Profiling of Pluripotency Factors in Single Cells and Early Embryos.

Cell fate decisions are governed by sequence-specific transcription factors (TFs) that act in small populations of cells within developing embryos. To understand their functions in vivo, it is important to identify TF binding sites in these cells. However, current methods cannot profile TFs genome-wide at or near the single-cell level. Here we adapt the cleavage under targets and release using nuclease (CUT&RUN) method to profile TFs in low cell numbers, including single cells and individual pre-implantation embryos. Single-cell experiments suggest that only a fraction of TF binding sites are occupied in most cells, in a manner broadly consistent with measurements of peak intensity from multi-cell studies. We further show that chromatin binding by the pluripotency TF NANOG is highly dependent on the SWI/SNF chromatin remodeling complex in individual blastocysts but not in cultured cells. Ultra-low input CUT&RUN (uliCUT&RUN) therefore enables interrogation of TF binding from rare cell populations of particular importance in development or disease.

Direct Promoter Repression by BCL11A Controls the Fetal to Adult Hemoglobin Switch.

Fetal hemoglobin (HbF, α2γ2) level is genetically controlled and modifies severity of adult hemoglobin (HbA, α2ß2) disorders, sickle cell disease, and ß-thalassemia. Common genetic variation affects expression of BCL11A, a regulator of HbF silencing. To uncover how BCL11A supports the developmental switch from γ- to ß- globin, we use a functional assay and protein binding microarray to establish a requirement for a zinc-finger cluster in BCL11A in repression and identify a preferred DNA recognition sequence. This motif appears in embryonic and fetal-expressed globin promoters and is duplicated in γ-globin promoters. The more distal of the duplicated motifs is mutated in individuals with hereditary persistence of HbF. Using the CUT&RUN approach to map protein binding sites in erythroid cells, we demonstrate BCL11A occupancy preferentially at the distal motif, which can be disrupted by editing the promoter. Our findings reveal that direct γ-globin gene promoter repression by BCL11A underlies hemoglobin switching.

PALI1 promotes tumor growth through competitive recruitment of PRC2 to G9A-target chromatin for dual epigenetic silencing.

PALI1 is a newly identified accessory protein of the Polycomb repressive complex 2 (PRC2) that catalyzes H3K27 methylation. However, the roles of PALI1 in cancer are yet to be defined. Here, we report that PALI1 is upregulated in advanced prostate cancer (PCa) and competes with JARID2 for binding to the PRC2 core subunit SUZ12. PALI1 further interacts with the H3K9 methyltransferase G9A, bridging the formation of a unique G9A-PALI1-PRC2 super-complex that occupies a subset of G9A-target genes to mediate dual H3K9/K27 methylation and gene repression. Many of these genes are developmental regulators required for cell differentiation, and their loss in PCa predicts poor prognosis. Accordingly, PALI1 and G9A drive PCa cell proliferation and invasion in vitro and xenograft tumor growth in vivo. Collectively, our study shows that PALI1 harnesses two central epigenetic mechanisms to suppress cellular differentiation and promote tumorigenesis, which can be targeted by dual EZH2 and G9A inhibition.

A unified atlas of CD8 T cell dysfunctional states in cancer and infection.

CD8 T cells play an essential role in defense against viral and bacterial infections and in tumor immunity. Deciphering T cell loss of functionality is complicated by the conspicuous heterogeneity of CD8 T cell states described across experimental and clinical settings. By carrying out a unified analysis of over 300 assay for transposase-accessible chromatin sequencing (ATAC-seq) and RNA sequencing (RNA-seq) experiments from 12 studies of CD8 T cells in cancer and infection, we defined a shared differentiation trajectory toward dysfunction and its underlying transcriptional drivers and revealed a universal early bifurcation of functional and dysfunctional T cell states across models. Experimental dissection of acute and chronic viral infection using single-cell ATAC (scATAC)-seq and allele-specific single-cell RNA (scRNA)-seq identified state-specific drivers and captured the emergence of similar TCF1+ progenitor-like populations at an early branch point, at which functional and dysfunctional T cells diverge. Our atlas of CD8 T cell states will facilitate mechanistic studies of T cell immunity and translational efforts.

Conserved Gsx2/Ind homeodomain monomer versus homodimer DNA binding defines regulatory outcomes in flies and mice.

How homeodomain proteins gain sufficient specificity to control different cell fates has been a long-standing problem in developmental biology. The conserved Gsx homeodomain proteins regulate specific aspects of neural development in animals from flies to mammals, and yet they belong to a large transcription factor family that bind nearly identical DNA sequences in vitro. Here, we show that the mouse and fly Gsx factors unexpectedly gain DNA binding specificity by forming cooperative homodimers on precisely spaced and oriented DNA sites. High-resolution genomic binding assays revealed that Gsx2 binds both monomer and homodimer sites in the developing mouse ventral telencephalon. Importantly, reporter assays showed that Gsx2 mediates opposing outcomes in a DNA binding site-dependent manner: Monomer Gsx2 binding represses transcription, whereas homodimer binding stimulates gene expression. In Drosophila, the Gsx homolog, Ind, similarly represses or stimulates transcription in a site-dependent manner via an autoregulatory enhancer containing a combination of monomer and homodimer sites. Integrating these findings, we test a model showing how the homodimer to monomer site ratio and the Gsx protein levels defines gene up-regulation versus down-regulation. Altogether, these data serve as a new paradigm for how cooperative homeodomain transcription factor binding can increase target specificity and alter regulatory outcomes.

Opposing Functions of BRD4 Isoforms in Breast Cancer.

Bromodomain-containing protein 4 (BRD4) is a cancer therapeutic target in ongoing clinical trials disrupting primarily BRD4-regulated transcription programs. The role of BRD4 in cancer has been attributed mainly to the abundant long isoform (BRD4-L). Here we show, by isoform-specific knockdown and endogenous protein detection, along with transgene expression, the less abundant BRD4 short isoform (BRD4-S) is oncogenic while BRD4-L is tumor-suppressive in breast cancer cell proliferation and migration, as well as mammary tumor formation and metastasis. Through integrated RNA-seq, genome-wide ChIP-seq, and CUT&RUN association profiling, we identify the Engrailed-1 (EN1) homeobox transcription factor as a key BRD4-S coregulator, particularly in triple-negative breast cancer. BRD4-S and EN1 comodulate the extracellular matrix (ECM)-associated matrisome network, including type II cystatin gene cluster, mucin 5, and cathepsin loci, via enhancer regulation of cancer-associated genes and pathways. Our work highlights the importance of targeted therapies for the oncogenic, but not tumor-suppressive, activity of BRD4.

RSC-Associated Subnucleosomes Define MNase-Sensitive Promoters in Yeast.

The classic view of nucleosome organization at active promoters is that two well-positioned nucleosomes flank a nucleosome-depleted region (NDR). However, this view has been recently disputed by contradictory reports as to whether wider (≳150 bp) NDRs instead contain unstable, micrococcal nuclease-sensitive ("fragile") nucleosomal particles. To determine the composition of fragile particles, we introduce CUT&RUN.ChIP, in which targeted nuclease cleavage and release is followed by chromatin immunoprecipitation. We find that fragile particles represent the occupancy of the RSC (remodeling the structure of chromatin) nucleosome remodeling complex and RSC-bound, partially unwrapped nucleosomal intermediates. We also find that general regulatory factors (GRFs) bind to partially unwrapped nucleosomes at these promoters. We propose that RSC binding and its action cause nucleosomes to unravel, facilitate subsequent binding of GRFs, and constitute a dynamic cycle of nucleosome deposition and clearance at the subset of wide Pol II promoter NDRs.

The CUT&RUN greenlist: genomic regions of consistent noise are effective normalizing factors for quantitative epigenome mapping.

Cleavage Under Targets and Release Using Nuclease (CUT&RUN) is a recent development for epigenome mapping, but its unique methodology can hamper proper quantitative analyses. As traditional normalization approaches have been shown to be inaccurate, we sought to determine endogenous normalization factors based on the human genome regions of constant nonspecific signal. This constancy was determined by applying Shannon's information entropy, and the set of normalizer regions, which we named the 'Greenlist', was extensively validated using publicly available datasets. We demonstrate here that the greenlist normalization outperforms the current top standards, and remains consistent across different experimental setups, cell lines and antibodies; the approach can even be applied to different species or to CUT&Tag. Requiring no additional experimental steps and no added cost, this approach can be universally applied to CUT&RUN experiments to greatly minimize the interference of technical variation over the biological epigenome changes of interest.

A single-cell multiomic analysis of kidney organoid differentiation.

Kidney organoids differentiated from pluripotent stem cells are powerful models of kidney development and disease but are characterized by cell immaturity and off-target cell fates. Comparing the cell-specific gene regulatory landscape during organoid differentiation with human adult kidney can serve to benchmark progress in differentiation at the epigenome and transcriptome level for individual organoid cell types. Using single-cell multiome and histone modification analysis, we report more broadly open chromatin in organoid cell types compared to the human adult kidney. We infer enhancer dynamics by cis-coaccessibility analysis and validate an enhancer driving transcription of HNF1B by CRISPR interference both in cultured proximal tubule cells and also during organoid differentiation. Our approach provides an experimental framework to judge the cell-specific maturation state of human kidney organoids and shows that kidney organoids can be used to validate individual gene regulatory networks that regulate differentiation.

A new CUT&RUN low volume-urea (LoV-U) protocol optimized for transcriptional co-factors uncovers Wnt/ß-catenin tissue-specific genomic targets.

Upon WNT/ß-catenin pathway activation, stabilized ß-catenin travels to the nucleus where it associates with the TCF/LEF transcription factors, constitutively bound to genomic Wnt-responsive elements (WREs), to activate target gene transcription. Discovering the binding profile of ß-catenin is therefore required to unambiguously assign direct targets of WNT signaling. Cleavage under targets and release using nuclease (CUT&RUN) has emerged as prime technique for mapping the binding profile of DNA-interacting proteins. Here, we present a modified version of CUT&RUN, named LoV-U (low volume and urea), that enables the robust and reproducible generation of ß-catenin binding profiles, uncovering direct WNT/ß-catenin target genes in human cells, as well as in cells isolated from developing mouse tissues. CUT&RUN-LoV-U outperforms original CUT&RUN when targeting co-factors that do not bind the DNA, can profile all classes of chromatin regulators and is well suited for simultaneous processing of several samples. We believe that the application of our protocol will allow the detection of the complex system of tissue-specific WNT/ß-catenin target genes, together with other non-DNA-binding transcriptional regulators that act downstream of ontogenetically fundamental signaling cascades.

Multiomic analysis implicates FOXO4 in genetic regulation of chick lens fiber cell differentiation.

A classic model for identification of novel differentiation mechanisms and pathways is the eye lens that consists of a monolayer of quiescent epithelial cells that are the progenitors of a core of mature fully differentiated fiber cells. The differentiation of lens epithelial cells into fiber cells follows a coordinated program involving cell cycle exit, expression of key structural proteins and the hallmark elimination of organelles to achieve transparency. Although multiple mechanisms and pathways have been identified to play key roles in lens differentiation, the entirety of mechanisms governing lens differentiation remain to be discovered. A previous study established that specific chromatin accessibility changes were directly associated with the expression of essential lens fiber cell genes, suggesting that the activity of transcription factors needed for expression of these genes could be regulated through binding access to the identified chromatin regions. Sequence analysis of the identified chromatin accessible regions revealed enhanced representation of the binding sequence for the transcription factor FOXO4 suggesting a direct role for FOXO4 in expression of these genes. FOXO4 is known to regulate a variety of cellular processes including cellular response to metabolic and oxidative stress, cell cycle withdrawal, and homeostasis, suggesting a previously unidentified role for FOXO4 in the regulation of lens cell differentiation. To further evaluate the role of FOXO4 we employed a multiomics approach to analyze the relationship between genome-wide FOXO4 binding, the differentiation-specific expression of key genes, and chromatin accessibility. To better identify active promoters and enhancers we also examined histone modification through analysis of H3K27ac. Specific methods included CUT&RUN (FOXO4 binding and H3K27ac modification), RNA-seq (differentiation state specific gene expression), and ATAC-seq (chromatin accessibility). CUT&RUN identified 20,966 FOXO4 binding sites and 33,921 H3K27ac marked regions across the lens fiber cell genome. RNA-seq identified 956 genes with significantly greater expression levels in fiber cells compared to epithelial cells (log2FC > 0.7, q < 0.05) and 2548 genes with significantly lower expression levels (log2FC < -0.7, q < 0.05). Integrated analysis identified 1727 differentiation-state specific genes that were nearest neighbors to at least one FOXO4 binding site, including genes encoding lens gap junctions (GJA1, GJA3), lens structural proteins (BFSP1, CRYBB1, ASL1), and genes required for lens transparency (HSF4, NRCAM). Multiomics analysis comparing the identified FOXO4 binding sites in published ATAC-seq data revealed that chromatin accessibility was associated with FOXO4-dependent gene expression during lens differentiation. The results provide evidence for an important requirement for FOXO4 in the regulated expression of key genes required for lens differentiation and link epigenetic regulation of chromatin accessibility and H3K27ac histone modification with the function of FOXO4 in controlling lens gene expression during lens fiber cell differentiation.

Intra-scan RF power amplifier drift correction.

PURPOSE: The drift in radiofrequency (RF) power amplifiers (RFPAs) is assessed and several contributing factors are investigated. Two approaches for prospective correction of drift are proposed and their effectiveness is evaluated. METHODS: RFPA drift assessment encompasses both intra-pulse and inter-pulse drift analyses. Scan protocols with varying flip angle (FA), RF length, and pulse repetition time (TR) are used to gauge the influence of these parameters on drift. Directional couplers (DICOs) monitor the forward waveforms of the RFPA outputs. DICOs data is stored for evaluation, allowing calculation of correction factors to adjust RFPAs' transmit voltage. Two correction methods, predictive and run-time, are employed: predictive correction necessitates a calibration scan, while run-time correction calculates factors during the ongoing scan. RESULTS: RFPA drift is indeed influenced by the RF duty-cycle, and in the cases examined with a maximum duty-cycle of 66%, the potential drift is approximately 41% or 15%, depending on the specific RFPA revision. Notably, in low transmit voltage scenarios, FA has minimal impact on RFPA drift. The application of predictive and run-time drift correction techniques effectively reduces the average drift from 10.0% to less than 1%, resulting in enhanced MR signal stability. CONCLUSION: Utilizing DICO recordings and implementing a feedback mechanism enable the prospective correction of RFPA drift. Having a calibration scan, predictive correction can be utilized with fewer complexity; for enhanced performance, a run-time approach can be employed.

Left Ventricular Vertical Run-Length Nonuniformity MRI Adds Prognostic Value to MACE in Patients with End-Stage Renal Disease.

BACKGROUND: Vertical run-length nonuniformity (VRLN) is a texture feature representing heterogeneity within native T1 images and reflects the extent of cardiac fibrosis. In uremic cardiomyopathy, interstitial fibrosis was the major histological alteration. The prognostic value of VRLN in patients with end-stage renal disease (ESRD) remains unclear. PURPOSE: To evaluate the prognostic value of VRLN MRI in patients with ESRD. STUDY TYPE: Prospective. POPULATION: A total of 127 ESRD patients (30 participants in the major adverse cardiac events, MACE group). FIELD STRENGTH/SEQUENCE: 3.0 T/steady-state free precession sequence, modified Look-Locker imaging. ASSESSMENT: MRI image qualities were assessed by three independent radiologists. VRLN values were measured in the myocardium on the mid-ventricular short-axis slice of T1 mapping. Left ventricular (LV) mass, LV end-diastolic and end-systolic volume, as well as LV global strain cardiac parameters were measured. STATISTICAL TESTS: The primary endpoint was the incident of MACE from enrollment time to January 2023. MACE is a composite endpoint consisting of all-cause mortality, acute myocardial infarction, stroke, heart failure hospitalization, and life-threatening arrhythmia. Cox proportional-hazards regression was performed to test whether VRLN independently correlated with MACE. The intraclass correlation coefficients of VRLN were calculated to evaluate intraobserver and interobserver reproducibility. The C-index was computed to examine the prognostic value of VRLN. P-value <0.05 were considered statistically significant. RESULTS: Participants were followed for a median of 26 months. VRLN, age, LV end-systolic volume index, and global longitudinal strain remained significantly associated with MACE in the multivariable model. Adding VRLN to a baseline model containing clinical and conventional cardiac MRI parameters significantly improved the accuracy of the predictive model (C-index of the baseline model: 0.781 vs. the model added VRLN: 0.814). DATA CONCLUSION: VRLN is a novel marker for risk stratification toward MACE in patients with ESRD, superior to native T1 mapping and LV ejection fraction. EVIDENCE LEVEL: 2 TECHNICAL EFFICACY STAGE: 2.

The functions of leg muscles, structures and mechanisms in running.

The actions of the major human leg muscles are well established; however, the functions of these muscle actions during steady running remain unclear. Here, leg structures and mechanisms are considered in terms of their functions in meeting the task of a vehicle acting as an effective machine, supporting body weight during translation with low mechanical work demand and in supplying mechanical work economically. Legs are modelled as a sequence of linkages that predict muscle actions and reveal the varying muscle functions within the integrated leg. Work avoidance is achieved with isometric muscles and linkages that promote a sliding of the hip over the ground contact, resulting in an approximately horizontal path of the centre of mass. Economical work supply requires, for muscle with constrained power, shortening over the entire stance duration; this function is achieved by the hamstrings without disrupting the linkages resulting in work avoidance. In late stance, the two functions occur through coactivation of antagonistic muscles, providing one answer to Lombard's paradox. Quadriceps and hamstring tensions result in opposing moments about both hip and knee joints, but by doing so perform the independent yet complementary roles of work avoidance during translating weight support and economical work supply.

Ice cage: new records and cryptic, isolated lineages in wingless snow flies (Diptera, Limoniidae: Chionea spp.) in German lower mountain ranges.

In Earth's history warm and cold periods have alternated. Especially, during the Pleistocene, the alternation between these different climatic conditions has led to frequent range expansions and retractions of many species: while thermophilic species dispersed during warm periods, cold adapted species retracted to cold refugia and vice versa. After the last Pleistocene cycle many cold adapted taxa found refuges in relict habitats in mountain ranges. One example for such a cold adapted relict is the flightless snow fly Chionea araneoides (Dalman, 1816). It can be found in lower mountain ranges of Central Europe exclusively in stone runs and stony accumulations which provide cold microclimates. Imagines develop only in winter. They have strongly restricted ranges and hence experienced strong isolation predicting that local populations may show local adaptation and hence also genetic differentiation. We investigated this for several middle mountain ranges of Germany using the COI barcoding gene. Our analyses revealed two distinct lineages, one in the Bavarian Forest and a second one in all other more northern locations up to Scandinavia. These lineages likely go back to post-Pleistocene isolation and should be studied in more detail in the future, also to confirm the taxonomic status of both lineages. Further, we confirmed former records of the species for Germany and report new records for the federal states of Saxony, Lower Saxony, Saxony-Anhalt and Thuringia. Finally, we provide the first evidence of two types of males for the species, a small and a larger male type.

Downregulation of MYBL1 in endothelial cells contributes to atherosclerosis by repressing PLEKHM1-inducing autophagy.

MYBL1 is a strong transcriptional activator involved in the cell signaling. However, there is no systematic study on the role of MYBL1 in atherosclerosis. The aim of this study is to elucidate the role and mechanism of MYBL1 in atherosclerosis. GSE28829, GSE43292 and GSE41571 were downloaded from NCBI for differentially expressed analysis. The expression levels of MYBL1 in atherosclerotic plaque tissue and normal vessels were detected by qRT-PCR, Western blot and Immunohistochemistry. Transwell and CCK-8 were used to detect the migration and proliferation of HUVECs after silencing MYBL1. RNA-seq, Western blot, qRT-PCR, Luciferase reporter system, Immunofluorescence, Flow cytometry, ChIP and CO-IP were used to study the role and mechanism of MYBL1 in atherosclerosis. The microarray data of GSE28829, GSE43292, and GSE41571 were analyzed and intersected, and then MYBL1 were verified. MYBL1 was down-regulated in atherosclerotic plaque tissue. After silencing of MYBL1, HUVECs were damaged, and their migration and proliferation abilities were weakened. Overexpression of MYBL1 significantly enhanced the migration and proliferation of HUVECs. MYBL1 knockdown induced abnormal autophagy in HUVEC cells, suggesting that MYBL1 was involved in the regulation of HUVECs through autophagy. Mechanistic studies showed that MYBL1 knockdown inhibited autophagosome and lysosomal fusion in HUVECs by inhibiting PLEKHM1, thereby exacerbating atherosclerosis. Furthermore, MYBL1 was found to repress lipid accumulation in HUVECs after oxLDL treatment. MYBL1 knockdown in HUVECs was involved in atherosclerosis by inhibiting PLEKHM1-induced autophagy, which provided a novel target of therapy for atherosclerosis.

Loss of viral genome with altered immune microenvironment during tumour progression of Epstein-Barr virus-associated gastric carcinoma.

Epstein-Barr virus (EBV) is one of the major drivers of gastric carcinogenesis. EBV infection is established before tumour initiation and is generally maintained throughout tumour development; however, the significance of EBV in tumour maintenance and progression remains to be elucidated. Here, we report eight cases of EBV-associated gastric carcinoma (EBVaGC) with intratumoural heterogenous expression of EBV-encoded small RNA (EBER), a highly expressed latent gene of EBV, and demonstrate clinicopathological characteristics of these rare cases. By performing detailed histological assessment of EBER-positive and -negative components of each case, detection of EBV genome in tumour cells by fluorescence in situ hybridisation, TP73 methylation analysis, whole exome sequencing, and targeted gene panel sequencing, we identified tumours in two patients to be collision tumours of different origins. In the other six patients, some genetic/epigenetic alterations were shared between EBER-positive and -negative components, suggesting that EBV was eliminated from tumour cells during progression. Interestingly, in both tumour types, programmed death ligand 1 and intratumoural infiltration of CD8+ T lymphocytes were lower in EBER-negative than in EBER-positive components, suggesting an immunogenic role of EBV. To the best of our knowledge, this study is the first to demonstrate the detailed histological features and genetic/epigenetic alterations in EBVaGC with heterogenous EBER expression; the loss of EBV may benefit tumour progression and immune evasion and might be clinically important for selecting treatment strategies for such cancers. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Global Run-On sequencing to measure nascent transcription in Saccharomyces cerevisiae.

Global Run-On sequencing is a reliable and widely used approach for monitoring nascent transcription on a genomewide scale. The assay has been successfully used for studying global transcription in humans, plants, worms, flies, and fission yeast. Here we describe a GRO-seq protocol for studying transcription in budding yeast, Saccharomyces cerevisiae. Briefly, the technique involves permeabilization of actively growing yeast cells, allowing transcription to proceed in permeabilized cells in the presence of brominated UTP, affinity purification of bromo-UMP incorporated nascent transcripts followed by cDNA library construction, deep sequencing, and mapping against the reference genome. The approach maps the position of transcriptionally active RNA polymerase on a genomewide basis. In addition to identifying the complete set of transcriptionally active genes in a cell under a given set of conditions, the method can be used to determine elongation rate, termination defect and promoter directionality at the genomewide level. The approach is especially useful in identifying short-lived unstable transcripts that are rapidly degraded even before they leave the nucleus.

Adjusting for false discoveries in constraint-based differential metabolic flux analysis.

One of the critical steps to characterize metabolic alterations in multifactorial diseases, as well as their heterogeneity across different patients, is the identification of reactions that exhibit significantly different usage (or flux) between cohorts. However, since metabolic fluxes cannot be determined directly, researchers typically use constraint-based metabolic network models, customized on post-genomics datasets. The use of random sampling within the feasible region of metabolic networks is becoming more prevalent for comparing these networks. While many algorithms have been proposed and compared for efficiently and uniformly sampling the feasible region of metabolic networks, their impact on the risk of making false discoveries when comparing different samples has not been investigated yet, and no sampling strategy has been so far specifically designed to mitigate the problem. To be able to precisely assess the False Discovery Rate (FDR), in this work we compared different samples obtained from the very same metabolic model. We compared the FDR obtained for different model scales, sample sizes, parameters of the sampling algorithm, and strategies to filter out non-significant variations. To be able to compare the largely used hit-and-run strategy with the much less investigated corner-based strategy, we first assessed the intrinsic capability of current corner-based algorithms and of a newly proposed one to visit all vertices of a constraint-based region. We show that false discoveries can occur at high rates even for large samples of small-scale networks. However, we demonstrate that a statistical test based on the empirical null distribution of Kullback-Leibler divergence can effectively correct for false discoveries. We also show that our proposed corner-based algorithm is more efficient than state-of-the-art alternatives and much less prone to false discoveries than hit-and-run strategies. We report that the differences in the marginal distributions obtained with the two strategies are related to but not fully explained by differences in sample standard deviation, as previously thought. Overall, our study provides insights into the impact of sampling strategies on FDR in metabolic network analysis and offers new guidelines for more robust and reproducible analyses.

Growing up without health insurance: Evidence from rural China.

This paper examines the long-term effects of health insurance on children's educational attainment in a developing country. Utilizing the county-by-county rollout of the New Rural Cooperative Medical Scheme in rural China, we find that exposure to the health insurance program in early life leads to improved educational attainment in adulthood. Empirical tests suggest that a short-term increase in health care utilization is unlikely to be a potential channel. We provide some evidence for the channel that health insurance reduces household financial burdens and increases household expenditure on children's nutrition.