hepatitis B virus (HBV), hepatitis C virus (HCV), human T-cell leukemia/lymphotropic virus type 1 (HTLV-1), human papilloma virus (HPV), Kaposi’s sarcoma herpesvirus ), and Merkel cell polyomavirus (MCV). These viruses are responsible for approximately 15% of all human cancers ,3, and tAlthough not ubiquitous, HTLV-1 is found worldwide, and clusters of high endemicity often exist near areas where the virus is rarely present. This could be explained by factors including a possible founder effect, predominance of mother-to-child transmission (MTCT), and cell-to-cell transmission mechanisms ,14. The Most ATL cases are attributed to MTCT ,16. The HTLV-1 virions are rarely detected in the extracellular environment . Thus, HRecent studies have shown that cell-free HTLV-1 can infect certain types of cells rather than being poorly infectious as previously thought . In vitrBecause DCs, monocytes, epithelial cells, macrophages, and B cells express these receptors, they can also infect each other in individuals with HTLV-1 infection ,35. CD4+Infected lymphocytes transmit HTLV-1 through intercellular contact with target cells, and viral components, including the single-stranded RNA genome of HTLV-1, are transferred to target cells through these junctions . HTLV-1 HBZ is the only gene that is encoded in the antisense strand and is transcribed from the 3’ LTR. The HTLV-1 genome has the potential to express multiple products using various strategies, such as frameshifting and alternative mRNA splicing.According to previous studies, immediately after infecting the cell, the HTLV-1 virus spreads from cell to cell. Later, during the chronic infection phase, the virus survives through clonal expansion as a provirus, which is incorporated into the host cell genome and replicates as the infected cells divide ,45. ReplTax and Rex are essential for viral replication. Tax promotes viral mRNA synthesis by transactivating the HTLV-1 promoter located in the 5’ LTR. Tax acts in a coordinated manner on various intracellular targets during cell transformation and is involved in immortalization, cell proliferation, and leukemogenesis. On the other hand, Tax is a major target antigen recognized by cytotoxic T lymphocytes (CTLs) . Ho. Ho54]. In Japan, methods such as limiting the duration of breastfeeding to three to six months or inactivating infected cells by freezing and thawing procedures  have been proposed as alternatives to ExFF for carrier mothers who wish to breastfeed their babies . In the In a recent technical report on HTLV-1, the WHO recommends that “available data should be further analyzed to better define the risk of HTLV-1 transmission associated with specific duration of breastfeeding, balanced with the risks of other adverse health outcomes that may result from reduced breastfeeding” . In thisA meta-analysis of 12 studies by Rosadas et al. in 2022 showed that the risk of MTCT with breastfeeding (of any duration) was approximately four times higher than that with ExFF , supportAccording to a review article by Millen et al., avoidance of breastfeeding is not an option in resource-limited areas or populations with few infected individuals . In partAlthough the precise mechanism of MTCT prevention by STBF is unknown, it is assumed to be due to the transplacental transfer of neutralizing antibodies from the mother to infant during pregnancy. The antibodies remain in the infant for several months after birth and may prevent MTCT during the first few months of life. The period of exposure to the infected cells is short, and the cumulative number of infected cells entering the digestive tract is small.In the Nagasaki Prefecture, the duration of STBF has been set at six months or less since the late 1980s. During this period, the MTCT rate was 2.4% (23/962) for ExFF, while it was significantly higher for STBF (≤6 months) at 8.3% (14/169) . Since tIn a Japanese prospective cohort study by Itabashi et al., the intention-to-treat (ITT) analysis showed that the MTCT rates for STBF (less than 90 days) and ExFF were 2.3% (4/172 children born to carrier mothers) and 6.4% (7/110), respectively, with no statistically significant difference between the two groups . Among 1A 2021 systematic review included a meta-analysis of the risk of MTCT of STBF ≤ 3 months and STBF ≤ 6 months compared with that of ExFF . The metIt is speculated that freeze–thaw treatment of breast milk destroys infected cells, thereby inactivating its infectivity in the infant . SpecifiFTBMF requires several work processes. If an infant born to an infected mother is admitted to the NICU, the mother’s work involves expressing and freezing breast milk, and then bringing the frozen breast milk to the NICU. However, if not admitted to the NICU, two additional processes are required: thawing the frozen breast milk and transferring it to a bottle. It is difficult to repeat a series of work processes on a daily basis.When newborn infants cannot be fed with their mother’s milk, such as preterm infants admitted to the NICU, human milk donated to human milk banks is an important resource for supporting their health. According to international guidelines , milk isBanked human milk should be screened for maternal HTLV-1 infections . TheoretThe method of supplementing the deficiency with infant formula in the case of decreased breast milk secretion is called mixed feeding. Some carrier mothers intentionally choose mixed feeding immediately after birth to reduce the amount of breast milk ingested by their infants, thus reducing the amount of virus transferred to them. However, the effect of this approach on MTCT is unknown. In recent studies, the rate of MTCT of HIV was extremely high (approximately 20%) compared to normal breastfeeding or infant formula feeding . Mixed fTo prevent the MTCT of HIV, antiretroviral prophylaxis, cesarean section, and avoidance of breastfeeding are now sentinel events in resource-rich countries . These aBittencourt et al. reported that when elective cesarean sections were performed in 81% of 41 HTLV-1 carrier pregnant women who opted for ExFF, no MTCT was observed in any of the 41 infants . AlthougTo date, no clinical trials have been conducted on ART during pregnancy, although in vitro studies have suggested efficacy of ART . In a caPrevious animal experimental and pilot studies have suggested that immunotherapy, such as neutralizing antibodies and vaccination with the HTLV-1 gene product, may protect against infection ,123,124.Introduction of an HTLV-1 antibody screening program for all pregnant women remains controversial ,91,126. HTLV-1 antibody screening is usually performed within 30 weeks of gestation, allowing carriers sufficient time to obtain more information from their healthcare providers before delivery and select the appropriate feeding regimen for their infants. Pregnant women with positive screening results undergo confirmatory antibody testing using an algorithm  54,129],12954,12Since 2017, the Japanese nutrition protocol for the prevention of postnatal MTCT via breast milk has changed from the three previous options of ExFF, STBF, and FTBMF to ExFF as the first choice with the most reliable preventive effect . Based oMore than 10 years have passed since the screening test for all pregnant women was introduced in Japan. However, carrier mothers were not satisfied with the status quo. Their main opinions were as follows: (i) it is difficult to say that medical providers adequately support carrier mothers’ choice of nutritional regimen, and (ii) there are no medical facilities close by where mothers can discuss their concerns about the onset of the disease or their children’s infection, and they do not know whom to consult. This might be due to limited experience and insufficient knowledge of obstetricians and pediatricians about HTLV-1 infection, as well as lack of collaboration among obstetricians, pediatricians, hematologists or neurologists, and local government officials. Therefore, establishment of a consultation and support system for carrier mothers and their families based on local medical resources, along with public awareness of HTLV-1 infection, is an urgent issue.It is unclear whether it is appropriate to select ExFF or STBF without considering risk factors for MTCT. Deprivation of long-term breastfeeding in infants at very low risk for MTCT may impact their future health . In addiAlthough HAM/TSP is generally considered an adult manifestation of HTLV-1, the possibility of early-onset HAM/TSP via MTCT has long been reported, mainly in South America ,134,135.Dermatological lesions, such as infectious dermatitis, atopic dermatitis, seborrheic dermatitis, acquired ichthyosis, candidiasis, palmar erythema, dermatophytosis, crusted scabies, and folliculitis decalvans, may be associated with HTLV-1 infection . CutaneoIf MTCT is obvious, parents should consider at what age the child will be informed and who will inform the child of this fact. Furthermore, if the child is anxious, counseling may be necessary.The perception of HTLV-1 infection as a “silent disease” has recently given way to concern that its presence may be having a variety of effects. Therefore, measures to prevent mother-to-child and horizontal transmissions are becoming increasingly important. Currently, no antiretroviral drugs or immunotherapies can be used clinically. More than 90% of MTCT cases involve trans-breastfeeding; therefore, the main preventive measures are avoidance of breastfeeding or reduction in infected cells in breast milk. Our study indicated that the MTCT rate of STBF within 90 days of birth in infants born to carrier mothers did not exceed that of ExFF. However, it is estimated that approximately 20% of mothers who choose STBF are unable to discontinue it by 90 days; therefore, adequate support from healthcare providers is essential. ExFF and STBF are available only in resource-rich areas with good sanitation. On the other hand, breastfeeding has various advantages. Accurate prediction based on risk factors for MTCT may curb more over-intervention cases for infants born to carrier mothers in resource-rich countries and reduce cases where the benefits of breastfeeding are traded off. However, in countries with limited medical resources, ExFF may not be a realistic option, particularly because it is directly associated with increased infant mortality. If antiretroviral drugs and immunotherapy, such as vaccines and neutralizing antibodies, are introduced in the future, it is expected that they may contribute to the prevention of MTCT after birth without compromising the advantages of breastfeeding and may even be useful for prenatal prevention of infection.


Mammalian target of rapamycin (mTOR) signaling pathway is a major regulator of cell proliferation and metabolism, playing significant roles in proliferation, apoptosis, inflammation, and illness. More and more evidences showed that the mTOR signaling pathway affects hair follicle circulation and maintains the stability of hair follicle stem cells. mTOR signaling may be a critical cog in Vitamin D receptor (VDR) deficiency-mediated hair follicle damage and degeneration and related alopecia disorders. This review examines the function of mTOR signaling in hair follicles and hair diseases, and talks about the underlying molecular mechanisms that mTOR signaling regulates. It also regulates a number of functions, such as gene transcription, autophagy, mRNA translation, protein synthesis, cell proliferation, and metabolism , 2. Targ2.The mTOR pathway is a crucial metabolic regulator. The mTOR protein exists in two complexes with distinct structures and functions, called mTOR complex 1 and mTOR complex 2 . The proThe functions of mTORC1 include mRNA translation, metabolism, protein turnover, etc. mRNA translation includes translation of the multi-protein exon junction complex (EJC)-associated transcripts and 5′ cap-dependent translation , 18. MetRapamycin-insensitive mTOR companion (RICTOR) and mammalian stress-activated Map kinase interacting 1 (mSIN1) are both found in mTORC2. Additionally, PI3K through PI  p3 activ3.Hair is a fiber of amazing tensile strength composed primarily of terminally differentiated, dead keratinocytes. The hair follicle is an important structure supporting hair growth, and its periodic structural changes are the basis of hair cycles. The disease and aging of hair follicles will lead to an imbalance of hair cycles, and eventually cause hair loss and other hair diseases. Hair follicles generally go through cycles of growth (anagen), regression (catagen), and rest (telogen) . The mos3.1.in vivo using the particular mTORC1 inhibitor, rapamycin, demonstrating that mTORC1 has a role in anagen entrance. The timing of the anagen beginning phase of the hair cycle may be regulated by mTORC1  in Graves’ orbitopathy  that their epithelium displays from the bulge area, the habitat for HF stem cells, downstream to the hair bulb . Alopecid people . Inhibitd people . Both TGd people . Rapamycitopathy . When th  number, induce immune tolerance, and reduce inflammation, which may help reduce the number of local inflammatory cells around the bulge and bulb and maintain relative IP. As a distinct marker for Tregs, the fork head transcription factor (Foxp3) was discovered. Only T cells expressing Foxp3 were able to prevent transplant rejection terparts . The ratpathways .2  promotes a stem cell state. Low pO2 inhibits the expression of glutaminase in a way that is reliant on mTORC2-Akt, thereby reducing glutamine hydroxylation to stop the glutamate from entering the Krebs cycles and promoting the return of progenitor cells to the HFSC state, thereby allowing progenitor cells to return to the low oxygen niche, restore stem cell status, and restore bulge to maintain hair follicle stem cells throughout the long run. Loss of mTORC2 impairs niche regeneration of progenitor cells and triggers hair follicle stem cell failure, and for HFSC destiny reversibility and long-term maintenance in vivo, mTORC2 is necessary  expression occurs in a tissue-specific manner. Heat stress stimulates the mTOR signaling pathway in skeletal muscle, but significantly inhibits the expression of the p-mTOR protein in hair follicle cells and regulates the development of hair follicle cells. By increasing the expression of BMP2 and BMP4, decreasing the mRNA levels of noggin, IGF1, and IGF1R, and increasing the protein level of p-mTOR, heat stress decreased the number of hair follicles . Hair foHair follicle damage and degeneration may have a great correlation with mTOR signaling in Vitamin D receptor (VDR) knockout mice. The follicular epithelium of VDR−/− mice is defective in the regression-to-rest morphogenetic stage. VDR−/− hair follicles exhibit DNA damage-inducible transcript 4 (Ddit4) stress compartments with increased Ddit4 during morphogenesis. In VDR−/− mice, abnormalities in Ddit4 signaling impact follicular integrity by disrupting follicular energy balance. In epidermal keratinocytes, the mTOR inhibitor Ddit4 is a direct transcriptional target of the VDR. Ddit4 suppresses mTOR signaling by activating TSC2, a GTP hydrolase activating protein, which subsequently prompts Rheb to inactivate mTOR in a GDP-bound form. In VDR-deficient hair follicles, mTOR signaling is reduced, BMP signaling is increased, the transition from catagen to telogen phase is advanced and prolonged, hair follicle cell differentiation is reduced, and eventually the hair follicle degenerates, which can cause various hair loss diseases  62)..62).4.3 affects mTOR function in the cytoplasm in multiple ways , which is also known as androgen-related progressive hair thinning. AGA is further classified into male-and female-pattern hair loss. Many studies have confirmed that the serum vitamin D level of AGA patients is lower than that of the control group and that vitamin D deficiency exists , 68. AGAol group , 72. Thehis view . The serhis view . In the his view . Vitamin5.+ and CD8+, MHC classes I and II cells, and migration inhibitory factor (MIF), is indicative of the function that these pathways play in the disease. Immunological mediators such macrophages, Langerhans’ cells, and cytokines, as well as CD4+, CD8+, and other immunological mediators, have all been linked to the onset and progression of AA  pathway, JAK inhibitors are promising drugs for managing AA in vivo (+T cells in the skin (Alopecia areata (AA) and primary cicatricial alopecia (PCA) are the results of an immune attack on the HF when this IP collapses. The relationship between the PI3K/Akt/mTOR pathways and the molecular markers that are specific to AA, such as NK cells, CD4on of AA . CD8+ T  in vivo . JAK1 an in vivo , 79. By  in vivo . It was  in vivo . CXCR3-bthe skin . The carthe skin , 84. γδTthe skin  85, 86), 86+ andAs a type of PCA, Lichen planopilaris (LPP) includes the classical form, frontal fibrosing alopecia (FFA), and Graham-Little syndrome. LPP and FFA can lead to permanent alopecia, which is considered to be related to hair follicle stem cell damage and the mTOR signaling pathway. The mTOR signaling pathway protein is found in all regions of healthy hair follicles, but it has abnormal expression in those of lichen planopilaris and frontal fibrosing alopecia patients. The expression of p-mTOR decreased in the interfollicular epidermis of the patients’ skin lesions, and increased in the lower part of the hair follicle in the ORS and the hair follicle bulb . It has Serenoa repens extracts encourage hair growth and repair in AGA mice models .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.


Pituitary adenoma (PA) is the third most common central nervous system tumor originating from the anterior pituitary, but its pathogenesis remains unclear. The Wnt signaling pathway is a conserved pathway involved in cell proliferation, Self-renewal of stem cells, and cell differentiation. It is related to the occurrence of various tumors, including PA. This article reviews the latest developments in Wnt pathway inhibitors and pathway-targeted drugs. It discusses the possibility of combining Wnt pathway inhibitors with immunotherapy to provide a theoretical basis for the combined treatment of PA. PAs are the most common tumor in the sellar region, accounting for 10–15% of the total intracranial tumors . There aThe Wnt signaling pathway is an ancient evolutionarily conserved pathway mediated by Wnt proteins. It involves multiple events during embryonic development and tissue homeostasis, including cell multiplication, stem cell self-renewal, and cell differentiation , 10.Historically, the Wnt signaling pathway has been divided into two broad categories: canonical and noncanonical , 11. CanThe atypical pathway is β-catenin independent and primarily activated by Wnt5a. It can be divided into two pathways, Wnt/Ca2+ and PCP, whose primary function is to regulate cell polarity and migration . The WntResearch has demonstrated that abnormal activation of the Wnt pathway is relevant to the progression and pathogenesis of many neoplasms, including breast carcinoma , melanomAs early as 2005, Moreno et al. found that altered expression of SFRP1, TLE2, PITX2, NOTCH3, and DLK1 was observed in NFPA. The Wnt pathway may be crucial to the progression of NFPA . The WntAs the core protein of the canonical Wnt pathway, β-catenin is a downstream protein of Wnt4. It is crucial in promoting cellular proliferation and invasion in the PAs. In 2002, Howng et al. discovered that mutations in β-catenin, E-cadherin, and Wnt signaling pathways have participated in the progression of cerebral neoplasms . In 2014Other downstream signaling targets, such as c-myc, have also been shown to be essential for forming PAs. C-myc may facilitate tumor growth, cell transformation, and evolution. Nevertheless, c-myc did not affect the recurrence of functional tumors . In 2017In addition, pituitary homeobox 2, one of the downstream signaling targets of the Wnt pathway, also plays a significant part in pituitary formation. In 2011, Acunzo et al. found that the Wnt pathway-induced PITX2 gene may participate in the emergence of gonadotropin-secreting adenoma and NFPA through anti-apoptosis . In 2013However, PAs often shares multiple signaling pathways with other tumors rather than being induced by wnt signaling alone. As the study by Hosseinkhan et al., in NFPA, Wnt, MAPK, TGF-b, Hippo, VEGF, JAK-STAT, PI3K-Akt, ErbB, TGF-b, and Ras signaling pathways are intertwined and jointly participate in the formation of tumors . CrosstaConventionally, inhibitors of Wnt signaling can be divided into two categories. The first class includes the SFRP family, WIF1, and Cerberus . They coThe SFRP family, located at 8q12-11.1, participates in the competitive binding of Wnt proteins through the N-terminal CRD structure (homologous to the FZD receptor). It incorporates five members (SFRP1-SFRP5). Melkonyan et al. identified SFRP1, SFRP2, and SFRP5 in mouse embryonic cells . By contWIF1, the first secretory antagonist found in the retina, is located at 12q14.3 and is a crucial regulator of Wnt signaling –52. HypeThe DKK family is located at 10q11 and is mainly related to the atypical Wnt pathway by binding LRP5/6 and may simultaneously affect the canonical inhibitors of the Wnt pathway . DKK4 beRecently, many researchers have focused on non-coding RNAs, which also have inhibitory effects on Wnt signaling. In 2018, Song et al. miRNAs, including miRNA-137, were found to affect Wnt signaling by regulating WIF1 methylation . In 2019According to previous studies, the abnormal expression of wnt pathway inhibitors may be related to PAs’ multiplication, aggressiveness, and typing. The research on Wnt pathway inhibitors will help to study the detailed mechanism of drugs targeting the Wnt pathway and treat PAs and their complications Table 1.Currently, prolactinomas are typically treated with drug therapy, whereas other types of PAs are often treated with surgery . If the in vitro experiments that fulvestrant is closely related to the atypical Wnt signaling pathway. Furthermore, they also suggest that fulvestrant can be used as a therapeutic agent  the Mechanism of the wnt signaling pathway in NFPA and prolactinomas. (2) achieving a deeper understanding of crosstalk among these pathways ; (3) Inhibitors of wnt signaling in PA should pay more attention to ncRNAs and to gain insight into their roles in wnt signaling in PA. (4) Targeting the wnt signaling pathway improves the TME in refractory PA. (5) Targeting the wnt pathway combined with other therapies covers more types of PAs.In summary, the wnt signaling pathway plays a significant part in guiding the development and pathogenesis of PA. Abnormal expression of Wnt-signaling inhibitors may be related to the cell multiplication, invasion, and recurrence of PA. Currently, there are few studies on the wnt signaling pathway in PA. Furthermore, studies on these drugs have yet to go into sufficient depth into the mechanism of action. Finding new therapeutic targets and combining them with traditional treatments will reveal novel directions for the individualized treatment of PA, which needs further research.WW conceived of the topic of the review, performed the literature searches and wrote the manuscript. LM, YZ, ML, WY, and XL contributed to intellectual development and helped to edit the final manuscript. All authors contributed to the article and approved the submitted version.


Nowadays, the rates of antidepressant prescription are high and increasing. In this context, the issue of whether these medications are addictive has been increasingly discussed.The aim of this review was to explore the debate about the addictive property of antidepressants.We conducted a literature review in the Pubmed database, using the search terms “antidepressants”, “SSRI”, “tricyclic”, “addiction”, “dependence” in various combinations, and narrowing the search to the last 10 years, to identify articles about the addiction to antidepressants.All the articles included in our study highlighted the fact that antidepressants were associated with withdrawal symptoms. These symptoms are heterogeneous, and long-lasting in some cases, and Paroxetine was shown to have particularly high rates of withdrawal symptoms.Some articles reported a psychological and physical dependence on antidepressants. However, the most recent studies agreed that, using established classification systems and concepts and after integrating neurobiological and behavioral criteria, antidepressants are shown to have no addictive property.Antidepressants are proven to be associated with withdrawal symptoms. Whether or not these symptoms are enough to constitute an “addiction” remains controversial, as recent studies agree that antidepressants should not be classified as addictive substances.None Declared


In this review, we summarized six major pathways involved in the odontogenic differentiation of DPSCs, Wnt signaling pathways, Smad signaling pathways, MAPK signaling pathways, NF-kB signaling pathways, PI3K/AKT/mTOR signaling pathways, and Notch signaling pathways. Various factors can influence the odontogenic differentiation of DPSCs through one or more signaling pathways. By understanding the interactions between these signaling pathways, we can expand our knowledge of the mechanisms underlying the regeneration of the pulp–dentin complex.Dental pulp stem cells (DPSCs) are a type of mesenchymal stem cells that can differentiate into odontoblast-like cells and protect the pulp. The differentiation of DPSCs can be influenced by biomaterials or growth factors that activate different signaling pathways  Dental pulp stem cells (DPSCs) are a type of mesenchymal stem cells (MSCs) that express mesenchymal markers, including CD29, CD44, CD59, CD90, CD105, CD146, and CD166 . Since tAPC), glycogen synthase kinase-3β (GSK-3β), Axin, and casein kinase 1 (Ck1). This complex then phosphorylates β-catenin and leads to its degradation. This inhibits signal transduction in the Wnt signaling pathway. When Wnts are present, they interact with the frizzled receptor and the low-density lipoprotein receptor-related proteins 5 and 6 (LRP5/6). This interaction leads to the destruction of the protein complex. This allows cytoplasmic β-catenin to accumulate and translocate into the nucleus. Once in the nucleus, β-catenin activates target genes and proteins that are involved in odontogenic differentiation, such as Runx2, ALP, DSPP, and OCN. In addition to the canonical pathways, there are also non-canonical pathways that are mediated by Wnt5a. The protein β-catenin is not involved in these pathways. Instead, non-canonical pathways are divided into two subtypes: the planar cell polarity (PCP) pathways and the Wnt/Ca2+ pathways. These non-canonical pathways play a role in tissue repair and regeneration as well  and the accumulation of β-catenin can enhance the odontogenic differentiation of DPSCs (SATB2) into DPSCs to induce odontogenic differentiation. The results showed that DPSCs transfected with wild-type SATB2 increased the expression of Runx2, osteopontin (OPN), and alkaline phosphatase (ALP) and also improved calcium nodule formation ability compared to those transfected with mutant SATB2. Dickkopf-1 (DKK1) is considered to be an inhibitor of the Wnt/β-catenin signaling pathways. Compared with wild-type ones, DPSCs that were transfected with mutant SATB2 increased the expression of DKK1 and decreased the expression of active β-catenin. Treated with XAV939 , the expression of ALP and Runx2 was decreased both in wild-type and mutant SATB2 transfection groups. This indicates that wild-type SATB2 enhanced the odontogenesis of DPSCs through the Wnt/β-catenin signaling pathways (DSPP) and dentin matrix protein 1 (DMP-1) in DPSCs after 14 days of odontogenic induction. Meanwhile, the expression of β-catenin and the phosphorylation level of GSK-3β were significantly decreased. Therefore, DANCR inhibits the differentiation of DPSCs into odontoblast-like cells by blocking the canonical Wnt/β-catenin signaling pathways   .SOX2, SSEA1, LIN28, NANOG, and REX1. As a result, the ability of DPSCs to proliferate, self-renew, and generate mature cell types becomes more efficient   .β-catenin by retrovirus infection, while others activated β-catenin by exogenous application. This suggests that transient activation of β-catenin can induce odontogenic differentiation of DPSCs. Differences in the level of Wnt activity, experimental conditions, timing, or duration of activation could contribute to these variations  superfamily transduces intracellular signals via Smad proteins (BMP), which belongs to the TGF-β superfamily, is most closely related to the Smad proteins , a MAPK kinase (MAPKK or MEK), and a MAPK kinase kinase (MAPKKK or MEKK). When a stimulus is detected, MAPKKKs phosphorylate and activate downstream MAPKKs, which then phosphorylate and activate the MAPKs. In turn, the activated MAPKs can phosphorylate numerous downstream substrates and activate cellular responses , P38MAPK, and c-Jun N-terminal kinase (JNKMAPK)  .ERK, P38MAPK, and JNK. However, leptin-induced DSPP protein expression levels and mineralization in DPSCs were blocked by ERK, JNK, or P38 inhibitors  expression and mineralization, as well as the phosphorylation levels of hibitors . Cui et  reduced .According to the aforementioned studies, when certain factors promote the odontogenic differentiation of DPSCs, the related protein of MAPK signaling pathways could be enhanced. MAPK signaling pathways are one of the crucial mechanisms that regulate cell proliferation and odontogenic differentiation . It has NF-κB proteins usually exist as inactive cytoplasmic complexes associated with inhibitors of the κB (IκB) family. NF-κB signaling pathways are divided into canonical and non-canonical signaling pathways (TAK1). TAK1 then phosphorylates IκB kinase-β (IKKβ), which in turn activates a complex called IκB kinase (IKK). The IKK complex is made up of two catalytic subunits (IKKα and IKKβ) and one regulatory subunit (IKKγ). Then, IκB family members, particularly IκBα, are phosphorylated by the IKK complex and eventually undergo ubiquitylation and proteasomal degradation. Consequently, various NF-κB complexes, predominantly the p50/RelA dimers, translocate to the nucleus.The NF-κB signaling pathways are involved in various biological processes, such as cell proliferation and apoptosis, inflammation, and immune response. The pathways . In canoTNFR), which can activate the kinase NF-κB-inducing kinase (NIK). IKKα is phosphorylated and activated by NIK, which then phosphorylates and degenerates p100 and generates p52. P52 and RELB translocate to the nucleus to activate target genes and proteins related to odontogenic differentiation, including Runx2, ALP, DSPP, and OCN , activated PI3K can convert phosphatidylinositol 4,5-bisphosphate (PIP2) into phosphatidylinositol 3,4,5-triphosphate (PIP3). This conversion can be reversed by the phosphatase and tensin homolog (PTEN). PIP3 then activates PI3K-dependent kinase 1 (PDK1). Subsequently, PDK1 and mTORC2 will, respectively, phosphorylate amino acid residues T308 and S473, leading to the activation of AKT to phosphorylate target proteins  could enhance the odontoblastic differentiation of DPSCs. After 14-day odontoblastic differentiation, the expression level of phosphorylated AMPK increased, while the expression level of phosphorylated mTOR decreased. This indicated that GOQDs would activate the AMPK signaling pathways but inhibit the mTOR signaling pathways in DPSCs . Xu et aThe aforementioned studies indicated that when a certain factor promotes the odontogenic differentiation of DPSCs, the related proteins of the PI3K/AKT/mTOR signaling pathways decreased. They concluded that the PI3K/AKT/mTOR signaling pathways negatively regulated the odontogenic differentiation of DPSCs. Osteogenic/dentinogenic differentiation of stem cells from apical papilla (SCAP) could be promoted by inhibiting PI3K/AKT/mTOR signaling pathways . ConsistNotch-1, Notch-2, Notch-3, and Notch-4) and two families of ligands (Delta and Jagged). When a receptor binds to a ligand from a neighboring cell, Notch receptors will undergo proteolytic cleavages and release the Notch intracellular domain (NICD). The NICD then moves into the nucleus and combines with members of the CSL  transcription factor family to activate Notch target genes  using retroviruses, they showed decreased levels of ALP activity and the formation of calcified nodules and dentin sialoprotein (DSP). Two months after transplanting cells into nude mice, the results revealed that control cells formed mineralized tissues, but DPSC/Jag cells did not generate any mineralized tissues in vivo. According to their results, since Notch signaling inhibited DSPP expression in DPSCs, it increases the possibility that Notch signaling could inhibit Runx2 transcriptional activities. In contrast, Manokawinchoke et al. discovered that after maintaining the DPSCs on indirect immobilized Jagged1 surfaces in an osteogenic medium, the odonto/osteogenic differentiation of DPSCs was enhanced , the activation of JNK activity and the expression of c-Jun mRNA were decreased. This indicates that Wnt6 activated JNK signaling in DPSCs. Meanwhile, they discovered that Wnt6 enhanced the level of DSPP, Runx2, and DMP-1 mRNA as well as upregulated the activity of ALP and the formation of calcium deposits. However, when DPSCs were treated with SP600125, these effects of Wnt6 on DPSCs were blocked.It has been reported that MAPK signaling pathways and Wnt signaling pathways can interact with each other . Li reve2+ could enhance the odontogenic differentiation of DPSCs by activating the ERK/BMP2/Smad signaling pathways. KEGG pathway analysis revealed that after odontogenic differentiation with high extracellular Mg2+, genes related to the MAPK and TGF-β signaling pathways in DPSCs were differentially expressed. Compared to DPSCs treated with 0 mM Mg2+, those treated with high extracellular Mg2+ showed increased levels of Runx2, DMP-1, DSPP, phosphorylated ERK, BMP2, BMPR1, and phosphorylated Smad1/5/9. Meanwhile, these effects could be reduced by 2-APB (inhibiting Mg2+entry) or U0126 . Li et aof DPSCs . In addiroblasts . Furtherroblasts ; Li et aroblasts  discoverP38MAPK signaling pathways can mediate BMP2 to enhance the levels of β-catenin in DPSCs. By treating DPSCs with rhBMP2, they observed an increase in the expression of β-catenin in both the cytoplasm and nucleus, as well as overall protein expression. Stimulation with BMP2 would increase the obtained TOPflash values, indicating that BMP2 could activate canonical Wnt signaling. They found that BMP2 could activate P38MAPK signaling, and when canonical BMP signaling pathways were blocked with LDN193189, the levels of p-P38MAPK were decreased. When they blocked P38MAPK signaling with SB203580, the levels of β-catenin were not changed after BMP2 stimulation, indicating that blockade of the P38MAPK pathway could prevent BMP2-induced activation of Wnt signaling pathways and differentiation of DPSCs  inhibited the odonto/osteogenic differentiation of DPSCs through the activation of the NF-κB and MAPK signaling pathways. However, when the NF-κB inhibitor or MAPK inhibitors were applied, it was observed that the odonto/osteogenic differentiation of DPSCs was enhanced, both in vivo and in vitro, in comparison to the group treated with IFN-γ alone. This study indicated that a certain factor could promote the odontogenic differentiation of DPSCs through NF-κB and MAPK signaling pathways.Wnt5A is involved in the Jagged1-induced mineralization in DPSCs (Jagged1-mediated Notch activation enhanced the expression of Wnt-related genes, including Wnt2B and Wnt5A. Treatment with Wnt5A enhances mineralization, indicating its potential involvement in the Notch-induced osteo/odontogenic differentiation of DPSCs.Previous studies have reported on the interplay between the Notch and Wnt signaling pathways . Kornsutin DPSCs . This inin vitro culture, emphasizing the need for improvement (At present, numerous types of stem cells have shown their clinical therapeutic potential . DPSCs crovement .in vitro using cell culture and have found that certain factors can increase the expression of odontogenic differentiation markers in DPSCs, as well as the phosphorylation or expression of key molecules in related signaling pathways. It indicated that these factors could be used to activate related signaling pathways to mediate the odontogenic differentiation of DPSCs.Many researchers have conducted mineralization experiments in vitro. We need to explore the odontoblast differentiation and repair capability of DPSCs through in vivo studies to understand the further mechanisms. Meanwhile, through mineralization experiments and gene expression analysis, most studies have only verified that a certain factor triggered or inhibited a certain signaling pathway to influence the odontogenic differentiation of DPSCs. As is known to all, each signaling pathway does not exist in isolation but rather has certain interactions between them. Nevertheless, a few literature works summarize all the signaling pathways related to the odontogenic differentiation of DPSCs.In this review, we introduced six signaling pathways with different effects on the odontogenic differentiation of DPSCs. Wnt and Notch signaling pathways have opposite influences on the odontogenic differentiation of DPSCs by distinct stimuli, and the detailed mechanisms need to be figured out. However, due to the complex mechanisms of the signaling pathways, most studies have only assessed the basic mechanisms in vivo and in vitro studies. Similarly, researchers should pay attention to the relationships between parallel multipath signaling pathways, when assessing the influence of different factors on the odontogenic differentiation of DPSCs.In brief, gaining a comprehensive understanding of the signaling pathways in the odontogenic differentiation of DPSCs will help researchers discover the most effective materials in promoting the regeneration of the pulp–dentin complex. We need further studies to figure out the detailed mechanisms of signaling pathways through


Osteoporosis, an age-related disease, develops in part because the rate of new bone formation does not catch up with the increased bone destruction caused by osteoclasts. A full understanding of how osteoclasts function is essential to identify novel drug targets for osteoporosis treatment. Previously, we demonstrated that mice lacking leucine rich repeat kinase 1 (LRRK1) had high bone mass due to defective resorption caused by dysfunctional osteoclasts. Little is known about how LRRK1 regulates osteoclast activity. Here, we found that LRRK1-deficient osteoclasts had an altered distribution of the acidic vacuoles/lysosomes, unlike the wild-type osteoclast. The lysosomes remained in the cytoplasm away from the extracellular lacunae of the bone. The apparent F-actin ring was large, but the sealing zone was weak. Albeit CTSK and v-ATPase were comparably expressed in LRRK1 deficient osteoclasts, CTSK and v-ATPase, two lysosome-associated proteins, were not stationed on the ruffled border. Our data indicate the LRRK1 positively controls osteoclast activity via regulating lysosomal distribution, acid secretion, and protease exocytosis.Lrrk1) gene were osteopetrotic due to the failure of osteoclasts to resorb bone. To determine how LRRK1 regulates osteoclast activity, we examined the intracellular and extracellular acidification with an acidotropic probe, acridine orange, in live osteoclasts on bone slices. We examined lysosome distribution in osteoclasts by localization of LAMP-2, cathepsin K, and v-ATPase by immunofluorescent staining with specific antibodies. We found that both vertical and horizontal cross-sectional images of the wild-type (WT) osteoclasts showed orange-staining of the intracellular acidic vacuoles/lysosomes dispersed to the ruffled border. By contrast, the LRRK1 deficient osteoclasts exhibited fluorescent orange staining in the cytoplasm away from the extracellular lacunae because of an altered distribution of the acidic vacuoles/lysosomes. In addition, WT osteoclasts displayed a peripheral distribution of LAMP-2 positive lysosomes with a typical actin ring. The clustered F-actin constitutes a peripheral sealing zone and a ruffled border which was stretched out into a resorption pit. The LAMP-2 positive lysosomes were also distributed to the sealing zone, and the cell was associated with a resorption pit. By contrast, LRRK1-deficient osteoclasts showed diffused F-actin throughout the cytoplasm. The sealing zone was weak and not associated with a resorption pit. LAMP-2 positive lysosomes were also diffuse in the cytoplasm and were not distributed to the ruffled border. Although the LRRK1-deficient osteoclast expressed normal levels of cathepsin K and v-ATPase, the lysosomal-associated cathepsin K and v-ATPase were not accumulated at the ruffled border in Lrrk1 KO osteoclasts. Our data indicate that LRRK1 controls osteoclast activity by regulating lysosomal distribution, acid secretion, and protease exocytosis.We previously demonstrated that mice with targeted deletion of the leucine repeat rich kinase 1 ( Osteoporosis develops with age in part because the rate of bone formation cannot catch up with the rate of bone loss. Currently, up to 8 million American women (one in five U.S. women) and 2 million American men have osteoporosis, and another 44 million Americans with low bone density are at risk . The totThe ideal drugs for the treatment of osteoporosis should not only promote bone formation but also have an anti-resorptive function. However, such medicines have not been developed yet. Currently, most available anti-osteoporosis drugs such as bisphosphonates function to inhibit bone turnover. However, treatment with bisphosphonates leads to the suppression of both osteoblast-mediated bone formation and osteoclast-mediated bone resorption ,4,5. LonLrrk1 gene were severely osteopetrotic in vertebral and long bones due to the functional failure of osteoclasts to resorb bone [Lrrk1 KO mice responded normally to anabolic PTH treatment and resisted ovariectomy (OVX)-induced bone loss [Lrrk1 has been identified in a human patient [Lrrk1 KO mice. The patient with a loss of LRRK1 function suffered from severe osteopetrosis at the metaphysis of the long and short tubular bones, as observed in the Lrrk1 KO mice [Lrrk1 KO mice was comparable to osteopetrosis-specific transmembrane protein 1 gene (Ostm1) and chloride channel 7 (Clc7) KO mouse lines [Lrrk1 KO and WT mice on bone slices and examined lysosome dispersion on the ruffled border and the intracellular and extracellular acidification in osteoclasts in vitro.Leucine rich repeat kinase 1 (LRRK1) belongs to a serine/threonine protein kinase family. The kinase activity relies on GTP binding . A pointorb bone . Osteoclorb bone . Lrrk1 Kone loss . More re patient . A parti patient . The cli KO mice ,18. The se lines ,20,21,22, Alexa fuor-488 conjugated phalloidin (A12379), and protein A/G Sepharose beds were purchased from Invitrogen . RANKL and macrophage colony-stimulating factor (M-CSF) proteins were purchased from R & D Systems . Monoclonal and polyclonal anti-actin and anti-flag (M2 and M5) antibodies, polyclonal anti-HA (SAB5600116), monoclonal anti-cathepsin K (MAB3324), and anti-M2 conjugated agarose resin were obtained from Sigma . Monoclonal LAMP2 antibody (ABL-93) was purchased from the Developmental Studies Hybridoma Bank . Anti-v-ATPase (GTX 32087) antibody was from GeneTex . Polyclonal anti-GFP antibody (SC-8334) was from Santa Cruz Biotechnology, Inc. . Polyclonal anti-OSTM1 (#14621-1-AP) antibody was from Proteintech . Rhodamine-conjugated second antibodies from mouse, rat, and rabbit  were from Jackson ImmunoResearch Laboratory . Plasmids of pUC57 harboring mouse Lrrk1, OSTM1-HA, and CLC-7-Myc coding sequences were synthesized by GenScript  and subcloned into the pRRLsin-cPPT-SFFV-GFP-wpre plasmid  by replacing GFP. The pRRLsin-cPPT-SFFV-mLrrk1/Flag-wpre was constructed previously [Acridine orange (A1301)loidin A179, and pn K MAB33, and ant2 incubator for 24 h, followed by harvesting and lysing in 0.6% CHAPS in 20 mM Bicine buffer (pH 7.5) for immunoprecipitation and Western blot.Lentiviral particles were generated by co-transfection of pRRLsin-cPPT-SFFV-OSTM1-wpre plasmid or pRRLsin-cPPT-SFFV-CLC-7-wpre plasmid with pMD2.G and Pax2 plasmids in 293T cells  by using FuGene 6  as described by the manufacturer’s instructions. Transduction of primary osteoclast precursors with Lenti-OSTM1 and Lenti-CLC-7 viruses at a ratio of 1:1 was carried out as described previously [Lrrk1 KO mice were generated as described [escribed . Mice we2 for 3 days. The cells were infected with lentivirus (Multiplicity Of Infection: 5) and differentiated in the presence of M-CSF (20 ng/mL) and RANKL (50 ng/mL) for 7 days. The media were changed every other day. Multinucleated osteoclasts  were harvested for immunofluorescent staining, cell imaging, or Western blot. TRAP staining and pit formation assays were reported previously [Primary monocytes were isolated from the spleen of 4-week-old WT and KO mice as described . The preeviously .Mature osteoclasts were fixed with 5% formalin in PBS at room temperature for 10 min, then rinsed 3 times with PBS (pH 7.4). The cells were permeabilized with 0.1% saponin, 1% BSA in PBS at room temperature for 30 min. The sections were then incubated with primary antibody at a dilution of 1:300. Negative control cells were incubated with normal rat, rabbit, or mouse IgG. After 1 h incubation at room temperature, the cells were rinsed with PBS and incubated with corresponding rhodamine conjugated secondary antibody at a dilution of 1:200 in PBS containing 5% FBS for an additional 30 min. The cells were washed with PBS again and mounted with Vectashield mounting medium with DAPI . Actin ring and 3D structure of the osteoclast were scanned and imaged by a confocal laser scanning microscope .293T cells overexpressing mLRRk1/Flag and OSTM1-eGFP were lysed with 0.6% CHAPS in 20 mM Bicine lysis buffer (pH 7.5). The lysate was precleaned with protein A/G Sepharose beds for 1 h at 4 °C, followed by incubation with anti-Flag M2 antibody or control mouse IgG for 1 h. Immunoprecipitation was carried out with protein A/G Sepharose beds at 4 °C overnight. After 4 times washing with lysis buffer, interaction of LRRK1/Flag with OSTM1-eGFP was detected by Western blot using polyclonal anti-GFP antibody. For Western immunoblotting analyses, 20 µg of total cellular lysates were separate on 4–12% SDS-NuPAGE and the protein was transferred to 0.45 µm PVDF membrane. The membrane was incubated with primary antibodies for Western blot as reported .2, 150 mM NaCl, 4M guanidine hydrochloride, and 5 mM TCEP-HCl for 20 min at room temperature. Lysate was centrifuged at 12,000 rpm for 10 min and the supernatant was collected. The cellular protein was reduced with 5 mM DTT, and the protein was precipitated with 4 volumes of acetone, followed by overnight trypsin digestion (0.1 mg/mL in 50 mM NH4HCO3) at 37 °C. Digested peptides were desalted with a Sep-Pac C18 cartridge and the phospho-peptides were enriched with the High- Select TM TiO2 Phospho-peptide Enrichment Kit  according to the manufacturer’s instruction. The enriched phospho-peptides were dried, resuspended in 0.1% formic acid, and subjected to LS/MS [Lrrk1 KO and WT osteoclasts were lysed in a lysis buffer containing 50 mM Tris-HCl (pH 8.0), 10 mM MgClto LS/MS .2 for 15 min, washed 3 times with the α-MEM medium, and chased in fresh acridine orange-free medium for 10 min, followed by observation under a confocal microscope.Mature osteoclasts on bone slices were incubated in α-MEM differentiation media containing 5 µg/mL of acridine orange at 37 °C in 5% CO12–18 in a 20 µL reaction volume to generate cDNA. The real time PCR reaction contained 0.5 µL of cDNA, 1x SYBR GREEN master mix (ABI), and 100 nM of forward and reverse primers in a 12 µL reaction volume. Data were normalized by the expression levels of peptidyl prolyl isomerase A. The primer sequences specific to the mouse Ostm1 gene are 5′-ACGCAAACTCATTCTACCCAA (forward) and 5′-AGACCTCAGCCTTTTCTGCT (reverse).RNA was isolated from differentiated osteoclasts . Reverset-test. Values were considered statistically significant when p < 0.05.Data are expressed as mean ± SEM (standard error of the mean) and were analyzed using Student’s Lrrk1 KO mice were large and flat on the bone surface with a disarranged cytoskeleton. The cells were not associated with a resorptive pit -2 primary antibody, followed by immunostaining with rhodamine-conjugated second antibody. F-actin of the osteoclasts was stained with Alexa fluor-488 conjugated phalloidin. As shown in To examine if the lack of LRRK1 in osteoclasts impairs lysosome distribution to the ruffled border, precursors derived from WT and Ostm1 and Clc-7 KO mice [It is now well established that protons produced by v-ATPase play a crucial role in lysosomal trafficking and protease exocytosis in osteoclasts. To determine if v-ATPase dysregulation of lysosome distribution affects exocytosis of lysosomal proteases, we determined the cellular distribution of lysosomal cathepsin K (CTSK) and v-ATPase in WT and LRRK1 KO osteoclasts A. We fou KO mice ,20,21,22Lrrk1 KO osteoclasts, we searched for proteins that are differentially phosphorylated between the WT and LRRK1-deficient osteoclasts. Total cellular proteins were extracted from osteoclasts, and the trypsin-digested peptides were then examined for phosphorylated serine/threonine residues by LC/MS analyses. One of the proteins identified was OSTM1, which was phosphorylated at threonine 328 and serine 329 in WT but not LRRK1-deficient osteoclasts . The Phos-tag is a functional molecule that can bind specifically to phosphate groups, which causes retarded migration of the phosphorylated protein on the acrylamide gel [Lrrk1 KO osteoclasts by Western blot. These studies suggest that activation of LRRK1 signaling might phosphorylate OSTM1 and stabilize OSTM1-HA to prevent it from degradation.Since LRRK1 belongs to a family of threonine/serine kinases, we theorized that a lack of LRRK1 would impair protein phosphorylation in osteoclasts. To identify the molecular cause for impaired lysosomal distribution in eoclasts A. To stuTo determine the protein level of endogenous OSTM1 in osteoclasts, total cellular proteins were extracted from WT and LRRK1 deficient osteoclasts for Western blot. Differentiated osteoclasts were also harvested for RNA extraction for real-time RT-PCR. The specificity of the antibody against OSTM1  was validated prior to use by recognizing the same recombinant OSTM1-HA fusion protein expressed in 293T cells with both anti-HA antibody anti-OSTM1 antibody. With the validated OSTM1 antibody, our immunoblotting analyses showed that the endogenous level of OSTM1 protein was significantly reduced by 80% in LRRK1-deficient mature osteoclasts as compared to the level in WT osteoclasts . HoweverLrrk1 KO osteoclasts were unable to accumulate OSTM1-containing lysosomes on the ruffled border. In addition, the LRRK1-deficient osteoclasts were not associated with a resorptive pit on bone slices with enlarged vacuoles in the cytoplasmic compartment -mediated lysosomal exocytosis in COS-7 cells [− concentration, and pH in osteoclasts placed on bone slices and the secreted CTSK levels in the culture media derived from LRRK1 deficient osteoclasts are needed to confirm our findings.Bone resorption relies on the extracellular acidification of vacuolar ATPase proton pumps positioned in the ruffled border of osteoclasts. In this study, we examined the lysosome distribution, the CTSK and v-ATPase localizations, and the intracellular and extracellular acidification in live osteoclasts. We found that both WT and KO osteoclasts have normal intracellular acid production, as evidenced by the presence of orange staining vacuoles in the cytoplasmic compartment. The WT osteoclast displayed orange staining of the intracellular acidic vacuoles/lysosomes positioned to the ruffled border toward the extracellular acidification. By contrast, the osteoclast deficient LRRK1 manifested fluorescent orange staining in the cytoplasm away from the extracellular lacunae, indicating that the distribution of the acidic vacuoles/lysosomes were altered in the absence of LRRK1. In addition, the WT osteoclasts demonstrated a peripheral distribution of LAMP-2 positive lysosomes with a typical actin ring. The clustered F-actin formed a peripheral sealing zone and a ruffled border that was expanded into a dent resorption pit. The LAMP-2 positive lysosomes were also distributed to the sealing zone and dug into the resorbed pit. By contrast, osteoclasts lacking LRRK1 showed diffused F-actin in the cytoplasm. The sealing zone was weak, and the cell membrane was not extruded into the bone. LAMP-2 positive lysosomes were also diffuse in the cytoplasm and were not distributed to the ruffled border. While no change in the expression levels of CTSK and V-ATPase was found in the LRRK1 deficient cells compared to those in WT osteoclasts, CTSK and v-ATPase, which are associated with lysosomes, were not positioned in the ruffled border in -7 cells , suggestOstm1 or Clc7 in mice, causing severe osteopetrosis [Lrrk1 KO mice was comparable to Ostm1 and Clc7 KO mouse lines [Ostm1 mutations exhibited severe osteopetrosis due to osteoclast dysfunction [Our proteomics study identified that OSTM 1, a β-subunit of the chloride channel 7 (ClC7), was highly phosphorylated in WT osteoclasts but not in Lrrk1 KO cells. We also demonstrated that LRRK1 protein physically interacted with OSTM1, and the lack of LRRK1 protein in osteoclasts caused a reduction in OSTM1 level and disposition of OSTM1 to the ruffled border. It is known that phosphorylation can modulate the nature and the strength of protein–protein interactions by either affecting the binding energy of the complex or changing the protein conformation . Our datpetrosis . The C-tpetrosis . More inse lines ,20,21,22function , stronglOstm1 in LRRK1-deficient osteoclasts. Therefore, it is likely that LRRK1 stabilization of OSTM1 plays a critical role in regulating osteoclast function either by their physical interaction or by phosphorylation of OSTM1 to enhance OSTM1 interaction with CLC7 or both. The relative contribution of LRRK1/OSTM1 interaction vs. LRRK1 phosphorylation of OSTM1 to OSTM1 stabilization needs to be studied in the future. We will evaluate whether the skeletal phenotypes in the Lrrk1 KO mice can be rescued by transgenic overexpression of OSTM1 or expression of phosphomimic OSTM1 in osteoclasts. We will also investigate if the OSTM1 is a direct biological substrate of LRRK1 using purified recombinant proteins in an in vitro kinase assay.In this study, we did not determine whether the OSTM1 is the direct biological substrate of LRRK1 nor examine how LRRK1 stabilizes OSTM1 protein in osteoclasts. While we detected phosphorylated OSTM1 in WT osteoclasts, the lack of detection of phosphorylated OSTM1 protein in the LRRK1 deficient cells could be due to the OSTM1 destabilization and degradation rather than the reduced phosphorylation of OSTM1. Consistent with this interpretation, our Western blot and gene expression studies demonstrated an 80% reduction in the endogenous OSTM1 protein and an approximately 2-fold compensatory increase in the mRNA expression of Lrrk1 KO osteoclasts. The apparent F-actin ring was large, but the sealing zone was weak. Although the LRRK1-deficient cells expressed comparable CTSK and v-ATPase protein levels, CTSK and v-ATPase were not stationed on the ruffled border in LRRK1-deficient osteoclasts. Our data indicate the LRRK1 positively modulates osteoclast activity via controlling lysosomal distribution, acid secretion, and exocytosis. Further studies are needed to evaluate if the skeletal phenotypes in the Lrrk1 KO mice can be rescued by transgenic overexpression of OSTM1 or expression of phosphomimic OSTM1 in osteoclasts.In this study, we found that the LRRK1-deficient osteoclast, unlike the WT osteoclast, has an altered distribution of the acidic vacuoles/lysosomes. The lysosomes remained in the cytoplasm away from the extracellular lacunae of the bone in


Infertility is one of the three major public health problems in the world, bringing immense physical and psychological damage to men and affecting the quality of men’s fertility life. Thus, the purpose of this study was to analyze the status of social support, fertility stress, mindfulness, and fertility quality of life in infertile men, and to explore the dual mediating effects of social support and fertility stress on mindfulness and fertility quality.A case–control group study was conducted, with 246 men in the case group and 149 in the control group. The Social Support Scale, Fertility Stress Scale, Mindfulness Scale, and Fertility Quality of Life Scale were used to establish a structural equation model using Mplus 8.3 to explore social support and fertility stress. Pathway relationships were drawn between mindfulness and fertility quality of life in infertile men.p < 0.05 in each case). Further, the fertility quality of life in infertile men was positively correlated with mindfulness and social support, and negatively correlated with fertility stress (p < 0.05); mindfulness could directly affect the core and treatment modules of fertility life quality, and indirectly affect the core of fertility life quality through social support (mediation effect accounted for 19.0%), while the treatment module (mediation effect accounted for 13.7%), and the core module indirectly affected fertility life quality through fertility stress (mediation effect accounted for 16.8%).There were significant differences between infertile and healthy men in each dimension of the core module of fertility quality of life, in the total score of the treatment module, in the total score of social support, in subjective and objective support, and in the total score of fertility stress, social pressure, sexual pressure, marital relationship, and childless pressure (The fertility quality of life of infertile men is not optimistic. Mindfulness-related interventions and programs can improve their fertility quality of life. According to the World Health Organization, male infertility is defined as the inability of women to conceive due to reasons pertaining to men, under the conditions of them sharing living arrangements for more than one year, having a normal sexual life, and not taking contraceptive measures during this period . The worThe concept of “quality of life” is defined by the World Health Organization as “a person’s perception of the culture and value system in which they live in life, and their relationship to their goals, expectations, standards and concerns” . The ferMindfulness is often defined as “a deliberate, non-judgmental awareness of the present moment” that arises through “purposeful, non-judgmental attention to the present moment” . Short eStudies found that social support enhances mechanisms of stress, has positive effects on health , and canFertility stress refers to the stress from social networks, marital relationships, as well as that related to physical and mental health due to infertility . MultiplTherefore, based on the aforementioned research, this study maintains that infertile men have lower fertility quality of life, lower social support, higher fertility stress, and lower levels of mindfulness. This study proposes that social support and fertility-related stress in infertile men could act as mediators between positive mindfulness and fertility quality of life, influencing the direct effect of positive mindfulness levels on both dimensions of quality of fertility life. Exploring the potential mechanisms between positive mindfulness and fertility quality of life in infertile men provides new ideas for clinical research that could help improve the fertility quality of life in infertile men by improving social support and fertility stress.n = 246) with infertility who visited the outpatient clinic of the Fertility and Infertility Centre of the First Affiliated Hospital of Xinjiang Medical University, China, from December 2020 to September 2022 were recruited as a case group and 149 healthy men who underwent health check-ups during the same period as a control group.The aim of this study was to examine the correlation between social support, fertility-related stress, positive mindset, and quality of life in fertility. For this purpose, men . ③ Participation in the study was voluntary and informed consent was obtained from each person prior to conducting their assessment.Excluded criteria: ① Patients with a major medical condition . ② A proven psychological disorder, a history of drug or substance use, alcohol addiction, experience in routine mind–body practice, psychological intervention, or who had used antipsychotic medication in the past 6 months. ③ Had difficulties in understanding and completing the questionnaire.The top four factors contributing to male infertility were, in order, abnormal semen, sexual dysfunction, prostatitis, and varicocele. All the subjects included in this study were men with abnormal semen quality leading to infertility, which may be accompanied by sexual dysfunction, prostatitis, and varicocele.The main contents of the questionnaire included items on participants’ age, ethnicity, place of residence, education level, weight, height, fertility history, past disease history, monthly family income, occupation, smoking history, drinking history, exercise time, sleep time, medical insurance classification, and environmental exposure history.almost always, to 6 = almost never). Participants rated how often they experienced states such as autopilot thinking or preoccupation. The minimum and maximum scores on this scale are 15 and 90 points, respectively. The higher the score, the higher the level of mindfulness.The Mindful Attention and Awareness Scale is a scale developed by Ryan and Brown in 2003 to measure mindfulness, which is individuals’ retention of present experiences in their daily lives—a general tendency to notice and be aware. The scale comprises 15 questions, which are rated on a 6-point Likert scale .completely disagree to 6 = completely agree), the total score ranges from 46 to 276 points—the higher the score, the higher the level of fertility stress.The fertility-related stress of infertile couples was measured using the Fertility Problem Inventory scale, which was specially designed by CR Newton in 1999 in Canada to evaluate fertility-related stress in infertile patients. The score and each subscale have high reliability and validity. The scale has a total of 46 items on 5 dimensions, including social pressure (10 items), sexual pressure (8 items), marital relationship (10 items), demand for parenthood (10 items), and rejection of a childless lifestyle (8 items). A Likert 6-point scale was used to calculate the score  indicator with Merck KGaA Darmstadt, Geneva, Switzerland . The overall goal of the FertiQoL project was to develop an international instrument to measure the quality of life of men and women with fertility problems. Its second purpose was to assess the psychometric properties of the tool. The scale consists of 2 items and 2 modules  of subjective overall quality of life and subjective overall health status, with a total of 36 items. The core module contains 24 items on 4 dimensions , with 6 items for each dimension; the optional treatment module contains 10 items, including the treatment environment (6 items) and tolerance (4 items) dimensions. Each item is scored from 0 to 4 points. By converting the scores of the total scale and subscales, the total score ranges from 0 to 100 points. The higher the score, the better the quality of life.The statistical analysis was performed in SPSS 26.0, and the model construction was performed using Mplus 8.3 software. First, the quantitative data were expressed as means, standard deviations, and interquartile ranges. R language was used to analyze the relationship between variables. The maximum likelihood estimation method was used to construct the model in Mplus 8.3 to explore the fit between the actual data and the theoretical model, as well as the direct and indirect influence paths and effect sizes. The Bootstrap program  was used to test whether the chain mediation effect was significant. The 95% confidence interval was used as the basis for judgment. If the interval did not contain 0, the mediation effect was significant, with the significance level at 0.05.The case group and the control group included 246 and 149 participants, respectively. The baseline data of the participants in the two groups are shown in p < 0.05). The case group obtained significantly higher total scores and scores on each dimension of fertility stress and positive thinking than the control group. The differences between fertility stress, social stress, sexual pressure, conjugal relations, and parental role needs were significant (p < 0.05).As shown in p < 0.001), whereas it was negatively correlated with the total and subscale scores of the fertility stress (p < 0.01).χ2 to degrees of freedom (χ2/df) = 3.35, CFI = 0.878, TLI = 0.843, RMSEA = 0.098 (p < 0.001), SRMR = 0.090, indicating that the fit of the structural equation model to the actual data was within the acceptable range. The mediation model diagram and path coefficients are shown in The correlation between fertility stress, mindfulness, social support, and fertility quality of life reached a significant level. Based on the existing research, this study used the maximum likelihood method to establish a model and analyzed the mediation effect. Considering mindfulness as an external latent variable, social support , fertility pressure  as dual mediating variables, fertility life quality core modules  and treatment modules  were used as internal latent variables to establish a mediating structure model. The results showed that the ratio of n = 246), and 95% confidence intervals of the mediation effect were estimated using the 2.5th and the 97.5th percentile. If the 95% confidence interval for the indirect effect does not include 0, the mediating effect is significant. The results of this study showed that social support had a significant mediating role in the core and treatment modules of mindfulness and fertility quality of life, with confidence intervals of  , respectively. The mediating role of fertility stress in the core modules of mindfulness and fertility quality of life were significant, with a  confidence interval, while the mediating effect of the treatment module was not significant, the confidence interval being . The mediating effect of social support on the core modules of mindfulness and fertility quality of life accounted for 19.0%, the mediating effect of mindfulness and fertility quality of life treatment modules accounted for 13.7%, and the mediating effect of fertility stress on the core modules of mindfulness and fertility quality of life accounted for 16.8% , the quality of life of infertile men being lower than that of Sexty’s study of 234 infertile men in Germany ; mindfulness → social support → fertility life quality core module (β = 0.067); mindfulness → fertility life quality treatment module (β = 0.262); mindfulness → social support → fertility life quality treatment module (β = 0.042), indicating that the indirect effect of mindfulness is weakened after being mediated by social support; and mindfulness → fertility stress → fertility quality of life core module (β = 0.058), indicating that the indirect effect of mindfulness is weakened after being mediated by fertility stress. Consistent with previous research findings  Infertile men had lower fertility quality of life and social support and higher fertility stress and positive thinking compared to healthy men. (ii) There is a significant correlation between mindfulness and fertility quality of life, which can affect fertility quality of life not only directly but also indirectly through social support and fertility stress. An intervention program is offered to improve the fertility quality of life of infertile men. (iii) Social support positively influences the core and treatment modules of fertility quality of life, and fertility stress negatively influences the core modules of fertility quality of life.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding authors.The studies involving human participants were reviewed and approved by Ethics Review Committee of the First Affiliated Hospital of Xinjiang Medical University (20210226-168). The patients/participants provided their written informed consent to participate in this study.MA contributed fully to the study, including study setup, data processing, manuscript writing, analysis review, manuscript drafting, and review. HX contributed to the discussion section and reviewed the paper. AA contributed to the data analysis and paper review. WZ contributed to the data collection and paper review. LH contributed to the methods section and reviewed and revised the paper. CZ contributed to the introduction section and reviewed the paper. All authors contributed to the article and approved the submitted version.This work was supported by Xinjiang Key Laboratory of Special Environment and Health Research Open Subjects (SKL-SEHR-2021-05), National Natural Science Foundation (82260651) and Post-doctoral Research Start-up Grant.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.


DNAH gene family and the IFT-complex. The examination of the parents showed that most of the genetic changes were inherited. In summary, this study indicates three genetically determined damage mechanisms for VACTERL with the potential to influence each other, namely Shh- and Wnt-signaling pathway disruption, structural cilia defects and disruption of the ciliary signal transduction.VACTERL association is a rare malformation complex consisting of vertebral defects, anorectal malformation, cardiovascular defects, tracheoesophageal fistulae with esophageal atresia, renal malformation, and limb anomalies. According to current knowledge, VACTERL is based on a multifactorial pathogenesis including genomic alterations. This study aimed to improve the understanding of the genetic mechanisms in the development of VACTERL by investigating the genetic background with a focus on signaling pathways and cilia function. The study was designed as genetic association study. For this, whole-exome sequencing with subsequent functional enrichment analyses was performed for 21 patients with VACTERL or a VACTERL-like phenotype. In addition, whole-exome sequencing was performed for three pairs of parents and Sanger-sequencing was performed for ten pairs of parents. Analysis of the WES-data revealed genetic alteration in the Shh- and Wnt-signaling pathways. Additional performed functional enrichment analysis identified an overrepresentation of the cilia, including 47 affected ciliary genes with clustering in the  Shh −/−, Gli2 −/−; Gli3 +/− mutant mice have different combinations of a VACTERL-phenotype and postulated that Sonic Hedgehog (Shh) signaling might be central to the pathogenesis of the disorder. Further, the VACTERL mimicking avc1Intraflagellar Transport 172 (Ift172) mutant mice, showed an association with ciliopathies  in a total volume of 20 μL. PCR reactions were performed on a Mastercycler Personal  under the following conditions: initial denaturation at 95 °C for 4 min, followed by 38 cycles of denaturation at 95 °C for 40 s, annealing at 59 °C for 40 s, elongation at 72 °C for 1 min, and final elongation at 72 °C for 10 min. DNA amplicon visualization was performed using 1% agarose gel electrophoresis and a Gel Jet Imager . Purification of PCR amplicons was performed with ExoSAP-IT . Sanger sequencing was performed on a 3730 48-Capillary Array DNA Analyzer  by the LMU Munich Sequencing Service ) using the BigDye Terminator Cycle Sequencing Kit . Sequence analysis was performed using Chromas .Sanger sequencing was used to confirm the detected variants and to investigate their inheritance. Polymerase chain reaction (PCR) amplification primers were designed using either the Helmholtz Center Munich primer design tool or Primer-BLAST (e 2020))  (Table Shttps://inkscape.org/ (accessed on 5 June 2022)), Microsoft Excel® and Microsoft PowerPoint® (Version Microsoft Office Home and Student 2016) were used.The database PubMed®  was usedThree patients were excluded from the study due to insufficient DNA samples. Twenty-one patients met the inclusion criteria. Their median age was 10.8 years (range: 1–31 years). The female-to-male ratio was 1:1.6 .https://data.mendeley.com/datasets/xkbb8wrmv4/1 (accessed on 12 July 2022)). A total of 2531 missense variants were excluded from the analysis due to their categorization as benign or possibly damaging in PolyPhen-2, SIFT, and PROVEAN scoring, resulting in 921 remaining damaging missense variants. No SNPs (MAF > 1%) were identified in the cluster of these damaging missense variants, as well as nonsense and frameshift variants  of Shh-signaling were detected. In total, 10 patients (48%) were affected by damaging genetic alteration in the Shh-pathway.The Shh-pathway is known to play a role in the VACTERL association and is involved in embryonic development ,48. A toaffected . In patiThe Wnt-pathway is also involved in embryonic development  and has Based on the model of an oligogenic event in holoprosencephaly by Kim et al. , we inveTo investigate for further pathways and biological processes involved in VACTERL we performed an enrichment analysis with DAVID v6.8. We identified an overrepresentation of cilia in all three investigated GoTerm categories.The terms centriole, ciliary part, cilium and microtubule cytoskeleton are cellular components (CC) of cilia. We have seen a fold enrichment (FE) for centriole of 2.5, for ciliary part of 2.2, for cilium of 1.8, and for microtubule cytoskeleton of 1.4 (random expectation of FE = 1.0) A. For thDNAH1, DNAH2, DNAH5, DNAH10, DNAH11, KIF19, KLC3) were associated to the term motor activity affecting nine patients.In the categories of molecular function (MF) and biological process (BP) we found cilia-associated properties. The term motor activity in the category MF showed a FE of 2.7 B. From oC2CD3, CENPJ, OFD1) were associated to the centriole replication affecting three patients, while twelve  were associated to the microtubule-based movement affecting twelve patients.The terms centriole replication (FE 5.3) and microtubule-based movement (FE 2.0) represent BP C. From oDynein heavy chain (DNAH), which includes the genes DNAH1, DNAH2, DNAH5, DNAH10, DNAH11, and affected nine (43%) patients  patients  and TCTN3 (VCK18) were found to be de novo.Further, we analyzed triplets of the revealed genetic variants from both, cilia and signaling pathways by Sanger sequencing of the parents. Seven families were excluded due to missing DNA or DNA of poor quality , the related patient only showing one cilia gene variant and no signaling pathway variants altered  and insufficient Sanger sequences (VCK19). In total, 13 pair of parents were examined successful. Most  genetic variants were inherited. Inheritance for signaling pathway genes and ciliary genes by both parents was seen in ten patients . Here, wobserved . The varAs previous studies suggested that VACTERL association is based on a multifactorial pathogenesis including genomic alterations , WES andGli gene family, which is central to the Shh-pathway. We identified ten patients (48%), almost half of our patient cohort, that were affected by a genetic alteration in Shh-signaling, whereas one patient was found to have a damaging GLI1 variant. This may indicate that in humans, as already shown in mice models, a disruption of the Shh-signaling pathway is associated with VACTERL phenotypes. This signaling disruption may be caused by alteration of activating components, as our results show half of the Shh-signaling-associated genes as having activation function and only PTCH2 working to inactivate  and in the IFT complex . The avc1Ift172 mutant mouse has already been associated with VACTERL as well as Shh-signaling, the IFT-complex, and ciliary defects in general [Ift172 mutation induced a global reduction in the expression of Ptch1, another gene of the Shh-signaling pathway [PTCH2 variant, a paralog to PTCH1 with related function [IFT172 mutation. This supports our theory introduced above, that single gene mutation may cause signaling pathway impairment, in this case by global reduction in expression of genes associated with the same pathway. Further, the researchers found structural, ciliary defects in the mouse embryos, also caused by a mutation-induced reduction of Ift172 expression. These findings are in line with our detection of altered genes associated with cilia structure as described previously. The DNAH gene family codes for chain proteins of the microtubule-associated motor protein dynein, which is found in cilia and flagella. Their gene products play an essential role for correct cilia function, including cilia motility [A closer look on the cilia genes revealed clustering in the  general . Within  pathway . Similarfunction , in addimotility ,74,75. Bmotility ,17,18. Tmotility ,18.Third, we investigated the inheritance of the signaling pathway and the cilia-associated genetic variants. The majority was inherited, in most patients (10 of 14 patients) even by both parents. The inheritance shows different compositions of the three damage mechanisms: signaling pathway disruption, structural cilia defect and disruption of the signal transduction. We assume the combination of these different damage mechanisms leading to the formation of complex malformations and to phenotypes of varying severity. The theory of heterogeneous malformation syndromes caused by a combination of different damage mechanisms, including disruption of the primary cilia, Shh- and Wnt/PCP-signaling pathways, was previously described by Kim et al.  as an olThe limitation of the study is its small number of patients with a total of 21 included patients. This is due to the rarity of the VACTERL association and the study design with only in-house patient recruitment. Accordingly, it is important that cell and animal models are realized as a next step to further investigate these molecular interactions during the development of VACTERL.In summary, this study indicates three different damage mechanisms for VACTERL association. The first damage mechanism is the general disruption of Shh- and Wnt-signaling pathways, known to mutually influence each other’s activity. Our results suggest a general downregulation of Shh-signaling by the genetic alteration of activating components, as well as an activation of the Wnt-signaling pathway by genetic alteration of inactivating components. The second and third damage mechanism affect the cilia structure or the ciliary signal transduction, respectively. We suggest these may result in a general disruption of the cilia function, as well as loss of control of the signaling pathway activity for the Shh- and Wnt-signaling pathways. All three mechanisms have been individually associated with VACTERL-like diseases or mouse models showing VACTERL(-like) phenotypes before. Interestingly, our patients showed different combinations of the three damage mechanisms, which may arise in part from the inheritance of genetic changes from patients’ mothers and fathers. Additionally, all three damaging mechanisms show the potential to influence each other, but further studies are needed.