Biology subjects

Chen, M.

Publications and source records attributed to Chen, M..

At least 343 records · Page 19Linked to original sources

bioRxiv2022

An international scholastic network to generate LexA enhancer-trap lines for Drosophila

Conditional gene regulation in Drosophila through binary expression systems like the LexA-LexAop system provides a superb tool for investigating gene and tissue function. To increase the availability of defined LexA enhancer trap insertions, we present molecular, genetic and tissue expression studies of 301 novel Stan-X LexA enhancer traps derived from mobilization of the index SX4 line. This includes insertions into distinct loci on the X, II and III chromosomes that were not previously associated with enhancer traps or targeted LexA constructs, an insertion into ptc, and eleven insertions into natural transposons. A subset of enhancer traps was expressed in CNS neurons known to produce and secrete insulin, an essential regulator of growth, development and metabolism. Fly lines described here were generated and characterized through studies by students and teachers in an international network of genetics classes at public, independent high schools, and universities serving a diversity of students, including those underrepresented in science. Thus, a unique partnership between secondary schools and university-based programs has produced and characterized novel resources in Drosophila, establishing instructional paradigms devoted to unscripted experimental science.

genetics↗

bioRxiv2022

Caught in the Act: targeted mutagenesis of the herpesvirus glycoprotein B central helix captures fusion transition states

Herpesviruses remain a burden for animal and human health, including the medically important varicella-zoster virus (VZV). Membrane fusion mediated by conserved core glycoproteins, the fusogen gB and the heterodimer gH-gL, enables herpesvirus cell entry. The ectodomain of gB orthologs has five domains and is proposed to transition from a prefusion to postfusion conformation but the functional relevance of the domains for this transition remains poorly defined. Structure-function studies of the VZV gB DIII central helix were performed targeting residues 526EHV528. Critically, a H527P mutation captured gB in a prefusion conformation as determined by cryo-EM, a loss of membrane fusion in a virus free assay, and failure of recombinant VZV to spread in cell monolayers. Importantly, two predominant cryo-EM structures of gB[H527P] were identified by 3D classification and focused refinement, suggesting they represented gB conformations in transition. These studies reveal gB DIII as a critical element for herpesvirus gB fusion function.

microbiology↗

bioRxiv2022

Cholecystokinin from the Rhinal Cortex Facilitates Motor Skill Learning

Cholecystokinin (CCK) is an essential modulator for neuroplasticity in sensory and emotional domains. Here, we investigated the role of CCK in motor learning using a single pellet reaching task in mice. Mice with a knockout of cck gene (CCK-/-) or blockade of CCK-B receptor (CCKBR) showed defective motor learning ability; the success rate of retrieving reward remained at the baseline level compared to the wildtype mice with significantly increased success rate. We observed no long-term potentiation (LTP) upon high-frequency stimulation (HFS) in the motor cortex of CCK-/- mice, indicating a possible association between motor learning deficiency and neuronal plasticity in the motor cortex. In vivo calcium imaging demonstrated that the deficiency of CCK signalling disrupted the refinement of population neuronal activity in the motor cortex during motor skill training. Anatomical tracing revealed direct projections from CCK-expressing neurons in the rhinal cortex to the motor cortex. Inactivating the CCK neurons in the rhinal cortex using chemogenetic methods significantly suppressed motor learning, and intraperitoneal application of CCK4, a tetrapeptide CCK agonist, rescued the motor learning deficits of CCK-/- mice. In summary, our results suggest that CCK, which could be provided from the rhinal cortex, enables neuroplasticity in the motor cortex leading to motor skill learning.

neuroscience↗

bioRxiv2022

USP10 strikes down Wnt/β-catenin signaling by dual-wielding deubiquitinase activity and phase transition potential

Wnt/{beta}-catenin signaling is a conserved pathway crucially governing development, homeostasis and oncogenesis. Discovery of novel regulators holds great values in both basic and translational research. Through screening, we identified a deubiquitinase (DUB) USP10 as a novel and critical modulator of {beta}-catenin. Mechanistically, USP10 binds to key scaffold Axin1 via conserved motifs and stabilizes Axin1 through K48-linked deubiquitination, and surprisingly, tethers Axin1 and {beta}-catenin physically while promoting phase separation for {beta}-catenin suppression regardless of its enzymatic activity. Functionally, USP10 prominently regulates embryonic development and intestinal homeostasis by antagonizing {beta}-catenin via DUB activity. In colorectal cancer, USP10 substantially represses cancer growth mainly through physical binding compensation and phase separation promotion and correlates with Wnt/{beta}-catenin magnitude clinically. Collectively, we discovered USP10 functioning in multiple biological processes against {beta}-catenin and unearthed a novel enzyme-dependent and -independent "dual-regulating" mechanism by which USP10 utilizes parallelly and context-dependently. USP10 inhibitor was suggested in treating certain Wnt-related diseases.

cell biology↗

bioRxiv2022

Cooperatively Designed Aptamer-PROTACs for Spatioselective Degradation of Nucleocytoplasmic Shuttling Protein for Enhanced Combinational Therapy

Nucleocytoplasmic shuttling proteins (NSPs) has emerged as a promising class of therapeutic targets for many diseases including cancer. However, most reported NSPs-based therapies largely rely on small molecule inhibitors with limited efficacy and off-target effects. Proteolysis targeting chimera (PROTAC) represents a revolutionary inhibitory modality for targeted protein degradation with many advantages, including substoichiometric catalytic activity, improved selectivity, and high efficacy. However, the majority of reported PROTACs so far are still limited to the degradation of cytoplasmic proteins and lack of tumor-specific targeting. To realize the full potential of the PROTAC technology and broaden its applications for the degradation of NSPs, we herein report a conceptual approach for the design of a new archetype of PROTAC (denoted as PS-ApTCs) by introducing phosphorothioate-modified AS1411 aptamer to a ligand of the CRBN E3 ligase, realizing tumor-targeting and spatioselective degradation with improved efficacy. We have demonstrated that PS-ApTCs is capable of effectively degrading nucleolin in target cell membrane and cytoplasm but not in the nucleus, in a CRBN- and proteasome-dependent manner. In addition, PS-ApTCs exhibits superior antiproliferation, pro-apoptotic, and cell cycle arrest potencies. Importantly, we demonstrate for the first time that combination of PS-ApTCs-mediated nucleolin degradation with aptamer-drug conjugates-based chemotherapy can enable an AND-gated synergistic effect on tumor inhibition. Collectively, our results suggest that PS-ApTCs possess the ability to expand the PROTACs toolbox to even wider range of targets in subcellular localization and accelerate the discovery of new combinational therapeutic approaches. For Table of Contents Only O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=99 SRC="FIGDIR/small/514682v1_ufig1.gif" ALT="Figure 1"> View larger version (47K): org.highwire.dtl.DTLVardef@ed8331org.highwire.dtl.DTLVardef@1400b91org.highwire.dtl.DTLVardef@1b795a6org.highwire.dtl.DTLVardef@1c1f141_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2022

The BRAIN Initiative Cell Census Data Ecosystem: A User's Guide

Characterizing cellular diversity at different levels of biological organization across data modalities is a prerequisite to understanding the function of cell types in the brain. Classification of neurons is also required to manipulate cell types in controlled ways, and to understand their variation and vulnerability in brain disorders. The BRAIN Initiative Cell Census Network (BICCN) is an integrated network of data generating centers, data archives and data standards developers, with the goal of systematic multimodal brain cell type profiling and characterization. Emphasis of the BICCN is on the whole mouse brain and demonstration of prototypes for human and non-human primate (NHP) brains. Here, we provide a guide to the cellular and spatial approaches employed, and to accessing and using the BICCN data and its extensive resources, including the BRAIN Cell Data Center (BCDC) which serves to manage and integrate data across the ecosystem. We illustrate the power of the BICCN data ecosystem through vignettes highlighting several BICCN analysis and visualization tools. Finally, we present emerging standards that have been developed or adopted by the BICCN toward FAIR (Wilkinson et al. 2016a) neuroscience. The combined BICCN ecosystem provides a comprehensive resource for the exploration and analysis of cell types in the brain.

bioinformatics↗

bioRxiv2022

Predicting individualized tissue gene expression profiles with multi-task learning

Predicting tissue expression profiles from peripheral surrogate samples, especially blood transcriptome, has become an effective alternative when invasive procedures are not ideal. However, existing approaches ignore tissue-shared intrinsic relevance, inevitably limiting predictive performance. Here, we propose a unified deep learning-based multi-task learning framework, Multi-tissue Transcriptome Mapping (MTM), enabling the prediction of individualized expression profiles from any available tissue of an individual. By jointly leveraging individualized cross-tissue information through multi-task learning, MTM achieves superior sample-level and gene-level performance. With the high prediction accuracy and the ability to preserve individualized biological variations, MTM could facilitate both fundamental and clinical biomedical research.

bioinformatics↗

bioRxiv2022

Intermittent hypoxia therapy engages multiple longevity pathways to double lifespan in C.elegans

Genetic activation of the hypoxia response robustly extends lifespan in C. elegans, while environmental hypoxia shows more limited benefit. Here we describe an intermittent hypoxia therapy (IHT) able to double the lifespan of wildtype worms. The lifespan extension observed in IHT does not require HIF-1 but is partially blocked by loss of DAF-16/FOXO. RNAseq analysis shows that IHT triggers a transcriptional state distinct from continuous hypoxia and affects down-stream genes of multiple longevity pathways. We performed a temperature sensitive forward genetic screen to isolate mutants with delayed nuclear localization of DAF-16 in response to IHT and suppression of IHT longevity. One of these mutations mapped to the enzyme Inositol Polyphosphate MultiKinase (IPMK-1). ipmk-1 mutants, like daf-16 mutants, partially suppress the benefits of IHT, while other effectors of phosphatidyl inositol signaling pathways (PLC{beta}4, IPPK, Go/i) more robustly suppress IHT longevity. One-Sentence SummaryIntermittent hypoxia therapy is frequency dependent, HIF independent, and requires FOXO, PLC{beta}, Go/i, IPMK, and IPPK.

physiology↗

bioRxiv2022

Increased public health threat of avian-origin H3N2 influenza virus during evolution in dogs

Influenza A viruses in animal reservoirs repeatedly cross species barrier to infect humans. Once an animal-borne virus with novel antigenicity acquired the efficient human to human transmissibility, it will become epidemic in the population. Dogs are the closest animal companions to humans and canine respiratory tract expresses both SA2,3-(avian type) and 2,6-Gal (human type) receptors. However, the role of dogs in the ecology of influenza viruses is unclear. H3N2 avian influenza viruses transmitted to dogs around 2006 and have formed stable lineages. The long-term epidemic of avian-origin H3N2 virus in canine offers the best models to investigate the effect of dogs on the evolution of influenza viruses. Here, we carried out a systematic and comparative identification of the biological characteristics of H3N2 canine influenza viruses (CIVs) isolated in the worldwide over 10 years. We found that during the adaptation of H3N2 CIVs to dogs, H3N2 CIVs became to recognize the human-like SA2,6-Gal receptor, gradually increased HA acid stability and replication ability in human airway epithelial cells, and acquired a 100% transmission rate via respiratory droplet in ferret model, which were essential hallmarks of being adapted to humans. We also identified that the frequency of substitutions related to human adaptation has gradually increased in H3N2 CIVs, and determined four cumulative molecular changes responsible for the increased airborne transmission ability in ferrets. Our results suggested that canine may serve as an intermediate for the adaptation of avian influenza virus to human. Continuous surveillance coordinated with risk assessment for CIVs is necessary.

microbiology↗

bioRxiv2022

Emergency hematopoiesis proceeds without contribution of hematopoietic stem cells

Hematopoietic stem cells (HSCs) are the ultimate source of blood and immune cells. Under homeostatic conditions, these cells are considered a quiescent reserve population. However, it is not clear to what extent HSCs participate in emergency responses. Herein, we use fate mapping and proliferation tracking mouse models, which cumulatively record HSC activity in situ. We observed no direct contribution of HSCs to mature blood cell regeneration in response to common hematopoietic emergencies, including inflammation or blood loss. Innate immune training, in which HSCs were proposed to store and integrate information on previous infections, did not alter HSC activity upon secondary exposure. Only severe myeloablation resulted in a robust increase of HSC contribution. Our data demonstrates that HSCs do not directly participate in the regeneration of mature blood cells and therefore do not represent a reserve population to compensate for physiological hematopoietic perturbations.

cell biology↗

bioRxiv2022

The protein phosphatases MoPtc1 and MoPtc2 are induced during pathogen-host interactions and play synergistic roles in regulating MAPK pathways in Magnaporthe oryzae

Reversible protein phosphorylation is essential in cellular signal transduction. The rice blast fungus Magnaporthe oryzae, contains six putative type 2c protein phosphatases namely; MoPtc1, MoPtc2, MoPtc5, MoPtc6, MoPtc7 and MoPtc8 respectively. In this study, we carried out transcription expression analysis and found that MoPtc1, MoPtc2 and MoPtc7 are significantly induced during pathogen-host interactions. Subsequent deletions of MoPtc1 or MoPtc2 not only resulted in increased sensitivity to cell wall stress mediated by Congo red but also increased phosphorylation of Mps1-MAPK. By immunoblot analysis, we found that deletion of both MoPtc1 and MoPtc2 resulted in overstimulation of both the HOG1 and Pmk1 pathways in M. oryzae. We also demonstrate that MoPtc1 is recruited directly to Osm1 by the adaptor protein MoNbp2 to inactivate the Osm1 during hypoosmotic stress unlike in budding yeast. Finally, we show that type 2c protein phosphatases are localized in different cellular compartments in the life cycle of Magnaporthe oryzae. Taken together type 2C protein phosphatases MoPtc1 and MoPtc2 play synergistic roles in regulating MAPK signaling pathways in M. oryzae. This work expands our understanding of the MAPK signaling regulation circuits in M. oryzae and the essential roles of type 2C phosphatases in fine-tuning phosphorylation levels of MAPK during fungal development.

molecular biology↗

bioRxiv2022

Metaplasticity: Dark exposure boosts excitability and visual plasticity in adult human cortex

AO_SCPLOWBSTRACTC_SCPLOWAn interlude of dark exposure for about one week is known to shift the excitatory/inhibitory (E/I) balance of the mammalian visual cortex, promoting cortical plasticity across species and accelerating visual recovery in animals that have experienced cortical lesions during development. However, the translational impact of our understanding of dark exposure from animal studies on humans remains elusive. Here, we use magnetic resonance spectroscopy as a probe for E/I balance in the primary visual cortex (V1) to determine the effect of 60-min dark exposure, and measure binocular combination as a behavioral assay to assess visual plasticity in 18 normally sighted human adults (13 females). To induce neuroplastic changes in the observers, 60-min monocular deprivation was performed, which is known to shift sensory eye balance in favor of the previously deprived eye. We report that prior dark exposure strengthens cortical excitability in V1 and boosts visually plasticity in normal adults. Our findings are surprising, given the fact that the interlude is very brief. We present direct evidence that an environmental manipulation that reduces intracortical inhibition can act as a metaplastic facilitator for visual plasticity in adult humans.

neuroscience↗

bioRxiv2022

Chemoenzymatic Measurement of Cell-surface Glycan in Single-cell Multiomics: LacNAc as an Example

Despite the rich information of a cells physiological state encoded in the dynamic changes of cell-surface glycans, methods of capturing glycosylation states at the single-cell level are quite limited. Here we report a chemoenzymatic single-cell N-acetyllactosamine (LacNAc) detection method via tagging the LacNAc with a specific DNA barcode. Compared to the lectin-based glycan detection, the chemoenzymatic labeling does not change the transcriptional status of immune cells and is more compatible with scRNA-seq. Integrated analysis of LacNAc and transcriptome of T cells at a single-cell level reveals that the quantity of cell-surface LacNAc is significantly upregulated in activated CD8+ T cells but maintained at the basal level in quiescent CD8+ T cells (i.e., naive and central memory T cells). Further analysis confirms that the LacNAc level is positively correlated to the glycolytic activity of CD8+ T cells at all statues. Taken together, our study demonstrates the feasibility of chemoenzymatic detection of cell-surface glycan in single-cell RNA sequencing-based multiomics with information of TCR sequence and cell-surface epitopes (i.e., scTCR and CITE-seq) and offers a new way to characterize the biological role of glycan in diversified physiological states.

immunology↗

bioRxiv2022

Regulation of iron transport is required for terminal erythroid differentiation even under iron-replete conditions that are sufficient for hemoglobinization

Iron metabolism drives key erythropoietic processes, including hemoglobinization, survival, and proliferation. Here, we developed in vivo methods to interrogate how iron regulates erythropoiesis and report that mitochondrial iron transport via MFRN1 is essential for erythroid cell cycle progression. mfrn1 embryos had severely decreased erythroid cell number caused by cell cycle arrest at G2/M. They had enlarged nuclei, suggesting a mitotic defect. Iron supplementation rescued the cell cycle defect, implicating mitochondrial iron deficiency as its cause. In contrast, fpn1 mutants, anemic from systemic iron deficiency, had less severe decreases in erythroid mitochondrial iron than mfrn1 mutants and no proliferative defects. scRNAseq and FACS analyses for cd41 (thrombocytic) and gata1 reporters indicated that developmental defects in mfrn1 mutants were largely erythroid restricted. mfrn1 mutant gata1+ erythroid progenitors were severely decreased at 3 dpf, and a further decrease in globin- expressing terminally differentiating erythroid cells. While wild-type erythroid cells mostly lost expression of the gata1 progenitor marker by 3 dpf, mfrn1 mutant erythroid cells retained gata1 expression. These data are consistent with a model where mitochondrial iron transport facilitates development of gata1+ erythroid progenitors and is required for the completion of erythropoiesis by facilitating mitosis in the terminal cell cycles. Key pointsO_LIMitochondrial iron import through MFRN1 is required for progress through the G2/M checkpoint in the terminal cell cycles leading to terminal erythroid differentiation. C_LIO_LIMitochondrial iron supply through MFRN1 is required for maintenance/proliferation of erythroid progenitors and terminal maturation into globin expressing erythroid cells. C_LI

biochemistry↗

bioRxiv2022

BCAA catabolism drives adipogenesis via an intermediate metabolite and promotes subcutaneous adipose tissue expansion during obesity

Branched-chain amino acids (BCAAs, including leucine, isoleucine, and valine) have emerged as major players in metabolic health and diseases, but the underlying mechanisms remain obscure. Here, we report that BCAA catabolism drives adipogenesis via an intermediate metabolite of leucine and promotes subcutaneous white adipose tissue (sWAT) expansion during obesity. Genetic analyses of humans and mice reveal that the BCAA catabolic pathway in WAT is strongly correlated with adipose physiology and obesity traits. Altering BCAA catabolism in mature adipocytes exerts minor effects on adiposity in mice. However, enhancing adipose BCAA catabolism via FABP4-Cre-mediated Bckdk deletion promotes diet-induced obesity while blocking adipose BCAA catabolism through Bckdha ablation does the opposite. Intriguingly, the catabolism of BCAAs elicits fat depot-specific responses and promotes sWAT extension and adipogenesis in a cell-autonomous manner. Mechanistically, BCAA catabolism drives adipocyte differentiation via an intermediate metabolite of leucine, which activates mTORC1 and polyamine synthesis from methionine to promote the expression of adipogenic master regulators. Together, these results demonstrate that BCAA catabolism promotes adipogenesis and sWAT expansion during obesity. The crosstalk between leucine and methionine metabolism driven by the catabolic intermediate highlights an unexpected regulatory role of amino acids in metabolic health and diseases.

physiology↗

bioRxiv2022

The genome of a vestimentiferan tubeworm (Ridgeia piscesae) provides insights into its adaptation to a deep-sea environment

Vestimentifera (Polychaeta, Siboglinidae) is a taxon of deep-sea worm-like animals living in the deep-sea hydrothermal vent and cold seep areas. The morphology and lifespan of Ridgeia piscesae, which is the only vestimentiferan tubeworm species found in the hydrothermal vents on the Juan de Fuca Ridge, vary greatly according to the endemic environments. Recent analyses have revealed the genomic basis of adaptation in three vent- and seep-dwelling vestimentiferan tubeworms. However, the evolutionary history and mechanism of adaptation in R. piscesae, a unique species in the family Siboglinidae, is remained to be investigated. Here we report a high-quality genome of R. piscesae collected at Cathedral vent of the Juan de Fuca Ridge. Comparative genomic analysis revealed that that the high growth rates of vent-dwelling tubeworms might derive from small genome size. The small genome sizes of these tubeworms are attributed to the repeat content but not the number of genes and intron sizes. Additionally, four genes involved in cell proliferation were subject to positive selection in the genome of R. piscesae, suggesting that, besides apoptosis, cell proliferation is important for regulating growth rate in this species.

genomics↗

bioRxiv2022

KAS-pipe2: a flexible toolkit for exploring KAS-seq and spKAS-seq data

Kethoxal-assisted ssDNA sequencing (KAS-seq) is gaining popularity as a robust and effective approach to study the dynamics of transcriptionally engaged RNA polymerases through profiling of genome-wide single-stranded DNA (ssDNA). Its latest variant, spKAS-seq, a strand-specific version of KAS-seq, has been developed to map genome-wide R-loop structures by detecting imbalances of ssDNA on two strands. However, user-friendly, open-source analysis pipelines for KAS-seq data are still lacking. Here we present KAS-pipe2 as a flexible and integrated toolkit to facilitate the analysis and interpretation of KAS-seq data. KAS-pipe2 can perform standard analyses such as quality control, read alignment, and differential RNA polymerase activity analysis. In addition, KAS-pipe2 introduces many novel features, including, but not limited to: calculation of transcriptional indexes, identification of single-stranded transcribing enhancers, and high-resolution mapping of R-loops. We use benchmark datasets to demonstrate that KAS-pipe2 provides a powerful framework to study transient transcriptional regulatory programs. KAS-pipe2 is available at https://github.com/Ruitulyu/KAS-pipe2.

bioinformatics↗

bioRxiv2022

Isoform-specific Disruption of the TP73 Gene Reveals a Critical Role for TAp73gamma in Tumorigenesis via Leptin

TP73, a member of the p53 family, is expressed as TAp73 and {Delta}Np73 along with multiple C-terminal isoforms (-{eta}). {Delta}Np73 is primarily expressed in neuronal cells and necessary for neuronal development. Interestingly, while TAp73 is a tumor suppressor and predominantly expressed in normal cells, TAp73 is found to be frequently altered in human cancers, suggesting a role of TAp73 C-terminal isoforms in tumorigenesis. To test this, the TCGA SpliceSeq database was searched and showed that exon 11 (E11) exclusion occurs frequently in several human cancers. We also found that p73 to p73{gamma} isoform switch resulting from E11 skipping occurs frequently in human prostate cancers and dog lymphomas. To determine whether p73 to p73{gamma} isoform switch plays a role in tumorigenesis, CRISPR technology was used to generate multiple cancer cell lines and a mouse model in that Trp73 E11 is deleted. Surprisingly, we found that in E11-deificient cells, p73{gamma} becomes the predominant isoform and exerts oncogenic activities by promoting cell proliferation and migration. In line with this, E11-deficient mice were more prone to obesity and B-cell lymphomas, indicating a unique role of p73{gamma} in lipid metabolism and tumorigenesis. Additionally, we found that E11-deficient mice phenocopies Trp73-deficient mice with short lifespan, infertility, and chronic inflammation. Mechanistically, Mechanistically, we showed that Leptin, a pleiotropic adipocytokine involved in energy metabolism and oncogenesis, was highly induced by p73{gamma}, necessary for p73{gamma}-mediated oncogenic activity, and associated with p73 to {gamma} isoform switch in human prostate cancer and dog lymphoma. Finally, we showed that E11-knockout promoted, whereas knockdown of p73{gamma} or Leptin suppressed, xenograft growth in mice. Our study indicates that the p73{gamma}-Leptin pathway promotes tumorigenesis and alters lipid metabolism, which may be targeted for cancer management.

cancer biology↗