The aim of the study was to examine the associations of uric acid (UA) in blood and urine with subclinical renal damage (SRD) and its progression in a Chinese cohort.1) 2342 participants from our previously established cohort who were followed up in 2017 were included. Cross-sectional analysis was used to examine the relationships between serum and urinary UA and the risk of SRD. 2) A total of 266 participants were recruited from the same cohort in 2013, and followed up in 2017. Longitudinal analysis was used to determine the relationships of serum and urinary UA with progression of SRD, which was defined as urinary albumin-to-creatinine ratio (uACR) progression or estimated glomerular filtration rate (eGFR) decline.In cross-sectional analysis, higher levels of uACR were associated with higher levels of serum uric acid (SUA) and urinary uric acid/creatinine ratio (uUA/Cre). Lower eGFR was associated with higher levels of SUA and fractional excretion of uric acid (FEUA) but lower uUA/Cre levels in all subjects. In addition, the multivariate-adjusted odds ratios for SRD compared with non-SRD were 3.574 (2.255–5.664) for uUA/Cre. Increasing uUA/Cre levels were associated with higher risk of SRD. In longitudinal analysis, 4-year changes of uUA/Cre and SUA were significantly associated with eGFR decline.This study suggested that urinary UA excretion was significantly associated with the risk of SRD in Chinese adults. Furthermore, 4-year changes of serum and urinary UA were associated with SRD progression. These findings suggest that UA, especially urinary UA, may be used as a simple, noninvasive marker for early detection of decreased renal function in otherwise healthy subjects. Chronic kidney disease (CKD) has become a major public health issue because of the global prevalence and the associated increase in the incidence of cardiovascular disease and premature death ,2. StudiUric acid (UA) is the final oxidation product of purine catabolism in humans ,6. For dAlthough multiple studies have shown that hyperuricaemia is common in CKD patients ,15,16, tIn the present study, we therefore conducted cross-sectional analysis based on our previously established cohort to investigate the relationships of UA in blood and urine with SRD. Furthermore, in the longitudinal analysis, we also aimed to determine the relationship between UA and the progression of SRD, which was defined as uACR progression or eGFR decline during 4-year follow-up.The study population was derived from the Hanzhong Adolescent Hypertension Study, which was established in 1987. This ongoing prospective, population-based cohort study of 4623 adolescents who regularly undergo several follow-ups for investigating the development of cardiovascular risk factors originating from children and young adults . DetailsIn order to explore the associations of UA in blood and urine with SRD, we used cross-sectional analysis of data from the large cohort that was followed up in 2017. The participant selection process is described in To further examine the relationships of changes in serum and urinary UA with SRD progression, a 4-year longitudinal analysis was also conducted in this study. We used data from a small cohort of 338 subjects that was created based on the large cohort in 2005. The detailed study design and procedures have been published previously ,26. We fWritten informed consent was obtained from each subject, and the study protocol was approved by the Ethics Committee of the First Affiliated Hospital of Xi’an Jiaotong University (code: 2015–128). This study was performed in accordance with the Declaration of Helsinki (2008). .2). Blood pressure (BP) was measured in the sitting position using a standard mercury sphygmomanometer as previously described , serum uric acid (SUA), serum creatinine, alanine aminotransferase (ALT), aspartate transaminase (AST) and fasting glucose were measured by an automatic biochemical analyser . Hyperuricemia was defined as SUA level of ≥ 420μmol/L in men or ≥ 360μmol/L in women. eGFR was calculated using the formula adapted from the Modification of Diet in Renal Disease equation on the basis of data from Chinese subjects with CKD ,24,29.2, as previously reported  and FEUA  were not associated with SRD . In the multivariate model, further adjusting for hypertension, diabetes, BMI, total cholesterol and triglycerides, the ORs (95% CIs) were 1.00, 1.122 (0.756–1.664), 1.362 (0.928–1.998) and 2.480 (1.719–3.578), respectively; linear trend P < 0.001.We further assessed the effect of UA excretion on the risk of SRD when uUA/Cre was modelled in quartiles . In an aThe characteristics of those who participated in both surveys are presented in vs. 1.0±6.1 mL/min/1.73m2/y, P < 0.001). However, uACR progression rate was not significantly different between men and women  and SUA  were significantly associated with eGFR decline. However, we did not find an association between changes in UA and uACR progression over a 4-year follow-up (The relationships of 4-year changes in serum and urinary UA levels with SRD progression (2013–2017) are shown in ollow-up .Several sensitivity analyses were performed. Firstly, we removed subjects with hyperuricemia under treatment in the cross-sectional study (n = 11) and longitudinal trial (n = 3) to exclude the potential influence of urate-lowering drugs. As shown in To the best of our knowledge, the present study is the first to evaluate the relationship between urinary UA excretion and the risk of SRD. Interestingly, we showed that a higher uUA/Cre category was significantly associated with an increased OR for the presence of SRD compared with the reference group. The observed positive association between uUA/Cre and SRD consistently occurred when uUA/Cre was considered a continuous variable. These data suggest that the urinary UA may act as a simple noninvasive, cost-effective, single biochemical marker for assessing the severity of early renal damage.UA homeostasis is determined by the dynamic balance between its production and excretion, and the latter mainly includes intestinal and renal pathway . In“renaTo our knowledge, this study is the first report concerning the association between urinary UA and the progression of SRD. We found that urinary UA change over 4 years was associated with eGFR decline in this Chinese population. Previous studies suggested that elevated SUA levels were significantly associated with GFR decline. For example, Akasaka et al.  attempteAlbuminuria not only an important prognostic indicator for renal damage, and but also a marker of endothelial dysfunction . IncreasSome limitations of our study deserve mention. Firstly, our results are obtained from northern Chinese individuals and consequently cannot be directly extrapolated to other ethnic groups. Secondly, we use spot urine specimens to determine urinary albumin excretion in present study. Nevertheless, 24-h urine is difficult to collect, and spot urine samples are more practical for use in large-scale studies. uACR has been verified to closely associate with estimates of microalbuminuria in 24-h urine and is effective in predicting kidney diseases ,45. ThirIn summary, our study shows that urinary UA excretion is significantly associated with the risk of early renal damage in Chinese adults. However, we failed to find a significant relationship between serum UA and SRD. Our results also indicate that 4-year changes of UA in blood and urine are associated with eGFR decline in this Chinese population. These findings suggest that UA, especially urinary UA, may be used as a simple, noninvasive marker for early detection of decreased renal function in otherwise healthy subjects. Further clinical trials targeting hyperuricosuria can be of much interest.S1 File(XLS)Click here for additional data file.S1 Fig(TIF)Click here for additional data file.S1 Table(DOC)Click here for additional data file.S2 Table(DOC)Click here for additional data file.S3 Table(DOC)Click here for additional data file.S4 Table(DOC)Click here for additional data file.S5 Table(DOC)Click here for additional data file.S6 Table(DOC)Click here for additional data file.S7 Table(DOC)Click here for additional data file.S8 Table(DOC)Click here for additional data file.S9 Table(DOC)Click here for additional data file.


The Hedgehog pathway (HhP) plays an important role in normal embryonic development and its abnormal function has been linked to a variety of neoplasms. Recently, the complex mechanisms involved in this pathway have been deciphered and the cross talks with other important pathways involved in carcinogenesis have been characterized. This knowledge has led to the development of targeted therapies against key components of HhP, which culminated in the approval of vismodegib for the treatment of advanced basal cell carcinoma in 2012. Since then, other compounds have been developed and evaluated in preclinical and clinical studies with interesting results. Today, several medications against components of the HhP have demonstrated clinical activity as monotherapies and in combination with cytotoxic treatment or other targeted therapies against mitogenic pathways that are linked to the HhP. This review aims to clarify the mechanism of the HhP and the complex crosstalk with others pathways involved in carcinogenesis and to discuss both the evidence associated with the growing number of medications and combined therapies addressing this pathway and future perspectives. The Hedgehog pathway (HhP) plays a fundamental role in embryonic development, tissue patterning, and wound healing . AberranThe idea that alterations in the HhP are linked to carcinogenicity came from the discovery of activating mutations in this pathway in patients with basal cell carcinoma (BCC), medulloblastoma and rhabdomyosarcoma ,5,6,7. TIn this review, we focus on the characterization of the HhP, its relation with other pathways related to the development of cancer and especially on treatment strategies as a way to fight cancer.The HhP is very complex and can be divided into two different pathways: canonical and noncanonical. The canonical HhP is initiated by the release of three ligands named Sonic Hedgehog (SHH), Desert Hedgehog (DHH), and Indian Hedgehog (IHH) . In the In the noncanonical HhP, activation of the GLI transcription factors can occur independently of the upstream components of the HhP by cross talk with other signaling cascades . MultiplCytotoxic agents, such as radiotherapy and cytokines, can also activate and upregulate the expression of GLI1 independent of canonical pathway activation. For example, tumor necrosis factor alpha (TNF-α) and interleukin-1β (IL-1β) can upregulate the expression of GLI1 through the nuclear factor kappa light chain enhancer of activated B cells (NF-κB) pathway in pancreatic and breast cancer cells ,20. InteThe RAS/RAF/MEK/ERK pathway can also upregulate and activate GLI transcriptional activity. An interesting study demonstrated that oncogenic KRAS was able to increase GLI activity in pancreatic adenocarcinoma cell lines, and this effect was inhibited by MEK inhibitors . AnotherAs mentioned above, upregulation and overexpression of the HhP are related to several cancer types, such as lung, head and neck, esophagus, colon, pancreas, glioma, breast, ovarian and cervical . RecentlBMI1, WNT-2, and Kruppel-like factor 4 (KLF4) [The HhP is also implicated in resistance to cancer therapies. For example, a study demonstrated the role of the HhP in chemoresistant ovarian cancer cell lines, and the use of SMO antagonists was able to sensitize chemotherapy-resistant cell lines to paclitaxel . One of 4 (KLF4) ,46.Preclinical data have shown that in HNSSC cells, the expression of GLI transcription factors is increased in the population of cells that were resistant to EGFR inhibitors and radiotherapy ,48. ThesA great deal of effort has been focused on targeting SMO in particular . To dateVeratrum californicum) and belongs to the family of steroidal alkaloids.Cyclopamine (11-deoxojervine), a name derived from the word cyclopia, is a naturally occurring chemical isolated from the corn lily  were similar for vismodegib and sonidegib (69% vs. 57%), but complete response rates were not (31% vs. 3%). In metastatic disease, the ORR of vismodegib was 2.7-fold higher than the ORR of sonidegib (39% vs. 15%). Side effects were similar in both groups, except for upper gastrointestinal symptoms, which were higher in patients receiving sonidegib .Regarding the tumors resistant to Hedgehog inhibitors, a study showed that patients with advanced BCCs who were previously resistant to treatment with vismodegib similarly demonstrated the same treatment resistance with sonidegib .Currently, sonidegib is under active clinical phase I/II investigation in both solid and hematologic malignancies. Studies that have examined sonidegib alone or in combination with targeted therapy have demonstrated a synergistic mediated effect ,108,109.Saridegib is a potent and specific inhibitor of SMO derived from cyclopamine. In vitro and in vivo studies have shown the activity of this drug in many types of cancer, such as ovarian, medulloblastoma and head and neck . FurtherA multicenter phase Ib study evaluated saridegib in combination with FOLFIRINOX in patients with advanced pancreatic cancer . The objTaladegib is an antagonist of the Hedgehog ligand cell surface receptor SMO with potential antineoplastic activity. Recently, a study demonstrated the in vivo efficacy of taladegib in a mouse medulloblastoma allograft model .Taladegib has been studied in several trials involving solid tumors, such as colon cancer, breast cancer and rhabdomyosarcoma. However, a phase 1 trial suggests that taladegib is effective only in BCC. This trial evaluated several advanced solid tumors, and collectively, the clinical responses in BCC patients had an estimated ORR of 46.8%. This study also suggested potential benefit of taladegib not only in Hedgehog treatment-naïve patients but also in patients who were previously treated with Hedgehog inhibitor therapy .Glasdegib is an oral, potent, selective inhibitor of the HhP that functions through binding to the SMO receptor . This moIts use seems to be attractive in myeloid malignancies since it has demonstrated that glasdegib inhibition of SMO reduces the expression of key intracellular leukemia stem cell regulators . In addiItraconazole, an FDA-approved antifungal drug, appears to act on the essential HhP component SMO by a mechanism distinct from that of cyclopamine and other known SMO antagonists .In clinical trials, itraconazole was tested in an open-label, phase II trial for the treatment of BCC in 29 patients. Itraconazole reduced cell proliferation by 45%, HhP activity by 65%, and tumor area by 24% . In anotAdditional retrospective studies supported the survival advantage of itraconazole treatment in refractory malignancies, including ovarian clear cell, triple-negative breast, pancreatic and biliary tract cancers, compared with previous reports ,119,120.As demonstrated above, the HhP can be activated downstream of SMO, and multiple cross talk between the HhP and other mitogenic pathways converges to amplify and activate GLI transcriptional factors. This cross talk is also one of the causes of resistance to SMO inhibitors, along with mutations in SMO ,122,123.GANT58 and GANT61 are GLI antagonists that can interfere with GLI translocation into the nucleus and can prevent DNA binding . GANT61 Arsenic trioxide (ATO) is known for its use in acute promyelocytic leukemia; however, it is also a GLI inhibitor . The mecFour Hedgehog pathway inhibitors (HPIs) with unique mechanisms of action downstream of SMO that are capable of modulating GLI activity have been recently described . HPI-1 lThe characterization of the noncanonical pathway elucidated cross talk between the HhP and other oncogenic pathways, such as mTOR, EGFR, MAPK, NF-κB and TGF-β. Consequently, several studies evaluated the combination therapy between agents targeting HhP components and agents addressing components of these other pathways. A promising combination is dual inhibition of the HhP and mTOR pathways. Two preclinical studies have demonstrated the efficacy of GANT61 and mTOR inhibitor combination in myeloid leukemia cells and in rhabdomyosarcoma cells ,154. In The combination of targeted therapy against the HhP and cytotoxic agents has also demonstrated promising results in preclinical trials. GANT61 was able to increase the radiosensitivity of renal cell carcinoma cells, and this was also noted in the combination therapy involving GANT61 and hypoxia-inducible factor 2α (HIF2α) inhibitor ,156. SimPreclinical evidence that Hedgehog inhibition may sensitize acute myeloid leukemia cells to cytarabine or azacytidine provided rationale for evaluating glasdegib in combination with chemotherapeutic agents. A recent phase 2 trial evaluated the combination of glasdegib with cytarabine and daunorubicin in patients with acute myeloid leukemia or high-risk myelodysplastic syndromes. This trial demonstrated that the combination is feasible and safe with clinical activity. An ongoing phase 3 trial will help clarify the activity of this combination . ItraconHowever, in some clinical trials, the results have been disappointing. Two phase II trials failed to show the benefit of the combination of HhP inhibitor and chemotherapy. One study failed to show improvement in PFS with vismodegib in combination with standard treatment (FOLFOX or FOLFIRI and bevacizumab) versus standard treatment alone (hazard ratio (HR): 125, p=0.28) in patients with previously untreated metastatic colorectal cancer . The othThe HhP is essential for embryogenesis and several other physiologic processes, such as wound healing. Recently, this pathway was also related to carcinogenesis in several cancer types. Targeted therapy against the HhP has shown impressive results for BCC, meduloblastoma and rhabdomyosarcoma but disappointing results in other histologies. In addition, resistance to SMO inhibitors in BCC is a reality that needs to be better comprehended. Recently, the elucidation and better understanding of this pathway have led to important discoveries. Multiple cross talk with other important oncogenic pathways opened new avenues to explore targeted therapy and overcome resistance. Several preclinical and animal models have revealed the efficacy and antitumor activity of targeted therapy against HhP components or combination therapy with other targeted agents, which have become promising in the fight against cancer.Future clinical trials have the potential to add more knowledge to this scenario.


The function of vascular endothelial growth factor (VEGF) in cancer extends beyond angiogenesis and vascular permeability. Specifically, VEGF-mediated signaling occurs in tumor cells and this signaling contributes to key aspects of tumorigenesis including the self-renewal and survival of cancer stem cells (CSCs). In addition to VEGF receptor tyrosine kinases, the neuropilins (NRPs) are critical for mediating the effects of VEGF on CSCs, primarily because of their ability to impact the function of growth factor receptors and integrins. VEGF/NRP signaling can regulate the expression and function of key molecules that have been implicated in CSC function including Rho family guanosine triphosphatases (GTPases) and transcription factors. The VEGF/NRP signaling axis is a prime target for therapy because it can confer resistance to standard chemotherapy, which is ineffective against most CSCs. Indeed, several studies have shown that targeting either NRP1 or NRP2 can inhibit tumor initiation and decrease resistance to other therapies. Despite advances in the diagnosis, prognosis, and treatment of solid tumors, multiple issues continue to drive morbidity and mortality. Key challenges include resistance to therapeutic strategies, tumor recurrence and metastasis. These concerns are complicated by tumor heterogeneity. Tumor cells within a given tumor differ in morphology, proliferative capacity, sensitivity to therapeutic agents and metastatic potential . One of 121, VEGF145, VEGF148, VEGF165, VEGF183, VEGF189, and VEGF206) that arise from alternative splicing and also differ in receptor specificity and function [VEGF was characterized initially as an endothelial-specific mitogen that has the capacity to induce physiological and pathological angiogenesis, as well as vascular permeability ,10. Thisfunction . Not surfunction ) and VEGfunction . This fiThe classical VEGF receptors are the receptor tyrosine kinases (RTKs) VEGFR1 (Flt-1), VEGFR2 (Flk-1/KDR), and VEGFR3 (Flt-4) . AlthougThere are two NRPs expressed in vertebrates (NRP1 and NRP2). These transmembrane glycoproteins that exhibit 44% identity at the amino acid level and they contain four distinct extracellular domains that mediate ligand binding and a short cytoplasmic domain that lacks known catalytic activity ,25,26,27The critical finding in the context of this review is that NRPs can function as VEGF receptors and that they are expressed on tumor cells . This seNRP2 transcription in prostate cancer by a mechanism that involves the Jun N-terminal kinase (JNK)/c-jun pathway, providing a direct link between the loss of a tumor suppressor and induction of NRP2 transcription [NRP2 promoter and may function in concert to regulate NRP2 transcription [NRP2 [The fact that the expression of NRP1 and NRP2 is absent or low in many epithelial tissues but increased in carcinomas that originate from these tissues provides correlative support for their involvement in oncogenic processes . Moreovecription . Interescription ,34. Expron [NRP2 ,38. COUPon [NRP2 , which ion [NRP2 , but othon [NRP2 . Other son [NRP2  and theron [NRP2 . CollectVEGFA in tumor cells of established tumors caused regression by reducing both microvascular density and the proliferation and renewal of CSCs. Moreover, genetic deletion of NRP1 prevented the ability of VEGF to promote stemness and self-renewal. A subsequent study described the importance of VEGF/VEGFR2/NRP1 signaling in the survival of glioma CSCs and tumor growth [Although earlier studies had recognized the importance of VEGF/NRP signaling to the behavior of aggressive tumor cells and tumor development , the invr growth . These fr growth . Althougr growth . NRP2 alr growth . Anotherr growth .One outstanding issue with respect to VEGF/NRP signaling in CSCs is that the expression of both VEGF and the NRPs is not limited to CSCs in many tumors. In TNBC, for example, a high frequency of tumor cells express both VEGF and NRP2 but only a fraction of these cells has CSC properties . This obAlthough not the focus of this review, a discussion of VEGF signaling in tumor cells must include mention of semaphorins, especially class III semaphorins (SEMA3s), because they are secreted by tumor cells, function as NRP ligands and have been implicated in tumor-associated functions . Taken together, the studies described above affirm the importance of VEGF/NRP signaling in the function of CSCs and tumor development. Given that NRPs lack known intrinsic signaling capacity and function primarily as co-receptors, an important issue is how NRPs signal in CSCs. As mentioned, there is evidence that NRP1 can function in concert with VEGFR2 to affect CSC function, but there is also compelling evidence that VEGF/NRP signaling in CSCs can occur independently of VEGFRs. In this context, there is evidence that NRPs can influence the signaling properties of specific integrins that contribute to the function of CSCs. For example, NRP2 can interact with and function as a co-receptor for the α6β1 integrin in breast cancer cells . This inThe possibility exists that NRPs signal independently. Specifically, the cytoplasmic domains of NRP1 and NRP2 contain a PDZ binding domain that can bind PDZ-containing proteins, especially neuropilin interacting protein . GIPC1 is a cytoplasmic scaffolding protein that interacts with a broad range of receptors and contributes to receptor trafficking and signal transduction ,56, and NRP2 [How VEGF/NRP signaling impacts the self-renewal and survival of CSCs is a problem of paramount importance that has been addressed in relatively few studies. One example of such a mechanism derives from work on VEGF/NRP2 activation of the α6β1 integrin. This activation induces a FAK/Ras signaling pathway that culminates in non-canonical hedgehog (Hh) signaling that activates Gli1, which induces expression of Bmi-1 , a PolycNRP2 ,35, creaNRP2 . More reNRP2 . This fiNRP2 ,63. ImpoNRP2 . An esseNRP2 . An interesting connection between VEGF-C/NRP2 signaling and oxidative stress in breast CSCs has also been reported . SpecifiAlthough the existing data provide some insight into the effector mechanisms that enable VEGF/NRP signaling to promote CSC properties, much remains to be learned and several key questions need to be addressed. More work is needed to determine if the known mechanisms and mechanisms to be determined can be integrated into a broad network. Clearly, however, these mechanisms may differ depending on tumor type and the nature of NRP signaling employed by specific tumor cell populations. Also, NRP1 and NRP2 likely differ in their ability to promote CSC function and this difference may reflect differences in their expression patterns. It is also possible that they differ in their signaling properties. If so, what is the structural basis for this difference given their high degree of homology. Another issue is how the different VEGF variants differ in their ability to affect CSC function. NRP1 and NRP2 can bind multiple spice variants of VEGF-A, as well as other VEGF family members including VEGF-C and PlGF . Indeed,Another area that has received scant attention is the contribution of the tumor microenvironment to VEGF/NRP signaling and the regulation of CSC function. The notion of a CSC ‘niche’ comprised of stromal cells and extracellular matrix proteins implies that this niche sustains CSC function . There iOne of the defining characteristics of CSCs is their resistance to standard chemo- and radiation therapy . For thiA more recent study implicated NRP1 in adaptive resistance to oncogene-targeted therapies . It was VEGF/NRP signaling appears to be a defining characteristic of CSCs in multiple tumor types that is necessary to sustain their self-renewal and survival. Moreover, the acquisition of VEGF/NRP signaling in tumor cells is intimately associated with oncogenic transformation and conditions in the tumor microenvironment. There is also compelling evidence that VEGF/NRP signaling is a prime target for therapy, especially the use of reagents that inhibit NRP function. These reagents may be most efficacious when used in combination with drugs that target non-CSCs or oncogene-addicted pathways as a means to overcome therapy resistance.Despite the progress that has been made in assessing the importance of VEGF/NRP signaling in CSCs, much remains to be learned. Key areas for future work include a better understanding of the nature of NRP signaling and how NRP1 and NRP2 signaling differ in this regard. One area in particular is a more rigorous investigation of how NRPs signal independently of VEGFRs and whether they possess intrinsic signaling capacity. Also, the mechanisms by which VEGF/NRP signaling impacts the self-renewal and survival of CSCs needs more study. In this direction, emphasis should be on the relationship of VEGF/NRP signaling to other mechanisms that have been implicated in CSC function.


The development of an autistic brain is a highly complex process as evident from the involvement of various genetic and non-genetic factors in the etiology of the autism spectrum disorder (ASD). Despite being a multifactorial neurodevelopmental disorder, autistic patients display a few key characteristics, such as the impaired social interactions and elevated repetitive behaviors, suggesting the perturbation of specific neuronal circuits resulted from abnormal signaling pathways during brain development in ASD. A comprehensive review for autistic signaling mechanisms and interactions may provide a better understanding of ASD etiology and treatment.UBE3A) has been reported to influence WNT, BMP, and RA signaling pathways, suggesting crosstalk between various signaling pathways during autistic brain development. Finally, the article comments on what further studies could be performed to gain deeper insights into the understanding of perturbed signaling pathways in the etiology of ASD.Recent studies on genetic models and ASD patients with several different mutated genes revealed the dysregulation of several key signaling pathways, such as WNT, BMP, SHH, and retinoic acid (RA) signaling. Although no direct evidence of dysfunctional FGF or TGF-β signaling in ASD has been reported so far, a few examples of indirect evidence can be found. This review article summarizes how various genetic and non-genetic factors which have been reported contributing to ASD interact with WNT, BMP/TGF-β, SHH, FGF, and RA signaling pathways. The autism-associated gene ubiquitin-protein ligase E3A (The understanding of mechanisms behind various signaling pathways in the etiology of ASD may help to facilitate the identification of potential therapeutic targets and design of new treatment methods. CHD8, ARID1B, DYRK1A, SYNGAP1, ADNP, ANK2, DSCAM, SCN2A, ASH1L, CHD2, KDM5B, and POGZ genes with ≥ 3 class 1 de novo protein-truncating variants (PTVs) in individuals with ASD [ANK2, CHD8, CUL3, DYRK1A, GRIN2B, KATNAL2, POGZ, SCN2A, and TBR1 genes are identified as high-confidence ASD risk genes, of which CHD8 is strongly associated with ASD because of the largest number of de novo loss-of-function (LOF) mutations observed in patients [NLGN), fragile X mental retardation 1 (FMR1), ubiquitin-protein ligase E3A (UBE3A), and DLX, which modulate BMP signaling, have been found to be associated with ASD [Autism spectrum disorder (ASD) is a multifactorial neurodevelopmental disorder characterized by impaired social interactions and elevated repetitive behaviors, in which various circuits in the sensory, prefrontal, hippocampal, cerebellar, striatal, and other midbrain regions are perturbed , 2. Compwith ASD . ANK2, Cpatients . Variouspatients –9. More with ASD –13. Dysrwith ASD , 15, fibwith ASD , transfowith ASD , 18, andwith ASD  signalinwith ASD . Due to WNT signaling is fundamental for neurodevelopmental and post-neurodevelopmental processes, such as CNS regionalization, neural progenitor differentiation, neuronal migration, axon guidance, dendrite development, synaptogenesis, adult neurogenesis, and neural plasticity –30, and CTNNB1) [APC) [PRICKLE2 [Several genetic loci/mutations linked to and/or reported in ASD patients are either core components of canonical WNT signaling, such as β-catenin CTNNB1) , 36, 39  , 36, 39PRICKLE2 , suggestWNT ligands, mutations in WNT1, WNT2, WNT3, and WNT9B have been linked with ASD. A rare WNT1 missense variant found in ASD patients has a higher capability than the wild-type WNT1 to activate WNT/β-catenin signaling [WNT2, WNT3, and WNT9B have also been found in ASD patients [WNT3 expression is elevated in the prefrontal cortex of ASD patients [Wnt1 is required for midbrain and cerebellar development [Wnt2 has been shown to be sufficient for cortical dendrite growth and dendritic spine formation, and its expression is regulated by a brain-derived neurotrophic factor (BDNF) [Wnt3 is essential for gastrulation and regulates hippocampal neurogenesis [T-brain-1 (Tbr1), a T-box transcription factor and one of the high-confidence ASD risk genes, in cortical layer 6 neurons (Tbr1layer6 mutant) during late mouse gestation has been reported to cause a decrease in inhibitory synaptic density and excitatory synapse numbers [Wnt7b expression rescues the synaptic deficit in Tbr1layer6 mutant neurons [Wnt9b promotes lip/palate formation and fusion [Among 19 ignaling . Rare vapatients –44. Notapatients , suggestelopment –48. Wnt2r (BDNF) , while ar (BDNF) . Wnt3 isogenesis , 51. Los numbers . It is i neurons . Wnt9b pd fusion , 54, whid fusion , 55, whoAPC inactivating gene mutations have been linked to ASD [Apc mice exhibit learning and memory impairment and autistic-like behaviors [Dkk1, Sp5, Neurog1, Syn2) are increased in Apc-cKO forebrain neurons [Apc-cKO mice showed higher β-catenin levels compared to those of control mice [The tumor suppressor APC is a key component of the β-catenin-destruction complex . Human Ad to ASD , 58. Comehaviors . β-catenCTNNB1 mutations have been reported in individuals with ASD, intellectual disability, microcephaly, motor delay, and speech impairment [CTNNB1 haploinsufficiency has been found to cause neuronal loss, craniofacial anomalies, and hair follicle defects in both humans and mice [Pax3 and Cdx2 at the dorsal posterior neuropore and leads to a decreased expression of the WNT/β-catenin signaling target genes T, Tbx6, and Fgf8 at the tail bud, resulting in spina bifida aperta, caudal axis bending, and tail truncation [β-Catenin is a key intracellular molecule in the canonical WNT signaling pathway and plays significant roles in development and disease , 60. De pairment , 61–63. and mice . Conditiuncation . Conditiuncation . These muncation .TCF7L2 have been found in ASD patients [TCF7L2 is one of the TCF/LEF1 transcription factors in the canonical WNT/β-catenin signaling pathway and is associated with type II diabetes in humans . De novopatients , 69. In patients , 71, sugPRICKLE2 variants (p.E8Q and p.V153I) have been reported in ASD patients [PRICKLE2’s role in ASD is further supported by the finding of a 3p interstitial deletion including PRICKLE2 in identical twins with ASD [Prickle2-deficient mice display ASD-like phenotypes, such as altered social interaction, learning abnormalities, and behavioral inflexibility [Prickle2-deficient mice [patients . PRICKLEwith ASD . Pricklexibility . PRICKLExibility . Reducedent mice . An in vent mice . Future ANK3) [CHD8) [UBE3A) [DIXDC1) [PGE2) [Several ASD-associated genes are direct or indirect modulators of WNT signaling, such as ankyrin-G (ANK3) –77, chro) [CHD8) , 6, HECT [UBE3A) , DIX dom[DIXDC1) , and Pro) [PGE2)  (Table 1) [PGE2)  ANK3 gene [ANK3 gene, localizes to the axon initial segment (AIS) and the nodes of Ranvier, where it has roles in the assembly and maintenance of the AIS and neuronal polarity [Whole-genome and whole-exome sequencing studies in ASD patients have identified mutations in NK3 gene –77. Ankypolarity , 83. Ankpolarity . Ankyrinpolarity . Ankyrinpolarity .Chd8 gene knockout (Chd8−/−) in mice is embryonic lethal [Chd8+/−) result in mice with macrocephaly, craniofacial abnormalities, and behavioral deficits [Chd8+/− mice, highlighting the critical role CHD8 plays in the regulation of WNT signaling in the NAc [CHD8, an ATP-dependent chromatin remodeler, interacts with β-catenin and negatively regulates the expression of β-catenin-targeted genes . CHD8 bic lethal , whereasdeficits . Its knodeficits . WNT sig the NAc .UBE3A is linked to autism, Angelman syndrome, and cancer [UBE3AT485A, a de novo autism-linked UBE3A mutant that disrupts phosphorylative control of UBE3A activity, ubiquitinates multiple proteasome subunits, reduces proteasome subunit abundance and activity, stabilizes nuclear β-catenin, and stimulates canonical WNT signaling more effectively than the wild-type UBE3A [Dysfunction of d cancer . UBE3AT4pe UBE3A .DIXDC1 were identified in ASD patients [Rare missense variants in patients . These vpatients . DIXDC1 patients . MARK1, patients . Dixdc1-patients , 92. ThePTGS2 expression and upregulates MMP9 and CCND1 expression, whereas in differentiating neuronal cells, PGE2 causes upregulation of WNT3, TCF4, and CCND1 [PGE2, an endogenous lipid molecule, has been linked to ASD and alters the expression of downstream WNT-regulated genes previously associated with neurodevelopmental disorders . The linnd CCND1 .NLGN3 and NLGN4 have been reported in autistic patients [Mutations in neuroligins patients . These tpatients . Chromatpatients . It will+Itpr3tf/J (BTBR) mice are widely used in the study of ASD [The TGF-β/activin and the bone morphogenetic protein (BMP)/growth and differentiation factor (GDF) are the two subgroups of TGF-β superfamily . BMPs coy of ASD . It has y of ASD  (Table 1y of ASD . ASD hasy of ASD  (Table 1y of ASD .DNlg4) LOF results in reduced growth of neuromuscular junctions (NMJs), with fewer synaptic boutons due to the reduction in the bone morphogenetic protein (BMP) type I receptor thickvein (Tkv) [Drosophila neuroligin 4 (in (Tkv) , suggestFMR1 [BMPR2) and activation of a non-canonical BMP signaling component LIM domain kinase 1 (LIMK1), which stimulates reorganization of actin to promote neurite outgrowth and synapse formation [BMPR2 and LIMK1 activity has been reported in the prefrontal cortex of FXS patients compared with that of healthy subjects [Fragile X syndrome (FXS) is the most common heritable form of intellectual disability and ASD, which is caused due to the silencing of FMR1 . FMR1 prormation . Increassubjects .Tkv [The inhibition of BMP signaling by Ube3a has been reported to play a role in the regulation of synapse growth and endocytosis . A direcTkv .DLX genes encoding homeodomain transcription factors have been associated with ASD [Bmper) has been found upregulated in a cell line overexpressing Dlx5 [DLX function in ASD patients may lead to altered BMP signaling.The with ASD –102. Thewith ASD . The BMPing Dlx5 , suggestSHH plays a crucial role in the organization of the vertebrate brain . SHH hasPTCHD1) have been reported in ASD and ID patients [Ptchd1 KO male mice exhibit cognitive alterations [Ptchd1−/y) induces global changes in synaptic gene expression, affects the expression of the immediate-early expression genes Egr1 and Npas4, and impairs excitatory synaptic structure and neuronal excitatory activity in the hippocampus, leading to cognitive dysfunction, motor disabilities, and hyperactivity [Mutations in the gene patched domain-containing 1 (patients . Ptchd1 erations . LOF experations . PTCHD1 erations . Ptchd1 activity .DHCR7) has been associated with the ASD [The impaired function of the cholesterol biosynthetic enzyme 7-dehydrocholesterol reductase  further support the susceptibility of EN2 gene for ASD [EN2 levels result in the elevated levels of the SHH expression as reported in post-mortem samples [SHH is one of the genes flanking EN2 which is coexpressed during brain development [The transcription factor engrailed2 (EN2) has been associated with ASD –116. The for ASD , 118. Th samples . SHH is elopment , 120.mGluR5) LOF results in aberrant dendritogenesis, one of the characteristics observed in autistic brains, in the cortical neurons by increasing nerve growth factor (NGF) and FGF10 mRNA levels [FGF signaling plays a crucial role in brain patterning, and its malfunction can result in various neurological disorders . There aA levels  Table 1mGluR5 LORORA variants have been associated with ASD [ALDH1A3) and the RORA-regulated FOXN1 to ASD [ALDH1A1 has been found in a subset of autistic patients [RERE) that encode a nuclear receptor coregulator for RA signaling may cause ASD and other defects associated with proximal 1p36 deletions [NLGN1 [ALDH1A2 and impairs RA-mediated synaptic plasticity in ASD, which can be alleviated by RA supplements [trans-RA can upregulate the reduced CD38 expression in lymphoblastoid cell lines from ASD, while CD38-deficient mice exhibit ASD-like behavior [Retinoic acid (RA), the functional metabolite of vitamin A, is an essential morphogen in vertebrate development , 127. RAwith ASD . Whole-e1 to ASD . Low levpatients . De novoeletions . Genome-s [NLGN1 . Intriguplements . All-trabehavior , 141. Bebehavior . A synthbehavior . These sVarious exogenous factors, such as prenatal exposure to viral infection or VPA, lead to several neurodevelopmental disorders with perturbed WNT signaling Fig. . The traWNT3, WNT7B, WNT8A) which further disrupt the transcription of downstream target genes [The infection with rubella in early pregnancy has been linked to autism in clinical and epidemiological studies . Prenataet genes , suggestThe use of the anticonvulsant valproic acid (VPA) during early pregnancy has been reported to cause autism in 11% children and autistic traits in an even larger number of children . VPA is Wnt2b, Wnt4, and Wnt7b is shown to be induced by Gli1 [UBE3A, an ASD-associated gene, influences both the WNT and BMP signaling pathways, suggesting possible crosstalk between them [UBE3A impairs RA-mediated neuronal synaptic plasticity in ASD probably by negative regulation of ALDH1A2, the rate-limiting enzyme of retinoic acid (RA) synthesis. [Nlgn3 is a direct target of WNT/β-catenin signaling, the ASD-associated gene may also regulate BMP signaling. These results suggest that signaling crosstalk among morphogenetic pathways is mediated by autistic causal genes, thereby demonstrating value in further in-depth studies on interactions between signaling molecules in normal physiological and diseased conditions. Evidence has suggested a tissue-specific mechanism behind WNT and BMP signaling crosstalk [ASD causal genes may act upstream or downstream of WNT, BMP/TGFβ, SHH, FGF, and RA signaling pathways in vertebrates and invertebrates Table , and 4.  by Gli1 . The cro by Gli1 , as well by Gli1 . Neuropi by Gli1 . Further by Gli1 , 169. Wh by Gli1 , its rol by Gli1 . A hiera by Gli1 . Crossta by Gli1  and mous by Gli1 . Recipro by Gli1 . WNT/β-c by Gli1 . Wnt has by Gli1 . Further by Gli1 . A LOF meen them , 78. Xu nthesis. . Medina nthesis.  suggesterosstalk . Moreoverosstalk , while Wrosstalk , suggest


Melanoacanthoma is a benign epithelial tumor composed of melanocytes and keratinocytes that can morphologically mimic malignant neoplasms. Two patients with melanoacanthoma that clinically masqueraded as melanoma are described: a 65-year-old African-American woman with a pigmented nodule on the right preauricular area and an 85-year-old Haitian-Creole man with a large exophytic nodule on his left lower abdomen. Melanoma was clinically suspected in both patients. Biopsies were performed, which established the diagnosis of melanoacanthoma. Complete removal of a melanoacanthoma should be considered since partial excision may result in recurrence. Melanoacanthoma is a benign, cutaneous neoplasm consisting of melanocytes and keratinocytes . It is aCase 1 A 65-year-old African-American woman with a history of lymphoma, currently in remission, presented with a black lesion on the right side of her face. The lesion had been progressively enlarging and had begun to irritate her skin. Her past medical history was remarkable for arthritis, asthma, depression, hypertension, and insomnia. She also had a history of hand dermatitis but denied any other personal or family history of skin cancer. She smoked one pack of cigarettes per day and did not drink alcohol. A complete cutaneous examination was performed. A 2 x 1-cm black plaque was present in the right preauricular area . However, six lesions also had melanoma-specific dermoscopic features  . TherefoMelanoacanthoma can mimic other skin lesions. The clinical differential diagnoses of melanoacanthoma are listed in Table Hematoxylin and eosin staining of melanoacanthoma shows papillomatosis, acanthosis, and hyperkeratosis with heavily pigmented melanocytes dispersed throughout all layers of the epidermis. Other features commonly seen include keratin-filled pseudocysts, inflammation, and Langerhans cells ,5. ElectDopa and Fontana-Masson silver stains can be used to differentiate melanoacanthoma from seborrheic keratosis. Both of these stains highlight melanin and melanocytes in all layers of the epithelium in melanoacanthoma. In contrast, positive staining is confined to the basal layers of the epidermis in seborrheic keratosis . Immunoperoxidase staining may be useful in establishing the diagnosis of a melanoacanthoma. Indeed, using a chromogen with a red indicator may also be helpful in identifying melanoacanthoma by differentiating melanocytes from melanin-laden keratinocytes. Cytokeratin such as CK7, which shows positive staining of the keratinocytes in melanoacanthoma, is a marker that can aid in differentiating melanoacanthoma from melanoma . Melanoma antigen recognized by T-cells 1 (MART-1) is an antigen that highlights melanocytes. It demonstrates positive staining of the melanocytes in a melanoacanthoma. However, since melanoma and benign nevi also demonstrate positive staining of melanocytes with MART-1, other pathologic features observed on hematoxylin and eosin staining are necessary to confirm the diagnosis of melanoacanthoma ,15.Many investigators consider melanoacanthoma to be a variant of seborrheic keratosis. Yet, the definitive pathogenesis of melanoacanthoma remains to be established. Electron microscopy studies suggest that the presence of highly dendritic, hyperpigmented melanocytes is due to a failure of melanin transfer from melanocytes to keratinocytes. Variation in the arrangement and speed of keratinocyte differentiation has been speculated as the etiology of this failed transfer of melanin. In addition, Langerhans cells, which are present throughout the Malpighian layers of the epidermis, have been found to affect the proliferation control of keratinocytes and may play a role in the failed transfer ,16. The proclivity of melanoacanthoma to occur in areas of prior trauma introduces the possibility of cutaneous injury as a factor in their pathogenesis. One of our patients not only had a melanoacanthoma but also two seborrheic keratoses. The observation of concurrent pigmented seborrheic keratoses and melanoacanthoma prompts speculation that the pathogenesis may be different than that of seborrheic keratosis.Excisional biopsies of melanoacanthomas can be both diagnostic and curative. Although these benign neoplasms do not need to be removed, most patients opt for their lesion to be excised for cosmetic purposes or because their skin is irritated by the tumor -- similar to our female patient. Incomplete removal of the lesion at the time of biopsy may result in recurrence and require subsequent intervention. Patients with melanoacanthomas have a good prognosis with low risk of recurrence following complete removal of the tumor. Melanoacanthoma is a benign tumor consisting of melanocytes and keratinocytes that typically occurs in older individuals and presents as a light-brown to black plaques or nodules ranging in size from two by two millimeters to 15 by 15 centimeters. Similar to our patients, the clinical differential diagnosis of melanoacanthoma includes not only melanoma, but also other melanocytic neoplasms and epithelial tumors; microscopic examination establishes the diagnosis. The concurrent observation of pigmented seborrheic keratosis and melanoacanthoma in one of our patients prompts the speculation that the pathogenesis of melanoacanthoma may be different than that of a seborrheic keratosis. Complete, conservative excision of the tumor is the treatment of choice; partial removal may result in recurrence as observed in one of our patients.


This study investigates the role of changing social relations for fertility decline during the European fertility transition. The growth of voluntary associations at the end of the nineteenth century entailed a radical shift in the landscape of social relations in Sweden. By combining micro-census data from 1890 to 1900 with local-level membership data for three voluntary association groups, this article assesses the effect of parish-level voluntary association size on net fertility in Sweden using mixed-effects Poisson regression models. The results show that the adoption of fertility limitation during the transition period was associated with the creation and diffusion of the idea of respectability within large social network organisations, an idea that has previously been shown to be connected to fertility limitation. Furthermore, by applying a social network perspective, the results show that the strength of the effect was dependent on the structure of the social networks in terms of size, density, and homogeneity. Voluntary association size had the strongest effect for the free churches, which created dense heterogeneous networks through systems of social control, while the size of the temperance association showed no effect on fertility because the connections between nodes were sparse. This study investigates the role of changing social relations for fertility decline during the European fertility transition in the late nineteenth century. Studies of post-transitional populations have shown that fertility decisions are always influenced by the social interactions on which they depend. Thus, researchers have focused on how social networks influence actors’ perceptions of reproductive practices and on the role played by social networks in diffusing ideational effects on fertility  within the network, and density refers to the number and strength of the connections between nodes. The homogeneity of the network refers to similarities between network partners Keim : 22. ThiA voluntary association in this study refers to an organisation that falls within the term “popular movement”, or Folkrörelsen in Swedish. Such an association (1) “arises in order to promote some common interest of its members”; (2) where “membership is voluntary”; and (3) “the organisation exists independently of the state” , which has pre-processed and standardised the censuses for research purposes. The censuses provide snapshots of the entire population at a given time, allowing for analysis of fertility using individual-level data. However, the censuses have some limitations. There is no information on all children born, so the numbers observed in the censuses only represent achieved fertility and are biased by differences in child mortality. Mother–child relations are obtained through information on their relationship to the household head. Although this type of relational linking is prone to errors in complex households, with children mismatched to stepmothers or grandmothers, the Swedish censuses have an explicit link between children to their own mothers, which minimises the risk of mismatching  is the distance between parish j and voluntary association j, and d is the distance between voluntary association k and parish j. Thus, k.In addition, the absolute number of members could not be used because some members might have come from the surrounding area. Therefore, the membership number for each movement was weighted by the population that might belong to that movement. The size of voluntary association type The parish-level voluntary association sizes were divided into four groups for each association type. The first consisted of parishes without a voluntary association presence, and the other three groups were divided into quantiles. The resulting groups were none, small, medium, and large; none was used as the reference.b is a matrix of random effects, and X and Z are the model matrices used to determine the conditional mean b through the log link function  between parishes with no free churches and those with large ones. The temperance movement had no significant effect in either rural or urban parishes, similar to the results in the previous regression models. For the workers’ movement, it is not clear whether the negative effect was present only in urban environments, and at most it shows a 7% difference in net fertility. Urbanisation can also be seen as a crude proxy for industrialisation because the two processes were often correlated during the time period studied here. The results can therefore be interpreted as showing how the effect of voluntary association size was modified by industrialisation and that the effect of the free churches existed independently of industrialisation, showing clear negative effects on fertility in rural environments.It is evident that the different effects of free churches and workers’ organisations were not only due to the organisations’ network structures, but also to the structural environment of the context in which they operated. The effects of voluntary association size in urban areas were nonlinear, suggesting a cut-off point. The proportion of the population the organisations needed to attract before their influence was visible in the whole population was larger in urban areas than in rural areas. As the population density increases, the number of possible social interactions between people increases. Within urban areas there was a wider range of social organisations and networks, which weakened the influence of any one organisation. As Gamm and Putnam  have hypSocial networks have been shown to influence reproductive behaviours in post-transitional societies (Balbo and Barban In the Swedish context, the emergence of voluntary association mass movements in the late nineteenth century represented a radical shift in the landscape of social relations (Ambjörnsson While the growth of the Swedish voluntary associations facilitated social change, the different movements had quite different effects on net fertility. In 1890 the free churches were the only voluntary association that displayed a clear negative relationship to net fertility, and net fertility was 5% lower within parishes with strong free churches than in parishes with no free churches. The relationship strengthened in 1900 to almost 10%, and at this point the effect of workers’ organisations on net fertility was also negative. The workers’ movement comprised quite homogenous networks because they consisted primarily of young men from the working classes, which strengthened the mechanisms. Although the free churches were not as socially homogenous, they created dense connections between their members through strong social control. The importance of the structure of the organisations for the effect of social network mechanisms is highlighted by the temperance organisations, which did not show any relationship to net fertility. This was likely because, although they were large associations and had morality and restraint central to their agenda, they were not as dense as the other groups because the connections between nodes were sparse and weak due to high membership turnover (Lundkvist In addition, the results question the importance of social homogeneity of networks because the effects on fertility were stronger for the socially heterogeneous free churches than the more homogenous temperance associations. However, this could indicate differences between types of diffusion processes. While the probability of joining an association was higher in homogenous networks during the time period studied here (Sandell and Stern The probability of joining a voluntary association was not only affected by the structure of the network, but also by the context in which these social relations were embedded (Sandell and Stern The connection between respectability and the adoption of birth control practices within voluntary associations found in qualitative studies is corroborated by the results in this article (Ambjörnsson Structural changes, such as mortality decline or economic modernisation, disrupted the pre-transitional demographic regime, creating new circumstances for family formation. How individuals acted in response to this depended on how they could incorporate new reproductive practices into their lives. Social class has been shown to have an impact on the pace of the transition by creating fertility differentials (Dribe and Scalone The choice of data involved a trade-off which introduced some important limitations. It is not possible to draw conclusions on causal links or the direction of causality from aggregate-level voluntary association size. Therefore, the results could be an effect of reverse causation, and it is easy to assume that having fewer children would make it easier, especially for women, to join an association. Another limitation is the possibility of confounding factors not controlled for in the regression model causing both the size of voluntary associations and fertility decline. Although the literature is not in agreement, several studies link voluntary association growth to industrialisation Anderson , which iIn conclusion, the results of this study show that the use of social network theory in the study of fertility change helps us to include norms and values in the explanation for such change by shifting the focus from individual attributes to practices in the context of social relations. The theory helps us to shift the theoretical focus from the innovation of birth control to the meaning and perception of birth control and thereby to assert the importance of the context in which human action is embedded. However, because this study lacks individual-level information on voluntary association connections and instead relies on aggregate-level information, the causal link between these social relations and changing reproductive practices cannot be determined and needs to be studied further. The urban and rural differences also suggest that further research using spatial analysis could uncover even more distinct geographical patterns. The scope of this article has moreover been limited to the beginning of the fertility transition, and studies on the following decades might reveal a different pattern, especially considering the growth of the political organisations and trade unions during the first decades of the twentieth century.


The tenacious prevalence of human pancreatic diseases such as diabetes mellitus and adenocarcinoma has prompted huge research interest in better understanding of pancreatic organogenesis. The plethora of signaling pathways involved in pancreas development is activated in a highly coordinated manner to assure unmitigated development and morphogenesis in vertebrates. Therefore, a complex mesenchymal–epithelial signaling network has been implicated to play a pivotal role in organogenesis through its interactions with other germ layers, specifically the endoderm. The Fibroblast Growth Factor Receptor FGFR2-IIIb splicing isoform (FGFR2b) and its high affinity ligand Fibroblast Growth Factor 10 (FGF10) are expressed in the epithelium and mesenchyme, respectively, and therefore are well positioned to transmit mesenchymal to epithelial signaling. FGF10 is a typical paracrine FGF and chiefly mediates biological responses by activating FGFR2b with heparin/heparan sulfate (HS) as cofactor. A substantial number of studies using genetically engineered mouse models have demonstrated an essential role of FGF10 in the development of many organs and tissues including the pancreas. During mouse embryonic development, FGF10 signaling is crucial for epithelial cell proliferation, maintenance of progenitor cell fate and branching morphogenesis in the pancreas. FGF10 is also implicated in pancreatic cancer, and that overexpression of FGFR2b is associated with metastatic invasion. A thorough understanding of FGF10 signaling machinery and its crosstalk with other pathways in development and pathological states may provide novel opportunities for pancreatic cancer targeted therapy and regenerative medicine. The Fibroblast Growth Factor (FGF) family of peptides and the corresponding family of receptor tyrosine kinases (RTKs) collectively constitute one of the most adaptable, complex, and diverse growth factor signaling systems that are involved in many developmental and repair processes in virtually all vertebrate and invertebrate tissues and cells . CurrentFGFR family of RTKs comprises of FGFR1, FGFR2, FGFR3, and FGFR4. As the name suggests, FGFRs bind to members of secreted FGFs along with the sequential formation of complexes with heparin/heparan sulfate (HS) cofactor-proteoglycans to propagate downstream signal transduction pathways, which include activation of PLCγ, MAPK, AKT, and STAT cascades. At the cellular level, paracrine FGF-FGFR-HS signaling engages in vital roles in regulating cell proliferation, migration, survival, and differentiation during the development of the embryo .FGF10, a FGF7 subfamily member, is a typical paracrine FGF and chiefly mediates its biological responses by activating FGFR2b. FGF10 is a potent morphogen and plays a crucial role in transmitting mesenchyme signaling to the epithelium. Genetic ablation of FGF10 in mice results in gross developmental defects characterized by agenesis and dysgenesis in a variety of organs and tissues highlighting an essential role of FGF10 signaling for the development of multiple organs including the pancreas . AlthougAlternative splicing of the extracellular IgIII loop of FGFR1-3 generates IIIb- and IIIc-variants of the receptors. Tissue- and cell-specific expression of these isoforms and modification in binding properties for the FGF ligands confer signaling specificity and functional diversity in regulating interactions in embryonic development, tissue homeostasis, repair, and cancer . FGFR2 gFormation of the FGF10-FGFR2b-HS (2:2:2) ternary complex results in the phosphorylation of intracellular tyrosine residues in FGFRs Figure . Phosphoin vitro  and Pdx1/Fgf10 null neonates revealing pancreatic hypoplasia, as early as E14, further implying a role for both signaling pathways in pancreatic growth  PDX1  and PTF1a (Pancreas transcription factor 1). Ablation of either rmations .Pdx1 at the focal apex in order to maintain pancreatic identity  can be similarly defined by several TFs such as SOX9, HNF6, NKX2.2, HNF1β, HES1, CAP1, and NKX6.1. At this juncture, MPCs not only have the potential to self-renew, but also can differentiate to form exocrine and endocrine progenitors with PDX1 functioning as the central node .The SOX9 interacts with the FGF signaling pathway in concert with PDX1 to maintain both expansion (in a dosage-dependent manner) and organ identity of MPCs . SOX9 an2, as well as the NOTCH target gene HES1 + cells that form the early α-cells.NGN3 (ii)+ that will remain proliferative and available to differentiate to other endocrine cell types  is the most common exocrine malignancy and represents one of the deadliest diseases with high mortality due to difficulties in its early diagnosis, metastasis and intrinsic resistance to conventional chemoradiotherapy. At a molecular level, cancer cells in PDAC are often characterized by mutations in the KRAS oncogene, SMAD4, and TP53. Several FGFs and FGFRs are expressed in stromal cells scattered around pancreatic cancer cells and their expression levels have been linked to increased cancer motility, proliferation and metastatic invasion . FGF7 anβ1 genes , these gβ1 genes . Moreoveβ1 genes .G12D-mediated ADM and PanIN formation , pancreatic intraepithelial neoplasia (PanIN), and eventually PDAC. Significantly, SOX9 is imperative for KRASormation . A more ormation .Our recent studies further showed that SOX9 could be induced by NFATC1 and NFATC4 in response to EGFR activation and pancreatitis, which promote ADM and PanIN . In a seFgf9 and Fgf18 are essential for the development of mesenchymal components, numerous studies highlight FGF10 as an indispensable mesenchyme to epithelium signal required for the development of epithelial components in multiple organs. Despite the interesting observations from previous reports, research on FGF10/FGFR2b in the pancreas is lagging behind compared to some other organs such as the lung. There remain some critical questions unanswered regarding how FGF/FGFR2b signaling influence acinar and ductal specification , as well as its impact on the endocrine system remain largely unexplored. More elegant and specifically targeted genetic models allowing better spatiotemporal manipulation of gene expression will be essential to better address these questions. During both embryonic development and oncogenic process, FGF10 acquires the ability for unique crosstalk with other pathways as exemplified by its inter-dependent expression with SOX9, which may represent a key knot linking oncogenic KRAS, inflammation and other growth factor signaling. Understanding of FGF10 signaling machinery and its crosstalk with other pathways may provide novel opportunities for PDAC precision therapy and regenerative medicine.Animal models lacking each of the secreted FGFs have been developed with diverse phenotypes ranging from mild abnormality in adult physiology to early embryonic lethality. Only three FGFs  upon knockout result in early postnatal lethality due to severe developmental defects in multiple organs. While RN, L-CD, JW, X-KL, and J-SZ conceived the study. RN and J-SZ wrote the manuscript. RN, L-CD, JW and J-SZ designed and drew the figures. J-SZ designed and edited the manuscript. J-SZ and X-KL supervised the study and acquired funding.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.


The fundamental roles for the Salvador–Warts–Hippo (SWH) pathway are widely characterized in growth regulation and organ size control. However, the function of SWH pathway is less known in cell fate determination. Here we uncover a novel role of the SWH signaling pathway in determination of cell fate during neural precursor  development. Inactivation of the SWH pathway in SOP of the wing imaginal discs affects caspase-dependent bristle patterning in an apoptosis-independent process. Such nonapoptotic functions of caspases have been implicated in inflammation, proliferation, cellular remodeling, and cell fate determination. Our data indicate an effect on the Wingless (Wg)/Wnt pathway. Previously, caspases were proposed to cleave and activate a negative regulator of Wg/Wnt signaling, Shaggy (Sgg)/GSK3β. Surprisingly, we found that a noncleavable form of Sgg encoded from the endogenous locus after CRISPR-Cas9 modification supported almost normal bristle patterning, indicating that Sgg might not be the main target of the caspase-dependent nonapoptotic process. Collectively, our results outline a new function of SWH signaling that crosstalks to caspase-dependent nonapoptotic signaling and Wg/Wnt signaling in neural precursor development, which might be implicated in neuronal pathogenesis. It has also been found to play important roles in cancer metastasis5. Originally identified as a prominent regulator of organ size in Drosophila, this pathway is highly conserved from fly to mammals. The regulation of SWH pathway depends on its response to various upstream stimuli through intercellular junctions, including adhesion cues through cell–cell contact, polarity, extracellular signal, mechanical signals, and cellular stress. Core components of the SWH pathway comprise a kinase cascade, which is the main regulation modulating SWH signaling. The Ste20 family kinase Hpo  forms a heterodimer with the adapter protein Sav , thereby promoting their interaction with the serine/threonine kinase Wts . Hpo subsequently activates Wts activity via phosphorylation7. The activated Wts kinase operates with its cofactor Mats  to phosphorylate Yorkie , which is a transcriptional coactivator and serves as the final effector of the Hippo signaling. Yki/YAP/TAZ lacks DNA-binding motif but binds to the promoters of target genes through interacting with Scalloped  or other transcription factors6. By activating target gene expression, Yki/YAP/TAZ plays important roles in controlling cell growth, proliferation, and survival. Wts kinase inhibits transcriptional activity of Yki/YAP/TAZ through nuclear export, cytoplasmic retention, and protein degradation10. Phosphorylation-independent regulations also exist. Yki can directly bind to Hpo, Wts and the FERM-domain containing adapter protein expanded (Ex), and sequesters Yki in the cytoplasm12. In addition to intrinsically regulates Yki activity, Ex, as an apical junctions-localized protein, also transduces signaling cues through binding to the apical membrane protein Crumbs 16. In addition, ex is a downstream target gene of Yki, thereby forming a feedback regulatory loop of Hippo pathway17. It has been demonstrated that activation of the SWH pathway through elevating expanded (ex) levels is required to eliminate the inappropriately differentiating neurons during development19. However, whether SWH pathway has any roles in normal neurogenesis remained unclear.The Salvador–Warts–Hippo (SWH) pathway has been recognized as a significant regulator for growth control, tissue regeneration, and stem cell pluripotencyex mutants often differentiate supernumerary sensory bristles18. Bristles are a component of the Drosophila peripheral nervous system and can be divided into macro- (large bristles) and microchaetae  according to their size and position. Drosophila notum is a classical model to study pattern formation because each macrochaetae develop in precise positions and microchaetae appears in a characteristic density pattern20. Each of these external sensory organs comprises five cells  that are generated through asymmetric cell divisions of single sensory organ precursor (SOP) cell22. The accuracy of bristle patterns on the adult body depends on the correct SOP cell positioning. The phenotype of ex mutations promoted us to study in depth how ex mediates sensory organ development.Intriguingly, hypomorphic 23. By studying how ex takes part in SOP development, we discovered a crosstalk between SWH pathway and caspase-dependent nonapoptotic signaling mediated through Wg pathway. Interestingly and unexpectedly, we found Sgg might not be the main target of the caspase-dependent nonapoptotic event.Caspase activation has been implicated in SOP development through a caspase-dependent nonapoptotic machinery. This caspase-dependent machinery is thought to be required for cleavage and activation of a negative regulator of Wingless (Wg)/Wnt signaling, Shaggy (Sgg)/GSK3β, in SOP cell formationex1, exe1, ex69724; Diap125; Diap1426; dsh327; arm2, arm328 are loss-of-function or null alleles. Other transgenes used in this study include UAS-ex-RNAi (BDSC BL#28703); UAS-yki, UAS-ykiS168A10; UAS-dTCF[DN] (BDSC BL#4784); UAS-dTCF-RNAi (BDSC BL#26743); dpp40C6-Gal429; ExIntron3-GFP18.30. Confocal imaging was performed using Leica SP2, SP8 and Zeiss LSM 880 microscopy. Primary antibodies used were anti-Sens ; GFP . Photograph of adult notum was carried out using Leica MZFLIII microscope and Nikon SMZ1500 microscope.Preparation of wing discs for immunostaining and adult notum for light microscope was performed as described previouslysgg-RD/RP/RQ isoforms were conducted in w1118 flies. Two single guide RNAs (sgRNAs) were used to introduce double strand breaks near by the edited genomic region and followed by homology-directed repair (HDR). The HDR donor plasmid was designed to harbor a DNA cassette containing the upstream homology arm of sgg, 3XP3-ScarlessDsRed flanking with PiggyBac terminal repeats, and the downstream homology arm of sgg with D235G/D300G mutations, which was constructed into the pUC57-Kan vector. The sgRNA and HDR donor plasmids used for microinjection were purified using the Plasmid Midi-prep kit (Qiagene). After validation of the CRISPR-knockin sgg alleles by genomic PCR coupled with Sanger sequencing, the ScarlessDsRed selection marker was then excised by PiggyBac transposon. The genomic PCR coupled with Sanger sequencing was performed to confirm the precise excision of ScarlessDsRed.By using CRISPR/Cas9-mediated genome editing, mutagenesis of the corresponding genomic sequences in both 235th and 300th Asp residues of ex function in Drosophila by using transheterozygous ex mutants caused the appearance of ectopic macrochaete on the notum . These data suggest that the SWH pathway is involved in ex-dependent bristle inhibition through modulating Diap1 activity. SWH activity was monitored by using ex intron 3 enhancer (referred to ExIntron-GFP) reporter19, and also fj-lacZ, both of which report Yki activity. It was difficult to see overall changes in the notum region, although both reporters were present in the ectopic aSC SOP cell .To determine the involvement of tum Fig. . These dachaete and scute, which is controlled by multiple cis-regulatory elements distributed throughout the achaete and scute transcription units to permit the precision of SOP cell specification37. Among the critical regulators in modulating proneural gene expression, previous studies have identified Wg/Wnt signaling in particular as affected by nonapoptotic caspase activity during thoracic bristle patterning23. Sgg/GSK3β is a negative regulator of Wg/Wnt signal transduction. ex and sgg mutations shared similar phenotypes in bristle formation . Independent evidence also points out that Wg signaling is altered in the absence of ex in eye discs39. Genetic analyses were performed to verify the involvement of Wg signaling cascade in ex-mediated SOP specification. Indeed, the extra bristle phenotype caused by downregulating ex was suppressed by removing one copy of Wg signaling components  expression was not changed when ex was mutated  protein42. Hence, we hypothesize that the caspase inhibitor Diap1 might be the critical effector connecting SWH pathway with Wg pathway through modulating kinase activity of Sgg to determine the correct number of SOP cells. To probe the involvement of Sgg in this regulation in depth, we generated an in vivo noncleavable form of Sgg (sggD235G/D300G) using CRISPR-Cas9 technique .A caspase-dependent cleavage resulting from caspase activation mediates nonapoptotic signaling in determining SOP cells. This caspase-dependent cleavage is thought to activate Sgg/GSK3β during SOP cell formationDNA Fig. . If casples Fig. , a much les Fig. . PenetraDiap1, which restrains caspase activity. Although this can regulate cell survival, here we report that SWH and Yki play a role in normal development in suppressing nonapoptotic caspase activity. In the Drosophila thorax, nonapoptotic caspase activity is needed to suppress activity of Wg signaling and restrain SOP cell specification23. This is here shown to depend on the SWH pathway, without which Diap1 expression is too high to permit normal patterning. While this paper was under review, another study reported that nonapoptotic caspase activity is also regulated by the SWH pathway, during tracheal development43.Unlike the well-known concept that the key roles for SWH pathway are in the regulation of cell proliferation and organ size, the present study reveals a novel function of SWH signaling in cell fate determination through nonapoptotic caspase signaling. As is well-known, deregulation of SWH pathway leads to the activation of the Yki target gene, Drosophila IKK-related kinase (DmIKKε)-dependent phosphorylation. In consistent with phenotype of high levels of Diap1, downregulation of DmIKKε led to extra macrochaetae formation44. Hence, the levels of Diap1 play determinant role in cell fate specification. Here we provide evidence that transcriptional regulation of Diap1 by Yki and the SWH pathway is also important for SOP cell determination, which is disrupted by hypomorphic mutations of ex, which affect the level of Yki activity.During SOP specification, caspase activity is transiently controlled by the turnover of Diap1. Diap1 degradation is triggered by the Drosophila GSK3β ortholog, Sgg, was identified as a potential substrate for caspase-dependent cleavage. One isoform, Sgg46, is inactive but can be cleaved into the active isoform Sgg10, which negatively regulates Wg signaling through the phosphorylation and degradation of Arm23, and also directly phosphorylate Scute and its activator Pannier in SOP cell specification45. Caspases potentially have hundreds of substrates, but Sgg46 was believed to be significant for Wg signaling and SOP patterning because overexpression of a form with mutated caspase sites, SggD235G/D300G, phenocopied blockade of nonapoptotic caspases by p35 overexpression23. In fact, SggD235G/D300G overexpression was quantitatively less effective than completely blocking caspases with p35, but this was attributed to the simultaneous presence of wild-type Sgg46 encoded by the endogenous locus23. It was presumed that the overexpressed, SggD235G/D300G protein behaved as a competitive inhibitor of Sgg46 cleavage. If this model was correct, we would expect that modifying the endogenous sgg locus to encode SggD235G/D300G (which would not affect other, shorter isoforms of Sgg, Fig. sggD235G/D300G mutant. This is not consistent with the model that Sgg46 is the main target of nonapoptotic caspase signaling in bristle patterning. Although we cannot exclude that Sgg46 might be activated by cleavage at another site, this would not explain why Sgg46 could not be activated when SggD235G/D300G protein was overexpressed. Therefore, we conclude that nonapoptotic caspases regulate one or more other substrates that are critical for Wg signaling and SOP patterning, and that SggD235G/D300G overexpression is a competitive inhibitor of cleavage of these other substrates.The 46. Aside from our finding that the caspase cleavage sites of Sgg46 are largely dispensable for bristle patterning, the model that Sgg46 is a Dronc target also does not fit with the observation that bristle patterning is disrupted by p35 overexpression23, since p35 does not inhibit Dronc. The ectopic p35 phenotype strongly suggests that the major regulators of bristle patterning are substrates of p35-dependent effector caspases, not direct Dronc targets. These data could explain how SggD235G/D300G overexpression is dominant negative, however, if SggD235G/D300G inhibits Dronc and Crinkled function, leading to deficient nonapoptotic signaling by downstream effector caspases.A recent study has reported that the unconventional myosin Crinkled acts as an adapter to facilitate Sgg46 cleavage and activation by DroncDiap1 transcription. In the Drosophila thorax this is required to restrain Wg signaling and bristle patterning (Fig. wg was a transcriptional target of Yki but it is now thought this reflects enhanced wg autoregulation when Wg signaling is elevated47. Crosstalk between SWH and Wnt signaling appears to be conserved, having also been reported in vertebrates54. The mechanisms that have been suggested in vertebrates are not yet known to involve nonapoptotic caspase signaling, however.Our main conclusion is that, in addition to its previously known roles, the SWH pathway is important for regulating nonapoptotic caspase signaling, presumably through Yki control of ing Fig. . It is pDrosophila and in mammals, SWH signaling and nonapoptotic caspase signaling both are implicated in neuronal morphogenesis. SWH signaling affects synapse development and dendrite morphogenesis59, while nonapoptotic roles of caspases remodel Drosophila dendritic arborization neurons and regulate axon degeneration in mammals. Defects of caspase-dependent nonapoptotic signaling affect plasticity and result in disease such as Alzheimer’s disease61. Wnt signaling is also involved in neuronal development and has been associated with neurological diseases including Alzheimer’s disease, Parkinson’s disease, schizophrenia, and autism66. Crosstalk between SWH signaling, Wnt signaling and caspase-dependent nonapoptotic signaling may contribute to the molecular mechanisms of neuronal pathogenesis. This crosstalk may occur in multiple processes during development, in light of the finding that DrICE has a nonapoptotic function, which acts downstream of the SWH signaling to regulate endocytic trafficking during tracheal morphogenesis43.It is striking that in both Extra scutellar bristle is not caused by growth defect in the depletion of exInactivation of caspase or Hippo pathway is involved in extra bristle formationExpression of Notch target is not changed in ex mutantSgg is not the only target of non-apoptotic caspase activity


Human T cell leukemia virus (HTLV-1) is an oncoretrovirus that infects at least 10 million people worldwide. HTLV-1 exhibits a remarkable genetic stability, however, viral strains have been classified in several genotypes and subgroups, which often mirror the geographic origin of the viral strain. The Cosmopolitan genotype HTLV-1a, can be subdivided into geographically related subgroups, e.g. Transcontinental (a-TC), Japanese (a-Jpn), West-African (a-WA), North-African (a-NA), and Senegalese (a-Sen). Within each subgroup, the genetic diversity is low. Genotype HTLV-1b is found in Central Africa; it is the major genotype in Gabon, Cameroon and Democratic Republic of Congo. While strains from the HTLV-1d genotype represent only a few percent of the strains present in Central African countries, genotypes -e, -f, and -g have been only reported sporadically in particular in Cameroon Gabon, and Central African Republic. HTLV-1c genotype, which is found exclusively in Australo-Melanesia, is the most divergent genotype. This reflects an ancient speciation, with a long period of isolation of the infected populations in the different islands of this region . Until now, no viral genotype or subgroup is associated with a specific HTLV-1-associated disease. HTLV-1 originates from a simian reservoir (STLV-1); it derives from interspecies zoonotic transmission from non-human primates to humans (ancient or recent). In this review, we describe the genetic diversity of HTLV-1, and analyze the molecular mechanisms that are at play in HTLV-1 evolution. Similar to other retroviruses, HTLV-1 evolves either through accumulation of point mutations or recombination. Molecular studies point to a fairly low evolution rate of HTLV-1 (between 5.6E−7 and 1.5E−6 substitutions/site/year), supposedly because the virus persists within the host via clonal expansion (instead of new infectious cycles that use reverse transcriptase). Retroviridae family, the Orthoretrovirinae subfamily and the Deltaretrovirus genus, which includes bovine leukemia virus (BLV) and T-lymphotropic viruses infecting primates (PTLV). PTLVs consist of simian T-lymphotropic viruses (STLVs) type 1 to 4, which infect non-human primates and human T-lymphotropic viruses type 1–4.The human T-cell lymphotropic virus (or T-cell leukemia virus) type 1 (HTLV-1), discovered in 1980, has been identified as the first human oncoretrovirus , 4140, 4The Senegalese subgroup represents, by definition, the major subgroup present in Senegal Fig.  25, 26], 2625, 2The North African subgroup is mainly present in Algeria, Morocco, Mauritania, Western Sahara, and Mali Fig.  21, 41], 4121, 4The initial classification comprised the Transcontinental A subgroup, the Japanese B subgroup, the West-African C subgroup, and the North African D subgroup; they are now referred as a-TC, a-Jpn, a-WA, and a-NA, respectively Table  19–21].–21.19–21The Central African b-genotype is most frequently found in Central Africa, i.e. Cameroon, Gabon, CAR, DRC, and Nigeria Fig. . It reprThe Australo-Melanesian c-genotype is the most divergent: the genetic nucleotide variability can reach 6–9% when compared to the reference ATK prototype. This reflects an ancient speciation, with a long period of isolation of infected populations living in the different islands of this Pacific region. HTLV-1c was first described in a small group of hunter-horticulturalists living in the fringe highlands of Papua New Guinea (PNG) –49 and aOther genotypes d, e, f and g have been reported in Central Africa, mainly in Cameroon, Gabon, DRC and CAR , 54, 55 hbz ORF. Valeri et al. [hbz. This virus could persist in the rabbit model, but could not persist in Macaques. Thus, the importance of accessory proteins may depend on the host species. Interestingly, some replication may still occur in the latter model as some revertants could appear. Some variability of these accessory proteins has been reported, particularly with regard to p12. Truncated forms of p12 have been described in Japan and South America [HTLV-1 is a complex retrovirus: in addition to structural and regulatory proteins, it encodes several accessory proteins . In the HTLV-1a genome, two open reading frames (ORFs) encode four accessory proteins: p12 is encoded by ORFI and can be cleaved into p8; p13 and p30 are encoded by ORFII and are obtained by alternative splicing . These pi et al.  generate America –63. More America . HoweverThe importance of accessory proteins in vivo has been regularly questioned. A HTLV-1a strain deleted for p12 has been described in 3 siblings, suggesting that this virus was transmitted by their mother and is therefore capable of transmission, replication, and persistence in vivo . SimilarThe absence of accessory ORFs, as suggested in the in silico analysis, may indicate that: (1) the encoded proteins are not essential for viral replication in vivo, (2) there are compensatory mutations in the HTLV-1b and HTLV-1c genomes that turn accessory proteins optional, or (3) there are alternative accessory proteins for these viral subtypes. The latter hypothesis is the most likely. Indeed, although the start codon is absent from the ORFII, the ORF contains no additional stop codon. This may suggest a selective pressure to keep the ORF open. The Franchini’s laboratory recently suggested that alternative splicing could lead to the synthesis of p16, an alternative protein to p12 . In conclusion, the genetic organization and accessory genes may be different between viral genotypes.Many non-human primates (NHPs) are endemic for STLV-1, the simian counterparts of HTLV-1: STLV-1 can be found in chimpanzees, gorillas, mandrills, baboons, several species of African monkey, a wide range of macaques, and orangutans –74. ClonInterspecies transmission can occur, and is currently ongoing in Central Africa. STLV-1 can be transmitted to humans through infected body fluids, such as saliva and blood. Epidemiological studies have recently found that a severe bite by an ape or a monkey is a major risk factor for HTLV-1 infection in NHP hunters  in West Central Africa , 80.C. nictitans in Central Africa [It is thus believed that the different HTLV-1 genotypes have originated from ancient interspecies transmission of STLV-1. It is supported by the fact that STLV-1 infecting chimpanzees and gorillas in South Cameroon cannot be distinguished from HTLV-1b strains –82. Simil Africa , 70, 73,l Africa , 83.However, the case is different for HTLV-1a and -1c. There is no known STLV-1 closely related to these two human genotypes. For HTLV-1a, it can be assumed that either the simian reservoir has not been described yet, or the simian ancestors may have disappeared since the virus was transmitted to humans. For HTLV-1c, the case is even more complex. Indeed, monkeys have never been present in the Australo-Melanesian region. As a result, interspecies transmission of STLV-1 to humans could not occur on these islands. Therefore, it is proposed that HTLV-1c was acquired by proto-Australo-Melanesians during their migration through Southeast Asia, and that populations that reached the highlands of Papua New Guinea were already HTLV-1 infected. The infected populations would then have disseminated, together with their virus, throughout the Australo-Melanesian region , 84–87.macaca artoides could constitute a novel genotype, called STLV-5 [In Asia, STLV-1 is found in many species of macaques , 74. Macd STLV-5 . Intrigud STLV-5 , 90. Thed STLV-5 .Both recombination and point mutations contribute to the genetic variation of retroviruses. However, until recently, recombination was disregarded when considering HTLV-1 evolution. Indeed, no recombination event had been identified for HTLV-1. The absence of recombination was supported by the fact that no superinfection at the cellular level had been described . Recentlenv, that 16 of the 19 samples displayed genetic variants. Many mutations could be linked to cytidine deaminase activity. Apart from the G>A transition, 7 samples (out of 19) were composed of multiple strains, suggesting the presence of HTLV-1 quasi-species (or multiple infection).Some intra-individual viral genetic diversity has been reported. Ehrlich et al.  found, wenv segment). Of note, the authors had followed only 3 individuals for 6 to 20 months, which explain why no mutation emerged. However, by studying the viral genetic diversity within (and between) infected cellular clones, Mortreux et al. [The origin of such diversity is often attributed to the RT. Indeed, the mutation rate of HTLV-1 RT is estimated at 7E−6 mutation/site/replication cycle , which ix et al.  suggesteIn a nutshell, the origin of intra-individual genetic diversity is mostly related to genetic instability and mutations that occur during proliferation of infected cells.There are two different methods for estimating the evolution rate of HTLV-1. Such an estimate only takes into account single point mutations, and recombinant strains should be excluded.env (522 bp-long) within 16 vertical transmission chains [env, respectively. In a similar study in Brazil, the estimation was found surprisingly high (2E−5 substitutions/site/year for LTR), supposedly because it was calculated on the basis of 1 mutation on a single mother–child pair [On the one hand, the mutation rate can be estimated by studying vertical/intrafamilial transmission chains of the virus. In this context, remarkable genetic stability was observed: first, a study in the DRC (ex-Zaïre) revealed that 10 related individuals carried the same virus, without mutation (in a 755-nt segment of the LTR), although one member was also co-infected with a second strain that differed in one nucleotide . This lan chains . As a reild pair . This vaild pair . This megag, pol, env) were informative for studying all PTLVs (PTLV-1–2 and 3). Similarly, when considering PTLV-1 only, the LTR sequences are also informative [On the other hand, the mutation rate can be estimated using phylogeny and an anthropological approach, using a dating anchor point for a given clade. Such analyses are based upon several assumptions: (1) the data set is informative, i.e. the genetic variability is not too high and the phylogenetic signal is not saturated. Salemi et al.  found thormative . (2) Theormative –102. Howormative  found thormative  found saThe calibration values for the molecular clock can be major points of debate, and are based on strong assumptions.The most commonly used date to estimate the time scale for PTLV evolution is the divergence date between HTLV-1c and PTLV1a/b, which is estimated between 40,000 and 60,000 years ago , 100–102Another possible date is the divergence between HTLV-2a and -2c (in studies combining the two types of viruses). Indeed, these two clades are composed exclusively of strains present in Amerindian populations. It was therefore proposed that they share a common ancestor who reached the Americas at the time of human migration on the Bering Strait. Thus, the HTLV-2a/c node is dated at 25,000 ± 5,000 years ago , 104.In conclusion, depending on the different models and assumptions, the estimated mutation rates vary from 5.6E−7  to 1.5E−Several European countries  regularly report cases of HTLV-1 infection (among blood donors or pregnant women) or HTLV-1 associated diseases –107. In HTLV-1 has been found in many native populations in the Americas, such as the Inuit in Canada and the USA, the Quetchua in Peru, the Mapuche in Chile, and indigenous groups from Argentina –115. MosThe modes of dissemination of HTLV-1 in the Middle East and Asia remain to be clarified. Regions of the Middle East (e.g. areas of Iran and Kuwait) have been found endemic for HTLV-1 , 122. A The importance of human migrations in the modern area will likely modify the distribution of HTLV-1 and lead to a mixing of genotypes and subtypes. Indeed, the Tokyo metropolitan area may become a hotspot of endemicity for HTLV-1 as individuals migrate from endemic areas such as the Kyushu-Okinawa region . In someThere is no clear evidence of specific mutations in the HTLV-1 genome that would render the virus more pathogenic , 129. HoDespite a good understanding of the genetic diversity and evolution mechanisms of HTLV-1, many questions remain concerning the origins of some groups infected with HTLV-1, and the pathogenicity of each genotype.


EOS plays an important role in maintaining the suppressive function of regulatory T cells (Tregs), and induces a regulated transformation of Tregs into T helper-like cells, which are capable of secreting proinflammatory cytokines in response to specific inflammatory signals. Meanwhile, significant reduction in Treg activity along with production of proinflammatory cytokines has been reported in patients with HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP).In this study, to examine whether there is an alteration in EOS expression in peripheral blood mononuclear cells (PBMCs) derived from HTLV-1-infected individuals especially HAM/TSP, we investigated the expression of HTLV-1 tax genotype, proviral load (PVL), and the mRNA expression of tax, HBZ and EOS in HTLV-1 infected individuals including adult T-cell leukemia/lymphoma (ATL), HAM/TSP, or asymptomatic carriers. The expression levels of EOS mRNA and protein in various HTLV-1-infected or uninfected human T-cell lines were also investigated.EOS was highly expressed at the protein level in most HTLV-1 infected T-cell lines, and was augmented after the HTLV-1 regulatory factor Tax was induced in a Tax-inducible JPX-9 cell line. Immunoprecipitation experiments demonstrated a physical interaction between EOS and the viral regulatory protein Tax, but not HBZ. Meanwhile, there was a significant decrease in EOS mRNA levels in PBMCs of HTLV-1 infected individuals irrespective of their clinical statuses. We found an inverse correlation between EOS mRNA levels and HTLV-1 PVL in ATL patients, and positive correlations between both EOS mRNA load and PVL, and EOS and HBZ mRNA load in HAM/TSP patients, whereas this correlation was not observed in other clinical statuses.These findings suggest that both Tax and HBZ can alter the expression of EOS through undetermined mechanisms, and dysregulated expression of EOS in PBMCs of HTLV-1 infected individuals may contribute to the pathological progression of HTLV-1-associated diseases, such as ATL and HAM/TSP. Human T-cell leukemia virus type 1 (HTLV-1) was the first human retrovirus identified as a causative agent of human diseases, including adult T-cell leukemia/lymphoma (ATL)  and HTLVEOS, which is encoded by the Ikzf4 gene, is a transcription factor that belongs to the Ikaros family and is primarily expressed in Tregs . It playThe results showed that there was a significant decrease in EOS mRNA levels in PBMCs of HTLV-1 infected individuals, irrespective of their clinical statuses. Furthermore, we found an inverse correlation between EOS mRNA levels and PVL in ATL, and positive correlations between both EOS mRNA load and PVL, and EOS and HBZ mRNA load in HAM/TSP, suggesting a possible role for EOS in the pathogenesis of HTLV-1-associated diseases.tax genotype, PVL, and mRNA expression of tax, HBZ and EOS in HTLV-1 infected individuals from Okinawa, which is located in the subtropical southern-most point and comprised of remote islands off the mainland of Japan, and is highly endemic for HTLV-1  × 104. All samples were examined in triplicate.To examine the HTLV-1 PVL, quantitative PCR (qPCR) using primers and probes for the most conserved HTLV-1 reported . The HTLreported . In thisTo estimate tax and HBZ mRNA expression levels, a real-time quantitative reverse transcription PCR  was performed as previously described . Human Ip values < 0.05.To test for significant differences among four different groups of subjects , the data were statistically analyzed using one-way analysis of variance (ANOVA). Inter-group comparisons were done by Scheffe’s post hoc multiple comparisons test. The Mann-Whitney U test was used to compare the data between two groups. Correlations between variables were examined using Spearman rank correlation analyses. The results shown represent the mean ± SD where applicable. Results were considered statistically significant with First, we evaluated EOS protein levels in HTLV-1-infected and -uninfected human T-cell lines. As shown in Fig. 2 to the culture medium . Western blot analysis showed that treatment of JPX-9 cells with CdCl2 induced Tax expression, and EOS expression was augmented approximately 48 h after Tax induction, suggesting that Tax is involved in the upregulation of EOS in a human leukemic T-cell line  by co-immunoprecipitation. As shown in Fig. n = 21; lymphoma-type, n = 6; smoldering-type, n = 5; chronic-type, n = 3), 17 patients with HAM/TSP, 26 ACs, and 14 NCs. The results showed that the expression level of EOS mRNA in HTLV-1 infected individuals was significantly lower than in NCs, irrespective of clinical statuses    transcription factors have a crucial role not only in immune cell development and homeostasis, but also pathological conditions in human , 44. It Our data indicated that most HTLV-1 infected T-cell lines  exhibited high expression of the EOS protein. In HTLV-1 infection, it is well established that both Tax and HBZ play a key role in the transactivation of viral and cellular genes for HTLV-1 replication and pathogenesis . To dateIn PBMCs of HTLV-1 infected individuals, EOS mRNA levels was considered low when compared with NCs, regardless of clinical statuses, i.e., ATL, HAM/TSP, or ACs. However, since ex vivo data obtained using patient samples may provide more valuable insight into the clinical significance of EOS with respect to the risk of HTLV-1 associated diseases, we assume that this is the most fascinating and important finding of the present study. In ATL patients, there was an inverse correlation between EOS mRNA levels and HTLV-1 PVL, suggesting lower levels of EOS mRNA expression in ATL cells through an as yet undetermined mechanism. ATL is divided into four clinical subtypes, i.e., acute, lymphoma, chronic, and smoldering , and patIn contrast to ATL patients, we found a positive correlation between EOS mRNA load and PVL in HAM/TSP patients. Moreover, consistent with our previous study , we alsoIt is well established that HTLV-1 infection induces an abnormal frequency and phenotype of Foxp3 + CD4+ T cells in infected individuals . In partMeanwhile, downregulation of EOS impaired and reduced Foxp3-mediated gene repression, thereby leading to Treg dysfunction and autoimmunity. EOS-deficient (EOS−/−) mice developed more severe experimental autoimmune encephalomyelitis, which is the most commonly used experimental model for the human inflammatory demyelinating disease called multiple sclerosis, which is pathologically similar to HAM/TSP and presents with an increased abundance of effector T cells in the periphery and central nervous system , FurtherIn conclusion, our study identified dysregulated expression of EOS in HTLV-1-infected individuals. Although further studies are needed to define the in vivo significance of EOS expression and its participation in disease processes during HTLV-1 infection, dysregulated expression of EOS may be the key to understanding the pathogenesis of HTLV-1 associated diseases.Additional file 1: Table S1. Characteristics of HTLV-1-infected and -uninfected study participants. Table S2. Primer sequences for plasmid construction.