Biology subjects

Liu, R.

Publications and source records attributed to Liu, R..

At least 217 records · Page 12Linked to original sources

bioRxiv2022

Circadian clock control of MRTF-SRF signaling suppresses beige adipocyte thermogenic recruitment

The morphological transformation of adipogenic progenitors into mature adipocytes requires dissolution of cytoskeleton driven by progressive loss of MRTF-SRF activity. Circadian clock confers temporal control in adipogenic differentiation, and MRTF-SRF inhibits beige adipocyte development. Here we identify that key components of the actin cytoskeleton-MRTF/SRF signaling cascade are direct transcriptional targets of circadian clock, and a clock-MRTF/SRF regulatory axis suppresses beige adipocyte thermogenic recruitment. Genetic loss- or gain-of-functions of the core clock regulator, Brain and Muscle Arnt-like 1 (Bmal1), altered actin cytoskeleton organization, cell shape and SRF activity. Genes involved in actin dynamic-MRTF-SRF pathway display diurnal expression profiles in beige adipose tissue, with identification of Bmal1 direct transcriptional regulation. Using beige adipocyte-selective genetic models together with pharmacological approaches, we demonstrate that Bmal1 inhibits beige adipogenesis and thermogenic capacity via the MRTF-SRF pathway. Selective ablation of Bmal1 induced beigeing, whereas its targeted overexpression attenuated the beige thermogenic program resulting in obesity. Collectively, our findings define a novel circadian clock control in actin cytoskeleton-MRTF-SRF signaling that suppresses beige adipogenesis to maintain metabolic homeostasis.

cell biology↗

bioRxiv2022

A full spectrum PNGase activity analysis of R328 mutations on NGLY1

In humans, N-glycanase 1 (NGLY1; Peptide: N-glycanase, PNGase) is responsible for the deglycosylation of misfolded glycoproteins. Pathogenic mutations in NGLY1 cause a clinical condition known as congenital disorder of deglycosylation (NGLY1-CDDG), a rare autosomal recessive disease first reported in 2012. Although NGLY1-CDDG was diagnosed through whole-exome or whole-genome sequencing and by evaluating the expression levels of NGLY1, the clinical relevance of a detected mutation in NGLY1 needs to be further confirmed. In this study, an in vitro enzymatic assay system was established to evaluate the thermal stability and substrate specificity of NGLY1, as well as the optimum reaction conditions for its activity. A panel of all mutations at the amino acid site R328 in NGLY1 was subjected to this assay. The results revealed that R328A, R328D, R328E, R328F, R328G, R328I, R328P, R328V, R328W, and R328Y were dysfunctional mutations (10/19); NGLY1 mutations with R328H and R328T exhibited similar activity as wild-type NGLY1 (2/19); and NGLY1 mutations with R328C, R328K, R328L, R328M, R328N, R328Q, and R328S showed decreased activity (7/19) compared to wild-type NGLY1. In addition, the effect of potential regulatory compounds, including N-acetyl-L-cysteine and dithiothreitol, on NGLY1 was examined. This in vitro assay may serve as a standard protocol to facilitate rapid diagnosis of all mutations on NGLY1 and a practical screening method for drugs and compounds with potential therapeutic value for NGLY1-CDDG patients.

biochemistry↗

bioRxiv2022

Identification and receptor mechanism of TIR-catalyzed small molecules in plant immunity

Plant nucleotide-binding leucine-rich-repeat receptors (NLRs) with an N-terminal toll/interleukin-1 receptor (TIR) domain sense pathogen effectors to enable TIR-encoded NADase activity for immune signaling. TIR-NLR (TNL) signaling requires conserved helper NLRs NRG1 and ADR1 and the lipase-like protein EDS1 that functions as a heterodimer with each of its paralogs PAD4 and SAG101. We show that TIR-containing proteins catalyze production of 2-(5-phosphoribosyl)-5-adenosine mono-/di-phosphate (pRib-AMP/ADP) in vitro and in planta. Biochemical and structural data demonstrate that EDS1-PAD4 is a receptor complex for pRib-AMP/ADP. pRib-ADP binding triggers a conformational change in the PAD4 C-terminal domain to allosterically promote EDS1-PAD4 interaction with ADR1-L1 but not NRG1A. Our study identifies TIR-catalyzed pRib-AMP/ADP as a missing link in TIR signaling via EDS1-PAD4 and as likely second messengers for plant immunity.

plant biology↗

bioRxiv2022

Antigen-dependent inducible T cell reporter system for PET imaging of breast cancer and glioblastoma

For the past several decades, chimeric antigen receptor T cell (CAR T) therapies have shown promise in the treatment of cancers. These treatments would greatly benefit from companion imaging biomarkers to follow the trafficking of T cells in vivo. Using synthetic biology, we engineered T cells with a chimeric receptor SyNthetic Intramembrane Proteolysis Receptor (SNIPR) that induces overexpression of an exogenous reporter gene cassette upon recognition of specific tumor markers. We then applied a SNIPR-based positron emission tomography (PET) reporter system to two cancer-relevant antigens, human epidermal growth factor receptor 2 (HER2) and epidermal growth factor receptor variant III (EGFRvIII), commonly expressed in breast and glial tumors respectively. Antigen-specific reporter induction of the SNIPR-PET T cells was confirmed in vitro using GFP fluorescence, luciferase luminescence, and the HSV-TK PET reporter with [18F]FHBG. T cells associated with their target antigens were successfully imaged using PET in dual xenograft HER2+/HER2- and EGFRvIII+/EGFRvIII-animal models, with > 10-fold higher [18F]FHBG signals seen in antigen-expressing tumors versus the corresponding controls. The main innovation described is therefore PET detection of T cells via specific antigen-induced signals, in contrast to reporter systems relying on constitutive gene expression.

synthetic biology↗

bioRxiv2022

Glutamate indicators with improved activation kinetics and localization for imaging synaptic transmission

The fluorescent glutamate indicator iGluSnFR enables imaging of neurotransmission with genetic and molecular specificity. However, existing iGluSnFR variants exhibit saturating activation kinetics and are excluded from post-synaptic densities, limiting their ability to distinguish synaptic from extrasynaptic glutamate. Using a multi-assay screen in bacteria, soluble protein, and cultured neurons, we generated novel variants with improved kinetics and signal-to-noise ratios. We also developed surface display constructs that improve iGluSnFRs nanoscopic localization to post-synapses. The resulting indicator, iGluSnFR3, exhibits rapid non-saturating activation kinetics and reports synaptic glutamate release with improved linearity and increased specificity versus extrasynaptic signals in cultured neurons. In mouse visual cortex, imaging of iGluSnFR3 at individual boutons reported single electrophysiologically-observed action potentials with high specificity versus non-synaptic transients. In vibrissal sensory cortex Layer 4, we used iGluSnFR3 to characterize distinct patterns of touch-evoked feedforward input from thalamocortical boutons and both feedforward and recurrent input onto L4 cortical neuron dendritic spines.

neuroscience↗

bioRxiv2022

sgcocaller and comapr: personalised haplotype assembly and comparative crossover map analysis using single gametes

Profiling gametes of an individual enables the construction of personalised haplotypes and meiotic crossover landscapes, now achievable at larger scale than ever through the availability of high-throughput single-cell sequencing technologies. However, high-throughput single-gamete data commonly have low depth of coverage per gamete, which challenges existing gametebased haplotype phasing methods. In addition, haplotyping a large number of single gametes from high-throughput singlecell DNA sequencing data and constructing meiotic crossover profiles using existing methods requires intensive processing. Here, we introduce efficient software tools for the essential tasks of generating personalised haplotypes and calling crossovers in gametes from single-gamete DNA sequencing data (sgcocaller), and constructing, visualising, and comparing individualised crossover landscapes from single gametes (comapr). With additional data pre-possessing, the tools can also be applied to bulk-sequenced samples. We demonstrate that sgcocaller is able to generate impeccable phasing results for high-coverage datasets, on which it is more accurate and stable than existing methods, and also performs well on low-coverage single-gamete sequencing datasets for which current methods fail. Our tools achieve highly accurate results with user-friendly installation, comprehensive documentation, efficient computation times and minimal memory usage.

bioinformatics↗

bioRxiv2022

Harringtonine has the effects of double blocking SARS-CoV-2 membrane fusion

Fusion with host cell membrane is the main mechanism of infection of SARS-CoV-2. Here, we propose a new strategy to double block SARS-CoV-2 membrane fusion by using Harringtonine (HT), a small-molecule antagonist. By using cell membrane chromatography (CMC), we found that HT specifically targeted the SARS-CoV-2 S protein and host cell TMPRSS2, and then confirmed that HT can inhibit pseudotyped virus membrane fusion. Furthermore, HT successfully blocked SARS-CoV-2 infection, especially in the delta and Omicron mutant. Since HT is a small-molecule antagonist, it is minimally affected by the continuous variation of SARS-CoV-2. Our findings show that HT is a potential small-molecule antagonist with a new mechanism of action against SARS-CoV-2 infection, and thus HT mainly targets the S protein, and thus, greatly reduces the damage of the S proteins autotoxicity to the organ system, has promising advantages in the clinical treatment of COVID-19.

pharmacology and toxicology↗

bioRxiv2022

Quantitative proteomics of the CDK9 interactome reveals a novel function of the HSP90-CDC37-P-TEFb complex for BETi-induced HIV-1 latency reactivation

Brd4 has been intensively investigated as a promising drug target because of its implicated functions in oncogenesis, inflammation and HIV-1 transcription. The formation of the Brd4-P-TEFb (CDK9/Cyclin T1) complex and its regulation of transcriptional elongation is critical for HIV latency reactivation and expression of many oncogenes. To further investigate the mechanism of the Brd4-P-TEFb complex in controlling elongation, mass spectrometry-based quantitative proteomics of the CDK9 interactome was performed. Upon treatment with the selective BET bromodomain inhibitor (BETi) JQ1, 535 proteins were successfully identified with high confidence as CDK9-interacting proteins. Among them, that increased bindings of HSP90 and CDC37 to CDK9 were particularly striking, and our data suggest that the HSP90-CDC37-P-TEFb complex is involved in controlling P-TEFbs dynamic equilibrium during BETi-induced HIV-1 latency reactivation. Furthermore, the HSP90-CDC37-P-TEFb complex directly regulates HIV-1 transcription and relies on the recruitment by heat shock factor 1 (HSF1) for binding to the HIV-1 promoter. These results advance the understanding of HSP90-CDC37-P-TEFb in HIV-1 latency reversal and enlighten the development of potential strategies to eradicate HIV-1 using a combination of targeted drugs.

molecular biology↗

bioRxiv2022

Pericentrin is a Kinesin-1 Activator that Drives Centriole Motility

Centrosome positioning is essential for their function. Typically, centrosomes are transported to various cellular locations through the interaction of centrosome nucleated microtubules with motor proteins. However, it remains unknown how centrioles migrate in cellular contexts in which centrioles do not nucleate microtubules. Here, we demonstrate that during interphase inactive centrioles move directly along the non-centrosomal microtubule network as cargo for the motor protein Kinesin-1. We identify Pericentrin-Like-Protein (PLP) as a novel Kinesin-1 interacting molecule essential for centriole motility. PLP directly interacts with the cargo binding domain of Kinesin-1 and they comigrate on microtubules in vitro. Finally, we demonstrate that PLP-Kinesin-1 dependent transport is essential for centrosome asymmetry age-dependent centrosome inheritance in asymmetric stem cell division.

cell biology↗

bioRxiv2022

PlasmidMaker: a Versatile, Automated, and High Throughput End-to-End Platform for Plasmid Construction

Plasmids are used extensively in basic and applied biology. However, design and construction of plasmids, specifically the ones carrying complex genetic information, remains one of the most time-consuming, labor-intensive, and rate-limiting steps in performing sophisticated biological experiments. Here, we report the development of a versatile, robust, automated end-to-end platform named PlasmidMaker that allows error-free construction of plasmids with virtually any sequences in a high-throughput manner. This platform consists of a most versatile DNA assembly method using Pyrococcus furiosus Argonaute (PfAgo)-based artificial restriction enzymes, a user-friendly frontend for plasmid design, and a backend that streamlines the workflow and integration with a robotic system. As a proof of concept, we used this platform to generate 101 plasmids from six different species ranging from 5 to 18 kb in size from up to 11 DNA fragments within 3 days. PlasmidMaker should greatly expand the potential of synthetic biology.

synthetic biology↗

bioRxiv2021

The microRNA miR-132 is a key regulator of lymphatic vascular remodelling

Lymphangiogenesis (growth of new lymphatic vessels), and lymphatic remodelling more broadly, are important for disease progression in cancer, lymphedema and the pulmonary disease lymphangioleiomyomatosis. Multiple molecular pathways which signal for aspects of lymphangiogenesis are known but little is understood about their co-ordinate regulation in lymphatic endothelial cells (LECs). Small RNA molecules co-ordinately regulate complex biological processes, but knowledge about their involvement in lymphangiogenesis is limited. Here we used high-throughput small RNA sequencing of LECs to identify microRNAs (miRs) regulating lymphatic remodelling driven by the lymphangiogenic growth factors VEGF-C and VEGF-D. We identified miR-132 as up-regulated by both growth factors, and demonstrated that inhibiting miR-132 in LECs in vitro blocked cell proliferation and tube formation, key steps in lymphangiogenesis. We showed that miR-132 is expressed in human LECs in vivo in the lymphatics of human breast tumours expressing VEGF-D. Importantly, we demonstrated that inhibiting miR-132 in vivo blocked many aspects of lymphangiogenesis in mice. Finally, we identified mRNAs regulated by miR-132 in LECs, by sequencing after RNA-protein cross-linking and Argonaute immunoprecipitation, which demonstrated how miR-132 co-ordinately regulates signalling pathways in lymphangiogenesis. This study shows miR-132 is a critical regulator of lymphangiogenesis and a potential target for therapeutically manipulating lymphatic remodelling in disease.

cell biology↗

bioRxiv2021

Colorimetric and fluorescent TRAP assays for visualising and quantifying fish osteoclast activity

Histochemical detection of tartrate-resistant acid phosphatase (TRAP) activity is a fundamental technique for visualizing osteoclastic bone resorption and assessing osteoclast activity status in tissues. This approach has mostly employed colorimetric detection, which has limited quantification of activity in situ and co-labelling with other skeletal markers. Here we report simple colorimetric and fluorescent TRAP assays in zebrafish and medaka, two important model organisms for investigating the pathogenesis of bone disorders. We show fluorescent TRAP staining, utilising the ELF97 substrate, is a rapid, robust and stable system to visualise and quantify osteoclast activity in zebrafish, and is compatible with other fluorescence stains, transgenic lines and antibody approaches. Using this approach, we show that TRAP activity is predominantly found around the base of the zebrafish pharyngeal teeth, where osteoclast activity state appears to be heterogeneous.

cell biology↗

bioRxiv2021

ME-ACP: Multi-view Neural Networks with Ensemble Model for Identification of Anticancer Peptides

Cancer remains one of the most threatening diseases, which kills millions of lives every year. As a promising perspective for cancer treatments, anticancer peptides (ACPs) overcome a lot of disadvantages of traditional treatments. However, it is time-consuming and expensive to identify ACPs through conventional experiments. Hence, it is urgent and necessary to develop highly effective approaches to accurately identify ACPs in large amounts of protein sequences. In this work, we proposed a novel and effective method named ME-ACP which employed multi-view neural networks with ensemble model to identify ACPs. Firstly, we employed residue level and peptide level features preliminarily with ensemble models based on lightGBMs. Then, the outputs of lightGBM classifiers were fed into a hybrid deep neural network (HDNN) to identify ACPs. The experiments on independent test datasets demonstrated that ME-ACP achieved competitive performance on common evaluation metrics.

bioinformatics↗

bioRxiv2021

A marine fungus efficiently degrades polyethylene

Plastics pollution has been a global concern. Huge quantities of polyethylene (PE), the most abundant and refractory plastic in the world, have been accumulating in the environment causing serious ecological problems. However, the paucity of microorganisms and enzymes that efficiently degrading PE seriously impedes the development of bio-products to eliminate this environmental pollution. Here, by screening hundreds of plastic waste-associated samples, we isolated a fungus (named Alternaria sp. FB1) that possessing a prominent capability of colonizing, degrading and utilizing PE. Strikingly, the molecular weight of PE film decreased 95% after the fungal treatment. Using GC-MS, we further clarified that a four-carbon product (named Diglycolamine) accounted for 93.28% of all degradation products after the treatment by strain FB1. We defined potential enzymes that involved in the degradation of PE through a transcriptomic method. The degradation capabilities of two representative enzymes including a laccase and a peroxidase were verified. Lastly, a complete biodegradation process of PE is proposed. Our study provides a compelling candidate for further investigation of degradation mechanisms and development of biodegradation products of PE.

microbiology↗

bioRxiv2021

Wnt1-Cre mediated deletion of BMP7 suggests a role for neural crest-derived BMP7 in retina development and function

Neural crest (NC) contributes to various structures of the eye including cornea, ciliary body and retina. The association of NC-derived cells with hyaloid vessels in the form of pericytes is established. Similarly, persistence of NC-derived cells in the inner retina layer of the mature retina has been suggested. To date, no specific function has been attributed to them. NC-derived Bone morphogenetic protein 7 (BMP7) controls neurogenic properties in the brain and regulates glia differentiation. Here, we assessed the role of NC-derived BMP7 in the adult retina. BMP7 expression was determined using Bmp7LacZ reporter mice. BMP7 was expressed in GCL, IPL, OPL, and photoreceptors in P0, P14 and P30 retinas. Lineage tracing confirmed the presence of NC-derived cells in the GCL, INL, and ONL. Some but not all cells associated with vasculature. To test the function of NC-derived Bmp7, Bmp7fl/flWnt1cre (Bmp7ncko) mice were assessed by histological and functional methods. Loss of NC-derived cells in the GCL and INL and mild structural abnormalities were observed in the Bmp7ncko retina. Electroretinography revealed reduced a wave under photopic conditions and b wave under both scotopic and photopic conditions. The neuronal circuitry in the inner retina appeared affected, evidenced by decreased Calbindin in the GCL, IPL and INL. In the outer retina, S-opsin was increased. BMP7 expression in the mutant retina was strongly decreased at birth, but increased expression from cells other than NC was observed in the adult retina. This was associated with an increase in IBA1, suggestive that loss of NC-derived BMP7 predisposes to development of gliosis-like changes in the adult retina. Overall, our data reveal an important contribution of NC-derived BMP7 for the development and function of the inner and outer retina.

physiology↗

bioRxiv2021

Tracking neural activity from the same cells during the entire adult life of mice

Recording the activity of the same neurons over the adult life of an animal is important to neuroscience research and biomedical applications. Current implantable devices cannot provide stable recording on this time scale. Here, we introduce a method to precisely implant nanoelectronics with an open, unfolded mesh structure across multiple brain regions in the mouse. The open mesh structure forms a stable interwoven structure with the neural network, preventing probe drifting and showing no immune response and neuron loss during the yearlong implantation. Using the implanted nanoelectronics, we can track single-unit action potentials from the same neurons over the entire adult life of mice. Leveraging the stable recordings, we build machine learning algorithms that enable automated spike sorting, noise rejection, stability validation, and generate pseudotime analysis, revealing aging-associated evolution of the single-neuron activities.

neuroscience↗

bioRxiv2021

Lin28a rejuvenates muscle stem cells via mitochondrial optimization

The well-conserved correlation between juvenility and tissue regeneration was first discussed by Charles Darwin. Ectopic Lin28 is known to play an important role in somatic reprogramming and tissue regeneration, but endogenous Lin28s role in tissue homeostasis and juvenility had remained unclear. Through lineage tracing, we found that a rare subset of muscle stem cells (MuSCs) expressing Lin28a can respond to acute injury by proliferating as Pax3+ or Pax7+ MuSCs, and contribute to all types of myofibers during muscle regeneration. Compared with conventional Pax7+ MuSCs, Lin28a+ MuSCs express more Pax3 and show enhanced myogenicity in vitro. In terms of the epigenetic clock, adult Lin28a+ MuSCs lie between adult Pax7+ MuSCs and embryonic Pax7+ myoblasts according to their DNA methylation profiles. We found that Lin28a+ MuSCs upregulate several embryonic limb bud mesoderm transcription factors and could maintain a juvenile state with enhanced stem cell self-renewal and stress-responsiveness in vitro and in vivo. When combined with telomerase and TP53 inhibition to biomimic mouse Lin28a+ MuSCs, we found that Lin28a can rejuvenate and dedifferentiate aged human primary myoblasts into engraftable, self-renewing MuSCs. Mechanistic studies revealed that Lin28a activated the HIF1A pathway by optimizing mitochondrial ROS (mtROS), thereby rejuvenating MuSC self-renewal and muscle regeneration. Our findings connect the stem cell factor Lin28, mtROS metabolism and stress response pathways to the process of stem cell rejuvenation and tissue regeneration.

cell biology↗

bioRxiv2021

Mutual Regulation between PFKP and VEGF Promotes Glioblastoma Tumor Growth

Glioblastoma (GBM) is highly vascular malignant brain tumor that overexpresses vascular endothelial growth factor (VEGF) and phosphofructokinase 1 platelet isoform (PFKP), which catalyzes a rate-limiting reaction in glycolysis. However, it remains unknown whether PFKP and VEGF are reciprocally regulated during GBM tumor growth. Here, we show that PFKP promotes EGFR activation-induced VEGF expression in HIF-1-dependent and -independent manners in GBM cells. Importantly, we demonstrate that EGFR-phosphorylated PFKP Y64 has critical roles in the AKT/SP1-mediated transcriptional expression of HIF-1 and in the AKT-mediated {beta}-catenin S552 phosphorylation, to fully enhance VEGF transcription and subsequent blood vessel formation and brain tumor growth. The levels of PFKP Y64 phosphorylation in human GBM specimens positively correlate with HIF-1 expression, {beta}-catenin S552 phosphorylation, and VEGF expression. Conversely, VEGF upregulates PFKP expression in a PFKP S386 phosphorylation-dependent manner, leading to increased PFK enzyme activity, aerobic glycolysis, and proliferation in GBM cells. These findings highlight a novel mechanism underlying the mutual regulation that occurs between PFKP and VEGF for promoting GBM tumor growth and provide the therapeutic potential of targeting the PFKP/VEGF regulatory loop for GBM patients.

cancer biology↗