Background: The human T-cell lymphotropic virus type-I (HTLV-I) is the first identified pathogenic human retrovirus. Breastfeeding has been reported to be the predominant route of vertical transmission of HTLV-I. The objective of this systematic review was to pool and evaluate the data on the transmission of HTLV-I with different infant-feeding practices on children born to HTLV-I-positive mothers. We conducted a systematic review of comparison of HTLV-I transmission risk to breastfed and bottle-fed babies.Methods: We searched the following databases: MEDLINE, SID, Magiran, and Cochrane Library. The search strategy was limited to articles in English. Initial screening identified 254 citations; of these, 96 potentially relevant articles were identified. After reviewing the 96 full-text articles in detail, 7 reports met the inclusion criteria for this review.Results: Pooled odds ratio (OR) and risk difference (RD) of HTLV-I transmission in the breastfed group compared to the bottle-fed infants were  and . So, we have evidence to support that exclusive breast feeding more than 6 months in comparison to bottle feeding highly increases transmission rate of HTLV-I infection. We have also enough evidence to support that exclusive breast feeding up to 6 months compared to bottle feeding does not increase transmission rate of HTLV-I infection .Conclusion: The current meta-analysis showed that short period (less than 6 months) of breastfeeding did not increase risk of HTLV-I infection transmission from mother to child among breastfeeders and more than 6 months of breastfeeding significantly increased the risk of HTLV-I infection. However, our meta-analysis shows that refraining from breastfeeding can decrease the risk of vertical HTLV-I transmission. Mother-to-child transmission is the first route, most likely due to ingestion of breast milk, breastfeeding has been reported to be the main route.6The first experimental evidence to support milk-borne infection of HTLV-I was the demonstration of infected cells in breast milk of carrier mothers. HTLV-I antigen was detected in milk from HTLV-I-seropositive mothers.7Infected cells transferred via breast milk may be abortive for infection in the presence of enough maternal antibodies. The addition of anti-HTLV-I-containing serum to cultures of infected and normal cells inhibited HTLV-I infection.The profits of breastfeeding, like immunity against non-HTLV-I-related infectious agents, have to be balanced against the risk of HTLV-I transmission and the illness related with other feeding practices.The optimal selection of feeding modality might be different for any individual mother and her infant by health providers about HTLV-I, and the most appropriate infant feeding choice is not determined.This systematic review assessed the accessible evidence and data to answer this important question: whether the protective profits are associated with breastfeeding or not.A conceptual framework was developed to recognize transmission pathways based on route of feeding in children born to HTLV-I-positive mothers. The search terms [free text not Medical Subject Headings (MeSH) terms] were “Breastfeeding OR breast milk OR bottle feeding OR formula AND HTLV-I”. We searched the following databases: MEDLINE, SID, Magiran, and Cochrane Library. The search strategy was limited to articles in English. No date limitation was obligatory on the search and meeting abstracts were also not excluded from our search. All studies which compared the seropositivity of breastfed and bottle-fed infants born to the HTLV-I-positive mothers were included. Studies without bottle-fed control group were excluded. Reference list of the included studies were searched for any additional missed citation. Data were abstracted for the subsequent factorsStudy characteristics: publication year, study design, follow-up duration, sample size, setting, country of study conduct, type of feeding of infants (breastfed or bottle-fed), and duration of breastfeeding.Outcome results at the three-month intervals till the longest duration of follow-up in each study (for example HTLV-I seropositivity of children by antigen or antibody detection method) was also extracted.A standardized data abstraction tool was developed in excels and calibrated with the team. The following data were extracted from the studies on to standardized data collection forms by the first author: characteristics of participants, study design, and study outcomes. Any disagreement was resolved by author’s discussion. The methodological quality of the included studies was appraised using the Newcastle-Ottawa Scale (NOS). Odds ratio (OR) and risk difference (RD) of seropositivity for HTLV-I in the breastfed infants as compared to the bottle-fed infants were used as the major effect size for meta-analysis. 2 index were used for heterogeneity evaluation. Sub-group analysis was used for evaluating the effect of breastfeeding and follow-up duration on the HTLV-I seropositivity. Random effects model was used for pooling effect size across studies. Cochran’s Q value  and IFunnel plots, Egger’s regression intercept, and Duval-Tweedie’s trim and fill method were used for publication bias evaluation. The Funnel plot is the plot of the effect size on the x-axis and the standard errors (SEs) of the included publications on the y-axis, and asymmetry of this plot can be due to publication bias. Egger’s regression intercept is the mathematical complement of this visual assessment plot. Statistically significant results of this test indicate an outsized asymmetry in the funnel plot. All statistical analyses were done by Comprehensive Meta-Analysis (CMA) software (version 2).Study selection: Initial screening identified 254 citations and 1 additional reference was found by screening reference lists; of these, 96 potentially relevant articles were identified. After reviewing the 96 full-text articles in detail, 7 reports  met the inclusion criteria for this review , and , respectively. Subgroup analyses according to duration of breastfeeding (< 6 months vs. > 6 months) showed pooled ORs of 0.91 (95% CI: 0.45-1.80) and 3.83 (95% CI: 1.80-8.10) and pooled RDs of -0.3% (95% CI: -3.8%-3.3%) and 11.9% (95% CI: 6.3%-17.5%), respectively. Subgroup analyses according to duration of follow up (12 months vs. 24 months) showed pooled ORs of 2.15 (95% CI: 0.15-3.00) and 15.34 (95% CI: 0.31-752.00) and pooled RDs of 15.1% (95% CI: -22.9%-53.2%) and 42.5% (95%CI: -19.4%-100%), respectively.2,7 It has been assumed that maternal HTLV-I antibody may protect children from HTLV-I infection through breast milk during the first 6 months of life.7 This inhibitory effect of the antibody has also been showed in an animal model.16 Based on our findings, shorter period of breastfeeding did not show increased risk of vertical HTLV-I transmission. The review found that there was enough evidence to support that exclusive breastfeeding up to 6 months compared to bottle feeding did not increase transmission rate of HTLV-I infection in children born to seropositive mothers. This is probably because of gradually declining level of maternal antibody (anti-HTLV-I-IgG) in babies after birth till 6-9 months of age.There was sufficient evidence to support that exclusive breastfeeding more than 6 months markedly increased the transmission rate of HTLV-I infection in comparison to bottle feeding group. Another important factor in seroconversion after each feeding method is duration of follow up. Subgroup analysis showed that serologic status between 12 and 24 months of follow up is different and it may affect the results of our pooled studies; moreover, it has great importance in interpreting the results of our study. 17 carrying a high viral load of HTLV-I-positive cells in breast milk,18-21 and expressing anti-pX40 seropositivity,22 breastfeeding that extends after the disappearance of the maternal antibody seems to be an important risk factor for mother-to-child transmission of HTLV-I. This view is concordant with the protective role of the maternal antibody to human immunodeficiency virus 1 (HIV-1) within the first 6 months in children born to HIV-seropositive mothers.23,24 Another approach to decrease the risk of vertical transmission of HTLV-I is freeze-thawing of breast milk from HTLV-I-seropositive mothers. Infectivity of HTLV-I in breast milk has been lost during freezing and thawing processes. This is especially important in the countries with limited resources.25Although many risk factors for mother-to-child transmission of HTLV-I have been mentioned, like the mothers bearing a high titer of anti-HTLV-I antibody, These findings suggest that length of breastfeeding should be relatively short in order to decrease the risk of HTLV-I infection among children for whom breastfeeding is necessary, especially in many developing countries all around the world. 4,8Other than duration of breastfeeding, mother-to-child transmission of HTLV-I is also related with older age of mother, maternal antigenemia, and high HTLV-I antibody titer level in particular to a specific immunogenic portion of gp46 envelope glycoprotein.Our study had some limitations. The main limitation of the systematic review was that the patient populations, follow up, and study design was not the same in all studies. We included meeting abstracts of studies in the meta-analysis, which can reduce publication bias despite this consideration, important limitation of our study is that publication bias, if present, greatly affects the results of this meta-analysis.However, the wider applicability of these findings is unclear, in particular to areas with non-Japanese population.The current meta-analysis showed that short period (less than 6 months) of the breastfeeding did not increase chance of HTLV-I infection transmission from mother to child among breastfeeders and more than 6 months of breastfeeding highly increased transmission rate of HTLV-I infection. This study highlighted the importance of breastfeeding no longer than six months in infants born to HTLV-I-positive mothers. These estimates can serve as a basis for regulation of policy, decision making, and control measures against vertical transmission of HTLV-I.


TRAFs [tumor necrosis factor (TNF) receptor associated factors] are a family of signaling molecules that function downstream of multiple receptor signaling pathways and play a pivotal role in the biology of innate, and adaptive immune cells. Following receptor ligation, TRAFs generally function as adapter proteins to mediate the activation of intracellular signaling cascades. With the exception of TRAF1 that lacks a Ring domain, TRAFs have an E3 ubiquitin ligase activity which also contributes to their ability to activate downstream signaling pathways. TRAF-mediated signaling pathways culminate in the activation of several transcription factors, including nuclear factor-κB (NF-κB), mitogen-activated protein kinases , and interferon-regulatory factors . The biological role of TRAFs is largely due to their ability to positively or negatively regulate canonical and non-canonical NF-κB signaling. While TRAF-mediated signaling regulates various immune cell functions, this review is focused on the recent advances in our knowledge regarding the molecular mechanisms through which TRAF proteins regulate, positively and negatively, inflammatory signaling pathways, including Toll–IL-1 receptors, RIG-I like receptors, and Nod-like receptors. The review also offers a perspective on the unanswered questions that need to be addressed to fully understand how TRAFs regulate inflammation. The Tumor-Necrosis Factor (TNF) Receptor Associated Factor (TRAF) family is comprised of cytoplasmic adaptor proteins involved in transducing downstream effects of a variety of receptors . TRAF1 aToll-like receptors (TLRs) are a family of transmembrane receptors lining both cellular and endosomal membranes that sense various pathogen-associated-molecular patterns (PAMPs), and danger-associated molecular patterns (DAMPS) , 9–11. TMyD88-dependent signaling is initiated with the recruitment of the IL-1 receptor-associated kinase (IRAK) 4 which, in turn, recruits and activates, through phosphorylation, IRAK1 and IRAK2 . IRAK1/2In TRIF-mediated signaling, TRIF recruits TRAF3, which catalyzes its own K63-linked polyubiquitination. This leads to the activation of the TBK1 and the non-canonical IKK, IKKε, which in turn phosphorylates IRF3 resulting in its nuclear translocation and the subsequent induction of type 1 IFNs (IFN-Is) , 23. WitIn addition to activating TLRs, TRAFs can also function as negative regulators of TLR signaling. TRAF3 negatively regulates TLR-mediated MAPK activity, possibly by preventing the release of the TRAF6:TAB1/2:TAK1 complex, but the negative regulation is inhibited by cIAP1/2 which catalyze K48-polyubiquitinated degradation of TRAF3  11, 23,,23, 24. TLR signaling can also be regulated by targeting the function, expression, or catalytic activity of certain TRAF proteins. Several deubiquitinases (DUB) have been shown to negatively regulate TLR signaling by removing ubiquitin chains from TRAFs or their targets. For instance, A20 is a key regulator of TLR signaling, whereby it targets several aspects of the signaling cascade. It can accomplish this, in part, by directly deubiquitinating TRAF6 . MonocytNOD-like receptors (NLRs) are a family of cytosolic receptors that sample intracellular PAMPs and DAMPs –44. ThesNOD1 and NOD2 are the most studied members of the NLR family [reviewed in Motta et al. ]. Upon a−/− bone marrow-derived macrophages (BMDMs) show normal inflammasome activation, suggesting that TRAF2 may not even be involved in this pathway  are capable of activating inflammasomes , 54. Inf pathway . Followi pathway .TRAF4 has been shown to act as a negative regulator in NOD2-mediated NF-κB signaling by direct interaction with NOD2. This interaction then allows IKKα to phosphorylate TRAF4, which results in its dissociation from NOD2 and inhibition of NOD2 signaling . NLRC3 hRIG-I-Like receptors (RLRs) are a family of DEAD box helicases that play a crucial role in the innate immune response to viral infections by detecting the presence of viral RNA in the cytosol. RIG-I and MDA5 are the two prototypical members of the RLR family . Upon seRIG-I/MDA5 employ TRAF3 to induce IRF3-mediated IFN-I production. Mechanistically, TRAF3 is recruited by MAVS, where it catalyzes its own K63 polyubiquitination followed by recruitment and subsequent activation of TBK1 and IKKε  67). TR. TR67). During bacterial infections, the E3 ligase HCTD3 adds K63-linked ubiquitin chains to TRAF3, which enhances the activation of the TBK1/IKKε complex and subsequent production of IFN-Is . ConversIn addition to cytosolic sensors of RNA, DNA sensors in the cytosol are equally crucial in detecting and mounting an inflammatory response against viral and bacterial pathogens. Stimulator of Interferon Genes (STING) is activated directly by second messengers like bacterial cyclic dinucleotides  –83 or byThere continues to be a great interest in understanding how TRAFs regulate innate immune signaling. Specifically, novel mechanisms have been recently identified to regulate TLR, NLR, and RLR pathways by modulating the ubiquitination status of TRAFs, their stability or their function. However, as most of these regulators seem to be non-redundant, investigating additional novel regulators, and their mechanism of action remains an active area of investigation.Each individual TRAF protein plays several, sometimes contradictory, roles that are pathway and/or cell specific. For example, a particular TRAF protein might induce lymphocyte survival and maturation while inhibiting a certain inflammatory pathway. Furthermore, most TRAFs function as E3 ligases as well as adapter proteins. Therefore, TRAFs are poor candidates for novel therapies since targeting a TRAF protein could lead to unintended consequences. For these reasons, future studies should focus on assessing the various roles of each TRAF protein in isolation from its other functions. This is especially important when designing therapies for complex inflammatory and autoimmune diseases by targeting TRAFs.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication. BD, FA and AA-S wrote the text. A-AS edited the manuscript and conceptualized the figures. ZA-S created the figures.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.


However, cellular stress responses trigger the reactivation of HTLV-1, enabling the virus to transmit to a new host cell. Using single-molecule RNA FISH, we measured the kinetics of the HTLV-1 transcriptional reactivation in peripheral blood mononuclear cells (PBMCs) isolated from HTLV-1+ individuals. The abundance of the HTLV-1 sense and antisense transcripts was quantified hourly during incubation of the HTLV-1-infected PBMCs ex vivo. We found that, in each cell, the sense-strand transcription occurs in two distinct phases: the initial low-rate transcription is followed by a phase of rapid transcription. The onset of transcription peaked between 1 and 3 hours after the start of in vitro incubation. The variance in the transcription intensity was similar in polyclonal HTLV-1+ PBMCs (with tens of thousands of distinct provirus insertion sites), and in samples with a single dominant HTLV-1+ clone. A stochastic simulation model was developed to estimate the parameters of HTLV-1 proviral transcription kinetics. In PBMCs from a leukemic subject with one dominant T-cell clone, the model indicated that the average duration of HTLV-1 sense-strand activation by Tax (i.e. the rapid transcription) was less than one hour. HTLV-1 antisense transcription was stable during reactivation of the sense-strand. The antisense transcript HBZ was produced at an average rate of ~0.1 molecules per hour per HTLV-1+ cell; however, between 20% and 70% of HTLV-1-infected cells were HBZ-negative at a given time, the percentage depending on the individual subject. HTLV-1-infected cells are exposed to a range of stresses when they are drawn from the host, which initiate the viral reactivation. We conclude that whereas antisense-strand transcription is stable throughout the stress response, the HTLV-1 sense-strand reactivation is highly heterogeneous and occurs in short, self-terminating bursts.The human T cell leukemia virus HTLV-1 establishes a persistent infection  + individuals by single-molecule RNA FISH. Viral transcripts were visualized as diffraction-limited spots and their abundance was quantified at one-hour intervals. The onset of the virus transcription peaked after one to three hours of incubation. In each cell, a short period of slow HTLV-1 transcription was followed by a phase of rapid transcription. Computer simulation, based on experimental data on PBMCs from a leukemic patient with a single dominant HTLV-1-infected T cell clone, indicated that this rapid transcription from the HTLV-1 sense-strand promoter activated by Tax was terminated in less than an hour. The HTLV-1 antisense transcript HBZ was constantly produced at a low level, and 50% ± 20% of HTLV-1+ cells were negative for HBZ at a given time. These results demonstrate how rapidly HTLV-1 is reactivated and potentially becomes infectious, once HTLV-1+ cells are transmitted into a new host.Human retroviruses such as HIV-1 and HTLV-1 (human T cell leukemia virus) can establish a latent infection in the host cell. However, these viruses need to be able to produce viral genome to propagate in a new host. HTLV-1-infected cells are transmitted through breastfeeding, blood transfusion and sexual contact, and HTLV-1 restores transcription once the infected cells are drawn from infected individuals. We measured the kinetics of the HTLV-1 transcriptional reactivation in blood cells isolated from HTLV-1 The provirus is not completely silent, but is intermittently transcribed to produce viral particles that infect new clones of cells . This inculation . Here, w+ T lymphocytes and causes adult T cell leukemia/lymphoma (ATL), a CD4+ T cell malignancy, in some 5% of HTLV-1-infected individuals. HTLV-1 also causes, in up to another 5%, HTLV-1-associated myelopathy/tropical spastic paraparesis  and other inflammatory disorders [4 HTLV-1+ T cell clones [It is estimated that 10 million people across the globe are living with HTLV-1 . HTLV-1 isorders , with anl clones , 8.tax is undetectable in vitro in freshly isolated cells from most HTLV-1-infected individuals [in vivo. By contrast, transcripts of the HTLV-1 bZIP factor (HBZ) from the antisense strand are usually detectable in vitro at the cell population level in peripheral blood mononuclear cells (PBMCs) obtained from HTLV-1-infected individuals [HTLV-1 transcribes both the sense and antisense strand of its 9 kb provirus. The long terminal repeats (LTRs) located at the 5′ and 3′ ends of the provirus serve as the promoters for sense and antisense transcription respectively. HTLV-1 Tax, produced from the sense strand, is a transcriptional transactivator protein that triggers NF-κB activation and other transcriptional cascades as well as its own promoter at the 5′ LTR  , 13 to qin vitro . We obseTo propagate between cells and between individuals, HTLV-1 must restore the sense-strand transcription from latency and produce viral components to form a virological synapse at the site of cell-to-cell contact . The inf+ cells to avoid CTL-mediated killing of HTLV-1-infected PBMCs that express sense-strand antigens (particularly Tax) during culture [HTLV-1-infected PBMCs carry a single copy of the provirus in the genome. HTLV-1 rapidly initiates transcriptional reactivation of the sense strand once the PBMCs are introduced into culture. We used smFISH to quantify the frequency and intensity of HTLV-1 reactivation at the single-cell level . We remo culture . The PBMgag  in gag  which isgag , indicatolecules .The population of PBMCs imaged by smFISH contained both HTLV-1-infected and uninfected cells. We wished to analyse the distribution of the HTLV-1 mRNA count within the HTLV-1-infected population. We used the proviral load to calculate the number of HTLV-1-infected cells in the total population .+ cell over time is shown in + subject examined , the total number of transcripts increased continuously during the 8 hours of incubation. The distribution in the sample from one patient with HAM (TBC) presented a long tail of cells that carried a larger number of sense transcripts (up to 250 molecules), with a continuous distribution of cells that carried fewer than 50 transcripts. By contrast, a single mode was identified at around 100 molecules in an ATL patient (LGZ) carrying a single dominant clone.The distribution of the number of sense-strand transcripts per HTLV-1The average count of the HTLV-1 sense-strand transcripts in the top 25% of all the infected cells was 100 to 300 molecules . A steep+ T cell clone would show more uniform distribution of the number of sense transcripts. We plotted the variance vs. the mean of the observed transcript count in the four HTLV-1+ subjects with observations at nine time points  were positive for sense-strand transcripts. After 8 h of incubation, the fraction of positive cells rose to between 0.2 and 0.8, depending on the HTLV-1+ subject examined. We assumed that the waiting time until we observed 3 or more molecules in a cell is an accumulation of single stochastic processes, and therefore fitted the plots presented in ex vivo culture. The timing of onset was more broadly distributed in samples from patients with HAM (TBC and TDZ) than from those with ATL (LGZ and LGB): the 75% quantiles of the probability distribution were 3.9 h and 4.9 h for TBC and TDZ respectively, whereas those for LGZ and LGB were less than 3 h. However, in each case the onset of the transcriptional reactivation was completed by 8 h ex vivo incubation in those cells that expressed detectable HTLV-1 mRNAs ; the remaining cells showed no sign of proviral transcription during the period of observation.To further characterize the transcriptional reactivation of the HTLV-1 sense strand, we quantified the fraction of cells that had become positive for sense transcripts at a given time during short-term culture of PBMCs . Cells wex vivo PBMCs during the short-term culture. (1) The timing of the onset of transcriptional reactivation peaks in the first few hours of ex vivo incubation. (2) The sense-strand transcription is restored with an initially low transcription rate. (3) Subsequently the rate of transcription of the sense strand increases rapidly: we postulate that this rapid sense-strand transcription is triggered by the strong positive feedback loop exerted by Tax protein. (4) Only a subset of HTLV-1+ PBMCs  restore the sense transcription.From the results presented above, we established the following characteristics of the transient and heterogeneous HTLV-1 transcription in k5 and k6, respectively (k5 < k6). We used the smFISH data from the ATL patient LGZ for the parameter optimization, because the single dominant clone of HTLV-1+ PBMCs in this leukemic subject requires only a single kinetic parameter for each reaction rate in the model, as opposed to a polyclonal population in which each clone would require a unique reaction rate to be assigned. The simulations of this three-state model are shown in k10. This model fitted the smFISH data well  (k10) in this putatively malignant clone. The duration of the rapid transcription phase was measured in each cell in the simulation by recording the interval between the initiation of the On-Tax promoter state and the direct transition from the On-Tax to the Off state  , indicatff state . These rHBZ. PBMCs isolated from one asymptomatic HTLV-1+ carrier (HBL), three patients with HAM  and four with ATL  were examined by smFISH (HBZ mRNA count per HTLV-1+ PBMCs (without incubation) is shown in HBZ mRNA transcripts carried per cell was very low: frequently only one or two molecules of HBZ mRNA were present per cell; and unexpectedly 20% to 70% of HTLV-1+ PBMCs in the subjects (except LFK) were negative for HBZ at a given time. In the subject LFK, almost all the HTLV-1+ PBMCs were negative for HBZ  falls within the range 0.0599 to 0.206 h-1 .HBZ expression by smFISH in five HTLV-1-infected T cell clones isolated by limiting dilution from HTLV-1+ patients and maintained in vitro [HBZ mRNA count in three of the clones showed a small but significant departure from the expectation derived from the Poisson distribution, the other two clones conformed to the Poisson distribution , suggesting that the HBZ production rate varies little with the genomic insertion site of the HTLV-1 provirus.In our previous study, we examined in vitro . While tribution . The slicription   restored the sense-strand transcription, it is possible that transcriptional interference—by the collision of two converging polymerase complexes—diminished the antisense transcription in this subject. However, in each of the remaining three subjects studied, there was no correlation between the sense-strand transcription and HBZ transcription. These results suggest that transcriptional interference between the sense- and antisense-strands does not play an important part in regulating HTLV-1 proviral transcription in most infected T cell clones.During the pression  in threever time , LGZ. Siex vivo PBMCs, as shown by three observations. First, only a subset of the HTLV-1+ PBMCs reactivated proviral transcription; second, the time of onset of transcription varied between cells; and third, the intensity of reactivation (i.e. the number of transcripts produced in a given period of time) varied widely in the population. Our results indicate two distinct phases of sense-strand transcription: the initial low-rate transcription produces ~10 to 20 mRNA molecules; thereafter a phase of rapid transcription occurs, probably attributable to the positive feedback exerted by the viral transactivator protein Tax. Transcriptional heterogeneity is likely to arise from the difference between clones in the HTLV-1 proviral integration site in the host genome. However, even in the ATL subjects examined in this study (LGZ and LGB), who carried one dominant clone in which (by definition) each cell shares the same integration site, a significant variance between cells in reactivation intensity was observed, which was equivalent to that of HTLV-1+ patients with polyclonal insertions  dynamics of HTLV-1 transcriptional bursting in primary, naturally-infected lymphocytes, whereas others studied Tat transcription from synthetic Tat circuits to analyze the mechanisms that produce bimodality in Tat expression over an extended parameter regime in a steady-state system –31.tax mRNA in the nucleus, which results in a reduction of Tax protein production, leading in turn to reduced viral transcription [ex vivo culture [The mechanism of this rapid termination of the sense-strand burst remains unknown, as in mammalian gene expression in general. The HTLV-1 accessory protein p30 retains cription . However culture . At leas culture  and meth culture . Second, culture . Third,  culture . Recent  culture , 38. HTL culture  and form culture . These oIn HIV-1 infection, the potential use of shock-and-kill treatment is still under debate because all approaches used to date have activated only a small fraction of the proviruses in a host. The results presented here demonstrate that the extent of heterogeneity in retrovirus reactivation is diverse in a population of naturally-infected cells and suggest a quantitative approach for examining the efficacy of the treatment.EDTA-anticoagulated blood from HTLV-1-infected patients was collected at St Mary’s Hospital . All donors gave written informed consent in accordance with the Declaration of Helsinki to donate blood samples to the Communicable Diseases Research Tissue Bank, approved by the UK National Research Ethics Service (15/SC/0089).+ subjects examined in this study  is available in Information on the HTLV-1+ cells were removed with Dynabeads  to avoid CTL killing against HTLV-1 Tax+ cells. Cells were suspended (1 × 106 cells/mL) in RPMI1640  supplemented with 2 mM L-glutamine , penicillin/streptomycin  and 10% fetal bovine serum . One mL of cell suspension (1 × 106 cells) was aliquoted and dispensed into 5 mL polystyrene round-bottom tubes  to keep the cell density consistent between assays. Cells were incubated in 5% CO2 at 37°C until sampled. One aliquot was sampled at each time point (every hour) and subjected to single-molecule RNA FISH.Patient-derived PBMCs were thawed and washed with PBS  containing 2 mM EDTA and 0.1% FBS. CD86 cells/mL. An aliquot (20 μL) of the suspension was spread on to a coverslip that had been coated with poly-L-lysine . Cells were fixed with 2% formaldehyde  on the coverslip for 15 min. Coverslips were immersed in 70% ethanol and stored at -20°C until hybridization.Cells were washed and suspended in PBS at a density of 5–10 × 10gag , tax  or HBZ  in hybridization buffer . Cells were washed on the coverslip twice with wash buffer A , and then wash buffer B . DAPI staining was included during the second wash with the buffer A. The cells were embedded in VECTASHIELD mounting medium  for imaging.Hybridization was performed as previously described . Namely,The coverslips were imaged with an Olympus IX70 inverted widefield microscope with a 100× 1.35NA UPlanApo oil objective lens (Olympus), a Spectra Light Engine illumination source (Lumencor) and an ORCA-Flash 4.0 V2 digital CMOS camera (Hamamatsu).10.17605/OSF.IO/R6Q8T). Dead cells were identified by the abnormal morphology of nucleus and omitted from the analysis. Spots in each of the three channels  were detected with FISH-QUANT [10.17605/OSF.IO/R6Q8T .Stacked TIFF images containing 54 z-slices with a 300 nm interval were obtained using Micro Manager . Each slSH-QUANT . A threspX locus . The copy number of a host gene RPP30 was determined using 1 μL of HEX-labeled PrimePCR RPP30  in the reaction to calibrate the amount of DNA input. Droplets were generated with QX200 Droplet Generator (Bio-Rad). PCR was performed  with C1000 Touch thermal cycler (Bio-Rad). Fluorescent signal was detected using QX200 Droplet Reader (Bio-Rad).DNA was extracted from unfixed cells using DNeasy Blood & Tissue Kit . The reaction of 20 μL volume was set up using ddPCR Supermix  containing 900 nM each of forward and reverse primers and 250 nM FAM-labeled probe to detect the HTLV-1 Two HTLV-1 infected T-cell clones  were trek1) and the number of sense transcripts observed prior to the rapid transcription (for k5 and k3). The sense-strand mRNA degradation rate  was determined experimentally in the present study (k9) was obtained from Rende et al. [10.17605/OSF.IO/R6Q8T.A Gillespie stochastic simulation algorithm was employed . The molnt study . The dege et al. . One milS1 Figin vitro incubation. The data from two patients with HAM (TBC and TDZ) and two with ATL (LGZ and LGB) are presented.The average number of HTLV-1 sense transcripts in the top 25% of all the infected cells at successive timepoints during (PDF)Click here for additional data file.S2 FigHistograms of the intensity of spots pre-detected with FISH-QUANT  with a c(PDF)Click here for additional data file.S3 Fig6 iterations of the models. We used the resulting 50 parameter sets to predict the data points at T = 5, 6, 7 and 8 hrs that were omitted from the parameter estimation. The mean predicted frequency and its standard deviation in each bin are plotted in green and overlaid on the experimental observation (solid grey line).To test the performance of the models, we used the experimental data (LGZ) from T = 1, 2, 3 and 4 hrs  to estimate the best 50 parameter sets from 10(PDF)Click here for additional data file.S4 Fig6 iterations with the above prior set and selected the best 50 parameter sets with the minimum distance function  To check the parameter convergence, we repeated the parameter search within a second set of prior distributions. (B) We performed 10(PDF)Click here for additional data file.S5 FigHBZ molecules per cell in in vitro maintained HTLV-1+ T cell clones. Histograms are reproduced from the graphs presented in Billman et al 2017 [HBZ molecules (<m>) is indicated in the inset. ; in the present figure the bin is shown as = 10 spots). The black line indicates the Poisson distribution with the parameter <m>, the observed mean number of HBZ molecules.The distribution of the number of  al 2017 . The mea(PDF)Click here for additional data file.S6 FigHBZ mRNA count per HTLV-1+ cell at successive timepoints during in vitro incubation. The data from two patients with HAM (TBC and TDZ) and two with ATL (LGZ and LGB) are presented.(PDF)Click here for additional data file.S7 Figin vitro HTLV-1-infected T cell clones were treated with actinomycin D to block transcription, and the abundance of HTLV-1 sense-strand transcripts was quantified at each time point  (see Two urs) see . The err(PDF)Click here for additional data file.S1 Text(PDF)Click here for additional data file.S2 Text(PDF)Click here for additional data file.S1 References(DOCX)Click here for additional data file.


Over the past 20 years, enhanced analyses of tumor-specific genomic alterations coupled with elegant biochemical approaches have helped to map essential signaling pathways in healthy and malignant cells. For example, the B cell lymphoma/leukemia 10 (BCL10) gene was identified in 1999 from a recurrent chromosomal translocation noted in non-Hodgkin lymphomas that arise in mucosa-associated lymphoid tissue (MALT). These studies demonstrated that BCL10 could oligomerize via its caspase recruitment domain (CARD) and induce robust activation of nuclear factor of kappaB (NF-κB), a critical family of transcription factors first implicated in lymphocyte differentiation. In <3 years, multiple groups discovered that BCL10 must partner with one of four CARD-containing scaffold proteins  and the MALT1 paracaspase  to drive NF-κB signaling in response to various receptor-mediated inputs. Building from murine knockout models, a growing spectrum of malignancies and human immune disorders have been genetically linked to altered signaling through this “CARD-BCL10-MALT1 (CBM) complex.” As this Research Topic attests, indeed this list has expanded exponentially in just the past few years. Drawing from leading investigators in the field, including many involved in the seminal discoveries summarized above, the 10 reviews, three original research articles, and one case report compiled in this Frontiers in Immunology eBook provide a comprehensive and timely examination of CBM complex assembly and signaling. Although we now appreciate that tissue-specific expression of CARMA/CARD proteins dictates function and underlying pathology of associated diseases, this collection also underscores the ubiquity and significance of the CBM signalosome as a central governor of receptor-mediated signaling to NF-κB and additional outputs important for cell proliferation, survival, differentiation, and function.Gehring et al. discuss the discovery and subsequent characterization of BCL10 as the central adaptor protein that channels various innate inflammatory and adaptive antigen receptor stimuli to NF-κB and c-Jun N-terminal kinase (JNK) activation in various immune cells. The formation of CARD-dependent cytoplasmic BCL10 filaments, “seeded” by CARD/CARMA scaffold proteins, constitute an essential platform for MALT1 protease activation and downstream signaling. Informed by studies of T and B cell receptor signaling in lymphocytes, the authors also review the complex post-translational modifications that ensure the orderly extension and degradation of putative BCL10 filaments in vivo that ultimately tune and regulate immune responses. Juilland and Thome provide an up-to-date overview of MALT1, which has been intensively interrogated in recent years as a potential therapeutic target for both lymphoma and autoimmune disease. MALT1 constitutively associates with BCL10, and functions both as a scaffold and protease activated upon CBM assembly and BCL10 filament construction. At least 10 MALT1 substrates have been described to date, with more likely to be identified in future studies. Proteolytic cleavage of many of these proteins , including MALT1 itself, typically serves to prolong canonical NF-κB activation, although other MALT1 substrates are implicated in regulating cellular adhesion, mRNA stabilization, and metabolic reprogramming in lymphocytes. Both reviews discuss how loss or mutation of BCL10 or MALT1 contribute to immune deficiency and dysregulation, highlighting the need for further mechanistic studies that will better define the essential functions these proteins fulfill in propagating receptor-directed signals in immune and non-immune cells, and maintaining vigorous yet controlled immune responses.Two reviews provide an in-depth look at the complex regulation and function of the two subunits shared by all CBM complexes: BCL10 and MALT1. BCL10 and MALT1 are rendered operational primarily through interactions with multi-domain scaffold proteins known as CARD-MAGUK 1-3 (CARMA1-3) and CARD9. These molecules were initially identified based on high sequence similarity shared between their CARD motifs and that of BCL10, facilitating heterotypic CARD-CARD interactions between the two. As illustrated in several salient articles collected in this eBook, these four CARD/CARMA proteins are structurally and functionally homologous in nucleating CBM complexes for downstream signaling. However, their discrete tissue-specific expression patterns dictate the upstream receptors through which these signals are conveyed in different cell types, resulting in distinct functions that help to shape immunity, inflammation, and tumorigenesis.Drummond et al. discuss the indispensable function of CARD9 in human anti-fungal immunity. CARD9 facilitates CBM-dependent activation of NF-κB, activation of ERK, and production of multiple pro-inflammatory cytokines upon recognition of fungal cell wall components by SYK-coupled C-type lectin receptors such as Dectin-1. Animals and humans lacking CARD9 are particularly susceptible to pathogenic fungal infections of the oral mucosa, skin, and central nervous system, as well as certain bacterial and viral infections. The authors broadly discuss the multi-faceted role of CARD9 in innate immunity, autoimmune disease, and cancer in humans, informed by both population-based studies and rare variants in patients suffering from uncontrolled, spontaneous fungal infections. This review is complimented by original research from De Bruyne et al. describing a CARD9 founder mutation in a cohort of 11 patients from five Turkish families suffering from various fungal infections. Consistent with a previous report, the authors demonstrate that this specific CARD9 variant  fails to stimulate NF-κB. Furthermore, CARD9 R70W also dominantly interferes with WT CARD9 signaling by disrupting BCL10 filament formation and attenuating NF-κB activity, although enough signaling may be preserved to explain the absence of pathology in heterozygous carriers. Remarkably, this dominant negative CARD9 mutant could also repress constitutive NF-κB signaling induced by gain-of-function variants of CARD10, CARD11, and CARD14. Although CARD9 is not co-expressed with these proteins in normal cells, this result beautifully illustrates the evolutionarily conserved requirements for scaffold multimerization and heterotypic CARD-CARD interactions in nucleating signal-competent CBM signalosomes.Two reviews examine the fundamental role of CARD9 in microbial recognition by myeloid lineage cells of the innate immune system. CARD9 is utilized by several membrane and intracellular pattern recognition receptors specific for molecules derived from both commensal and pathogenic bacteria, fungi and viruses. Based on recent studies of human patients with CARD9 deficiency, Hartjes and Ruland focuses primarily on the importance of CARD9 signaling in maintaining homeostasis and a healthy microbiome in the gut. Over the past 10 years, single nucleotide polymorphisms in the human CARD9 gene have been shown to influence susceptibility to inflammatory bowel disease (IBD) and colorectal cancer development. A detailed overview of several studies using murine colitis models reveals how loss or perturbation of Card9 signaling can alter the composition and function of resident myeloid cell populations and contribute to microbial dysbiosis, intestinal inflammation, and tumorigenesis. Future studies that delineate CARD9-dependent signaling pathways in greater molecular detail will hopefully open new therapeutic avenues for treating invasive fungal infections, IBD and colon cancer.In contrast, the review from Bedsaul et al. review the complex events surrounding TCR/BCR-induced CARD11 signaling. This process involves an elaborate intramolecular regulation of closed and open CARD11 protein conformations, which dictates recruitment and subsequent modification of BCL10, MALT1, and additional co-signaling proteins that serve to either activate the IκB kinase (IKK) complex  or terminate CARD11 signaling . A brief overview of human somatic and germline CARD11 mutations found in lymphoma and immunodeficient patients, respectively, culminates in a list of salient unanswered questions about CARD11 signaling that prompt further structure-function studies. A clever original article by Seeholzer et al. answers this call by implementing a creative approach to investigate the molecular details of CBM assembly in lymphocytes. A fusion protein joining BCL10 directly to the N-terminus of CARD11 promotes chronic MALT1 protease activation in CARD11-deficient cell lines, with differential effects on downstream NF-κB activation in B vs. T cells. Structure-guided mutagenesis of either CARD domain within the BCL10-CARD11 fusion protein helped to clarify how BCL10-MALT1 recruitment to CARD11 and formation of BCL10 CARD-dependent filaments cooperate in a highly integrated process for CBM signaling.Four articles in this collection highlight CARD11 (a.k.a. CARMA1) as a critical regulator of adaptive immune responses. The expression of CARD11 is largely restricted to lymphocytes, where it primarily functions to bridge antigen recognition through T and B cell receptors (TCR/BCR) with downstream activation of NF-κB, JNK, and mTOR. Lu et al. build upon this foundation by providing an exhaustive review of recently described human congenital immune disorders attributed to inherited mutations in CARD11, BCL10, and MALT1. As expected, null mutations in all three genes result in severe combined immunodeficiency, underscoring the importance of CBM signaling for mature B and T cell functions. However, various missense mutations, insertions, and/or deletions that preserve CARD11 protein expression contribute to a surprising spectrum of disease presentations in human patients. Whereas, gain-of-function (GOF) CARD11 mutations congruent with those found in B cell lymphoma cause a selective B cell lymphoproliferative disorder, loss-of-function (LOF)/dominant negative (DN) CARD11 variants were recently discovered in patients with severe atopic disease. The authors compare and contrast the expanding list of phenotypic manifestations described in these patients, revealing shared features of immunodeficiency  that relate back to dysregulated CBM-dependent signaling to NF-κB and mTOR. An intriguing case report offered by Desjardins et al. adds to this complicated picture by challenging the conventional classification of a single CARD11 mutation as gain or loss-of-function. This study describes a four-generation family that exhibits both B cell lymphocytosis and atopy, linked to a unique inherited CARD11 variant (p.His234_Lys238delinsLeu) that exhibits both GOF and DN activity in T cell signaling assays. Collectively, these articles describe how CARD11 mutations can manifest in distinct and blended features of immune dysregulation, depicting the CARD11 scaffold as a critical fulcrum for balanced T and B cell responses.Zotti et al. consider how three alternative splice variants of CARD14 affect NF-κB triggering in response to pathogen-associated molecular patterns (and stimuli yet to be identified), with a short CARMA2 isoform (CARMA2sh) lacking the C-terminal SH3 and guanylate kinase (GUK) domains predominating in human skin. Various GOF CARMA2sh variants linked to psoriasis, pityriasis and other skin disorders are also discussed in relation to an amplified pro-inflammatory NF-κB-dependent gene program in the skin. Importantly, the authors also discuss emerging roles for several proteins in enabling  or modulating  CARD14 signaling, which may present novel avenues for therapeutic intervention. The article by Israel and Mellett presents a deeper dive into the molecular genetics and clinical features of inflammatory skin disorders caused by numerous human CARD14 mutations described to date. Informed by detailed phenotyping and clinical management of patient kindreds, mouse models of psoriasis harboring synonymous Card14 mutations, and in vitro molecular signaling studies, this review summarizes how different human CARD14 variants can manifest in a heterogeneous spectrum of psoriatic skin diseases. Biologic therapies targeting TNF and the IL-12/IL-23/IL-17 cytokine axis have already proven beneficial in treating these diseases, whereas future specific inhibitors of CARD14 or MALT1 protease may prove to be attractive tools for tamping down inflammation driven by CARD14 GOF mutations.Two reviews delve into CARD14/CARMA2 as the pivotal scaffold for CBM signaling in epithelial cells and keratinocytes, focusing on how dysregulation of CARD14 signaling contributes to inflammatory skin disorders such as psoriasis. CARD14/CARMA2 was perhaps the least characterized member of the CARMA/CARD protein family, but recent insights have illuminated how dysregulation of CARD14 can result in skin inflammation. Zhang and Lin provide a general overview of CARD10/CARMA3 in CBM signaling induced by multiple G-protein coupled receptors and receptor tyrosine kinases (RTKs) such as the epidermal growth factor receptor (EGFR), culminating in NF-κB activation in several cell types. The authors also discuss newly recognized partners in CARD10 signaling , as well as a recently uncovered role for CARD10 in the antiviral interferon response. McAuley et al. discuss knowns and unknowns about how CARD10-dependent signals downstream of a variety of GPCR/RTKs promote solid tumor pathogenesis in non-immune cells. CARD10 overexpression has been noted in many tumor types, affected in part by failed miRNA-mediated modulation. Both reviews also comment on recent reports of receptor-independent CBM signaling in response to DNA damage, and reminds readers of the many unanswered questions surrounding the mechanisms connecting CARD10-dependent CBM signaling to various receptors, and how these might be specifically targeted for anti-cancer therapy.In contrast to the other CARD/CARMA proteins, CARD10/CARMA3 is ubiquitously expressed in many non-lymphoid tissues and may play an important role in the pathogenesis of several types of cancer. Two salient reviews explore this emerging paradigm, offered from investigators that have led the characterization of CARD10 signaling since its initial discovery. Staal et al. describes the ancient evolutionary origin of CBM signaling in invertebrates, revealing co-evolution of CARD-CC homologs with Bcl10, Syk-like kinases, and type I paracaspases. Careful structure/function analysis of paracaspases from invertebrates and lower vertebrates suggests that MALT1 functions for NF-κB activation evolved recently in jawed vertebrates, despite the appearance of conserved, independent scaffold and protease functions in older invertebrates. Their results highlight the utility of a comparative biology approach for studying CBM complex functions, and point to fascinating alternative CBM-independent roles for MALT1, including neuronal function in C. elegans.Finally, an original article from In summary, the articles and reviews in this eBook constitute the most comprehensive, current assessment of CBM complex function in normal and pathologic settings. We hope this collection will inspire future mechanistic studies to identify novel therapeutic approaches to treating myriad diseases connected to dysregulation of this ubiquitous and critical signalosome.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.FB is an employee and shareholder of Novartis. The remaining author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Hedgehog (Hh) signaling is an essential growth factor signaling pathway especially in the regulation of epithelial–mesenchymal interactions (EMI) during the development of the urogenital organs such as the bladder and the external genitalia (EXG). The Hh ligands are often expressed in the epithelia, affecting the surrounding mesenchyme, and thus constituting a form of paracrine signaling. The development of the urogenital organ, therefore, provides an intriguing opportunity to study EMI and its relationship with other pathways, such as hormonal signaling. Cellular interactions of prostate cancer (PCa) with its neighboring tissue is also noteworthy. The local microenvironment, including the bone metastatic site, can release cellular signals which can affect the malignant tumors, and vice versa. Thus, it is necessary to compare possible similarities and divergences in Hh signaling functions and its interaction with other local growth factors, such as BMP (bone morphogenetic protein) between organogenesis and tumorigenesis. Additionally, this review will discuss two pertinent research aspects of Hh signaling: (1) the potential signaling crosstalk between Hh and androgen signaling; and (2) the effect of signaling between the epithelia and the mesenchyme on the status of the basement membrane with extracellular matrix structures located on the epithelial–mesenchymal interface. Hedgehog (Hh) signaling is an effective and critical regulatory growth factor signal for organogenesis. There are three ligands found in mammals: Sonic hedgehog (Shh), Indian hedgehog (Ihh), and Desert hedgehog (Dhh). Among these three, Shh is the best studied Hh ligand. During development, the expression of Shh is found often in the epithelia ,2,3. In The downstream effects of the Hh signaling pathway are mediated by the Gli family of transcription factors ,10,11,12The Hh ligands are secreted molecules, often from the epithelia. These signals can be received by the neighboring mesenchymal layer, and the presence of both negative and positive regulators of the signaling pathway has been reported in these signal-receiving cells. This establishes a signaling crosstalk between the epithelia and mesenchyme, known as epithelial–mesenchymal interactions (EMI), which drives cell proliferation, differentiation, migration, and other developmental processes. The role of Hh signaling in EMI in both development and cancer will be discussed below.Loss of Hh signaling during mammalian development results in several defects such as polydactyly, cyclopia, and limb malformation, indicating the essential functions of Hh signaling pathway in organogenesis. In several cases, other growth factor signals are also involved in Hh signaling regulation during development. These factors are released from either the epithelia or the mesenchyme, depending on the developmental context. Urogenital organ development is a dynamic process, involving several signaling pathways which changes depending on tissue contexts. The urogenital system includes the urinary tract: composed of the kidney, the ureter, the bladder, and the urethra; and the reproductive tract: composed of the testes, the accessory glands, and the external genitalia (EXG). Recently, an increasing number of regulatory genes for EXG development has been reported ,15,16,17During urogenital tissue formation, especially in the EXG, Hh signaling and its role during EMI is essential. One of its ligands, Shh, is expressed in the endodermal epithelia of the embryonic urethra ,19. The Several works report on the role of Shh signaling and EMI during urogenital tract development. In these organs, the Hh ligands are typically expressed in the epithelia; and the expressed growth factor ligands emanate signals towards the mesenchyme, which, in turn, relays signals to the epithelia. This can induce cellular proliferation or differentiation and, in some cases, may also result in the expression of other growth factor signaling genes. Such signaling between the two tissue layers constitutes an essential crosstalk during organogenesis. The growth factor ligands that have been reported to be responsive to Hh include BMP (bone morphogenetic protein), fibroblast growth factor, and Wnt signaling ,24,25,26CreERT2 is a tamoxifen inducible Cre recombinase gene, and it is knocked into the Gli1 gene locus by homologous recombination. Thus, taking advantage of the inducible nature of Gli1 gene expression, gene modulation in the target mesenchyme cells was performed using a Gli1-CreERT2 modifier mouse strain [Because Shh is expressed in the developing cloacal and ureteric epithelia, the interaction between epithelia-derived Hh signal and corresponding BMP signaling from the nearby mesenchyme was investigated . To anale strain . Followie strain ,33. Bmpre strain . As a ree strain . Thus, se strain . This suHh signaling has also been often implicated in tumorigenesis. Over the recent years, prostate cancer (PCa) has been recognized as one of the life-threatening cancers in men. PCa arises in the prostatic epithelia, and it has been reported to interact with the nearby mesenchyme ,36,41. ISeveral studies have been published on the autocrine and paracrine modes of Hh signaling. As Hh signaling proteins can either be expressed solely in the epithelia, or in both epithelia and mesenchyme, there is still no strong consensus on the primary type of Hh signaling in PCa ,66. TherComparing paracrine Hh signaling in organogenesis and tumorigenesis may provide new insights for future research directions ,69. BMP In addition to the above investigations, application of recent analytical techniques may be required. Many PCa tumors contain various types of cancer cells. The mesenchymal cells adjacent to the PCa cells are often termed as cancer associated fibroblasts (CAF). Interactions between PCa and CAFs has been noted to be essential for PCa pathogenesis . This inIn addition to such experimental models, information on the dysregulation of BMP signaling in some metastatic sites is available. Changes in the expression levels of several BMP ligands and BMP receptors have been implicated in bone metastasis . BMPRs aLocally released BMPs and its relationship with Hh signals can also be noted in other reproductive organ tumors, such as ovarian cancer. BMP signals have been reported to function in cell differentiation in the ovary, particularly in the maturation of ovarian follicles  and has Overall, various Hh ligands and its signals have been detected in the epithelia in both urogenital organ development and reproductive organ cancers, such as PCa and ovarian cancer. BMP is often expressed in the mesenchyme of these systems, and signals between these two tissue layers are important for both development and pathogenesis. This putative Hh-Bmp interaction and its role in cancer can be an interesting research topic, based on the transduction by the epithelial Hh signal interpreted at the level of the adjacent mesenchyme during urogenital organ development .What kind of consequences are expected as a result of such signaling crosstalk? The promotion and inhibition of PCa cell proliferation has been investigated in the field of cancer biology, but whether such environment-derived BMP render positive or negative effects for PCa cell proliferation requires further investigation. Recent studies indicate that the addition of recombinant BMP protein inhibits PCa growth in vitro . AdditioRecently, progress on the mechanism of sexual differentiation has been described , and sexIn mice, EXG development occurs independently of androgen during the early stages. In the male, this is subsequently followed by an androgen-dependent masculinization process ,88,89. RAndrogens, such as testosterone and 5α-dihydrotestosterone (DHT), are produced locally in both embryonic and adult tissues. Both androgens possess essential roles for GT organogenesis. In fact, androgen administration is used during treatment of several conditions in human patients. As administration of androgen can promote penis elongation, it is used as a treatment for micropenis . SimilarThe regulation of cell proliferation in the different stages of PC by androgens has been reported. In contrast to the developmental process, PCa progression is initially dependent on androgens but may switch into androgen-independent during the advanced stages. It has long been established that the proliferation and tumor growth of PCa is driven by androgen ,98. LNCaDue to the dependence of tumor growth on androgens, castration and administration of androgen signaling inhibitors are frequent treatments for PCa . AlthougChanges in steroid levels due to local steroidogenesis has been reported in CRPC using mass spectrometry , and thiOf note, it has also been suggested that paracrine type Shh signaling is involved in the local steroidogenesis in PCa . AlthougAndrogen signaling is mediated by the nuclear androgen receptor AR. Gli1, the major Hh downstream transcriptional regulator, may interact with AR in some types of PCa cell lines . BMP sigPreviously, the description of EMI has been limited to the cellular layers, but recently, the participation of the basement membrane (BM) in EMI has been suggested . When thThe proper development of the BM is essential during organogenesis: its formation and subsequent removal is an essential step in the development of several organs. This removal is particularly essential in cases wherein there is tissue fusion in the midline region, such as in the formation of the male-type urethra and the palate. Morphological and loss of function studies on palatal fusion revealed the necessity of regulating the status of the BM during this process . It has Shh is one of the important soluble factors involved in the maintenance of the BM during organogenesis. The BM is composed of several extracellular matrix proteins, including laminin and collagen. During urogenital organ development, loss of the α5 chain of laminin has been reported to result in the formation of an abnormal urethra . As mentHow the BM participates in EMI is a growing research field. As the disappearance of the epithelia is associated with the breakdown of the BM, it is likely that the changes in the BM are mediated, at least in part, by the epithelia. As mentioned, epithelial Shh can induce the deposition of laminin in the BM. Loss of Hh signaling results in defective myotome in both mice and zebrafish ,124. HowAnother emerging possibility for the role of BM integrity in EMI is its potential to regulate protein diffusion between the epithelia and the mesenchyme. A study performed on MCF10A acini showed that the status of the BM is positively correlated with molecular permeability . As the Cell migration is a key event during metastasis, and it is important to consider the role of the BM during this event. The proper regulation and dysregulation of the BM can be related with PCa progression, as there is differential expression of BM proteins in normal and malignant prostate cells ,126. In Modulating Hh signal may lead to altered laminin production in the BM. Matrix metalloproteinases (MMPs), which can cleave extracellular matrix proteins, have been suggested to be involved in tumorigenesis in several cancers, including PCa ,131. WheEMI  is a fundamental concept which governs both organogenesis and cancer progression. In this text, we use the development of the EXG and prostate cancer as representatives of these events. Hh signaling has been shown to play a vital role in both processes, and it is worth noting that the effect of Hh signaling might either be positive or detrimental depending on the tissue and developmental context. This review offers a summary of the possible and known roles of Hh signaling during EMI. Although there are several other signaling pathways by which Hh can interact, we show here the importance of the Hh/Bmp signaling interactions in the two systems. This crosstalk is necessary for the proper development of the urogenital system and is also responsible for driving part of PCa malignancy. Furthermore, Shh can induce local steroidogenesis of androgens, a hormone that is critical for both events of interest. This local action is currently a concern as it offers possible therapeutic targets for PCa. Lastly, we discussed the potential role of the BM during EMI. The BM has long been believed to function as a barrier between the epithelia and the mesenchyme, but we describe that Hh can participate in EMI via the BM. Insights from the role of Hh in each biological event may contribute further understanding of this complex signaling pathway. Studying this pathway using comparative and interdisciplinary viewpoints would be necessary to understand even better the Hh signaling function and its implications in EMI.


Considering China is facing a precipitous decline in its population, there is an emerging trend of developing baby robots to encourage people’s willingness to become “parents”. Based on the decomposed theory of planned behavior and the theory of uncanny valley, this study empirically investigated whether a baby robot could perform as a prominent antecedent of fertility intention in China, and how this relates to its visual appearance. Consistent with prior research, the current study used a between-subjects design to show (1) a baby robot could significantly improve people’s fertility attitude through temporal visual stimulation; (2) fertility attitude, subjective norms from peers, and perceived behavioral control of finance could significantly contribute to fertility intention. Theoretical contributions and implications are discussed in this study. A falling birth rate could lead to widespread social and economic problems. Like many countries in the post fertility transition, China is facing a precipitous decline in its population, setting the stage shortly for potential demographic and economic issues. According to a recent report by the Chinese Academy of Social Sciences , China’sDeclining populations are creating many social problems in countries where this phenomenon is occurring ,4,5. To The attempt of baby robots as infant simulators for fertility education can be traced to the early pilot program of virtual infant parenting . It was Based on the well-defined theory of planned behavior in fertility research , this stChina is not alone in confronting a shrinking population. It has been reported that nearly half of the world’s population now live in countries with fertility rates at or below replacement level, and nearly all countries will reach low fertility levels in the next two decades ,4,5. TheAmong these, there is an increasingly growing interest in the infant simulator-based approach to promoting fertility intention ,29. VariPrior research has reported a controversial relationship between infant simulators and teenage pregnancy behavior. The theory of fertility and infant simulators dates back to the Virtual Infant Parenting (VIP) program aiming at preventing teenage pregnancy in Australia and the US, and often referred to as “Baby Think It Over” . ScholarTo address the complex users’ reactions to robots, scholars argue that the visual appearance of humanoid robots is effective in influencing users’ attitudes towards an interactive system and predicting consequent behavioral response ,21,22. TMore recent research has revealed that negative reactions to imperfect humanoid robots may be caused by the well-known phenomenon of UV proposed by Mori . It is aBaby robots to enhance parenthood experience strongly rely on their humanness and lifeness. The facial presence on a baby robot is assumed to be a crucial component of the overall visual appearance. However, the current design application is various without a clear guideline to indicate its relative effects on users’ responses. Therefore, incorporating the theory of UV and baby robot design is significant in revealing whether the humanoid appearance can influence users’ attitudinal reactions and the consequent decision of having a child.Much of the previous research into fertility promotion is underpinned by the theory of fertility intention ,43, alsoIn recent years, however, the inconsistency between fertility intention and actual behavior has gained much attention . Schoen More recent research suggests a psychological perspective to explain the path from fertility intention to parenting behavior . ProposeThough TPB has been applied in understanding the psychological path to fertility behavior in the Chinese context, the prominent antecedents of the three factors predicting fertility intention are limited in previous studies. Luo and Mao , for insAs the aforementioned functions of baby robots in improving the willingness of having a child, this study assumes that an individual’s fertility intention is improved by the accompaniment of a baby robot following a psychological path of decomposed TPB see . The resConsidering the theory of planned behavior ,57, we uAlthough there are not many infant simulators in our daily lives, the robot stimuli used in the current study were still adapted by a professional designer from the real infant robots in the current market. For example, No.2 infant robot was adapted from Toyota’s Kirobo Mini, which aimed to encourage couples to become “parents” ,60. No.4In order to explore the relationship among robot stimuli to the cognitive intention of fertility, we recruited a sample group from an anonymous Chinese “survey monkey”, Wen Juan Xing, to collect data. Wen Juan Xing is a reliable survey collection resource, specializing in recruiting Chinese participants and a large number of studies in the literature of Chinese social science have been conducted via this platform  ,63,64. TThe reference number of the ethical approval for research involving human subjects in Anhui College of Traditional Chinese Medicine is HSERS2019050001. We recruited a total of 180 subjects in this study. To be more specific, the participants were initially exposed to our task. After acknowledging the purpose of this study and the screening criteria, they could decide whether to participate in this study. The selection criteria were that they should be in the status of either being married, divorced, separated or widowed. As for the experiment procedure, participants first met the screening criteria and consented to take part in this survey. Then they needed to fill in the demographic information and were informed that they were going to evaluate a baby robot. Each robot stimulus was assigned with 30 participants and they were all asked to look at the stimulus (a baby robot) and to consider taking care of it. After that, they were required to answer a few questions and a manipulation check . p < 0.05), suggesting experiment manipulation was successful.MANOVA and Logit Regression were performed to analyze the data through SPSS 22.0. There were no missing or incomplete responses in the data set. Consistent with our predicted extent of human resemblance in the robot stimuli, manipulation check confirmed the extent of human resemblance gradually increased from No.1 robot to No.6 robot . ANOVA analysis showed a significant difference in the human resemblance between No.1 and No.5/6  = 4.863; Moreover, different robot stimuli did not significantly influence people’s subjective norm  = 0.730, ns; and, for peers: F  = 0.200, ns) and perceived behavioral control  = 1.277, ns; for energy: F  = 2.225, ns). As for the fertility intention, a binary logistic regression was introduced to explore the factors that contribute to fertility intention. According to the theory of planned behavior, an empirical analysis was further developed to investigate the effect of fertility attitude, subjective norms (family and peers), and perceived behavioral control (finance and energy).chi-square (8) = 31.445; p <0.05). The current model accounted for 21.5% of the variance in fertility intention with 68.9% identification accuracy. According to the result, fertility attitude was positively associated with fertility intention. While subjective norms from peers significantly contributed to fertility intention, subjective norms from family did not have a significant impact on fertility intention. Similarly, while perceived behavioral control of finance was positively related to fertility intention, behavioral control of energy only showed a marginal influence on fertility intention. In addition, age worked as a significantly negative indicator of fertility intention. Men showed somehow greater fertility intention compared with women however the effect was only marginal. Besides, no other significant effects were found.Given the ambiguous relationship between the visual appearance of baby robots and people’s fertility intention, this paper introduced a behavioral experiment approach and tried to obtain a deeper understanding of the effect of a baby robot on people’s fertility behavior. Based on the decomposed theory of planned behavior, results show that the visual appearance of a baby robot could increase people’s fertility attitude. Under a temporal visual stimulation, the uncanny valley effects in baby robot design are associated with people’s fertility attitude, in which the degree of human-resemblance in a baby robot and fertility attitude followed a U-shape relationship. In other words, people tend to have a temporally higher fertility attitude when facing the baby robot with the most or the least human features, compared with the baby robot with the medium human features. However, the temporal visual effects of baby robots do not significantly contribute to the subjective norms (family and peers) and perceived behavioral control (finance and energy). This result in the current study is consistent with prior research: the temporal visual reaction towards stimuli would be more associated with individual cognition and attitude formation, rather than with long-term social cognition and control beliefs ,67. SubjRegarding the relationship between fertility attitude, subjective norms, perceived behavioral control, and fertility intention, results show that fertility attitude is a significant indicator of promoting fertility intention. This is consistent with the prior research ,44,58. BMoreover, age was also a reliable indicator of fertility intention: older people tended to give up having children more easily. Gender tends to work as a marginal factor for fertility intention: compared with men’s fertility intention, women showed a marginally weaker intention to have children. That might be caused by Chinese women’s increased education level and work participation .This study tended to have the following theoretical contributions. To begin with, although some prior research tried to address the antecedents of fertility intention and birth-intervention policies in the context of China ,59,71,72This study also has some limitations. First, this research explored the relationship between the uncanny valley and people’s fertility intentions, providing the preliminary guidelines for robot designers. The robot No.6 was adapted from a real infant picture which by far cannot be made at the current state of the robot industry. Thus, it might work as a confounding factor since people might be biased due to its realistic appearance. Future studies should try to use different actual social robots to validate the current result.Second, there are many other features of a social robot that could be explored and work as promoting factors for people’s fertility intention, such as specific facial features , expressThird, the main goal of this study was to explore the effect of the baby robot on fertility intention. Though this study recruited sufficient participants to take part in the experiment, the sample size might be relatively small when exploring the relationship between fertility attitude, subjective norms, perceived behavioral control, and fertility intention. Future studies should try to introduce a sizeable domestic survey to validate the conclusions in the current study.Fourth, the measurement of behavioral control and fertility intention might be limited. The current study did not differentiate in the difference between the extensive and intensive margin of fertility, which might work as a cofounding factor. Further studies should try to examine the factors in detail and validate the current finding. Though the current study adapted the items from two perspectives, namely finance and physical energy ,55, ther


Helicobacter pylori (H. pylori), a pathogen that colonizes the stomach and is the main risk factor for gastric cancer. In addition to experimental data that link Wnt signaling to carcinogenesis, we discuss that Wnt signaling is affected in a substantial proportion of patients with gastric cancer, and provide examples for potential clinical implications for altered Wnt signaling in gastric cancer. The Wnt signaling pathway is one of the most prominent developmental signals. In addition to its functions in development, there is emerging evidence that it is also crucial for various organ functions in adult organisms, where Wnt signaling controls tissue stem cell behavior, proliferation and differentiation. Deregulation of Wnt signaling is involved in various pathological conditions and has been linked to malignant tissue transformation in different organ systems. The study of the Wnt signaling pathway has revealed a complex regulatory network that tightly balances the quality and strength of Wnt signaling in tissues. In this context, R-spondins are secreted proteins that stabilize Wnt receptors and enhance Wnt signaling. In this review we focus on new insights into the regulatory function of Wnt and R-spondin signaling in the stomach. In addition to its function in the healthy state, we highlight the connection between Wnt signaling and infection with  Wnt, originally named as int1, was discovered in 1982 when Roel Nusse and Harold Varmus performed research on oncogenic retroviruses ,4. SubseWhile it is crucial for development, more recently the role of Wnt signaling in adult tissues has become a major focus of research. In this context, it emerged as a critical pathway that regulates stemness, and self-renewal and cellular differentiation in several tissues . In addiIn the healthy stomach, Wnt signaling is induced by an interaction of secreted lipid-modified glycoproteins that act as ligands for Wnt receptors. The lipid modification, so-called palmitoylation, is thought to be important for the short range signaling of Wnt ligands, allowing spatial diversity within the tissues . There aIn the gastrointestinal tract, the canonical pathway has been extensively studied and has been shown to regulate cell fate and proliferation . It has The Wnt-receptor LRP5/6 is predominantly expressed in epithelia that are highly proliferative and in which cells that derive from dividing stem cells are constantly pushed along the epithelial unit, such as gland or crypt. Importantly, those epithelia are dependent on sufficient Wnt signaling for maintaining healthy homeostasis . Most of the research on Wnt signaling in the gastrointestinal tract has been done in the intestine and colon. This is probably due to the fact that mutations in Wnt signaling are found in almost all patients with colorectal cancer . The rolAs Wnt signaling has a large influence on the overall epithelial architecture, the tight regulation of Wnt activity within the tissue is essential for its integrity. While Wnt ligands are present throughout the stomach tissue, Wnt signaling, reflected by the expression of target genes, is limited to the base of the glands in the antrum .To clarify this discrepancy, R-spondin3 was identified as a critical stromal niche factor, produced by myofibroblasts of the lamina muscularis mucosae, specifically in the vicinity of the gland base . Four different R-spondin homologues have been described. All of them are secreted proteins, produced in the endoplasmic reticulum, and each of them can bind to all three Lgr5 homologues  . NeverthIn the absence of R-spondin, activation of the Wnt signaling pathway results in activation of target genes, including the transmembrane E3 ubiquitin ligases ring finger 43 (RNF43) and zinc and ring finger 3 (ZNRF3), which are functional homologues ,22. UponR-spondin binds to the extracellular domain of RNF43/ZNRF3 and to Lgr4/5/6, leading to their physical association and internalization into the cytoplasm ,23. ThisWhile R-spondin molecules potentiate Wnt signaling, the functional impact of Wnt ligands interacting with the Fzd/LRP5/6 receptor and of R-spondin interacting with Lgr5 has been shown to be synergistic but non-redundant in the context of small intestinal stem cells . InhibitThe stomach is anatomically divided into two main parts: the stomach body or corpus, which contains acid producing parietal cells, and the distal part, or antrum.The antral epithelium is highly proliferative and shows a rapid migration of differentiating cells, which are constantly shed into the lumen, resulting in a full gland turnover within one to two weeks ,27. AntrSince both Axin2+/Lgr5+ as well as Axin2+/Lgr5− cells are capable of repopulating the gland, the stem cell compartment seems to contain different non-homogenous populations of cells. In fact, the Lgr5+ stem cells have a rather slow turnover time of 10–14 days and a low proliferation rate of 10% to 20% . In contThe heterogeneity of cells in the gland base that are able act as stem cells demonstrates the cellular plasticity in this compartment, and it is likely that interconversion between the two cell types occurs. Of note, as both Lgr5 and Axin2 are Wnt target genes, Wnt signaling could be crucial for the stem cell identity, irrespectively of the proliferative state of the cell. It is possible that different subpopulations of gastric antrum stem cells are differentially regulated, allowing activation or deactivation of a particular population based on the environmental context, which may be particularly important for a rapid response to epithelial damage and an efficient repair of injured gastric epithelium. Accordingly, R-spondin3 leads to an expansion and increased proliferation of Axin2+/Lgr5− cells, whereas Axin2+/Lgr5+ cell proliferation is rather silenced. Since the expression of Rspo3 increases in the context of injury (see below), Lgr5+ cell silencing may represent a strategy to protect this cell population in the context of injury. In this context, further investigations may shed light onto which other signals are involved in fine-tuning the gland base and regulating the differential behavior of different stem cell subpopulations, which finally determines the architecture of the gland.In the corpus, the gland organization differs from the one in the antrum, which is particularly reflected by the presence of acid-producing parietal cells throughout the corpus gland, as well as by secretory chief cells in the base of the glands. Although Lgr5+ cells were identified throughout the gastrointestinal tract, they were not detected in the corpus in the original studies that used Lgr5− EGFP-ires-CreERT2 mice to follow the fate of Lgr5-expressing cells ,29. HoweIn the context of Wnt signaling, an alternative stem cell marker used was Troy, which is also a Wnt target gene and is furthermore enriched in Lgr5+ intestinal stem cells . Using tMore recently, the availability of a new reporter for Lgr5 has confirmed that, under homeostatic conditions, corpus Lgr5+ cells only rarely contribute to the gland homeostasis . In contNonetheless, it remains unclear how the functional switch of gland base chief cells to proliferative stem cells occurs upon injury. Leushacke et al. characterized the expression pattern in Lgr5+ cells upon injury and found an upregulation of Wnt related genes such as Mmp7, which is a direct target gene of beta catenin/TCF4, and Sostdc1, which is a Wnt inhibitor . These rHelicobacter pylori (H.pylori) is a WHO class I carcinogen and the main risk factor for gastric cancer [H. pylori is able to colonize gastric glands, and once colonization is established the bacterium has evolved to persist for decades [H. pylori infection will develop gastric cancer [The gram-negative bacterium c cancer . H. pylo decades . Howeverc cancer ,35. H. pylori. Upon adherence to epithelial cells, H. pylori uses its type four secretion system, which acts as a molecular syringe, to inject CagA into host cells. Upon translocation, CagA is phosphorylated and interferes with signal transduction within the host cells. In the context of Wnt signaling, it has been shown that CagA can interfere with GSK3beta-induced degradation of beta-catenin and thereby lead to the stabilization of beta-catenin, which is then translocated to the nucleus and initiates the expression of Wnt target genes [H. pylori positive gastric cancer samples have a significantly higher beta-catenin expression than those of H. pylori negative cancer tissues [H. pylori induces upregulation of stem cell associated markers such as Axin2 [There are several host-related factors as well as bacterial virulence factors that are linked to an increased risk for pathology : CagA iset genes . This is tissues . Further tissues . Yet othas Axin2 , Nanog aas Axin2  and theras Axin2 . H. pylori on Wnt signaling, infection also interferes with Wnt signaling on the tissue level through intercellular communication. As pointed out above, Wnt signaling in the stomach is not a cell intrinsic feature of the cells but is instead largely controlled and induced by the microenvironment. In this context, infection with H. pylori has been shown to interfere with the homeostatic division of stem cells within the antral gland, resulting in an increased number and division rate of Axin2+ cells [H. pylori that are able to invade the gland and colonize the apical junctions of the stem cell and progenitor cell pool [H. pylori are more pronounced when cells are grown in media with Wnt and R-spondin3, whereas the response of differentiated cells grown without Wnt is diminished [H. pylori infection need to be investigated in more detail. Apart from direct effects of CagA and 2+ cells . This is2+ cells . In cont2+ cells . Of noteell pool . This inminished ,43. WhilNew studies reveal that not only in the colon but also in the stomach the activation of Wnt signaling could represent a critical step in the carcinogenic cascade. Thus, pathologic activation or mutation of the Wnt signaling cascade has been found in around 30% of gastric cancer tissues . VariousWithin the group of upregulated molecules, different Wnt molecules are enhanced in gastric cancer: For example, Wnt5A has been shown to be significantly upregulated , and hisApart from that, loss-of-function mutations of genes that inhibit or limit Wnt signaling were observed: Mutations in the APC gene have been reported in 7% , 15–18% Epigenetic changes have also been reported to play a significant role in gastric cancer. For instance, APC is not only often mutated but is also commonly hypermethylated. Accordingly, methylation of APC occurs in 37.7% of healthy tissues but in 52.9% of gastric cancer samples . The freAnother mechanism prominent in gastric cancer is the pathologic activation or inactivation of Wnt target genes via microRNAs . A revieStudies also revealed the existence of single nucleotide polymorphisms (SNPs) in the CTNNB1 gene  as well Among the investigations of Wnt signaling in gastric cancer, several studies have also focused on its role in the development of premalignant lesions. For instance, APC mutations were found already in low-grade dysplasia and were less frequent during the progression of gastric cancer . These fH. pylori infection, mice with RNF43 mutations presented higher levels of gastric gland atrophy, hyperplasia and the gastric stem cell marker CD44 [H. pylori induced gastritis.Further experimental mouse work showed that upon ker CD44 . ConsequAdditionally, histological studies that examined the nuclear staining of Wnt pathway components found nuclear staining for beta-catenin in one-third of gastric tumors . As thisExperimental models have been developed to study the impact of aberrant Wnt signaling on the development of gastric pathology: K19-Wnt1 mice overexpressing Wnt1 in gastric epithelial cells developed “small preneoplastic lesions consisting of undifferentiated epithelial cells” in the stomach after 7 weeks and furthermore, the number of those lesions increased over the time . Another group performed subcutaneous injection of AGS-Wnt1 cells in nude mice, which then developed significantly larger tumors compared to mice injected with AGS cells alone .In a different study, upregulation of the Wnt signaling pathway was achieved via three different mechanisms, either by deletion of APC or GSK3, which are Wnt signaling inhibitors, or by inhibition of beta-catenin degradation in mice and they consistently found that small antral microadenomas appeared already 4 days post induction, which then enlarged over the course of time and overexpressed Lgr5 and Axin2 . AdditioF/F mice that develop intestinal-type gastric cancer were treated with a Fzd inhibitor leading to an inhibited tumor growth [In addition to its procarcinogenic effect, beneficial effects of inhibition of Wnt signaling in already established gastric tumors have been demonstrated in a xenograft model where injection of a Wnt5a-inhibitor into mice reduced the amount of liver metastases compared to mice that did not receive the inhibitor treatment . Similarr growth . The expression of Wnt target genes has been shown to correlate with the prognosis of gastric cancer patients. Wnt5A expression in gastric cancer patients is associated with more advanced stages of the tumor and a poor prognosis for the patient .A meta-analysis performed by Huang et al. revealed furthermore that Lgr5 overexpression is significantly correlated to the T-stage of the TNM classification, as well as to the N-stage . They foFurthermore, hypermethylation of Dkk3 has been found to be associated with higher mortality . AdditioDue to its involvement in gastric carcinogenesis, modulating the (aberrant) Wnt signaling pathway could be a suitable therapeutic target, and several compounds are under investigation.For example, DKN-01, a monoclonal antibody against the Wnt signaling antagonist Dkk1 is currently in phase 1 of a clinical trial for patients with gastric adenocarcinoma . Other mAnother promising drug is IWP, a small molecule that targets Porcupine , an endoFurthermore, colorectal cancer research revealed that a combination of peptide vaccines and anti-cancer drugs induced upregulation of the Wnt-inhibitor RNF43 , and thiWhile new strategies to target Wnt signaling in gastric cancer are emerging, so far there is no Wnt pathway targeting drug that has successfully passed all phases of clinical trials and is implemented as a therapeutic agent. This is probably because Wnt signaling is crucial for many physiological functions of the organism. However, new approaches, as outlined above, take advantage of specific alterations found in cancer tissue and are more likely to succeed. Alterations of Wnt signaling in patients with gastric cancer are heterogenous, and therefore, it will be important to apply diagnostic tools to identify the individual patient mutations to be then able to apply personalized strategy to target aberrant Wnt signaling. H. pylori infection. While this adaptation is beneficial for epithelial regeneration and injury repair, a deregulated Wnt signaling is a critical driver of gastric carcinogenesis. Alterations in the Wnt signaling pathway not only initiate cancer progression but can also be determinants for the prognosis of cancer patients. Therefore, Wnt signaling remains a promising therapeutic target in gastric cancer, with many new compounds that are being developed and currently investigated in clinical trials. Wnt signaling has been shown to be a key mechanism for maintaining homeostasis of the gastric gland. Wnt signaling is essential for stem cell identity, epithelial turnover, and is a determinant of cell fate and cellular diversification within the gland. R-spondin3 is a critical regulator of Wnt signaling in the stomach, and its anatomically restricted expression in the gland base enables the maintenance of the Wnt gradient in the gland. This system allows a rapid adaptation of the tissue proliferation and its function, as has been demonstrated in the context of