Biology subjects

Lee, H.

Publications and source records attributed to Lee, H..

At least 469 records · Page 26Linked to original sources

bioRxiv2019

Cardiac nitric oxide synthase 1 worsens heart failure with preserved ejection fraction through S-nitrosylation of histone deacetylase 2

Although the clinical importance of heart failure with preserved ejection fraction (HFpEF), which makes up half of heart failure, has been extensively explored, most therapeutic regimens, including nitric oxide (NO) donors, lack therapeutic benefit1-12. Here we report that neuronal nitric oxide synthase (nNOS, also known as NOS1) induces HFpEF by S-nitrosylation of histone deacetylase 2 (HDAC2). HFpEF animal models--SAUNA (SAlty drinking water/Unilateral Nephrectomy/Aldosterone)13,14 and mild transverse aortic constriction (TAC) mice14,15--showed increased nNOS expression and NO production, which resulted in the S-nitrosylation of HDAC2. HFpEF was alleviated in S-nitrosylation-dead HDAC2 knock-in mice. Pharmacologic intervention by either nNOS inhibition or HDAC2 denitrosylation attenuated HFpEF. Our observations are the first to demonstrate a completely new mechanistic aspect in HFpEF, which may provide a novel therapeutic approach to HFpEF. In addition, our results provide evidence for why conventional NO-enhancement trials have not been effective for improving HFpEF.

molecular biology↗

bioRxiv2019

Rho-kinase mediates the anorexigenic action of melanocortin by suppressing AMPK

ObjectiveMelanocortin action is essential for the maintenance of energy homeostasis. However, knowledge of the signaling mechanism(s) that mediates the effect of melanocortin remains incomplete.\n\nMethodsROCK1 is a key regulator of energy balance in the hypothalamus. To explore the role of ROCK1 in the anorexigenic action of melanocortin, we deleted ROCK1 in MC4R neurons in mice. Next, we studied the metabolic effects of MC4R neuron-specific ROCK1-deficiency and following treatment with -melanocyte-stimulating hormone (MSH).\n\nResultsHere we show that -MSH increases Rho-kinase 1 (ROCK1) activity in the hypothalamus. Deficiency of ROCK1 in MC4R-expressing neurons results in increased body weight in mice fed normal chow diet. This is likely due to increased food intake and decreased energy expenditure. Importantly, we find that ROCK1 activation in MC4R expressing neurons is required for melanocortin action, as evidenced by the fact that - MSHs ability to suppress food intake is impaired in MC4R neuron-specific ROCK1-deficient mice. To elucidate the mechanism by which ROCK1 mediates melanocortin action, we performed in vitro studies in hypothalamic cells expressing MC4R. We demonstrate that -MSH promotes the physical interaction of ROCK1 and G12, and this results in suppression of AMPK activity.\n\nConclusionsOur study identifies ROCK1 as a novel mediator of melanocortins anorexigenic action and uncover a new MC4R[->]G12[->]ROCK1[->]AMPK signaling pathway. Targeting Rho-kinase in MC4R-expressing neurons could provide a new strategy to combat obesity and its related complications.

neuroscience↗

bioRxiv2019

Global analysis of epigenetic heterogeneity identifies divergent drivers of esophageal squamous cell carcinoma

Epigenetic landscapes can shape physiologic and disease phenotypes. We used integrative, high resolution multi-omics methods to characterize the oncogenic drivers of esophageal squamous cell carcinoma (ESCC). We found 98% of CpGs are hypomethylated across the ESCC genome and two-thirds occur in long non-coding (lnc)RNA regions. DNA methylation and epigenetic heterogeneity both coincide with chromosomal topological alterations. Gene body methylation, polycomb repressive complex occupancy, and CTCF binding sites associate with cancer-specific gene regulation. Epigenetically-mediated activation of non-canonical WNT signaling and the lncRNA ESCCAL-1 were validated as potential ESCC driver alterations. Gene-specific cancer driver roles of epigenetic alterations and heterogeneity are identified.

cell biology↗

bioRxiv2019

Identification of Pathogenic Structural Variants in Rare Disease Patients through Genome Sequencing

PurposeClinical whole genome sequencing is becoming more common for determining the molecular diagnosis of rare disease. However, standard clinical practice often focuses on small variants such as single nucleotide variants and small insertions/deletions. This leaves a wide range of larger \"structural variants\" that are not commonly analyzed in patients.\n\nMethodsWe developed a pipeline for processing structural variants for patients who received whole genome sequencing through the Undiagnosed Diseases Network (UDN). This pipeline called structural variants, stored them in an internal database, and filtered the variants based on internal frequencies and external annotations. The remaining variants were manually inspected and then interesting findings were reported as research variants to clinical sites in the UDN.\n\nResultsOf 477 analyzed UDN cases, 286 cases ({approx} 60%) received at least one structural variant as a research finding. The variants in 16 cases ({approx} 4%) are considered \"Certain\" or \"Highly likely\" molecularly diagnosed and another 4 cases are currently in review. Of those 20 cases, at least 13 were identified originally through our pipeline with one finding leading to identification of a new disease. As part of this paper, we have also released the collection of variant calls identified in our cohort along with heterozygous and homozygous call counts. This data is available at https://github.com/HudsonAlpha/UDN_SV_export.\n\nConclusionStructural variants are key genetic features that should be analyzed during routine clinical genomic analysis. For our UDN patients, structural variants helped solve {approx} 4% of the total number of cases ({approx} 13% of all genome sequencing solves), a success rate we expect to improve with better tools and greater understanding of the human genome.

genomics↗

bioRxiv2019

Chromosome-level de novo assembly of the pig-tailed macaque genome using linked-read sequencing and HiC proximity scaffolding

Old world monkey species share over 93% genome homology with humans and develop many disease phenotypes similar to those of humans, making them highly valuable animal models for the study of numerous human diseases. However, the quality of genome assembly and annotation for old world monkeys including macaque species lags behind the human genome effort. To close this gap and enhance functional genomics approaches, we employed a combination of de novo linked-read assembly and scaffolding using proximity ligation assay (HiC) to assemble the pig-tailed macaque (Macaca nemestrina) genome. This combinatorial method yielded large scaffolds at chromosome-level with a scaffold N50 of 127.5 Mb; the 23 largest scaffolds covered 90% of the entire genome. This assembly revealed large-scale rearrangements between pig-tailed macaque chromosomes 7,12, and13 and human chromosomes 2, 14, and 15.

genomics↗

bioRxiv2019

Accurate forecasts of the effectiveness of interventions against Ebola may require models that account for variations in symptoms during infection

Epidemiological models are routinely used to predict the effects of interventions aimed at reducing the impacts of Ebola epidemics. Most models of interventions targeting symptomatic hosts, such as isolation or treatment, assume that all symptomatic hosts are equally likely to be detected. In other words, following an incubation period, the level of symptoms displayed by an individual host is assumed to remain constant throughout an infection. In reality, however, symptoms vary between different stages of infection. During an Ebola infection, individuals progress from initial non-specific symptoms through to more severe phases of infection. Here we compare predictions of a model in which a constant symptoms level is assumed to those generated by a more epidemiologically realistic model that accounts for varying symptoms during infection. Both models can reproduce observed epidemic data, as we show by fitting the models to data from the ongoing epidemic in the Democratic Republic of Congo and the 2014-16 epidemic in Liberia. However, for both of these epidemics, when interventions are altered identically in the models with and without levels of symptoms that depend on the time since first infection, predictions from the models differ. Our work highlights the need to consider whether or not varying symptoms should be accounted for in models used by decision makers to assess the likely efficacy of Ebola interventions.

epidemiology↗

bioRxiv2019

Identification of Microbiota-Induced Gene Expression Changes in the Drosophila melanogaster Head

Symbiotic microorganisms exert multifaceted impacts on the physiology of their animal hosts. Recent discoveries have shown the gut microbiota influence host brain function and behavior, but the host and microbial molecular factors required to actuate these effects are largely unknown. To uncover molecular mechanisms that underlie the gut-microbiota-brain axis, we used Drosophila melanogaster and its bacterial microbiota as a model to identify microbiota-dependent gene expression changes in the host brain and head. Specifically, we employed RNA-seq and nanoString nCounter technology to identify Drosophila genes that exhibit altered transcript levels in fly heads upon elimination of the microbiota. The identified genes, some of which exhibited sex-specific differences, have demonstrated or inferred functional roles in the immune response, metabolism, neuronal activity, and stress resistance. Overall, this study reveals microbiota-responsive genes in the fly head, an anatomical structure not previously investigated in this context. Our results serve as a foundation for future investigations of how microbe-driven gene expression changes impact Drosophila biology.

microbiology↗

bioRxiv2019

Link clustering explains non-central essential genes in protein interaction networks

Essential genes (EGs) often form central nodes in protein-protein interaction (PPI) networks. However, many reports have shown that numerous EGs are non-central, suggesting that another principle governs gene essentiality. We propose link clustering as a distinct indicator of the essentiality for non-central nodes. Specifically, in various human and yeast PPI networks, we found that 29 to 47% of EGs were better characterized by link clustering than by centrality. Such non-central EGs with clustered links have significant impacts on communities at lower hierarchical levels, suggesting that their essentiality derives from functional dependency among relevant local neighbors, rather than their implication on global connectivity. Moreover, these non-central EGs exhibited several distinct characteristics: they tend to be younger and fast-evolving, and likely change their essentiality across different human cell lines and between human and mouse than central-EGs. Author summaryThe centrality in network structure was thought to be the cause of gene essentiality, as it conveys nodes importance in given networks. However, many essential genes are also found to be non-central, which leads us to ask whether a principle other than centrality may govern gene essentiality. Here we demonstrate that link clustering conveying functional dependency between nodes is the other principle characterizing gene essentiality. The link clustering explains numerous essential genes that are non-central, which are distinct from central ones regarding their molecular function and evolution. Our results clearly establish a two-dimensional principle governing gene essentiality.

systems biology↗

bioRxiv2019

Distinct role of flexible and stable encoding in sequential working memory

The serial-position effect in working memory is considered important for studying how a sequence of sensory information can be retained and manipulated simultaneously in neural memory circuits. Here, via a precise analysis of the primacy and recency effects in human psychophysical experiments, we propose that stable and flexible coding take distinct roles of retaining and updating information in working memory, and that their combination induces serial-position effects spontaneously. We found that stable encoding retains memory to induce the primacy effect, while flexible encoding used for learning new inputs induces the recency effect. A model simulation based on human data, confirmed that a neural network with both flexible and stable synapses could reproduce the major characteristics of serial-position effects. Our new prediction, that the control of resource allocation by flexible-stable coding balance can modulate memory performance in sequence-specific manner, was supported by pre-cued memory performance data in humans.

neuroscience↗

bioRxiv2019

The symmetrical pattern of base-pair substitutions rates across the chromosome in Escherichia coli has multiple causes

Mutation accumulation experiments followed by whole-genome sequencing have revealed that for several bacterial species the rate of base-pair substitutions is not constant across the chromosome but varies in a wave-like pattern symmetrical about the origin of replication. The experiments reported here demonstrate that in Escherichia coli several interacting factors determine the wave. Perturbing replication timing, progression, or the structure of the terminus disrupts the pattern. Biases in error-correction by proofreading and mismatch repair are major factors. The activities of the nucleoid binding proteins, HU and Fis, are important, suggesting that mutation rates increase when highly structured DNA is replicated. These factors should apply to most bacterial, and possibly eukaryotic, genomes, and imply that different areas of the genome evolve at different rates.

microbiology↗

bioRxiv2019

Scarless Enriched selection of Genome edited Human Pluripotent Stem Cells Using Induced Drug Resistance

An efficient gene editing technique for use in human pluripotent stem cells (hPSCs) would have great potential value in regenerative medicine, as well as in drug discovery based on isogenic human disease models. However, the extremely low efficiency of gene editing in hPSCs is a major technical hurdle that remains to be resolved. Previously, we demonstrated that YM155, a survivin inhibitor developed as an anti-cancer drug, induces highly selective cell death in undifferentiated hPSCs. In this study, we demonstrated that the high cytotoxicity of YM155 in hPSCs, which is mediated by selective cellular uptake of the drug, is due to high expression of SLC35F2 in these cells. Consistent with this, knockout of SLC35F2 with CRISPR-Cas9 or depletion with siRNAs made hPSCs highly resistant to YM155. Simultaneous gene editing of a gene of interest and transient knockdown of SLC35F2 following YM155 treatment enabled genome-edited hPSCs to survive because YM155 resistance was temporarily induced, thereby achieving enriched selection of genome-edited clonal populations. This precise and efficient genome editing approach took as little as 3 weeks without cell sorting or introduction of additional genes.

developmental biology↗

bioRxiv2019

Tumor mutational landscape is a record of the pre-malignant state

Chromatin structure has a major influence on the cell-specific density of somatic mutations along the cancer genome. Here, we present a pan-cancer study in which we searched for the putative cancer cell-of-origin of 2,550 whole genomes, representing 32 cancer types by matching their mutational landscape to the regional patterns of chromatin modifications ascertained in 104 normal tissue types. We found that, in almost all cancer types, the cell-of-origin can be predicted solely from their DNA sequences. Our analysis validated the hypothesis that high-grade serous ovarian cancer originates in the fallopian tube and identified distinct origins of breast cancer subtypes. We also demonstrated that the technique is equally capable of identifying the cell-of-origin for a series of 2,044 metastatic samples from 22 of the tumor types available as primaries. Moreover, cancer drivers, whether inherited or acquired, reside in active chromatin regions in the respective cell-of-origin. Taken together, our findings highlight that many somatic mutations accumulate while the chromatin structure of the cell-of-origin is maintained and that this historical record, captured in the DNA, can be used to identify the often elusive cancer cell-of-origin.

genomics↗

bioRxiv2019

Reversible silencing of endogenous receptors in intact brain tissue using two-photon pharmacology

The physiological activity of proteins is often studied with loss-of-function genetic approaches, but the corresponding phenotypes develop slowly and can be confounding. Photopharmacology allows direct, fast and reversible control of endogenous protein activity, with spatiotemporal resolution set by the illumination method. Here, we combine a photoswitchable allosteric modulator (alloswitch) and two-photon excitation (2PE) using pulsed near-infrared lasers to reversibly silence mGlu5 receptor activity in intact brain tissue. Endogenous receptors can be photoactivated in neurons and astrocytes with pharmacological selectivity and with an axial resolution between 5 and 10 m. Thus, two-photon pharmacology (2PP) using alloswitch allows investigating mGlu5-dependent processes in wild type animals, including synaptic formation and plasticity, and signaling pathways from intracellular organelles.

pharmacology and toxicology↗

bioRxiv2019

Nedd4 E3 ligase and beta-arrestins regulate ubiquitination, trafficking, and stability of the mGlu7 receptor

The metabotropic glutamate receptor 7 (mGlu7) is a class C G protein-coupled receptor (GPCR) that modulates excitatory neurotransmitter release at the presynaptic active zone. Although post-translational modification of cellular proteins with ubiquitin is a key molecular mechanism governing protein degradation and function, mGlu7 ubiquitination and its functional consequences have not been elucidated yet. Here, we report that Nedd4 ubiquitin E3 ligase and {beta}-arrestins regulate ubiquitination of mGlu7 in heterologous cells and neurons. Upon agonist-stimulation, {beta}-arrestins recruit Nedd4 to mGlu7 and facilitate Nedd4-mediated ubiquitination of mGlu7. Nedd4 and {beta}-arrestins regulate constitutive and agonist-induced endocytosis of mGlu7 and are required for mGlu7-dependent MAPK signaling in neurons. In addition, Nedd4-mediated ubiquitination results in the degradation of mGlu7 by both the lysosomal and proteasomal degradation pathways. These findings provide a model in which Nedd4 and {beta}-arrestin act together as a complex to regulate mGlu7 surface expression and function at the presynaptic terminals.

neuroscience↗

bioRxiv2018

Genomic perspectives on adaptation and conservation in the endangered long-tailed goral (Naemorhedus caudatus).

Native to the mountains of East Asia, the long-tailed goral (Naemorhedus caudatus) is a vulnerable wild ungulate in the tribe Caprini. To understand key conservation issues related to fragmentation and subsequent endangerment of this montane species, we sequenced and analyzed the genome of a long-tailed goral to explore historical demography, contemporary levels of genetic diversity, and potential immune response. When compared to ten additional mammalian reference genomes, we identified 357 positively selected genes (PSGs) in the long-tailed goral and 364 PSGs in the Caprini lineage. Gene Ontology analyses showed statistical enrichment in biological processes related to immune function and in genes and pathways related to blood coagulation. We also identified low levels of heterozygosity (0.00114) in the long-tailed goral along with decreases in population size relative to other species of Caprini. Finally, we provide evidence for positive selection on the muscle development gene myostatin (MSTN) in the Caprini lineage, which may have increased the muscle development and climbing ability in the caprine common ancestor. Low effective population size and decreased heterozygosity of the long-tailed goral raise conservation concern about the effects of habitat fragmentation, over harvest, and inbreeding on this vulnerable species.

bioinformatics↗

bioRxiv2018

Identifying the Biosynthetic Gene Cluster for Triacsins with an N-hydroxytriazene Moiety

Triacsins are a family of natural products containing an N-hydroxytriazene moiety not found in any other known secondary metabolites. Though many studies have examined the biological activity of triacsins in lipid metabolism, the biosynthesis of triacsins has remained unknown. Here, we report the identification of the triacsin biosynthetic gene cluster in Streptomyces aureofaciens ATCC 31442. Bioinformatic analysis of the gene cluster led to the discovery of the tacrolimus producer Streptomyces tsukubaensis NRRL 18488 as a new triacsin producer. In addition to targeted gene disruption to identify necessary genes for triacsin production, stable isotope feeding was performed in vivo to advance the understanding of N-hydroxytriazene biosynthesis.

biochemistry↗

bioRxiv2018

Commensal gut bacteria convert the immunosuppressant tacrolimus to less potent metabolites

Tacrolimus exhibits low and variable drug exposure after oral dosing, but the contributing factors remain unclear. Based on our recent report showing a positive correlation between fecal abundance of Faecalibacterium prausnitzii and oral tacrolimus dose in kidney transplant patients, we tested whether F. prausnitzii and other gut abundant bacteria are capable of metabolizing tacrolimus. Incubation of F. prausnitzii with tacrolimus led to production of two compounds (the major one named M1), which was not observed upon tacrolimus incubation with hepatic microsomes. Isolation, purification, and structure elucidation using mass spectrometry and nuclear magnetic resonance spectroscopy indicated that M1 is a C-9 keto-reduction product of tacrolimus. Pharmacological activity testing using human peripheral blood mononuclear cells demonstrated that M1 is 15-fold less potent than tacrolimus as an immunosuppressant. Screening of 22 gut bacteria species revealed that most Clostridiales bacteria are extensive tacrolimus metabolizers. Tacrolimus conversion to M1 was verified in fresh stool samples from two healthy adults. M1 was also detected in the stool samples from kidney transplant recipients who had been taking tacrolimus orally. Together, this study presents gut bacteria metabolism as a previously unrecognized elimination route of tacrolimus, potentially contributing to the low and variable tacrolimus exposure after oral dosing.

pharmacology and toxicology↗

bioRxiv2018

Non-canonical Drosophila X chromosome dosage compensation and repressive topologically-associated domains

BackgroundIn animals with XY sex chromosomes, X-linked genes from a single X chromosome in males are imbalanced relative to autosomal genes. To minimize the impact of genic imbalance in male Drosophila, there is a dosage compensation complex (MSL), that equilibrates X-linked gene expression with the autosomes. There are other potential contributions to dosage compensation. Hemizygous autosomal genes located in repressive chromatin domains are often de-repressed. If this homolog-dependent repression occurs on the X, which has no pairing partner, then de-repression could contribute to male dosage compensation.\n\nResultsWe asked whether different chromatin states or topological associations correlate with X chromosome dosage compensation, especially in regions with little MSL occupancy. Our analyses demonstrated that male X chromosome genes that are located in repressive chromatin states are depleted of MSL occupancy, however they show dosage compensation. The genes in these repressive regions were also less sensitive to knockdown of MSL components.\n\nConclusionsOur results suggest that this non-canonical dosage compensation is due to the same trans-acting de-repression that occurs on autosomes. This mechanism would facilitate immediate compensation during the evolution of sex chromosomes from autosomes. This mechanism is similar to that of C. elegans, where enhanced recruitment of X chromosomes to the nuclear lamina dampens X chromosome expression as part of the dosage compensation response in XX individuals.

genomics↗