Biology subjects

Lee, J.

Publications and source records attributed to Lee, J..

At least 667 records · Page 37Linked to original sources

bioRxiv2021

Astrocytic urea cycle detoxifies Aβ-derived ammonia while impairing memory in Alzheimers Disease

Alzheimers disease (AD) is one of the foremost neurodegenerative diseases, characterized by beta-amyloid (A{beta}) plaques and significant progressive memory loss. In AD, astrocytes are known to take up and clear A{beta} plaques. However, how A{beta} induces pathogenesis and memory impairment in AD remains elusive. We report that normal astrocytes show non-cyclic urea metabolism, whereas A{beta}-treated astrocytes show switched-on urea cycle with upregulated enzymes and accumulated entering-metabolite aspartate, starting-substrate ammonia, end-product urea, and side-product putrescine. Gene-silencing of astrocytic ornithine decarboxylase-1 (ODC1), facilitating ornithine-to-putrescine conversion, boosts urea cycle and eliminates aberrant putrescine and its toxic by-products ammonia, H2O2, and GABA to recover from reactive astrogliosis and memory impairment in AD model. Our findings implicate that astrocytic urea cycle exerts opposing roles of beneficial A{beta} detoxification and detrimental memory impairment in AD. We propose ODC1-inhibition as a promising therapeutic strategy for AD to facilitate removal of toxic molecules and prevent memory loss.

neuroscience↗

bioRxiv2021

An arsenic stress signaling code in yeast

Arsenic is an environmental toxin that exists mainly as pentavalent arsenate and trivalent arsenite. Both forms activate the yeast SAPK Hog1, but with different consequences. We describe a mechanism by which cells distinguish between these arsenicals through one-step metabolism to differentially regulate the bidirectional glycerol channel Fps1, an adventitious port for arsenite. Cells exposed to arsenate reduce it to thiol-reactive arsenite, which modifies a set of cysteine residues in target proteins; whereas cells exposed to arsenite metabolize it to methylarsenite, which modifies an additional set of cysteine residues. Hog1 becomes arsenylated, which prevents it from closing Fps1. However, this block is overcome in cells exposed to arsenite through methylarsenylation of Acr3, an arsenite efflux pump that we found also regulates Fps1 directly. This adaptation allows cells to restrict arsenite entry through Fps1, but also allows its exit when produced from arsenate exposure. These results have broad implications for understanding how SAPKs activated by diverse stressors can drive stress-specific outputs. Summary (for JCB table of contents) Jongmin Lee, David E. Levin Lee and Levin investigate the question of how different stressors can drive divergent outputs from an active SAPK. The work describes the mechanism by which two forms of arsenic that both activate the yeast SAPK Hog1 differentially regulate the glycerol channel Fps1.

molecular biology↗

bioRxiv2021

Taxonomic classification methods reveal a new subgenus in the paramyxovirus subfamily Orthoparamyxovirinae

As part of a broad One Health surveillance effort to detect novel viruses in wildlife and people, we report several paramyxoviruses sequenced primarily from bats during 2013 and 2014 in Brazil and Malaysia, including seven from which we recovered full-length genomes. Of these, six represent the first full-length paramyxovirus genomes sequenced from the Americas, including two sequences which are the first full-length bat morbillivirus genomes published to date. Our findings add to the vast number of viral sequences in public repositories that have been increasing considerably in recent years due to the rising accessibility of metagenomics. Taxonomic classification of these sequences in the absence of phenotypic data has been a significant challenge, particularly in the paramyxovirus subfamily Orthoparamyxovirinae, where the rate of discovery of novel sequences has been substantial. Using pairwise amino acid sequence classification (PASC), we describe a novel genus within this subfamily tentatively named Jeishaanvirus, which we propose should include as subgenera Jeilongvirus, Shaanvirus, and a novel South American subgenus Cadivirus. We also highlight inconsistencies in the classification of Tupaia virus and Mojiang virus using the same demarcation criteria and show that members of the proposed subgenus Shaanvirus are paraphyletic. Importantly, this study underscores the critical importance of sequence length in PASC analysis as well as the importance of biological characteristics such as genome organization in the taxonomic classification of viral sequences.

microbiology↗

bioRxiv2021

Intravital imaging of cerebral microinfarct reveals an astrocyte reaction led to glial scar

Cerebral microinfarct increases the risk of dementia. But how microscopic cerebrovascular disruption affects the brain tissue in cellular-level are mostly unknown. Herein, with a longitudinal intravital imaging, we serially visualized in vivo dynamic cellular-level changes in astrocyte, pericyte and neuron as well as microvascular integrity after the induction of cerebral microinfarction for 1 month in mice. At day 2-3, it revealed a localized edema with acute astrocyte loss, neuronal death, impaired pericyte-vessel coverage and extravascular leakage indicating blood-brain barrier (BBB) dysfunction. At day 5, edema disappeared with recovery of pericyte-vessel coverage and BBB integrity. But brain tissue continued to shrink with persisted loss of astrocyte and neuron in microinfarct until 30 days, resulting in a collagen-rich fibrous scar surrounding the microinfarct. Notably, reactive astrocytes appeared at the peri-infarct area early at day 2 and thereafter accumulated in the peri-infarct. Oral administration of a reversible monoamine oxidase B inhibitor significantly decreased the astrocyte reactivity and fibrous scar formation. Our result suggests that astrocyte reactivity may be a key target to alleviate the impact of microinfarction.

neuroscience↗

bioRxiv2021

Lomitapide, a cholesterol-lowering drug, is an anticancer agent that induces autophagic cell death via inhibiting mTOR

Autophagy is a biological process that maintains cellular homeostasis and regulates the internal cellular environment. Hyperactivating autophagy to trigger cell death has been a suggested therapeutic strategy for cancer treatment. Mechanistic target of rapamycin (mTOR) is a crucial protein kinase that regulates autophagy; therefore, using a structure-based virtual screen analysis, we identified lomitapide, a cholesterol-lowering drug, as a potential mTOR complex 1 (mTORC1) inhibitor. Our results showed that lomitapide directly inhibits mTORC1 in vitro and induces autophagy-dependent cancer cell death by decreasing mTOR signaling, thereby inhibiting the downstream events associated with increased LC3 conversion in various cancer cells (e.g., HCT116 colorectal cancer cells) and tumor xenografts. Lomitapide also significantly suppresses the growth and viability along with elevated autophagy in patient-derived colorectal cancer organoids. Furthermore, a combination of lomitapide and immune checkpoint blocking antibodies synergistically inhibits tumor growth in murine MC-38 or B16-F10 pre-clinical syngeneic tumor models. These results elucidates the direct, tumor-relevant immune-potentiating benefits of mTORC1 inhibition by lomitapide, which complement the current immune checkpoint blockade. This study highlights the potential repurposing of lomitapide as a new therapeutic option for cancer treatment.

cancer biology↗

bioRxiv2021

Intraspecific de novo gene birth revealed by presence absence variant genes in Caenorhabditis elegans

Genes embed their evolutionary history in the form of various alleles. Presence-absence variants (PAVs) are extreme cases of such alleles, where a gene present in one haplotype does not exist in another. Since PAVs may result from either birth or death of a gene, PAV genes and their alternative alleles, if available, can represent a basis for rapid intraspecific gene evolution. Here, we traced a possible evolution of PAV genes in the PD1074 and CB4856 C. elegans strains as well as their alternative alleles found in other 14 wild strains, using long-read sequencing technologies. We updated the CB4856 genome by filling 18 gaps and identified 50 novel genes and 7,460 novel isoforms from both strains. We verified 328 PAV genes, out of which 48 were C. elegans-specific. Among these possible newly-born genes, 13 had alternative alleles in other wild strains and, in particular, alternative alleles of three genes showed signatures active transposons. Alternative alleles of four other genes showed another type of signature reflected in accumulation of small insertions or deletions. Our results exemplify that research on gene evolution using both species-specific PAV genes and their alternative alleles is expected to provide new perspectives for how genes evolve.

genomics↗

bioRxiv2021

HCN2 in cholinergic interneurons of the nucleus accumbens mediates reward response

Cholinergic interneurons (ChIs) of the nucleus accumbens (NAc) are important for mediating the behavioral response to rewarding stimuli. A major role for these cells is to regulate dopamine (DA) transmission by activating cholinergic receptors at local DAergic nerve terminals. However, the mechanisms that enable cholinergic neurons to enhance DA release in response to reward remain unknown. Here we report that the hyperpolarization-activated cyclic nucleotide-gated channel 2 (HCN2) in NAc ChIs mediates an enhancement in DA signaling in response to rewarding stimuli. The HCN current in NAc ChIs and its modulation by DA, as well as the increase in cholinergic efflux by local cocaine infusion were impaired in mice with deletion of HCN2 in cholinergic cells. Enhancement in the DA efflux and signaling in the NAc in response to rewarding stimuli, as well as cocaine conditioning were also dependent on HCN2 in ChIs. These results provide a mechanistic link between the activity of NAc ChIs and reward encoding.

neuroscience↗

bioRxiv2021

A-Prot: Protein structure modeling using MSA transformer

In this study, we propose a new protein 3D structure modeling method, A-Prot, using MSA Transformer, one of the state-of-the-art protein language models. For a given MSA, an MSA feature tensor and row attention maps are extracted and converted into 2D residue-residue distance and dihedral angle predictions. We demonstrated that A-Prot predicts long-range contacts better than the existing methods. Additionally, we modeled the 3D structures of the free modeling and hard template-based modeling targets of CASP14. The assessment shows that the A-Prot models are more accurate than most top server groups of CASP14. These results imply that A-Prot captures evolutionary and structural information of proteins accurately with relatively low computational cost. Thus, A-Prot can provide a clue for the development of other protein property prediction methods.

bioinformatics↗

bioRxiv2021

Structural anatomy of C1 domain interactions with DAG and other agonists

Diacylglycerol (DAG) is a versatile lipid whose 1,2-sn-stereoisomer serves both as second messenger in signal transduction pathways that control vital cellular processes, and as metabolic precursor for downstream signaling lipids such as phosphatidic acid1,2. DAG effector proteins compete for available lipid using conserved homology 1 (C1) domains as DAG-sensing modules. Yet, how C1 domains recognize and capture DAG in the complex environment of a biological membrane has remained unresolved for the 40 years since the discovery of Protein Kinase C (PKC)3 as the first member of the DAG effector cohort. Herein, we report the first high-resolution crystal structures of a C1 domain (C1B from PKC{delta}) complexed to DAG and to each of four potent PKC agonists that produce different biological readouts and that command intense therapeutic interest. This structural information details the mechanisms of stereospecific recognition of DAG by the C1 domains, the functional properties of the lipid-binding site, and the identities of the key residues required for the recognition and capture of DAG and exogenous agonists. Moreover, the structures of the five C1 domain complexes provide the high-resolution guides for the design of agents that modulate the activities of DAG effector proteins.

biophysics↗

bioRxiv2021

Drosophila centrocortin is dispensable for centriole duplication but contributes to centrosome separation

Centrosomes are microtubule-organizing centers that duplicate exactly once to organize the bipolar mitotic spindle required for error-free mitosis. Prior work indicated that Drosophila centrocortin (cen) is required for normal centrosome separation, although a role in centriole duplication was not closely examined. Through time-lapse recordings of rapid syncytial divisions, we monitored centriole duplication and the kinetics of centrosome separation in control versus cen null embryos. Our data suggest that although cen is dispensable for centriole duplication, it contributes to centrosome separation.

cell biology↗

bioRxiv2021

Cholinergic interneurons mediate cocaine extinction through similar plasticity across medium spiny neuron subtypes

Cholinergic interneurons (ChINs) in the nucleus accumbens (NAc) have been implicated in the acquisition and extinction of drug associations, as well as related plasticity in medium spiny neurons (MSNs). However, since most previous work has relied on artificial manipulations, if and how endogenous patterns of cholinergic signaling relate to drug associations is unknown. Moreover, despite great interest in the opposing effects of dopamine on MSN subtypes, whether ChIN-mediated effects are similar or different across MSN subtypes is also unknown. Here, we find that endogenous acetylcholine event frequency during extinction negatively correlates with the strength and persistence of cocaine-context associations across individuals, consistent with effects of artificial manipulation of ChIN activity during extinction. Moreover, ChIN activation during extinction produces a reduction in excitatory synaptic strength on both MSN subtypes, similar to the effect of multiple extinction sessions in the absence of ChIN manipulations. Together, our findings indicate that natural variation in NAc acetylcholine may contribute to individual differences in drug-context extinction by modulating glutamatergic presynaptic strength similarly at both D1R and D2R MSN subtypes. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=146 SRC="FIGDIR/small/458113v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@14b4cc1org.highwire.dtl.DTLVardef@9c5224org.highwire.dtl.DTLVardef@d4bff3org.highwire.dtl.DTLVardef@1862150_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2021

Wear and Tear of the Intestinal Visceral Musculature by Intrinsic and Extrinsic Factors

The gut visceral musculature plays essential roles in not only moving substances through the lumen but also maintaining the function and physiology of the gut. Although the development of the visceral musculature has been studied in multiple model organisms, how it degenerates is poorly understood. Here, we employ the Drosophila midgut as a model to demonstrate that the visceral musculature is disrupted by intrinsic and extrinsic factors, such as aging, feeding, chemical-induced tissue damage, and oncogenic transformation in the epithelium. Notably, we define four prominent visceral musculature disruption phenotypes, which we refer as sprout, discontinuity, furcation, and crossover of the longitudinal muscle. Given that the occurrence of these phenotypes is increased during aging and under various stresses, we propose that these phenotypes can be used as quantitative readouts of deterioration of the visceral musculature. Intriguingly, administration of a tissue-damaging chemical dextran sulfate sodium (DSS) induced similar visceral musculature disruption phenotypes in zebrafish larvae, indicating that ingestion of a tissue-damaging chemical can disrupt the visceral musculature in a vertebrate as well. Our study provides insights into the deterioration of the gut visceral musculature and lays a groundwork for investigating the underlying mechanisms in Drosophila as well as other animals.

developmental biology↗

bioRxiv2021

Yeast-expressed Recombinant SARS-CoV-2 Receptor Binding Domain, RBD203-N1 as a COVID-19 Protein Vaccine Candidate

BackgroundSARS-CoV-2 protein subunit vaccines are being evaluated by multiple manufacturers to fill the need for low-cost, easy to scale, safe, and effective COVID-19 vaccines for global access. Vaccine candidates relying on the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein have been the focus of our development program. In this paper, we report on the generation of the RBD203-N1 yeast expression construct, which produces a recombinant protein that when formulated with alum and the TLR-9 agonist, CpG1826 elicits a robust immune response and protection in mice. MethodThe RBD203-N1 antigen was expressed in the yeast Pichia pastoris X33. After fermentation at the 5 L scale, the protein was purified by hydrophobic interaction chromatography followed by anion exchange chromatography. The purified protein was characterized biophysically and biochemically, and after its formulation, the immunogenicity and efficacy were evaluated in mice. Results, Conclusions, and SignificanceThe RBD203-N1 production process yielded 492.9 {+/-} 3.0 mg/L of protein in the fermentation supernatant. A two-step purification process produced a >96% pure protein with a recovery rate of 55 {+/-} 3% (total yield of purified protein: 270.5 {+/-} 13.2 mg/L fermentation supernatant). The protein was characterized as a homogeneous monomer with well-defined secondary structure, thermally stable, antigenic, and when adjuvanted on alum and CpG, it was immunogenic and induced robust levels of neutralizing antibodies against SARS-CoV-2 pseudovirus. These characteristics show that this vaccine candidate is well suited for technology transfer with feasibility of its transition into the clinic to evaluate its immunogenicity and safety in humans.

molecular biology↗

bioRxiv2021

Multi-organ analysis of low-level somatic mosaicism reveals stage- and tissue-specific mutational features in human development

Most somatic mutations arising during normal development present as low-level in single or multiple tissues depending on the developmental stage and affected organs1-4. However, it remains unclear how the human developmental stages or mutation-carrying organs affect somatic mutations features. Here, we performed a systemic and comprehensive analysis of low-level somatic mutations using deep whole-exome sequencing (WES; average read depth: [~]500x) of 498 multiple organ tissues with matched controls from 190 individuals. We found that early-stage mutations shared between multiple organs are lower in number but showed higher allele frequencies than late-stage mutations [0.54 vs. 5.83 variants per individual: 6.17% vs. 1.5% variant allele frequency (VAF)] along with less nonsynonymous mutations and lower functional impacts. Additionally, early- and late-stage mutations had unique mutational signatures distinct from tumor-originate mutations. Compared with early-stage mutations presenting a clock-like signature across all studied organs or tissues, late-stage mutations show organ, tissue, and cell-type specificity in mutation count, VAFs, and mutational signatures. In particular, analysis of brain somatic mutations shows bimodal occurrence and temporal-lobe-specific mutational signatures. These findings provide new insight into the features of somatic mosaicisms dependent on developmental stages and brain regions.

genomics↗

bioRxiv2021

A Meta-Analytic Single-Cell Atlas of Mouse Bone Marrow Hematopoietic Development

The clinical importance of the hematopoietic system makes it one of the most heavily studied lineages in all of biology. A clear understanding of the cell types and functional programs during hematopoietic development is central to research in aging, cancer, and infectious diseases. Known cell types are traditionally identified by the expression of proteins on the surface of the cells. Stem and progenitor cells defined based on these markers are assigned functions based on their lineage potential. The rapid growth of single cell RNA sequencing technologies (scRNAseq) provides a new modality for evaluating the cellular and functional landscape of hematopoietic stem and progenitor cells. The popularity of this technology among hematopoiesis researchers enables us to conduct a robust meta-analysis of mouse bone marrow scRNAseq data. Using over 300,000 cells across 12 datasets, we evaluate the classification and function of cell types based on discrete clustering, in silico FACS sorting, and a continuous trajectory. We identify replicable signatures that define cell types based on genes and known cellular functions. Additionally, we evaluate the conservation of signatures associated with erythroid and monocyte lineage development across species using co-expression networks. The co-expression networks predict the effectiveness of the signature at identifying erythroid and monocyte cells in zebrafish and human scRNAseq data. Together, this analysis provides a robust reference, particularly marker genes and functional annotations, for future experiments in hematopoietic development. Key PointsO_LIMeta-analysis of 9 mouse bone marrow scRNAseq identifies markers for cell types and hematopoietic development C_LIO_LICharacterize a replicable functional landscape of cell types by exploiting co-expression C_LI

genomics↗

bioRxiv2021

Circadian Clock Core Component Bmal1 Dictates Cell Cycle Rhythm of Proliferating Hepatocytes during Liver Regeneration

Following partial hepatectomy (PH), the majority of remnant hepatocytes synchronously enter and rhythmically progress through the cell cycle for three major rounds to regain lost liver mass. Whether and how the circadian clock core component Bmal1 modulates this process remains elusive. We performed PH on Bmal1+/+ and hepatocyte-specific Bmal1 knockout (Bmal1hep-/-) mice and compared the initiation and progression of the hepatocyte cell cycle. After PH, Bmal1+/+ hepatocytes exhibited three major waves of nuclear DNA synthesis. In contrast, in Bmal1hep-/-hepatocytes, the first wave of nuclear DNA synthesis was delayed by 12 h, and the third such wave was lost. Following PH, Bmal1+/+ hepatocytes underwent three major waves of mitosis, whereas Bmal1hep-/-hepatocytes fully abolished mitotic oscillation. These Bmal1-dependent disruptions in the rhythmicity of hepatocyte cell cycle after PH were accompanied by suppressed expression peaks of a group of cell cycle components and regulators, and dysregulated activation patterns of mitogenic signaling molecules c-Met and EGFR. Moreover, Bmal1+/+ hepatocytes rhythmically accumulated fat as they expanded following PH, whereas this phenomenon was largely inhibited in Bmal1hep-/-hepatocytes. In addition, during late stages of liver regrowth, Bmal1 absence in hepatocytes caused the activation of redox sensor Nrf2, suggesting an oxidative stress state in regenerated liver tissue. Collectively, we demonstrated that during liver regeneration, Bmal1 partially modulates the oscillation of S-phase progression, fully controls the rhythmicity of M-phase advancement, and largely governs fluctuations in fat metabolism in replicating hepatocytes, and eventually determines the redox state of regenerated livers. New and NoteworthyWe demonstrated that Bmal1 centrally controls the synchronicity and rhythmicity of the cell cycle and lipid accumulation in replicating hepatocytes during liver regeneration. Bmal1 plays these roles, at least in part, by ensuring formation of the expression peaks of cell cycle components and regulators, as well as the timing and levels of activation of mitogenic signaling molecules. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=187 SRC="FIGDIR/small/455958v1_ufig1.gif" ALT="Figure 1"> View larger version (17K): org.highwire.dtl.DTLVardef@16dc661org.highwire.dtl.DTLVardef@1fb1a65org.highwire.dtl.DTLVardef@820e1dorg.highwire.dtl.DTLVardef@6cc264_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract C_FIG

pathology↗

bioRxiv2021

In vitro activity of bedaquiline and imipenem against actively growing, nutrient-starved, and intracellular Mycobacterium abscessus

Mycobacterium abscessus lung disease is difficult to treat due to intrinsic drug resistance and the persistence of drug-tolerant bacteria. Currently, the standard of care is a multi-drug regimen with at least 3 active drugs, preferably including a {beta}-lactam (imipenem or cefoxitin). These regimens are lengthy, toxic, and have limited efficacy. The search for more efficacious regimens led us to evaluate bedaquiline, a diarylquinoline licensed for treatment of multidrug-resistant tuberculosis. We performed in vitro time-kill experiments to evaluate the activity of bedaquiline alone and in combination with the first-line drug imipenem against M. abscessus under various conditions. Against actively growing bacteria, bedaquiline was largely bacteriostatic and antagonized the bactericidal activity of imipenem. Contrarily, against nutrient-starved persisters, bedaquiline was bactericidal, while imipenem was not, and bedaquiline drove the activity of the combination. In an intracellular infection model, bedaquiline and imipenem had additive bactericidal effects. Correlations between ATP levels and the bactericidal activity of imipenem and its antagonism by bedaquiline were observed. Interestingly, the presence of Tween 80 in the media affected the activity of both drugs, enhancing the activity of imipenem and reducing that of bedaquiline. Overall, these results show that bedaquiline and imipenem interact differently depending on culture conditions. Previously reported antagonistic effects of bedaquiline on imipenem were limited to conditions with actively multiplying bacteria and/or the presence of Tween 80, whereas the combination was additive or indifferent against nutrient-starved and intracellular M. abscessus, where promising bactericidal activity of the combination suggests it may have a role in future treatment regimens.

microbiology↗

bioRxiv2021

Can AlphaFold2 predict protein-peptide complex structures accurately?

AO_SCPLOWBSTRACTC_SCPLOWIn this preprint, we investigated whether AlphaFold2, AF2, can predict protein-peptide complex structures only with sequence information. We modeled the structures of 203 protein-peptide complexes from the PepBDB DB and 183 from the PepSet. The structures were modeling with concatenated sequences of receptors and peptides via poly-glycine linker. We found that for more than half of the test cases, AF2 predicted the bound structures of peptides with good accuracy, C-RMSD of a peptide < 3.0 [A]. For about 40% of cases, the peptide structures were modeled with an accuracy of C-RMSD < 2.0 [A]. Our benchmark results clearly show that AF2 has a great potential to be applied to various higher-order structure prediction tasks.

bioinformatics↗