Biology subjects

Zhu, W.

Publications and source records attributed to Zhu, W..

At least 127 records · Page 7Linked to original sources

bioRxiv2022

Predicting the structure of large protein complexes using AlphaFold and sequential assembly

AlphaFold can predict the structure of single- and multiple-chain proteins with very high accuracy. However, the accuracy decreases with the number of chains, and the available GPU memory limits the size of protein complexes which can be predicted. Here we show that one can predict the structure of large complexes starting from predictions of subcomponents. We assemble 91 out of 175 complexes with 10-30 chains from predicted subcomponents using Monte Carlo tree search, with a median TM-score of 0.51. There are 30 highly accurate complexes (TM-score [≥]0.8, 33% of complete assemblies). We create a scoring function, mpDockQ, that can distinguish if assemblies are complete and predict their accuracy. We find that complexes containing symmetry are accurately assembled, while asymmetrical complexes remain challenging. The method is freely available and accesible as a Colab notebook https://colab.research.google.com/github/patrickbryant1/MoLPC/blob/master/MoLPC.ipynb.

bioinformatics↗

bioRxiv2022

Medial prefrontal cortex Notch1 signalling mediates methamphetamine-induced psychosis via Hes1-dependent suppression of GABAB1 receptor expression.

Methamphetamine (METH), a widely abused stimulant drug, induces psychosis in approximately half of abusers; this effect is becoming a major concern for society. Although the Notch1 signalling pathway has been shown to play a part in the pathogenesis of some psychiatric disorders, its role in METH-induced psychosis (MIP) is still unknown. Here, we showed that Notch1 signalling was downregulated in the medial prefrontal cortex (mPFC) in a MIP mouse model. Direct genetic and pharmacological manipulations of Notch1 signalling bidirectionally altered MIP-related behaviours and governed neuronal activity in the mPFC. Moreover, Notch1 signalling negatively regulated GABAB1 receptor expression in the mPFC of MIP mice through Hes1, a transcriptional repressor in Notch1 signalling. Further, we show that Hes1 can directly bind to the GABAB1 receptor promoter. Notably, pharmacological regulation of the GABAB receptor in the mPFC reversed the changes in MIP-related behaviours caused by the dysfunction of Notch1 signalling. Together, our findings uncover a previously unrecognised Notch1-Hes1-GABAB1 receptor-dependent mechanism involved in regulating mPFC neuronal activity and behavioural phenotypes in MIP. Our work provides mechanistic insight into the aetiology and pathophysiology of MIP.

molecular biology↗

bioRxiv2022

Gene Expression and Tracer-Based Metabolic Flux Analysis Reveals Tissue-Specific Metabolic Scaling in vitro, ex vivo, and in vivo

Metabolic scaling, the inverse correlation of metabolic rates to body mass, has been appreciated for more than 80 years. Studies of metabolic scaling have almost exclusively been restricted to mathematical modeling of oxygen consumption. The possibility that other metabolic processes scale with body size has not been studied. To address this gap in knowledge, we employed a systems approach spanning from transcriptomics to in vitro and in vivo tracer-based flux. Gene expression in livers of five species spanning a 30,000-fold range in mass revealed differential expression of genes related to cytosolic and mitochondrial metabolic processes, in addition to detoxication of oxidative damage. This suggests that transcriptional scaling of damage control mechanisms accommodates increased oxidative metabolism in smaller species. To determine whether flux through key implicated metabolic pathways scaled, we applied stable isotope tracer methodology to study multiple cellular compartments, tissues, and species. Comparing mice and rats, we demonstrate that while scaling of metabolic fluxes is not observed in the cell-autonomous setting, it is present in liver slices and in vivo. Together, these data reveal that metabolic scaling extends beyond oxygen consumption to numerous other metabolic pathways, and is likely regulated at the level of gene expression and substrate supply.

physiology↗

bioRxiv2022

A detailed map of mitochondrial repeat-mediated recombination in Arabidopsis msh1 mutants using long-read sequencing

The abundant repeats in plant mitochondrial genomes can cause rapid genome rearrangements and are also a major obstacle in short-read sequencing studies. Nuclear-encoded proteins such as MSH1 are known to suppress the generation of repeat-associated mitochondrial genome variants, but our understanding of these mechanisms has been constrained by the limitations of short-read technologies. Here, we used highly accurate long-read sequencing (PacBio HiFi) to characterize mitochondrial and plastid genome variants in Arabidopsis thaliana msh1 mutant individuals. The HiFi reads provided a global view of recombination dynamics with detailed quantification of parental and crossover recombination products for both large and small repeats. We found that recombination breakpoints were distributed relatively evenly across the length of repeated sequences and detected widespread internal exchanges of sequence variants between pairs of imperfect repeats in the mitochondrial genome of msh1 mutants. Long-read assemblies of mitochondrial genomes from seven other Arabidopsis thaliana wild-type accessions differed by repeat-mediated structural rearrangements similar to those observed in msh1 mutants, but they were all in a simple low-heteroplasmy state. The Arabidopsis plastid genome generally lacks small repeats and exhibited a very different pattern of variant accumulation in msh1 mutants compared with the mitochondrial genome. Our data illustrate the power of HiFi technology in studying repeat-mediated recombination in plant organellar genomes and improved the sequence resolution for recombinational processes suppressed by MSH1. SignificancePlant organellar genomes can undergo rapid rearrangements. Long-read sequencing provides a detailed and quantitative view of mitochondrial and plastid genome variants normally suppressed by MSH1, advancing our understanding of plant organellar genome dynamics.

bioinformatics↗

bioRxiv2021

Physico-chemical principles of HDL-small RNA binding interactions.

Extracellular small RNAs (sRNA) are abundant in many biofluids, but little is known about their mechanisms of transport and stability in RNase-rich environments. We previously reported that high-density lipoproteins (HDL) of mice were enriched with multiple classes of sRNA derived from the endogenous transcriptome, but also exogenous organisms. Here, we show that human HDL transports tRNA-derived sRNAs (tDRs) from host and non-host species which were found to be altered in human atherosclerosis. We hypothesized that HDL binds to tDRs through apolipoprotein A-I (apoA-I) and these interactions are conferred by RNA-specific features. We tested this using microscale thermophoresis and electrophoretic mobility shift assays and found that HDL bind tDRs and other single-stranded sRNAs with strong affinity, but not doublestranded RNA or DNA. Natural and synthetic RNA modifications influenced tDR binding to HDL. Reconstituted HDL bound tDRs only in the presence of apoA-I and purified apoA-I alone was sufficient for binding sRNA. Conversely, phosphatidylcholine vesicles did not bind tDRs. In summary, HDL preferentially binds to single-stranded sRNAs likely through non-ionic interactions with apoA-I. These studies highlight binding properties that likely enable extracellular RNA communication and provide a foundation for future studies to manipulate HDL-sRNA for therapeutic approaches to prevent or treat disease.

molecular biology↗

bioRxiv2021

Non-rapid eye movement sleep and wake neurophysiology in schizophrenia

Motivated by the potential of objective neurophysiological markers to index thalamocortical function in patients with severe psychiatric illnesses, we comprehensively characterized key NREM sleep parameters across multiple domains, their interdependencies, and their relationship to waking event-related potentials and symptom severity. In 130 schizophrenia (SCZ) patients and controls, we confirmed a marked reduction in sleep spindle density in SCZ and extended these findings to show that only slow spindles predicted symptom severity, and that fast and slow spindle properties were largely uncorrelated. We also describe a novel measure of slow oscillation and spindle interaction that was attenuated in SCZ. The main sleep findings were replicated in a demographically distinct sample, and a joint model, based on multiple NREM components, predicted disease status in the replication cohort. Although also altered in patients, auditory event-related potentials elicited during wake were unrelated to NREM metrics. Consistent with a growing literature implicating thalamocortical dysfunction in SCZ, our characterization identifies independent NREM and wake EEG biomarkers that may index distinct aspects of SCZ pathophysiology and point to multiple neural mechanisms underlying disease heterogeneity. This study lays the groundwork for evaluating these neurophysiological markers, individually or in combination, to guide efforts at treatment and prevention as well as identifying individuals most likely to benefit from specific interventions.

neuroscience↗

bioRxiv2021

A Precision Medicine Approach to Metabolic Therapy for Breast Cancer in Mice

Increasing evidence highlights the possibility for approaches targeting metabolism as potential adjuvants to cancer therapy. Sodium-glucose transport protein 2 (SGLT2) inhibitors are the newest class of antihyperglycemic therapies, and have recently been highlighted as a novel therapeutic approach to breast cancer. To our knowledge, however, SGLT2 inhibitors have not been applied in the neoadjuvant setting as a precision medicine approach to combining metabolic therapy with standard of care therapy for this devastating disease. In this study we combine the SGLT2 inhibitor dapagliflozin with paclitaxel chemotherapy in both lean and obese mice. We show that dapagliflozin enhances the efficacy of paclitaxel, reducing tumor glucose uptake and prolonging survival in an insulin-dependent manner in some but not all breast tumors. Our data find a genetic signature for breast tumors most likely to respond to dapagliflozin in combination with paclitaxel. Tumors driven by mutations upstream of canonical insulin signaling pathways are likely to respond to such treatment, whereas tumors driven by mutations downstream of canonical insulin signaling are not. These data demonstrate that dapagliflozin enhances the response to chemotherapy in mice with breast cancer and suggest that breast cancer patients with driver mutations upstream of canonical insulin signaling may be most likely to benefit from this neoadjuvant approach. A clinical trial is currently in preparation, with an application recently submitted for Yale Human Investigations Committee approval, to test this hypothesis in breast cancer patients. One Sentence SummaryWe identify a driver mutation signature by which glucose-wasting metabolic therapy (dapagliflozin) enhances the efficacy of chemotherapy in mice with breast cancer.

cancer biology↗

bioRxiv2021

Metabolitin-based molecular drug delivery by targeting GPR158 in glioblastoma

Glioblastoma multiforme (GBM) is a lethal form of intracranial tumor. One of the obstacles to treat GBM is the blood-brain barrier which limit the transportation of drugs into the tumor site. Here, based on our previous study on metabolitin (MTL) and osteocalcin, we generated a molecular drug delivery system that consisted of metabolitin and small molecules such as fluorescent dye or peptide drugs for diagnosis and treatment. And we designed a GBM diagnostic probe (MTL-ICG) and therapeutic peptide drug (MTL-NBD) that can cross the blood-brain barrier (BBB). In a NIR animal live imaging system, we found MTL-ICG can penetrate cross BBB and label the GBM site. The in vitro experiment showed that MTL-NBD had inhibitory effect on GBM cell line (U87-MG). Besides, after orthotopic transplantation of GBM into mouse cortex, treatment of MTL-NBD intravenously showed inhibition trend, which were similar with the effect of NBD, a known anti-tumor polypeptide drug. In addition, we found the GPR158, the receptor of osteocalcin, was also high expressed in grafting site. Taken together, these findings suggest that MTL is a promising cell penetrating peptide targeting GPR158 in GBM, which provide a novel delivery tool for GBM.

cancer biology↗

bioRxiv2021

Towards a structurally resolved human protein interaction network

All cellular functions are governed by complex molecular machines that assemble through protein-protein interactions. Their atomic details are critical to the study of their molecular mechanisms but fewer than 5% of hundreds of thousands of human interactions have been structurally characterized. Here, we test the potential and limitations of recent progress in deep-learning methods using AlphaFold2 to predict structures for 65,484 human interactions. We show that higher confidence models are enriched in interactions supported by affinity or structure-based methods and can be orthogonally confirmed by spatial constraints defined by cross-link data. We identify 3,137 high confidence models, of which 1,371 have no homology to a known structure, from which we identify interface residues harbouring disease mutations, suggesting potential mechanisms for pathogenic variants. We find groups of interface phosphorylation sites that show patterns of co-regulation across conditions, suggestive of coordinated tuning of multiple interactions as signalling responses. Finally, we provide examples of how the predicted binary complexes can be used to build larger assemblies. Accurate prediction of protein complexes promises to greatly expand our understanding of the atomic details of human cell biology in health and disease.

bioinformatics↗

bioRxiv2021

Nuclear PHGDH promotes neutrophil recruitment to drive liver cancer progression

Metabolic dysregulation and the communications between cancer and immune cells are emerging as two essential features of malignant tumors. In this study, we observed that nuclear localization of phosphoglycerate dehydrogenase (PHGDH) associates with poor prognosis of liver cancer patients, and Phgdh is required for liver cancer progression in a mouse model. Unexpectedly, the impairment of Phgdh enzyme activity exerts a slight effect on liver cancer model, indicating PHGDH contributes to liver cancer progression mainly depending on its non-metabolic roles with nuclear location. PHGDH uses its ACT domain to bind cMyc in nuclear and forms a transactivation axis "PHGDH/p300/cMyc/AF9" which drives CXCL1/8 gene expression. Chemokines CXCL1/8 promotes neutrophils recruitment and then supports tumor associated macrophages (TAMs) filtration in liver, thereby urging liver cancer into advanced stages. Forced cytosolic location of PHGDH or destruction of the PHGDH/cMyc interaction abolishes the oncogenic function of nuclear PHGDH. Collectively, this study reveals a non-metabolic role of PHGDH with altered cellular location in liver cancer, and suggests a promising drug target for liver cancer therapy by targeting the interaction between PHGDH and "undruggable" cMyc.

cancer biology↗

bioRxiv2021

Antibacterial 3D-printed PMMA/ceramic composites

Following the rising interested on 3D-printed technologies, this research explores the possibility to use stereo-lithography to 3D print PMMA resins reinforced with up to 15% in weight of antibacterial ceramic powders. Three different reinforcements were tested, following previous literature data: aluminum nitride, titanium oxide and barium titanate. Between the three powders, the most uniform dispersion was achieved using aluminum nitride. Initial screenings with mixed and cured composite resins showed that between the three composite materials, only aluminum nitride or barium titanate PMMA showed a clear antibacterial effect when compared to the pristine reference, with aluminum nitride being the most effective against E. coli. When 3D printed using stereo-lithography, the composite containing aluminum nitride showed an even higher degree of dispersion and comparable antibacterial effects. Moreover, aluminum nitride reinforced PMMA resins showed good mechanical properties, comparable to the basic resin, and could be further strengthened by a standard post-curing process.

bioengineering↗

bioRxiv2021

A high throughput screening assay for inhibitors of SARS-CoV-2 pseudotyped particle entry

Effective small molecule therapies to combat the SARS-CoV-2 infection are still lacking as the COVID-19 pandemic continues globally. High throughput screening assays are needed for lead discovery and optimization of small molecule SARS-CoV-2 inhibitors. In this work, we have applied viral pseudotyping to establish a cell-based SARS-CoV-2 entry assay. Here, the pseudotyped particles (PP) contain SARS-CoV-2 spike in a membrane enveloping both the murine leukemia virus (MLV) gag-pol polyprotein and luciferase reporter RNA. Upon addition of PP to HEK293-ACE2 cells, the SARS-CoV-2 spike protein binds to the ACE2 receptor on the cell surface, resulting in priming by host proteases to trigger endocytosis of these particles, and membrane fusion between the particle envelope and the cell membrane. The internalized luciferase reporter gene is then expressed in cells, resulting in a luminescent readout as a surrogate for spike-mediated entry into cells. This SARS-CoV-2 PP entry assay can be executed in a biosafety level 2 containment lab for high throughput screening. From a collection of 5,158 approved drugs and drug candidates, our screening efforts identified 7 active compounds that inhibited the SARS-CoV-2-S PP entry. Of these seven, six compounds were active against live replicating SARS-CoV-2 virus in a cytopathic effect assay. Our results demonstrated the utility of this assay in the discovery and development of SARS-CoV-2 entry inhibitors as well as the mechanistic study of anti-SARS-CoV-2 compounds. Additionally, particles pseudotyped with spike proteins from SARS-CoV-2 B.1.1.7 and B.1.351 variants were prepared and used to evaluate the therapeutic effects of viral entry inhibitors.

pharmacology and toxicology↗

bioRxiv2021

A structural biology community assessment of AlphaFold 2 applications

Most proteins fold into 3D structures that determine how they function and orchestrate the biological processes of the cell. Recent developments in computational methods have led to protein structure predictions that have reached the accuracy of experimentally determined models. While this has been independently verified, the implementation of these methods across structural biology applications remains to be tested. Here, we evaluate the use of AlphaFold 2 (AF2) predictions in the study of characteristic structural elements; the impact of missense variants; function and ligand binding site predictions; modelling of interactions; and modelling of experimental structural data. For 11 proteomes, an average of 25% additional residues can be confidently modelled when compared to homology modelling, identifying structural features rarely seen in the PDB. AF2-based predictions of protein disorder and protein complexes surpass state-of-the-art tools and AF2 models can be used across diverse applications equally well compared to experimentally determined structures, when the confidence metrics are critically considered. In summary, we find that these advances are likely to have a transformative impact in structural biology and broader life science research.

biophysics↗

bioRxiv2021

Zika virus causes placental pyroptosis and associated adverse fetal outcomes by activating GSDME

Zika virus (ZIKV) can be transmitted from mother to fetus during pregnancy, causing adverse fetal outcomes. Several studies have indicated that ZIKV can damage the fetal brain directly; however, whether the ZIKV-induced maternal placental injury contribute to adverse fetal outcomes are sparsely defined. Here, we discovered that ZIKV causes the pyroptosis of placental cells by activating the executor Gasdermin E (GSDME) in vitro and in vivo. Mechanistically, caspase-8 undergoes activation upon the recognition of 5 untranslated region of viral RNA by RIG-I, followed by the stimulation of caspase-3 to ultimately escalate the GSDME cleavage. Further analyses revealed that the ablation of GSDME in ZIKV-infected pregnant mice attenuates placental pyroptosis, which consequently confers protection against adverse fetal outcomes. In conclusion, our study unveils a novel mechanism of ZIKV-induced adverse fetal outcomes via causing placental cell pyroptosis, which could be employed for developing new therapies for ZIKV-associated diseases. Significance statementSeveral studies have elucidated the link between ZIKV infection and congenital ZIKV syndroms (CZS), but the pathogenesis yet needs further study. Here, we reported a novel pathogenic mechanism of ZIKV which leads to pyroptosis of placental cells through activating the pyroptotic executor GSDME, rather than GSDMD. Upon ZIKV infection, GSDME-mediated pyroptosis damages the structure and function of the placenta, thereby affecting the development of the fetus and contributing to the adverse fetal outcomes. Our study highlights the importance of pyroptotic executor GSDME in regulate ZIKV pathogenicity and further confirms that placental injury caused by ZIKV infection is a key factor for CZS.

microbiology↗

bioRxiv2021

Identification of TAZ as the essential molecular switch in orchestrating SCLC phenotypic transition and metastasis

Small cell lung cancer (SCLC) is a recalcitrant cancer featured with high metastasis. However, the exact cell type contributing to metastasis remains elusive. Using Rb1L/L/Trp53L/L mouse model, we identify the NCAMhiCD44lo/- subpopulation as SCLC metastasizing cell (SMC), which is progressively transitioned from non-metastasizing NCAMloCD44hi cell (Non-SMC). Integrative chromatin accessibility and gene expression profiling studies reveal an important role of SWI/SNF complex, and knockout of its central component, Brg1, significantly inhibits such phenotypic transition and metastasis. Mechanistically, TAZ is silenced by SWI/SNF complex during SCLC malignant progression, and its knockdown promotes SMC transition and metastasis. Importantly, ectopic TAZ expression reversely drives SMC-to-Non-SMC transition and alleviates metastasis. Single-cell RNA-sequencing analyses identify SMC as the dominant subpopulation in human SCLC metastasis, and immunostaining data show a positive correlation between TAZ and patient prognosis. These data uncover high SCLC plasticity and identify TAZ as key molecular switch in orchestrating SCLC phenotypic transition and metastasis.

cancer biology↗

bioRxiv2021

Botrytis cinerea BcPTP1 is a late infection phase, cysteine rich protein cytotoxic effector

Botrytis cinerea is a broad-host-range necrotrophic phytopathogen responsible for serious crops diseases. To facilitate infection, B. cinerea secretes a large number of effectors that induce plant cell death. In screening secretome data of B. cinerea during infection stage, we identified a phytotoxic protein (BcPTP1) that can also induce immune resistance in plants. BcPTP1 is a small (90 aa), cysteine rich protein without any known domains. Transiently expression of BcPTP1 in leaves caused chlorosis that intensifies with time and eventually lead to cell death. Point mutations in eight of the 10 cysteine residues of BcPTP1 abolished the toxic effect, however residual toxic activity remained after heating the peptide, suggesting contribution of unknown epitopes to protein phytotoxic effect. The transcript level of the bcptp1 gene was low during the first 36 h after inoculation and increased sharply upon transition to the late infection stage, suggesting a role of BcPTP1 in lesion spreading. While statistically insignificant, deletion of the bcptp1 gene led to slightly smaller lesions on bean leaves. Further analyses indicated that BcPTP1 is internalized into plant cells after secreting into the apoplast and its phytotoxic effect is negatively regulated by the receptor-like kinases BAK1 and SOBIR1. Collectively, our findings show that BcPTP1 is a virulence factor that toxifies the host cells and facilitates lesion spreading during the late infection stage.

pathology↗

bioRxiv2021

Lentiviral vector optimization enhances the expression and cytotoxicity of chimeric antigen receptors

Adoptive chimeric antigen receptor (CAR)-modified T or NK cells (CAR-T/NK) have emerged as a novel form of disease treatment. Lentiviral vectors (LVs) are commonly employed to engineer T/NK cells for the efficient expression of CARs. This study reported for the first time the influence of single-promoter and dual-promoter LVs on the CAR expression and cytotoxicity of engineered NK cells. Our results demonstrated that the selected CAR exhibits both a higher expression level and a higher coexpression concordance with the GFP reporter in HEK-293T or NK92 cells by utilizing the optimized single-promoter pCDHsp rather than the original dual-promoter pCDHdp. After puromycin selection, the pCDHsp produces robust CAR expression and enhanced in vitro cytotoxicity of engineered NK cells. Therefore, infection with a single-promoter pCDHsp lentivector is recommended to prepare CAR-engineered cells. This research will help to optimize the production of CAR-NK cells and improve their functional activity, to provide CAR-NK cell products with better and more uniform quality.

cell biology↗

bioRxiv2021

Exposure route, sex, and age influence disease outcome in a golden Syrian hamster model of SARS-CoV-2 infection

The emergence of Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the resultant pandemic of coronavirus disease 2019 (COVID-19) has led to over one hundred million confirmed infections, greater than three million deaths, and severe economic and social disruption. Animal models of SARS-CoV-2 are critical tools for the pre-clinical evaluation of antivirals, vaccines, and candidate therapeutics currently under urgent development to curb COVID-19-associated morbidity and mortality. The golden (Syrian) hamster model of SARS-CoV-2 infection recapitulates key characteristics of severe COVID-19, including high-titer viral replication in the upper and lower respiratory tract and the development of pathogenic lesions in the lungs. In this work we examined the influence of the route of exposure, sex, and age on SARS-CoV-2 pathogenesis in golden hamsters. We report that delivery of SARS-CoV-2 primarily to the nasal passages (low-volume intranasal), the upper and lower respiratory tract (high-volume intranasal), or the digestive tract (intragastric) results in comparable viral titers in the lung tissue and similar levels of viral shedding during acute infection. However, low-volume intranasal exposure results in milder weight loss during acute infection while intragastric exposure leads to a diminished capacity to regain body weight following the period of acute illness. Further, we examined both sex and age differences in response to SARS-CoV-2 infection. Male hamsters, and to a greater extent older male hamsters, display an impaired capacity to recover from illness and a delay in viral clearance compared to females. Lastly, route of exposure, sex, and age were found to influence the nature of the host inflammatory cytokine response, but they had a minimal effect on both the quality and durability of the humoral immune response as well as the susceptibility of hamsters to SARS-CoV-2 re-infection. Together, these data indicate that the route of exposure, sex, and age have a meaningful impact SARS-CoV-2 pathogenesis in hamsters and that these variables should be considered when designing pre-clinical challenge studies.

microbiology↗