Biology subjects

Bae, H.

Publications and source records attributed to Bae, H..

15 recordsLinked to original sources

bioRxiv2026

Experimental Methods for CRISPR Enzyme Assays with Fluorescence Readout

Fluorescence-based CRISPR diagnostic assays have become a popular platform for nucleic acid detection due to their programmability, configurability, specificity, and compatibility with standard laboratory equipment. However, reported enzymatic kinetic rates and limits of detection for CRISPR trans-cleavage assays vary by several orders of magnitude across the literature. This variation in performance parameters is coupled with and exacerbated by inconsistent calibration, incomplete correction of measurement biases, and nonstandardized or incomplete data-analysis procedures. We present an experimental protocol and quantitative analysis framework for fluorescence-based enzyme assays using routine laboratory instrumentation, including thermocyclers and fluorescence microplate readers. Building on previous studies of CRISPR enzyme kinetics and fluorescence calibration, we describe procedures for flat-field and background correction; comprehensive fluorescence calibration including correction for inner-filter-effect; quantification and implications of reporter degradation; extraction of Michaelis-Menten kinetic parameters; and determination of assay limits of detection. We provide step-by-step experimental guidelines and open-source Python implementations for each stage of the workflow. Using representative Cas12 trans-cleavage datasets, we demonstrate that explicit fluorescence calibration and correction procedures substantially reduce systematic bias in measured kinetic rates and improve consistency between experiments. Our framework aims to establish standardized practices for quantitative fluorescence-based CRISPR assays and provides researchers with practical tools for reproducible kinetic characterization and rational assay design.

biochemistry↗

bioRxiv2026

Metabolic control of smooth muscle cell phenotype switching in atherosclerosis

The loss of smooth muscle cell (SMC) contractile phenotype contributes to various diseases including atherosclerosis. However, its metabolic basis is not entirely elucidated. Since the transforming growth factor beta (TGF{beta}) signaling is among principal regulators of SMC contractility, we studied metabolic regulation of TGF{beta} signaling in SMCs in vitro and atherosclerotic mouse models and human lesions. We found that TGF{beta} induced Ac-CoA synthetase 2 (ACSS2)-dependent Ac-CoA production, by suppressing pyruvate dehydrogenase kinase 4 (PDK4). This stabilized R-SMADs and TGF{beta} receptor 1, preserving SMC contractile phenotype. SMC-specific PDK4 knockout mimicked the effect of TGF{beta} signaling both metabolically and phenotypically, increasing glucose-derived synthesis of Ac-CoA and SMC contractile phenotype. SMC-specific Pdk4 knockout in ApoE knockout mice reduced atherosclerosis. Furthermore, human specimens demonstrated a strong correlation between PDK4 level and atherosclerosis severity. These findings indicate that continuous TGF{beta} signaling, critical to the maintenance of the normal SMC contractile state and is regulated by PDK4 and carbohydrate metabolism. TeaserReducing PDK4 metabolically restricts aortic plaque growth via TGF{beta}-dependent SMC contractility.

cell biology↗

bioRxiv2026

Genetic Modifiers of Cognitive Function: Novel Genetic Associations with Memory, Executive Function, and Attention in the Long Life Family Study

Cognitive performance is central to health and quality of life. Studying the factors that sustain performance may offer insights into maintaining cognitive function into advanced ages and may inform strategies to promote healthy cognitive aging. To identify single nucleotide polymorphisms (SNPs) underlying cognitive function, we performed genome-wide association studies (GWAS) of nine neuropsychological test scores capturing performance in three cognitive domains in 2,455 participants of the Long Life Family Study (LLFS). We identified 12 variants in seven tests and three domains that reached genome-wide significance (p < 5x10-). Three rare (minor allele frequency, European population MAF<0.01) protective, intronic variants, rs190287985 (CCSER1), rs75730801 (FHOD3), and rs552842447 (LINC00508), were uniquely associated with semantic fluency, phonemic fluency and number span forward, respectively. Two rare deleterious variants, rs556333682 and rs188304645, were associated with performance on the Hopkins Verbal Learning Test-Revised (HVLT-R) learning trials and lie in ischemia-related genes SH3TC1 and RPH3A. Another variant, rs180691759, associated with HVLT-R delayed recall, was proximal to RSPO3 and linked to decreased ECHDC1 expression, implicating ischemic and unexplained Ethylmalonic acid (EMA) pathways. We compared the results with GWASs of a general cognitive factor (GCF) and reaction time (RT) in the UK Biobank and meta-analysis results of the UK Biobank, CHARGE and COGENT by Davies et al. We found that rs535509651 associates with HVLT-R delayed recall in the LLFS and nominally associates (p<0.05) with GCF, and that rs10424537 associates with Immediate Logical Memory in the LLFS and nominally associates with RT. Furthermore, we identified 5 genome-wide significant loci in Davies et al. that reached loci adjusted significance in the LLFS (GCF p<0.05/128 and RT p<0.05/39). Each locus was associated with a single cognitive domain in the LLFS. We annotated the genome-wide significant results with quantitative trait loci (QTL) analyses of whole blood transcriptomic, serum metabolomic data, and gene set enrichment analyses (GSEA) using all nominally significant transcripts (p < 0.05/12). QTL analyses discovered 1 SNP-transcript (ECHDC1, p < 3 x 10-6), no SNP-lipid (p < 2 x 10-4), and no SNP-polar metabolite (p < 2 x 10-4) associations. Gene set enrichment analyses identified three pathways at FDR < 0.05. Our findings provide insight into the domain-specific genetic architecture of cognitive function.

genetics↗

bioRxiv2025

Intestinal catabolism of dietary fructose promotes obesity and insulin resistance via ileal lacteal remodeling

High-fructose corn syrup (HFCS) consumption is a risk factor for obesity and metabolic syndrome, yet the underlying mechanisms are incompletely understood. Catabolism of dietary fructose primarily occurs in the small intestine and liver, with fructose breakdown in the liver being pathological, while small intestinal fructose clearance protects the liver. Here, we unexpectedly found that inhibition of fructose catabolism specifically in the small intestine mitigates fructose-induced obesity and insulin resistance. Mechanistically, blocking intestinal fructose catabolism reduces dietary fat absorption, which is associated with a decrease in the surface area of the ileal lacteals and alterations in gut microbiome. Fecal transplantation experiments revealed that such a microbiome stimulates the intestine-resident macrophages, promoting lacteal growth and boosting dietary fat absorption. Given the preclinical and clinical studies reporting the effect of fructose catabolism suppression on mitigating diet-induced obesity, our data suggest that such effects are partly mediated by intestinal lacteal remodeling. Significance StatementHere, we uncover a previously unappreciated link between intestinal fructose catabolism and ileal lacteal remodeling, suggesting the mechanisms by which fructose intake promotes obesity. Using mice lacking the fructose-processing enzyme specifically in the intestine, we show that blocking intestinal fructose metabolism protects against diet-induced obesity by reducing fat absorption. Changes in gut microbiome and immune cell interactions drive this effect.

physiology↗

bioRxiv2025

Absolute Membrane Potential Recording with ASAP-Type Genetically Encoded Voltage Indicators Using Fluorescence Lifetime Imaging

The electrical membrane voltage (Vm) characterizes the functional state of biological cells, thus requiring precise, non-invasive Vm-sensing techniques. While voltage-dependent fluorescence intensity changes from genetically encoded voltage indicators (GEVIs) indicate Vm changes, variability in sensor expression confound determination of absolute Vm. Fluorescence lifetime imaging microscopy (FLIM) promises a solution to this problem, as fluorescence lifetime is expected to be unaffected by sensor expression and excitation intensity. By examining ASAP1, ASAP3, JEDI-1P, rEstus, and rEstus-NI (G138N:T141I) with one-photon excited FLIM measurements, we demonstrate that all sensors display a voltage-dependent lifetime. With the highest lifetime change in the Vm range of -100 to 50 mV of about 730 ps, ASAP3 and rEstus-NI are preferred for FLIM recordings. At a physiologically relevant Vm of -30 mV, the voltage sensitivity of rEstus-NI (6.6 ps/mV) is 3.6 and 1.4 times greater than that of ASAP1 and rEstus, respectively. As a proof of concept, we successfully used rEstus-NI to estimate absolute resting Vm in HEK293T, A375 melanoma, and MCF7 breast cancer cells and quantified spontaneous Vm fluctuations in A375 cells. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=89 SRC="FIGDIR/small/669310v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@1eaff8borg.highwire.dtl.DTLVardef@12f1a54org.highwire.dtl.DTLVardef@169ad83org.highwire.dtl.DTLVardef@fe9f44_HPS_FORMAT_FIGEXP M_FIG C_FIG

physiology↗

bioRxiv2025

Cardiac metabolite exchange measured in pigs during myocardial ischemia and reperfusion

Myocardial ischemia and reperfusion (I/R) injury are the primary contributors to death in patients with cardiovascular disease. While decades of research have elucidated the molecular players and biochemical mechanisms underlying I/R injury, how these pathologies influence the metabolic activities of the heart remains incompletely understood. Such a knowledge gap hampers the development of therapies aimed at mitigating the metabolic stresses of the heart during injury. Using comprehensive arteriovenous metabolomics in a highly relevant porcine I/R model, we report the metabolic landscape of cardiac metabolic changes after ischemia and during the reperfusion time course. Paradoxically, ischemia increases the cardiac uptake of circulating fatty acids while reperfusion for 60 minutes reverses this activity. By 120 minutes of reperfusion, the hearts resume the uptake of fatty acids, suggesting restoration of their metabolism. On the other hand, we found a strong release of amino acids by the heart only after 60-minute reperfusion, but not after 120-minute reperfusion, implicating I/R-induced transient protein degradation. In addition to these findings, we identified several previously unrecognized changes in cardiac metabolic inputs and outputs during I/R, including nucleotides, TCA cycle intermediates and creatine/creatinine. These data highlight the dynamic alterations in cardiac metabolism in response to I/R, providing insights into how to mitigate myocardial I/R injury.

biochemistry↗

bioRxiv2025

Increased Genetic Protection Against Alzheimer's Disease in Centenarians

We constructed a polygenic protective score specific to Alzheimers disease (AD PPS) based on the current literature among the participants enrolled in five studies of healthy aging and extreme longevity in the US, Europe, and Asia. This AD PPS did not include variants on Apolipoprotein E (APOE) gene. Comparisons of AD PPS in different data sets of healthy agers and centenarians showed that centenarians have stronger genetic protection against AD compared to individuals without familial longevity. The current study also shows evidence that this genetic protection increases with increasingly older ages in centenarians (centenarians who died before reaching age 105 years, semi-supercentenarians who reached age 105 to 109 years, and supercentenarians who reached age 110 years and older). However, the genetic protection was of modest size: the average increase in AD PPS was approximately one additional protective allele per 5 years of gained lifetime. Additionally, we show that the higher AD PPS was associated with better cognitive function and decreased mortality. Taken together, this analysis suggests that individuals who achieve the most extreme ages, on average, have the greatest protection against AD. This finding is robust to different genetic backgrounds with important implications for universal applicability of therapeutics that target this AD PPS.

genetics↗

bioRxiv2025

Robust Serum Proteomic Signatures of APOE2

We previously identified a signature of 16 serum proteins that highlighted a role of the e2 allele of APOE in lipid regulation via apolipoprotein B (APOB) and apolipoprotein E (APOE), and in inflammation. The serum proteins were profiled using the aptamer-based Somalogic technology. Here, we validate and expand the serum protein signature of APOE using a combination of mass-spectrometry, ELISA, Luminex, antibody-based Olink proteomics, and blood transcriptomics. We replicate the association between APOB and the e2 allele of APOE, we correct the pattern of association between APOE genotypes and serum level of APOE, and we detect new associations between APOE genotypes and the complex of apolipoproteins APOC1, APOC4, APOC2, APOC3, APOE, APOF and APOL1. In addition, we discover 13 new proteins that correlate with APOE genotypes. This extended signature includes granule proteins CAMP, CTSG, DEFA3, and MPO secreted from neutrophils and points to olfactomedin 4 (OLFM4) as a new target for the prevention of Alzheimers disease.

genetics↗

bioRxiv2024

Sex-dependent metabolic remodeling of kidneys revealed by arteriovenous metabolomics

Sex is a fundamental biological variable important in biomedical research, drug development, clinical trials, and prevention approaches. Among many organs, kidneys are known to exhibit remarkable structural, histological, and pathological differences between sexes. However, whether and how kidneys display distinct metabolic activities between sexes is poorly understood. By developing kidney-specific arteriovenous (AV) metabolomics combined with transcriptomics, we report striking sex differences in both basal metabolic activities and adaptive metabolic remodeling of kidneys after a fat-enriched ketogenic diet (KD), a regimen known to mitigate kidney diseases and improve immunotherapy for renal cancer. At the basal state, female kidneys show highly accumulated aldosterone and various acylcarnitines. In response to the KD, aldosterone levels remain high selectively in females but the sex difference in acylcarnitines disappears. AV data revealed that, under KD, female kidneys avidly take up circulating fatty acids and release 3-hydroxybutyrate (3-HB) whereas male kidneys barely absorb fatty acids but consistently take up 3-HB. Although both male and female kidneys take up gluconeogenic substrates such as glycerol, glutamine and lactate, only female kidneys exhibit net glucose release. Kidney transcriptomics data incompletely predict these sex differences, suggesting post-transcriptional/translational regulation mechanisms. This study provides foundational insights into the sex-dependent and diet-elicited metabolic flexibility of the kidneys in vivo, serving as a unique resource for understanding variable disease prevalence and drug responses between male and female kidneys.

biochemistry↗

bioRxiv2024

Cross-platform proteomics signatures of extreme old age

In previous work we used a Somalogic platform targeting approximately 5000 proteins to generate a serum protein signature of centenarians that we validated in independent studies that used the same technology. We set here to validate and possibly expand the results by profiling the serum proteome of a subset of individuals included in the original study using liquid chromatography tandem mass spectrometry (LC-MS/MS). Following pre-processing, the LC-MS/MS data provided quantification of 398 proteins, with only 266 proteins shared by both platforms. At 1% FDR statistical significance threshold, the analysis of LC-MS/MS data detected 44 proteins associated with extreme old age, including 23 of the original analysis. To identify proteins for which associations between expression and extreme-old age were conserved across platforms, we performed inter-study conservation testing of the 266 proteins quantified by both platforms using a method that accounts for the correlation between the results. From these tests, a total of 80 proteins reached 5% FDR statistical significance, and 26 of these proteins had concordant pattern of gene expression in whole blood. This signature of 80 proteins points to blood coagulation, IGF signaling, extracellular matrix (ECM) organization, and complement cascade as important pathways whose protein level changes provide evidence for age-related adjustments that distinguish centenarians from younger individuals.

bioinformatics↗

bioRxiv2023

Leveraging genetic correlation structure to target discrete signaling mechanisms across metabolic tissues

Abstract/IntroductionInter-organ communication is a vital process to maintain physiologic homeostasis, and its dysregulation contributes to many human diseases. Beginning with the discovery of insulin over a century ago, characterization of molecules responsible for signal between tissues has required careful and elegant experimentation where these observations have been integral to deciphering physiology and disease. Given that circulating bioactive factors are stable in serum, occur naturally, and are easily assayed from blood, they present obvious focal molecules for therapeutic intervention and biomarker development. For example, physiologic dissection of the actions of soluble proteins such as proprotein convertase subtilisin/kexin type 9 (PCSK9) and glucagon-like peptide 1 (GLP1) have yielded among the most promising therapeutics to treat cardiovascular disease and obesity, respectively1-4. A major obstacle in the characterization of such soluble factors is that defining their tissues and pathways of action requires extensive experimental testing in cells and animal models. Recently, studies have shown that secreted proteins mediating inter-tissue signaling could be identified by "brute-force" surveys of all genes within RNA-sequencing measures across tissues within a population5-9. Expanding on this intuition, we reasoned that parallel strategies could be used to understand how individual genes mediate signaling across metabolic tissues through correlative analyses of gene variation between individuals. Thus, comparison of quantitative levels of gene expression relationships between organs in a population could aid in understanding cross-organ signaling. Here, we surveyed gene-gene correlation structure across 18 metabolic tissues in 310 human individuals and 7 tissues in 103 diverse strains of mice fed a normal chow or HFHS diet. Variation of genes such as FGF21, ADIPOQ, GCG and IL6 showed enrichments which recapitulate experimental observations. Further, similar analyses were applied to explore both within-tissue signaling mechanisms (liver PCSK9) as well as genes encoding enzymes producing metabolites (adipose PNPLA2), where inter-individual correlation structure aligned with known roles for these critical metabolic pathways. Examination of sex hormone receptor correlations in mice highlighted the difference of tissue-specific variation in relationships with metabolic traits. We refer to this resource as Gene-Derived Correlations Across Tissues (GD-CAT) where all tools and data are built into a web portal enabling users to perform these analyses without a single line of code (gdcat.org). This resource enables querying of any gene in any tissue to find correlated patterns of genes, cell types, pathways and network architectures across metabolic organs.

physiology↗

bioRxiv2023

Deficiency in neprilysin causes laminar impairments in the visual cortex

1Neprilysin (Nep) is a membrane-bound zinc-dependent endopeptidase that cleaves a wide variety of small peptides in the body. In the brain, it has gained fame as an important endogenous degrader of amyloid beta peptide, responsible for the pathophysiology of Alzheimers disease. Nep is expressed specifically by parvalbumin (PV)-expressing inhibitory neurons in the adult cortex, the maturation of which regulates critical period plasticity. Given that PV neuron soma and proximal dendrites are primary sites of perineuronal net (PNN) formation, we investigated the role that Nep plays in PNN and PV neuron maturation and cortical development in the mouse visual cortex, using mice whose Nep expression is constitutively knocked out (Nep KO mice). Nep expression is high in young wildtype mice (P10) and is downregulated rapidly throughout postnatal development. It is especially prominent in layer 5 where it is highly expressed by inhibitory neurons and also expressed at low levels by many excitatory neurons. Contrary to our hypothesis, Nep KO mice did not show an alteration in the relative density or gross morphology of PNNs. Instead, Nep KO mice showed reduced maximal activation of L5 after white matter stimulation and decreased number of inhibitory neurons in L4. These laminar defects may lead to impaired development of optomotor acuity in Nep KO mice.

neuroscience↗

bioRxiv2023

Heat-induced seizures, premature mortality, and hyperactivity in a novel Scn1a nonsense model for Dravet syndrome

Dravet syndrome (Dravet) is a severe congenital developmental genetic epilepsy caused by de novo mutations in the SCN1A gene. Nonsense mutations are found in ~20% of the patients, and the R613X mutation was identified in multiple patients. Here we characterized the epileptic and non-epileptic comorbidities of a novel preclinical Dravet mouse model harboring this nonsense Scn1a mutation. Heterozygous Scn1a R613X mutation on a mixed C57BL/6J:129S1/SvImJ background exhibited spontaneous seizures, susceptibility to heat-induced seizures, and premature mortality, recapitulating the core epileptic phenotypes of Dravet. In addition, these mice, available as an open-access model, demonstrated increased locomotor activity in the open-field test, mimicking some non-epileptic Dravet-associated comorbidities. Conversely, Scn1aWT/R613X mice on the pure 129S1/SvImJ background had a normal life span and were easy to breed. Homozygous Scn1aR613X/R613X mice died before P16. Our molecular analyses of hippocampal and cortical expression demonstrated that the premature stop codon induced by the R613X mutation reduced Scn1a mRNA and Nav1.1 protein levels to ~50% in heterozygous Scn1aWT/R613X mice, with marginal expression in homozygous Scn1aR613X/R613X mice. Together, we introduce a novel Dravet model carrying the R613X Scn1a nonsense mutation that can. be used to study the molecular and neuronal basis of Dravet, as well as the development of new therapies associated with SCN1A nonsense mutations in Dravet.

neuroscience↗

bioRxiv2022

PH Domain-Mediated Autoinhibition and Oncogenic Activation of Akt

Previously, we reported that the conformation of autoinhibited Akt, a Ser/Thr protein kinase that plays a central role in metabolism and cancer, may be shifted by small molecule allosteric inhibitors limiting the mechanistic insights from existing X-ray structures that have relied on such compounds (Chu, Viennet, et al, 2020). Here we discover unexpectedly that a single mutation, R86A, in Akts PH domain exhibits intensified autoinhibitory features with enhanced PH domain-kinase domain affinity. Structural and biochemical analysis uncovers the importance of a key interaction network involving Arg86, Glu17, and Tyr18 that controls Akt conformation and activity. Our studies also shed light on the molecular basis for E17K Akt activation as an oncogenic driver.

biochemistry↗

bioRxiv2022

Hyperactivation of YAP/TAZ drives alterations in mesangial cells through stabilization of N-MYC in diabetic nephropathy

Mesangial cells (MCs) in the kidney are central to maintaining glomerular integrity, and their impairment leads to major glomerular diseases including diabetic nephropathy (DN). Although high blood glucose elicits abnormal alterations in MCs, the underlying molecular mechanism is poorly understood. Here, we show that YAP and TAZ, the final effectors of the Hippo pathway, are highly increased in MCs of patients with DN and of Zucker diabetic fatty rats. Moreover, high glucose directly induces activation of YAP/TAZ through the canonical Hippo pathway in cultured MCs. Hyperactivation of YAP/TAZ in mouse model MCs recapitulates the hallmarks of DN, including excessive proliferation of MCs and extracellular matrix deposition, endothelial cell impairment, glomerular sclerosis, albuminuria, and reduced glomerular filtration rate. Mechanistically, activated YAP/TAZ bind and stabilize N-Myc protein, one of the Myc family of oncogenes. N-Myc stabilization leads to aberrant enhancement of its transcriptional activity and eventually to MC impairments and DN pathogenesis. Together, these findings shed light on how high blood glucose in diabetes mellitus leads to DN and support a rationale that lowering blood glucose in diabetes mellitus could delay DN pathogenesis.

pathology↗