Molecular and epidemiological studies of transmission routes and risk factors for infection by HTLV-1 are extremely important in order to implement control measures, especially because of the high prevalence of HTLV-1 in several regions of the world. San Salvador de Jujuy, Northwest Argentina, is a highly endemic area for HTLV-1 and foci of tropical spastic paraparesis/HTLV-1-associated myelopathy.To gain further insight into the role of intrafamilial transmission of HTLV-1 in a highly endemic region in Argentina.Cross-sectional study in Northwest Argentina. Epidemiological data and blood samples were collected from 28 HTLV-1 infected subjects (index cases) and 92 close relatives/cohabitants. HTLV-1 infection was diagnosed by detection of antibodies and proviral DNA. The LTR region was sequenced and analyzed for genetic distances (VESPA software), in addition to determination and identification of polymorphisms to define HTLV-1 family signatures.Fifty seven of the 120 subjects enrolled had antibodies against HTLV-1 and were typified as HTLV-1 by PCR. The prevalence rate of HTLV-1 infection in family members of infected index cases was 31.52% (29/92). The infection was significantly associated with gender, age and prolonged lactation. Identity of LTR sequences and presence of polymorphisms revealed high prevalence of mother-to-child and interspousal transmission of HTLV-1 among these families.There is an ongoing and silent transmission of HTLV-1 through vertical and sexual routes within family clusters in Northwest Argentina. This evidence highlights that HTLV-1 infection should be considered as a matter of public health in Argentina, in order to introduce preventive measures as prenatal screening and breastfeeding control. Human T-cell Lymphotropic virus type 1 (HTLV-1) has been associated with lymphoproliferative disorders, inflammatory and degenerative diseases of the central nervous system, and some disorders of the immune system . HTLV-1 Infection is considered lifelong and most of the infected subjects remain asymptomatic, becoming viral reservoirs and continuing the chain of transmission . The effFamilial clustering has been studied in Japan, Caribbean and Brazil , 9,10,11In 2004, we reported diverse prevalence rates of HTLV-1 infection according to different geographic areas, through an overview of the seroepidemiology of HTLV-1 in Argentina. In fact, the endemic zone in the North area of the country has a prevalence of 0.6–1.2% in blood donors while in non-endemic parts , the prevalence in blood donors is <0.1% .There are few published data about family transmission of HTLV-1 in Argentina based on epidemiological and molecular evidence. While we have shown that HTLV-1 is familiarly transmitted in a non-endemic area of Argentina , reliablIn a cross-sectional study, epidemiological data and blood samples were collected from 28 HTLV-1 infected subjects (index cases) randomly selected from the records of San Roque Hospital, San Salvador de Jujuy and 92 members of their family groups. Samples were codified as J-family number-order of sampling. HTLV-1 infection in these 28 index cases had been diagnosed by detection of antibodies against HTLV-1 and amplification of proviral DNA. The 92 family members were included in the study based on the criteria of being a close relative of an index case or cohabiting in the same household.This study complied with the principles outlined by the Declaration of Helsinki and was approved by the Ethics Committee of San Roque Hospital, San Salvador de Jujuy, Argentina . Written informed consent was signed by all the participants or their parents/legal guardians prior to sample collection. HTLV-1 infected subjects were counseled by a multidisciplinary team that offered information about the infection and medical support.At enrolment, a structured questionnaire was performed by trained interviewers to collect epidemiological and social data, parenteral exposure and sexual history. Each subject was tested for HTLV-1 and HTLV-2 antibodies by ELISA 3.0  and Particle Agglutination (PA) assay . Reactive samples were further tested with an ‘‘in house” indirect immunofluorescence assay (IFA) on MT-2 and Mo-T cell lines  and Westtax gene following the protocols described by Vandamme et al. 1997 [tax region [High molecular weight DNA was extracted from blood samples of all 120 subjects. Polymerase chain reaction (PCR) was carried out to amplify 219-bp of the al. 1997 . All HTLx region . PCR proAmplification of 672 bp of the LTR region of the viral genome was carried out in all positive samples, as previously described . PCR proFrequency distribution of each variable was evaluated by Chi-square, Fisher’s exact test or Contingency Coefficient and Cox-Mantel-Haenszel tests.A bivariate analysis was also carried out. Odds ratios (ORs) and exact 95% confidence intervals (CIs) were calculated to determine the association between selected variables and HTLV-1 infection. In addition, test of difference between proportions was applied. The statistical package R version 3.2.4 software (R Core Team) was used for statistical analyses. P-values <0.05 were considered significant.Fifty-seven of the 120 subjects enrolled in this study (including the 28 HTLV-1 index cases) had antibodies against HTLV-1. All these cases were confirmed and typified as HTLV-1. The prevalence of HTLV-1 infection in close relatives/cohabitants of the infected index cases was 31.52% (29/92).The overall genetic distance observed on HTLV-1 LTR (552bp) considering all infected family members ranged from 0.0 to 1.4%.Except for two families (J54 and J11), genetic distance between viral isolates of each family group was 0.0%, showing 100% identity. Epidemiological data, along with molecular results evidenced that familial transmission of HTLV-1 occurred in 19 of the 28 (67.9%) family groups. In all these families, at least two members were infected with HTLV-1 .Isolates from members of Family J01 showed 100% identity. Furthermore, a common family polymorphism at position 746 on LTR respect to ATK reference strain was found . In thisSexual transmission was demonstrated in Families J02 and J47 showing 100% sequence identity between spouses.The isolates from Family J03 were 100% identical. Furthermore, a common family polymorphism at position 561 on LTR respect to ATK reference strain was observed . In thisThe isolates from the infected brothers of Family J11  showed 100% sequence identity between them and with their HTLV-1 positive mother ; all of them had a common polymorphism at position 212 on LTR respect to ATK reference strain . DespiteGenetic distance was 0.0% between J13/01 (HTLV-1 infected mother) and J13/05, demonstrating vertical transmission; moreover, the epidemiological data showed that daughter J13/05 had been breastfed for more than 1 year.Mother (J14/01-index case) and son (J14/02) of Family J14 presented 100% viral sequence identity, demonstrating vertical transmission. Moreover, a common family polymorphism at position 711 on LTR respect to ATK reference strain was found . The sonIn Family J16, isolates from mother (J16/01-index case) and son (J16/03) had 100% viral sequence identity, indicating vertical transmission. Moreover, a common family polymorphism at position 632 on LTR respect to ATK was identified . AlthougThe isolates from spouses of Family J17 had 100% sequence identity, showing a family polymorphism at position 441 on LTR respect to ATK . Thus, sVertical transmission was demonstrated also in the mother of Family J19 (J19/01-index case) and her son (J19/03). They had 100% sequence identity and the epidemiological data showed that the child had been breastfed for 2 years.In Family J20, LTR sequence identity of 100% was found between mother (J20/02) and daughter (J20/01-index case), who had been breastfed for more than 1 year. Moreover, common polymorphisms at positions 212 and 646 on LTR respect to ATK were detected .In Family J22, sexual transmission between spouses J22/01 and J22/02 was observed (0.0% genetic distance) and two common polymorphisms at positions 233 and 628 on LTR respect to ATK were found . Both spIn Family J25, vertical and sexual transmission of the virus (100% sequence identity) was demonstrated. Epidemiological data showed that the infected son (J25/05) had been breastfed by his mother (J25/01) for more than 2 years.Vertical transmission was demonstrated from mother (J28/02-index case) to son (J28/03) in Family J28 (100% sequence identity); the son had been breastfed during 2 years.In Family J29, vertical transmission was established since the daughters (J29/02 and J29/03) had 100% sequence identity compared to their mother (J29/01); they were breastfed for more the 1 year.In Family J30, there was 100% identity between virus from mother (J30/02-index case) and daughter (J30/01), demonstrating vertical transmission. The same route of transmission was observed in Family J35 between mother (J35/02-index case) and daughter (J35/03), also with 100% of LTR sequence identity.Sexual transmission was demonstrated in Family J43, with 100% sequence identity between spouses (J43/01 and J43/02). Moreover, an insertion of 20 nucleotides at positions 663–682 on LTR was exclusively observed in both isolates .Higher genetic distance was noticed between isolates of Family J54, in which virus sequence from mother (J54/03) and daughter (J54/01) were 100% identical and the genetic distance between the virus from mother (index case) and son (J54/02) was 1.4%. Thus, the son J54/02 would have acquired the infection outside the family .From the 9 family groups that had only one infected subject (data not shown), familial transmission was demonstrated only for the index case of Family J15 (J15/01) and individual transmission for Family J41 (J41/02).Index case J15/01 was daughter of the index case of Family J14 (J14/01) . There wOn the other hand, individual transmission of HTLV-1 was demonstrated in one of the subjects of Family J41 by the absence of HTLV-1 infection in all family members (parents and brothers of J41/02). J41/02 probably acquired infection through the sexual route, since his partner had history of intravenous drug usage . No dataVertical and sexual transmission occurred in 14 (73.7%) and 7 (36.8%) of the 19 families, respectively. Vertical transmission was confirmed in 20 (40%) of the 50 mother/children pairs, while sexual transmission occurred in 8 (50%) of the 16 couples. Thereby, there were not significant differences between sexual and vertical routes for HTLV-1 transmission in this population (95% CI: 0.0034 to 0.5299 p = 0.057). Moreover, no significant differences were found in the frequency of transmission from mothers to daughters  compared to mothers to sons  (95% CI: 0.1333 to 0.4904 P = 0.3228) .Most of the infected subjects were females , mean age: 36.3 years (SD±22.1), ranging from 3 to 83 years; the majority of them (49.1%) were aged between 30 and 60 years.p = 0.0026)  .Despite the fact that prevalence of HTLV-1 in wives  was higher than in husbands , this difference was not significant (95% CI: 0.2158 to 0.673 P = 0.4642) . All theThe HTLV-1 genetic analysis determined that all sequences belonged to Cosmopolitan subtype HTLV-1a Transcontinental subgroup A, in Latin American cluster (data not show). These results are in agreement with data previously reported by our group .In this study, a systematic research concerning familiar transmission of HTLV-1 infection in an endemic area of Northwest Argentina was performed for the first time. We evaluated 28 HTLV-1 infected subjects (index cases) selected from the records of San Roque Hospital, San Salvador de Jujuy, in addition to 92 members of their family groups.As HTLV-1 infection had been suspected to spread silently within families in North Argentina based on the observations of physicians about the usual outcome of TSP in family groups, we aimed to investigate the aggregation of this infection among close relatives/cohabitants of the index cases. Previous studies from Colombia, Chile and Brazil have shown high rates of HTLV-1 infection in relatives of TSP/HAM patients , 21–23.The results presented herein reflect a high prevalence of HTLV-1 infection (31.5%) in people cohabiting with the index cases, pointing out the great importance of familial dissemination of HTLV-1 infection in North Argentina. In agreement with what happens in the rest of the world, the highest rate of infection was found in women  2, 24–2–224–27.High prevalence rates of HTLV-1 infection in viral-carrier families have been demonstrated in several regions worldwide. Previous studies in high-endemic areas of Brazil and Africa have reported prevalence rates similar to values found in this study , 28, corThe seroprevalence rate for HTLV-1 described herein for family members of HTLV-1 carriers is markedly higher than the mean rate of infection of blood donors in Jujuy (1.0%) . This fiRegarding the routes of transmission, during years it was suspected that in Jujuy, HTLV-1 infection was acquired through lactation and sexual routes, similar to other regions in the world, but so far, no systematic studies had been carried out to clarify the importance of these routes for HTLV-1 dissemination in North Argentina. Herein, we present epidemiological and molecular evidence of this fact, evidencing high rates of intrafamilial transmission of HTLV-1, since transmission of the virus was demonstrated inside 19 of the 28 (67.9%) families studied .High levels of vertical (73.7%) and sexual (36.8%) transmission were demonstrated for family groups with more than one infected subject. Rates of HTLV-1 infection found in mother-child pairs (40.0%) were higher than values reported in Brazil, where the overall prevalence of HTLV infection in mother-child pairs is 20.4% [We found a high rate (70%) of HTLV-1 infection among children breastfed for more than 1 year . MoreoveSeveral studies have suggested that vertical transmission is more prevalent in women than in men , 35. NevOur results clearly corroborated that infection by HTLV-1 was mainly concentrated among women  and despEven though there were not significant differences between vertical and sexual transmission of HTLV-1 in our population and both routes proved to be relevant, we consider that the importance of sexual transmission might be underestimated. This may be due in part to the limitation of our study to include all sexual partners (husbands). These subjects could not be studied because it is a cultural trait that men of this population live away from their homes, working in diverse temporary jobs.Molecular analysis demonstrated that HTLV-1 isolates from this region of Argentina are highly preserved . NucleotMolecular and epidemiological analysis showed that most of the infected subjects, except for two of them, acquired the infection from a close relative , Table 1Although prevention of viral spread from the clusters depends on the identification of cases in the population, in Jujuy, as in overall Argentina, infection by HTLV is not currently considered as a public health problem and has been largely neglected. Regarding this fact, we agree with Costa et al. when they say that “HTLV is an example of the key neglected infectious diseases, which have been identified as priorities for research in Latin America and the Caribbean” and “Overall, the general ignorance of the virus increases the risk of transmission considerably” .Since year 2000, we have been carrying out a systematic investigation of HTLV infection in families of virus carriers detected in blood banks in the province of Córdoba (main inland province of Argentina). We demonstrated that intrafamily transmission of HTLV-1 also occurs in this non-endemic area of the country  and thanDespite North Argentina is recognized as a high endemic area for HTLV, no prevention is being carried out and although HTLV is regularly tested in blood banks, pregnant women are not systematically evaluated. We think, based on the observation of sociodemographic characteristics of the studied population (data not shown) and in agreement with other authors , 38, 39,The results presented herein demonstrate a strong familial dissemination of HTLV-1 in North Argentina and point out that this infection should be considered a matter of public health in this country.


Mammalian limb development is driven by the integrative input from several signaling pathways; a failure to receive or a misinterpretation of these signals results in skeletal defects. The brachydactylies, a group of overlapping inherited human hand malformation syndromes, are mainly caused by mutations in BMP signaling pathway components. Two closely related forms, Brachydactyly type B2 (BDB2) and BDB1 are caused by mutations in the BMP antagonist Noggin (NOG) and the atypical receptor tyrosine kinase ROR2 that acts as a receptor in the non-canonical Wnt pathway. Genetic analysis of Nog and Ror2 functional interaction via crossing Noggin and Ror2 mutant mice revealed a widening of skeletal elements in compound but not in any of the single mutants, thus indicating genetic interaction. Since ROR2 is a non-canonical Wnt co-receptor specific for Wnt-5a we speculated that this phenotype might be a result of deregulated Wnt-5a signaling activation, which is known to be essential for limb skeletal elements growth and patterning. We show that Noggin potentiates activation of the Wnt-5a-Ror2-Disheveled (Dvl) pathway in mouse embryonic fibroblast (MEF) cells in a Ror2-dependent fashion. Rat chondrosarcoma chondrocytes (RCS), however, are not able to respond to Noggin in this fashion unless growth arrest is induced by FGF2. In summary, our data demonstrate genetic interaction between Noggin and Ror2 and show that Noggin can sensitize cells to Wnt-5a/Ror2-mediated non-canonical Wnt signaling, a feature that in cartilage may depend on the presence of active FGF signaling. These findings indicate an unappreciated function of Noggin that will help to understand BMP and Wnt/PCP signaling pathway interactions. Limb bud development and the concomitant formation of limb skeletal structures are regulated by the intricate interplay and integration of various signaling pathways, with major roles played by the Shh, BMP, FGF, and Wnt/β-catenin pathways  were derived from Ror1 flox/flox Ror2 flox/flox MEF cells as described previously .Lysates for western blotting were prepared as follows: Growth medium was removed and cells were directly lysed in 100 mM Tris/HCl (pH 6.8), 20% glycerol, 1% SDS, 0.01% bromophenol blue and 1% 2-mercaptoethanol. Western blotting was performed according to manufacturer's instructions with minor adjustments . Antibodies were from Santa Cruz Biotechnologies: anti-Dvl2 (dephospho-Dvl2)–sc8026, anti-beta-Actin–sc1615-R, anti-Dvl3 sc8027 and from Cell Signaling Technologies: anti-Dvl2–CS3224. Anti-Ror2 antibody was a gift from Henry Ho (UC Davis)  in 1200 μl of DMEM. Cells were treated by 0.3% collagenase type II  before transfection, 50 μl of transfection mixture and 500 μl of collagenase treated RCS cells in DMEM were used per 1 well of 24 well plate. Transfection was carried out overnight, cells were treated according to the experimental scheme for 20 h, and samples were processed by Dual-Luciferase® Reporter Assay System according to the manufacturer instructions .+/− mice  and the zeugopod (radius and ulna) showed a consistent small lateral expansion  as well as the interphalangeal joints separating the phalanges are present  and frequently in BDB2 (NOG mutation). In addition, joint fusions are the hallmark of proximal symphalangism 1A (SYM1A) and multiple synostosis syndrome (SYSN1), two conditions caused by a different set of NOG mutations  2, an event dependent on the Ror1 and Ror2 receptors  cell line. RCS chondrocytes maintain a fully differentiated proliferating chondrocyte phenotype in culture, manifested by abundant production of cartilaginous extracellular matrix rich in sulfated proteoglycans and collagen type 2, but not collagen type 10 characteristic for hypertrophic chondrocytes , we have used an alternative readout—electrophoretic mobility shift of Dvl induced by Wnt-5a. Such mobility shift indeed represents a phosphorylation and can be effectively abrogated by alkaline phosphatase (AP) treatment Figure . Using tSignaling pathways do not operate as standalone units but functionally cooperate and interact. Inspired by the phenotypic resemblance of BDB1 and BDB2, inheritable syndromes caused by mutations in ROR2 or NOGGIN, respectively, we decided to study how Noggin, an inhibitor of BMP pathway, and non-canonical Wnt signaling, driven by Ror2 receptor, can interact. We could show that Noggin increased biological activity of Wnt-5a and rendered cells sensitive to Wnt-5a concentrations otherwise not causing cellular responses. This function was dependent on the presence of Ror2, but Noggin did not elicit a signal on its own via Ror2.In vitro, Ror2 and Bmpr1b were shown to interact and Ror2 is phosphorylated by Bmpr1b , which is mutated in BDA2 (Lehmann et al., The importance of the BMP pathway and its tight regulation by antagonists for digit development is underscored by the fact that the majority of human brachydactylies are caused by mutations in different members of this signaling network (reviewed in Stricker and Mundlos, Our work on RCS chondrocytes, a cell model for chondrocyte growth and differentiation that to some extent recapitulate the behavior of developing limb growth plate cartilage (Krejci et al., in vivo? In limb cartilage development, Wnt/PCP signaling appears to be involved at two steps: during condensation of cartilage elements, especially the digits (Gao et al., In vitro, FGFs inhibit chondrogenesis (Buchtova et al., in vivo (Montero et al., Where can such FGF-dependent Noggin-induced activation of Wnt-5a-Ror2 signaling pathway in chondrocytes take place In summary our data pinpoint a novel, yet unappreciated role for Noggin in sensitizing cells to Wnt-5a. The cellular mechanism by which Noggin accomplishes this effect on the Wnt-5a-Ror2 pathway remains to be elucidated.PK, SS, and VB designed research; MB, TR, OB, ZD, FW, AM, NC, and PK performed research; all authors analyzed data; and OB, SS, and VB wrote the paper.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.


Human sociality depends upon the benefits of mutual aid and extensive communication. However, diverse norms and preferences complicate mutual aid, and ambiguity in meaning hinders communication. Here we demonstrate that these two problems can work together to enhance cooperation through the strategic use of deliberately ambiguous signals: covert signaling. Covert signaling is the transmission of information that is accurately received by its intended audience but obscured when perceived by others. Such signals may allow coordination and enhanced cooperation while also avoiding the alienation or hostile reactions of individuals with different preferences. Although the empirical literature has identified potential mechanisms of covert signaling, such as encryption in humor, there is to date no formal theory of its dynamics. We introduce a novel mathematical model to assess when a covert signaling strategy will evolve, as well as how receiver attitudes coevolve with covert signals. Covert signaling plausibly serves an important function in facilitating within-group cooperative assortment by allowing individuals to pair up with similar group members when possible and to get along with dissimilar ones when necessary. This mechanism has broad implications for theories of signaling and cooperation, humor, social identity, political psychology, and the evolution of human cultural complexity. Even without incentives for individuals to defect, mutualism provides a different dilemma. When individuals differ in preferences or norms, it is harder to efficiently coordinate. The formation of reliable expectations of partner behavior that make coordination possible is therefore essential for the evolution of mutualism5.Much of the research on human cooperation has focused on the free-rider problem: how to maintain cooperation when individuals’ interests are opposed to those of the group. However, individual interests are often 6. If such mismatches are sufficiently frequent, Chris and Pat might be better off finding new partners with better-aligned interests. Successful cooperation requires resolution of this clash of preferences. Human societies are replete with dilemmas of this kind7, and the need to efficiently coordinate extends to many forms of collective action8. Institutions like punishment convert other social dilemmas into coordination dilemmas, expanding their importance. If individuals could assort on preferences and norms, cooperative payoffs may be increased. But often these traits are impossible to directly observe. When preferences are consciously held, individuals can merely signal them. But often individuals are not conscious of their preferences or realize their relevance too late to signal them.Consider, for example, a couple planning their Saturday. Chris wants to go to the opera; Pat wants to go to the monster truck rally. Each would rather do something with their partner than go it alone, but each has a different preference9. An extensive formal literature has developed exploring how arbitrary signals can facilitate assortment on unconscious norms and preferences15. Language can also serve as a marker for social coordination15, as can visible purchasing and fashion choices17.One solution is the evolution of ethnic marking. Anthropologists have long argued that ethnic markers may signal group membership and improve cooperative outcomes20. Previous work has illustrated how leaders may use ambiguous language to rally diverse followers18, politicians may use vague platforms to avoid committing to specific policies21, and suitors may mask their flirtations to be viewed innocuously (or at least to provide plausible deniability) if their affections are unreciprocated22.Communication is implicated in all these solutions. However, much communication is ambiguous. Is this ambiguity merely the result of constraints on the accuracy of communication? It may naïvely appear that communication should have clarity as its goal. However, purposeful ambiguity may allow signalers flexibility and plausible deniability27. To be clear, we focus on those aspects of individual variation for which similarity enhances cooperation—these include norms, values, and identity29. Similarity on these dimensions matter even when the cooperative benefit is maximal for diverse rather than homogenous groups30. Scenarios in which individuals are unable to effectively assort on norms or attitudes are common, especially in complex societies (for example in business or education settings), but also in smaller societies. A signaling system that enables group members to communicate relative similarity only when similarity is high while retaining a shroud of ambiguity when similarity is low is likely to have been advantageous for much of human history.We propose more broadly that ambiguity may enable coordination and thereby enhance cooperation. Consider again the use of signals for assortment on cooperative norms. Overt signals like ethnic markers are useful in some contexts, where the adaptive problem is to delimit a set of partners who subscribe to the same broad behavioral norms and to categorically avoid interaction with those who do not. However, the “all-or-nothing” character of such signals can also foreclose valuable partnerships in different contexts. Signals that communicate similarity can also communicate difference, and this can be damaging for within-group cooperation. Individuals may benefit from not foreclosing relationships with less similar group members, so as to successfully cooperate with them in other contexts. Although any two individuals within a group can cooperate when it is mutually beneficial, pairs who are more similar can cooperate more effectively, generating larger benefitsCovert signaling is the transmission of information that is accurately received by its intended audience but obscured when perceived by others. A common example is “dog-whistling,” in which statements have one meaning for the public at large and a more specialized meaning for others31. Such language attempts to transmit a coded message while alienating the fewest listeners possible. A possibly much more common form of covert signaling is humor. According to the encryption model of humor33, a necessary component of humorous production is the presence of multiple, divergent understandings of speaker meaning, some of which are dependent on access to implicit information. Only listeners who share access to this information can “decrypt” the implicit understandings and understand the joke. Because the successful production of a joke requires access to that implicit information, humor behaves in manner similar to “digital signatures” in computer cryptography, verifying the speaker’s access to that information without explicitly stating it. Laughter may also serve as an honest signal of a shared sense of humor34, and thus of similarity on a variety of traits. While not all humor has this form, a substantial amount of spontaneous, natural humor does33. We allow that other types of identity signals16 may be also be covert.We propose that covert signaling serves an important function in facilitating effective cooperation within groups by allowing individuals to assort on norms when possible while avoiding conflict with dissimilar individuals when their assistance is necessary. In the remainder of this paper, we define the logic of covert signaling. We analyze the conditions for selection to favor covert signaling over overt signaling, in which information about an individual’s traits is more transparent. Using a formal model, we show that covert signaling can be favored. It sacrifices transparency for the sake of maintaining working relationships with dissimilar individuals. Although covert signals are less accurate than overt signals, we show that the increased ambiguity can in some cases be advantageous. This characterization of covert signaling in terms of cooperative assortment may therefore help to explain forms of communication and coordination such as coded speech and humor, as well as for the flexibility of human sociality more generally. Nevertheless, covert signaling is not always advantageous. For example, if it is possible to freely choose cooperative partners from a large pool, overt signaling may be more advantageous, as individuals will be better able avoid dissimilar partners. Our model yields specific predictions about default attitudes toward strangers in the absence of clear signals, with implications for understanding differences between contemporary political affiliations.similar. Otherwise they are deemed dissimilar. Pairs of similar individuals can more effectively coordinate and obtain higher payoffs. The probability that two randomly selected individuals have similar trait profiles is given by s.We consider a large population of individuals who have already solved the first-order cooperation problem of suppressing free riders, and can instead focus on maximizing the benefit generated by cooperation. Although individuals all belong to the same group, they also vary along many trait dimensions and thus share more in common with some individuals than others. Pairs of individuals whose trait profiles overlap to some threshold degree are deemed Our model proceeds in discrete generations, with each generation subdivided into two stages. In the first stage, individuals signal information about their trait profiles to the other members of the group, and those who receive signals form attitudes about their senders. In the second stage, individuals interact in one of two ways and receive payoffs conditional upon similarity as well as attitudes formed in the first stage.p of the time. Overt signals are received by a fraction R of the population and explicitly signal similarity or dissimilarity. Covert signals in contrast are received by a fraction r < R of the population and have content contingent upon the similarity of the sender and receiver. See Fig. Individuals produce either an overt or covert signal of their underlying traits. We study a family of continuous signaling strategies in which covert signals are produced a fraction aXY indicates the probability of mapping signal X ∈ {Similar, None, Dissimilar} to attitude Y ∈ {Like, Neutral, Dislike}. The total receiver strategy can be represented by a table . As we show later, these synergistic costs are very important to the overall signaling dynamics.In the forced choice context, dyads form at random with respect to attitudes, but attitudes directly influence payoffs. This context entails a baseline payoff of 1 for both individuals. However, attitudes adjust payoffs, because negative attitudes make it harder to interact. When one individual dislikes the other, he makes the interaction more difficult than it must be and thereby imposes a cost −q be the relative importance of the free choice context and 1−q the relative importance of the forced choice context. These two contexts are starkly different, presenting the clearest investigation of the hypothesis that covert signals trade worse performance in assortment contexts, in which norms influence payoffs, for better performance in forced contexts in which attitudes influence payoffs. Real contexts are some mix of these extremes, and the parameter q allows us to explore the range of mixes. We note also that payoffs depend on similarity and attitudes, which result in part due to sender and receiver strategies, but the payoffs are not directly derived from those strategies. When dyads form for cooperation, both parties are senders and receivers, each having had the opportunity to signal to and receive from one another. While we can imagine cases of sender-receiver asymmetries in terms of cooperative payoffs, we have chosen not to model these in this particular analysis.Let p′ and receiver strategy matrix a′ in a population with common-type strategy {p, a}. The expected payoff to this individual is:p′, a′), Pr, and Pr. In the mathematical appendix, we show how to define these probabilities, using the assumptions above. The resulting general payoff expression is very complicated. In the following section, however, we are nevertheless able to analyze it by considering invasion and stability of relevant combinations of signaling and receiver strategies.With the assumptions above, we can define a general payoff expression for a rare individual with signal strategy r is not too large) and avoidance of disliked individuals is not too efficient .Covert signals require a sufficient proportion of receivers to default to neutral attitudes. If everyone defaults to disliking, then covert signals can produce no benefit. Defaulting to neutral is favored under a wide range of conditions, provided that covert signals are sufficiently hard to receive (s) must be sufficiently low, because this creates the risk of being disliked by dissimilar individuals.Covert signals require that the cost of being disliked in the forced choice context be sufficiently high. This also means that baseline similarity in the population  = 0:And this is never satisfied, for any α is above this value, overt signals are favored. When it is below it, covert signals are favored. Why? When receivers are relatively generous, and there is sufficient dissimilarity in the population, covert signals reduce costs by avoiding being disliked. If generous receivers are relatively rare, however, then covert signals can actually do worse than overt signals, because they are received less often than overt signals, r < R. If r/R is sufficiently small, overt signals are favored for a wide range of values of α. If however r = R and covert signals have no disadvantage in audience size, then overt signals are never favored in this context, no matter the amount of similarity s.When α will fall below this threshold α, in the forced choice context only, is just δ > 0. Therefore the forced choice context always favors smaller values of α, provided there is any negative synergy between disliking and being disliked. Otherwise α is neutral and does not move at all, based on payoff dynamics. Why does this context always favor generous receivers? There is no advantage to be had in disliking people in this context, because assortment does not depend upon attitudes. Payoffs depend upon attitudes, however, and mutual dislike results in poor payoffs. Therefore, it pays to be generous in attitudes towards those one has no information about.Now the crucial question is when α = 1, is favored by this context, because it also increases assortment with similar individuals. Therefore this context is hostile to covert signals and to the receiver strategy that favors them.In the free choice context, attitudes influence assortment but do not directly influence payoffs. Instead, hidden norm similarity influences payoffs. The free choice context favors overt signals over covert signals, because overt signals increase assortment—such signals are easier to receive and more effectively discriminate similarity. The churlish receiver strategy, p in this context is always negative and that the gradient for α in this context is always positive. The gradients in this context are:Z is a normalizing term and the symbols vD = 1 − wD and vL = wL − 1 are used for compactness of notation. By inspection, every term after the leading (−1) in g(p) is positive, for all allowed values of the variables, and so the gradient is always negative. Similarly, every term in g(α) is positive, and so the gradient is always positive. Therefore payoff incentives in the free choice context never favor covert signals and always favor churlish receivers.To support the above statements, we demonstrate that the gradient for s(1 − s). This indicates that intermediate similarity more strongly favors overt signals, unlike the situation in the forced choice context, in which high similarity favored overt signals. The reason that the variance matters now is that payoffs depend directly upon similarity, not upon attitudes. The more variance in similarity in the population, the greater the advantage of efficient assortment.While this context always favors overt signalers and churlish receivers, the strength of the incentives may vary. First, both gradients are proportional to the variance in similarity, R, the probability an overt signal is received, and the assortment proportional odds wD and wL. Reducing R reduces overt signalers’ advantage, because it makes signals less valuable overall. Making either wD or wL closer to 1 makes assortment, based on attitudes, less effective. This also reduces overt signalers’ advantage.Overt signals have the advantage in this context, because they are better at assortment. Therefore, any change in variables that reduces the accuracy of assortment overall will reduce overt signalers’ advantage. The important variables are q = 0.5. The other parameters then shift the strength of incentives in each context to influence overall dynamics. For other values of q, the strength of incentives would have to shift as well to overcome weight given to each context.When both contexts matter, the joint dynamics take on one of three characteristic regimes. First, covert signals both invade and are evolutionarily stable. Second, overt signals both invade and are evolutionary stable. Third, a mixed equilibrium exists at which covert and overt signals coexist in the population. Figure r/R low), similarity is sufficiently rare (s low), and assortment  not too efficient, covert signals can both invade and are an ESS. This situation is shown in the lefthand plot. While dynamics do not favor covert signals when α is large, near the top of the phase space, dynamics in that region favor smaller values of α. Eventually, α becomes small enough to allow covert signals to invade and reach fixation. In many cases, a small amount of churlish receiver strategy, α > 0, persists.When covert signals are sufficiently noisy |p=1 = 0 and g(α)|p=1 = 0. These cannot in general be solved analytically. So we solve the system numerically, in order to illustrate the range of joint dynamics. Values of q less than q greater than p. Therefore A more general view of the dynamics is available by considering the boundary conditions that make covert signaling an ESS. Recall that d, is needed to favor covert signals. Therefore increasing d makes it easier for covert signals to be an ESS. However, the rate of similarity, s, favors overt signals. It is of value to note that d cannot compensate for s—if s is large, then steeply increasing costs d will not favor covert signals. We show this relationship in Fig. s, the impact of increasing d diminishes rapidly. Therefore some cost d is necessary for covert signals to evolve and be stable, but these costs cannot easily compensate when similarity is sufficiently common.We use r/R in covert/overt signals and the efficiency of assortment, as measured by wL and wD. Figure wL = 1/wD, as functions of r/R. Covert signals are favored when r/R is small, as explained in the previous sections. But when assortment is very efficient, such as wL = 2 near the bottom of the plot, it requires very low values of r/R to compensate in favor of overt signaling.Consider another important pair of dimensions: the ratio of transmission rates 39.The dynamics of cooperation are more complicated than implied by models in which maximal benefits accrue to those who can simply avoid free riders. Not all cooperators are equal. Individuals vary, making assortment among cooperators important. Circumstances also vary. When individuals must occasionally collaborate with those outside their circles of friends, it can be critical to avoid burning bridges with dissimilar members of one’s group. Covert signaling makes this possible, and this may be why phenomena like humor are observed in all human societies, at both small and large scalesWe have shown that covert signaling is favored when forced choice scenarios are common, when similarity is low, when the cost of being disliked is high, and when covert signals are sufficiently noisy to make the meaning of a signal’s absence ambiguous. We emphasize that covert signaling can be favored even though it is less effective than overt signaling at communicating similarity, because it simultaneously avoids communicating dissimilarity. Although we have focused our attention on the initial establishment of cooperative relationships via signaling, we also note that people can change over time; they may grow more similar to one another or further apart. Covert signals may be important for the continued maintenance of a relationship, or for its reestablishment after prolonged absence.13. In fact, it might be argued that burning bridges with dissimilar out-group members is as much a goal of overt signals like ethnic markers as is attracting similar in-group members.Our model points to interesting transitions from inter- to intra-group assortment dynamics. As noted, overt signaling systems are favored when the ability to assort on attitudes is high, and when being disliked by dissimilar individuals carries little risk. This is precisely the kind of situation that is assumed to obtain in inter-group assortment, where overt signals such as ethnic markers are used to discriminate between similar and dissimilar individuals. In these between-group contexts, the difference between similar and dissimilar individuals is so great that attempting to coordinate with dissimilar others is not worth the effort, and one can afford to burn bridges with them in order to ensure that similar others are aware of their similarityIntra-group assortment, however, is not simply a matter of scaling down inter-group dynamics. Rather, we must already presume some baseline level of similarity resulting from inter-group assortment; for there to be a group within which to assort, some degree of similarity should already be in place that defines that group, such as the shared interaction norms, communication systems, etc. that ethnic markers are thought to ensure. The benefits of further assorting on the basis of more nuanced similarity are therefore likely to be marginal relative to random assortment within the group. When such benefits are small but the costs of being disliked are high, covert signaling is favored.s, need not reflect some number of discrete types in the population, but can instead refer to a level of selectivity in how much a given pair of individuals must have in common to be considered “similar.” That is, s refers to the proportion of the population that could be considered similar to a focal individual in a given context, with higher values indicating a looser concept of “similar” than lower ones. Changes to s can have a significant impact on the overall dynamics of the system. When s is large, the focus is on avoiding rare dissimilar individuals, and overt signals will be favored. As s decreases, the criteria for considering a potential partner sufficiently similar to reap the benefits of enhanced coordination become stricter, and the utility of covert signals increases.Relatedly, we emphasize that the probability of similarity, 40. Consequently, expected similarity will be lower, signal fidelity will be noisier, and assortment on attitudes will be less efficient. These are precisely the conditions in our model associated with the evolution of covert signaling. In large, diverse populations, covert signaling may sustain social cohesion and prevent burning bridges between individuals or groups that must occasionally collaborate. That said, covert signals are not necessarily rare in small-scale societies. Our own experiences in the field and conversations with other researchers indicate that they occur with some regularity. Our model can help to identify contexts in which covert signaling should or should not be expected.An interesting direction for future exploration is how these dynamics might respond to increased social complexity. In the larger and more complex societies associated with the development of agriculture, and particularly in the last few centuries, interactions with strangers are more frequent and occur across many contexts, necessitating strategies for temporary assortment29. If the need to cooperate with dissimilar individuals is unlikely or if similar individuals are common, then overt declarations of identity should be expected. On the other hand, if burning bridges is both costly and likely given an overt signaling strategy, we should expect identity to be signaled much more subtly. In reality, increasing levels of specificity may be signaled in increasingly covert ways, and without all received signals actively inducing a change in disposition toward the sender. A related signaling strategy, not covered by our model, might facilitate liking between similar individuals but only indifference otherwise. Casual, coarse-grain identity signaling may often take this form, as in cases of fashion adoption or pop culture allegiances. It would be interesting to investigate how common these “semi-covert” signals are in small-scale communities, as they seem pervasive in complex industrialized societies.Identity signaling, whether overt social markers or more covert communication, can be used by individuals looking to find others similar to themselves and to avoid being mistaken for something they are not43. In our model, a default attitude to dislike was linked with overt signaling, which we in turn associate with the preservation of strong between-group boundaries. In contrast, a default attitude of neutral was associated with covert signaling, and with the avoidance of burned bridges to facilitate more widespread within-group cooperation. As a broad generalization, our analysis suggests that conservatives may be operating under the assumptions of stronger ingroup/outgroup boundaries, increased expectations of similarity toward those they signal, and lower costs to being disliked by dissimilar individuals. In contrast, liberals may be operating under the assumptions of a more broadly defined ingroup, limited expectations for similarity toward those they signal, and higher costs to being disliked by dissimilar individuals. Lending modest support to this idea is the finding that conservatives appear to have a stronger “need for cognitive closure” (reviewed in ref.42), which is associated with, among other things, a distaste for uncertainty and ambiguity. The modeling framework we present in this paper may thus be useful in understanding patterns of differences between groups, including but not limited to political affiliation.Our model additionally helps make sense of findings from political psychology suggesting that people in the industrialized West who identify as conservative or right-leaning tend to view ambiguous people as hostile, while those identifying as liberal or left-leaning tend view ambiguous people as neutralare able to detect the signal some of the time, and therefore update their disposition to disliking the covert signaler. Adding an additional parameter to account for this possibility does not qualitatively change our analysis. But it may create conditions where a non-signaling “quiet” strategy could invade. In addition, we ignore the possibility of strategic action on the part of the receiver to either improve coordination or to avoid partnering with dissimilar individuals entirely. We assumed that a pairing of dissimilar partners would simply lead to an unsuccessful collaboration, but such a pairing might instead lead each individual to pursue more individualistic interests. At the population level, we assumed that all individuals had an equal probability of encountering similar individuals, and that all similar and dissimilar individuals were equivalent. In reality, some individuals may be more or less likely to encounter similar individuals, perhaps related to differences in the tendency to be conformity- versus distinctiveness-seeking44, or reflecting minority-majority dynamics46. Exploration of this variation opens the door to evaluating signaling and assortment strategies in stratified groups. All of these limitations provide avenues for future research that will build upon the central findings reported here.Ours is the first formal model of covert signaling. As such, it necessarily involves simplifying assumptions concerning the nature of signaling and cooperative assortment. For example, while we have allowed for covert signaling errors in the form of failed transmission to similar individuals, we have not included the converse form of error, where dissimilar individuals In a population where individuals vary and burning bridges is costly, overtly announcing precisely where one stands entails venturing into a zone of danger. Covert signaling, as in the case of humor or otherwise encrypted language, allows individuals to effectively assort when possible while avoiding burned bridges when the situation calls for partnerships of necessity.Supplementary Information


TNBC) remains challenging due to the underlying heterogeneity of this disease coupled with the lack of predictive biomarkers and effective targeted therapies. Intratumoral heterogeneity, particularly enrichment for breast cancer stem cell‐like subpopulations, has emerged as a leading hypothesis for systemic therapy resistance and clinically aggressive course of poor prognosis TNBC. A growing body of literature supports the role of the stem cell renewal Hedgehog (Hh) pathway in breast cancer. Emerging preclinical data also implicate Hh signaling in TNBC pathogenesis. Herein, we review the evidence for a pathophysiologic role of Hh signaling in TNBC and explore mechanisms of crosstalk between the Hh pathway and other key signaling networks as well as their potential implications for Hh‐targeted interventions in TNBC.Treatment of triple‐negative breast cancer ( Triple‐negative breast cancer (TNBC), defined immunohistologically by the absence of estrogen (ER), progesterone (PR), and HER2 receptors, is a heterogeneous entity comprising subtypes with distinct clinical and molecular features The advent of high‐throughput sequencing technologies and bioinformatics has improved our understanding of the molecular diversity of TNBC and opened new avenues for potential targeted therapeutic strategies +/CD24−/low phenotype The identification of breast cancer cells with self‐renewal and multilineage differentiation capacity led to the hypothesis that tumor growth and heterogeneity are driven by subpopulations of breast cancer stem cells (BCSCs) Because of their intrinsic chemoresistance β signaling and embryonic pathways, Wnt, Hedgehog (Hh), and Notch have been recognized as key signaling events driving tumorigenesis in BCSCs Deregulation of receptor tyrosine kinase (RTK), TGF‐Herein, we review the evidence supporting a pathogenic role for Hh signaling in TNBC. We also discuss mechanisms of Hh pathway activation highlighting the critical contribution of extrinsic mediators and other key oncogenic pathways to deregulated Hh signaling in TNBC. Insight into these mechanisms and their potential implication in bypass signaling promoting resistance to Hh inhibitors is crucial for the design of effective Hh‐ and BCSC‐targeted therapeutic strategies in TNBC.The Hh pathway plays an essential role in embryonic patterning and is involved in stem cell renewal, tissue regeneration, and repair A) or as truncated repressors (GLIR), whereas GLI1, lacking a repression domain, exists only as a full‐length transcriptional activator (GLIA) Three glioma‐associated oncogene (GLI) transcription factors, GLI1, GLI2, and GLI3, are the effectors of Hh signaling and regulate the expression of the pathway target genes A and GLI3R are considered to be the primary transcriptional activator and repressor of Hh signaling during embryogenesis, respectively; conversely, GLI1A is dispensable for development R‐mediated suppression of Hh target gene transcription is a critical event for mammary bud formation R in the mammary gland parenchyma GLI2In contrast, GLI1 and GLI2 are considered to be the main oncogenic effectors of Hh signaling, with lack of data supporting the contribution of GLI3 to breast carcinogenesis β), and casein kinase 1 (CK1)—creates binding sites for adaptor protein Beta‐transducin repeat containing E3 ubiquitin protein ligase (β‐TrCP), and marks GLI proteins for ubiquitin‐mediated proteasomal degradation or processing into repressor forms The GLI‐driven transcriptional program is modulated by intrinsic and extrinsic regulation of GLI activity through post‐translational modification, such as phosphorylation, ubiquitin‐mediated degradation, acetylation, as well as regulation of nucleocytoplasmic shuttling  or SMO (type II) independent of GLI, are classically considered as noncanonical signaling mechanisms Under this definition, mechanisms of canonical Hh signaling in carcinogenesis include ligand‐dependent signaling, further classified as paracrine, autocrine or reverse paracrine, and ligand‐independent signaling Fig. .Ligand‐dependent autocrine signaling occurs when cancer cells are activated by their secreted Hh ligand GLI amplification or extrinsic potentiation of GLI transcriptional activity) Ligand‐independent Hh signaling results in GLI activation independent of the presence of Hh ligand via mutational (loss of function PTCH mutations or activating SMO mutations) SHH, PTCH, and GLI1Current evidence suggests that Hh signaling in breast cancer overall, and TNBC specifically, is not mutation‐driven; infrequent somatic mutations in α, and downstream ERK1/2 signaling has been reported in mammary luminal epithelial cells in response to noncanonical PTCH‐dependent Hh pathway activation α signaling likely exert a mitogenic effect on luminal progenitor cells leading to mammary ductal elongation from terminal end buds at puberty Noncanonical Hh signaling mediated by PTCH (type I) has been shown to exert an antiapoptotic effect in nonmammary cells via disruption of proapoptotic DRAL‐caspase activity or inhibition of H‐Ras‐induced apoptosis by modulation of tumor suppressor protein Tid1 SMO‐dependent (type II) noncanonical Hh signaling promotes endothelial cell tubulogenesis, fibroblast and non‐mammary cancer cell migration by activation of small GTP‐ases Rho and Rac1 in vitro and tumor initiation in vivo has been reported GLI1 expression is characteristically observed in claudin‐low breast cancer subtype mammospheres and tumors known to be highly enriched for BCSCs Extensive preclinical data highlight the key contribution of Hh signaling in cancer stem cell reprogramming in TNBC The role of Hh signaling in EMT regulation has been described in several malignancies including ovarian In vivo studies in murine mammary glands with constitutive Hh signaling reveal prominent ductal hyperplasia and dysplasia caused by expansion of cell populations exhibiting basal cytokeratin, P63, and progenitor marker expression along with loss of basolateral polarity reminiscent of EMT GLI1 expression further corroborates the implication of the Hh pathway in EMT‐mediated tumorigenesis Expression of mesenchymal markers and EMT transcription factors in TNBC cells is under direct regulation of Hh effector GLI1; abrogation of Hh signaling leads to loss of the mesenchymal phenotype and restores E‐cadherin and epithelial marker keratin expression Preclinical studies provide strong evidence that deregulated Hh signaling confers a more aggressive tumor phenotype in TNBC. Activation of the Hh pathway enhances the proliferation, invasion, and migration of TNBC cells In vitro resistance to doxorubicin, paclitaxel, and cisplatin was shown to be mediated by GLI1 and was associated in TNBC cells with upregulation of multidrug resistance protein 1 (MDR1) and breast cancer resistance protein (BCRP), both transcriptional targets of Hh signaling , highly proliferative tumors and TNBC histology has been reported β, RAS/MAPK signaling pathways, the extracellular matrix protein osteopontin (OPN), and transcription factors—NF‐kB, FOXC1, and Hypoxia‐induced factor (HIF)‐1α—contribute to deregulated Hh signaling, thereby, leading to acquisition of the mesenchymal phenotype, enhanced growth, and invasion in TNBC TGF‐β/SMAD signaling, plays a key role in the pathogenesis of osteolytic metastases in TNBC IHH transcription, it further potentiates the vicious cycle of tumor growth and osteolysis mediated by TGF‐β/GLI2 Increased GLI2 expression and upregulation of its target gene PTH‐rP, in response to TGF‐α in response to tumor microenvironment hypoxia promotes the mesenchymal switch in TNBC cells via Hh signaling activation α‐Hh crosstalk and hypoxia‐induced EMT. It is unclear, though, whether increased GLI1 expression in response to hypoxia involves direct regulation of GLI1 expression by HIF‐1α downstream of SMO, as was reported in other cancers αβ by OPN was shown to enhance the nuclear translocation and transcriptional activity of GLI1 The tumor microenvironment is a key contributor to tumor growth and progression, and the importance of stromal signaling in regulating epithelial‐mesenchymal plasticity in breast cancer cells is increasingly being recognized Peiris‐Pagès et al.Paracrine Hh signaling has been implicated in stromal‐dependent tumor growth in breast cancer in vivo studies in murine models of basal‐like TNBC also demonstrate stromal‐specific Hh target gene expression driven by tumor‐secreted ligand contributing to tumor growth, invasion, and metastasis Hh ligand secreted by TNBC cells was shown to activate paracrine Hh signaling in neighboring bone marrow stromal cells promoting their release of pro‐tumorigenic cytokines Hh‐targeted therapies currently in use, and those that have been or are presently the subject of clinical investigation, consist of inhibitors directed against the most “druggable” pathway target, SMO . Although clinical benefit from SMO inhibitors has been established in basal cell carcinoma (BCC) Failure of SMO inhibitors in these malignancies can be at least partly explained in that aberrant Hh signaling in these cancers, unlike medulloblastoma or BCC, is not driven by mutational mechanisms effectively targeted by SMO inhibition. Instead, deregulation of Hh signaling in these malignancies, not unlike breast cancer, is likely due to multiple coexisting mechanisms of pathway activation comprising SMO‐dependent autocrine and/or paracrine signaling and SMO‐independent signaling through direct regulation of GLI expression or activity Table . LikewisFew SMO inhibitor trials on solid tumors have allowed enrollment of breast cancer patients and only three studies  investigated the use of SMO inhibitors, alone or in combination, exclusively in breast cancer. Pending formal presentation of all study results, preliminary findings suggest limited therapeutic efficacy, and with early closure of two of the breast cancer trials, it is unlikely that we will gain further insight into the clinical value of SMO inhibitors in TNBC , hedgehog pathway inhibitors (HPI) 1‐4, arsenic trioxide (ATO) and indirect inhibitors, arcyriaflavin C, physalin B/F, staurosporinone, pyrvinium, selective HDAC1/2 inhibitors, I‐BET151, and JQ1.A  and disruption of ciliary processes required for GLI function (HPI 4) These drugs directly bind to and antagonize GLI transcription factors by interfering with their processing, ciliary trafficking, or DNA binding. GANT58, GANT61, and Glabrescione B (GLaB) are thought to inhibit the transcriptional output of Hh signaling by interfering with GLI DNA‐binding α‐mediated GLI1/2 degradation, respectively, δ/MAPK‐mediated GLI activation GLI transcription Drugs in this category target GLI proteins indirectly by interfering with mechanisms affecting their transcriptional activity, including post‐translational modifications leading to GLI activation/degradation, and epigenetic regulation. For instance, forskolin and pyrvinium enhance PKA and CK1Of all GLI inhibitors, only GANT58 and GANT61 have been evaluated in TNBC cells with more compelling data on the activity of the latter drug Indirect GLI inhibitors have emerged as attractive Hh targets mostly due to their favorable characteristics for druggability compared to GLI transcription factors per se, however, their Hh inhibitory activity has not been investigated in TNBC. Taking into account the existence of several distinct oncogenic signals leading to GLI activation in TNBC and the uncertainty that extrinsic GLI activation is subject to similar regulatory mechanisms as endogenous GLI, the efficacy of indirect inhibitors remains unclear.Furthermore, the functional redundancy of GLI proteins and the implication of both GLI1 and GLI2 in the pathogenesis of TNBC may limit the application of any direct or indirect inhibitor exhibiting preferential activity against one or the other but not both GLI proteins. Therefore, demonstration of potent GLI1/GLI2 inhibitory activity at cytotoxic concentrations will be an important criterion when selecting drugs for clinical development in TNBC.β, RAS/MAPK signaling, OPN, NF‐kB, FOXC1, and HIF‐1α, in driving Hh pathway activation downstream of SMO in TNBC was also reviewed.In this review, we have discussed the current evidence and recent studies highlighting the role of the Hh pathway in the pathogenesis of TNBC and explored mechanisms of deregulated Hh signaling in this breast cancer subtype. The key contribution of extrinsic oncogenic signals, mediated by TGF‐Although we have limited information on the use of SMO inhibitors in breast cancer in the clinical setting, the preclinical data we presented suggest resistance to SMO inhibitors in TNBC and instead provide the rationale for a GLI‐targeted approach.The contribution of GLI activation in circumventing SMO inhibition in BCC or medulloblastoma and the recognition of the role of direct mechanisms of GLI activation downstream of SMO in drug resistance, tumor growth, and progression in other solid tumors have provided impetus for identifying potent GLI inhibitors. So far, several direct and indirect inhibitors of GLI have been discovered; yet, their suitability for clinical use is still uncertain. Based on currently available data, the use of direct inhibitors with GLI1/GLI2 activity such as GANT61 may represent the most effective approach at targeting Hh signaling in TNBC. Determining whether epigenetic and post‐translational regulatory mechanisms targeted by indirect GLI inhibitors can be overcome by extrinsic GLI activation will be important to ascertain the potential activity of these drugs in TNBC.Additional questions warranting further investigation include:Which TNBC patients might benefit from Hh‐targeted interventions?Hh pathway deregulation may not necessarily play a pathogenic role in all subtypes of TNBC; hence, Hh inhibition may prove to be of limited value among unselected patients. A recent analysis of a dataset of over 330 tumor samples yielded evidence for preferential GLI1 expression in claudin‐low relative to other basal tumors and higher sensitivity to GLI inhibition was observed in claudin‐low cells lines. Recognizing the heterogeneity of TNBC and limiting future studies to specific molecular subtypes, such as claudin‐low subtype, enriched for BCSCs may potentially identify subgroups of TNBC patients who might benefit from Hh‐targeted interventions.In addition, no biomarker predictive of clinical benefit from SMO antagonists in other solid tumors has been identified to date. It is uncertain as to whether or not this endeavor has been unsuccessful due to ineffective Hh signaling inhibition in these cancers, which, not unlike TNBC, are characterized by downstream pathway activation not targeted by SMO antagonists. As the development of GLI inhibitors moves forward, integration of putative predictive biomarkers in clinical studies evaluating the use of these drugs in TNBC will be of paramount importance.When should Hh‐targeted intervention be timed during treatment?Data presented in this review implicate chemotherapy‐induced Hh signaling activation in BCSC expansion and tumor regrowth in TNBC. It is unclear whether chemotherapy “selects” for the survival of BCSCs subpopulations in TNBC or promotes stromal metabolic stress leading to the acquisition of stem‐like phenotype by differentiated tumor cells. Nevertheless, based on the current evidence, targeting Hh signaling after completion of chemotherapy seems intuitive and the application of Hh‐targeted strategies in the future may conceivably be limited to TNBC patients who have failed to achieve clinical or pathologic complete response to neoadjuvant chemotherapy. Studies evaluating mechanisms of deregulated Hh signaling following chemotherapy in TNBC are scarce. In the absence of these data, one cannot assume that these mechanisms are identical to those driving Hh signaling in chemotherapy‐naïve TNBC. Further insight into the signaling machinery contributing to deregulated Hh signaling following chemotherapy in TNBC, both in preclinical models of residual tumor and clinical tumor samples after neoadjuvant chemotherapy, is therefore necessary.Should drug combination strategies be used when planning Hh‐targeted therapeutic interventions in TNBC?Hh, TGF‐β, PI3K/AKT, RAS/MAPK signaling possess overlapping functionality in TNBC; potential crosstalk between Hh and these signaling pathways could potentially circumvent pharmacological Hh inhibition. Thus, uncovering mechanisms of crosstalk between Hh signaling and other key tumorigenic pathways in TNBC can provide the basis for the development of effective combination therapies. A synergistic therapeutic effect has been reported in non‐mammary solid tumors when combined targeting of GLI and PI3K/AKT or GLI and EGFR signaling was used Is there a role for stromal Hh signaling inhibition in TNBC?Despite the potential resistance of TNBC cells to SMO modulation, stromal paracrine signaling is, on the other hand, SMO‐dependent and consequently may be amenable to modulation by SMO antagonists. Classically, Hh signaling in the stromal microenvironment was thought to foster tumor growth and invasion, and corroborative preclinical data have revealed potential benefit for the use of SMO modulation and stromal inhibition in TNBC The authors declare they have no competing interests.


Molecular and cellular study demonstrated that Asp-UA significantly down-regulated the expression of cell adhesion and invasion molecules including integrin α6β1, CD44, MMP-2, MMP-9, COX-2, EGFR and ERK proteins, and up-regulated the epithelial markers “E-cadherin” and “β-catenin”, and PTEN proteins. Furthermore, Asp-UA (80 mg/kg) reduced lung metastasis in a 4T1 murine breast cancer metastasis model more efficiently, which was associated with a decrease in the expression of CD44. More importantly, we did not detect side effects with Asp-UA in mice such as weight loss and main viscera tissues toxicity. Overall, our research suggested that co-drug Asp-UA possessed potential metastasis chemoprevention abilities via influencing EMT and EGFR-mediated pathways and could be a more promising drug candidate for the prevention and/or treatment of breast cancer metastasis.Metastasis currently remains the predominant cause of breast carcinoma treatment failure. The effective targeting of metastasis-related-pathways in cancer holds promise for a new generation of therapeutics. In this study, we developed an novel Asp-UA conjugate, which was composed of classical “old drug” aspirin and low toxicity natural product ursolic acid for targeting breast cancer metastasis. Our results showed that Asp-UA could attenuate the adhesion, migration and invasion of breast cancer MCF-7 and MDA-MB-231 cells in a more safe and effective manner  Breast cancer is the second leading cause of cancer mortality worldwide . Every yMetastasis is thought to begin with the epithelial–mesenchymal transition (EMT), a cascade of events in which tumor cells lose many of their ‘epithelial’ characteristics and become more like mesenchymal cells with the ability to spread and invade into the extracellular matrix (ECM) . Extensiet al. reported that ursolic acid can inhibit the metastasis of colorectal cancer through the suppression of multiple biomarkers linked to invasion, angiogenesis, and metastasis + calculated for C39H54O6Na, 641.3813, found, 641.3820. NMR spectra of Asp and Asp-UA with indicated peaks were illustrated in To search for more safe and effective drug candidates for the prevention and treatment of cancer metastasis, we firstly synthesized the conjugate of the “old drug” aspirin and natural product UA. UA and Asp were used as the parent compounds and the structure modifications were performed at the C-28 position of UA. The synthesis route of conjugate Asp-UA was depicted in Figure 50 value of 72.16, 63.94 and 62.03 μM, respectively , respectively after treatment of 10 and 20 μM Asp-UA. Meanwhile, the adhesion of MCF-7 cells treated with 20 μM of UA were inhibited only by 34% (p < 0.05). Compared with the control group, the adhesion of MDA-MB-231 cells was reduced by 38% and 60% (p < 0.01) in 10 and 20 μM Asp-UA-treated groups, respectively. Interestingly, Asp alone exerts a slight effect in the tested concentrations  and 9.7%, respectively  on breast cancer cell surface were evaluated after exposure to UA, Asp, and Asp-UA, respectively. The protein expressions were quantitatively analyzed by flow cytometry. Among all the detecting proteins, we found integrin α6, β1 and CD44 proteins in MCF-7 and MDA-MB-231 cells were significantly down-regulated after treatment with Asp-UA for 24 h. As shown in Figure P < 0.01). UA and Asp also inhibited the expression of the corresponding mRNA, while the level of these mRNA showed no prominent change compared with Asp-UA.In addition, quantitative real-time PCR method Figure  also verWe further detected the expression levels of integrin α6, β1 and CD44 by western blot method. As shown in Figure Due to the fact that Asp-UA could decrease the expression of CAMs, we further assessed the expression of crucial proteins including MMP-2, MMP-9 and COX-2, which are critical functional molecules within the tumor invasion process in breast cancer cells. As shown in Figure To investigate whether Asp-UA suppress EMT-related signaling pathway, we detected the relevant molecules in protein level with flow cytometry and western blot. FACS analysis Figure  showed tTo verify if Asp-UA can suppress EGFR-mediated signaling pathway, we detected several pivotal proteins in this signaling pathway by western blot assay. It turned out that Asp-UA could enhance the expression level of PTEN and reduce the expression levels of EGFR and ERK Figure . CompareTo confirm whether Asp-UA could inhibit the activity of EGFR, confocal imaging was performed to visualize its expression in MCF-7 cells. As a result, we found EGFR was mainly localized in the plasma membrane, with high expression in red fluorescence Figure  and the in vivo, we examined the anti-metastatic efficacy of Asp-UA with a 4T1 murine artificial pulmonary metastatic model. The 4T1 cell line is a highly metastatic murine breast cancer with a proclivity for lung metastasis. As shown in Figure in vivo. In addition, the H&E staining further confirmed that the Asp-UA therapy reduced the metastatic tumor burden in the lungs in a disseminated breast cancer model  is an initial process which is associated with cancer metastasis . EMT-relet al. demonstrated that one of the mechanisms that UA used to inhibit metastasis was that UA might block the EGFR-mediated pathway within tumor cells [Activation and over-expression of EGFR play significant roles in the invasion and metastasis cascade of breast cancer. Prasad S or cells . In our in vitro findings, the in vivo anti-metastasis effects of Asp-UA were further investigated in a mouse model of established pulmonary metastasis. We found the Asp-UA treatment resulted in remarkable reduction of lung metastasis . Mouse anti-human beta-actin (β-actin) antibody was purchased from Cell Signaling Technology . Matrigel were obtained from BD BiocoatTM . The primary anti-EGFR, anti-ERK, anti-MMP-2, anti-MMP-9, anti-COX-2, anti-E-cadherin, anti-β-catenin, anti-integrin α6, anti-integrin β1, anti-PTEN and anti-CD44 antibodies were obtained from Abcam Biotechnology . The secondary antibody goat-anti-mouse-IgG or goat-anti-rabbit-IgG horseradish peroxidase was obtained from Promega . All other reagents used in this study were of the highest purity commercially available.Ursolic acid  was purchased from Xi'an Ocean Biological Engineering Co. . Aspirin  was purchased from Aladdin Reagent Company . UA, Asp and Asp-UA were dissolved in dimethyl sulfoxide (DMSO) and were used in all experiments. PE-labeled mouse anti-human E-cadherin, vimentin, EpCAM and integrin α1, α3, α5, α6 and β1 integrin mAbs, PE/FITC mouse IgG1 kappa isotype control and APC mouse IgG1 kappa isotype control antibody were all purchased from Becton Dickinson (BD) PharmingenTo a solution of 0.5 g Asp (2.78 mmol), 2 mL oxalyl chloride and one drop of dimethyl formamide in 20 mL dry dichloromethane was stirred at 25°C for 10 h. The reaction mixture was concentrated under reduced pressure to remove solvent. Then 20 mL dry dichloromethane was added in reaction mixture. Add a solution of 1.27 g of UA (2.78 mmol) and 0.1 eq DMAP in 20 mL of pyridine from the dropping funnel over a period of 30 minutes. The reaction mixture was stirred at 25°C for 10 h. After stirring, the reaction mixture was concentrated under reduced pressure to remove dichloromethane. 100 mL water was added and stirred at 25°C for 20 minutes. Then the reaction mixture was filtered to get crude product. The reaction mixture was purified on silica gel column using ethyl acetate: petroleum ether  as eluents to give compound Asp-UA. All reactions were performed in oven-dried glassware. Yields refer to crystallised material or homogeneous products (TLC) obtained by column chromatography. All final compounds were purified to > 95% purity, as determined by a HPLC Breeze from Waters Co. using an Atlantis T3 5.0 μM 4.6 mm × 150 mm reverse phase column .2 at 37°C.Human breast cancer MDA-MB-231, MCF-7 cells, mouse 4T1 breast tumor cells and the normal human mammary epithelial cell line HMEC were purchased from the cell Bank of Shanghai Institute of Cell Biology. MDA-MB-231 and MCF-7 cells were cultured in L-15 and DMEM medium separately and the other cells were cultured in RPMI-1640 medium supplemented with fetal bovine serum , penicillin (100 U/mL) and streptomycin (100 μg/mL) in a humidified atmosphere of 5% COFemale BALB/C mice  were purchased from Shanghai SLAC Laboratory. These mice were maintained with free access to pellet food and water in a controlled environment of 20–25°C, with lighting on from 6:00 AM to 6:00 PM. All animals used in these investigations were handled in accordance with protocol procedures, approved by the Institutional Animal Care and Use Committee of Fuzhou University. All efforts were made to minimize the animals suffering and to reduce the number of animals used.The cytotoxicity of UA, Asp and Asp-UA was investigated by the MTT assay as previously described by this lab , 53. VarThe adhesion assay of MCF-7 cells to the matirgel was assessed according to the method described previously by this lab with minor modifications , 54. Mat5/mL, and then cell suspension (200 μL) containing UA, Asp or Asp -UA were seeded onto the upper chamber wells. After 24 h incubation, the cells that had invaded through the matrigel membrane were fixed in methanol and stained with 0.1% crystal violet for 20 min. Soft remove the cells on the inner layer with a cotton swab. The filters were washed with PBS and images were taken. The invading cells were counted and photographed under a light microscope  at ×200 magnification. Five fields were counted per filter in each group and the experiment was conducted in triplicate.Inhibition capacity against invasion of MCF-7 and MDA-MB-231 cells was estimated by transwell invasion assay as we described previously . Brieflyin vitro, as described in our previous work [Scratch assay was performed to analyze cell migration ous work . The MCFThis experiment was conducted as we described previously . MCF-7 aWestern blotting was performed as previously described . Briefly® RT reagent Kit  according to the manufacturer's instructions. The first strand cDNA products were used as PCR templates and the PCR amplificationwas performed in a 25 μL reaction volume using a SYBR®Premix Ex Taq™ PCR kit , using SYBR-Green real-time PCR method on the CFX96TM Real-Time PCR Detection Systems (Bio-Rad). The sequences are 5′-TCCCTGAA CCTAACGGAGTCT-3′ and 5′-ATGTCCAAGTAGTTCA GTTTG-3′ for integrin α6, 5′-ACAGCAGAGAAGCTGA AGCCA-3′ and 5′-GAGCTTAGCTGGTGTTGTGC-3′ for integrin β1, 5′-AGACGAAGACAGTCCCTGGATCAC-3′ and 5′-TGTGTTTGCTCCACCTTCTTGACTC-3′ for CD44, 5′-TGGCACCCAGCACAATGAA-3′ and 5′-CATA GTCATAGTCCGCCTAGAAGCA-3′ for β-actin. Target mRNA expression levels were normalized with the β-actin mRNA and the relative changes of gene expression were measured using the 2−ΔΔCT method. Results are expressed as the mean ± standard deviation of three independent experiments.Total RNA was isolated from cells using TRIZOL Reagents (Invitrogen). A sample (1 μg) of total RNA was used to generate cDNA templates by RT reaction with the PrimeScript5 cells/mL. Then inject 200 μL into the tail vein of female BALB/C mice. On the following day (Day 1), mice were randomly divided into six groups (n = 8 for each group) and then normal solvent (CMC-Na (control)), UA (80 mg/kg), Asp (80 mg/kg) and Asp-UA , respectively, were administered to mice for 28 days via orally gavage. Mice weight were measured every other day. After the treatment, the mice were sacrificed by cervical dislocation after euthanizing with isoflurane (3%). The heart, liver, spleen, lung, kidney and small intestine were dissected, washed with PBS and fixed in 10% (v/v) buffered formaldehyde. These tissues were then paraffin embedded and stained with hematoxylin and eosin (H&E). The number of surface tumor nodules was counted visually with the aid of a dissecting microscope and histological observations were performed under a microscope .Subconfluent 4T1 cells were harvested and resuspended in PBS to a final density of 1 × 10n = 8 for each group) and then normal solvent (CMC-Na (control)), UA (80 mg/kg), Asp (80 mg/kg) and Asp-UA , respectively, were administered to mice for 7 days via orally gavage. After the treatment, the mice were sacrificed by cervical dislocation after euthanizing with isoflurane (3%). Cut the stomach across the greater curvature and expose the gastric mucosal, followed by washing with PBS and examination under a light microscopy.On the first day (Day 1), mice were randomly divided into six groups  for 12 h in 4°C. The cells were then rewashed, and incubated with CY3-conjugated goat anti-rabbit IgG  for 1 h in room temperature. Finally, the cells were washed and restained with DAPI. Blue fluorescence of DAPI, red fluorescence of EGFR was observed using a Zeiss LSM 780 confocal microscope .MCF-7 cells grown on 35 mm cell culture dish with 10t-test and one-way analysis of variance using least significance difference test (IBM SPSS Statistics 19.0). A probability (P) value < 0.05 was considered statistically significant, and P < 0.01 was highly statistical significance.Data were presented as the means ± standard deviations (SD) for three independent experiments. Statistical analysis was done by student's in vitro and in vivo compared with UA and Asp alone. Asp-UA managed to significantly inhibit the adhesion-invasion-migration cascade of MCF-7 and MDA-MB-231 cells by down-regulating the relevant functional molecules. These effects were accompanied by suppressing the EMT and EGFR-mediated pathways. Moreover, Asp-UA also undermined the progression of pulmonary metastasis for 4T1 cells in female BALB/C mice without obvious side effects such as weight loss and main viscera tissues. Taken together, this newly-discovered activity of Asp-UA makes it best-suited for the development of chemo-preventive drugs for cancer metastasis. Our results will promote better use of existing drugs and facilitate the design of more complex therapeutic approaches to target advanced malignancies, and more importantly, provide a molecular basis for clinical evaluation of Asp-UA as a potential cancer metastatic chemopreventive agent.In the present study, for the first time, we demonstrated that a novel co-drug Asp-UA had better inhibitory effect on breast cancer metastasis and low toxicity both


Giardia duodenalis is a common human and animal pathogen. It has been increasingly reported in wild and captive non-human primates (NHPs) in recent years. However, multilocus genotyping information for G. duodenalis infecting NHPs in southwestern China is limited. In the present study, the prevalence and multilocus genotypes (MLGs) of G. duodenalis in captive NHPs in southwestern China were determined. We examined 207 fecal samples from NHPs in Sichuan and Guizhou provinces, and 16 specimens were positive for G. duodenalis. The overall infection rate was 7.7%, and only assemblage B was identified. G. duodenalis was detect positive in northern white-cheeked gibbon , crab-eating macaque  and rhesus macaques . Multilocus sequence typing based on beta-giardin (bg), triose phosphate isomerase (tpi) and glutamate dehydrogenase (gdh) revealed nine different assemblage B MLGs (five known genotypes and four novel genotypes). Based on a phylogenetic analysis, one potentially zoonotic genotype of MLG SW7 was identified in a northern white-cheeked gibbon. A high degree of genetic diversity within assemblage B was observed in captive northern white-cheeked gibbons in Southwestern China, including a potentially zoonotic genotype, MLG SW7. To the best of our knowledge, this is the first report using a MLGs approach to identify G. duodenalis in captive NHPs in Southwestern China. Giardia duodenalis is the etiological agent of giardiasis, a gastrointestinal infection that is typically asymptomatic, but may also be severe in some individuals [G. duodenalis (A-H), assemblages A and B frequently infect humans and animals, assemblages C and D have been described in domestic and wild canines, assemblage E have been widely reported in ruminants but sporadically detected in NHPs and humans, assemblage F in cats, assemblage G in rodents and assemblage H in seals and gulls [ividuals –3. At prnd gulls . Assemblnd gulls –6.G. duodenalis have a monoxenous life cycle and can spread rapidly in captive NHPs [G. duodenalis has been widely investigated in NHPs. Assemblages A, B and E are found in NHPs and assemblage B is dominant [NHPs are valuable wildlife resources. Owing to their high genetic homology to humans, NHPs are important experimental models for clinical research and public health research. ive NHPs . Geneticdominant , 6. Moledominant , 5.G. duodenalis infecting NHPs based on multi-locus genotyping. Molecular analyses to date have typically focused on a single genetic locus [G. duodenalis, multi-locus genotyping (MLG) employing beta-giardin (bg), triose phosphate isomerase (tpi) and glutamate dehydrogenase (gdh) has been used for genotyping and subtyping G. duodenalis in humans and animals [G. duodenalis in captive NHPs in Southwestern China. These findings improve our understanding of the genetic diversity and the transmission routes of G. duodenalis in NHPs.However, little is known about genetic variation in ic locus , 8, 9. Iic locus , 10. To  animals –13. The This study was reviewed and approved by the Institutional Animal Care and Use Committee of Sichuan Agricultural University under permit number DYY-S20156703. Prior to the collection of fecal specimens from NHPs, permission was obtained from owners.From March to May 2016 and September to November 2016, 207 fecal specimens from NHPs were collected from Sichuan and Guizhou provinces. Fresh fecal specimens were collected immediately after defecation on the ground and separately stored in 50-mL centrifuge tubes. The specimens were kept cool during transport and arrival at the Sichuan Agricultural University. Specifically, 101 samples were obtained from rhesus macaques from the National Experimental Macaque Reproduce Laboratory in Southwest China (n = 31), Chengdu Gaoxin rhesus macaque farm (n = 30), Chengdu zoo (n = 20) and Bifengxia zoo (n = 20). Thirty-six samples were from northern white-cheeked gibbons from zoos in Guiyang (n = 30), Chengdu (n = 2) and Bifengxia (n = 4). Nine samples were from Golden snub-nosed monkeys in the Chengdu zoo. Sixty samples were from crab-eating macaques in the National Experimental Macaque Reproduce Laboratory in Southwest China and the Chengdu Gaoxin rhesus macaque farm . Samples® DNA Isolation Kit  following the manufacturer`s instructions. DNA samples were stored in 100 μL of the kit's Solution Buffer at 20°C until use.Before extracting DNA, the fecal samples were washed with distilled water until potassium dichromate was removed. Genomic DNA was extracted using the PowerSoilG. duodenalis by nested PCR amplification of the beta-giardin (bg) gene [bg-positive specimens were further characterized by PCR amplification of the tpi and gdh genes [Each specimen was examined for bg) gene , The bg-dh genes . SecondaG. duodenalis assemblage, the sequences were aligned with sequences downloaded from the GenBank database based on a BLAST analysis (http://blast.ncbi.nlm.nih.gov) using ClustalX. For the phylogenetic analysis, sequences obtained in this study were used to construct a neighboring-joining tree using Mega 5 (http://www.megasoftware.net/). A total of 1000 replicates were used for the bootstrap analysis.The amplified products of the expected size were sequenced by Invitrogen . To determine the Differences in infection rates among NHPs and among animals in different areas were assessed using the chi-square test implemented in SPSS version 17.0 . P < 0.05 was considered significant.bg-based PCR analysis of 207 specimens from 4 NHP species, 16 (7.7%) samples from 3 species were positive for G. duodenalis. All the positive specimens were successfully amplified and sequenced for the bg, tpi and gdh genes. Sequences were deposited in the GenBank database under the accession numbers KY696790-KY696821.In the G. duodenalis. Northern white-cheeked gibbons showed the highest infection rate . All golden snub-nosed monkeys (n = 9) were negative for G. duodenalis. The difference in infection rates among 4 species was significant (P<0.05). In China, six studies have examined G. duodenalis infection in NHPs in parks, zoos, farms and laboratories to date, and the overall infection rate was between 1.3% and 18.6% in these studies [G. duodenalis infection is common in wild and captive NHPs and has a wide geographic distribution in China.The infection rates ranged from 0% to 38.9% in the 4 species . Specifi studies , 15–19.  studies , and was studies  and two  studies , 18. OurG. duodenalis infection in NHPs showed a similar trend to that observed in China. The overall infection rate of G. duodenalis in NHPs is between 2.2% and 47.0% [In other countries, nd 47.0% , indicatnd 47.0%  and Thaind 47.0% , and it nd 47.0%  and Croand 47.0% . This rend 47.0% , 8, 20.G. duodenalis, which was also higher than the infection rate in a previous study  [G. duodenalis than wild animals. This might be explained by the single-host life cycle and the resilient infectious cysts of G. duodenalis [In this study, the infection rate for captive NHPs in Sichuan province was 0.6% (1/106), which is almost identical to that in a comprehensive parasite study performed in 2009–2015 in Sichuan  . The infodenalis . Captiveodenalis , 15.G. duodenalis may explain the low infection diversity of assemblages [To date, assemblages A, B and E have been detected in NHP species in China. Assemblage A and B have both been found in captive and free-range NHPs, but assemblage E has only been found in captive NHPs . In thisemblages , and sugemblages , assemblgdh sequences. All of the 16 sequences were 100% similar to sequences of human isolates from Brazil  [bg and tpi loci showed high levels of sequence polymorphism. 5 and 6 subtypes were identified in the 16 strains, including two new subtypes. At the bg locus, four known subtypes Bb-4 (KJ888977) [tpi locus, five known subtypes B7 (JQ863259) [No genetic variation was observed among the 16 F507682) . The bg J888977) , BIII-1 J888977) , and B1 J888977)  were fouQ863259) , WB8 (KFQ863259) , BIV (ABQ863259)  and WB6 A total of 16 NHPs specimens  were classified as assemblage B and nine MLGs were identified among the 16 positive specimens. The subtype identities and geographical and host distributions of the nine MLGs are listed in G. duodenalis between animals [G. duodenalis from this species [Phylogenetic analyses showed that MLG SW7 belonged to a zoonotic group. Given the zoonotic potential of this subtype, epidemiological and source tracking investigations as well as strict surveillance in captive NHPs in southwestern China are needed. MLG SW9 was closely related to the sequences obtained from NHPs in other studies . MLG SW1 animals . Other M species .G. duodenalis in captive NHPs in Southwestern China. One genotype of the potentially zoonotic assemblage B of MLG SW7 strain was identified in a northern white-cheeked gibbon. This suggests that the zoonotic transmission of Giardia might occur between the northern white-cheeked gibbon and humans. Additionally, high degree genetic diversity of assemblage B MLGs (7 MLGs) was detected in captive northern white-cheeked gibbons in Southwestern China. Additional MLGs studies of captive NHPs are needed, to better characterize genetic diversity and the routes of transmission of G. duodenalis between NHPs and humans or other animals.The results of the present study confirm previous findings that assemblage B is dominant in northern white-cheeked gibbons. We first used a MLGs approach to identify