Biology subjects

Bai, F.

Publications and source records attributed to Bai, F..

50 records · Page 3Linked to original sources

bioRxiv2020

Discovery of baicalin and baicalein as novel, natural product inhibitors of SARS-CoV-2 3CL protease in vitro

Human infections with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) cause coronavirus disease 19 (COVID-19) and there is currently no cure. The 3C-like protease (3CLpro), a highly conserved protease indispensable for replication of coronaviruses, is a promising target for development of broad-spectrum antiviral drugs. To advance the speed of drug discovery and development, we investigated the inhibition of SARS-CoV-2 3CLpro by natural products derived from Chinese traditional medicines. Baicalin and baicalein were identified as the first non-covalent, non-peptidomimetic inhibitors of SARS-CoV-2 3CLpro and exhibited potent antiviral activities in a cell-based system. Remarkably, the binding mode of baicalein with SARS-CoV-2 3CLpro determined by X-ray protein crystallography is distinctly different from those of known inhibitors. Baicalein is perfectly ensconced in the core of the substrate-binding pocket by interacting with two catalytic residues, the crucial S1/S2 subsites and the oxyanion loop, acting as a "shield" in front of the catalytic dyad to prevent the peptide substrate approaching the active site. The simple chemical structure, unique mode of action, and potent antiviral activities in vitro, coupled with the favorable safety data from clinical trials, emphasize that baicalein provides a great opportunity for the development of critically needed anti-coronaviral drugs.

molecular biology↗

bioRxiv2020

Structure-Based Design, Synthesis and Biological Evaluation of Peptidomimetic Aldehydes as a Novel Series of Antiviral Drug Candidates Targeting the SARS-CoV-2 Main Protease

SARS-CoV-2 is the etiological agent responsible for the COVID-19 outbreak in Wuhan. Specific antiviral drug are urgently needed to treat COVID-19 infections. The main protease (Mpro) of SARS-CoV-2 is a key CoV enzyme that plays a pivotal role in mediating viral replication and transcription, which makes it an attractive drug target. In an effort to rapidly discover lead compounds targeting Mpro, two compounds (11a and 11b) were designed and synthesized, both of which exhibited excellent inhibitory activity with an IC50 value of 0.05 M and 0.04 M respectively. Significantly, both compounds exhibited potent anti-SARS-CoV-2 infection activity in a cell-based assay with an EC50 value of 0.42 M and 0.33 M, respectively. The X-ray crystal structures of SARS-CoV-2 Mpro in complex with 11a and 11b were determined at 1.5 [A] resolution, respectively. The crystal structures showed that 11a and 11b are covalent inhibitors, the aldehyde groups of which are bound covalently to Cys145 of Mpro. Both compounds showed good PK properties in vivo, and 11a also exhibited low toxicity which is promising drug leads with clinical potential that merits further studies.

biochemistry↗

bioRxiv2020

Structure-based drug design, virtual screening and high-throughput screening rapidly identify antiviral leads targeting COVID-19

A new coronavirus (CoV) identified as COVID-19 virus is the etiological agent responsible for the 2019-2020 viral pneumonia outbreak that commenced in Wuhan1-4. Currently there is no targeted therapeutics and effective treatment options remain very limited. In order to rapidly discover lead compounds for clinical use, we initiated a program of combined structure-assisted drug design, virtual drug screening and high-throughput screening to identify new drug leads that target the COVID-19 virus main protease (Mpro). Mpro is a key CoV enzyme, which plays a pivotal role in mediating viral replication and transcription, making it an attractive drug target for this virus5,6. Here, we identified a mechanism-based inhibitor, N3, by computer-aided drug design and subsequently determined the crystal structure of COVID-19 virus Mpro in complex with this compound. Next, through a combination of structure-based virtual and high-throughput screening, we assayed over 10,000 compounds including approved drugs, drug candidates in clinical trials, and other pharmacologically active compounds as inhibitors of Mpro. Six of these inhibit Mpro with IC50 values ranging from 0.67 to 21.4 M. Ebselen also exhibited promising antiviral activity in cell-based assays. Our results demonstrate the efficacy of this screening strategy, which can lead to the rapid discovery of drug leads with clinical potential in response to new infectious diseases where no specific drugs or vaccines are available.

biochemistry↗

bioRxiv2020

Selecting for Chlamydomonas reinhardtii fitness in a liquid algal growth system compatible with the International Space Station Veggie plant growth chamber

A biological life support system for spaceflight would capture carbon dioxide waste produced by living and working in space to generate useful organic compounds. Photosynthesis is the primary mechanism to fix carbon into organic molecules. Microalgae are highly efficient at converting light, water, and carbon dioxide into biomass, particularly under limiting, artificial light conditions that are a necessity in space photosynthetic production. Although there is great promise in developing algae for chemical or food production in space, most spaceflight algae growth studies have been conducted on solid agar-media to avoid handling liquids in microgravity. Here we report that breathable plastic tissue culture bags can support robust growth of Chlamydomonas reinhardtii in the Veggie plant growth chamber, which is used on the International Space Station to grow terrestrial plants. Live cultures can be stored for at least one month in the bags at room temperature. The gene set required for growth in these photobioreactors was tested through a short-wave ultraviolet light (UVC) mutagenesis and selection experiment with wild-type (CC-5082) and cw15 mutant (CC-1883) strains. Genome sequencing identified UVC-induced mutations, which were enriched for transversions and nonsynonymous mutations relative to natural variants among laboratory strains. Genes with mutations indicating positive selection were enriched for information processing genes related to DNA repair, RNA processing, translation, cytoskeletal motors, kinases, and ABC transporters. These data suggest modification of signal transduction and metabolite transport may be needed to improve growth rates in this spaceflight production system.

plant biology↗

bioRxiv2019

Enhancing Intracellular Concentration and Target Engagement of PROTACs with Reversible Covalent Chemistry

Current efforts in the proteolysis targeting chimera (PROTAC) field mostly focus on choosing an appropriate E3 ligase for the target protein, improving the binding affinities towards the target protein and the E3 ligase, and optimizing the PROTAC linker. However, due to the large molecular weights of PROTACs, their cellular uptake remains an issue. Through comparing how different warhead chemistry, reversible noncovalent (RNC), reversible covalent (RC), and irreversible covalent (IRC) binders, affects the degradation of Brutons Tyrosine Kinase (BTK), we serendipitously discover that cyano-acrylamide-based reversible covalent chemistry can significantly enhance the intracellular accumulation and target engagement of PROTACs and develop RC-1 as a reversible covalent BTK PROTAC with a high target occupancy as its corresponding kinase inhibitor and effectiveness as a dual functional inhibitor and degrader, a different mechanism-of-action for PROTACs. Importantly, this reversible covalent strategy is generalizable to improve other PROTACs, opening a path to enhance PROTAC efficacy.

pharmacology and toxicology↗

bioRxiv2019

Dynamic production and loss of flagellar filaments during the bacterial life cycle

Bacterial flagella are large extracellular protein organelles that drive bacteria motility and taxis in response to environmental changes. Previous research has focused mostly on describing the flagellar assembly, its rotation speed and power output. However, whether flagella are permanent cell structures and, if not, the circumstances and timing of their production and loss during the bacterial life cycle remain poorly understood. Here we used the single polar flagellum of Vibrio alginolyticus as our model and, using in vivo fluorescence imaging, revealed that the percentage of flagellated bacteria (PFB) in a population varies substantially across different bacterial growth phases. In the early-exponential phase, the PFB increases rapidly in respect to incubation time, mostly through widespread flagella production. In the mid-exponential phase, the PFB peaks at around 76% and the partitioning of flagella between the daughter cells is 1:1 and strictly at the old poles. After entering the stationary phase, the PFB starts to decline, mainly because daughter cells stop making new flagella after cell division. Interestingly, we discovered that bacteria can actively abandon flagella after prolonged stationary culturing, though cell division has long been suspended. Lack of glucose was found to be a major factor promoting flagellar disassembly. We also revealed that the active loss of flagella was initiated by breakage in the rod connecting the extracellular filament to the basal body formed by MS- and C-rings. Our results highlight the dynamic production and loss of flagellar filaments during the bacterial life cycle.

microbiology↗

bioRxiv2019

Effects of Shift-Work on the Carotid Artery and Cerebral Blood Flow of SHR Rats and WKY Rats

ObjectiveThe objective was to investigate the effects of shift-work (SW) on the carotid arteries.\n\nMethodsThis study used two inverted photoperiods (inverted light:dark [ILD]16:8 and ILD12:12) to create the SW model. We recorded the rhythm and performed serological tests, carotid ultrasound, magnetic resonance imaging, and carotid biopsy.\n\nResultsSW induced elevated blood pressure and increased angiotensin-II, apolipoprotein E, blood glucose, and triglycerides. SW increased the carotid intima-media thickness. SW led to the development of carotid arterial thrombosis, reduced cerebral blood flow, and increased the number of collagen fibers, expression of angiotensin receptor and low-density lipoprotein receptor in the carotid arteries. SW decreased 3-hydroxy-3-methylglutaryl-CoA reductase and nitric oxide. SW induced the atherosclerotic plaque in the aorta. Multiple results of SHR were worse than WKY rats.\n\nConclusionSW can induce metabolic disorders and elevated blood pressure. SW can cause intima-media thickening of the carotid artery and aorta atherosclerosis. SW may result in carotid arterial thrombosis and affect cerebral blood flow. Hypertension can aggravate the adverse effects of SW.

pathology↗

bioRxiv2019

Comparison of Escherichia coli surface attachment methods for single-cell, in vivo microscopy

For in vivo, single-cell imaging bacterial cells are commonly immobilised via physical confinement or surface attachment. Different surface attachment methods have been used both for atomic force and optical microscopy (including super resolution), and some have been reported to affect bacterial physiology. However, a systematic comparison of the effects these attachment methods have on the bacterial physiology is lacking. Here we present such a comparison for bacterium Escherichia coli, and assess the growth rate, size and intracellular pH of cells growing attached to different, commonly used, surfaces. We demonstrate that E. coli grow at the same rate, length and internal pH on all the tested surfaces when in the same growth medium. The result suggests that tested attachment methods can be used interchangeably when studying E. coli physiology.

systems biology↗

bioRxiv2019

Escherichia coli’s physiology can turn membrane voltage dyes into actuators

The electrical membrane potential (Vm) is one of the components of the electrochemical potential of protons across the biological membrane (proton motive force), which powers many vital cellular processes, and Vm also plays a role in signal transduction. Therefore, measuring it is of great interest, and over the years a variety of techniques has been developed for the purpose. In bacteria, given their small size, Nernstian membrane voltage probes are arguably the favourite strategy, and their cytoplasmic accumulation depends on Vm according to the Nernst equation. However, a careful calibration of Nernstian probes that takes into account the trade-offs between the ease with which the signal from the dye is observed, and the dyes interactions with cellular physiology, is rarely performed. Here we use a mathematical model to understand such trade-offs and, based on the knowledge gained, propose a general work-flow for the characterization of Nernstian dye candidates. We demonstrate the work-flow on the Thioflavin T dye in Escherichia coli, and identify conditions in which the dye turns from a Vm probe into an actuator.\n\nSIGNIFICANCE STATEMENT\n\nThe phospholipid bilayer of a biological membrane is virtually impermeable to charged molecules. Much like in a rechargeable battery, cells harness this property to store an electrical potential that fuels life reactions but also transduces signals. Measuring this electrical potential, also referred to as membrane voltage, is therefore of great interest and a variety of techniques have been employed for the purpose, starting as early as the 1930s. For the case of bacteria, which are smaller in size and possess a stiffer cell wall, arguably the most popular approach to measuring membrane voltage are Nernstian probes that accumulate across the bacterial membrane according to the Nernst potential. The present study characterizes the undesired effects Nernstian probes can have on cell physiology, which can be crucial for the accurate interpretation of experimental results. Using mathematical modelling and experiments, the study provides a general, simple workflow to characterise and minimise these effects.

biophysics↗

bioRxiv2018

A lysosomal dimmer switch regulates cellular quiescence depth

Numerous physiological and pathological phenomena are associated with the quiescent state of a cell. Cellular quiescence is a heterogeneous resting state; cells in deep than shallow quiescence require stronger growth stimulation to exit quiescence and reenter the cell cycle. Despite the importance of quiescent cells such as stem and progenitor cells to tissue homeostasis and repair, cellular mechanisms controlling the depth of cellular quiescence are poorly understood. Here we began by analyzing transcriptome changes as rat embryonic fibroblasts moved progressively deeper into quiescence under increasingly longer periods of serum starvation. We found that lysosomal gene expression was significantly upregulated in deep than shallow quiescence, which compensated for gradually reduced autophagy flux observed during quiescence deepening. Consistently, we show that inhibiting lysosomal function drove cells deeper into quiescence and eventually into a senescence-like irreversibly arrested state. By contrast, increasing lysosomal function progressively pushed cells into shallower quiescence. That is, lysosomal function modulates quiescence depth continuously like a dimmer switch. Mechanistically, we show that lysosomal function prevents quiescence deepening by reducing oxidative stress in the cell. Lastly, we show that a gene expression signature developed by comparing deep and shallow quiescent cells can correctly classify senescent and aging cells in a wide array of cell lines in vitro and tissues in vivo, suggesting that quiescence deepening, senescence, and aging may share common regulatory mechanisms.

cell biology↗

bioRxiv2018

RNA Binding Motif Protein 48 is required for U12 splicing and maize endosperm differentiation

The last eukaryotic common ancestor had two classes of introns that are still found in most eukaryotic lineages. Common U2-type and rare U12-type introns are spliced by the major and minor spliceosomes, respectively. Relatively few splicing factors have been shown to be specific to the minor spliceosome. We found that the maize RNA Binding Motif Protein48 (RBM48) is a U12 splicing factor that functions to promote cell differentiation and repress cell proliferation. RBM48 is coselected with the U12 splicing factor, ZRSR2/RGH3. Protein-protein interactions between RBM48, RGH3, and U2 Auxiliary Factor (U2AF) subunits suggest major and minor spliceosome factors may form complexes during intron recognition. Human RBM48 interacts with ARMC7. Maize RBM48 and ARMC7 have a conserved protein-protein interaction. These data predict that RBM48 is likely to function in U12 splicing throughout eukaryotes and that U12 splicing promotes endosperm cell differentiation in maize.

plant biology↗

bioRxiv2018

Reduced but not Enhanced Default Mode Network Functional Connectivity in Major Depressive Disorder: Evidence from 25 Cohorts in the REST-meta-MDD Project

Major Depressive Disorder (MDD) is common and disabling, but its neural pathophysiology remains unclear. Functional brain network studies in MDD have largely had limited statistical power and data analysis approaches have varied widely. The REST-meta-MDD Project of resting-state fMRI (R-fMRI) addresses these issues. The 25 research groups in China composing the REST-meta-MDD Project contributed R-fMRI data of 1,300 patients with MDD and 1,128 normal controls (NCs). The data were preprocessed locally with a standardized protocol prior to aggregated group analyses. We focused on functional connectivity (FC) within the default mode network (DMN), frequently reported to show increased FC in MDD. We found decreased instead of increased DMN FC when comparing 848 MDDs with 794 NCs from 17 sites after data exclusion. We found FC reduction only in recurrent MDD, not in first-episode drug-naive MDD. Decreased DMN FC was associated with medication usage but not with MDD duration. DMN FC was also positively related to symptom severity but only in recurrent MDDs. Exploratory analyses also revealed alterations of local intrinsic activity in MDD. We confirmed the key role of DMN in MDD but found reduced rather than increased FC within the DMN. Future studies should test whether decreased DMN FC mediates treatment response. This manuscript announces the publicly available resting-state fMRI indices of the REST-meta-MDD consortium shared via the R-fMRI Maps Project.\n\nSIGNIFICANCE STATEMENTFunctional connectivity within the default mode network in major depressive disorder patients has been frequently reported abnormal but with contradicting directions in previous small sample size studies. By creating the REST-meta-MDD consortium containing neuroimaging data of 1,300 depressed patients and 1,128 normal controls from 25 research groups in China, we found decreased default mode network functional connectivity in depressed patients, driven by patients with recurrent depression, and associated with current medication treatment but not with disease duration. These findings suggest that default mode network functional connectivity remains a prime target for understanding the pathophysiology of depression, with particular relevance to revealing mechanisms of effective treatments.

neuroscience↗

bioRxiv2018

Perforin-2 Permeabilizes the Envelope of Phagocytosed Bacteria

Perforin-2, the product of the MPEG1 gene, limits the spread and dissemination of bacterial pathogens in vivo. It is highly expressed in murine and human phagocytes, and macrophages lacking Perforin-2 are compromised in their ability to kill phagocytosed bacteria. In this study we used Salmonella typhimurium as a model intracellular pathogen to elucidate the mechanism of Perforin-2 s bactericidal activity. In vitro Perforin-2 was found to facilitate the degradation of antigens contained within the envelope of phagocytosed bacteria. In contrast, degradation of a representative surface antigen was found to be independent of Perforin-2. Consistent with our in vitro results a protease sensitive, periplasmic superoxide disumutase (SodCII) contributed to the virulence of S. typhimurium in Perforin-2 knockout but not wild-type mice. In aggregate our studies indicate that Perforin-2 breaches the envelope of phagocytosed bacteria facilitating the delivery of proteases and other antimicrobial effectors to sites within the bacterial envelope.

immunology↗

bioRxiv2017

Dynamic protein aggregation regulates bacterial dormancy depth critical for antibiotic tolerance

The ability of some bacteria within a population to tolerate antibiotic treatment is often attributed to prolonged bacterial infection1-3. Unlike antibiotic resistance, which generally results from genetic mutations or plasmid transfer4,5, antibiotic tolerance usually refers to the phenomenon that a subgroup of cells can survive high dose antibiotic treatment as a result of phenotypic heterogeneity6,7. Previous studies mainly associate antibiotic tolerance with cell dormancy, by hypothesizing that the lethal effects of antibiotics are disabled due to the extremely slow metabolic and proliferation rates in dormant bacteria 8,9. However, less is known about how surviving bacteria subsequently escape from the dormant state and resuscitate, which is equally important for disease recurrence. Here we monitored the process of bacterial antibiotic tolerance and regrowth at the single-cell level, and found that each individual survival cell shows different dormancy depth, which in return regulates whether and when it can resume growth after removal of antibiotic. The persister cells are considered to be in shallow dormancy depth, while the viable but non-culturable cells (VBNC cells) are in deep dormancy depth. We further implemented time-lapse fluorescent imaging and biochemical analysis to establish that dynamic endogenous protein aggregation is an important indicator of bacterial dormancy depth. For cells to leave the dormant state and resuscitate, clearance of cellular protein aggregates and recovery of proteostasis are required. Through additional mutagenesis studies, we found the ability to recruit functional DnaK-ClpB machineries, which facilitate protein disaggregation in an ATP-dependent manner, determines the timeline (whether and when) for bacterial regrowth. Better understanding of the key factors regulating bacterial regrowth after surviving antibiotic attack could lead to new therapeutic strategies for combating bacterial antibiotic tolerance.

microbiology↗