Biology subjects

Lee, Y.

Publications and source records attributed to Lee, Y..

17 recordsLinked to original sources

bioRxiv2018

Analysis of contributory gut microbiota and lauric acid against necrotic enteritis in Clostridium perfringens and Eimeria side-by-side challenge model

Gut microbiota has been demonstrated to be involved in intestinal nutrition, defense, and immunity, as well as participating in disease progression. This study was to investigate gut microbiota changes in chickens challenged with netB-positive Clostridium perfringens strain 1 (CP1) and/or the predisposing Eimeria species (Eimeria). In addition, the effects of lauric acid, a medium-chain fatty acid (MCFA), on NE reduction and modulation of microbiota were evaluated. The results demonstrated that microbial communities in the jejunum were distinct from those in the cecum, and the microbial community change was more significant in jejunum. Challenge of CP1 in conjunction with Eimeria significantly reduced species diversity in jejunal microbiota, but cecal microbiota remained stable. In the jejunum, CP1 challenge increased the abundance of the genera of Clostridium sensu stricto 1, Escherichia Shigella, and Weissella, but significantly decreased the population of Lactobacillus. Eimeria infection on its own was unable to promote NE, demonstrating decrements of Clostridium sensu stricto 1 and Lactobacillus. Co-infection with CP1 and Eimeria reproduced the majority of NE lesions with significant increment of Clostridium sensu stricto 1 and reduction in Lactobacillus. The changes of these two taxa increased the severity of NE lesions. Further analyses of metagenomeSeq, STAMP, and LEfSe showed significant overgrowth of Clostridium sensu stricto 1 was associated with NE and Eimeria infection than C. perfringens challenge alone. The supplementation of lauric acid did not reduce NE incidence and severity but decreased the relative abundance of Escherichia Shigella. In conclusion, significant overgrowth of Clostridium sensu stricto 1 in the jejunm is the major microbiota contributory to NE. Controlling proliferation of this taxon in the jejunum should be the niche for developing effective strategies against NE.

microbiology↗

bioRxiv2018

RUNX3 regulates cell cycle-dependent chromatin dynamics by functioning as a pioneer factor of the restriction point

The cellular decision regarding whether to undergo proliferation or death is made at the restriction (R)-point, which is disrupted in nearly all tumors. The identity of the molecular mechanisms that govern the R-point decision is one of the fundamental issues in cell biology. We found that early after mitogenic stimulation, RUNX3 bound to its target loci, where it opened chromatin structure by sequential recruitment of Trithorax group proteins and cell-cycle regulators to drive cells to the R-point. Soon after, RUNX3 closed these loci by recruiting Polycomb repressor complexes, causing the cell to pass through the R-point toward S phase. If the RAS signal was constitutively activated, RUNX3 inhibited cell cycle progression by maintaining R-point-associated genes in an open structure. Our results identify RUNX3 as a pioneer factor for the R-point and reveal the molecular mechanisms by which appropriate chromatin modifiers are selectively recruited to target loci for appropriate R-point decisions.

cell biology↗

bioRxiv2018

Yeast Chd1p remodels nucleosomes with unique DNA unwrapping and translocation dynamics

Chromodomain-helicase-DNA-binding protein 1 (CHD1) remodels chromatin by translocating nucleosomes along DNA, but its mechanism remains poorly understood. Here, we employ a single-molecule fluorescence approach to characterize nucleosome remodeling by yeast CHD1 (Chd1p). We show that Chd1p translocates nucleosomes in steps of multiple base pairs per ATP. ATP binding to Chd1p induces a transient unwrapping of the exit-side DNA, and facilitates nucleosome translocation. ATP hydrolysis induces nucleosome translocation, which is followed by the rewrapping upon the release of the hydrolyzed nucleotide. Multiple Chd1ps binding to a single nucleosome sequentially moves a histone octamer with a preference to the center of DNA fragments, suggesting a new mechanism for regularly spaced nucleosome generation by Chd1p. Our results reveal the unique mechanism by which Chd1p remodels nucleosomes. Significance StatementThere are four major ATP-dependent chromatin remodeler families: SWI/SNF, ISWI, CHD, and INO80/SWR1. The remodeling mechanisms of SWI/SNF and ISWI chromatin remodelers have been elucidated through extensive single-molecule studies, but it remains poorly understood how CHD chromatin remodeler operate. We use single-molecule FRET techniques, and show that Yeast CHD1 uses unique mechanisms to remodel a nucleosome.

biophysics↗

bioRxiv2018

Selection of an Appropriate Empiric Antibiotic Regimen in Culture-Negative Hematogenous Vertebral Osteomyelitis

The aim of this study was to determine which antibiotic combinations are appropriate for culture-negative hematogenous vertebral osteomyelitis (HVO), based on the antibiotic-susceptibility pattern of organisms isolated from cases of culture-proven HVO. We conducted a retrospective chart review of adult patients with microbiologically proven HVO in five tertiary-care hospitals over a 7-year period. The appropriateness of empiric antibiotic regimens was assessed based on the antibiotic susceptibility profiles of isolated bacteria. In total, 358 cases of microbiologically proven HVO were identified. The main causative pathogens identified were methicillin-susceptible Staphylococcus aureus (33.5%), followed by methicillin-resistant S. aureus (MRSA) (24.9%), aerobic gram-negative bacteria (21.8%), and Streptococcus species (11.7%). Extended spectrum {beta}-lactamase (ESBL)-producing Enterobacteriaceae and anaerobes accounted for only 1.7% and 1.4%, respectively, of the causative pathogens. Based on the susceptibility results of isolated organisms, levofloxacin plus rifampicin was appropriate in 73.5%, levofloxacin plus clindamycin in 71.2%, and amoxicillin-clavulanate plus ciprofloxacin in 64.5% of cases. These oral combinations were more appropriate for treating community-acquired HVO (85.8%, 84.0%, and 80.4%, respectively) than healthcare-associated HVO (52.6%, 49.6%, and 37.6%, respectively). Vancomycin combined with ciprofloxacin, ceftriaxone, ceftazidime, or cefepime was similarly appropriate (susceptibility rates of 93.0%, 94.1%, 95.8%, and 95.8%, respectively). In conclusion, in a setting with a high prevalence of MRSA HVO, oral antibiotic combinations may be suboptimal for treatment of culture-negative HVO and should be used only in patients with community-acquired HVO. Vancomycin combined with fluoroquinolone or a broad-spectrum cephalosporin was appropriate in most cases of HVO in this study.

epidemiology↗

bioRxiv2018

C. elegans Mediator 15 permits low temperature-induced longevity via regulation of lipid and protein homeostasis

Low temperatures slow aging and extend lifespan in many organisms, including Caenorhabditis elegans. However, the metabolic and homeostatic aspects of low temperature-induced longevity remain poorly understood. Here, we show that changes in lipid composition regulated by MDT-15/Mediator 15, transcriptional co-regulator, is essential for low temperature-induced longevity and proteostasis in C. elegans. We find that inhibition of mdt-15 prevents animals from living long at low temperatures. We show that MDT-15 up-regulates fat-7, a fatty acid desaturase, at low temperatures, which increases the ratio of unsaturated to saturated fatty acids. We further demonstrate that maintaining this increased fatty acid ratio is essential for protein homeostasis and longevity at low temperatures. Thus, the homeostasis of lipid composition by MDT-15 appears to be a limiting factor for C. elegans proteostasis and longevity at low temperatures. Our findings highlight the crucial roles of fat regulation in maintaining normal organismal physiology under different environmental conditions.

developmental biology↗

bioRxiv2018

Exacerbation of blood brain barrier dysfunction and brain injury after aneurysmal subarachnoid hemorrhage in endothelial specific S1PR1 knock out mice.

Blood brain barrier (BBB) dysfunction upon ischemia and hypoxia has been implicated in the exacerbation of neuronal injury in stroke. Despite the therapeutic potential of the cerebrovascular endothelium, the limited understanding of the endothelial signaling pathways governing BBB function restricts progress towards developing novel therapeutic approaches specifically targeting the endothelium. Sphingosine-1-phosphate (S1P) is a potent modulator of endothelial function via its receptors (S1PR). Recent human and mouse studies indicate that vasoprotective endothelial S1P signaling via S1PR1 may be impaired in cardiovascular and inflammatory diseases. Herein, we investigated the expression of S1PR1 in the mouse and human cerebrovascular endothelium and the role of endothelial-specific S1PR1 signaling in brain injury in a mouse model of aneurysmal subarachnoid hemorrhage (SAH), the most devastating type of stroke. We found that S1PR1 is the most abundant S1PR transcript in the mouse brain and in mouse and human brain endothelial cells (20-100 mRNA copies per cell). S1PR1 transcripts were significantly enriched (~6 fold) in mouse cortical microvessels compared to total brain. Using the S1PR1-eGFP knock in mouse, we found that S1PR1-eGFP is abundantly expressed in the cerebrovascular endothelium in the mouse brain. A similar pattern of expression was observed in human brain samples. Endothelial specific deletion of S1PR1 in adult mice (S1pr1 flox/floxxCdh5-CreERT2, referred to as S1pr1iECKO), resulted in exacerbation of brain edema, neuronal injury and worsened neurological outcomes upon SAH compared to S1pr1 flox/flox littermates. No differences in the subarachnoid blood, hemostasis or cerebral blood flow changes during and after SAH were found between groups. Mechanistically, S1pr1iECKO exhibited aggravated BBB dysfunction and increased phosphorylation of myosin light chain (MLC) in isolated cortical microvessels, a downstream effector of the Rho-ROCK pathway implicated in endothelial inflammation and barrier dysfunction. Taken together, our data indicate that S1PR1 is an endogenous protective signaling pathway in the endothelium, critical to maintain BBB function and to mitigate neuronal injury in pathological conditions. Thus, the therapeutic and diagnostic potential of the endothelial sphingosine-1-phosphate pathway in stroke deserves further study.

neuroscience↗

bioRxiv2018

Bayesian Multi-SNP Genetic Association Analysis: Control of FDR and Use of Summary Statistics

Multi-SNP genetic association analysis has become increasingly important in analyzing data from genome-wide association studies (GWASs) and molecular quantitative trait loci (QTL) mapping studies. In this paper, we propose novel computational approaches to address two outstanding issues in Bayesian multi-SNP genetic association analysis: namely, the control of false positive discoveries of identified association signals and the maximization of the efficiency of statistical inference by utilizing summary statistics. Quantifying the strength and uncertainty of genetic association signals has been a long-standing theme in statistical genetics. However, there is a lack of formal statistical procedures that can rigorously control type I errors in multi-SNP analysis. We propose an intuitive hierarchical representation of genetic association signals based on Bayesian posterior probabilities, which subsequently enables rigorous control of false discovery rate (FDR) and construction of Bayesian credible sets. From the perspective of statistical data reduction, we examine the computational approaches of multi-SNP analysis using z-statistics from single-SNP association testing and conclude that they likely yield conservative results comparing to using individual-level data. Built on this result, we propose a set of sufficient summary statistics that can lead to identical results as individual-level data without sacrificing power. Our novel computational approaches are implemented in the software package, DAP-G (https://github.com/xqwen/dap), which applies to both GWASs and genome-wide molecular QTL mapping studies. It is highly computationally efficient and approximately 20 times faster than the state-of-the-art implementation of Bayesian multi-SNP analysis software. We demonstrate the proposed computational approaches using carefully constructed simulation studies and illustrate a complete workflow for multi-SNP analysis of cis expression quantitative trait loci using the whole blood data from the GTEx project.

genetics↗

bioRxiv2018

Constructing and visualizing cancer genomic maps in 3D spatial context by phenotype-based high-throughput laser-aided isolation and sequencing (PHLI-seq)

A spatially resolved analysis of the heterogeneous cancer genome, in which the data are connected to the three-dimensional space of a tumour, is crucial to understand cancer biology and the clinical impact of cancer heterogeneity on patients. However, despite recent progress in spatially resolved transcriptomics, spatial mapping of genomic data in a high-throughput and high-resolution manner has been challenging due to current technical limitations. Here, we describe a novel approach, phenotype-based high-throughput laser-aided isolation and sequencing (PHLI-seq), which enables high-throughput isolation of a single-cell or a small number of cells and their genome-wide sequence analysis to construct genomic maps within cancer tissue in relation to the phenotypes of the cells. By applying PHLI-seq, we reveal the heterogeneity of breast cancer tissues at a high resolution and map the genomic landscape of the cells to their corresponding spatial locations and phenotypes in the tumour mass. Additionally, with different staining modalities, the genotypes of the cells can be connected to corresponding phenotypic information of the tissue. Together with the spatially resolved genomic analysis, we can infer the histories of heterogeneous cancer cells in two or three dimensions, providing significant insight into cancer biology and precision medicine.

genomics↗

bioRxiv2018

Cryo-EM structure of the adenosine A2A receptor coupled to an engineered heterotrimeric G protein

The adenosine A2A receptor (A2AR) is a prototypical G protein-coupled receptor (GPCR) that couples to the heterotrimeric G protein GS. Here we determine the structure by electron cryo-microscopy (cryo-EM) of A2AR at pH 7.5 bound to the small molecule agonist NECA and coupled to an engineered heterotrimeric G protein, which contains mini-GS, the {beta}{gamma} subunits and nanobody Nb35. Most regions of the complex have a resolution of ~3.8 [A] or better. Comparison with the 3.4 [A] resolution crystal structure shows that the receptor and mini-GS are virtually identical and that the density of the side chains and ligand are of comparable quality. However, the cryo-EM density map also indicates regions that are flexible in comparison to the crystal structures, which unexpectedly includes regions in the ligand binding pocket. In addition, an interaction between intracellular loop 1 of the receptor and the {beta} subunit of the G protein was observed.

biophysics↗

bioRxiv2017

Free energy landscape for the entire transport cycle of triose-phosphate/phosphate translocator

Secondary active transporters translocate their substrates using the electrochemical potentials of other chemicals, undergoing large-scale conformational changes. Despite extensive structural studies, the atomic details of the transport mechanism still remain elusive. Here we performed a series of all-atom molecular dynamics simulations of the triose-phosphate/phosphate translocator (TPT), which exports organic phosphates in the chloroplast stroma in strict counter exchange with inorganic phosphate (Pi). Biased sampling methods, including string method and umbrella sampling, successfully reproduced the conformational changes between the inward- and outward-facing states, along with the substrate binding. The free energy landscape of this entire TPT transition pathway demonstrated the alternating access and substrate translocation mechanisms, which revealed Pi is relayed by positively charged residues along the transition pathway. Furthermore, the conserved Glu207 functions as a \"molecular switch\", linking the local substrate binding and the global conformational transition. Our results provide atomic-detailed insights into the energy coupling mechanism of antiporter.

biophysics↗

bioRxiv2017

Structure of the triose-phosphate/phosphate translocator reveals the basis of substrate specificity

The triose-phosphate/phosphate translocator (TPT) catalyzes the strict 1:1 exchange of triose phosphate, 3-phosphoglycerate and inorganic phosphate across the chloroplast envelope, and plays crucial roles in photosynthesis. Despite rigorous studies for more than 40 years, the molecular mechanism of TPT is poorly understood due to the lack of structural information. Here we report crystal structures of TPT bound to two different substrates, 3-phosphoglycerate and inorganic phosphate, in occluded conformations. The structures reveal that TPT adopts a 10-transmembrane drug/metabolite transporter fold. Both substrates are bound within the same central pocket, where conserved lysine, arginine, and tyrosine residues recognize the shared phosphate group. A structural comparison with the outward-open conformation of the bacterial drug/metabolite transporter suggests a rocking-type motion of helix bundles, and molecular dynamics simulations support a model in which this helix rocking is tightly coupled to the substrate binding, to ensure strict 1:1 exchange. These results reveal the unique mechanism of sugar phosphate/phosphate exchange by TPT.

biochemistry↗

bioRxiv2017

Spatial and temporal distribution of genome divergence among California populations of Aedes aegypti.

In the summer of 2013, Aedes aegypti Linnaeus was first detected in three cities in central California (Clovis, Madera and Menlo Park). It has now been detected in multiple locations in central and southern CA as far south as San Diego and Imperial Counties. A number of published reports suggest that CA populations have been established from multiple independent introductions. Here we report the first population genomics analyses of Ae. aegypti based on individual, field collected whole genome sequences. We analyzed 46 Ae. aegypti genomes to establish genetic relationships among populations from sites in California, Florida and South Africa. We identified 3 major genetic clusters within California; one that includes all sample sites in the southern part of the state (South of Tehachapi mountain range) plus the town of Exeter in central California and two additional clusters in central California. A lack of concordance between mitochondrial and nuclear genealogies suggests that the three founding populations were polymorphic for two main mitochondrial haplotypes prior to being introduced to California. One of these has been lost in the Clovis populations, possibly by a founder effect. Genome-wide comparisons indicate extensive differentiation between genetic clusters. Our observations support recent introductions of Ae. aegypti into California from multiple, genetically diverged source populations. Our data reveal signs of hybridization among diverged populations within CA. Genetic markers identified in this study will be of great value in pursuing classical population genetic studies which require larger sample sizes.

genomics↗

bioRxiv2017

Computational correction of copy-number effect improves specificity of CRISPR-Cas9 essentiality screens in cancer cells

The CRISPR-Cas9 system has revolutionized gene editing both on single genes and in multiplexed loss-of-function screens, enabling precise genome-scale identification of genes essential to proliferation and survival of cancer cells. However, previous studies reported that an anti-proliferative effect of Cas9-mediated DNA cleavage confounds such measurement of genetic dependency, particularly in the setting of copy number gain1-4. We performed genome-scale CRISPR-Cas9 essentiality screens on 342 cancer cell lines and found that this effect is common to all lines, leading to false positive results when targeting genes in copy number amplified regions. We developed CERES, a computational method to estimate gene dependency levels from CRISPR-Cas9 essentiality screens while accounting for the copy-number-specific effect, as well as variable sgRNA activity. We applied CERES to sets of screens performed with different sgRNA libraries and found that it reduces false positive results and provides meaningful estimates of sgRNA activity. As a result, the application of CERES improves confidence in the interpretation of genetic dependency data from CRISPR-Cas9 essentiality screens of cancer cell lines.

genomics↗

bioRxiv2017

Multiple reference genome sequences of hot pepper reveal the massive evolution of plant disease resistance genes by retroduplication

Transposable elements (TEs) provide major evolutionary forces leading to new genome structure and species diversification. However, the role of TEs in the expansion of disease resistance gene families has been unexplored in plants. Here, we report high-quality de novo genomes for two peppers (Capsicum baccatum and C. chinense) and an improved reference genome (C. annuum). Dynamic genome rearrangements involving translocations among chromosome 3, 5 and 9 were detected in comparison between C. baccatum and the two other peppers. The amplification of athila LTR-retrotransposons, members of the gypsy superfamily, led to genome expansion in C. baccatum. In-depth genome-wide comparison of genes and repeats unveiled that the copy numbers of NLRs were greatly increased by LTR-retrotransposon-mediated retroduplication. Moreover, retroduplicated NLRs exhibited great abundance across the angiosperms, with most cases lineage-specific and thus recent events. Our study revealed that retroduplication has played key roles in the emergence of new disease-resistance genes in plants.

plant biology↗

bioRxiv2016

Discovery of an autoimmunity-associated IL2RA enhancer by unbiased targeting of transcriptional activation

The majority of genetic variants associated with common human diseases map to enhancers, non-coding elements that shape cell type-specific transcriptional programs and responses to specific extracellular cues 1-3. In order to understand the mechanisms by which non-coding genetic variation contributes to disease, systematic mapping of functional enhancers and their biological contexts is required. Here, we develop an unbiased discovery platform that can identify enhancers for a target gene without prior knowledge of their native functional context. We used tiled CRISPR activation (CRISPRa) to synthetically recruit transcription factors to sites across large genomic regions (>100 kilobases) surrounding two key autoimmunity risk loci, CD69 and IL2RA (interleukin-2 receptor alpha; CD25). We identified several CRISPRa responsive elements (CaREs) with stimulation-dependent enhancer activity, including an IL2RA enhancer that harbors an autoimmunity risk variant. Using engineered mouse models and genome editing of human primary T cells, we found that sequence perturbation of the disease-associated IL2RA enhancer does not block IL2RA expression, but rather delays the timing of gene activation in response to specific extracellular signals. This work develops an approach to rapidly identify functional enhancers within non-coding regions, decodes a key human autoimmunity association, and suggests a general mechanism by which genetic variation can cause immune dysfunction.

genetics↗

bioRxiv2016

L-DOPA reduces model-free control of behavior by attenuating the transfer of value to action

Dopamine is a key neurotransmitter in reinforcement learning and action control. Recent findings suggest that these components are inherently entangled. Here, we tested if increases in dopamine tone by administration of L-DOPA upregulate deliberative \"model-based\" control of behavior or reflexive \"model-free\" control as predicted by dual-control reinforcement-learning models. Alternatively, L-DOPA may impair learning as suggested by \"value\" or \"thrift\" theories of dopamine. To this end, we employed a two-stage Markov decision-task to investigate the effect of L-DOPA (randomized cross-over) on behavioral control while brain activation was measured using fMRI. L-DOPA led to attenuated model-free control of behavior as indicated by the reduced impact of reward on choice and increased stochasticity of model-free choices. Correspondingly, in the brain, L-DOPA decreased the effect of reward while prediction-error signals were unaffected. Taken together, our results suggest that L-DOPA reduces model-free control of behavior by attenuating the transfer of value to action.

neuroscience↗

bioRxiv2016

Effects of of dimethyl sulfoxide on surface water near phospholipid bilayers

Despite much effort to probe the properties of dimethyl sulfoxide (DMSO) solution, effects of DMSO on water, especially near plasma membrane surfaces still remain elusive. By performing molecular dynamics (MD) simulations at varying DMSO concentrations (XDMSO), we study how DMSO affects structural and dynamical properties of water in the vicinity of phospholipid bilayers. As proposed by a number of experiments, our simulations confirm that DMSO induces dehydration from bilayer surfaces and disrupts the H-bond structure of water. However, DMSO enhanced water diffusivity at solvent-bilayer interfaces, an intriguing discovery reported by a spin-label measurement, is not confirmed in our simulations. In order to resolve this discrepancy, we examine the location of the spin-label (Tempo), relative to the solvent-bilayer interface. In accord with the evidence in the literature, our simulations, which explicitly model Tempo-PC, find that the Tempo moiety is equilibrated at ~ 8 - 10 [A] below the bilayer surface. Furthermore, the DMSO-enhanced surface water diffusion is confirmed only when water diffusion is analyzed around the Tempo moiety that is immersed below the bilayer surface, which implies that the experimentally detected signal of water using Tempo stems from the interior of bilayers, not from the interface. Our analysis finds that the increase of water diffusion below the bilayer surface is coupled to the increase of area per lipid with an increasing XDMSO ([lsim] 10 mol%). Underscoring the hydrophobic nature of Tempo moiety, our study calls for careful re-evaluation of the use of Tempo in the measurement on lipid bilayer surfaces.

biophysics↗