Biology subjects

Lee, J. H.

Publications and source records attributed to Lee, J. H..

At least 73 records · Page 4Linked to original sources

bioRxiv2023

Genome assembly of Hibiscus sabdariffa L. provides insights into biosynthetic pathways of medicinal natural products

Hibiscus sabdariffa L. is a widely cultivated herbaceous plant with diverse applications in food, tea, fiber, and medicine. In this study, we present a high-quality genome assembly of H. sabdariffa using more than 33 Gbps of high-fidelity (HiFi) long-read sequencing data at 20X coverage. The primary assembly and haplotype-resolved genomes exhibit an N50 contig length of 26.25 Mbps, 11.96 Mbps, and 14.50 Mbps, with a coverage of 141.3%, 86.0%, and 88.6%, respectively. By aligning 26 Gbps of total RNA sequencing data (PE100) extracted from leaves, we predicted 154k, 79k, and 87k genes in the respective genome. The completeness of the genomes and predicted genes was confirmed by benchmarking universal single-copy orthologs analysis on a eudicots_odb10 database, with a 99.3% complete rate for the primary assembly. The high-quality genome resource allowed the construction of genetic networks for phenylpropanoid and flavonoid metabolism, thereby facilitating the identification of candidate genes involved in the biosynthesis of key intermediates for roselle-specific medicinal natural products. Comprehensive genomic and functional analysis opens avenues for further exploration and application of valuable natural products derived from H. sabdariffa.

genomics↗

bioRxiv2023

Poly(ADP-ribose) mediates bioenergetic defects and redox imbalance in neurons following oxygen and glucose deprivation

PARP-1 over-activation results in cell death via excessive PAR generation in different cell types, including neurons following brain ischemia. Glycolysis, mitochondrial function, and redox balance are key cellular processes altered in brain ischemia. Studies show that PAR generated after PARP-1 over-activation can bind hexokinase-1 (HK-1) and result in glycolytic defects and subsequent mitochondrial dysfunction. HK-1 is the neuronal hexokinase and catalyzes the first reaction of glycolysis, converting glucose to glucose-6-phosphate (G6P), a common substrate for glycolysis, and the pentose phosphate pathway (PPP). PPP is critical in maintaining NADPH and GSH levels via G6P dehydrogenase activity. Therefore, defects in HK-1 will not only decrease cellular bioenergetics but will also cause redox imbalance due to the depletion of GSH. In brain ischemia, whether PAR-mediated inhibition of HK-1 results in bioenergetics defects and redox imbalance is not known. We used oxygen-glucose deprivation (OGD) in mouse cortical neurons to mimic brain ischemia in neuronal cultures and observed that PARP-1 activation via PAR formation alters glycolysis, mitochondrial function, and redox homeostasis in neurons. We used pharmacological inhibition of PARP and adenoviral-mediated overexpression of wild-type HK-1 (wtHK-1) and PAR-binding mutant HK-1 (pbmHK-1). Our data show that PAR inhibition or overexpression of HK-1 significantly improves glycolysis, mitochondrial function, redox homeostasis, and cell survival in mouse cortical neurons exposed to OGD. These results suggest that PAR binding and inhibition of HK-1 during OGD drives bioenergetic defects in neurons due to inhibition of glycolysis and impairment of mitochondrial function.

neuroscience↗

bioRxiv2023

Liquid plug propagation in computer-controlled microfluidic airway-on-a-chip with semi-circular microchannels

This paper introduces a two-inlet, one-outlet lung-on-a-chip device with semi-circular cross-section microchannels and computer-controlled fluidic switching that enables a broader systematic investigation of liquid plug dynamics in a manner relevant to the distal airways. A leak-proof bonding protocol for micro-milled devices facilitates channel bonding and culture of confluent primary small airway epithelial cells. Production of liquid plugs with computer-controlled inlet channel valving and just one outlet allows more stable long-term plug generation and propagation compared to previous designs. The system also captures both plug speed and length as well as pressure drop concurrently. In one demonstration, the system reproducibly generates surfactant-containing liquid plugs, a challenging process due to lower surface tension that makes the plug formation less stable. The addition of surfactant decreases the pressure required to initiate plug propagation, a potentially significant effect in diseases where surfactant in the airways is absent or dysfunctional. Next, the device recapitulates the effect of increasing fluid viscosity, a challenging analysis due to higher resistance of viscous fluids that makes plug formation and propagation more difficult particularly in airway-relevant length scales. Experimental results show that increased fluid viscosity decreases plug propagation speed for a given air flow rate. These findings are supplemented by computational modeling of viscous plug propagation that demonstrate increased plug propagation time, increased maximum wall shear stress, and greater pressure differentials in more viscous conditions of plug propagation. These results match physiology as mucus viscosity is increased in various obstructive lung diseases where it is known that respiratory mechanics can be compromised due to mucus plugging of the distal airways. Finally, experiments evaluate the effect of channel geometry on primary human small airway epithelial cell injury in this lung-on-a-chip. There is more injury in the middle of the channel relative to the edges highlighting the role of channel shape, a physiologically relevant parameter as airway cross-sectional geometry can also be non-circular. In sum, this paper describes a system that pushes the device limits with regards to the types of liquid plugs that can be stably generated for studies of distal airway fluid mechanical injury.

bioengineering↗

bioRxiv2023

Evolutionary spread of protein L-(iso)aspartyl O-methyltransferases guides the discovery of distinct isoaspartate-containing peptides, pimtides

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a structurally diverse class of natural products with a distinct biosynthetic logic, the enzymatic modification of genetically encoded precursor peptides. Although their structural and biosynthetic diversity remains largely underexplored, the identification of novel subclasses with unique structural motifs and biosynthetic pathways has been challenging. Here, we report that protein L-(iso)aspartyl O-methyltransferases (PIMTs) present in several RiPP subclasses are highly homologous. Importantly, we discovered that the apparent evolutionary transmission of the PIMT gene could serve as a basis to identify a novel RiPP subclass. Biochemical and structural analyses suggest that these homologous PIMTs commonly convert aspartate to isoaspartate via aspartyl-O-methyl ester and aspartimide intermediates, and often require cyclic or hairpin-like structures for modification. By conducting homology-based bioinformatic analysis of PIMTs, we identified over 2,800 biosynthetic gene clusters (BGCs) for known RiPP subclasses in which PIMTs install a secondary modification, and over 1,500 BGCs in which PIMTs function as a primary modification enzyme, thereby defining a new RiPP subclass, named pimtides. Our results suggest that the genome mining of proteins with secondary biosynthetic roles could be an effective strategy for discovering novel biosynthetic pathways of RiPPs. Insert Table of Contents artwork here O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=114 SRC="FIGDIR/small/540355v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@1f8afadorg.highwire.dtl.DTLVardef@1d5b2f3org.highwire.dtl.DTLVardef@d78d2eorg.highwire.dtl.DTLVardef@176070a_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2023

In Vitro Modeling of CD8 T Cell Exhaustion Enables CRISPR Screening to Reveal a Role for BHLHE40

Identifying novel molecular mechanisms of exhausted CD8 T cells (Tex) is a key goal of improving immunotherapy of cancer and other diseases. However, high-throughput interrogation of in vivo Tex can be costly and inefficient. In vitro models of Tex are easily customizable and quickly generate high cellular yield, offering an opportunity to perform CRISPR screening and other high-throughput assays. We established an in vitro model of chronic stimulation and benchmarked key phenotypic, functional, transcriptional, and epigenetic features against bona fide in vivo Tex. We leveraged this model of in vitro chronic stimulation in combination with pooled CRISPR screening to uncover transcriptional regulators of T cell exhaustion. This approach identified several transcription factors, including BHLHE40. In vitro and in vivo validation defined a role for BHLHE40 in regulating a key differentiation checkpoint between progenitor and intermediate subsets of Tex. By developing and benchmarking an in vitro model of Tex, we demonstrate the utility of mechanistically annotated in vitro models of Tex, in combination with high-throughput approaches, as a discovery pipeline to uncover novel Tex biology.

immunology↗

bioRxiv2023

Methylglyoxal is the main culprit to impairing neuronal function: mediated through tryptophan depletion.

Depression is a common and prevalent illness and the exact cause of major depressive disorder is not known. Here, we investigated how methylglyoxal (MGO) stress induces depression and unveiled the potential molecular mechanism. Our in vivo results suggested that MGO caused depression in mice, confirmed by several behavioral tests. Interestingly, it halted the mices brains tryptophan levels and its related neurotransmitters. In addition, MGO induced a reduction in the number of cells in different hippocampal regions. Moreover, it decreased tryptophan hydroxylase 1 (TPH1) and tryptophan hydroxylase 1 (TPH2) levels in the brain and large intestine. Surprisingly, MGO showed the highest affinity and trapping ability toward tryptophan. Most importantly, combined treatment with MGO-tryptophan displayed similar effects as those exhibited by the tryptophan-null treatment in neuronal cells, which included neuronal apoptosis, decrease TPH1 and TPH2 levels, and inhibition of neuronal outgrowth. However, tryptophan treatment improved MGO induced depression-like behavior of mice and recovered the loss of neuronal and hippocampal cells. Subsequently, it also induced MGO detoxifying factors, tryptophan levels, and reduces inflammation in the intestine. Collectively, our data revealed that MGO induced depression facilitated by neuronal and synaptic dysfunction is mediated through the disturbance of tryptophan metabolism in the brain and intestine.

neuroscience↗

bioRxiv2023

FBP1 is a nonenzymatic safety valve that curtails AKT activation to prevent insulin hyperresponsiveness

Insulin inhibits gluconeogenesis and stimulates glucose conversion to glycogen and lipids. How these activities are coordinated to prevent hypoglycemia and hepatosteatosis is not clear. Fructose-1,6 bisphosphatase (FBP1) is rate controlling for gluconeogenesis. However, inborn human FBP1 deficiency does not cause hypoglycemia unless accompanied by fasting or starvation, which also trigger paradoxical hepatomegaly, hepatosteatosis, and hyperlipidemia in affected individuals. Hepatocyte FBP1-ablated mice exhibit identical fasting-conditional pathologies along with AKT hyperactivation, whose inhibition reversed hepatomegaly, hepatosteatosis and hyperlipidemia but not hypoglycemia. Surprisingly, fasting-mediated AKT hyperactivation is insulin-dependent. FBP1 prevents insulin hyperresponsiveness, independently of its catalytic activity, by interacting with AKT, PP2A-C and Aldolase-B (ALDOB) to specifically accelerate AKT dephosphorylation. Enhanced by fasting and weakened by elevated insulin, FBP1:AKT:PP2A-C:ALDOB complex formation, which is disrupted by human FBP1 deficiency mutations or a C-terminal FBP1 truncation, prevents insulin-triggered liver pathologies and maintains lipid and glucose homeostasis. Conversely, a complex disrupting peptide reverses diet-induced insulin resistance.

molecular biology↗

bioRxiv2023

Oomycete effector AVRblb2 targets cyclic nucleotide-gated channels through calcium sensors to suppress pattern-triggered immunity

Transient, rapid increase of cytosolic Ca2+ upon pathogen infection is essential for plant pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI). Several cyclic nucleotide-gated channels (CNGCs) have been implicated; however, their regulatory mechanisms remain elusive. Here, the Phytophthora infestans RXLR effector AVRblb2 family targeted NbCNGC18-20 at the plasma membrane, inhibiting Ca2+ influx and PTI. AVRblb2 required calmodulin (CaM) and calmodulin-like (CML) proteins as co-factors to interact with N. benthamiana CNGCs (NbCNGCs), forming the AVRblb2-CaM/CML-NbCNGCs complex. After recognizing PAMPs, NbCNGC18 formed active heteromeric channels with other CNGCs, potentially providing selectivity for diverse signals to fine-tune cytosolic Ca2+ levels and responses. AVRblb2 suppressed the Ca2+ influx and oxidative burst induced by NbCNGC18 heteromeric complexes. Silencing CNGC18, CNGC20, and CNGC25 compromised the effect of AVRblb2 on P. infestans virulence, confirming that AVRblb2 contributed to virulence by targeting CNGCs. Our findings delineated the regulatory mechanism and role of effector-targeted Ca2+ channels in plant innate immunity.

plant biology↗

bioRxiv2022

Circadian reprogramming of adipose progenitor cells regulates intermittent fasting-mediated adipose tissue remodeling and metabolic improvement

White adipose tissue (WAT) fibrosis is a hallmark of dysfunctional WAT that is directly linked to metabolic abnormalities. Recent studies have highlighted the role of dysfunctional adipose progenitor cells (APCs) in WAT fibrosis and impaired adaptive tissue plasticity, leading to systemic insulin resistance. However, therapeutic options for WAT fibrosis are limited. Intermittent fasting (IF) is an effective dietary regimen for weight control and metabolic improvement through various mechanisms, including healthy remodeling of WAT. However, whether IF is effective in improving age-associated WAT fibrosis and metabolic homeostasis is unknown. Here, we show that IF confers therapeutic benefits in aged and obese mice through reduction of WAT fibrosis. Single-cell analyses revealed that IF significantly reduces pro-fibrotic signatures within APCs along with upregulation of the circadian pathways, suggesting that the circadian clock of APCs mediates IF-induced WAT remodeling. Importantly, mice lacking core circadian gene exhibited increased fibrotic signatures in WAT and diminished beneficial response to IF, further supporting the importance of circadian rhythm in IF-mediated metabolic benefits. Lastly, insulin resistance in humans also presented with dysregulated circadian rhythm signatures in APC populations. Collectively, our findings highlight the novel role of the APC circadian rhythm in plasticity of WAT and metabolic response to IF.

molecular biology↗

bioRxiv2022

Emission of floral volatiles is facilitated by cell-wall non-specific lipid transfer proteins

For volatile organic compounds (VOCs) to be released from the plant cell into the atmosphere, they have to cross the plasma membrane, the cell wall, and the cuticle. However, how these hydrophobic compounds cross the hydrophilic cell wall is largely unknown. Using biochemical and reverse-genetic approaches combined with mathematical modeling, we show that cell-wall localized non-specific lipid transfer proteins (nsLTPs) facilitate VOC emission. Out of three highly expressed nsLTPs in petunia petals, which emit high levels of phenylpropanoid/benzenoid compounds, only PhnsLTP3 contributes to the VOC export across the cell wall to the cuticle. A decrease in PhnsLTP3 expression reduces volatile emission and leads to VOC redistribution with less VOCs reaching the cuticle without affecting their total pools. This intracellular build-up of VOCs lowers their biosynthesis by feedback downregulation of phenylalanine precursor supply to prevent self-intoxication. Overall, these results demonstrate that nsLTPs are intrinsic members of the VOC emission network, which facilitate VOC diffusion across the cell wall.

plant biology↗

bioRxiv2022

Physics of sliding on water predicts morphological and behavioral allometry across a wide range of body sizes in water striders (Gerridae)

Laws of physics shape morphological and behavioral adaptations to locomotion at different body sizes. Water striders serve as a model taxon to study how simple physical constraints of water-surface habitats affect their behavior and morphology, and hydrodynamics of rowing by midlegs on the surface is well understood. However, the physics of the subsequent passive sliding has been less explored. We created a model of sliding on the water surface to simulate the effect of body mass, striding type, and wetted leg lengths on an insects ability to float on the surface and on the sliding resistance. The model predicts that to support their weight on the surface during sliding, the heavy species should either develop long forelegs that support the frontal part of its body during symmetrical striding (when two midlegs thrust) or use asymmetrical striding (when one forward-extended midleg supports the body while the other midleg and contra-lateral hindleg thrust). These predictions are confirmed by the behavior and morphology of various Gerridae species. Hence, the results illustrate how simple physical processes specific to a certain habitat type have far-reaching consequences for the evolution of morphological and behavioral diversification associated with body size among biological organisms in these habitats.

biophysics↗

bioRxiv2022

SARS-CoV-2 multi-antigen protein microarray for detailed characterization of antibody responses in COVID-19 patients

Antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) target multiple epitopes on different domains of the spike protein, and other SARS-CoV-2 proteins. We developed a SARS-CoV-2 multi-antigen protein microarray with the nucleocapsid, spike and its domains (S1, S2), and variants with single (D614G, E484K, N501Y) or double substitutions (N501Y/Deletion69/70), allowing a more detailed high-throughput analysis of the antibody repertoire following infection. The assay was demonstrated to be reliable and comparable to ELISA. We analyzed antibodies from 18 COVID-19 patients and 12 recovered convalescent donors. S IgG level was higher than N IgG in most of the COVID-19 patients, receptor-binding domain of S1 showed high reactivity, but no antibodies were detected against heptad repeat domain 2 of S2. Furthermore, antibodies were detected against S variants with single and double substitutions in COVID-19 patients who were infected with SARS-CoV-2 early in the pandemic. Here we demonstrated that SARS-CoV-2 multi-antigen protein microarray is a powerful tool for detailed characterization of antibody responses, with potential utility in understanding the disease progress and assessing current vaccines and therapies against evolving SARS-CoV-2.

biochemistry↗

bioRxiv2022

EquiFold: Protein Structure Prediction with a Novel Coarse-Grained Structure Representation

Designing proteins to achieve specific functions often requires in silico modeling of their properties at high throughput scale and can significantly benefit from fast and accurate protein structure prediction. We introduce EquiFold, a new end-to-end differentiable, SE(3)-equivariant, all-atom protein structure prediction model. EquiFold uses a novel coarse-grained representation of protein structures that does not require multiple sequence alignments or protein language model embeddings, inputs that are commonly used in other state-of-the-art structure prediction models. Our method relies on geometrical structure representation and is substantially smaller than prior state-of-the-art models. In preliminary studies, EquiFold achieved comparable accuracy to AlphaFold but was orders of magnitude faster. The combination of high speed and accuracy make EquiFold suitable for a number of downstream tasks, including protein property prediction and design.

bioinformatics↗

bioRxiv2022

DNA-Stimulated Liquid-Liquid Phase Separation by Eukaryotic Topoisomerase II Modulates Catalytic Function

Type II topoisomerases modulate chromosome supercoiling, condensation, and catenation by moving one double-stranded DNA segment through a transient break in a second duplex. How DNA strands are chosen and selectively passed to yield appropriate topological outcomes - e.g., decatenation vs. catenation - is poorly understood. Here we show that at physiological enzyme concentrations, eukaryotic type IIA topoisomerases (topo IIs) readily coalesce into condensed bodies. DNA stimulates condensation and fluidizes these assemblies to impart liquid-like behavior. Condensation induces both budding yeast and human topo IIs to switch from DNA unlinking to active DNA catenation, and depends on an unstructured C-terminal region, the loss of which leads to high levels of knotting and reduced catenation. Our findings establish that local protein concentration and phase separation can regulate how topo II creates or dissolves DNA links, behaviors that can account for the varied roles of the enzyme in supporting transcription, replication, and chromosome compaction.

biochemistry↗

bioRxiv2022

Design of a highly specific glutamine sensor by splitting the glutamine-binding protein

Studies on glutamine (Gln) metabolism have illuminated the vital role of Gln in cellular functions and its potential as a biomarker for disease detection. Despite the increasing interest in Gln metabolism, in-depth evaluations are challenging owing to limitations of conventional Gln-measuring methods. Thus, we developed a ligand-induced dimerization-based sensor for Gln, termed Q-SHINE, by splitting a glutamine binding protein into two separate domains. Q-SHINE enables highly accurate and convenient measurement of Gln concentration in bio-fluid samples, and the detection range is optimal for physiological Gln levels. Genetically encoded Q-SHINE sensors could also visualize intracellular Gln levels and quantify cytoplasmic and mitochondrial Gln change in living cells, which enabled detection of various cell responses to extracellular Gln supplement.

bioengineering↗

bioRxiv2022

Efficient isolation of rare B cells using next-generation antigen barcoding

The ability to efficiently isolate antigen-specific B cells in high throughput will greatly accelerate the discovery of therapeutic monoclonal antibodies (mAbs) and catalyze rational vaccine development. Traditional mAb discovery is a costly and labor-intensive process, although recent advances in single-cell genomics using emulsion microfluidics allow simultaneous processing of thousands of individual cells. Here we present a streamlined method for isolation and analysis of large numbers of antigen-specific B cells, including next generation antigen barcoding and an integrated computational framework for B cell multi-omics. We demonstrate the power of this approach by recovering thousands of antigen-specific mAbs, including the efficient isolation of extremely rare precursors of VRC01-class and IOMA-class broadly neutralizing HIV mAbs.

immunology↗

bioRxiv2022

Glia-mediated cerebellar pain modulation

Clinical studies have revealed that the cerebellum is activated by noxious stimuli or pathological pain, and its removal results in somatosensory dysfunction. However, the neural circuits and molecular mechanisms underlying the processing of noxious information in the cerebellum remain unknown. Using two-photon microscopy and optogenetics in mice, we found that the locus coeruleus (LC) terminals in the cerebellar cortex release noradrenaline (NA) in response to cutaneous noxious electrical stimuli. Most Bergmann glia (BG) accumulated this LC-NA noxious information by increasing intracellular calcium in an integrative manner. This global calcium activation of BG, referred to as "flare," was also elicited in response to an intraplantar capsaicin injection. Chemogenetic inactivation of LC terminals or BG in the cerebellar cortex suppressed BG flares and reduced licking, a nocifensive behavior associated with capsaicin-induced pain. BG-specific knockdown of -1 adrenergic receptors also suppressed capsaicin-induced BG flares and licking. Chemogenetic activation of BG or an intraplantar capsaicin injection reduced Purkinje cell firings, which disinhibited the output activity of the deep cerebellar nuclei. These results suggest that BG in the cerebellar cortex play an essential role in computing noxious information ascending from the LC and modulate pain-related behaviors by controlling the activity of the cerebellar neural circuits. One Sentence SummaryBergmann glia mediate noxious information processing in the cerebellum

neuroscience↗

bioRxiv2022

SARS-CoV-2 Spike N-Terminal Domain modulates TMPRSS2-dependent viral entry and fusogenicity

Over 20 mutations have been identified in the N-Terminal Domain (NTD) of SARS-CoV-2 spike and yet few of them are fully characterised. Here we first examined the contribution of the NTD to infection and cell-cell fusion by constructing different VOC-based chimeric spikes bearing B.1617 lineage (Delta and Kappa variants) NTDs and generating spike pseudotyped lentivirus (PV). We found the Delta NTD on a Kappa or WT background increased spike S1/S2 cleavage efficiency and virus entry, specifically in Calu-3 lung cells and airway organoids, through use of TMPRSS2. We have previously shown Delta spike confers rapid cell-cell fusion kinetics; here we show that increased fusogenicity can be conferred to WT and Kappa variant spikes by transfer of the Delta NTD. Moving to contemporary variants, we found that BA.2 had higher entry efficiency in a range of cell types as compared to BA.1. BA.2 showed higher fusogenic activity than BA.1, but the BA.2 NTD could not confer higher fusion to BA.1 spike. There was low efficiency of TMPRSS2 usage by both BA.1 and BA.2, and chimeras of Omicron BA.1 and BA.2 spikes with a Delta NTD did not result in more efficient use of TMRPSS2 or cell-cell fusogenicity. We conclude that the NTD allosterically modulates S1/S2 cleavage and spike-mediated functions such as entry and cell-cell fusion in a spike context dependent manner, and allosteric interactions may be lost when combining regions from more distantly related spike proteins. These data may explain the lack of successful SARS-CoV-2 inter-variant recombinants bearing breakpoints within spike.

microbiology↗