Considerable research in western, low-fertility contexts has examined minority-vs.-majority fertility differentials, typically focusing on minority groups’ cultural idiosyncrasies and on socioeconomic disadvantages associated with minority status. However, the formation and functioning of ethnic complexities outside the western world often diverge from the standard western model and so may their impact on fertility preferences, behavior, and outcomes. We expand on the previous research by analyzing ethnic variation in completed and desired fertility in the multiethnic transitional setting of Kyrgyzstan, where ethnic groups and their ethnolinguistic subparts are characterized by both different stages of the demographic transition and different positioning in the socioeconomic and political hierarchies. Using combined data from two rounds of a nationally representative survey, we find that ethnic-specific levels of completed fertility generally align with culturally shaped group-level normative propensities. In contrast, in desires to have a(nother) child, the ranking of the ethnic segments is more reflective of their collective societal positioning, with more disadvantaged segments having lower fertility desires, regardless of actual number of children and various other characteristics. We also find that ethnic homophily of respondents’ social milieu and their optimism about the future of their ethnic group are positively associated with fertility desires, even though these associations are more potently present among women, compared to men. We relate our findings to the extant scholarship and reflect on their implications for a better understanding of ethno-racial fertility dynamics and differentials in transitional contexts. Considerable scholarship has examined ethnic and racial variations in fertility behavior and outcomes. The most common initial assumption of this literature is that such variations may reflect ethnoracial differences in individual characteristics, especially those that define socioeconomic status such as educational attainment, employment, income, and wealth. Beyond the ‘characteristics’ perspective, the literature has focused on inter-ethnic cultural differences that may be consequential for the norms and practices that are seen as proximate determinants of fertility such as pre/non-marital sexual partnership, timing of marriage, and use of contraception and abortion e.g., . This ‘cHowever, most research on ethnoracial variations in fertility has dealt with contemporary western contexts, with a particular focus on native vs. immigrant comparisons, typically attributing higher fertility among immigrants and their descendants to their greater traditionalism/familism, often sustained by barriers to their cultural assimilation and social integration—i.e., a combination of cultural and minority status factors. Yet, ethnic diversity in many non-western settings is fundamentally different as it usually preceded the formation of nation states and corresponding natives-immigrants encounters. While in most such settings one ethnic group may dominate numerically, culturally, and politically, and, in fact, the state may deploy policies aimed at effacing the unique ethnolinguistic and other cultural idiosyncrasies of minority groups, the assimilationist framework, which typically drives the research on fertility of ethnic minorities in the West, is not fully applicable to such settings. At the same time, research on the connections between societal uncertainty and fertility in western settings typically focuses on general labor market and other economic aspects of such uncertainty, without fully addressing possible ethnoracial variations in its nature and levels e.g., . In thisSubstantial research in western settings has focused on fertility of minority groups, especially those of non-native origin. Much of the evidence generated by this research points to higher fertility among minorities, compared to majorities, and the literature generally entertains two competing explanations—the cultural explanation, which typically emphasizes greater traditional pronatalism of minority groups, and the minority status explanation, which stresses various aspects of systemic societal disadvantages of minority group members.A large share of the research on ethnic and racial variations in fertility comes from the United States. Although the majority-minority differentials in completed fertility there have existed continuously, they have been declining recently, and most of the remaining variation is concentrated in the timing of childbearing, the prevalence of non-marital childbearing, contraceptive and abortion use, and the intendedness of births . Yet, thBeyond the U.S.A., several studies have looked at fertility of immigrants and their descendants in Western Europe. In the United Kingdom, Beyond individual-level ethnicity, studies in western settings have also looked at the ethnic community context. Thus, While most studies typically document higher fertility of immigrant minority groups, at least in the earlier stages of their incorporation into the host society, there is also evidence of the opposite direction of the immigrant vs. native fertility divide. Thus, The scholarship on ethnicity and fertility outside of the western world is much more limited but nonetheless informative. Thus, While focusing on a somewhat different dimension of societal complexity, studies linking fertility to religion also inform our understanding of group-level fertility differentials in non-western contexts. Thus Research has also looked into ethnic heterogeneity in fertility levels and patterns in transitional post-Communist multi-ethnic contexts, including Central Asia. While several studies documented fertility differences across ethnic groups, typically explaining them in terms of cultural factors and corresponding stages in the process of the demographic transition e.g., , some ofThe Kyrgyz Republic (Kyrgyzstan) is a country of over 6.5 million people in Central Asia that, became an independent nation after the dissolution of the Soviet Union in 1991. After a sharp decline in the early years following post-Soviet collapse, the country’s economy grew throughout much of the first two decades of this century; yet, with a GNI per capita equivalent to c. 1200 USD, Kyrgyzstan remains one of the poorest Soviet successor states. Like many post-Soviet nations, Kyrgyzstan is a multiethnic country. Ethnic Kyrgyz, the country’s titular group of Turkic origin, form the majority of its population (c. 74%). Kyrgyzstan’s largest ethnic minority, Uzbeks (15%), are also of Turkic linguistic background and are indigenous to the region; Uzbeks reside primarily in the country’s south. Both groups have nomadic roots; however, Uzbeks, unlike Kyrgyz, have long been sedentary. While both groups are Sunni Muslim, Uzbeks are characterized by a generally stronger Muslim identity. Although the two largest groups share Turkic linguistic roots and the Islamic religion, the relations between them have been marked by tensions that occasionally erupted into open confrontations, most recently culminating in massive anti-Uzbek violence in 2010 . NotablyWhile the ethnic labeling, systematically introduced and enforced in Central Asia in the Soviet era, has remained remarkably rigid and stable after its end cf. , the shiThe total fertility rate (TFR) in Kyrgyzstan, which hovered close to four children per women in the late Soviet era, dropped dramatically following the disintegration of the U.S.S.R. and the generalized crisis that it triggered, paralleling trends in other parts of the former Soviet empire e.g., . After tOur conceptualization engages, juxtaposes, and contrasts the cultural and minority status perspectives. For the cultural perspective, we suggest a holistic approach to the cultural realm and its connection to fertility, which we define in terms of group-level normative propensities. While group normative propensities are multidimensional and situational, the norms and corresponding behaviors that are most consequential for fertility evolve through the process of the demographic transition, of which fertility regulation and limitation are a central part. Collectively shared family and gender norms, marital practices, and related aspirations and expectations are core mechanisms of group cultural reproduction. Importantly, these norms, practices, aspirations, and expectations are produced and reproduced irrespective of individual variations among group members along such axes as urbanity, education, or religiousness.We adapt the scholarship on minority group status, and especially its ethnoracial stratification lens, to what we define as group societal positioning. This definition is rooted in, but also expands upon, the minority group status concept as it encompasses all ethnic groups and their sub-groups, regardless of the exact nature of their collective identity and of their relative size. Ethnic groups, and their segments, are stratified and hierarchized in any contemporary multi-ethnic society, and group societal positioning is established, reaffirmed, and reassessed in a constantly evolving ethno-economic and ethno-political context reflecting the dynamic allocations of collective economic and political powers. These variations in group positioning on different stratification axes are specific to country’s macro socio-political environment cf., . ImportaWhile normative propensities and societal positioning are understandably interconnected, they nonetheless capture different aspects of group identity and experiences. We therefore treat them as two competing group-level perspectives in theorizing ethnic variations in reproductive dynamics in a context of above-replacement, and even slightly rising, fertility, where the ethnic makeup is reflective both of cultural diversity and of status inequalities. Unlike most western countries, where comparisons are almost exclusively drawn between the majority and minorities, the ethnoracial hierarchies in Kyrgyzstan, as in many other non-western settings, do not fit neatly into this dichotomy. Specifically, the heterogeneity of the ethnic majority, both in terms of ethnocultural characteristics and social positioning of its subgroups, may be consequential for demographic behavior and outcomes, regardless of other individual-level variations among them. At the same time, in such settings ethnic minorities are often indigenous, rather than immigrant-origin, groups, and share considerable ethnocultural and religious affinity with majorities; in fact, they became ‘minorities’ historically recently, e.g., during the ethno-administrative partition of the former Russian empire following the Soviet takeover. As for outside-origin groups, such as Europeans in Kyrgyzstan, their initial arrival was part of a state-organized or sanctioned population transfer that typically resulted in their superior economic and even ethno-social placement, again, differing greatly from the entry and incorporation trajectories of immigrant-origin groups in the West, where such groups are typically both economically disadvantaged and less advanced in the process of the demographic transition, compared to the host country’s majority.Our study measures and models the outcomes that are commonly examined in fertility research—completed fertility and desired fertility—and uses them for a better understanding of the ethnic medley and corresponding societal hierarchies in this transitional post-Communist context and of their demographic implications and manifestations. While the two outcomes are closely and causally interconnected, i.e., fertility desires both predict and are predicted by actual fertility (even though the strength of this interconnection is still debated in the demographic scholarship), in this analysis we see them as complementary reflections of group normative propensities and societal positioning. And again, our conceptualization assumes the effects of group normative propensities and societal positioning above and beyond the effects of such universal individual determinants of actual and desired fertility as marital status, education, urbanity, employment, wealth, and religiosity—i.e., the factors that are typically entertained under the ‘characteristics’ perspective.Our hypotheses for both outcomes of interest focus on ethnic Kyrgyz, and their cultural and status heterogeneity based on the degree of Russification (as proxied by Russian language use), Uzbeks, the largest and relatively economically advantaged yet also most conspicuously vulnerable autochthonous minority, and Europeans, a culturally distinct group of outside origin that has retained some of its Soviet-era economic advantages but, at the same time, has been increasingly, even if indirectly, marginalized in the post-independence political environment. Although we include other ethnic minorities of both indigenous and non-indigenous origin (summarily labeled as Others hereafter) in the analyses, we do not explicitly situate this residual category in our conceptual model because of its small size and ethnic heterogeneity.completed fertility, we look at women’s number of children ever born. From the normative propensity perspective, fertility levels are expected to reflect ethnic cultural norms that impact childbearing: members of culturally more traditional groups would have higher lifetime fertility than members of culturally and demographically less traditional groups. Given the historical association of demographic modernity with the European-origin population and its cultural influence in the region, we hypothesize that ethnic Russians and other Europeans will display lower completed fertility than members of native groups, net of other factors. In the same vein, considering the persistent legacy of the Euro-Russian cultural influence, we also expect that more Russified Kyrgyz will have lower completed fertility than their less Russified co-ethnics. Finally, the normative propensity perspective suggests that fertility levels of less Russified Kyrgyz and Uzbeks, the two most traditional segments of society in the conventional demographic-cultural sense, will be similar.In the analysis of The societal positioning perspective, as formulated above, suggests alternative hypotheses. Thus, more Russified Kyrgyz, who combine the advantage of titular ethnicity with practical benefits of Russian language use, will have higher completed fertility than their less Russified co-ethnics. At the same time, members of the directly threatened, even if economically advantaged group, i.e., Uzbeks, will have lower completed fertility than members of the other groups. Finally, the societal position of Europeans, which blends the residual economic advantages with gradual, even if subtle, societal marginalization, suggests that their fertility will be closer to that of less Russified Kyrgyz.fertility desires, we consider both the overall desire to have a(nother) child and the desired timing—within two years or later—of the next birth. At the individual level, our hypotheses echo those proposed for completed fertility. Thus, the normative propensity perspective suggests ethno-linguistic distinctions in fertility desires that would be comparable to those in the number of children ever born: net of other factors (including the number of living children), European ethnicity will be associated with lower desire to have a child, regardless of the time horizon. The cultural Russification premise is also extended to hypothesize the variation in fertility desires among the titular group: more Russified Kyrgyz are expected to be less likely to want a child than their less Russified counterparts, and this difference should not be affected by preferred timing of future fertility. At the same time, the normative propensity perspective, as in the cases of completed fertility, suggests no difference between less Russified Kyrgyz and Uzbeks in desired fertility.For the analysis of Again, as in the case of completed fertility, the group societal positioning lens entails an alternative set of hypotheses. Specifically, if a group’s position in the societal hierarchy is positively associated with pronatalism, then more Russified Kyrgyz, the de facto privileged segment of society, should be more likely to desire another child than the other segments, including their less Russified co-ethnics, and this difference should be particularly pronounced in longer-term fertility desires. In comparison, ethnic Uzbeks, the minority group of residually high socioeconomic status that has been a target of systematic marginalization and direct violence, are hypothesized to be least likely to want a child. Moreover, given the relatively recent shock of ethnic violence that this group experienced, this tendency will be especially noticeable in shorter-term fertility desires. And finally, Europeans’ continuing economic advantages yet also gradual status decline should situate their fertility desires between those of Uzbeks and less-Russified Kyrgyz, net of other individual characteristics.In the analyses of desired fertility, we also look beyond the individual-level markers of ethnic identity. At what we consider the meso-level of ethnic belonging, here represented by the ethnic makeup of individuals’ non-family social milieu, we hypothesize that having friends/close acquaintances of same ethnicity will be associated with a higher likelihood of wanting a(nother) child, either sooner or later, compared to having an ethnically diverse close social circle, after individual ethnic self-identification as well as other relevant characteristics are controlled. Finally, at what we define as the macro-level ethnic comfort, we look at individuals’ expectations for the general situation of their ethnic group in the near future. We hypothesize that regardless of ethnic self-identification and other factors, greater optimism about the near-future prospects of own ethnic group will be associated with increased fertility desires, net of other factors. We also expect that the positive effect of such optimism will manifest itself most strongly with respect to shorter-term fertility desires.Following the dominant tradition of fertility research, the analyses of desired fertility focus primarily on women. However, we also conduct complementary analyses for men. Men’s fertility attitudes and desires may differ from women’s, reflecting different levels of gendered power, nature and scale of labor force participation, as well as established cultural norms and expectations e.g., . We alsoWe use combined data from two rounds of the Social, Economic, and Migration Processes in Kyrgyzstan (SEMPK) survey, a nationally representative household survey conducted in 2011 (round I) and 2017 (round II). Both rounds used a multistage cluster design and took place in the same communities (villages or city boroughs), selected at the time of the first round with a probability proportional to size. In those communities, households were randomly selected in each round, and in each selected household one randomly chosen resident, woman or man, aged 18–49 was interviewed. In each round, the sample size was 2030, and no individuals were interviewed in both rounds. The survey questionnaires, which were nearly identical in the two rounds, were administered face-to-face in the language of respondent’s choice and contained a variety of questions, including those on ethnicity, language proficiency and use, number of everborn and surviving children, and long- and short-term fertility desires. The survey design and the questionnaire content were approved by the Institutional Review Boards of Arizona State University and the University of Kansas (USA).Our first outcome, women’s completed fertility, is the total number of children a woman aged between 18 and 49 has had by the time of the survey, regardless of the survival status of her children. The second outcome is whether a respondent, woman or man, wants to have more children anytime in the future. It is operationalized as a dichotomy—wants to have a(nother) child vs. does not want to have a child. The third, and last, outcome of interest is a refinement of the second one: it breaks down individuals who want to have a child in the future into those who would like to have a child within the next two years and those who would like to have a child later. The fertility desires analyses exclude female respondents who considered themselves infecund. They also exclude female and male respondents who did not know or were unsure whether they wanted to have another child. We acknowledge potential heterogeneity of the subsample that provided “don’t know/unsure” responses as such responses may reflect a fairly wide spectrum of fertility preferences cf., . HoweverBased on the survey responses, we classify ethnic self-identification into four categories—Kyrgyz, Uzbek, European , and Other. Notably, although respondents could choose multiple ethnicities, almost all of them picked only one. Next, following our conceptual interest and guided by prior research e.g., , we subdWe first fit a set of two Poisson regression models predicting the number of children ever born. These analyses include all female respondents aged 18–49 . In the first model, ethnic groups are compared. The second model accounts for the degree of linguistic Russification among Kyrgyz. For the analysis of desired fertility, we fit separate models for women  and men . For each gender, we start with a pair of binomial logistic regression models predicting the desire to have a(nother) child any time in the future. Then, we fit a pair of multinomial logistic regression models with the following outcome: wants a child within the next two years, wants a child later, and does not want a child (reference). As we do for the two previous outcomes, we fit both the ethnicity-only and ethno-linguistic  models. All the desired fertility models include ethnic homophily of personal network and ethnic macro-optimism as predictors.All models control for respondent’s age; the fertility desire model also includes age squared to account for possible non-linearity in the association of age with desired fertility. All models control for educational level , current marital status (married vs. not), and marital history (married before vs. not). We do not control for marital partner’s ethnicity  because the overwhelming share of marital partnerships were of same ethnicity. The models also control for respondent’s current employment status operationalized into four categories: not employed; self-employed; employed in the state sector; employed in a private company/organization. Household’s socioeconomic standing is defined based on the household monthly income and is operationalized into four categories: low tier; medium tier; high tier; and unknown/unreported. All models also control for respondent’s self-assessed religiosity (very religious vs. not) and area of residence . Survey round (I or II) is also included as a control in all models: although the two rounds had the same sampling design, used nearly identical instruments, and were separated by only six years, the timing of each round and the related socio-economic, political, and ethno-cultural context could have potential implications for the outcomes of interest. The fertility desire models also include a control for the number of living children.We acknowledge that some of the covariates in the completed fertility model may not be fully exogenous to the outcome. We also acknowledge that men may under-report the number of their children, but we have no reason to expect any systematic variation across ethno-linguistic boundaries in such underreporting. We do not match women’s desires with those of their partners, and vice versa, because partner’s fertility desires are only available from the respondents’ reports and because considering partner’s desires would exclude currently unpartnered individuals from the analysis. All multivariable models account for the survey cluster design and are fitted in Stata.Next, we fit the models that predict fertility desires among women and men. These models, in addition to testing for variations across ethnic self-identification, also address the role of what we defined as meso- and macro-level ethnic belonging dimensions. The results of the gendered models predicting the desire to have a(nother) child at any time in the future are shown in p = 0.07) for the assessment of own group’s prospects.When we look at the effects of meso- and macro-level ethnic belonging markers, we find that women who have friends/close acquaintances of only their own ethnicity are more likely to want a child than women with ethnically mixed friendships, regardless of individual ethnic self-identification and other factors. Similarly, with respect to group macro-level prospects, women who expect the situation of their ethnic group to improve in the next few years are more likely to want a child. These results support our hypotheses. In comparison, among men, the effects of the two predictors point in the same direction, yet they are not significant for the ethnic composition of friends/acquaintances and marginally significant (p = 0.07).p = 0.06).At the meso-level of ethnic belonging, the results show that having friends/acquaintances of same ethnicity positively affects longer-term desire to have a child, relative to wanting no more children, and this effect is, again, statistically significant among women only . Finally, as we expected, the anticipation of the improvement of own ethnic group’s situation in the next few years is positively associated with shorter-term fertility desires. This association is fully statistically significant for women, but it also approaches the conventional threshold of significance in the men’s model (The effects of some other covariates are also noteworthy. Not surprisingly, being currently married and having been in marriage before both have a positive association with completed fertility. In comparison, for fertility desires, being currently married has a positive effect while previous marital experience has a negative effect; however, both effects are statistically significant only among women. Education only marginally affects women’s completed fertility and has no association with fertility desires. Employment status generally is not related to either outcome among women but shows some statistically significant relevance to men’s fertility desires. In comparison, household income is negatively associated with completed fertility but displays no association with fertility desires for either gender. Rural residents have higher completed fertility than urban dwellers, although this difference is statistically significant only in the model where Kyrgyz are treated as a single group. Interestingly, rural residence is negatively associated with fertility desires, but it is so for men only. High religiosity is a strong predictor of women’s completed fertility; however, surprisingly, it shows no net association with fertility desires. Finally, the models display an increase in both completed fertility and fertility desires in the second round of the survey, compared to the first round conducted six years earlier. While these patterns generally fit with the longer-term trends in fertility in Kyrgyzstan available from other sources  and may In this study, we projected the extant scholarship on ethnicity and fertility, which typically contrasts the demographically more advanced and economically privileged majorities with demographically more traditional and economically disadvantaged minorities in western post-transitional contexts e.g., , onto a The detected variations in completed fertility corresponded clearly to the normative propensity perspective, as Europeans, the group historically most advanced in the process of fertility transition, had by far the smallest number of children ever born, followed, at a statistically significant distance, by more-Russified Kyrgyz, the segment of the titular majority that has been greatly impacted by the Euro-Russian cultural influence. The less-Russified Kyrgyz and members of the Uzbek minority had the highest numbers of children among the examined groups, regardless of other factors.The desired fertility analysis produced a rather different ethno-linguistic lineup. Most conspicuously, Uzbeks, the ethnic minority that could be expected to be highly ‘pronatalist’ if judged through the prism of conventional demographic transition reasoning rooted in the degree of cultural traditionalism, displayed remarkably weak desires for additional children, even after controlling for the number of living children and other potential individual-level determinants of desired fertility. Notably, this pattern, at least with respect to the contrast between Uzbeks and Kyrgyz, echoes that reported by Although our data do not allow us to further investigate the divergence in ethnic-specific patterns of completed vs. desired fertility, some tentative explanations can be proposed. Thus, completed fertility reflects cumulative experiences, including those of a relatively distant past, whereas fertility desires and corresponding future expectations and preferences, while impacted by those experiences, are more influenced by current circumstances. In addition, fertility desires may reflect ethnic group members’ more general confidence and optimism, which, in turn, are likely to be influenced by both perceived and experienced vulnerabilities associated with ethnic group membership cf. . AlthougOur findings also help to expand the research on ethnicity and fertility beyond individual markers of ethnic identity. Thus, regardless of specific group membership and other individual-level factors, the ethnic homophily of women’s personal networks is associated with greater pronatalism, and this pattern is particularly strong with respect to longer-term fertility desires. With appropriate caveats, this finding resonates with those of studies that examined the effects of community ethnic or religious homogeneity on individual fertility behaviors and outcomes e.g., . Also amWe acknowledge several limitations of our study. Thus, the cross-sectional nature of the data constrains causal inferences, especially with respect to earlier fertility behavior and its proximate determinants, such as premarital sexual partnership, frequency of sexual intercourse, or use of contraceptive and abortion services, which may account for some of the observed patterns cf., . ImportaThese limitations notwithstanding, our findings usefully contribute to a better understanding of the association between ethnicity and fertility in transitional multi-ethnic settings. Our findings can also inform policies and actions in the areas of family and fertility: while such policies and actions are unlikely to focus explicitly on specific ethnic or ethno-linguistic segments of society, they should heed and reflect group-level cultural propensities and structural vulnerabilities, in addition to, and in conjunction with, other factors shaping reproductive and family outcomes.


Despite the fact that roughly 40% of all US Food and Drug Administration (FDA)-approved pharmacological therapeutics target G protein–coupled receptors (GPCRs), there remains a gap in our understanding of the physiologic and functional role of these receptors at the systems level. Although heterologous expression systems and in vitro assays have revealed a tremendous amount about GPCR signaling cascades, how these cascades interact across cell types, tissues, and organ systems remains obscure. Classic behavioral pharmacology experiments lack both the temporal and spatial resolution to resolve these long-standing issues. Over the past half century, there has been a concerted effort toward the development of optical tools for understanding GPCR signaling. From initial ligand uncaging approaches to more recent development of optogenetic techniques, these strategies have allowed researchers to probe longstanding questions in GPCR pharmacology both in vivo and in vitro. These tools have been employed across biologic systems and have allowed for interrogation of everything from specific intramolecular events to pharmacology at the systems level in a spatiotemporally specific manner. In this review, we present a historical perspective on the motivation behind and development of a variety of optical toolkits that have been generated to probe GPCR signaling. Here we highlight how these tools have been used in vivo to uncover the functional role of distinct populations of GPCRs and their signaling cascades at a systems level.G protein–coupled receptors (GPCRs) remain one of the most targeted classes of proteins for pharmaceutical intervention, yet we still have a limited understanding of how their unique signaling cascades effect physiology and behavior at the systems level. In this review, we discuss a vast array of optical techniques that have been devised to probe GPCR signaling both in vitro and in vivo. I.Throughout the history of research in neuroscience and psychology, the mechanisms underlying brain function have primarily been treated as a “black box.” Although inputs and outputs to and from the brain can be reliably tracked, how these networks ultimately map onto the function of the brain in behavior is often inferred through correlative studies. As in other domains of science, both theoretical and technological advancements have been necessary to expand and gain a deeper understanding of the brain within this limited conceptual framework.A pivotal breakthrough in our understanding of the principals of neural signaling was the precise description of the electrically excitable nature of neurons, exquisitely detailed in a series of seminal manuscripts by Alan Hodgkin and Andrew Huxley ,b. TheseHowever, a critical component from these experiments that limited our conceptual understanding was the establishment of causal links between neural activity and perception. For example, could one manipulate activity of an orientation-selective neuron to interfere with that specific aspect of visual perception while sparing the processing of color or contrast? The inability to manipulate the activity of functionally distinct neural populations was not unique to these studies. Indeed, this represented a significant methodological barrier that hindered neuroscience for several decades. Although lesions or intracranial electrical stimulation studies provided a gross understanding of the function of general brain regions, they offered no insight into the unique function of intermingled neural populations within these brain regions. This caveat was understood early on and explicitly stated in the 1979 article by Francis Crick entitled “Thinking about the Brain” . In thisDrosophila photoreceptor system into cultured rat hippocampal neurons, visible light irradiation led to increased excitability and action potential firing.Although this speculation was not lost the neuroscientific community, the first demonstration of using light to control the activity of specific neural populations did not arrive to the field for more than two decades. A study by This study represented a crucial step forward, but the technique was not widely adopted due to both the multicomponent transfection and the slower kinetics of this neuromodulatory approach via G protein–coupled receptor (GPCR)-based opsin. This precluded easy genetic targetability as well as the millisecond-scale temporal precision necessary to faithfully manipulate neural activity. In 2005, a study led by Ed Boyden and Karl Deisseroth described a novel optical toolkit that seemingly met both of these requirements. This system made use of a single component blue light–gated cation channel called channelrhodopsin 2 (ChR2), which is natively expressed in certain species of algae to drive phototaxis . This maAlthough the ChR2-based optogenetic toolkit has been revolutionary for researchers to gain direct access to control the excitability of neurons, the brain does not function solely as a binary digital processor (on/off) of information flow. Indeed, the brain produces dozens of neuromodulatory gain signals which fine-tune, stabilize, and alter distinct neural circuits through G protein–coupled receptors (GPCRs). Direct actuation of ion channels would be insufficient to recapitulate the functional properties many of these neuromodulatory systems. GPCRs represent one of the most targeted protein families for pharmaceutical intervention, with roughly 40% of all US Food and Drug Administration (FDA)-approved drugs interacting with these receptors . AlthougII.The photoreceptive protein rhodopsin was discovered almost 150 years ago by the German scientist Franz Christian Boll . Given tA.t protein signaling pathway. The now liberated Gt subunit subsequently activates cGMP phosphodiesterase, leading to decreased cGMP levels  generation and subsequent calcium release and neuronal depolarization  for efficient optogenetic silencing . Althoug release . Similar release . Notablyartments . This startments .C.β2AR)  . Specifiα1AR) and rhodopsin-β2AR receptor chimeras, demonstrating comparable levels of G protein recruitment to native receptors after light irradiation. Uniquely, this study illustrated for the first time the use of these chimeras in vivo. Not only did optical activation of the opto-β2AR alter innate neural excitability and firing properties, but it also drove robust conditioned place preference when it was expressed and activated on neurons in the shell of the nucleus accumbens (NAc). Other groups have recapitulated the in vivo viability of opto-β2AR, displaying its ability to drive anxiety-like behaviors and alter contextual encoding of fear memories  family of proteins  . Notably, the study that generated the opto-D1R showed that activation of this receptor on natively D1R neurons on the NAc could recapitulate the behavioral effects of dopamine terminal stimulation in this region  . These ereceptor  and dopas region . This exs region , metabots region , opioid s region , adenosis region , 5HT2A   . This drfficacy) .i/o recruitment rather than a receptor-specific effect. To address this concern, chimeras can be specifically expressed under   , adreneror (NAM) , and dopor (NAM) . Additioor (NAM) . A studyoeruleus .5 NAM raseglurant. This compound, JF-NP-26, exhibited similar pharmacological properties to the native compound after exposure to UV light. In preclinical models, raseglurant was capable of inducing analgesia in a model of neuropathic pain, but the locus of action in the brain for this effect remained unresolved. The authors first demonstrated that systemic injection of the caged compound JF-NP-26 had no effect in any pain assay. Then, after UV uncaging of this compound in the ventrobasal thalamus, a critical somatosensory relay center, they elicited robust antinociception comparable to that of systemically administered raseglurant. This study illustrates the power of photopharmacological techniques for determining the precise locus of action of pharmacological modulators of GPCR signaling   , chemoki2A) , 2019, a2A) (2A .C.α-(trimethylammonium)methyl] azobenzene (Bis-Q). Work by Erlanger and colleagues demonstrated that the native trans configuration of this compound functioned as a competitive antagonist at the muscarinic acetylcholine receptor (mAChR)  converts the compound from an inactive to active form. These modified compounds are then either perfused over the sample in in vitro experiments or directly injected into the animal for in vivo experiments . The dev (mAChR) . However (mAChR) . AlthougMuch subsequent development of photoswitchable ligands has been focused on selective modulators of GPCR action. Recent advances in chemical synthesis have allowed for the creation of novel photoswitchable compounds based on highly lipophilic molecules such as the cannabinoid receptor 1 (CB1) agonist Δ-9 tetrahydrocannabinol (THC), the principal psychoactive constituent of marijuana. Frank and colleagues synthesized two unique azobenzene-conjugated THC derivatives that displayed high efficacy at the CB1 receptor in either the cis (Azo-THC-3) or trans (Azo-THC-4) configuration, respectively . In this4 NAM  function by creating a compound that could be photoconverted from an antagonist to an agonist. In the dark, the trans configuration of the compound they synthesized, VF16216, was capable of antagonizing CXCR3 in vitro , 2019. A4 NAM , mGluR5 4 NAM -3′- [α-(trimethylammonium) methyl]azobenzene (trans-QBr) to the extracellular moieties of receptors. QBr had previously been demonstrated to be a potent photoswitchable cholinergic agonist. Consistent with previous reports, the group demonstrated that light-induced photoisomerization was capable of increasing the potency of QBr several fold  in larval zebrafish and assayed for the acoustic startle response (ASR). First, they demonstrated that pharmacological activation of native mGluR2s was sufficient to potentiate the ASR. Then, using LimGluR2, they showed bidirectional control over the ASR by activation or inhibition of the receptor using their photoswitchable tethered ligands. These pioneering studies have spurred the development of various photoswitchable tethered ligands for a number of GPCRs alongside nAChRs  is conjugated to the receptor far from the orthosteric site  Fig. 2DFig. 2D. cificity . By dire agonism . Thus, tism  domain . FlavoprF domain   domains represent another subclass of flavoproteins widely used for optogenetic control of second messenger cascades , 1999. Lractions . A recenractions , p. 1. Btivation . This pltivation      . This feor C1BN) .The success of this family of new optically sensitive protein-domain techniques has led to the development of a fairly robust phytochrome toolkit. These light-sensing domains found in plants, fungi, and bacteria are sensitive to longer wavelengths of light (650–760 nm), which improve compatibility with in vivo experiments due to increased tissue penetrance and decreased phototoxicity. Similar to the cryptochromes, the phytochrome system consists of a pair of heterologous subunits, phytochrome B (PhyB) and PIF6, that display light-dependent dimerization , p. 3. AB.α and βγ subunits, which each couple to unique effectors. Termination of G protein signaling occurs when the GTP in the alpha subunit is hydrolyzed to GDP, an event catalyzed by regulators of G protein signaling (RGS) proteins, leading to reconstitution of the inactive αβγ heterotrimer , which drive release of bound GDP from the G proteins, which is then replaced with GTP. This nucleotide swap induces the dissociation of the rotrimer . Multiplα subunits. These molecules have the ability to quickly curtail canonical G protein signaling. Early studies demonstrating the feasibility of optical approaches for manipulating RGS signaling used the CRY2-C1BN system to generate a CRY2-RGS4 fusion protein , which catalyze the hydrolysis of GTP in G protein . The RGSi/o and Gq proteins. A subsequent study circumvented this caveat by engineering a CRY2 dimer with RGS2, which shows selectivity for the Gq subunit . Using a similar technique to prevent plasma membrane localization of RGS2 in the dark, they demonstrated that light-catalyzed translocation of the CRY2-RGS2 fusion to membrane-bound C1B1 acceptor resulted in reduced intracellular calcium through inhibition of the Gq signal transduction pathway.One limitation to the CRY2-RGS4 technique is that RGS4 catalyzes nucleotide exchange from both G subunit . A commoα-binding and activating (GBA) motif to the C terminus of LOV2 . In addition to phosphorylating active GPCRs, GRK2 contains a pleckstrin homology domain in its C terminus, enabling it to directly interact with the Gβγ subunit. This domain has been shown to be sufficient for sequestration of free Gβγ subunits in vitro and was thus selected for generation of a photoswitchable tool for blockade of Gβγ signaling. Importantly, because Gβγ has a higher affinity for GDP-bound Gα than GRK, the activation cycle of the G protein remains intact, as does Gα signaling. A study by Gautam and colleagues demonstrated the feasibility of this approach using a chemoattractant to promote cell migration in a microphage-like cell line (3) gradient and cell migration toward the unstimulated side of the cell, demonstrating localized Gβγ sequestration. Although these elegant studies demonstrate the ability to manipulate Gβγ signaling using light, this system will uniformly inhibit all Gβγ subtypes, limiting the selectivity of the technique. Mammals express 5 Gβ and 12 Gγ subtypes, resulting in a myriad of possible combinations with different physiologic functions. In the future, it will be crucial to design tools to specifically target unique β-γ combinations to gain a more granular understanding of the varying signal transduction cascades that can be mediated by these proteins in a spatiotemporally precise manner.The feasibility of this approach for controlling individual Gell line . Here, aC.Arrestins were initially characterized as intracellular molecules that drove cessation of GPCR signaling, either through steric hinderance of heterotrimeric G protein recruitment or by driving receptor internalization via clathrin-coated pits. However, decades of subsequent research demonstrate that arrestins represent a unique effector arm of GPCR signaling, driving a distinct cascade of intracellular signaling events . Arrestiβ2-AR that displayed biased arrestin signaling through mutation of key residues involved in G protein recruitment . Therefore, to gain a more precise understanding of the consequences of GPCR activation, it would be quite powerful to be able to individually manipulate these propagated second messenger signals with independent spatiotemporal specificity. Multiple optical tools have since been developed to accomplish this goal.Second messengers are ubiquitous signaling molecules that propagate or amplify an incoming “first message”  to a host of intracellular effectors. Second messengers of GPCR signaling include but are not limited to PIPs effector arm, and its production is regulated through GPCR-dependent adenylyl cyclase (AC) activity (Beggiatoa (Drosophila (Catenaria anguillulae (cAMP is a predominant second messenger of the Gactivity . Intereseggiatoa . This paosophila . A subseuillulae . By seleuillulae . Howeveruillulae . Furtheruillulae . Overexpuillulae .q proteins exert their primary physiologic effects via activation of phospholipase C (PLC), which cleaves membrane-bound phosphatidylinositol biphosphate (PIP2) to generate soluble IP3 and membrane-bound DAG  channels through the use of a photoswitchable DAG. Despite the limited use of light-sensing protein domains for directly manipulating these small second messengers, optical control of heterotrimeric G proteins or kinases can offer indirect control over second messenger signaling cascades. For example, a study by 3K) demonstrated optical control over PIP3 levels. Several subsequent studies have built upon this approach by expanding the suite of optical tools to modulate the activity of kinases and other intracellular signaling proteins.Gound DAG . This acound DAG . This ofE.α subunits of heterotrimeric G proteins in that they act as molecular switches and have intrinsic GTPase activity , utilizing the dimeric protein pdDronpa . Further refinement and tailoring of the specificity and sensitivity of these tools, as well as careful assessment of their expression in neural systems, will be necessary for them to be widely adopted for systems neuroscience applications.A principal caveat that has slowed the implementation of many of GPCR-targeting tools is the fact that we still lack the ability to optically control native subtype-selective GPCR signaling in a genetic or cell type–specific manner. Conventional photopharmacology techniques such as ligand uncaging or photoswitchable ligands provide temporally precise localization to manipulate ligand-receptor interactions. However, for incorporation into modern systems neuropharmacology approaches, these benefits still remain insufficient for disentangling the functional role of GPCRs on specific neural populations.An example would be an experiment in which one might attempt to investigate the role of endocannabinoid signaling in the medial prefrontal cortex (mPFC), making use of the previously developed photouncageable 2-arachidonylglycerol (2-AG) . After cA work-around would be to devise a tethered photoswitchable ligand strategy in which the CB1 receptors in the region of interest  express the necessary mutations on the N terminus to allow for cysteine-maleimide conjugation so that the ligand can bind to the receptor. However, this would either necessitate the generation of a knockin mouse or heterologous expression of the mutant receptor using a viral method. In the latter case, heterologous expression can yield several unintended effects and will not necessarily recapitulate native receptor expression or function. This same pitfall applies to remotely tethered photoswitchable ligands and even an engineered Opto-CB1 based on the Opto-XR platform.B.For in vivo systems, we ultimately need to gain optogenetic control over native receptors in a cell-type specific manner. Over the last five years, significant progress has been made toward this goal. A seminal 2017 paper by Tadross and colleagues described the generation of a novel pharmacological tool called drugs acutely restricted by tethering (DART) . In this2 signaling, specifically in neurons that expressed the SNAP-tag. Although not characterized in vivo, this approach provides an important proof of concept for genetically targetable photoswitchable control over native GPCR populations. Given the comparative ease of synthesis of the tethered photoswitchable ligands compared with generation of knockin mice or chimeric GPCRs, this technique represents a crucial technical advancement for the investigation of functionally specific populations of natively expressed GPCRs in vivo. Other groups have implemented this orthogonal tethering approach to probe dopamine receptor , which served as the binding site for its cognate ligand benzylguanine (BG) . BG was receptor  and GPR5receptor .2 activity. This approach benefits from the fact that nanobodies can be easily tailored to bind motifs of interest  and they can either be directly administered or be genetically encoded. In the future, one can imagine an alternative system in which newly developed CRISPR/Cas9 mutagenesis techniques could be harnessed to induce specific point mutations on a receptor of interest, allowing for direct conjugation of a tethered photoswitchable ligand (Complementary approaches using nanobody-conjugated photoswitchable compounds are being developed to improve the efficiency and applicability of these techniques. A study by e ligand . This coOne possible reason for a lack of widespread adoption of these photopharmacology techniques in vivo is that many of these tethered photoswitchable ligands are too large or are otherwise unable to cross the blood-brain barrier (BBB). Therefore, in vivo experiments would require the coimplantation of both a fiber optic and a drug cannula, which must be connected to a laser power source and an infusion pump, respectively. Although certainly within the realm of feasibility, these experiments are complex and often prove to have detrimental effects on mouse behavior due to the number of distinct components that must be fastened to the animal’s head. Beginning in the early 2010s, work by the Rogers and Bruchas laboratories began developing wireless single component integrated devices for simultaneous drug delivery and optical stimulation . These “Another method of delivering these compounds into the brain would be via Trojan horse or nanoparticle-mediated delivery systems. As mentioned above, the majority of photopharmacology compounds are either too large or do not contain the right chemical properties to allow for transport across the BBB. Trojan horse methodologies rely on the conjugation of the drug of interest to a molecule or peptide (such as transferrin) that binds to specific receptors on the endothelial lining of capillaries that make up the BBB, allowing for transport of the drug-peptide conjugation into the brain . One draVI.Despite the fact that roughly 40% of FDA-approved pharmacological compounds target GPCRs, there remains a noted lack of understanding as to the physiologic and functional role of these receptors at the systems level across many biologic fields. The brain in particular represents a massively complex puzzle in which understanding GPCR signaling with cellular and even subcellular resolution will require the continued advancement of our techniques and methods for probing these unique biochemical signaling cascades. In this review, we attempted to outline several of the principal technological advancements that have occurred over the last half century that have significantly aided in our quest to better understand everything from molecular interactions between ligand and GPCR orthosteric binding domain all the way to transcriptional regulation in the nucleus. We hope that this summary of key optical methods will serve as a resource for future researchers interested in investigating the intricacies of GPCR signaling in vitro and in vivo across biologic systems.


Neuronal plasticity has for a long time been considered important for the recovery from depression and for the antidepressant drug action, but how the drug action is translated to plasticity has remained unclear. Brain-derived neurotrophic factor (BDNF) and its receptor TRKB are critical regulators of neuronal plasticity and have been implicated in the antidepressant action. We have recently found that many, if not all, different antidepressants, including serotonin selective SSRIs, tricyclic as well as fast-acting ketamine, directly bind to TRKB, thereby promoting TRKB translocation to synaptic membranes, which increases BDNF signaling. We have previously shown that antidepressant treatment induces a juvenile-like state of activity in the cortex that facilitates beneficial rewiring of abnormal networks. It is important to note that enhanced plasticity does not necessarily promote recovery, but may also be maladaptive if the environment is adverse. Our findings open a new framework for the antidepressant action and for treatment of depression: antidepressants directly bind to TRKB and allosterically promote BDNF signaling, thereby inducing a state of plasticity that allows re-wiring of abnormal networks for better functionality, when optimal supportive therapy is provided at the time of enhanced plasticity.E. Castrén Speakers bureau of: Janssen-Cilag


The name extracellular signal-regulated kinase (ERK) was first used for a cell cycle regulating Ser/Thr protein kinase cloned in mammalian cells , which wAs the critical focal point of the Raf/MEK/ERK pathway signaling, ERK1 and ERK2 (referred to as ERK1/2) have essential roles throughout development and adulthood. Capolongo et al. introduce different roles of ERK1/2 in embryonic kidney development, focusing on renal morphogenesis and differentiation, and several renal diseases, including polycystic kidney disease, glomerulonephritis, diabetic nephropathy, and unilateral ureteral obstruction . They reAlbert-Gascó et al. review the effects of ERK1/2 signaling on developing social and emotional behaviors, focusing on the molecular mechanisms that the kinases mediate in the amygdala and amygdala-related areas . The autEpithelial-mesenchymal transition (EMT) describes the trans-differentiation of epithelial cells into a mesenchymal phenotype. EMT is necessary for the normal development of higher eukaryotes and is involved in certain pathophysiological conditions. For example, different oncogenic signaling pathways and tumor microenvironment signals often trigger EMT to promote tumorigenesis. Olea-Flores et al. review three significant mechanisms of EMT, with a focus on the type 3 EMT associated with cancer metastasis and progression . In thisAlthough mainly known as cell-proliferative kinases, ERK1/2 can mediate cell cycle arrest and death. For example, ERK1/2 activation, in response to oncogenic RTK, Ras, or Raf mutants, can induce growth inhibition in normal cells and specific tumor cells. Wu et al. review recent updates on the molecular mechanisms underlying the Raf/MEK/ERK pathway-mediated growth inhibitory signaling, focusing on the mechanisms that determine the magnitude of pathway activity, spatial-temporal regulation, and non-canonical functions of the molecular switches in this pathway . The groViral pathogenesis requires the manipulation of signaling pathways in host cells. Notably, DNA viruses manipulate ERK1/2 signaling in cells to promote viral internalization, dysregulate the cell cycle, regulate viral replication through the host-cell machinery, and prevent host-cell death. Thus, elucidating how DNA viruses regulate the ERK1/2 pathway is critical for a better understanding of viral pathogenesis. DuShane and Maginnis review the dynamic nature of ERK1/2 signaling and its importance in regulating cellular fates that influence viral infection . InteresThis topic series also accommodates several research articles that suggest novel ERK pathway effects in different biological contexts. First, the hepatocyte growth factor exerts cardioprotective effects against the cardiotoxicity caused by the chemotherapeutic agent doxorubicin. Gallo et al. explore the molecular mechanism underlying this beneficial effect in the H9c2 cell culture model and demonstrate the role of ERK1/2 signaling in mediating the growth factor’s anti-genotoxic and pro-survival effects . Using t


Kt/V and URR (urea reduction ratio) measurements represent dialysis adequacy. Single-pool Kt/V is theoretically a superior method and is recommended by the Kidney Disease Outcomes Quality Initiative guidelines. However, the prognostic value of URR compared with Kt/V for all-cause mortality is unknown. The effect modifiers and cut-off values of the two parameters have not been compared. We investigated 2615 incident hemodialysis patients with URR of 72% and Kt/V (Daugirdas) of 1.6. The average patient age was 59 years, 50.7% were female, and 1113 (40.2%) died within 10 years. URR and Kt/V were both positively associated with nutrition factors and female sex and negatively associated with body weight and heart failure. In Cox regression mod-els for all-cause mortality, the hazard ratios (HRs) of high URR groups  and the URR < 65% group were 0.748 (0.623–0.898), 0.693 (0.578–0.829), and 0.640 (0.519–0.788), respectively. The HRs of high Kt/V groups  and the Kt/V < 1.2 group were 0.711 (0.580–0.873), 0.656 (0.540–0.799), and 0.623 (0.498–0.779), respec-tively. In subgroup analysis, Kt/V was not associated with all-cause mortality in women. The prognostic value of URR for all-cause mortality is as great as that of Kt/V. URR > 70% and Kt/V > 1.4 were associated with a higher survival rate. Kt/V may have weaker prognostic value for women. Dialysis inadequacy can be evaluated in multiple manners, including clearance of small and middle molecules4, acid–base and electrolyte balances, and fluid status5. The clearance of urea remains the most strongly recommended measurement of dialysis adequacy in clinical practice5, with measurements including Kt/V and URR (urea reduction ratio). Kt/V  was developed by Frank Gotch and John Sargent6 and further equilibrated by Daugirdas in the 1990s7;it remains the primary indicator of dialysis adequacy8. A landmark randomized controlled trial in 2002, the HEMO study, found that single-pool Kt/V (spKt/V) > 1.2 is associated with lower mortality in HD patients9. The KDOQI  guidelines recommend a target spKt/V of 1.4 per HD session for patients treated three times weekly, with a minimum delivered spKt/V of 1.25. URR is calculated using a relatively simple equation developed by Lowrie and Lew in 199110. The recommended dosage of URR ranges from > 65% to > 75% according to different studies12. Taiwan Society of Nephrology uses URR in clinical practice and suggests URR > 65% as a minimum requirement.Dialysis inadequacy affects the morbidity and mortality of hemodialysis (HD) patients13 and URR decreases substantially during continuous renal replacement therapy8. Kt/V has long been preferred to URR as the standard for prescribing HD dosage and is recommended by the KDOQI8. However, Kt/V also has its potential disadvantages which may overcome its benefits and has been discussed in recent decades.Due to reasons include the narrower range of doses achieved during HD for URR compare to Kt/V, the variation of curvilinear relationship between the two parameters (because Kt/V considers urea distribution volume and UF)15. For example, for patients with lower mass, high Kt/V occurs more easily due to lower V, which causes overestimation of dialysis dosage17. Although studies have attempted to establish an association between Kt/V and URR, measurements cannot be converted accurately due to ultrafiltration (UF) level and dialysis time 18. The prognostic value of Kt/V is theoretically greater than that of URR, but head-to-head comparison has not been made.Kt/V, which considers volume of urea distribution in the body, is thought to be more accurate than URR. However, V and Kt are both considered survival-associated factors, which may cause an offset effect19, but the results of some studies are inconsistent12. Some studies also showed gender, body size and physical activity can all influent the dialysis dose and mortality21. Others have found that, female sex, but not low body weight, modified this effect22. Nutritional status may also modify this association23, and cardiovascular disease may modify the association between Kt/v and cardiac outcomes24. We thus investigated whether low V modified the association between URR and all-cause mortality and compared the results of subgroup analysis between URR and Kt/v.The prognostic value of Kt/V is modified in some subgroups. Patients with low V may have high Kt/v. Studies have shown that body weight modifies the association between Kt/v and all-cause mortalityWe thus examined and compared the prognostic value of Kt/V with that of URR for all-cause mortality and compared the factors modifying this effect in incident HD patients in southern Taiwan. In subgroup analysis, we stratified the patients according to sex, age, body weight, comorbidities, and laboratory data. We used the averaged dialysis dose between months 4 and 9 in incident HD patients to prevent bias.Table After stratifying the cohort in Table Table 26, we performed Cox survival analysis using age at dialysis, sex, entry year, DM, hypertension, hepatitis, postdialysis BW, nPCR, UF/BW ratio, creatinine, hemoglobin, WBC, albumin, AC, log-transformed cholesterol, and phosphorus (Table P < 0.001) and 0.640  in the URR group and 0.656  and 0.623  in the Kt/V group, compared with the URR < 65% and Kt/V < 1.2 groups. Although no matter higher Kt/V or URR groups both associated with lower HR, the effect of reduction of HR attenuating as in higher Kt/V or URR group. Moreover, each1SD increase in URR and Kt/V was associated with HRs of 0.896  and 0.885, respectively  occurred during a median 5.02-year follow-up period. Based on the literature3.1% occuFigure We examined the prognostic value of URR versus Kt/V for all-cause mortality and compared the factors modifying this effect in 2615 incident HD patients in southern Taiwan. We determined that higher dialysis dose  were both associated with lower all-cause mortality compared with lower dialysis dose . Moreover, in subgroup analysis, we found that higher dialysis dose was significantly associated with mortality in those with Kt/V < 1.4 but not in those with Kt/V ≥ 1.4. Kt/V was not associated with mortality in women.27. The clearance of small molecules such as urea, Kt/V, and URR is critical for dialysis adequacy evaluation. Optimal dialysis and the relation between dialysis adequacy and survival have been heavily researched, although no randomized studies investigating minimum dialysis dosage have been undertaken. Several retrospective studies have demonstrated that Kt/V > 1.2 is associated with better survival29. Studies have also suggested targeting Kt/V > 1.4 to achieve a minimum of Kt/V > 1.2 due to barriers to adequate delivery, such as lower blood flow, shorter time, recirculation, and use of a catheter for vascular access31. The KDOQI Clinical Practice Guideline for Hemodialysis Adequacy8 suggests a minimum URR dose of > 65% and a target dose of > 70% for patients receiving HD three times per week with treatment times less than 5 hours8. Barriers to URR and Kt/V correlation have been indicated, including higher UF, which may cause increased Kt/V, and long dialysis time, which may cause decreased URR18. Another study found that after stratifying patients into three BMI (body mass index) groups , the relative risk (RR) decreased when URR increased. Furthermore, patients treated with URR > 75% had a substantially lower RR than patients treated with URR 70–75% (P < 0.005 for medium and low BMI groups)12. Although patients benefit from higher Kt/V or URR, Chertowet al. demonstrated that patients with extremely high URR (> 75%) or single-pooled Kt/V (> 1.6) may be more severely malnourished, which may increase mortality and limit the utility of URR or Kt/V32. In our study, we found that URR > 70% or Kt/V > 1.4 was associated with better survival. In the subgroup analysis, we found that higher dialysis dose was significantly associated with mortality in those with Kt/V < 1.4 but not in those with Kt/V ≥ 1.4 which is compatible with the attenuation of HR we found in higher Kt/V or URR group in Table Dialysis inadequacy is one of the main causes of mortality in HD patients13. However, Kt/V has some disadvantages compared with URR. First, V and Kt are both considered survival-associated factors, which may cause an offset effect15. For example, in patients with lower mass, high Kt/V is easier to achieve due to low V, which causes overestimation of dialysis dose16, even when compared with using Kt only17. Second, models of Kt/V assume stable outpatient status with end-stage renal disease, which does not apply in situations such as those of critically ill patients with acute kidney injury whose V may exceed total body water and may vary significantly15. Therefore, when outcomes, including death, are correlated with URR and Kt/V, no difference in degree of correlation is detectable8.Kt/V has long been preferred to URR as the standard for prescribing HD dosage and is recommended by the KDOQI. The reasons include the narrow range of doses achieved during HD, the curvilinear relationship between the two parameters (because Kt/V considers urea distribution volume and UF), that URR decreases substantially during continuous renal replacement therapy or daily dialysis whereas the given Kt/V remains the same, and the inability to account for the effect of residue kidney function with URR21. In another recent study in Gulf Cooperation Council (GCC) population, they found low Kt/V was strongly related to higher mortality in women, but not in men33. Perez-Garcia et al. demonstrated that patients with a higher Kt/V, which occurs more easily in women, had lower survival rates than others. Higher Kt/V values are due to a lower V and poorer nutritional status34. Another study determined that the increased mortality with extremely high URR (> 75%) or single-pooled Kt/V (> 1.6) values (manifested in a lower V) due to malnutrition outweighed the benefits of greater urea clearance32. Moreover, studies also found that female HD patients tended to have more severe malnutrition35 and lower albumin levels36,outweighing the benefits of relatively high Kt/V. Due to this malnutrition factor, it is difficult to evaluate the actual benefits of increased dialysis dosage in women by using Kt/V. Our study found that sex modified the association between either Kt/V or URR and all-cause mortality, but BW did not. However, the conversion of BW to V was based on the Watson formula, which was designed in relation to a healthy Western population and might not reflect actual V in our study population38. In a later analysis in the HEMO study, increasing dialysis dose (double-pool Kt/V 1.53 vs double-pool Kt/V 1.16) in a subgroup of women reduced mortality by 19%; it did not cause a significant difference in men. This result persisted after adjustment for the interaction of dosage with body water volume or with other mass parameters, including weight and body mass index, which indicates that factors other than body size may have contributed to this result22. Another study found a similar sex difference in mortality benefit for women on HD in Japan with spKt/V levels ≥ 1.639. Although the previous studies were all favorable of underdialysis of woman in Kt/V, which is opposite to our result that no obvious prognosis value for woman with higher Kt/V, but in man. However, these studies all undeniably point out that sex difference did exist when using Kt/V. The mechanism of the sex difference modifying the association between Kt/V and all-cause mortality in our study remains unclear and maybe relate to race or specific population in our study. However, an optimal indicator for mortality should not generally be affected by age, sex, or comorbidities. Therefore, URR may have prognostic value for mortality equal to or greater than Kt/V, as shown in this study.In subgroup analysis, we found that factors such as age, BW, and other comorbidities did not modify the association between either Kt/v or URR and all-cause mortality, except for sex in the Kt/V group. We found that increased Kt/V was not associated with mortality in women. To explain this sex difference, studies have indicated that underdialysis occurs more in women. In recent studies, the various minimum target dialysis does of Kt/V was found in sedentary patients by gender and body size after using alternative parameters such as Kt/TEE and Kt/BSA(Body surface area), woman and smaller man were found generally underdialysis by using Kt/V2 with considerably low urine output40. Third, some other data were absent, such as blood pressure, medication, and BMI. Fourth, Kt, BSA, TEE records were absent and thus could not be compared with Kt/V.This study enrolled incident HD patients and measured average dialysis dose between months 4 and 9 of HD. This may have prevented survival bias and ensured stable measurement of dialysis dose. This study has limitations. First, it was an observational study, and a causal relationship between Kt/V or URR and clinical outcomes could not be established. Second, residual urine was not measured. However, as in our previous report, our patients with chronic kidney disease started dialysis late, at a mean eGFR of 5 mL/mL/1.73mOur study demonstrated that in a head-to-head comparison between URR and Kt/V in 2651 incident HD patients, URR has as much prognostic value for mortality as Kt/V in this study population (Asian or Taiwanese population). Moreover, compared with URR, Kt/V appeared to have less prognostic value in the female subgroup. Finally, higher URR (URR > 70%) and Kt/V (Kt/V > 1.4) were both associated with lower all-cause mortality in incident HD patients. Larger studies to compare the prognostic value of URR and Kt/V for mortality and more application of URR in the future studies are necessary. In addition, the prognostic value of Kt/V in women may warrant further investigation.The Kaohsiung Hemodialysis Study was designed as a prospective cohort study to investigate the quality of patient care on the basis of the HD, Operation, Plan, Executive management system developed by the Taiwan Society of Nephrology. Between 1 January 1997 and 31 December 2009, 2748 consecutive incident HD patients who had stable thrice-weekly dialysis for more than 90 days and were aged more than 18 years from three affiliated hospitals and nine associated HD clinics of Kaohsiung Medical University in southern Taiwan were included and followed up until 31 May 2013. Of these, 94 patients discontinued follow-up within 6 months, and 39 patients had more than 10% data missing. The final study population consisted of 2615 incident HD patients. The study was conducted according to the guidelines of the Declaration of Helsinki, and ap-proved by the Institutional Review Board of Kaohsiung Medical University Hospital (KMUH-IRB-990198). We have confirmed that informed consent was obtained from all subjects and/or their legal guardian(s).2 and a mean residual urine of 560 mL. In total, 178 patients (6.1%) were receiving incident peritoneal dialysis at the same period. The National Health Insurance Administration provides total coverage of HD treatment and erythropoiesis-stimulating agent treatment with a fixed payment. The attending physicians rotated between the dialysis centers, and dialysis machines were also rotated; artificial kidneys and water management were similar.Dialysis initiation was carried out according to the regulations of the National Health Insurance Administration of Taiwan, which specify required laboratory data, nutritional status, uremic status, and estimated glomerular filtration rate (eGFR). Dialysis initiation was late with a mean eGFR of 4.9 mL/min/1.73 mIn addition, we do not lower the surface area dialyzer if target URR(70%) or Kt/V (> 1.4) as suggested by KDIGO achieved. However, we will increase the surface area dialyzer if the minimal requirement of URR(> 65%) or Kt/V (> 1.2) were not achieved according to the guideline of Taiwan Society of Nephrology.8: (1) The ultrafiltration rate was set to zero, (2) the blood pump was slowed to 100 mL/min for 10–20 s, (3) the pump was then stopped and (4) a sample was drawn either from an arterial blood line sampling port or from tubing attached to the arterial needle42.Baseline variables included demographic features , medical history . We collected and averaged examination findings , laboratory data  albumin assay via Roche cobas® 6000 analyzer), white blood cell count [WBC], hemoglobin, total cholesterol, cardiothoracic ratio [CTR], iron saturation, and glucose[AC]) and HD parameters between months4 and 9. Demographic features were obtained from baseline records, and medical history was obtained by reviewing doctors’ charts. DM and hypertension were defined by clinical diagnosis. Laboratory data were collected monthly, and the mean data from6 months after stable dialysis were used in analysis. Dialysis adequacy wasmeasured using the single-pool Daugirdas formula Kt/V =  − 1n((postBUN/preBUN) − 0.008 × t) + [(4 − 3.5 × (postBUN/preBUN)) × UF/BW] and URR = (preBUN − postBUN)/preBUN(BUN: mg/dL). Postdialysis BUN was drawn according to Kidney Disease Outcomes Quality Initiative (KDOQI) guidelines as followsPatients were followed up from month 4 to the end of month 20 of HD or death. All-cause mortality was ascertained by review of death certificates using charts or the National Death Index.P < 0.05 in univariate analysis were included in these models, and skewed distribution continuous variables were log transformed to attain normal distribution. These were age at dialysis initiation, sex, entry year, DM, hypertension, hepatitis, CHF, postdialysis BW, nPCR, UF:BW ratio, creatinine, hemoglobin, WBC, albumin, AC, log-transformed cholesterol, and phosphorus. Prespecified subgroup analyses were also performed in patients stratified by sex, age (≥ 65 years), DM, CHF, hepatitis, hypertension, anemia (hemoglobin < 10 g/dL), albumin (< 3.5 g/dL), UF (mean), and BW (mean). Interactions between subgroups were tested. A P value < 0.05 indicated significance. Statistical analysis was performed using R 4.1.3 software  and SPSS version 20.0 .The PA Baseline characteristics were expressed as percentages for categorical data, mean ± standard deviation (SD) for continuous variables with approximately normal distribution, and median and interquartile ranges for continuous variables with skewed distribution. The Markov chain Monte Carlo method was used to mitigate the effects of missing covariates . Multivariate linear regression was used to evaluate the relationship between URR, Kt/V, and the significant factors listed in Table The study was conducted according to the guidelines of the Declaration of Helsinki, and ap-proved by the Institutional Review Board of Kaohsiung Medical University Hospital (KMUH-IRB-990198).