Biology subjects

Peng, H.

Publications and source records attributed to Peng, H..

At least 109 records · Page 6Linked to original sources

bioRxiv2021

Reduced graphene oxide membrane as supporting film for high-resolution cryo-EM

Although single-particle cryogenic electron microscopy (cryo-EM) has been applied extensively for elucidating many crucial biological mechanisms at the molecular level, this technique still faces critical challenges, the major one of which is to prepare the high-quality cryo-EM specimen. Aiming to achieve a more reproducible and efficient cryo-EM specimen preparation, novel supporting films including graphene-based two-dimensional materials have been explored in recent years. Here we report a robust and simple method to fabricate EM grids coated with single- or few-layer reduced graphene oxide (RGO) membrane in large batch for high-resolution cryo-EM structural determination. The RGO membrane has decreased interlayer space and enhanced electrical conductivity in comparison to regular graphene oxide (GO) membrane. Moreover, we found that the RGO supporting film exhibited nice particle-absorption ability, thus avoiding the air-water interface problem. More importantly, we found that the RGO supporting film is particularly useful in cryo-EM reconstruction of sub-100 kDa biomolecules at near-atomic resolution, as exemplified by the study of RBD-ACE2 complex and other small protein molecules. We envision that the RGO membranes can be used as a robust graphene-based supporting film in cryo-EM specimen preparation.

biophysics↗

bioRxiv2021

SUFFICIENT ACTIVITY OF THE UBIQUITIN PROTEASOME SYSTEM IN AGED MICE AND DURING RETINAL DEGENERATION SUPPORTS DHFR-BASED CONDITIONAL CONTROL OF PROTEIN ABUNDANCE IN THE RETINA

The Escherichia coli dihydrofolate reductase (DHFR) destabilizing domain (DD) serves as a promising approach to conditionally regulate protein abundance in a variety of tissues. In the absence of TMP, a DHFR stabilizer, the DD is degraded by the ubiquitin proteasome system (UPS). To test whether this approach could be effectively applied to a wide variety of aged and disease-related ocular mouse models, which may have a compromised UPS, we evaluated the DHFR DD system in aged mice (up to 24 mo), a light-induced retinal degeneration (LIRD) model, and two genetic models of retinal degeneration (rd2 and Abca4-/- mice). Aged, LIRD, and Abca4-/- mice all had similar proteasomal activities and high-molecular weight ubiquitin levels compared to control mice. However, rd2 mice displayed compromised chymotrypsin activity compared to control mice. Nonetheless, the DHFR DD was effectively degraded in all model systems, including rd2 mice. Moreover, TMP increased DHFR DD-dependent retinal bioluminescence in all mouse models, however the fold induction was slightly, albeit significantly, lower in Abca4-/- mice. Thus, the destabilized DHFR DD-based approach allows for efficient control of protein abundance in aged mice and retinal degeneration mouse models, laying the foundation to use this strategy in a wide variety of mice for the conditional control of gene therapies to potentially treat multiple eye diseases.

physiology↗

bioRxiv2021

Structural basis for enhanced infectivity and immune evasion of SARS-CoV-2 variants

Several fast-spreading variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have become the dominant circulating strains that continue to fuel the COVID-19 pandemic despite intensive vaccination efforts throughout the world. We report here cryo-EM structures of the full-length spike (S) trimers of the B.1.1.7 and B.1.351 variants, as well as their biochemical and antigenic properties. Mutations in the B.1.1.7 protein increase the accessibility of its receptor binding domain and also the binding affinity for receptor angiotensin-converting enzyme 2 (ACE2). The enhanced receptor engagement can account for the increased transmissibility and risk of mortality as the variant may begin to infect efficiently infect additional cell types expressing low levels of ACE2. The B.1.351 variant has evolved to reshape antigenic surfaces of the major neutralizing sites on the S protein, rendering complete resistance to some potent neutralizing antibodies. These findings provide structural details on how the wide spread of SARS-CoV-2 enables rapid evolution to enhance viral fitness and immune evasion. They may guide intervention strategies to control the pandemic.

biochemistry↗

bioRxiv2021

Transcriptome-based phylogeny and whole-genome duplication in Theaceae

Theaceae, with three tribes and nine genera, is a family of great economic and ecological importance. Recent phylogenetic analyses based on plastid genome resolved the relationship among three tribes and the intergeneric relationships within Gordonieae and Stewartieae. However, generic level relationships within the largest tribe Theeae were not fully resolved and potential hybridization among genera within Theeae revealed previously also remains to be tested further. Here we conducted a comprehensive phylogenomic study of Theaceae based on transcriptomes and low-depth whole-genome sequencing of 57 species as well as additional plastome sequence data from previous work. Phylogenetic analyses suggested that Stewartieae was the first-diverging clade in Theaceae, consistent with previous study using plastomic data. Within Theeae, the highly supported Apterosperma-Laplacea clade grouped with Pyrenaria with maximum support based on the partitioned and unpartitioned concatenation analyses using the 610 low-copy nuclear genes, leaving Camellia and Polyspora as another sister genera in the tribe. PhyloNet analyses suggested one reticulation event within Camellia and Pyrenaria respectively, but no intergeneric reticulations were detected in Theeae. Another introgression was found between Gordonia lasianthus and the common ancestor of Gordonieae during the Late Oligocene. The existing land bridges (e.g. Bering land bridge) might have facilitated this ancient introgression. Further researches need to be conducted to uncover the interspecific introgression pattern within Camellia. Ks distribution analyses supported the tea family shared one whole-genome duplication (WGD) event Ad-{beta}, which was recently mapped to the clade containing core Ericales, Primuloids, Polemonioids and Lecythidaceae.

evolutionary biology↗

bioRxiv2021

MR-APSS: a unified approach to Mendelian Randomization accounting for pleiotropy and sample structure using genome-wide summary statistics

Mendelian Randomization (MR) is a valuable tool for inferring causal relationships among a wide range of traits using summary statistics from genome-wide association studies (GWASs). Existing summary-level MR methods often rely on strong assumptions, resulting in many false positive findings. To relax MR assumptions, ongoing research has been primarily focused on accounting for confounding due to pleiotropy. Here we show that sample structure is another major confounding factor, including population stratification, cryptic relatedness, and sample overlap. We propose a unified MR approach, MR-APSS, which (i) accounts for pleiotropy and sample structure simultaneously by leveraging genome-wide information; and (ii) allows to include more genetic variants with moderate effects as instrument variables (IVs) to improve statistical power without inflating type I errors. We first evaluated MR-APSS using comprehensive simulations and negative controls, and then applied MR-APSS to study the causal relationships among a collection of diverse complex traits. The results suggest that MR-APSS can better identify plausible causal relationships with high reliability. In particular, MR-APSS can perform well for highly polygenic traits, where the IV strengths tend to be relatively weak and existing summary-level MR methods for causal inference are vulnerable to confounding effects.

genetics↗

bioRxiv2021

Multiple nucleotide polymorphism DNA markers for the accurate evaluation of genetic variations

DNA markers are an essential tool for the detection and evaluation of genetic variations, a central theme in genetics and biology. Effective markers must be highly reproducible, polymorphic, accurate and efficient to profile. We developed multiple dispersed nucleotide polymorphism (MNP) DNA marker and an efficient MNP genotyping method called MNP-Seq. The MNP marker was 17.48% more polymorphic than the highly polymorphic marker of microsatellites on a collection of hybrid rice plants. When applied to genotype more than 80,000 individual MNP markers of diploid rice and polyploidy hybrid cotton varieties which were notoriously difficult to genotype accurately, MNP-Seq finished in two days and achieved accuracies of 99.999% and 99.988%, respectively. We adopted MNP-Seq to reveal the ubiquitous, albeit subtle and neglected, genetic heterogeneities in homonyms of Nipponbare rice, a popular model organism for plant biology. This result raised a question on the consistency of the published results using the model plant. We also used MNP-Seq to accurately and efficiently determine the identities of plant varieties, a key but difficult problem for the protection of plant intellectual property rights. While being applied to plants in the current study, the MNP marker and MNP-Seq are general and readily applicable to similar problems in animals and micro-organisms.

molecular biology↗

bioRxiv2021

Defining proximity proteomics of post-translationally modified proteins by antibody-mediated protein A-APEX2 labeling

Proximity labeling catalyzed by promiscuous enzymes, such as APEX2, has emerged as a powerful approach to characterize multiprotein complexes and protein-protein interactions. However, current methods depend on the expression of exogenous fusion proteins and cannot be applied to post-translational modifications. To address this limitation, we developed a new method to label proximal proteins of interest by antibody-mediated protein A-APEX2 labeling (AMAPEX). In this method, a modified protein is bound in situ by a specific antibody, which then tethers a protein A-APEX2 (pA-APEX2) fusion protein. Activation of APEX2 labels the nearby proteins with biotin; these proteins are then purified using streptavidin beads and are identified by mass spectrometry. We demonstrate the utility of this approach by profiling the binding proteins of histone modifications including H3K27me3, H3K9me3, H3K4me3, H4K5ac and H4K12ac, and we verified the genome-wide colocalization of these identified proteins with bait proteins by published ChIP-seq analysis. Overall, AMAPEX is an efficient tool to identify proteins that interact with modified proteins.

molecular biology↗

bioRxiv2021

Petabyte-Scale Multi-Morphometry of Single Neurons for Whole Brains

Recent advances in neuroscience make the extraction of full neuronal morphology at whole brain dataset available. To produce quality morphometry at large scale, it is highly desirable but extremely challenging to efficiently handle petabyte-scale high-resolution whole brain imaging database. Here, we developed a multi-level method to produce high quality somatic, dendritic, axonal, and potential synaptic morphometry, which was made possible by utilizing necessary petabyte hardware and software platform to optimize both the data and workflow management. Our method also boosts data sharing and remote collaborative validation. We highlight a petabyte application dataset involving 62 whole mouse brains, from which we identified 50,233 somata of individual neurons, profiled the dendrites of 11,322 neurons, reconstructed the full 3-D morphology of more than one thousand neurons including their dendrites and full axons, and detected million scale putative synaptic sites derived from axonal boutons. Analysis and simulation of these data indicate the promise of this approach for modern large-scale morphology applications.

neuroscience↗

bioRxiv2020

Optimising a Simple Fully Convolutional Network (SFCN) for accurate brain age prediction in the PAC 2019 challenge

Brain age prediction from brain MRI scans not only helps improve brain ageing modelling generally, but also provides benchmarks for predictive analysis methods. Brain-age delta, which is the difference between a subjects predicted age and true age, has become a meaningful biomarker for the health of the brain. Here, we report the details of our brain age prediction models and results in the Predictive Analysis Challenge 2019. The aim of the challenge was to use T1-weighted brain MRIs to predict a subjects age in multicentre datasets. We apply a lightweight deep convolutional neural network architecture, Simple Fully Convolutional Neural Network (SFCN), and combined several techniques including data augmentation, transfer learning, model ensemble, and bias correction for brain age prediction. The model achieved first places in both of the two objectives in the PAC 2019 brain age prediction challenge: Mean absolute error (MAE) = 2.90 years without bias removal, and MAE = 2.95 years with bias removal.

neuroscience↗

bioRxiv2020

Structural impact on SARS-CoV-2 spike protein by D614G substitution

Substitution for aspartic acid by glycine at position 614 in the spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the ongoing pandemic, appears to facilitate rapid viral spread. The G614 variant has now replaced the D614-carrying virus as the dominant circulating strain. We report here cryo-EM structures of a full-length S trimer carrying G614, which adopts three distinct prefusion conformations differing primarily by the position of one receptor-binding domain (RBD). A loop disordered in the D614 S trimer wedges between domains within a protomer in the G614 spike. This added interaction appears to prevent premature dissociation of the G614 trimer, effectively increasing the number of functional spikes and enhancing infectivity. The loop transition may also modulate structural rearrangements of S protein required for membrane fusion. These findings extend our understanding of viral entry and suggest an improved immunogen for vaccine development.

biochemistry↗

bioRxiv2020

Evaluating spiny mice (Acomys) as a model for cardiac research

Complex tissue regeneration is extremely rare among adult mammals. An exception, however, is the superior tissue healing of multiple organs in spiny mice (Acomys). While Acomys species exhibit the remarkable ability to heal complex tissue with minimal scarring, little is known about their cardiac structure and response to cardiac injury. In this study, we first examined baseline Acomys cardiac anatomy and function in comparison with the commonly used laboratory Mus strains (C57BL6 and SWR). Our results demonstrated comparable cardiac anatomy and function between Acomys and Mus, but Acomys exhibited a higher percentage of cardiomyocytes exhibiting immature characteristics. In response to myocardial infarction, all animals experienced a comparable level of initial cardiac damage. However, Acomys demonstrated superior ischemic tolerance and cytoprotection in response to injury as evidenced by cardiac functional stabilization, higher survival rate and smaller scar size 50 days after injury compared to the inbred and outbred mouse strains. Overall, these findings demonstrate augmented myocardial preservation in spiny mice post-MI and establish Acomys as a new adult mammalian model for cardiac research.

developmental biology↗

bioRxiv2020

A trimeric human angiotensin-converting enzyme 2 as an anti-SARS-CoV-2 agent in vitro

Effective intervention strategies are urgently needed to control the COVID-19 pandemic. Human angiotensin-converting enzyme 2 (ACE2) is a carboxypeptidase that forms a dimer and serves as the cellular receptor for SARS-CoV-2. It is also a key negative regulator of the renin-angiotensin system (RAS), conserved in mammals, which modulates vascular functions. We report here the properties of a trimeric ACE2 variant, created by a structure-based approach, with binding affinity of ~60 pM for the spike (S) protein of SARS-CoV-2, while preserving the wildtype peptidase activity as well as the ability to block activation of angiotensin II receptor type 1 in the RAS. Moreover, the engineered ACE2 potently inhibits infection of SARS-CoV-2 in cell culture. These results suggest that engineered, trimeric ACE2 may be a promising anti-SARS-CoV-2 agent for treating COVID-19.

microbiology↗

bioRxiv2020

Comprehensive characterization of single cell full-length isoforms in human and mouse with long-read sequencing

Alternative splicing shapes the phenotype of cells in development and disease. Long-read RNA-sequencing recovers full-length transcripts but has limited throughput at the single-cell level. Here we developed single-cell full-length transcript sequencing by sampling (FLT-seq), together with the computational pipeline FLAMES to overcome these issues and perform isoform discovery and quantification, splicing analysis and mutation detection in single cells. With FLT-seq and FLAMES, we performed the first comprehensive characterization of the full-length isoform landscape in single cells of different types and species and identified thousands of unannotated isoforms. We found conserved functional modules that were enriched for alternative transcript usage in different cell populations, including ribosome biogenesis and mRNA splicing. Analysis at the transcript-level allowed data integration with scATAC-seq on individual promoters, improved correlation with protein expression data and linked mutations known to confer drug resistance to transcriptome heterogeneity. Our methods reveal previously unseen isoform complexity and provide a better framework for multi-omics data integration.

genomics↗

bioRxiv2020

De novo design of ACE2 protein decoys to neutralize SARS-CoV-2

There is an urgent need for the ability to rapidly develop effective countermeasures for emerging biological threats, such as the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes the ongoing coronavirus disease 2019 (COVID-19) pandemic. We have developed a generalized computational design strategy to rapidly engineer de novo proteins that precisely recapitulate the protein surface targeted by biological agents, like viruses, to gain entry into cells. The designed proteins act as decoys that block cellular entry and aim to be resilient to viral mutational escape. Using our novel platform, in less than ten weeks, we engineered, validated, and optimized de novo protein decoys of human angiotensin-converting enzyme 2 (hACE2), the membrane-associated protein that SARS-CoV-2 exploits to infect cells. Our optimized designs are hyperstable de novo proteins ([~]18-37 kDa), have high affinity for the SARS-CoV-2 receptor binding domain (RBD) and can potently inhibit the virus infection and replication in vitro. Future refinements to our strategy can enable the rapid development of other therapeutic de novo protein decoys, not limited to neutralizing viruses, but to combat any agent that explicitly interacts with cell surface proteins to cause disease.

biochemistry↗

bioRxiv2020

Mutations from bat ACE2 orthologs markedly enhance ACE2-Fc neutralization of SARS-CoV-2

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) protein mediates infection of cells expressing angiotensin-converting enzyme 2 (ACE2). ACE2 is also the viral receptor of SARS-CoV (SARS-CoV-1), a related coronavirus that emerged in 2002-2003. Horseshoe bats (genus Rhinolophus) are presumed to be the original reservoir of both viruses, and a SARS-like coronavirus, RaTG13, closely related SARS-CoV-2, has been isolated from one horseshoe-bat species. Here we characterize the ability of S-protein receptor-binding domains (RBDs) of SARS-CoV-1, SARS-CoV-2, and RaTG13 to bind a range of ACE2 orthologs. We observed that the SARS-CoV-2 RBD bound human, pangolin, and horseshoe bat (R. macrotis) ACE2 more efficiently than the SARS-CoV-1 or RaTG13 RBD. Only the RaTG13 RBD bound rodent ACE2 orthologs efficiently. Five mutations drawn from ACE2 orthologs of nine Rhinolophus species enhanced human ACE2 binding to the SARS-CoV-2 RBD and neutralization of SARS-CoV-2 by an immunoadhesin form of human ACE2 (ACE2-Fc). Two of these mutations impaired neutralization of SARS-CoV-1. An ACE2-Fc variant bearing all five mutations neutralized SARS-CoV-2 five-fold more efficiently than human ACE2-Fc. These data narrow the potential SARS-CoV-2 reservoir, suggest that SARS-CoV-1 and -2 originate from distinct bat species, and identify a more potently neutralizing form of ACE2-Fc.

microbiology↗

bioRxiv2020

In-Vivo Toxicity Studies and In-Vitro Inactivation of SARS-CoV-2 by Povidone-iodine In-situ Gel Forming Formulations

To curb the spread of SARS-CoV-2, the etiologic agent of the COVID-19 pandemic, we characterize the virucidal activity of long-acting Povidone Iodine (PVP-I) compositions developed using an in-situ gel forming technology. The PVP-I gel forming nasal spray (IVIEW-1503) and PVP-I gel forming ophthalmic eye drop (IVIEW-1201) rapidly inactivated SARS-CoV-2, inhibiting the viral infection of VERO76 cells. No toxicity was observed for the PVP-I formulations. Significant inactivation was noted with preincubation of the virus with these PVP-I formulations at the lowest concentrations tested. It has been demonstrated that both PVP-I formulations can inactivate SARS-CoV-2 virus efficiently in both a dose-dependent and a time-dependent manner. These results suggest IVIEW-1503 and IVIEW-1201 could be potential agents to reduce or prevent the transmission of the virus through the nasal cavity and the eye, respectively. Further studies are needed to clinically evaluate these formulations in early-stage COVID-19 patients.

microbiology↗

bioRxiv2020

Distinct conformational states of SARS-CoV-2 spike protein

The ongoing SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) pandemic has created urgent needs for intervention strategies to control the crisis. The spike (S) protein of the virus forms a trimer and catalyzes fusion between viral and target cell membranes - the first key step of viral infection. Here we report two cryo-EM structures, both derived from a single preparation of the full-length S protein, representing the prefusion (3.1[A] resolution) and postfusion (3.3[A] resolution) conformations, respectively. The spontaneous structural transition to the postfusion state under mild conditions is independent of target cells. The prefusion trimer forms a tightly packed structure with three receptor-binding domains clamped down by a segment adjacent to the fusion peptide, significantly different from recently published structures of a stabilized S ectodomain trimer. The postfusion conformation is a rigid tower-like trimer, but decorated by N-linked glycans along its long axis with almost even spacing, suggesting possible involvement in a mechanism protecting the virus from host immune responses and harsh external conditions. These findings advance our understanding of how SARS-CoV-2 enters a host cell and may guide development of vaccines and therapeutics.

biochemistry↗

bioRxiv2020

Prohibitin protects against cigarette smoke extract-induced cell apoptosis in cultured human pulmonary microvascular endothelial cells

Prohibitin is an evolutionarily conserved and ubiquitously expressed protein in eukaryocyte. It mediate many important roles in cell survival, apoptosis, autophagy and senescence. In the present study, we aimed to explore the role of prohibitin in cigarette smoke extract (CSE)-induced apoptosis of human pulmonary microvascular endothelial cells (HPMECs). For this purpose, HPMECs were trasfected with prohibitin and challenged with CSE. Our results showed that CSE exposure inhibited prohibitin expression in a dose-dependent manner in HPMECs. Overexpression of prohibitin could protect cell from CSE-induced injury by inhibiting CSE-induced cell apoptosis, inhibiting reactive oxygen species (ROS) production, increase mitochondrial membrane potential, increase the content of mitochondrial transcription factor A (mtTFA), IKK/{beta} phosphorylation and I{kappa}B- degradation. CSE decreases prohibitin expression in endothelial cells and restoration of prohibitin expression in these cells can protect against the deleterious effects of CSE on mitochondrial and cells. We identified prohibitin is a novel regulator of endothelial cell apoptosis and survival in the context of cigarette smoke exposure.

pharmacology and toxicology↗