Fibroblast growth factor (FGF) 19 subfamily, also known as endocrine fibroblast growth factors (FGFs), is a newly discovered metabolic regulator, including FGF19, FGF21 and FGF23. They play significant roles in maintaining systemic homeostasis, regulating the balance of bile acid and glucolipid metabolism in humans. Osteoporosis is a chronic disease, especially in the current status of aging population, osteoporosis is the most prominent chronic bone disease, leading to multiple complications and a significant economic burden that requires long-term or even lifelong management. Members of the FGF family have been shown to be associated with bone mineral density (BMD), fracture repair and cartilage regeneration. Studies of the FGF19 subfamily in different populations with osteoporosis have been increasing in recent years. This review summarizes the role of the FGF19 subfamily in bone metabolism, and provides new options for the treatment of bone diseases such as osteoporosis. Osteoporosis is a systemic skeletal disease caused by disorders of bone metabolism. An article published in Lancet in 2019 showed that fractures resulting from osteoporosis became increasingly common in women after age 55 years and men after age 65 years, leading to increased substantial bone-associated morbidities, mortality and health-care costs . TherefoEarlier studies have suggested that osteoporosis is likely to be caused by complex interactions among local and systemic regulators of bone cell function . Recent FGF-19 is secreted by ileal epithelial cells and plays a regulatory role in the maintenance of bile acid and corresponding metabolic homeostasis Table 1.FGF-21 plays a key role in the metabolic process as a hepatic, adipokine and myokine. It appears to function through FGFR1c-mediated binding to β-klotho to form complexes . In receFGF-23 is known as a bone-derived endocrine hormone, mainly secreted by osteocytes and osteoblasts, mediated by the FGFR  and combined with α-Klotho to stimulate phosphate excretion and inhibit formation of 1, 25(OH)2D3, active vitamin D . ParathyMetS is a global health problem increasing worldwide, which includes a set of common metabolic abnormalities such as insulin resistance, impaired glucose tolerance, abdominal obesity, dyslipidemia, and hypertension . The intVitamin D is an important factor in bone metabolism. Animal studies have shown that vitamin D3 supplementation has a positive effect on fracture healing . A cliniThe decline of renal function in CKD is accompanied by chronic kidney disease-mineral and bone disorder (CKD-MBD), which can lead to renal osteodystrophy and osteoporosis . When kiThe risk of bone fracture increases with age, especially in postmenopausal women, who have the highest prevalence of osteoporosis of all bone metabolic diseases, so women should be screened for osteoporosis starting at age 65 , 45. As Previous studies have shown that FGF21 enhanced PPAR-γ activity by inhibiting osteoblastogenesis and increasing bone marrow adipogenesis in bone marrow mesenchymal stem cells (BMSCs) to increase bone resorption and decrease bone formation . RecombiHowever, Studies on the relationship between FGF21 levels and BMD have yielded inconsistent results in humans. A cross-sectional analysis of healthy postmenopausal women showed that their circulating FGF-21 levels were positively correlated with lumbar spine BMD . One stuA cross-sectional study of 28 patients with PMO, 32 with postmenopausal osteopenia and 30 healthy control subjects  found that significantly higher levels of FGF-23 in the PMO group compared to the postmenopausal osteopenia and control groups . In addiin vivo deletion of FXR resulted in a significant reduction in bone mass in mice  receptors by granulosa cells, and proliferation of ovarian germinal cells . Among tOverall, the FGF19 subfamily has been the focus of attention since its discovery. A large number of studies in recent years have revealed that the FGF19 subfamily is associated with bone metabolism, which has caused controversies and debates. Serum FGF19 and bile acid levels are positively correlated with BMD, while both levels are reduced in the serum of PMO patients. Therefore, FGF19 may provide a new therapeutic target for PMO patients by participating in the enterohepatic circulation of bile acid. The relationship between FGF21 and BMD is controversial in both mouse and human studies, but FGF21 does participate in the metabolic activity of MetS, PMO and POCS patients, and more basic and clinical experiments are needed to further clarify the relationship subsequently. As a bone-derived hormone, FGF23 can act on kidneys and parathyroid glands to regulate bone metabolism activity, which may offer new options for the treatment of osteoporosis and other bone diseases. In addition, scientific consensus has unanimously established that the FGF19 subfamily, especially FGF21, is a new key player in bone metabolism and its role is emerging as a possible therapeutic option to treat bone diseases.JJ and GY designed the study. XY wrote the paper. XY and YX made the figure and table. JJ and GY revised the paper. All authors read and approved the final manuscript.This study was supported by the National Natural Science Foundation of China , the Social Development Project of Jiangsu Province (BE2018692), the Natural Science Foundation of Jiangsu Province, China (BK20191222), the Youth Medical Talent Project of Jiangsu Province (QNRC2016842), the High Caliber Medical Personnel Foundation of Jiangsu Province (LGY2016053), the Six Talent Peaks Project in Jiangsu Province (2015-WSN-006), the Jiangsu University Affiliated Hospital “5123” Talent Plan (51232017305), the sixth “169 “ Talent Project of Zhenjiang, the Science and Technology Commission of Zhenjiang City (FZ2020038), Doctoral Research Initiation Fund (jdfyRC2020010) and Clinical Medical Science and Technology Development Foundation of Jiangsu University (JLY2021209).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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. 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In low and middle income countries macroeconomic volatility is common, and severe negative economic shocks can substantially increase poverty and food insecurity. Less well understood are the implications of these contractions for child acute malnutrition (wasting), a major risk factor for under-5 mortality. This study explores the nutritional impacts of economic growth shocks over 1990–2018 by linking wasting outcomes collected for 1.256 million children from 52 countries to lagged annual changes in economic growth. Estimates suggest that a 10% annual decline in national income increases moderate/severe wasting prevalence by 14.4–17.8%. An exploration of possible mechanisms suggests negative economic shocks may increase risks of inadequate dietary diversity among children. Applying these results to the latest economic growth estimates for 2020 suggests that COVID-19 could put an additional 9.4 million preschoolers at risk of wasting, net of the effects of preventative policy actions. Economic shocks may lead to food insecurity and therefore acute child malnutrition (wasting). Here, the authors use data from Demographic Health Surveys to estimate impacts of past economic shocks on wasting and project possible effects of shocks related to the COVID-19 pandemic. Other estimates suggest 140 million people may have fallen into $1.90/day poverty in 20202.Macroeconomic volatility is far more common in low and middle income countries (LMICs) for a variety of economic, political, and environmental reasons. However, the economic impacts of the COVID-19 pandemic have been exceptional, forcing governments throughout the world to shut down large parts of their domestic and international economies in both 2020 and 2021, thereby transforming a health crisis into a complex economic crisis of exceptional scale, scope, and duration. Virtually all LMIC economies contracted sharply in 2020, and most have experienced further shocks due to the Delta variant in 20213. Infants and young children are at the greatest risk of wasting – and of mortality due to wasting–because of their immature immune system and their high nutrient requirements for growth and development. Although less prevalent than stunting–an indicator of chronic undernutrition–wasting is a much stronger predictor of child mortality. A pooled analysis of ten prospective cohort studies estimated that severe wasting had a hazard ratio of 11.6 compared to 5.5 for severe stunting (height-for-age Z-scores < −3), while moderately wasted children (−2 > WHZ > −3) were 3.4 times and mildly wasted children (−1 > WHZ > −3) 1.6 times as likely to die before their fifth birthday compared to non-wasted children4.While there is not yet any evidence to suggest that COVID-19 has directly resulted in significantly higher mortality risks for young children in LMICs, the indirect effects of COVID-related economic shocks pose serious risks for malnutrition, morbidity, and mortality, particularly among children under 5 years of age. In the short term, nutritional insults often manifest in the form of acute weight loss, typically measured as low weight-for-height z scores (WHZ) that are used to classify wasting into different levels of severity. Wasting is usually the result of both severe reductions in food intake and recent or repeated episodes of infectious diseases, and the dynamic interaction between poor diets and infections11, or the numerous studies estimating the impacts of negative economic shocks on child mortality.9 Moreover, while LMICs have made significant progress in the past few decades in reducing stunting, progress in reducing wasting is uneven at best, with some programmatic improvements in treating wasting12, but limited improvement in prevention. Globally, more than 45 million under-5 children were estimated to be moderately (WHZ < −2) or severely (WHZ < −3) wasted in 2020, and as much as 75% of wasted children residing in South Asia and sub-Saharan Africa (particularly the Sahel and Horn of Africa), where prevalence of wasting remains remarkably high13.Despite the serious risks that wasting poses for morbidity and mortality in young children, the underlying economic causes of wasting are under-researched compared to the many studies linking longer-term economic growth to stunting14 that collected information on 1.256 million children in 52 countries over three decades (1990–2018). We link these child and household level data to national level estimates of short- and medium-term changes in Gross Domestic Product per capita (GDP) or Gross National Income per capita (GNI), which is equal to GDP plus net income earned abroad. With some modifications, we follow the empirical strategies of previous papers16 that combined macroeconomic data with multiple rounds of DHS data to examine the mortality effects of economic shocks: fixed effects estimators that control for region-specific time trends to net out the biasing effects of many potential confounding factors.In this study, we explore the impacts of short-term economic growth shocks on the risks of child wasting. While are data are historical and pre-date COVID-19, understanding whether the economic shocks currently being experienced in LMICs are likely to significantly increase risks of wasting is an important motivation of this study. To do so we use an extensive set of 177 Demographic Health Surveys (DHS)1 predicts that an extra 9.4 million children could become wasted in 2021 as a result of COVID-related economic contractions in 2020.We show that departures of annual economic growth from long term growth trends  increase the risk of child wasting in the subsequent calendar year, and by a significant margin: the model predicts that a 10% decline in GNI per capita predicts a 14.4–17.8% increase in moderate/severe wasting in children under-5. Our findings also support the hypothesis that increases in child wasting are associated with increased risks of children having poor dietary diversity and infectious diseases symptoms such as diarrhea or fever. Moreover, applying these growth elasticities to the latest GDP growth estimates for 2020Although this estimate may be an upper bound given the various interventions that governments have implemented to protect their economies during the pandemic, the effectiveness of these interventions in preventing household economic hardship and wasting remains uncertain. Unless protected urgently and holistically, children in LMICs could well be neglected victims of an economic pandemic affecting poor populations throughout LMICs, with no immediate end in sight.Our results are structured in three parts: (1) Descriptive results designed to outline the data structure and patterns in wasting and economic shocks; (2) Main regression results for child wasting; (3) predicted impacts of COVID-19 on wasting; (4) sensitivity tests and extensions; and (5) an exploration of potential mechanisms.17 Wasting is very common in South Asia (notably India) as well as the Sahel and parts of the Horn of Africa, but generally much lower in the rest of sub-Saharan Africa despite many African countries having high rates of stunting, a measure of chronic malnutrition. Supplementary Fig. 18 (27%), but then declines steadily to level off at around 15% from age 3 onwards. In sub-Saharan Africa wasting progression follows a very different pattern: children are commonly born wasted, wasting rates are then typically steady for the first few months of life, but then rise and peak in the 10-12 months of age (at 11–17% in most sub-regions) before declining thereafter. However, in the Sahel these dynamics are noticeably accentuated: 21% of children are born wasted, but by 11 months wasting prevalence reaches 30% and thereafter declines to 10% by 36 months.Our sample of 1.256 million children 0-59 months of age spread across 52 LMICs exhibits large variation in wasting prevalence , country fixed effects, and various region-specific temporal effects to control for wasting-age dynamics . Results from an earlier version of this study were also used to estimate the potential global change in numbers of wasted children using September 2020 GNI growth projections from IFPRI19.In total, the model estimates that an addition 9.37 million pre-school children could have become wasted in 2021 as a result of the growth shocks experienced in 2020. It is also notable that despite the use of a slightly different growth elasticity (GDP instead of GNI), a different measure of growth shocks , and a slightly different sample size, our results are very close to the 9.3 million projection previously reported using GNI shocks in combination with IFPRI MIRAGRODEP GNI projections for 2020One commonality across projections is the huge impact of large negative economic growth in India, which suffered an unusually large GDP shock in 2020 (−14.9%), had exceptionally high under-5 wasting prevalence prior to COVID-19 (20.8%), and an exceptionally large population of children under the age of 5 . Clearly, the massive risk of increased wasting in India is an issue that warrants closer empirical investigation and extraordinary policy responses, especially in light of the extremely severe third wave of COVID-19 cases in the second quarter of 2021.A final point of some note is that these estimates likely represent upper bounds, as they reflect historically derived wasting-growth elasticities. The COVID-19 crisis motivated a number of extraordinary social protection measures designed to bolster household incomes, employment, and small businesses, and these may have protected nutritionally vulnerable households and their children to some extent.Additional results testing robustness to variations in specifications and samples are reported in the Supplementary Information. Supplementary Fig. 22, while rural populations  tend to be more protected because of the inelastic demand for food. For GDP shocks, but not GNI shocks, we also find some evidence that the wasting status of girls is somewhat more sensitive to shocks than for boys. This heightened vulnerability of girls to economic shocks is consistent with an earlier literature suggest that girls in India are more adversely affected during times of deprivation23.Supplementary Tables Table The results in Table In addition to that suggestive evidence on dietary impacts, GDP shocks are also significantly and negatively associated with diarrhea and fever prevalence, although only at the 10% level, and the result does not robustly extend to GNI shocks. Elasticities of low maternal BMI with respect to growth shocks were imprecisely estimated and not statistically different from zero. Hence, evidence from this analysis on the hypothesis that economic shocks affect child infections or maternal weight is inconclusive.This study examined the impact of macroeconomic shocks–defined as annual differences from long-run economic growth–on the risk of child wasting in LMICs. We find robust evidence that the elasticity of wasting with respect to lagged growth shocks is negative and relatively large in magnitude. The results are robust to variations in specification, although point estimates for GDP shocks tend to be larger than those of GNI shocks (though less precisely estimated). We find some evidence of significantly greater sensitivity of urban children to macroeconomic shocks, and some evidence that girls are more sensitive to GDP shocks. We also find some indications that poor diets and increased infectious disease risks might be important mechanisms linking growth shocks to wasting, bearing in mind that the results are sensitive to the choice of GNI or GDP shock measures.11. Moreover, since wasting is a major risk factor in early childhood mortality24, our results help explain previous findings demonstrating that adverse economic shocks in LMICs significantly increase child mortality rates9.This study makes a novel contribution to the existing literature on economic growth and nutrition, which has been largely confined to studying the impacts of longer term economic growth on child stunting4. The true extent of these economic shocks has become increasingly apparent, with incorporation of April 2021 IMF GDP growth estimates predicting that approximately 9.37 million children could become wasted in 2021, but also some further deterioration in economic output in LMICs as the Delta variant has spread far and wide. Globally, the impacts of COVID-19 on wasting very much depend on what happens in India (and some other large countries). Disconcertingly, previous research suggests that state-level macroeconomic shocks in India do indeed predict higher levels of mortality among young children, especially girls15.Our findings are also highly relevant in the context of the complex and ongoing COVID-19 crisis because they predict that aggregate income shocks alone–even without COVID-related health disruptions–will have severe impacts on wasting and increase the risks of elevated child mortality rates25 as well as welfare evidence from earlier macroeconomic crises in LMICs populations22. This is likely because the farm economy is relatively resilient to macro shocks, with demand for food being less adversely affected than demand for most non-farm goods and services.Our empirical results also suggest that macroeconomic shocks might raise wasting risks among urban children more than they do among rural children, consistent with recent evidence on the impacts of COVID-19This study has limitations. Although we have a large sample of children, the number of economic shocks we study (country-year dyads) is relatively small and offers limited opportunities for exploring heterogeneity across shocks or across countries. Macroeconomic shocks can be caused by very diverse underlying factors , and the effects of any given shock could affect the welfare of different socioeconomic groups quite heterogeneously, depending on factors such as economic structure and the extent of social protection measures. Of course, if the impacts of economic shocks are indeed highly heterogenous, this might lead to attenuation of the relevant regression coefficient, perhaps towards statistical insignificance. The fact that we do find such statistically significant coefficients in the wasting regressions gives us some confidence that acute malnutrition is indeed generally quite sensitive to recent macroeconomic shocks in a general sense, especially where wasting is prevalent in normal times.26, and indeed some early country case studies of COVID-19’s economic impacts suggest that losses in household income will be much larger than losses in national income averaged over the population27. Contextually, the COVID-19 crisis has also generated unprecedently large government responses to limit the economic damage to households, although the scale of the response in LMICs has thus far been inadequate relative to the economic damage28.Another limitation is that it is also well known that national income measures are imperfect predictors of household welfare30. Reassignment of health staff to COVID-19 tasks, restrictions on mobility, lack of transport, and fear of using health services during lock-downs will also affect access to and utilization of health and nutrition services. A recent COVID-19 study estimated that different scenarios of reduced coverage of health and nutrition services combined with assumptions about increases in wasting could result in 168,000 additional deaths in under-five children in LMICs 19. That study estimated that increased wasting would account for close to 25% of the additional deaths.A final limitation is that this study only assessed the impacts on wasting due to economic shocks. While economic channels are clearly important in 2020 and 2021, the prolonged COVID-19 crisis will also affect child wasting and mortality through health system disruptions, including suspension of a range of essential nutrition and health actions such as antenatal care, vitamin A supplementation, and immunization, and the prevention and treatment of severe acute malnutrition and infectionsOur study complements these findings by identifying an important channel of impact: negative economic shocks substantially increasing the risk of all types of wasting. This implies that efforts to protect health systems also need to be bolstered by nutrition-sensitive social protection programs to protect incomes of poor and vulnerable populations, including newly poor populations who will often reside in urban areas. Another potential problem is that wasting is often seen as a public health issue rather than a broader problem of underdevelopment (unlike stunting), although our results suggest that wasting is very much affected by economic shocks, not just health-related problems. Wasting and other important forms of malnutrition are multidimensional problems requiring concerted multisectoral solutions, especially in the wake of COVID-19. Unfortunately, COVID-19 continued to ravage many LMICs in 2021 and early 2022 (the time of writing). Much stronger international support is needed to solve the immediate health and nutrition crises affecting these countries, as well as the ongoing economic crises affecting LMICs.To explore the impact of macroeconomic shocks on child wasting we combined a large multi-country child-level DHS dataset with national-level macroeconomic data. The DHS are well suited for this kind of analysis because of their high degree of standardization and coverage of a wide range of LMICs, their collection of a rich array of nutrition, health, demographic and socioeconomic data, their representativeness at both national and subnational levels, and their repeated application within countries over different periods of time. Our dataset comprises 177 DHS rounds that collected data on child weight in multiple rounds for children 0–59 months of age in 52 LMICs between 1990 and 2018 (See Supplementary Table 31. We then defined three standard measures of wasting: any wasting (WHZ < −1), moderate/severe wasting (WHZ < −2), and severe wasting (WHZ < −3).DHS data were used to calculate WHZ scores relative to WHO reference weight-for-height measures of healthy breastfed children in multiple countriesr = 0.90), is imperfect. In this study we sourced both of these indicators from the UN National Accounts Database,32 although we also tested robustness to a World Bank source for these same indicators33.Our key explanatory variable is the lagged annual change in economic growth, although economic growth can be measured through both GNI and GDP. GDP measures the value of goods and services produced within a country and includes national output, expenditures, and income, while GNI equals GDP plus wages, salaries, and property income of the country’s residents earned abroad and at home. A priori, it is not obvious which is a better predictor of changes in household incomes, and the correlation between the two indicators in our sample, while high ), medical facility births and vaccinations (% children fully immunized for age), improved sanitation and water supplies, household demographics , child sex and rural location. In addition to these DHS controls we also followed a previous cross-country DHS study on growth shocks by testing sensitivity to two additional national-level controls that could influence economic growth but independently affect child health: climate shocks, as captured by the one-year lag of total annual rainfall and average monthly temperatureFinally, we used child morbidity symptoms and dietary indicators, as well as low maternal body mass index (BMI < 18.5) to explore whether disease, dietary or maternal nutrition mechanisms might explain the impacts of growth shocks on child wasting. For morbidity symptoms we use the DHS measure of whether a child was reported to have had diarrhea or fever-only in the past two weeks. For diets we use the standard minimum dietary diversity (MDD) indicator corresponding to a child having consumed at least four of seven food groups in the previous 24 h measured for children 6–35 months. We also use the individual food groups to test sensitivity of their consumption to growth shocks.Our analysis of these data was conducted in three stages. All analysis was conducted in STATA™ Version 16.First, we estimated a new set of household weights to make the regressions more representative of under-5 children in the DHS countries in our analysis. In a typical single-country analysis one need only use the standard DHS survey weights for each household to render statistics nationally representative, but in a multi-country analysis a more complex approach is needed, especially when the main explanatory variable of interest is itself measured at the country-year level. This is because: (a) countries differ in terms of their total population of under-5 children; (b) different countries have different numbers of rounds, and survey sizes that do not reflect population sizes; and (c) different rounds within a country can have very different sample sizes. Were we not to construct explicit weights to reflect these facts then the regression coefficients for growth shocks would remain implicitly weighted by the somewhat arbitrary number of observations in each individual survey and the number of surveys in each country, resulting in some growth episodes being over-weighted or under-weighted relative to their country’s population of under-5 children or relative to the number of survey rounds per country.35 statistics on the population of children <5 years of age to create a country-level population weight. India, for example, accounts for 20% of all child-level observations in our sample, but India contains 34% of all under-5 children in our DHS sample of countries  to plot wasting by child age and region, and examined the distribution of growth shocks across DHS rounds.In the second step of our analysis we used different descriptive analysis techniques to explore patterns and trends in the data by mapping moderate/severe wasting, using non-parametric regressions , but also mathematically appropriate since a WHZ sample that is distributed more closely to the various wasting thresholds should see larger absolute changes in wasting. This specification also has a benefit for interpretation since the coefficient represents the elasticity of wasting prevalence with respect to economic growth shocks. The one-year lag in growth shocks is also justified on several grounds. First, the timing of wasting measurement varies within years but growth shocks can occur at different times within a year; specifying a lagged growth shock always ensures that the macroeconomic shock precedes wasting measurements. Second, caregivers may take actions to prevent or delay the translation of economic shocks into nutritional insults for their children, so the impacts of economic shocks may be quite delayed. Third, although diets and diseases could emerge quickly in the wake of economic shocks, changes in weight can a longer period of time.16, although the application to wasting poses some additional challenges highlighted below.The remaining variables in the model are control variables. The regression model closely approximates a difference-in-difference model in that it uses fixed and temporal effects to net out numerous time-invariant factors that influence wasting as well as common trends over time, such as improvements and expansions in programs aimed at preventing and treated acute malnutrition. This approach is similar to a previous study examining the impacts of growth shocks on retrospective mortality measures from the DHS38, whereas in some parts of the Sahel and Horn of Africa it is reported to increase in drier seasons40. Second, and relatedly, wasting may have quite different etiologies in different regions. For example, low birth weight is more prevalent in South Asia than in sub-Saharan Africa despite similar poverty levels.41 Moreover, DHS evidence suggests that there may be difference in long-term wasting trends across regions; wasting rates have also been stubbornly immune to longer-term economic growth in South Asia, but have declined notably in some West African countries, such as Ghana.The first is that wasting is seasonal and seasonal variations in wasting are region-specific. In South Asia, the available evidence suggests that wasting sharply increases in the monsoonTo control for these complex differences in the trends and timing of wasting, we first created a more refined series of regional dummy variables that account for the diversity of wasting prevalence within sub-Saharan Africa: the Sahel and Horn of Africa , Western and Central Africa , and Eastern and Southern Africa . The remaining regions are more standard: South Asia, South-East Asia, Middle East, North Africa, Eastern Europe, Central Asia, Latin America, and the Caribbean. We then interacted these regional dummy variables with three kinds of temporal effects: 5-year time brackets to flexibly control for secular changes in wasting in different regions; month of child measurement dummies to control for seasonality of wasting; and child age dummies (in months) to control for differences in the region-specific progression of wasting in early life.w) for child i in country c and region r at time t is a function of lagged annual economic growth (gt-1) interacted with average wasting prevalence across all rounds , country fixed effects (C), and three types of region-specific temporal effects: child age effects (A.R), seasonality effects (S.R), and trend effects (T.R). Since the specification includes country fixed effects, the coefficient on economic growth represents the elasticity of wasting risks with respect to deviations of lagged growth from long-term growth rates, which we, therefore, define as growth shocks. Standard errors (ε) are clustered at the country level for the calculation of 95% confidence intervals (CIs) in all tables, although several graphs report 90% CIs to reflect one-sided tests of the null hypothesis that the growth elasticities are significantly below zero.The remaining variables in Eq.  include Although all our regressions follow the basic structure of Eq.  we conduFirst, we explored the influence on key results of excluding various sets of control variables, as well to exclusion of extreme growth outliers. Our assumption is that many DHS controls are pre-determined and not influenced by recent growth shocks, including household assets, parental education, sanitation, and water sources, but access to health services might indeed be seriously affected by shocks.42. However, little variation across age ranges was uncovered, so those results are not reported herein (results are available on request).Second, we tested variation in wasting impacts by child age since previous research has shown that wasting prevalence is higher in young children (e.g. <2 years) and that wasting risks in younger children appear are more sensitive to various nutritional insults43 and that urban populations are typically more vulnerable to economic crises than rural populations22.Third, we introduced interactions with urban and girl dummies to test for differential impacts of growth shocks on boys and girls and rural and urban children since previous research on the health and nutrition impacts of economic shocks suggests impacts may differ by genderFinally, we explored potential mechanisms linking growth shocks to wasting, including symptoms of common childhood infections such as diarrhea and fevers, maternal nutrition (low BMI), as well as minimum dietary diversity for children 6–35 months of age, and its individual food group components. These regressions follow the same structure as Eq. , but witFurther information on research design is available in the Supplementary InformationPeer Review FileReporting Summary


At early stages of development, the brain is shaped by communication via trophic factors and other extracellular signaling, and by contact‐mediated cell–cell interactions including chemical synapses. The patterns of early neuronal impulses and spontaneous and regulated neurotransmitter release guide the precise topography of axonal projections and contribute to determining cell survival. The study of the role of specific proteins of the synaptic vesicle release machinery in the establishment, plasticity, and maintenance of neuronal connections during development has only recently become possible, with the advent of mouse models where various members of the N‐ethylmaleimide‐sensitive factor attachment protein receptor (SNARE) complex have been genetically manipulated. We provide an overview of these models, focusing on the role of regulated vesicular release and/or cellular excitability in synaptic assembly, development and maintenance of cortical circuits, cell survival, circuit level excitation–inhibition balance, myelination, refinement, and plasticity of key axonal projections from the cerebral cortex. These models are important for understanding various developmental and psychiatric conditions, and neurodegenerative diseases.Neural communication in the adult nervous system is mediated primarily through chemical synapses, where action potentials elicit Ca The earliest communication is through gap junctions and electrical synapses between newly born, still migrating neurons.Next, neurotransmitters start being released by spontaneous fusion of neurotransmitter vesicles with the cell membrane  during development in vivo Harris, . TTX bin2+ regulated evoked release and spontaneous vesicular release, and constitutive nonvesicular release of neurotransmitters all make use of specific components of the synaptic machinery, eliminating individual components allows the investigation of the relative contributions of these different forms of neuronal signaling , Munc13 and Munc18, Syntaxin, and Synaptobrevins/vesicle‐associated membrane proteins (VAMPs) in cortical development. We will also describe the implications of the findings from mouse studies for the understanding of neurodevelopmental and neurodegenerative diseases. We conclude with outstanding questions that should be studied with newly available techniques in the years to come.In this review, we aim to give an overview of historic and recent studies on the role of regulated and spontaneous vesicle release in cortical development. We discuss findings from in 1.1The soluble N‐ethylmaleimide fusion protein attachment protein receptor (SNARE) complex is universally involved in membrane fusion  by N‐ethylmaleimide sensitive factor (NSF) is necessary  is found in three isoforms in neurons, namely Snap23, which is also expressed in nonneuronal cells, Snap25a and Snap25b. In cultured neurons, deletion of Snap25 leads to a reduction in survival, arborization, and spontaneous vesicle release, and complete elimination of evoked synaptic vesicle release  mouse, which has a spontaneous missense mutation in Snap25b, unlike the Snap25 knockout mouse that has a complete abolition of the expression of all Snap25 protein isoforms. Bdr animals show impaired synaptic vesicle transport but survive until adulthood and display behavioural abnormalities including ataxia and sensorimotor gating deficits 0‐3 mouse brains), thalamocortical axons grew into the cortex, extended to the region of layer 4 and started to develop branches normally  protein family consists of four genes  , known as sec1/Munc18‐like proteins or SM proteins, are important for intracellular fusion. Munc18‐1 is essential for all transmitter release, but early developmental synaptogenesis is not altered in its absence  Munc18‐1 affect neurotransmitter release and vesicle recycling  and short (M18S) isoforms that are critical for the modulation of neurotransmission  are other components of the synaptic machinery and have been knocked out in various mouse models. Homozygous deletion of synaptobrevin‐2/VAMP2 results in perinatal lethality in mice, and the mutants have a noticeably different body shape including a shoulder hump  of vertebrates. Myelin insulates most nerve fibers, and the myelin sheath contains periodic breaks known as nodes of Ranvier that facilitate fast, saltatory conduction of neural signals. Interestingly, brain activity may modulate myelination  in recycling and late endosomes . The levels of BDNF increase during the first 3 weeks after birth  which suggests its developmental role in axonal growth, synaptogenesis, and synaptic maturation. BDNF increases the expression of myelin proteins in oligodendrocytes via activation of the tropomyosin‐related receptor kinase‐B/mitogen‑activated protein kinase (TrkB/MAPK) pathway  corresponds to the region typically deleted in Williams Beuren syndrome and the deletion of the syntaxin1‐A gene has been proposed to underlie certain neurological and cognitive deficits of this neurodevelopmental disorder such as impaired verbal, visuospatial, memory, and numerical skills . Moreover, in a major mouse model of schizophrenia, in which a human variant of the disrupted in schizophrenia (DISC1) gene is expressed transgenically, the hDISC1 mouse, Snap25 levels are concomitantly decreased. This model shows similarities to the negative symptoms of schizophrenia, including altered social interaction, anxiety, and reduced spatial memory supporting the translational applicability of this mouse model . In the 1.11Munc13 isoform 1 can be an alternative target of the intracellular diacylglycerol second messenger pathway in Alzheimer's diseases and plays a role in the regulation of amyloid precursor protein metabolism  or inhibitory optogenetic channels such as archaerhodopsin (ArchT). The difference between chronic and acute silencing can then be explained by the effects of regulated synaptic activity in the developing versus in the adult brain.The selective ablation of Snap25 in different populations of cortical projection neurons resulted in different timelines of synaptic loss, demyelination, and axonal degeneration suggesting that the development and maintenance of these different cortical projection neuronal populations might be influenced differentially by mechanisms that require Snap25. It would be interesting to control spatial expression of Snap25 with even greater specificity and to investigate whether its presence is necessary for vesicular release from axonal shafts or from synapses.vivo than In vitro. This suggests that there might be compensatory mechanisms in play that can reduce the impact of a loss of Snap25. What these mechanisms involve and whether they are also activated when spontaneous abnormalities in Snap25 function occur is a question for future studies.Remarkably, the effects of conditional knockout of Snap25 are less severe in The authors declare no conflict of interest.


Here, we review the development of LXR inverse agonists that were originally optimized for their ability to enable recruitment of corepressors leading to silencing of genes that drive de novo lipogenesis. Such compounds have efficacy in animal models of MAFLD, dyslipidemia, and cancer. Several classes of LXR inverse agonists have been identified and one is now in clinical trials for treatment of severe dyslipidemia.Activation of LXR activity by synthetic agonists has been the focus of many drug discovery efforts with a focus on treatment of dyslipidemia and atherosclerosis. Many agonists have been developed, but all have been hindered due to their ability to efficaciously stimulate  Metabolic-associated fatty liver disease (MAFLD) is a relatively new classification of disease to incorporate the metabolic dysfunction that often occurs within patients presenting with non-alcoholic fatty liver disease (NAFLD) . NAFLD hThe pathophysiology of chronic liver diseases is quite complex, and with the multitude of factors than may contribute to the development of fatty liver, the term MAFLD allows physicians to distinguish between the term “non-alcohol” and be inclusive of key metabolic factors contributing to the disease and potential therapeutic options , 7.PNPLA3), Membrane bound O-acyltransferase domain containing 7 gene (MBOAT7), transmembrane 6 superfamily member 2 gene (TM6SF2), and glucokinase regulator gene (GCKR) have been the most recognized genes involved in the pathogenesis of fatty liver diseases  identified that displays a robust association with the development and severity of NAFLD. This gene is highly expressed in the liver and white adipose tissues and is regulated by insulin signaling via LXR and sterol regulatory binding protein 1c (SREBP1c) pathways  , 5, 8. Tdiseases , 10. PNPt rs73840; I148M ipathways , 12. NorMBOAT7 functions to remodel phosphatidyl inositol with arachidonic acid and is primarily expressed in the liver –15. The de novo lipogenesis  in the liver. DNL is the metabolic pathway that synthesizes saturated fatty acids and monounsaturated fatty acids (MUFAs) from acetyl-coA  oxidation, which can stimulate etyl-coA , 19. In Within hepatocytes, FFAs can be esterified to produce TGs, which are either stored as lipid droplets in the liver or packaged as VLDLs into circulation. Because of this, MAFLD patients often present with pro-atherogenic lipid profiles  , 22, 24.MTTP) and TM6SF2. Several studies have described an increase in VLDL particle size and number due to increased ChREBP and SREBP1 activity, which was observed due to pharmacological activation of the nuclear receptor LXR, a known regulator of DNL  production via ChREBP activation of microsomal triglyceride transfer protein , which is the enzyme that synthesizes palmitate from acetyl-CoA and malonyl-CoA, can suppress hepatic steatosis in a variety of mouse models of fatty liver disease , 31, 32.Ldlr knockout rodents are prone to develop hepatic steatosis particularly when fed a western or high fat diet  in this disease as well. VLDLR is typically expressed at low levels in healthy liver and mediates the clearance of triglyceride-rich particles. During MAFLD development, in both mouse models and humans, the expression levels of VLDLR in liver increases enhancing the development of the disease. Recent data demonstrated that in mice lacking the proprotein convertase subtilisin/kexin type 9 (PCSK9) protein have increased expression of VLDLR, LDLR, and fatty acid transporters including CD36, which enhance the development of MAFLD. Loss of PCSK9 also results in increased hepatic lipid accumulation, impaired beta cell function, and decreased plasma insulin levels.clerosis –43. LikeMAFLD is a highly complex and systemic disease associated with a variety of metabolic changes and has similarities in the development of progression between mouse models and humans. Like NAFLD, MAFLD often begins with the accumulation of lipids in the hepatocytes, driven by a variety of biological factors  which is often mediated by VLDL secretion, fatty acid and lipoprotein uptake and processing, and DNL. These altered metabolic signaling processes and downstream effects on insulin signaling/regulation share features with NAFLD, cardiovascular diseases, and T2DM. While there is likely no single therapeutic target that can fully alleviate the complex metabolic dysfunction occurring in MAFLD, the nuclear receptors have proven to be a rich target class for targeting metabolic and cardiovascular diseases.Nuclear receptors (NRs) are ligand-regulated transcription factors that orchestrate numerous physiological processes including metabolism, immunity, and development . In humaSeveral NRs respond to changes in cellular levels of lipids and other metabolic signals including LXRs, farnesoid X receptor (FXR), and peroxisome proliferated-activated receptors (PPARs), and have been identified as therapeutic targets for a variety of metabolic diseases , 50. FXRLXRα and LXRβ were originally identified as orphan members of the NR superfamily , 57. BotLXRs have a significant role in the regulation of physiological processes involved in the development of MAFLD. As described earlier, numerous physiological pathways can contribute to MAFLD in both mice and humans. This disease is systemic in its development and pathogenesis, beginning with altered lipid storage and metabolism, and progressing in part, due to abnormal metabolic functioning in a variety of tissues and cell types . Here, we will focus on the physiological processes that LXR regulates, that are distinctively known for enhancing the development and progression of MAFLD.Lxrα-null mice, which accumulated significant amounts of cholesterol in the liver when challenged with a high cholesterol diet due to their inability to activate an LXR-dependent mechanism for excess cholesterol to be converted to bile acids (Abcg5 and Abcg8 (The role of LXRs functioning as “cholesterol sensors” was confirmed utilizing le acids . Subseqund Abcg8 . Reversend Abcg8 . Activatnd Abcg8 –70. LXR’inducible degrader of LDLR (IDOL) (It is well known that high levels of LDL cholesterol (LDL-C) contribute to the development of cardiovascular diseases including atherosclerosis. LDLR is responsible for the uptake of LDL-C and maintenance of systemic cholesterol levels and can be regulated at both the transcriptional and post-transcriptional levels. The sterol regulatory element-binding protein 2 (SREBP2) is the main transcription factor that regulates the expression of LDLR and is activated in response to low cholesterol levels in the cells. LXR however, can control the post-transcriptional regulation of LDLR through its direct target gene, R (IDOL) –73. The LXRs are not only important in maintaining cholesterol homeostasis, but they are intricately involved in the regulation of DNL in the liver. Numerous studies have demonstrated the enhancement of fatty acid biosynthesis and VLDL secretion due to LXR agonist treatment. LXR directly controls the transcription of SREBP1c, FASN, and SCD1, and modulates the expression of ChREBP, all of which are directly involved in the pathogenesis of MAFLD and have been discussed earlier.LXRs are master regulators of lipid and cholesterol metabolism and have remarkable anti-inflammatory activities. Because of their multiple roles, they are very interesting drug targets. The major classes of LXR modulators are agonists and antagonists. Agonists bind the LBD of the receptor and recruit coactivator proteins leading to receptor activation and increased expression of downstream target genes. Three LXR agonists are currently in clinical trials for the treatment of atopic dermatitis and advanced solid tumors and lymphoma. LXR antagonists block the binding of agonists and have yet to demonstrate therapeutic utility. A third type of modulator is LXR inverse agonists that was first developed by our group. LXRs have been demonstrated to recruit either coactivators or corepressors depending on the physiological context. We envisioned that development of a LXR ligand that bound to the LBD and selectively enhanced the ability of the receptor to recruit corepressor and suppress the expression of LXR target genes, such as those encoding the DNL enzyme genes, would have the potential to be used in the treatment of metabolic disorders such as MAFLD. The LXR antagonist scaffold  was used50 = 210 nM) and LXRβ (IC50 = 40 nM) in a co-transfection assay with a multimerized LXRE luciferase reporter in HEK293T cells  were also significantly reduced consistent with SR9238 improving hepatic function.When administered to diet induced obese (DIO) mice, SR9238 (i.p.) drove a decrease in expression of teatosis . The redob/ob leptin deficient mice with similar results, but the disease is accelerated in ob/ob mice possibly due to their increased intake of the diet. In the ob/ob mice provided this NASH diet, we observed similar effects of SR9238 as we did in the DIO mice with decreased expression of genes encoding DNL enzymes and decreased hepatic steatosis  were lowered significantly. In the DIO mice there was a ∼20% decrease whereas in the NASH model there was a ∼50% decrease. We also observed that SR9238 analog, SR9243, that has systemic exposure also displayed similar effects on plasma LDL-C in mice on a normal chow diet (∼50% decrease)  is not typically noted in this model. However, mice provided a diet high in cholesterol, fructose and trans-fat do develop NASH  and we eteatosis . Hepaticteatosis . Althougecrease) . At thisecrease) . This suecrease) . Huang eecrease) . LDL-C lSrebpf1c and Fasn. Interestingly, treatment also led to an increase in expression of ethanol metabolizing enzymes Cyp2e1, Adh2, and Adh3, suggesting that not only did the LXR inverse agonist suppress DNL but also increased ethanol clearance  diet) leads to substantial hepatic steatosis but does not lead to significant hepatic fibrosis. However, addition of “binge” ethanol doses near the end of the chronic ethanol consumption does lead to fibrosis and is a model of alcoholic hepatosteatosis (ASH). We assessed the effects of SR9238 in both models and observed that the drug reduced both fat content in the liver and inflammation (and fibrosis in the ASH model) . Like inlearance . Given tlearance . Importalearance .in vivo was that there was a significant decrease in LDL-C. When examining the expression of intestinal genes that changed with SR9238 or SR9243 treatment (i.p.) we found that sterol O-acyltransferase 2 (Soat2) gene expression was suppressed by ∼95%  in HepG2 cells. These compounds drove the recruitment of corepressor proteins to the LXRs, but they did not appear to be very potent as doses greater than 1 μM were required for activity) . The structure of TLC-2,716 is not directly disclosed.After the description of the SR9238/SR9243 series of LXR inverse agonists additional chemical scaffolds with similar pharmacological profiles have been described. Burton et al. discovered that several cholestenoic acid analogs displayed LXR inverse agonist activity  90, 91), 9190, 9ctivity)  (92). Chctivity) . These cctivity) . Their mctivity) , effectise model . Workingse model . This inde novo lipogenesis. With these compounds in hand we were able to demonstrate that were effective in treatment of NASH, ASH, hypercholesterolemia, and cancer in animal models. Several LXR inverse agonist chemical scaffolds have now been identified that display similar pharmacology and even one has entered phase I clinical trials for treatment of severe dyslipidemia.LXRs have long been the focus of synthetic ligand development for the purpose of treating dyslipidemia and atherosclerosis. However, this focus has almost entirely been on the development of LXR agonists attempting to drive RCT. It was rapidly determined that such compounds had limiting on-target toxicity associated stimulating DNL resulting in hepatic steatosis and hypertriglyceridemia. Work from our lab focused on utilizing this observed side effect to develop and characterize LXR inverse agonists that actively silence LXR target genes, particularly those that drive KG and TB wrote and edited the manuscript. Both authors contributed to the article and approved the submitted version.


This indicates that Rtbdn plays a neuroprotective role during retinal degeneration. Herein, we discussed the specific function of Rtbdn and its neuroprotective role in retinal metabolic homeostasis and its role in maintaining retinal health.Dysregulation of retinal metabolism is emerging as one of the major reasons for many inherited retinal diseases (IRDs), a leading cause of blindness worldwide. Thus, the identification of a common regulator that can preserve or revert the metabolic ecosystem to homeostasis is a key step in developing a treatment for different forms of IRDs. Riboflavin (RF) and its derivatives (flavins), flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), are essential cofactors for a wide range of cellular metabolic processes; hence, they are particularly critical in highly metabolically active tissues such as the retina. Patients with RF deficiency (ariboflavinosis) often display poor photosensitivity resulting in impaired low-light vision. We have identified a novel retina-specific RF binding protein called retbindin (Rtbdn), which plays a key role in retaining flavin levels in the neural retina. This role is mediated by its specific localization at the interface between the neural retina and retinal pigment epithelium (RPE), which is essential for metabolite and nutrient exchange. As a consequence of this vital function, Rtbdn’s role in flavin utilization and metabolism in retinal degeneration is discussed. The principal findings are that Rtbdn helps maintain high levels of retinal flavins, and its ablation leads to an early-onset retinal metabolic dysregulation, followed by progressive degeneration of rod and cone photoreceptors. Lack of Rtbdn reduces flavin levels, forcing the neural retina to repurpose glucose to reduce the production of free radicals during ATP production. This leads to metabolic breakdown followed by retinal degeneration. Assessment of the role of Rtbdn in several preclinical retinal disease models revealed upregulation of its levels by several folds prior to and during the degenerative process. Ablation of Rtbdn in these models accelerated the rate of retinal degeneration. In agreement with these  The two most commonly mutated genes in IRD patients are the rod visual pigment rhodopsin (RHO) and peripherin 2 , a retinal-specific tetraspanin protein that is vital for the structure of the photoreceptor outer segment (OS)  represent a highly variable group of blinding retinal diseases, which are known to be caused by mutations in more than 300 different genes or loci (ent (OS) . To inveent (OS) . AlthougOS)   , the parent molecule for the cofactors, flavin mononucleotide , and flavin adenine dinucleotide   supplies the photoreceptor cells with essential metabolites by transferring nutrients and oxygen from the choroidal blood supply to the neural retina (reviewed by N9O15P2) . FMN andN9O15P2) , are subN9O15P2) . RF and N9O15P2) . RF is aN9O15P2) ). Some oN9O15P2)  and MoseN9O15P2) ]. OcularN9O15P2) .in vitro  and the ensuing reduction in retinal flavins levels led to significant misregulation in the levels of multiple metabolites involved in the citric acid cycle prior to the onset of degeneration . Furthermore, Rtbdn was ablated in these models, and the change in the rate of retinal degeneration was determined. To investigate the neuroprotective effects of Rtbdn, its levels were determined in mouse models of retinal degeneration (RhoRetinitis pigmentosa (RP) represents inherited retinal dystrophy caused by the progressive loss of photoreceptors, with no effective therapy available thus far. RP affects over 1.5 million patients worldwide, with a prevalence of 1:4,000 (reviewed by P23H/+)  , with a P23H/+) . The mouP23H/+) .P23H/+ mice at P15 (before the onset of degeneration), P30  accelerated the onset of degeneration  was generated and showed a retinal phenotype similar to that of the patients  and during (P90) retinal degeneration (R172W/Rtbdn−/−) exacerbated functional and structural decline  and delivery systems to carry therapeutic genes to the retina are very promising. These vectors can deliver the normal gene in cases where gene augmentation is sufficient or neuroprotective genes. Delivery of a specific gene will only provide therapy for those IRDs caused by that mutant gene; however, delivery of a neuroprotective gene allows for a therapy that spans many different IRDs. Therefore, the search for neuroprotective genes is critical for the accomplishment of this goal. We found that the RF-binding protein, Rtbdn, is significantly increased during retinal degeneration, irrespective of the causative mutation or mutant gene. Furthermore, we have shown that ablation of Rtbdn exacerbates the degenerative process. Finally, the addition of Rtbdn-containing membrane preparations to the 661W cells, which are known to be light-sensitive, protected these cells from light-induced cell death. Combined, these data support a protective role for Rtbdn in the retina, making it a promising candidate in the development of a pan-mutant therapy.via suprachoroidal means, which prevents the retinal detachment that occurs with subretinal delivery. We have previously shown that some proteins present in the retina are expressed by the RPE (Going forward, Rtbdn expressing vectors packaged into non-viral nanoparticles will be used in gene therapy experiments in mouse models of retinal degenerative diseases. Since Rtbdn is extracellular in nature, it will be interesting to investigate whether targeting Rtbdn expression to the RPE is as effective as targeting it to the neural retina. This strategy is motivated by the RPE’s ability to take up therapeutic vectors much more readily than the neural retina. Furthermore, delivery to the RPE can be achieved  the RPE . In addiTo summarize, future investigations on Rtbdn and the mechanism of flavin regulation in the retina are needed to establish how it exerts its neuroprotective effects. However, that should not prevent attempts to determine the protective effects of Rtbdn through gene therapy approaches.


Ferroptosis plays an essential role in the pathology of numerous diseases and has emerged as a key area of focus in studies of chronic kidney disease (CKD). CKD is a major public health problem with high incidence and mortality that is characterized by a gradual loss of kidney function over time. The severity and complexity of CKD combined with the limited knowledge of its underlying molecular mechanism(s) have led to increased interest in this disease area. Here, we summarize recent advances in our understanding of the regulatory mechanism(s) of ferroptosis and highlight recent studies describing its role in the pathogenesis and progression of CKD. We further discuss the potential therapeutic benefits of targeting ferroptosis for the treatment of CKD and the major hurdles to overcome for the translation of  Other biochemical features of ferroptosis include an increased production of reactive oxygen species (ROS), an accumulation of lipid peroxides and aberrant iron metabolism  (Chronic kidney disease (CKD) is a major global public health problem that afflicts 8%–16% of the population and is estimated to contribute to 5–10 million deaths annually . CKD is 2) . Cardiov2) . Despite2)  and its 2) . A deepeIn recent years, ferroptosis has been widely studied in the context of kidney disease  and has Herein, we review our latest understanding of ferroptosis in the kidneys and discuss its regulatory genes and links to CKD. We further describe challenges and future perspectives for the use of modulators of ferroptosis as much needed anti-CKD therapeutics.− (Cysteine/Glutamate Antiporter), GPX4, iron homeostasis, ROS and lipid signaling. In this section, we summarize the role of these pathways during ferroptosis /glutamate (Glu) antiporter, first identified by SLC7A11 and SLC3A2, respectively  synthesis , extracellular glutamate, sorafenib and sulfasalazine, can block system Xc− and trigger ferroptosis (System Xcectively . System ynthesis . Inhibitroptosis .GPX4 is a peroxide-degrading enzyme that uses GSH as a substrate to produce glutathione disulfide (GSSG), ultimately preventing lipid peroxidation and maintaining redox homeostasis . The gen2+) to ferric iron (Fe3+) avoiding the occurrence of the Fenton reaction and subsequent oxidative damage  and lipid remodeling gene Lysophosphatidylcholine acyltransferase 3 (LPCAT3), regulate the insertion of PUFAs into cellular membranes and have been identified as pivotal biomarkers of ferroptosis (Lipid metabolism is closely associated with ferroptosis and can be triggered by non-enzymatic (Fenton chemistry) and enzymatic mechanisms [lipoxygenases (LOXs)] . Polyunsfunction . In thisroptosis . ACSL4 sroptosis . Increasroptosis .FSP1 , as a novel ferroptosis resistance gene. FSP1 was shown to complement the loss (GPX4 KO) or inhibition (RLS3-treated) of the master regulator GPX4  transport iron and metabolites and are essential to ferroptosis . ErastinCKD is a major public health issue . The twoThe kidney is an iron metabolism-related organ. A loss of GPX4 activity , throughFerroptosis regulates CKD and kidney function. The altered expression of genes that regulate ferroptosis may influence the incidence and predisposition to CKD. loci have been identified, with around 50% showing relevance to kidney function in the context of CKD , renal fibrosis and autosomal dominant polycystic kidney disease (ADPKD). These are summarized in in vitro , in vivo [streptozotocin (STZ)-induced DN mice model] and ex vivo (kidney biopsy samples) models, a significant reduction in the mRNA and protein expression of the ferroptosis-related molecules SLC7A11 and GPX4 were observed, leading to increased lipid peroxidation in DN compared to non-DN models , a well-characterized inhibitor of ferroptosis  leading to the growth of cysts in the kidneys . Zhang eA range of preclinical studies have shown the potential of inhibitors of ferroptosis to prevent kidney disease. These include Fer-1, Rosiglitazone, Deferasirox (DFX) and Deferoxamine mesylate (DFO), and are summarized in via hypoxia-inducible factor 1-alpha (HIF-1α)/HO-1 signaling was demonstrated in diabetic mouse models . Consistent with these studies, UUO-mouse models administered tectorigenin showed attenuated tubular cell damage and fewer fibrotic lesions in the kidneys as a result of the inhibition of Smad3-mediated ferroptosis and fibrosis. More specifically, tectorigenin inhibited Smad3 phosphorylation and the expression of Nox4, a downstream modulator of ferroptosis. Moreover, isoflavone could indirectly restore the expression of GPX4, a negative regulator of ferroptosis. Collectively, these studies highlight the potential of tectorigenin as a therapeutic strategy for CKD. Future studies are now required to dissect the mechanisms of action and potential therapeutic applications of this compound.Under physiological conditions, iron homeostasis is maintained by hepcidin and iron regulatory proteins . In CKD e models . In Pdk1e models . Treatmee models . In moree models . In vitrin vitro and in vivo CKD experiments  Ferroptosis is not an isolated event and is closely associated with other forms of cell death . The molecular mechanism(s) regulating this cross-talk require characterization to reveal potential antagonistic or synergistic roles in the context of kidney disease, and more specifically CKD.2) Although a range of regulatory factors of ferroptosis have been described, they do not represent specific markers of ferroptosis. In this regard, reliable biomarkers, specifically in the context of kidney disease remains a challenge.[99] However, in kidney disease, ferroptosis may negatively impact kidney health. Further mechanistic studies are therefore warranted.3) Accumulating evidence indicates that ferroptotic cell death can inhibit tumor growth and improve the efficacy of chemotherapeutic drugs. in vitro or in vivo models. However, as in vitro cell culture conditions and animal models of kidney diseases drastically differ, the interpretation and establishment of a link between ferroptosis and CKD can be challenging. Methods to translate scientific research into clinical applications should therefore be developed.4) Although the link between ferroptosis and acute kidney injury (AKI) has been widely explored and characterized, studies on ferroptosis and CKD remain limited. Indeed, the majority of studies to-date have used Numerous lines of evidence now support a role for ferroptosis in CKD, which is mediated by the iron-dependent accumulation of lipid peroxidation. Accordingly, we have discussed the major signaling pathways that regulate ferroptosis, its regulatory genes, the role of ferroptosis in CKD and potential therapeutic strategies to inhibit ferroptosis. Despite obvious progress, future studies must address the following concerns:


Starch and cellulose are the principal components in diets for dairy cows worldwide, providing the primary energy to the rumen microorganisms as well as the host. Starch and cellulose degradation in the rumen have always been of key importance for dairy cows to obtain high production performance. To improve the starch- and cellulose-degrading activities in the rumen, the amylolytic and cellulolytic microbes and the related enzymes need to be well understood. As the rapid development of sequencing technologies, bioinformatic tools and reference databases, the rumen metagenomics have made great progress in mining the rumen microbial community for novel enzymes, such as the carbohydrate active enzymes (CAZymes). This review will summarize the ruminal microbes and enzymes involved in starch and cellulose degradation. Recent studies with metagenomics techniques on CAZymes related to starch and cellulose degradation will be discussed.Carbohydrates  are the main energy source in the diets of dairy cows. The ruminal digestion of starch and cellulose is achieved by microorganisms and digestive enzymes. In order to improve their digestibility, the microbes and enzymes involved in starch and cellulose degradation should be identified and their role(s) and activity known. As existing and new analytical techniques are continuously being developed, our knowledge of the amylolytic and cellulolytic microbial community in the rumen of dairy cows has been evolving rapidly. Using traditional culture-based methods, the main amylolytic and cellulolytic bacteria, fungi and protozoa in the rumen of dairy cows have been isolated. These culturable microbes have been found to only account for a small fraction of the total population of microorganisms present in the rumen. A more recent application of the culture-independent approach of metagenomics has acquired a more complete genetic structure and functional composition of the rumen microbial community. Metagenomics can be divided into functional metagenomics and sequencing-based computational metagenomics. Both approaches have been applied in determining the microbial composition and function in the rumen. With these approaches, novel microbial species as well as enzymes, especially glycosyl hydrolases, have been discovered. This review summarizes the current state of knowledge regarding the major amylolytic and cellulolytic microorganisms present in the rumen of dairy cows. The ruminal amylases and cellulases are briefly discussed. The application of metagenomics technology in investigating glycosyl hydrolases is provided and the novel enzymes are compared in terms of glycosyl hydrolase families related to amylolytic and cellulolytic activities. The rumen ecosystem harbours a vast number of microorganisms fermenting the ingested feedstuffs and producing various metabolites to meet the host’s nutritional requirement . NutritiUnlike ruminants in the wild, starch and cellulose are the principal components in diets for commercial dairy cows worldwide, providing the primary energy to the rumen microorganisms as well as the host . Starch The exploration of the species and enzyme activities involved in ruminal cellulose and starch digestion has been hampered by the limited number of rumen bacteria that can be cultured . MetagenStarch-rich grain is the primary energy component used in the modern diet for dairy cows, accounting for 20–40% of the ration of high-yielding cows. Due to the relatively high price of starch-containing ingredients, dietary starch should be used wisely to achieve cost-effectiveness and efficient production. Starch is a heterogeneous polysaccharide containing two structurally distinct α-linked polymers of glucose: amylose and amylopectin. The former is a linear D-glucose polymer containing ~99% α-1,4 links and the latter is the most abundant component of starch with 95% α-1,4 links and 5% α-1,6 links .Unlike non-ruminants, starch degradation mainly occurs in the rumen, partly in the small intestine with the remainder fermented in the hindgut of ruminants. Starch degradation in each segment of the gastrointestinal tract is influenced by starch sources  and processing  of the grain . Data frStreptococcus bovis, Ruminobacter amylophilus, Succinimonas amylolytica, Selenomonas ruminantium and Bifidobacterium spp. , amylopectin  and pullulan  [Succinimonas amylolytica is an anaerobic, Gram-negative and straight rod with rounded ends which can be motile with polar flagella. This species is less abundant among the ruminal bacteria when cattle are fed forage rations but is among the predominant bacteria when dietary starch is offered in the form of a grain mixture [Selenomonas ruminantium is anaerobic and Gram-negative, and it consists of motile rods of 0.8–1.0 μm in width and 2–7 μm in length. This species was found to be more abundant in the rumen when animals were fed cereal grains compared to that fed roughage [Fibrobacter succinogenes, Butyrivibrio fibrisolvens and Clostridium spp. are also capable of unitizing starch under certain conditions [he rumen . Streptod is low . When co6 bonds) , mainly 6 bonds) . Succini mixture . This sproughage . Most stroughage . Besidesnditions ,18.2, CO2, acetate, butyrate and glycerol as products. However, the rate of uptake of starch grains varies greatly between species. The protozoa with high amylolytic activities include Eremoplastron bovis, Diploplastron affine, Ophryoscolex caudatus and Polyplastron multiesiculatum. The breakdown rate of starch by protozoa is by approximation determined by the initial starch or amylopectin concentration inside the protozoa [The protozoa are also involved in degrading starch in the rumen. Between 20–45% of the amylolytic activities in the rumen have been attributed to protozoa . The amyprotozoa . Protozoprotozoa .Neocallimastix frontalis was reported to hydrolyze starch by generating an endo-hydrolytic α-amylase from which maltose, maltotriose and maltotetraose were the major products [Orpinomyces joyonii, Neocallimastix patriciarum and Piromyces communis, were also observed to be capable of digesting cereal grains [Fungi account for a small proportion (~8%) of the rumen biomass where they are involved in degrading structural carbohydrates by producing a wide range of enzymes . Neocallproducts . Anotherl grains .Due to their small size, bacteria cannot directly ingest starch granules or high-molecular-weight starch , but generate enzymes which specifically cleave the α-1,4 or α-1,6 bonds of amylose and amylopectin. These amylases can be typically classified into three main categories of hydrolytic activity: endoamylases, exoamylases and debranching enzymes .Endoamylases cleave the α-1,4 glucosidic linkages in the interior of the starch polymer or oligosaccharides in a random manner leading to the production of linear and branched oligosaccharides . α-AmylaThe rate and content of ruminal starch degradation vary with the type of cereal grains. Usually, wheat and barley starch are degraded more rapidly in the rumen than corn or sorghum starch . RuminalPhysical processing is another factor influencing ruminal starch degradation. Generally, processed grains are more digestible in the rumen . With thMoreover, starch degradation in the rumen is influenced by intricate interrelations of multiple factors including diet composition, amount of feed consumed per unit time, mechanical alterations, chemical alterations and adapting degree of ruminal microbiota to the starch ratios in diet .The rations for dairy cows are predominantly plant-based. The plant cell walls are primarily composed of cellulose which accounts for 20–30% of the dry weight of the primary cell wall. Cellulose is a homopolymer of glucose linked by linear 1,4-β-glycosidic bonds. Cellulose molecules associate with each other to form microfibrils in the form of crystalline formulations.A large number of anaerobic bacteria, protozoa and fungi possess very efficient cellulolytic machinery which enables them to improve the feed conversion efficiency of cellulose. Cellulolytic organisms are those microbes predominantly digesting cellulose present in the diet, which were dominated mainly by bacteria, fungi and to a lesser extent the protozoa .Ruminococcus flavefaciens, Ruminococcus albus and Fibrobacter succinogenes are the major cellulolytic bacteria [Fibrobacter succinogenes is one of the most widespread cellulolytic bacteria in the rumen, which contributes 5–6% of the total prokaryotic 16S rRNA in the rumen contents of cattle [Fibrobacter succinogenes strains have been reported to degrade cellulose, glucose and cellobiose mainly producing acetate and succinate [Ruminococcus flavefaciens. Ruminococcus flavefaciens are usually Gram-positive or Gram-variable and often generate a characteristic yellow pigment, particularly when grown on cellulose. Most Ruminococcus flavefaciens strains are able to degrade quite recalcitrant forms of cellulose which is difficult to digest by other species [Ruminococcus flavefaciens mainly attach to the cut edges of the epidermis, sclerenchyma and phloem cells when incubated with ryegrass leaves [Ruminococcus flavefaciens mostly degrade cellulose and cellobiose, while some strains can also utilize glucose and other carbon compounds including maltose, lactose, xylose and starch. The main end products include acetate, succinate, formate and lactate, together with traces of hydrogen and CO2. Ruminococcus albus cells are usually single or diplococcic, 0.8–2.0 μm in diameter and Gram-negative to Gram-variable. Generally, in the rumen, Ruminococcus albus is more abundant than Ruminococcus flavefaciens [Ruminococcus albus strains are able to degrade cellulose and cellobiose but cannot utilize glucose or other sugars. The main end-products of this degradation include acetate, ethanol, formate, lactate, hydrogen and CO2 with different combinations and proportions as the major products. Ruminococcus albus can produce ethanol, while the Ruminococcus flavefaciens produce succinate instead. The abovementioned three cellulolytic bacteria share some common features: (1) their growth needs a strict pH range from 6 to 7, (2) they are all strictly anaerobic and cannot survive when exposed to oxygen, (3) they digest cellulose by attachment to the cell surface through an extracellular glycocalyx and (4) these bacteria are majorly restricted to cellulose or the hydrolyzed products of cellulose.The bacteria   endoglucanases , (2) exoglucanases  and (3) β-glucosidases (β-D-glucosidases)  Table 4Table 4. All these abovementioned cellulases have been isolated from the ruminal cellulolytic microbes and classified into specific GH families, for example, the endoglucanases mainly belong to the GH family 5 and 9, whereas, exoglucanases are mostly present in the GH family 6, with the β-glucosidases mainly classified into GH family 3 .To date, only a relatively small fraction of rumen microorganisms has been successfully isolated and cultured. Therefore, the largely unexplored microorganisms represent a significant untapped source of novel enzymes, especially those with multiple functions. Thanks to the development of next-generation sequencing technologies and bioinformatics tools together with the rapid progress in reference databases, metagenomics has become a powerful tool to study the rumen microbiome.With metagenomics technologies, we can acquire the collective genetic structure and functional composition of rumen microorganisms without culturing their inhabitants. According to amino acid sequence similarity, GH and related enzymes are classified into specific families with all members in one family possessing the conserved catalytic mechanism. The public database of Carbohydrate Active enZyme (CAZy) describes the present knowledge on the enzyme families which build and breakdown complex carbohydrates and glycoconjugates. These families are classified based on experimentally characterized proteins and populated by sequences from public databases with significant similarity ,56,57. TRumen metagenomics analysis comprises two areas, including (1) functional metagenomics, in which the high-throughput screening technique is used for investigating gene products out of cloned expression libraries established by rumen metagenome DNA and (2) sequencing-based metagenomics in which the genomes and genes present in rumen microbes are explored through high-throughput next-generation sequencing.Ferrer et al.  first apFibrobacter succinogenes S85 and Ruminococcus albus. The publications since 2015 on metagenomics application related to ruminal starch- and cellulose-degrading enzymes of cows are summarized in Sequencing-based metagenomics provides the collective genetic composition and functional activities of a microbial community at the DNA level. The first publication using next-generation sequencing-based rumen computational metagenomics for cataloguing the genes and activities involved in ruminal fibre degradation was reported in 2009 . Later, In total, with the assistance of metagenomics tools, comprehensive studies as illustrated above will broaden our knowledge of the ruminal microbial structure and enzymatic activities, which in turn would allow for rumen manipulations to achieve a more efficient fibre and starch degradation. For instance, (1) as more microbial amylases and cellulases are identified out of the ruminal microbiome, it will be foreseeable to regulate the ruminal microbial amylolytic and cellulolytic activities through supplementing exogenous enzymes in the form of feed additive, (2) newly identified species will promote the process of isolating microbes out of the rumen and improve the development of microbe-culture techniques and (3) it will facilitate the commercial applications of rumen enzymes in various industries including feed additives and biofuel production.This review has summarized the microbes and enzymes involved in starch and cellulose degradation and discussed the state of metagenomics technology in mining novel cellulase and amylase GH families in the rumen of dairy cows. To date, a number of amylolytic and cellulolytic microorganisms, their characteristics and their metabolic mechanisms in the rumen of dairy cows have been described. Still, uncharacterized microbes and enzymes need to be identified. The recently emerging technologies such as metagenomics have become more efficient in exploring new microbial species and strains, mining novel enzymes and monitoring microbial and enzymatic activities. This will improve the development of new culturing techniques. In turn, the advancement of our knowledge into the functioning of the microbiota of the rumen can facilitate the directed regulation of specific microbial activities or supplementation of exogenous enzymes.


The colonic mucus bilayer is an integral innate host defense mechanism that provides a physical barrier separating the lumen and its contents from the underlying epithelium. This essential barrier is produced by specialized secretory goblet cells of which Muc2 mucin is its primary product. IgG-Fc-binding protein (Fcgbp) is the second most abundant protein produced by goblet cells, which has a suggested function of crosslinking with Muc2 to stabilize the structural integrity of mucus. FCGBP is observed to decrease preceding the onset of inflammation in ulcerative colitis patients, leading to spatially distinct structural mucus weakening to contribute to the pathogenesis of the disease.Fcgbp is altered regionally in the gut and plays a role in the pathogenesis of dextran sulfate sodium (DSS)-induced colitis. The specific aims are:To characterize the spatial expression of Muc2 and Fcgbp basally in goblet cellsTo quantify alterations in Muc2 and Fcgbp in response to DSS-induced colitis and at restitution of diseaseMuc2+/+ and Muc2-/- C57BL/6 littermates were analyzed by RT-qPCR and Western blotting, respectively. Mucin granules were isolated from colonic goblet cells and the proteome quantified by liquid chromatography and tandem mass spectrometry (LC-MS/MS). Colitis was induced in Muc2+/+ mice with 3.5% (w/v) DSS in tap water for five days, whereas Muc2-/- littermates were given 1.5% (w/v) DSS in tap water for three days, ad libitum. Mice were given regular tap water for the remainder of the experiment to allow restitution of inflammation. Disease activity index (DAI) was scored based on weight loss. Mice were sacrificed at various time points up to 10 days, and colons excised and sectioned for histopathology analysis.mRNA and protein expression in Muc2-/- littermates. In response to DSS-induced colitis in Muc2+/+ mice, Muc2 transcription rapidly increased in all regions of the colon with highest expression in the mid colon. In contrast, Fcgbp transcription increased in the mid and distal colons and peaked during highest disease activity. Interestingly, Fcgbp protein expression was abrogated in the mid colon even at restitution. In Muc2-/- mice, Fcgbp transcription decreased during disease onset but returned to normal levels following removal of DSS.LC-MS/MS of mucin granules run under reducing and non-reducing conditions confirmed that Muc2 and Fcgbp were the most abundant proteins in mucin granules and were non-covalently bound to each other. mRNA and protein expression of Muc2 and Fcgbp were highly expressed in the mid colon, and regulation of Fcgbp was unaffected in Muc2+/+ littermates, despite Muc2 restoration, suggests that the mucus barrier remains structurally altered and functionally impaired at restitution. Supported by CIHRThis study demonstrates that in response to DSS, Fcgbp expression was spatially degraded at the onset of disease and remained low at restitution. The disappearance of Fcgbp in the mid colon of None Declared


However, substantial variation in individual immune responses to vaccination exists2. Lower vaccine immunogenicity, especially to oral vaccines, is often reported in developing countries5. Many intrinsic and extrinsic factors, such as age, genetics, pre-existing immunity, nutritional status and comorbidities contribute to the variations in vaccine responses6.Vaccination is the most cost-effective life-saving medical intervention7, influences responses to vaccination10. Studies which have investigated this have shown that a higher relative abundance of the phylum Actinobacteria is consistently associated with higher vaccine responses and a higher relative abundance of Bacteroides with lower responses, while the association between the relative abundance of the phyla Firmicutes and Proteobacteria and vaccine responses varies for different genera and species18. Almost all of these studies investigated the association for oral vaccination18, only three studies investigated responses after intramuscular vaccination20.Growing evidence shows that the composition of the intestinal microbiota, which is known to play an important role in the development and regulation of immune responses26, and whether the changes induced by vaccination, in turn, influence vaccine immunogenicity20. These studies have reported inconsistent results. In humans, the administration of oral typhoid or an intranodal and intramuscularly human immunodeficiency virus (HIV)-1 vaccine was not reported to significantly affect the composition of the intestinal microbiota one week to several months after vaccination22. The first two studies used amplicon sequencing, while the third used shotgun metagenomic sequencing. A recent study using metagenomic sequencing, reported that the intramuscular administration of an inactivated and an mRNA SARS-CoV-2 vaccine was associated with lower bacterial diversity, an increase in the relative abundance of Bacteroides caccae and a decrease in the relative abundance of Clostridiales  in the intestinal microbiota one month after vaccination20. Animal studies have also shown that the administration of vaccines can lead to changes in the intestinal microbiota: in mice, an increase in the relative abundance of Bacteroides has been reported after intramuscular vaccination with a Mycobacterium tuberculosis vaccine25 and compared with mice vaccinated with a placebo, mice vaccinated intramuscularly with an HIV T-cell immunogen had a higher relative abundance of Clostridiales 26. In rhesus macaques, the intradermal administration of a combined HIV-1 DNA/protein vaccine has been shown to lead to an increase in the Firmicutes/Bacteroidetes ratio and a decreased relative abundance of Prevotella, Alloprevotella, Bacteroides, Acetobacteroides, Falsiporphyromonas and Anaerocella. Interestingly, the abundance of Prevotella negatively correlated with rectal HIV-1 specific immunoglobulin G levels24. In piglets, the administration of an oral Lawsonia interacellularis vaccine led to an increased relative abundance of Streptococcus and Ruminococcus, and a decreased relative abundance of Clostridium23.Only a few studies have investigated whether the administration of vaccines is associated with changes in the composition of the intestinal microbiota10. The latter includes the stimulation of pattern recognition receptors through bacterial components, such as the stimulation of toll-like receptors (TLR)-5 through flagellin27 or nucleotide-binding oligomerization domain (NOD)-2 receptors through muramyl dipeptide which is part of peptidoglycan28. A recent study shows that “silent” flagellins, which are weak TLR-5 agonists are abundant in Lachnospiraceae in the intestinal microbiota and overrepresented in non-industrialised populations29. The microbiota has also been shown to regulate type I interferon production by plasmacytoid dendritic cells, which stimulates conventional dendritic cells to more efficiently prime antigen-specific T cell responses30. Moreover, in mice, SCFAs  produced by microbes of the intestinal microbiota through fermentation of dietary fibres have been shown to enhance B cell metabolism and regulate gene expression to promote B cell differentiation into antibody-producing cells. Mice with low dietary fibre intake or microbial insufficiency were defective in producing pathogen-specific antibody responses, which could be restored by the intake of SCFAs31. As a further example, the abundance of the genus Roseburia, a butyrate-producer, has been positively correlated with circulating interleukin (IL)-27 levels and T cell responses to an HIV T cell vaccine26.The proposed mechanisms behind the influence of the composition of the intestinal microbiota on vaccine immunogenicity include cross-reactive epitopes between microbes and vaccine antigens, modulation of B cell responses through microbial metabolites such as short-chain fatty acids (SCFAs), and the provision of natural adjuvants through certain microbes Fig. 10. The lRoseburia in the intestinal microbiota26. If T cell responses play an important role in inducing changes in the microbiota after vaccination, a further potentially important vaccine which could change the composition of the intestinal microbiota, is Bacillus Calmette-Guérin (BCG) vaccine. BCG is one of the most widely used vaccines globally and it is known to have many non-specific effects.Little is known about the mechanisms by which vaccination might induce changes in the intestinal microbiota. Immune cells and inflammation mediators induced by vaccination, likely migrate with the blood flow from the vaccination side to the intestine, where they interact with microbes Fig. . As an eThe limitations of the studies investigating the association between vaccination and changes in the composition of the intestinal microbiota and vice versa include small cohort size; the lack of longitudinal sampling which would allow for better assessment of temporal variations; a large heterogeneity in study design, including different vaccine and vaccine schedules, and timing of collection between vaccination and stool and blood samples. Furthermore, different analysis techniques have been used, and results have been reported on different taxonomic levels. Many studies have used amplicon sequencing which might not be sensitive enough to detect small differences in microbial composition. Moreover, there is a lack of investigations on the mechanisms by which vaccination might induce changes in the intestinal microbiota. This would require not only to investigate the composition of the intestinal microbiota, but also translational approaches, including in vitro and in vivo experiments.32. Bacterial species identified to positively influence vaccine responses could be administered as probiotics before vaccination in individuals who are at risk for low vaccine immunogenicity33. Further interventions to possibly optimise the microbiota composition to improve vaccine efficacy include the administration of synbiotics (a mixture of pro- and prebiotics), faecal transplants and small molecules that interact with specific bacterial processes. Importantly, as the composition of the intestinal microbiota has been associated with the risk of many immune- and non-immune-mediated diseases, vaccination could also be used to optimise the composition of the microbiota, for example in infants who are at risk for allergic diseases and asthma34.Understanding how vaccines might change the composition of the intestinal microbiota and how these changes could influence vaccine responses, is crucial to optimise vaccine responses, especially in settings or individuals in which/whom lower immunogenicity is expected, such as in developing countries or after antibiotic exposure


Nature by Munsunuru and colleagues explores the possibility of permanently disrupting PCSK9 expression via in vivo CRISPR gene editing in non-human primates, with long-lasting reductions in LDL cholesterol.In patients with high cholesterol and at risk of cardiovascular disease, inhibitors of PCSK9 are useful in lowering lipid levels but must be dosed regularly. A recent study in  PCSK9-targeted antisense oligonucleotides are also being tested in animal and human studies. However, all of these therapies require routine dosing to maintain lower cholesterol levels, with the risk of patient non-adherence to treatment.High LDL cholesterol is a causal risk factor for atherosclerotic cardiovascular disease, the leading cause of deaths globally. The liver protein PCSK9 regulates the level of circulating LDL and is an established target for lipid-lowering therapies, including monoclonal antibodies and small interfering RNAs. PCSK9 expression. In vivo CRISPR base editing enables the precise and stable modification of single nucleotides in the genome, correcting causative mutations or inactivating disease genes. To this end, Munsunuru and colleagues evaluated the feasibility, safety and efficacy of targeting wild-type PCSK9 using a CRISPR adenine base editor in cells grown in vitro and in living mice and non-human primates1.Therapeutic gene editing—which holds enormous potential for a number of clinical indications—might provide a solution to this problem: a strategy to permanently reduce PCSK9. After first confirming disruption of this site in primary human hepatocytes in vitro, they administered a range of doses to mice intravenously and achieved an editing efficiency of 70% within the liver, the main site of PCSK9 expression in the body.The authors formulated lipid nanoparticles containing the ABE8.8 adenine base editor, which can replace certain adenine residues with guanine, combined with guide RNAs to direct the base editor to a specific splice site within Editing efficiency was similar in the livers of cynomolgus monkeys two weeks after dosing with the nanoparticles and was accompanied by an 81% reduction in the level of PCSK9 in the blood and a 65% decrease in LDL cholesterol. In a longer-term study, which is still ongoing, the authors have achieved a stable 90% reduction in circulating PCSK9 and a 60% reduction in LDL up to 8 months post-dosing, which matches or surpasses the effect of current therapies used in patients.PCSK9 editing, and no adverse health effects were observed. Sequencing studies found that off-target editing was minimal.Markers of liver damage were transiently increased by the treatment, although this was determined to be a response to the nanoparticles themselves rather than PCSK9 and durably reduce cholesterol levels. Although further evaluation of the safety and risks of in vivo gene editing in humans will be required, these data represent an important step towards translation of PCSK9 editing into the clinic, whereby a ‘once-and-done’ approach would bring significant benefits for patients.These findings represent the first efficient delivery of a CRISPR base editor to non-human primates and indicate that in vivo base editing is a precise and effective strategy to target