Resistance to therapy is the major hurdle in the current cancer management. Cancer cells often rewire their cellular process to alternate mechanisms to resist the deleterious effect mounted by different therapeutic approaches. The major signaling pathways involved in the developmental process, such as Notch, Hedgehog, and Wnt, play a vital role in development, tumorigenesis, and also in the resistance to the various anticancer therapies. Understanding how cancer utilizes these developmental pathways in acquiring the resistance to the multi-therapeutic approach cancer can give rise to a new insight of the anti-therapy resistance mechanisms, which can be explored for the development of a novel therapeutic approach. We present a brief overview of Notch, Hedgehog, and Wnt signaling pathways in cancer and its role in providing resistance to various cancer treatment modalities such as chemotherapy, radiotherapy, molecular targeted therapy, and immunotherapy. Understanding the importance of these molecular networks will provide a rational basis for novel and safer combined anticancer therapeutic approaches for the improvement of cancer treatment by overcoming drug resistance. The emergence of resistance to anticancer therapeutics is one of the major barriers that limit the efficacy of cancer therapy . ResistaNotch, Hedgehog, and Wnt form the major developmental signaling pathways which play a fundamental role in the dynamic transformation of a single-celled zygote into a highly complex multicellular organism . These sThe Notch is an evolutionarily conserved signaling pathway involved in many developmental and cellular processes, starting from the germ layer formation to the differentiation of specialized cell types in the embryo . In the Notch mediates short-range intercellular communication through interaction with ligands present on the neighboring cells. The binding of Notch ligands to the Notch receptors triggers S2 cleavage by ADAM10 and ADAM17 in the extracellular part of the receptor, leading to the shedding of the extracellular part. This is followed by the S3 cleavage in the transmembrane portion by γ-secretase. After S3 cleavage, the Intracellular domain (ICN) is released from the plasma membrane, which translocates to the nucleus. Here, it interacts with RBPJ—recombining binding protein suppressor of hairless/CBF1/suppressor of hairless/Lag-1 (RBPJK/CSL) and convert/transform the repressor complex into coactivator complex, thus promoting the transcription of the target genes .In the recent past, increasing evidences suggest the existence of non-canonical Notch signaling that is independent of canonical Notch ligand or transcription factor CSL/RBPJ . In thisAs Notch signaling is involved in many crucial cellular functions, its aberrant regulation/expression often leads to pathological events ranging from developmental disorders to cancer. Indeed, many observations suggest that alterations in Notch signaling are associated with many human cancers. Moreover, Notch receptors and ligands have been found as prognostic markers in human cancers. Initially, the oncogenic role of Notch signaling was documented in T cell Acute Lymphoblastic Leukemia (T-ALL), where the activating mutations in NOTCH1 were suggested to be a major mediator for the development of malignancy . Later, Hedgehog signaling is an evolutionarily conserved pathway that regulates the morphogenesis of various organs during embryogenesis and postnatal development. It regulates diverse cellular processes in the adult, including proliferation, tissue differentiation, and repair of normal tissues. In addition, it is also involved in stem cell renewal and organ homeostasis. The major components of the Hedgehog signaling pathways are the Hh ligands Sonic hedgehog (Shh), Indian Hedgehog (IHH) and Desert hedgehog (DHH), Patched (PTCH), which is a 12-transmembrane domain receptor protein, which locates in the primary cilium, the 7-transmembrane G-protein coupled receptor Smoothened (SMO), the suppressor of fused protein (SUFU) in the cytoplasm, and the glioma-associated oncogene (GLI) transcription factors.In the absence of ligands, Ptch1 inhibits SMO accumulation in primary cilia, thereby resulting in the block of the pathway activity. The binding of the Hedgehog ligands to Ptch1 causes internalization and degradation of the Ptch receptor, thereby releasing SMO to enter the primary cilia where it promotes the dissociation of a SUFU– GLI complex. This activates GLI transcriptional activators and their translocation into the nucleus to activate the expression of Hedgehog target genes such as GLI1 and Ptch . In addiEctopic activation of the Hedgehog signaling is implicated in several cancers, including hematological malignancies and solid tumors, where it is associated with tumor development, progression, and recurrence after anticancer therapy. In hematological malignancies, the role of Hh signaling in maintaining leukemic stem cells has been well established. Thus, inhibition of HH signaling reduces the stem cell potential to initiate leukemia . This efWnt signaling pathway is another evolutionally conserved pathway that directs developmental processes, stem cell proliferation, and tissue homeostasis throughout the metazoans . Hence, The canonical Wnt signaling pathway is a well-studied pathway that is activated by the interaction of Wnt with a Frizzled (Fz) receptor and LRP5/LRP6, where LRP stands for lipoprotein receptor-related protein (which is a single-span trans-membrane receptor) . FolloweIn the inactivated/un-stimulated state, GSK-3 phosphorylates the transcriptional coactivator β-catenin that renders it in an active state. Inactivation of β-catenin is characterized by the formation of a “destruction complex” that comprises of GSK3, adenomatosis polyposis coli (APC), Axin, and casein kinase Iα (CKIα)  which leWnt signaling has a prominent role in carcinogenesis and has been widely studied in colorectal cancer. Indeed, the first evidence of dysregulated Wnt signaling came from the studies of hereditary colorectal cancer. It was found that this aberrant activation of Wnt signaling in colorectal cancer was due to the loss of function mutations in the APC gene or point mutation in the N-terminal sites of β-catenin that leads to the stabilization of β-catenin. Later, studies from several groups showed the activating mutation in Wnt signaling components in various cancer such as breast cancer, pancreatic cancer, melanoma, lung cancer, prostate, etc. . MoreoveSeveral seminal studies over the past decade have demonstrated that Notch, Hh, and Wnt signaling pathways crosstalk with one another, with one pathway acting as an upstream or downstream effector of another. This affects their transcriptional output in the specific cellular context in which they operate .in vitro and in vivo , enrichment or induction Cancer Stem Cell (CSC) phenotype, modification of the tumor microenvironment, etc. Interestingly, Notch, Hedgehog, and Wnt are involved in modulating nearly all of these mechanisms . In the Drug efflux forms the primary mechanism of chemo-resistance in cancer. Emerging evidences suggest that Notch, Hedgehog, and Wnt signaling can directly modulate the drug efflux by regulating the expression of the transporter involved in drug efflux.In prostate and breast cancer, the chemotherapeutic drug such as docetaxel or doxorubicin induces the activation of Notch signaling, which in turn increases the expression of ABCB1 and multidrug-resistant associated Protein1 (MRP1), thus increasing the drug efflux and contributing to chemo-resistance. This chemo-resistance effect is reversed by the Notch inhibitors .Likewise, Hedgehog signaling directly regulates the expression of ABCB1 and ABCG2 in ovarian cancer. The inhibition of Gli1 expression decreases ABCB1 and ABCG2 gene expression levels, thus enhancing the response of ovarian cancer cells to certain chemotherapeutic drugs . MoreoveSimilarly, the Wnt/β-catenin pathway is known to regulate the transporters involved in drug efflux. Several TCF4/LEF binding motifs are present in the promoter region of the ABCB1 gene in humans, suggesting it as a target gene of the β-catenin/TCF4 transcriptional regulators, thus the activation of β-catenin augments ABCB1 expression. LGR5, the target molecules for the Wnt pathway in colorectal cancer cells, was found to confer resistance to chemotherapy. ABCB1 higher expression was found to associated LGR5 expression in chemoresistance to CRCs. Cancer stem cell property in CRCs is regulated by LGR5 that leads to chemoresistance to oxaliplatin and 5-fluorouracil . PygopusDown-regulation of apoptosis induced by chemotherapeutic drugs is another mechanism that contributes to chemo-resistance in cancer. Events that lead to a block in apoptosis enhances chemo-resistance. Notch, Hedgehog, and Wnt signaling have been well known to modulate apoptosis in cancer.Notch signaling regulates apoptosis through its interaction with the key players of apoptosis pathways . TherefoHedgehog signaling pathways has also been implicated in the regulation of apoptosis in cancer . Emerginin vitro and in vivo anti-tumor, apoptosis, and anti-metastasis activities of CoQ0 (0–20 μM) through inhibition of Wnt/β-catenin signaling pathway.Several inhibitors that target apoptosis regulation by modulating Wnt signaling have been investigated. For example, Belinostat is a histone deacetylase inhibitor that induces apoptosis by decreasing the Wnt/β-catenin, CCND2, and Myc in MCF-7 cells . LanatosIn the recent past, it has become increasingly apparent that the cell cycle plays a critical role in chemosensitivity for the given chemotherapy, and the role of developmental pathways such as Notch, Hedgehog, and Wnt signaling in this phenomenon has been well established .Notch signaling regulates the cell cycle by inducing the expression of some of the critical cell-cycle related genes such as p21, p27, E2F etc. Therefore cell-cycle perturbation due to dysregulated Notch signaling in cancer forms one of the factors that impart chemoresistance to the drugs. For example, in T-cell transformation and chemoresistance, Notch1 signaling is associated with the promotion of G1-S through upregulation of CDK4 and CDK6 and downregulation of p27/KIP1 and p18/INK4C cell cycle inhibitors . TherefoSimilarly, the Hedgehog signaling pathway modulates the cell cycle progression via directly regulating the core cell cycle components such as D-type cyclins, Cip1, p57KIP2 . MoreoveWnt/β−catenin signaling regulates cell proliferation by modulating the cell cycle via β -catenin, which is a well-known regulator of cell cycle progression . MoreoveCell survival forms an important factor that attenuates the effectiveness of conventional chemotherapy. The role of Notch, Hedgehog, and Wnt signaling in regulating cell survival in the face of chemotherapy treatment has gained much interest due to its therapeutic importance.Report from multiple laboratories has shown the involvement of Notch signaling in the regulation of cell survival in normal development and also in cancer. Moreover, recent reports suggest that Notch signaling form the key mediator of increased cancer cell survival in the context of chemotherapy. For example, activated Notch protected T-ALL cells from glucocorticoid-mediated apoptosis, which was mediated from Notch dependent upregulation of Akt, leading to nuclear export of glucocorticoid receptor . Thus thHh signaling also has been shown to promotes cancer cell survival, which forms a selective growth advantage to tumor cells and has been implicated in multidrug resistance. In rhabdomyosarcoma (RMS) or Ewing sarcoma (EWS), HH pathway activity and GLI1 expression contribute to cell survival and proliferation. Chemotherapeutic drugs such as Vincristine (VCR), was shown to significantly upregulate Gli1 expression in these cells. Thus treatment with small molecule inhibitor GANT61 or siRNA against GLI1 together with vincristine significantly decreased cell viability . SimilarSimilarly, Wnt signaling is involved in regulating the drug resistance of various cancer via cell survival and proliferation. Thus inhibiting Wnt signaling can sensitizes cancer cells to chemotherapy. For example, in myeloid leukemia cells, the Wnt inhibitor (FH535) sensitizes it to chemotherapeutic drug imatinib and potentiated its chemotherapeutic effect . A similROS plays a crucial role in cancer progression and resistance to radio-and chemotherapy . Their rNotch and ROS can modulate each other by directly regulating their core components. Nrf2 is a well-established regulator of anti-oxidants and its overexpression cause resistance to both radio-and chemo-therapy in cancer . Notch cSimilarly, the hedgehog signaling pathway can crosstalk with ROS-induced signaling to regulate chemoresistance in various cancer. For example, in hepatocellular carcinoma HCC cells, ROS-induced NRF2 activation upregulated the expression of sonic hedgehog homolog (SHH), ultimately activating sonic hedgehog pathway. This mediated the tumor initiating function, ultimately leading to sorafenib resistance in HCC. Moreover natural compound such as, resveratrol and curcumin was shown to inhibit hypoxia-mediated activation of the Hh signaling pathway in pancreatic cancer, ultimately affecting the EMT phenotype, which has been known to play an important role in inducing chemoresistance in cancer . In contThe interplay between Wnt signaling and ROS has investigated in different cancer that leads to the development of resistance to therapy. Activation of Wnt canonical signaling aids the ubiquitination and proteasomal degradation of Nrf2 protein via axin1/GSK-3b complex . ROS/WntEpithelial to mesenchymal transition (EMT) is a process where the epithelial cells lose their epithelial characteristics and acquire mesenchymal characteristics. This dramatic cell transposition process not only plays critical roles in governing embryonic development and maintaining adult tissue hemostasis  but also contributes to pathological conditions, such as fibrosis, cancer progression, and drug resistance.Notch signaling can directly regulate the expression of genes involved in EMT . It is iThe role of the Wnt/β-catenin pathway has also been implicated in the EMT process. Runt-related transcription factor 1 (RUNX1) plays the roles of an oncogene and an anti-oncogene in epithelial tumors, and abnormally high expression of RUNX1 is associated with metastasis and EMT. It directly interacts with catenin and targeting the promoter and enhancer regions of KIT . FurtherCancer Stem Cell (CSC) is a small subset of tumor cells that has the potential to self-renew and give rise to a heterogeneous population in the tumor. Many studies have demonstrated that CSC is more resistant to chemotherapy because of the higher expression of the anti-apoptotic protein, multidrug resistance gene, etc. These studies have been extensively reviewed elsewhere . The rolThe Numb-/low population of castration-resistant prostate cancer has been shown to up-regulate Notch and Hedgehog signaling. This is associated with increased expression of genes linked to stem cells and greater resistance to androgen deprivation therapy (ADT). Inhibition of Notch and Hedgehog signaling pathways significantly increases apoptosis in Numb-/low cells in response to ADT . Consequ+ CSCs express higher levels of miR-9 and activate the SHH/PTCH1/MDR1 axis, which imparts resistance against TMZ  down-regulates the expression of CSC markers and sensitizes tumors to chemotherapy drugs such as docetaxel . HedgehoLikewise, the Wnt ligand or the growth factor secreted by stromal cells of TME leads to the stimulation of Wnt signaling in tumor cells, that leads to the EMT . For exaRadiotherapy is used in the treatment of approximately 50% of all malignancies. Resistance to radiation therapy is polymodal and is associated with several biological alterations both within the tumor and in the surrounding microenvironment. Moreover, like chemotherapy, radiotherapy also induces EMT and CSC-like phenotype in cancer cells, which contributes to the radio-resistance. As discussed earlier, Notch, Hedgehog, and Wnt have a prominent role in EMT and CSC, and inhibition of these pathways enhances radio-sensitivity through down-regulation of EMT and CSC .It has now been well established that Notch signaling gets activated upon irradiation and plays an important role in the radio-resistance of both stem and non-stem cancer cell populations . MoreoveThe role of Hedgehog signaling in the radioresistance of tumors has been well established. GLI proteins are the functional transcription activators of the Hh pathway, and the Inhibition of its activity can interfere with almost all DNA repair types in human cancer, indicating that Hh/GLI functions may play an important role in radiation-induced DNA damage . Indeed,in vitro and in vivo  cell lines , which u in vivo . Thus, tCancer immunotherapy has recently emerged as a new and promising option for the treatment of many malignancies. However, despite the recent successes of cancer immunotherapies, most patients are still refractory with tumors demonstrating resistance to this therapy. Notch, Hedgehog, and Wnt signaling pathways are well known to play an important role in the development, homeostasis, and function of various immune cells. Although limited studies have been carried out, available evidence suggests that these signaling pathways can mediate cancer immune evasion and resistance to immunotherapies through various mechanisms .Notch signaling plays multiple roles in the crosstalk between systemic inflammation, myeloid cells in the tumor microenvironment , the canHedgehog signaling pathway exerts complex and diverse effects on the tumor immune micro-environment. Emerging evidence suggests that the hedgehog signaling pathway has an immunosuppressive action in many cancers by modulating different components of tumor microenvironments. In Pancreatic ductal adenocarcinoma (PDAC), dense fibrosis is often observed, which acts as a barrier to immune cell infiltration into the tumor, thus making the tumor resistant to immunotherapy. As hedgehog signaling plays an important role in fibrosis, their inhibition has shown a positive effect on immunotherapy. For example, the Hedgehog signaling inhibitor, the Patched 1-interacting peptide has been found to inhibit proliferation and migration of cancer-associated fibroblasts and cancer cells and also increase the infiltration of immune cells by reducing fibrosis of PDAC, thus enhancing the effect of immunotherapy . MoreoveAlthough the role of Wnt signaling in immune cell development remains controversial , recent A deep understanding of the critical molecular drivers of cancer has led to the development of targeted therapies that strike the core molecules involved in many malignancies. However, after the initial response, many cancers outsmart such efforts, and thus therapeutic resistance follows, which contributes to cancer mortality. The strategies employed by cancer cells to gain resistance to targeted therapies include activating signals upstream or downstream of oncogenes, direct target reactivation, adaptive survival mechanisms, and engagement of parallel oncogenic pathways. As many of these processes are directly regulated by the Notch, Hedgehog, and Wnt signaling pathways, cancer cells can utilize these signaling pathways to modulate the downstream effectors of the targeted molecules, thereby becoming resistant to the targeted therapies .+ AML patients. Unfortunately, most often, they acquire resistance to TK inhibitors. Mechanistically, Notch signaling was upregulated following treatment with FLT3-TKIs, which resulted in alternative ERK activation. The addition of Notch inhibitor (GSI) abrogated the alternative activation of ERK, resulting in extreme repression of ERK activity, thereby showing a synergistic antitumor effect , often show upregulation of the key signaling pathways. For example, genetic alternations of internal tandem duplication (ITD) and mutations of FMS-like tyrosine kinase-3 (FLT3) are most frequently observed in AML. Consequently, FLT3 TKIs are widely used in the treatment of FLT3/ITDr effect . Likewissurvival . Adminissurvival . BRAF issurvival . Small-msurvival . In melasurvival .Hedgehog is known to co-operate with the epidermal growth factor receptor (EGFR) signaling pathway during embryogenesis. Therefore, it is not surprising that it is implicated in the resistance against TKIs. The EGFR-TKI resistance NSCLC cells show hyperactivation of hedgehog signaling. This results in EMT induction and ABCG2 up-regulation, and inhibition of hedgehog signaling increases the sensitivity to EGFR-TKIs in resistant NSCLC cells . MoreoveWnt signaling has also been attributed to the development of resistance against targeted therapy. Androgen deprivation therapy (ADT) is the most widely used therapy for the advanced and metastatic prostate cancer (PCa), who cannot be cured by surgery or radiation therapy. Wnt signaling, which is known to be involved in the late stage of PCa, was shown to be activated upon the inhibition of androgen receptor (AR), resulting in therapy resistance to the ADT. Mechanistically, it was shown that the activation of Wnt signaling was not by mutation but through cross talk with other signaling pathways, growth factors, and cytokines produced in response to damaged TME followed by inhibition of androgen receptor (AR) . The inhInhibitors of gamma-secretase were the first to enter clinical trials and has been evaluated extensively . Their mDrugs targeting Wnt signaling that has been evaluated in clinics include porcupine inhibitors, agents targeting β-catenin/CBP, and Frizzled receptors . Drugs tAlthough clinical trials of drugs to target the three developmental signaling pathways associated with the CSCs was initiated more than a decade back, it has not translated into clinical benefit both due to their inability to reduce the tumor recurrence linked to the CSCs as well as off-target toxicity . TherefoResistance to anticancer therapy stems from complex and multifactorial processes and has still remained the main reason behind the failure of various therapies. Evolutionary conserved developmental signaling pathways such as Notch, Hedgehog, and Wnt signaling are well recognized for their role in regulating many cellular functions that play key roles in tumor development and progression. These signaling pathways are often upregulated in tumors, which often hijack these pathways to evolve continuously under the pressure induced by the therapy, thereby enabling them to become resistant to various therapies. Many inhibitors developed to target these signaling pathways have shown promising efficacy in preclinical cancer models, and some have even advanced to clinical trials with modest efficacy seen so far. However, as these signaling pathways are broadly active in normal tissue also, targeting these pathways with pan inhibitor often leads to undesirable off-target effects, particularly in the gastrointestinal tract. Therefore, one of the major challenges in the future will be to develop more selective inhibitors that will not only be more effective on tumors but have minimal normal tissue toxicity. Efforts are also required in developing synergistic drug combinations so that lower dosages can be used to improve the therapeutic index.VK and BSD conceptualized the manuscript. VK, MV, and BSD wrote the manuscript. VK and MV prepared the figures. LK, XW, CG, and JL reviewed the draft of the manuscript. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The adoption of contraception often coincides with market integration and has transformative effects on fertility behavior. Yet many parents in small-scale societies make decisions about whether and when to adopt family planning in an environment where the payoffs to  have smaller families are uncertain. Here we track the fertility of Maya women across 90 years, spanning the transition from natural to contracepting fertility. We first situate the uncertainty in which fertility decisions are made and model how childbearing behaviors respond. We find that contraception, a key factor in cultural transmission models of fertility decline, initially has little effect on family size as women appear to hedge their bets and adopt fertility control only at the end of their reproductive careers. Family planning is, however, associated with the spread of lower fertility in later cohorts. Distinguishing influences on the origin versus spread of a behaviour provides valuable insight into causal factors shaping individual and normative changes in fertility. However, the emphasis on declining average family size can mask underlying changes in the structure of fertility within a population6. The distribution of fertility across the transition has been described as moving from relatively low8 to high variance and then stabilizing back to lower levels9. Unknown, however, is how this plays out longitudinally or within a population. Under natural fertility (absence of parity-specific fertility control) 1011 family size within a population varies largely due to stochastic individual differences in fecundity12. What follows with the onset of the fertility transition is of interest because parents diversify their childbearing strategies13, with some having much smaller and others larger families than in previous cohorts. This change in the distribution of fertility gives new norms a chance to emerge. While the elevation of fertility variance has been described at the population level, to our knowledge the mechanisms motivating it are unidentified, yet are crucial to understand how new family size norms take hold in a population and provide insight into factors affecting the origin versus spread of declining fertility14.The trend toward smaller families, ongoing today in small-scale societies worldwide, is broadly linked to market integration15) often accompanies market integration . It is in this context that parents are also exposed to new fertility options. Under premarket, natural fertility conditions, fertility is correlated with traditional measures of wealth  due to their physiological correlation with energy balance and fecundity22. While cultural factors also mediate family size on both sides of the transition, through for example, norms about sexual access, exposure to modernizing influences affects energy balance and constraints on fertility24. The introduction of calorie-rich market foods, mechanized farming, water pumps and other domestic appliances relaxes physiological constraints on female fecundity. At the same time, modernizing influences open up new options to reduce family size and invest in child quality26.In small-scale societies with no prior experience with the labor market economy, uncertainty  For example, when high status or prestigious individuals adopt new ideas or innovations that limit fertility, others are likely to imitate these behaviours, resulting in a shift in cultural norms toward lower fertility preferences45. Diffusion and cultural transmission approaches share in common an emphasis on changes in the ideal number of children, the perceived value of children, and norms regarding reproductive behavior58.The uncertainty introduced by market development may be alleviated by uncovering payoff structures through social learning and exposure to new ideas . An individual may emulate the behaviours of others to short-cut the time required to learn about potential costs and benefits of having smaller families. Propinquity helps to explain, for example, why even after controlling for structural factors, fertility decline occurs more rapidly in densely populated urban areas than in isolated rural areas61. Debate persists whether cultural transmission or economic influences offer competing explanations or are codrivers of the fertility transition58, and the extent to which cultural transmission models explain the origin versus the spread and maintenance of low fertility14. To further this discussion, we track the role of social learning across the transition to differentiate the effects that the adoption versus spread of family planning have on fertility decline within a small-scale society.The adoption of fertility-limiting family planning is a widely used proxy measure of cultural transmission in the spread of low-fertility norms9, and in population-level comparisons7, empirical longitudinal data that span natural to contracepting fertility are needed to demonstrate that this is indeed a within-population characteristic of fertility transitions. To characterize variance across the fertility transition, we use known determinants of fertility  to model changes in childbearing behavior. We then model the influence that family planning has in driving changes in the distribution of fertility. Our community census data include women who were at risk of conception from 1932 to 2018, a time period that brackets the transition from a natural fertility to a contracepting population, and from an egalitarian subsistence farming economy to a mixed-market economy. A paved road, which linked this remote, rural community to larger market towns62, was built in the early 2000s and initiated sweeping economic changes including the adoption of family planning. This longitudinal perspective leveraging  detailed reproductive histories and household economic data allow us to detect factors underlying changes in fertility behavior and to distinguish the influence that family planning has on the origin versus the spread of low fertility.We use longitudinal individual- and household-level fertility, social network and economic data for indigenous rural Maya women living in Campeche, Mexico to first characterize the distribution of fertility across the transition, and determine whether within-population variance precedes a change to low fertility. While the rise in variance has been described with cross-sectional data for populations already amidst the transitionTo assess how the distribution of fertility has changed over time, our census data includes Maya women born from 1912–1999 (n = 161) and their 600 children (birth years 1927–2018). Women were subset into four cohorts . However, the variance significantly increases . The distribution of completed fertility in Cohort 1 expands both at the upper end as some women have larger families, and at the lower end as other women have much smaller families compared to the baseline cohort  compared to the baseline pre-road cohort. Thus, the first cohort following the completion of the road shows a marked increase in variance with no significant change in mean fertility, which only becomes evident two decades later.We first establish whether an increase in variance proceeds a significant decline in fertility and find that in the first decade after the introduction of the road Cohort 1), mean completed fertility does not change in a statistically significant way  and appears to have stalled at high levels  and sharing-group size . Land under cultivation was determined both through economic interviews and accompanying farmers to their fields and taking GPS measurements of the perimeters their cultivated fields. Sharing groups were identified through detailed social network and economic interviews, and define groupings of biological families that are observed and self-identify as sharing food, labor, capital and other resources. Because sharing group is independent of biological family size, a decline in fertility over the four cohorts may not affect sharing group size 64  and wage labor (a binary variable indicating whether 50% or more of the adult members of the household engage in wage-labor). Since most households engaged in some form of occasional or part-time paid labor after the construction of the road and because the amount of time each laborer spends is quite variable, we chose to use a dummy variable, which indicates whether a household has a particularly strong commitment to wage-labor.  begin to have negative effects. At the population level, the combination of these contrasting influences on the probability of giving birth reflects a diversification of childbearing strategies and underlies the substantial increase in fertility variance in Cohort 1.67, and the prominent method in Latin America, with sharp increases in prevalence through the early 2000s68. In Mexico, tubal ligation is the most common form of fertility control, utilized among 58% of contracepting married women69, 2006 data, Tables As elsewhere in Latin America, the introduction of fertility-limiting family planning in the Maya community took the form of tubal ligation. Female sterilization is the most widely used contraceptive method in developing countriesdo early adopters of family planning drive the lower bound of family size down below what is feasible under natural fertility conditions, thereby increasing fertility variance?If the increase in fertility variance early in the transition is driven by the adoption of family planning, in this case by abruptly shortening the reproductive window, we expect to see a significant effect on the probability of giving birth in Cohort 1. In other words, the question we address is after they have had six to seven children , and at higher parities than those who do not. Having a TL appears initially to reflect the decision not to go on to have families of seven or more children, and to maintain an upper limit of fertility consistent with women’s preferences at baseline. Other reproductive traits do not significantly differ between women who elect to have a TL and those who do not  increases over time  was 4.1 (SD = 2.1), and 3.2 (SD = 1.1) for women in Cohort 3. Clearly, in later cohorts a TL substantially shortens women’s average reproductive careers by significantly lowering age at last birth  to an over-dispersed Poisson (where the variance becomes signifiantly greater than the mean) after the onset of the transition9. Further, our analyses provide evidence that increased variance is a characteristic of the fertility transition observable both as fertility rates across different populations7 as well as within populations .As a well-established metric for the influence of cultural transmission on low fertility norms72 and Gambian women73, who are more likely to adopt contraception at higher than lower parities. The relationship between contraception and fertility is complex in that it depends whether the population is viewed longitudinally across cohorts or within cohorts. Longitudinally, lower fertility is associated with contraceptive use, while within cohorts it may not be; higher than desired fertility may in fact be what motivates women to adopt contraception. Because Cohort 3 women have not yet completed their reproductive careers, younger women may go on to have another child before committing to a TL, and the difference in fertility between the two groups likely reflects age differences and comparing completed fertility (women who have had a TL) with  ongoing fertility (non-TL women).While this pattern has been described, the trigger mechanisms have remained unidentified. We find that family planning was adopted as it became available with market integration. But initially it has little influence on fertility variance, lowering total fertility or distinguishing the reproductive traits of those women who received a tubal ligation and those who did not. For the first cohort of women, family planning serves to maintain the upper limit of fertility, rather than pushing down the lower bound of fertility. This finding is consistent, for example, with studies among rural Ethiopian womenspread of lower fertility later in the transition. Exposure to outside social influences and learning increase over time with market integration, and by Cohort 2 nearly all Maya women who completed fertility had tubal ligations, which then has a clear effect on lowering fertility through earlier stopping behaviors. The lag time between the adoption of family planning in Cohort 1 and its effect on reproduction in Cohort 3 suggests that family planning itself does not instigate a change in fertility norms. The extent that the uptake of contraception represents cultural transmission73, this finding has implications for the debate concerning whether social versus economic influences explain fertility decline. While our model is not designed to evaluate economic influences per se, the uptake of family planning, as an indicator of social learning, is not initially associated with fertility decline.While the adoption of family planning does not appear to explain the origin of fertility decline, it contributes to the While initial exposure to new childbearing options and market opportunities was relatively homogenous, we expect individual responses to vary. The two-decade delay between the adoption of family planning and evidence of a fertility decline, we propose reflects the uncertainty of parents to commit to smaller families until they gain sufficient knowledge to weigh the costs and benefits of having fewer children. On a novel fitness landscape, given that the payoff to have either small or large families is yet unknown, we suggest that the response to this uncertainty is diversification of childbearing strategies at the population level, e.g. some parents double  down on what they know and have  the same or more children, while others take advantage of new investments in child quality and have fewer children.75. For example, environmental uncertainty can favor bet-hedging in reproductive strategies whereby parents vary their investments and reproductive output in attempts to match offspring phenotypes with alternative future environments77. Some evidence shows that when resource constraints on reproduction are diminished, as occurs when animals are relocated from wild environments to captivity, they exhibit unexpected increases in morphological variability78. Models of human fertility also have connected uncertainty and reproductive responses. When unpredictability is high or environmental circumstances calamitous, models predict both over-reproduction70 and low fertility80. Our empirical results suggest that during environmental fluctuation, both high and low fertility (e.g. increased variance within a population) are responses to environmental uncertainty.Variance in reproductive and behavioral strategies as a response to uncertainty has been studied nonhuman animals81. We suggest that this situation of uncertain payoffs characterizes the origin of many fertility transitions, and is the one that many small-scale societies find themselves in now, and has played out repeatedly over human history when technological innovations introduce new costs and benefits to family size decisions70.When the costs and benefits to family size adjustments are unknown, we propose that parents diversified childbearing strategies. Prior to market integration, parents know the value of goods and services and can make informed decisions about the number of children to have based on their anticipated food requirements, labor and production needs, and goals for child success. With market integration, in the Maya case instigated by a paved road, this known payoff structure is replaced with uncertainty such that parents cannot estimate the cost that smaller families may have on reducing the family work force or the benefit it may have for their children to pursue schooling and new economic opportunities. For instance, parents in the Dominican Republic who have no experience with formal education lack the prior knowledge to weigh the benefits to school their children versus the costs to relinquish their economic productionDifferent strategies may begin to emerge and then be perpetuated as some families gain access to market wealth, education or other new forms of status. Our results show that fertility tends to decline faster in those Maya households where wage-labor has become the dominant livelihood strategy (50% or more of the adults engage in some form of wage labor), than in mixed-economy households engaged in both agriculture and wage labor. Although new reproductive norms have yet to coalesce (e.g. variance is still high), women in predominantly wage labor households had the lowest yearly probability of giving birth. The trend toward smaller families, thus, is nuanced. While average family size is declining at the population level, it begs the future question whether divergent childbearing strategies will persist such that different fertility norms will form around distinct economic strategies.84. Over the course of four cohorts of Maya women, sharing-group size, land wealth and education all move from having relatively positive effects on fertility to having negative influences on fertility behaviors.Finally, our cohort analysis captures the well-known reversal of wealth effects on fertility, where wealth and status become decoupled from fertilityIn small indigenous communities world-wide, market integration can instigate transformative changes in fertility behavior. The Maya data show that during the early stages of market integration, before mean changes in fertility are evident, a diversification of childbearing strategies appears to be a first response to a new, yet uncertain environment. At the community-level, before the effects of family planning takes hold, childbearing strategies transition from being relatively homogenous to markedly heterogeneous. We highlight uncertainty as a key mechanism motivating the increase in variance, which results from the contrasting negative effects of novel determinants  pushing down fertility for some, and the positive effects of traditional determinants (land & labor pool) raising fertility for others. While the swing from low to high variance may settle down in the future, an important implication is that increased variance gives the foothold for inequality to emerge, and fertility to coalesce around different norms as parents pursue divergent childbearing strategies.87. Prior to 2000, all community members were subsistence maize farmers and the household was the unit of production and consumption. Because of the lack of roads and vehicles, little means existed to engage in cash cropping, formal schooling, wage-labor, or the regional economy. Wealth was measured by maize production, and high fertility was an asset because the size of the household labor force was directly related to the amount of maize that could be produced88.The Maya study population is located in the Puuc region of the central Yucatan Peninsula, Mexico. Multigenerational economic, demographic, and social network data have been collected in this community since the early 1990sA paved road was built into the community in the early 2000s, which introduced access to mechanized farming and means to transport crops to market, children to schools, and adults to wage labor jobs. However, costs and benefits to these new opportunities were clouded with uncertainty. Many households intensified maize production, putting more land under cultivation , the year of initial data collection. Relevant variables include age at first birth, age at last birth, year of each live birth, interbirth interval, total fertility, and year women had a tubal ligation. We recognize that a decline in infant mortality may lead to an increase in the number of surviving offspring, affecting changes in the variance. Infant mortality has been low in rural Yucatan throughout much of the historic period , and over the 26-year study period no significant changes in infant mortality have been documented in the communityn = 3), who were very elderly in 1992 and their reproductive histories could not be validated in later censuses (n = 2), or who had known fertility issues . This approach offers several advantages to analyzing aggregate counts (e.g. births per woman). First, recurrent event analysis increases the power to detect effects by drawing on information about the timing of births across a woman’s reproductive career, rather than simply her total fertility. Second, recurrent event analysis permits estimating covariate effects that are comparable for women who both have completed their fertility (Cohorts 0–2) and who are still in their reproductive careers (Cohort 3), which allows us to make inferences about current fertility trends.To model the influence that traditional  and novel (education and wage labor) fertility determinants have on changes in the distribution of fertility across the transition, we employ a recurrent event analyses, using an independent Cox proportional hazard model for each of the four cohorts. To account for correlated observations (births) within women, we employ a robust semiparametric analysis using a cluster id for women in each cohort using the ‘Survival’ package in RTotal fertility is summarized as the sum of live births a woman has had up to the year 2017, the last year of the survey. For women in Cohorts 0–2, this represent completed fertility, because all women have reached a terminal event. Because women in Cohort 3 are still of reproductive age (ages 20–39), they are right censored at end of survey in 2017. While fertility data have been collected annually since 1992, model covariates were collected at three time points, in 1993, 2013 and 2017; descriptive statistics of each covariate by cohort are given in Table Women census into the model at age 15 and reach a terminal event when they are no longer at risk of birth, either at age 50 or when they have a tubal ligation. Nulliparious women are included in the recurrent event history analyses in modeling changes in determinants of fertility. They were not included in modeling family planning influences (described below) since only women who have given birth have tubal ligations. Sensitivity analyses show that the results do not alter when nulliparous are or are not included  using a non-parametric Mann–Whitney U test among women with and without a tubal ligation within a cohort. Further, we evaluate whether women receiving tubal ligations differ significantly in terms of the wealth, education, or market engagement compared to women who never received one Table .The Maya research has been approved by the Human Subjects IRB at Stony Brook University (20074791), Harvard University (F18643-101-104) and the University of Utah , and by members and leaders of the Maya community. Informed consent was obtained from all subjects or, if subjects are under 18, from a parent and/or legal guardian.Supplementary Information


Background and Objectives: Hyperuricemia is associated with several comorbidities. The association between uric acid (UA) and pulmonary function is still a controversial issue. This study evaluated the gender-specific association of serum UA and pulmonary function. Materials and Methods: A total of 3177  participants aged 40 years or older were selected from the 2016 Korean National Health and Nutrition Examination Survey and included. Results: Female participants with hyperuricemia were older than participants with normouricemia. Body mass index (BMI), mean arterial pressure (MAP), hemoglobin A1c (HbA1c), and estimated glomerular filtration rate (eGFR) were significantly associated with UA levels in both males and females. Hyperuricemia and increase in UA quartile were significantly associated with decreased forced expiratory volume in 1 second (FEV1) and forced vital capacity (FVC) in females after adjustment for age, income, region, education, marital status, alcohol consumption, smoking, BMI, MAP, HbA1c, and eGFR. There was no significant association between UA levels and lung function in males. After additional adjustment for respiratory disease including pulmonary tuberculosis, asthma, and lung cancer, the association between hyperuricemia and decreased FEV1 and FVC in females was revealed. Conclusions: Hyperuricemia was associated with decreased FVE1 and FVC in the female general population.  Serum uric acid (UA) is the end product of the metabolism of purine compounds in humans and higher primates. The UA level is determined by several factors including genetics, diet, alcohol, renal function, and drugs. Hyperuricemia is caused by overproduction of UA or underexcretion of UA or a combination of both processes. UA homeostasis is maintained in the human body by the kidneys and intestinal excretion. Decreased efficiency of renal UA excretion in patients is responsible for about 85% to 90% of cases of hyperuricemia. Hyperuricemia is associated with an increased risk of gout. Furthermore, hyperuricemia is related to hypertension, coronary heart disease, stroke, diabetes, obesity, and metabolic syndrome ,2,3,4,5.n = 19,770,902) participants were selected for analysis. Written informed consent was obtained from participants before completing the survey. This study was approved by the Institutional Review Board of Soonchunhyang University Bucheon Hospital .The Korea National Health and Nutrition Examination Survey (KNHANES) is a nationwide survey conducted periodically by the Korea Centers for Disease Control and Prevention to investigate health and nutritional status of the Korean population . The KNHThe KNHANES was conducted by four special research teams, each comprising of eight experts including nutritionists, nurses, and students with majors in public health. The selected professional investigator was placed at the investigation site after completion of education and practice for one month. Subsequently, the ability to conduct research was verified through regular education and an on-site quality management system. A standardized interview was performed in participants’ homes, and an established questionnaire was used to collect information about demographic variables and socioeconomic characteristics. Specifically, data on age, sex, income, region, education, marital status, alcohol consumption, and smoking status were collected from the participants. Alcohol consumption was divided into the following four groups based on the frequency of alcohol consumption during the past year: never, ≤1/week, 2 to 3/week, and ≥4/week. Income level was categorized into quartiles based on average individual monthly income. Urban and rural areas were classified by the administrative district . Serum uBlood pressure was measured by nurses by using a standard protocol. Blood pressure was measured on the right arm using a mercury sphygmomanometer after at least five minutes of rest and a sitting position. Blood pressure was measured three times and the mean value of the second and third blood pressure was used for analysis. Mean arterial pressure (MAP) was calculated using the following formula:2). Pulmonary tuberculosis, asthma, and lung cancer were defined in the questionnaire as ʻpulmonary tuberculosis diagnosed by a physicianʼ through a standardized interview. The question was “Was your pulmonary tuberculosis diagnosed by a physician?”. The questionnaire consisted of three responses . Participants who chose 1 (Yes) were classified under pulmonary tuberculosis. Each interview was conducted individually by a trained professional investigator. Blood samples were collected from participants after at least eight hours of fasting. The blood samples were immediately refrigerated and transported in cold storage to the central testing facility. Height and weight were assessed using standardized techniques and equipment. Briefly, height was measured to the nearest 0.1 cm using a portable stadiometer SECA 225 . Weight was measured to the nearest 0.1 kg using a GL-6000-20 . Body mass index (BMI) was calculated by dividing weight by height squared . The subjects were required to be in a sitting posture with their upper body tilted about 15 degrees and asked to maintain the posture until the completion of the test. The test was demonstrated in easy-to-understand terms and the subjects were encouraged to exhale through exercises. Spirometry was conducted at least three times per subject (maximum up to eight times). Tests with acceptable curves and without errors were performed twice or more, and the best graph was stored, according to the American Thoracic Society/European Respiratory criteria for standardization . Spiromep-value outcomes ≤0.05 in univariable analysis were included in the multivariable analysis. Multivariable linear regression analysis was performed as model 1 adjusting for age, income, region, education, marriage, smoking, alcohol consumption, BMI, eGFR, hemoglobin A1c (HbA1c), and MAP. Model 2 was obtained after adjusting model 1 plus pulmonary tuberculosis, asthma, and lung cancer. Simple correlation analysis was performed to evaluate the association between lung function and UA levels. Analyses were performed using SPSS statistics version 20.0 . All p-values were two-sided, and a p-value of less than 0.05 was considered to indicate statistical significance.To reflect representative estimates of the non-institutionalized Korean civilian population, survey sample weights were applied in the analyses. Sample weights were calculated by considering the sampling rate, response rate, and age/sex proportions of the reference population . Clinical characteristics were analyzed according to dichotomized serum UA or quartiles of UA. Due to the sex difference in UA levels, the definition of hyperuricemia was different in males and females  ,17. To aThe KNHANES data were released after anonymization and the study population was not involved in the design of this study.n = 19,770,902) participants aged 40 years or older were selected from the 2016 KNHANES and analyzed. The mean age was 55.60 ± 0.27 years old. Among 3177 participants, 1401 (49.9%) were males and 1776 (50.1%) were females. Mean serum uric acid levels were 5.03 ± 0.02 mg/dL. The clinical characteristics of the subjects are summarized according to gender and the presence of hyperuricemia in A total of 3177  and IL-6 has been observed . UA stimIn addition, elevated UA is associated with endothelial dysfunction . PulmonaAlthough the sample size was not sufficient to analyze the effect of UA on pulmonary function in subjects with all pulmonary disease in the present study, we found that there was no significant association between UA and lung function among participants with pulmonary tuberculosis in both males and females. Previous studies showed that hyperuricemia was associated with poor prognosis in patients with COPD. In the German COPD cohort study, UA was significantly associated with reduced FEV1, reduced 6 min walk distance, and acute exacerbations . HyperurIn the current study, there was no association between UA and pulmonary function in male participants. The cause of the sexual difference between pulmonary function and UA is not certain. In females, serum UA levels are lower than in males, but UA levels increase around menopause. Sex hormones may impact UA metabolism and the relative physiologic impact of hyperuricemia may be stronger among females than males . This study has several limitations. First, although we analyzed pulmonary disease including asthma, pulmonary tuberculosis, and lung cancer, other pulmonary diseases such as interstitial lung disease and bronchiectasis were not identified in this survey. Second, it was not possible to analyze medications such as UA lowering agents or diuretics that could affect the serum UA levels. Third, biochemical characteristics that closely related serum uric acid levels were included in the analysis. Inflammatory biochemical parameters which could affect pulmonary function were not included in this study. Fourth, the causality could not be determined due to the cross-sectional design of this study. Despite all these limitations, the strength of this study lies in the fact that we analyzed the relationship between UA and pulmonary function using a nationwide representative sample of the general adult population. In conclusion, serum UA was negatively associated with FEV1 and FVC in the Korean female general population. Results of this study suggest that hyperuricemia may decrease pulmonary function. Further investigations are required to clarify the relationship between serum UA levels and pulmonary function.


Isolated rapid eye movement (REM) sleep behavior disorder (iRBD) is a prodromal stage of Lewy-type synucleinopathies (LTS), which can present either with an initial predominant parkinsonism (Parkinson’s disease (PD)) or dementia (dementia with Lewy bodies (DLB)). To provide insights into the underlying pathogenic mechanisms, the lipoprotein and protein glycosylation profile of 82 iRBD patients, collected before and/or after their conversion to an overt LTS, and 29 matched control serum samples were assessed by nuclear magnetic resonance (NMR) spectroscopy. Data were statistically analyzed to identify altered metabolites and construct predictive models. Univariant analysis detected no differences between iRBD patients with an LTS compared to controls. However, significant differences were found when the analysis distinguished between iRBD patients that manifested initially predominant parkinsonism (pre-PD) or dementia (pre-DLB). Significant differences were also found in the analysis of paired iRBD samples pre- and post-LTS diagnosis. Predictive models were built and distinguished between controls and pre-DLB patients, and between pre-DLB and pre-PD patients. This allowed a prediction of the possible future clinical outcome of iRBD patients. We provide evidence of altered lipoprotein and glycosylation profiles in subgroups of iRBD patients. Our results indicate that metabolic alterations and inflammation are involved in iRBD pathophysiology, and suggest biological differences underlying the progression of LTS in iRBD patients. Our data also indicate that profiling of serum samples by NMR may be a useful tool for identifying short-term high-risk iRBD patients for conversion to parkinsonism or dementia. The biological basis underlying this clinical heterogeneity remains poorly understood.Neurodegeneration associated with aggregated α-synuclein in the form of Lewy bodies and neurites  manifests clinically as a parkinsonism, usually associated with late onset dementia )4. Although iRBD patients have no overt neurological diseases, long-term prospective studies show that over 90% of them develop an LTS within 14 years after iRBD diagnosis, and the remaining disease-free patients show short-term risk markers for developing an LTS6. As such, iRBD is a clinical marker of the prodromal stage of LTS7, and provides an excellent opportunity to study LTS at early stages. This is important for management at clinical onset, development of early disease-modifying interventions, and selection and stratification in future neuroprotective trials.Isolated rapid eye movement (REM) sleep behavior disorder (iRBD) is a parasomnia characterized by vigorous dream-enacting behaviors and loss of REM sleep muscle atonia11 and inflammatory processes13. Nuclear magnetic resonance (NMR)-based tests are a robust method to characterize lipoproteins and protein glycosylation profiles in intact serum samples14, which have been used to study metabolic changes18 and systemic inflammatory processes20. However, this type of approach has not been explored before in the context of iRBD.iRBD and LTS have been associated with cardiovascular risk factorsHerein we evaluated the lipoprotein and protein glycosylation profiles of serum samples from iRBD patients by NMR and assessed whether the baseline profile identifies the future development of an LTS, and whether that manifests initially with parkinsonism or dementia.p value = 0.0373). There were also no differences in the age at iRBD nor LTS diagnosis, assessed by mean ranks comparison and corrected for multiple comparison. Moreover, there were no differences of time between iRBD and LTS diagnosis, sample collection and LTS diagnosis (pre-groups), and LTS diagnosis and sample collection (post-groups). There were only age differences at sample collection between controls and post-LTS , controls and post-DLB samples , pre-LTS and post-LTS samples , and pre-DLB and post-DLB samples . Comparably, there were differences in the time between iRBD diagnosis and sample collection between pre-LTS and post-LTS , and pre-DLB and post-DLB samples . These differences are inherent to the timeline of iRBD and LTS onset. Similar results were obtained when considering only the paired samples from each group , and 29 from controls (Table 21 (considered as controls)  compared to control subjects (431 ± 115 µmol/L), with a false discovery rate (FDR)-corrected p value of 0.006 , and post-LTS (which comprise post-DLB and post-PD) iRBD patients. No significant differences were found between pre-LTS or post-LTS iRBD patients compared with controls  higher levels of area glycB, and lower levels of medium LDL-P and LDL-TG were found in iRBD patients after the diagnosis of an LTS compared to the same patients before the diagnosis  towards lower levels of LDL-TG, LDL-Z, medium LDL-P, and large HDL-P, and higher levels of area glycB were found in iRBD patients after the diagnosis of DLB, compared to the same patients before the diagnosis  iRBD patients for which we had paired samples, from before and after their diagnosis of LTS, were compared. Significantly , a sensitivity of 53.33%, and a specificity of 96.55% at a cut-off of y > −0.233. Second, an equation was obtained distinguishing between pre-DLB and pre-PD patients that included small HDL-P and HDL-Z , a sensitivity of 72.22%, and a specificity of 86.67% at a cut-off of y > 0.201. Finally, we applied the model that distinguished between pre-DLB and pre-PD patients to 33 iRBD patients that remained disease-free at the end of the study . This allowed the prediction that six individuals would classify as pre-DLB patients, while the rest would classify as pre-PD patients  or dementia (pre-DLB) at onset.22, altered DAT-SPECT23, and pareidolias24, the latter being more associated to DLB. Also, few biochemical peripheral biomarkers have been suggested to identify iRBD from healthy controls, including impaired ghrelin excretion25, serum N-glycan composition26, and serum proteins, such as dopamine β-hydrolase12. Nevertheless, all these marker studies compared iRBD patients with controls, without considering that the patients might be a heterogeneous group including individuals close to phenoconversion to an LTS manifesting primarily with parkinsonism or dementia.Currently, existing short-term conversion biomarkers in iRBD to LTS include hyposmiaarea glycB could be considered as a biomarker for iRBD patients that develop a dementia onset LTS. This measurement corresponds to the amount of N-acetylneuraminic acid in glycoproteins. N-acetylneuraminic acid, the most abundant form of sialic acid in human cells, has a negative charge and hydrophilicity, which enable its structural and modulatory roles, such as blood cell charge repulsion or neural plasticity27. Sialylation changes have been associated with infectious diseases, stress, inflammation, autoimmune diseases, cancer, and neurodegeneration28. As such, changes in area glycB have been described as an inflammatory marker associated with insulin resistance and adiposity29, obesity20, and rheumatoid arthritis19 (reviewed in ref. 30). In our data set, although there were no differences in the prevalence of cardiovascular risk factors , area glycB was decreased in pre-DLB patients compared to controls, but then there was a trend to increased area glycB in post-DLB compared to pre-DLB condition. This parameter is not modified in any of the analysis considering our PD cohort, implying possible biochemical differences in the patients manifesting these two different phenotypes. A few recent studies reported differences in peripheral cytokines between iRBD patients and healthy controls33, in particular, increased serum/plasma TNF-alpha levels in iRBD compared to controls. Furthermore, their data showed higher levels at baseline in those patients who later converted to LTS compared to those iRBD non-converters. Although there are no studies specifically on the inflammatory profile of iRBD patients before developing dementia onset LTS, peripheral inflammation has been seen in early stages of manifest DLB35. This phenomenon could be associated with the observed trend to increased inflammatory markers during the neurodegeneration that spans from iRBD to DLB conversion, although the post-DLB levels are like those in control individuals. Further studies are needed to elucidate the role of peripheral immune changes in the pathogenesis of neurodegeneration in the prodromal stage of LTS.Here, using two different analysis methods, we observed that the parameter area glycB, and lower levels of medium LDL-P and LDL-TG after the diagnosis of an LTS. Higher levels of area glycB would point towards a more inflammatory profile when patients have an overt neurological condition, as also suggested by our previously discussed results. On the other hand, lower levels of medium LDL-P and LDL-TG would point towards a lower cardiovascular risk37, which is in disagreement with previous findings associating iRBD with higher cardiovascular risk factors8. It should be noted that these three parameters showed the same trend when analyzing the DLB, but not the PD group. Since DLB patients (n = 12) outnumbered the PD patients (n = 7) in the paired samples, it is possible that these changes are not common for both LTS manifestations, but rather specific for DLB. Studies with a larger cohort are needed to address these ambiguities and draw functional conclusions about the results.The analysis of samples from the same iRBD patients before and after their LTS diagnosis revealed higher levels of We built a model to distinguish between iRBD patients that will eventually evolve into an LTS with an initial parkinsonism or dementia. The identification of a predictive biomarker for the conversion of iRBD patients to parkinsonism or cognitive impairment is important for the clinical management and prognosis of both conditions. When applying our model to iRBD patients without an overt neurological condition (33 iRBD-only samples), we classified six individuals as pre-DLB patients while the rest would classify as pre-PD patients. Remarkably, five of the putative pre-PD patients, were classified as such with a sensitivity of 100%. Hence, we will examine the potential future clinical utility of our model by following closely the clinical evolution of these five individuals in the ensuing years at our institution.HDL-Z) and the number of small HDL particles . The parameter HDL-Z, which measures HDL particle profile and its heterogeneity, is inversely associated with cardiovascular risk38. Nevertheless, these differences are not statistically significant in a univariate analysis and there are no studies to confirm or deny the differences in cardiovascular risk between iRBD patients that will initially manifest parkinsonism or dementia, which should be further tested in future validation studies in independent iRBD cohorts.We have found that the putative prediction to distinguish iRBD patients that will manifest parkinsonism or dementia was mainly based on the changes in two inversely related parameters: the mean size of HDL particles  showed different gender distributions. Still, the models here described correspond to comparisons without any statistically significant gender bias.This study has some limitations. First, the metabolic profiling was done in serum samples from non-fasting individuals, which adds variability and represents a confounding factor that could modify the results. Still, we have used stringent statistical cut-off values to avoid false-positive and -negative results. Also, one would expect that the putative alterations would be present in all groups under study since all samples were taken in non-fasting conditions, as well as the fact that variability between individuals is greater than the one expected by fastingStrengths of our study include (1) the analyses of 101 samples from iRBD patients, which can be considered a large number in the iRBD field, (2) that the iRBD diagnosis was confirmed by video-polysomnography in all cases, (3) that some samples were obtained from the same patients longitudinally before and after conversion to LTS, and (4) that machine learning techniques were used to build predictive models rather than using simple statistical comparisons between groups.In conclusion, this study provides evidence of altered lipoprotein and protein glycosylation profiles in iRBD patients. Our results support that metabolic alterations and inflammation are involved in iRBD pathophysiology, and suggest biological differences underlying the progression of LTS in iRBD patients. We found that the baseline profiling of plasmatic lipoproteins and protein glycosylation in serum samples could potentially distinguish between iRBD patients that will eventually evolve to an LTS with either an initial development of parkinsonism or dementia. This information in combination with other clinical  and neuroimaging  markers could allow to better identify subjects with a high risk for short-term conversion to an overt LTS. Although this study requires an independent validation with larger and longitudinal cohorts to confirm the putative predictive value of the models here described, it provides an approach to a more accurate classification of iRBD patients in subtypes or variants with similar prognosis and underlying biology. If confirmed, this approach will help the design of future intervention studies to target more homogeneous subsets of LTS at early stages.39. Patients and controls were Caucasians of Spanish origin. In all patients, iRBD was diagnosed by increased electromyography activity in the four limbs and in the chin associated with abnormal behaviors during REM sleep . After iRBD diagnosis, patients were periodically followed at least every 6–12 months by a neurologist. Blood sample donation occurred during these routine visits in non-fasting conditions. If PD or DLB were suspected in the routine visits, iRBD patients were then examined by movement disorders or dementia expert neurologists to confirm the diagnosis. Diagnosis criteria were those accepted for PD1 and DLB2. When iRBD patients converted to PD or DLB, they were asked to donate a blood sample, either again if already participating in the study (paired samples) or for the first time if newly recruited.Polysomnographic-confirmed iRBD patients were recruited prospectively between 1996 and 2015 at the center for sleep disorders of the Neurology Service from the Hospital Clínic of Barcelona, SpainSamples from a total of 82 iRBD patients were obtained. From these, the following age- and sex-matched groups were distinguished: (i) 33 individuals that remained disease-free at the end of the study (July 2019) (iRBD-only), (ii) 33 iRBD individuals that had no overt neurological disease at sample collection but later on converted to LTS (pre-LTS), among which there were (ii-a) 15 iRBD individuals that later on converted to DLB (pre-DLB), and (ii-b) 18 iRBD individuals that later on converted to PD (pre-PD), and (iii) 35 iRBD individuals that at sample collection had already converted to an LTS (post-LTS), among which there were (iii-a) 20 individuals that had already converted to DLB (post-DLB), and (iii-b) 15 individuals that had already converted to PD (post-PD). From the 33 iRBD patients who had no overt neurological disease at sample collection (ii), 19 converted later  and a second sample was then collected and included within the 35 samples from the iRBD group with a diagnosed LTS (iii). Thus, from these 19 patients paired samples were available before and after the LTS diagnosis. We also included 29 samples from sex- and age-matched healthy controls without evidence of neurological or sleep disorders, which were selected from a database of healthy controls made of non-consanguineous attendants and volunteers who donate samples for research studies at our institution. All samples were processed and stored by the same person to avoid differences that could affect the results.Demographic data are presented in Table The ethical committee at the Hospital Clinic de Barcelona, Spain, approved the study and all participants gave written informed consent. Samples were registered at the biobank of IDIBAPS (S080327-01NL). Usage of the blood samples for research into disease biomarkers was approved by the Hospital Clínic Research Ethics Committee (HCB/2014/1065) and the Vall d’Hebron Hospital Research Ethics Committee (PR(AG)370/2014).g for 10 min at 4 °C. Serum volumes of 2 ml were removed from supernatant, aliquoted in polypropylene CryoTubes , flash frozen, and stored at −80 °C.Five to 10 ml of blood were collected in tubes without anticoagulant , preserved for 30 min at room temperature, and centrifuged at 1500 × 1H NMR spectroscopy to estimate the size (-Z) of the three main types of lipoproteins (very low-density lipoprotein (VLDL), low-density lipoprotein (LDL), and high-density lipoprotein (HDL)), and the concentration (-P) of particles of three subtypes  of the main types of lipoproteins, as well as the lipid content [cholesterol (-C) and triglycerides (-TG)] of the three main classes, together with intermediate-density lipoproteins (IDL)40. The glycoscale uses the same technique to determine the presence of glycosylated proteins in serum, which is an indicator of systemic inflammatory processes20. More specifically, it determines N-acetlyglucosamine and N-acetylgalactosamine bound to protein (glycA), N-acetylneuraminic acid bound to protein (glycB), and any of the three acetyl groups not bound to protein (glycF). From these three peaks, it distinguishes their area (associated with concentration) and the ratio hight/width (describing the peak shape).The lipoprotein profile and the presence of glycosylated proteins were measured in the 130 serum samples at the same time using the Liposcale® and the Glycoscale tests , respectively. The Liposcale® test is a CE marked and previously reported method based on 2D diffusion-ordered p value < 0.05). Results are expressed as median ± interquartile range.Differences between groups were assessed for those informative comparisons in terms of disease pathophysiology and with a diagnostic or prognostic value. These include control vs. pre-LTS, pre-DLB, pre-PD, post-LTS, post-DLB and post-PD, pre-LTS vs. post-LTS, pre-DLB vs. pre-PD, post-DLB vs. post-PD, pre-DLB vs. post-DLB, and pre-PD vs. post-PD. Since individuals in the iRBD-only group are a heterogeneous mixture of possible pre-LTS and individuals who might not convert during their lifetime, we excluded comparisons between them and any other group for being non-informative. The differences between comparisons were determined by a Wilcoxon signed-rank test combined with the Benjamini–Hochberg procedure to correct for false discovery rate (FDR) for any given comparison .Paired samples  were (re)analyzed independently using the paired and non-parametric test Wilcoxon matched-pairs signed-rank test. Correction for multiple comparisons was done by controlling the FDR with the Benjamini–Hochberg procedure . Next, elastic net (R function cv.glmnet) was used on all variables from the chosen comparisons for variable selection and model building41. The elastic net mixing parameter was tested from 0.1 to 0.9, at intervals of 0.05, and a 10-fold cross-validation was performed. The model with the lowest mean cross-validated error was kept, and a receiver operating characteristics curve was made. Internal validation of the model was achieved by bootstrapping42, using the R function vboot.glm. Internal bootstrapping validation logistic model was done with 1000 bootstrap samples, and 10-fold cross-validation with 100 replicates. The AUC from each model was corrected by subtracting the optimism value obtained by bootstrapping, and only those models with a corrected AUC > 0.75 were kept.Variables that allowed distinguishing between all groups were determined by non-parametric Kruskal–Wallis test correcting for multiple comparisons by controlling the FDR with the Benjamini–Hochberg procedure. Those variables significant with an FDR-corrected 43 and GraphPad Prism version 8.0.1 for Windows .Data analyses were performed in RFurther information on research design is available in the Supplementary Table 1REPORTING SUMMARY


Aberrant expression of the immune checkpoint molecule, CD276, also known as B7-H3, is associated with tumorigenesis. In this review, we aim to comprehensively describe the role of CD276 in malignancies and its potential therapeutic effect.Database including PubMed, EMbase, Cochrane Library, CNKI, and Clinical Trails.gov were searched for eligible studies and reviews. Study selection: Original studies and review articles on the topic of CD276 in tumors were retrieved.CD276 is an immune checkpoint molecule in the epithelial mesenchymal transition (EMT) pathway. In this review, we evaluated the available evidence on the expression and regulation of CD276. We also assessed the role of CD276 within the immune micro-environment, effect on tumor progression, and the potential therapeutic effect of CD276 targeted therapy for malignancies.CD276 plays an essential role in cell proliferation, invasion, and migration in malignancies. Results from most recent studies indicate CD276 could be a promising therapeutic target for malignant tumors. Immune Checkpoint Therapy (ICT) is a novel treatment for malignancy which shows great efficacy by enhancing anti-tumor immune response of T cells. The B7 family, which is important in regulating T cell immune response, has been highlighted for its potential therapeutic effect on cancers. B7 family members, including B7-H1 PD-L1/CD274), B7-DC (PD-L2/CD273), B7-H2 (ICOSL), B7-H3 (CD276), B7-H4 (B7S1/B7x/Vtcn1), B7-H5 (VISTA/GI24/Dies/PD-1H), B7-H6 (NCR3LG1), B7-H7 (HHLA2), B7.1 (CD80), and B7.2 (CD86), could be categorized into (a) co-stimulatory, (b) co-inhibitory, or (c) mixed co-stimulatory and co-inhibitory subtype according to how they influence T cell activation  is characterized as a malignant adrenal tumor with a high recurrence rate and a short disease-free survival, and 91.7% (44/48) of ACCs expressed cytoplasmic and membranous CD276 in neoplastic or tumor associated vascular cells. CD276 expression in tumor blood vessels was significantly positively correlated with local invasion (higher T stage) and with advanced ENSAT stage . Cytoplasmic CD276 expression is negatively correlated with both the overall survival and disease-free survival of ACC patients. Gene set enrichment analysis demonstrated that CD276 expression is closely related to immune regulation and malignant biological behavior of ACC .http://ualcan.path.uab.edu/index.html). UALCAN is an online web-server for facilitating tumor subgroup gene expression and survival analyses based on TCGA RNA sequencing data and clinical data. CD276 expression level in cancer and corresponding normal tissues is shown To give a more intuitive aspect of the expression level of CD276 in malignancies, we performed a pan-cancer analysis to evaluate CD276 expression level in multiple cancers on UALCAN database (in vivo study and found that in mouse dendritic cells CD276 expression could be enhanced by interferon-γ (IFN-γ) and suppressed by interleukin 4 (IL-4). They concluded that CD276 was a negative regulator of T cell function preferentially affecting type-1 T helper cell responses (+CD25 regulatory T cells (Tregs) have immunosuppressive effects and can inhibit the proliferation of effector T cells both in vivo and in vitro  or PD-L2 (B7-DC) bind to PD-1, and B7-H6 binds to NKp30 , 41. HowIn 2019, Husain et al.  implemenCD276 was initially described as a co-stimulatory molecule for T cell activation . FurtherProgrammed Cell Death-1 (PD-1) is an immunosuppressive receptor, first reported in 1992 . PD-1, aPD-1 is one of the checkpoint receptors, which is tightly associated with immune escaping of tumor cells. Such a characteristic renders PD-1/PD-L1/PD-L2 as a significant target for immunotherapy, which has been applied to cancer therapy and promising results were reported. Even so, PD-1/PD-L1/PD-L2 molecules are still only a small part of immunological checkpoints with therapeutic values. There are more potential immune checkpoints which may be used as therapeutic targets. PD-1 and CD276 are both members of B7/CD28 family and had similar effects on the tumor growth micro-environment. The binding of CD276/PD-1 and corresponding receptors inhibits the proliferation of T cells and the secretion of IFN-γ, TNF-α, and other cytokines, exerting synergistic effect with PD-1/PD-L1/PD-L2 , 48. ComAnother promising molecule for immune regulation is CTLA4 (CD152). CTLA4 is an intracellular glycoprotein that acts as a functional inhibitor of T cell responses . MechaniBased on the information above, CD276 inhibits T cell activation and has synergistic effects with CTLA4. Leitner et al. believe that CD276 exerts its inhibitory effect on T cells by inhibiting IL-2 secretion. Exogenous supplementation with IL-2 can reverse this effect, suggesting that CD276 acts as an upstream regulatory molecule for IL-2 . CTLA4 iCD276 can directly participate in the regulation of immunity and various malignant behaviors of tumor cells.in vivo and in vitro. Thus, even in aerobic environments, cancer cells still produce energy by metabolizing glucose into lactic acid (anaerobic glycolysis), which is called Warburg effect and this is promoted by CD276. CD276 also increases the expression level of HIF-1α, as well as the expression of its downstream signaling molecules and the key enzymes in the glycolysis pathway (LDHA and PDK1). Furthermore, it is reported that CD276 promotes the stability of active oxygen-dependent HIF-1α by inhibiting the stress-activated transcriptional factor (Nrf2) and its targets . Finally, a metabolic imaging experiment further confirmed that CD276 enhances the glucose uptake by tumor cells, thereby promoting tumor growth in a mouse breast cancer xenograft model. These findings provided a new perspective of immunomodulatory B7 family on tumor progression.CD276 is involved in the regulation of proliferation, apoptosis, invasion, cell cycle, cell differentiation, cell autophagy, and epithelial-mesenchymal transition. To examine the role of CD276 in metabolic reprogramming of cancer cells, Lim et al.  conducteIn vivo, silencing CD276 with shRNA prolongs the asymptomatic survival period. CD276 shRNA inhibits the expression of metastasis-related proteins in melanoma cells, such as matrix metalloproteinase MMP-2, tissue inhibitors of metalloproteinase (TIMP1 and TIMP2), signal transduction and transcription activator (STAT3), and IL-8  and enhanced SMAD4 expression . In vivor growth . In anotr growth .Nutrition is essential for tumor cell proliferation. New blood vessels are required in tumors to ensure substrate provision to cells distant from the normal vasculature. Normal tissue blood supply cannot guarantee the stable support of tumors. Therefore, angiogenesis markers are considered to be another characteristic of tumors . AngiogeIn vitro and in vivo experiments demonstrated that CD276 promotes the secretion of vascular endothelial growth factor (VEGF), which further promotes the formation of blood vessels. CD276 could also activate NF-κB signaling through a TLR-4-dependent mechanism, and this could cause subsequent up-regulation of VEGF and IL-8. Elevated expression levels of VEGF and IL-8 further promote tumor invasion and angiogenesis  targeting PD-1/PD-L1, CTLA-4 have been brought into clinical application and great efficacy was shown for numerous malignant tumors. As CD276 expression level was observed to be correlated to tumor growth, invasion, and metastasis, CD276 could be a potential target for immunotherapy. In previous studies, CD276 targeted therapies including monoclonal antibodies blocking CD276, CD276 specific antibody-dependent cell-mediated cytotoxicity (ADCC), antibody drug conjugates (ADC); bispecific antibodies against CD276/CD3, CD276–specific small–molecule inhibitor, and chimeric antigen receptor (CAR) T-cell therapy have been discussed , 72, 73.Drug vectors targeting the expression of CD276 may be an ideal therapeutic approach. Lupu et al.  injectedTo date, anti-CD276 antibodies have been developed, tested in animals, and are in clinical trials as immunotherapy agents. Xu et al.  confirmeIn vitro studies showed that mAb 376.96 combined with the tyrosine kinase inhibitor sunitinib significantly inhibits tumor growth. Ma et al.  T cell therapy is popular in immunotherapy. The objective response rate of CAR-T cell therapy in patients with hematologic malignancy is high. In solid tumors, due to the lack of tumor-specific antigens and the immunosuppressive effect of tumor microenvironment, there are some obstacles for CAR T cells to overcome. The gene module programming technology can improve the efficacy and specificity of CAR T, which is expected to target solid tumors for CAR T cells.in vitro and in vivo. CD276 CAR-T showed stronger anti-tumor activity when targeted at tumor cells expressing PD-L1. Du et al. believed that CAR T cells significantly inhibit tumor growth without obvious toxicity or side effects. These evidence all support the prospect of CD276 CAR-T cell therapy being promising in patients. CAR-T cell therapy has shown great success in the treatment of recurrent childhood acute lymphocytic leukemia. However, due to the lack of reliable, targeted, and differentially expressed molecular cell surface markers in solid tumors, CAR-T cell technology has not yet been applied. Majzner et al. (in vivo, leading to regression of solid tumors in xenograft models including osteosarcoma, medulloblastoma, and Ewing’s sarcoma. They also demonstrated that the efficacy of CD276 CAR-T cells depended on the density of target antigens on the tumor surface, while the activity of cells expressing low levels of antigens is significantly reduced. In a recent study, Johanna et al. (In 2019, Du et al.  construcr et al.  developea et al.  found CDThe functionality of CD276 is complicated. Although great progress has been made in the past two decades, further research is still needed. The regulation of CD276 expression is closely related to tumor growth, invasion, metastasis, and immune escape. Due to increased expression levels of CD276 in multiple tumors and low expression in a variety of normal tissues, numerous studies used CD276 as the target for tumor gene therapy and monoclonal antibody therapy. Overall, CD276 does have great potential as an immunotherapeutic target, but its clinical value must be confirmed by further studies and clinical trials.Conception and design: XW and YL. Acquisition of data: SL and JL. Analysis and interpretation of data: ZL, CZha, YW, and CZho. Drafting of the manuscript: SL and JL. Critical revision of the manuscript for important intellectual content: XW and YL. Administrative, technical, or material support: AW, FZ, KW, and FXZ. Supervision: XW and YL. All authors contributed to the article and approved the submitted version.This paper was supported by the following grants: China Scholarship Council 202006240301 to SL.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.