Biology subjects

Wan, Z.

Publications and source records attributed to Wan, Z..

47 records · Page 3Linked to original sources

bioRxiv2022

Single-Objective Evanescent Scattering Microscopy for Imaging Single Proteins and Binding Kinetics

Plasmonic scattering microscopy has advanced the evanescent detection approaches by offering wide-field single-molecule imaging capability. However, two limitations prevent the broader application of plasmonic single-molecule imaging. One is the heating effect accompanying the plasmonic enhancement, and the other is the complicated system structure resulting from the two-objective optical arrangement. Here, we report single-objective evanescent scattering microscopy. The evanescent field is created by total internal reflection instead of the surface plasmon resonance on the gold film. As a result, the sensing substrate without gold film produces little heat, and allows excitation and observation using one objective. In addition, this system enables quantification of protein binding kinetics by simultaneously counting the binding of individual molecules and recording their binding sites with nanometer precision, providing a digital method to measure binding kinetics with high spatiotemporal resolution. This work may pave a road for label-free single protein analysis in conventional microscopy. TeaserLabel-free single-molecule imaging on a total internal reflection fluorescence objective.

biophysics↗

bioRxiv2021

Personalized models of breast cancer desmoplasia reveal biomechanical determinants of drug penetration

Breast cancer desmoplasia heterogeneity contributes to high disease mortality due to discrepancies in treatment efficacy between patients. Personalized in vitro breast cancer models can be used for high throughput testing and ranking of therapeutic strategies to normalize the aberrant microenvironment in a patient-specific manner. Here, tumoroids assembled from patient-derived cells cultured in microphysiological systems including perfusable microvasculature reproduce key aspects of stromal and vascular dysfunction. Increased hyaluronic acid and collagen deposition, loss of vascular glycocalyx and reduced perfusion, and elevated interstitial fluid pressure in the models result in impaired drug distribution to tumor cells. We demonstrate the application of these personalized models as tools to rank molecular therapies for the normalization of the tumoroid microenvironment and to discover new therapeutic targets such as IL8 and CD44, which may ultimately improve drug efficacy in breast cancer patients.

bioengineering↗

bioRxiv2021

Three-dimensional tracking of tethered particles for probing nanometer-scale single-molecule dynamics using plasmonic microscope

Three-dimensional (3D) tracking of surface-tethered single-particle reveals the dynamics of the molecular tether. However, most 3D tracking techniques lack precision, especially in axial direction, for measuring the dynamics of biomolecules with spatial scale of several nanometers. Here we present a plasmonic imaging technique that can track the motion of [~]100 tethered particles in 3D simultaneously with sub-nanometer axial precision at millisecond time resolution. By tracking the 3D coordinates of tethered particle with high spatial resolution, we are able to determine the dynamics of single short DNA and study its interaction with enzyme. We further show that the particle motion pattern can be used to identify specific and non-specific interactions in immunoassays. We anticipate that our 3D tracking technique can contribute to the understanding of molecular dynamics and interactions at the single-molecule level.

biophysics↗

bioRxiv2021

Comparative genomics of Chinese and international isolates of Escherichia albertii: population structure and evolution of virulence and antimicrobial resistance

Escherichia albertii is a newly recognized species in the genus Escherichia that causes diarrhea. The population structure, genetic diversity and genomic features has not been fully examined. Here, 169 E. albertii isolates from different sources and regions in China were sequenced and combined with 312 publicly available genomes for phylogenetic and genomic analyses. The E. albertii population was divided into 2 clades and 8 lineages, with lineage 3 (L3), L5 and L8 more common in China. Clinical isolates were observed in all clades/lineages. Virulence genes were found to be distributed differently among lineages: subtypes of the intimin encoding gene eae and the cytolethal distending toxin (Cdt) gene cdtB were lineage associated, the second type three secretion system (ETT2) island was truncated in L3 and L6. Seven new eae subtypes and 1 new cdtB subtype (cdtB-VI) were found. Alarmingly, 85.9% of the Chinese E. albertii isolates were predicted to be multidrug resistant (MDR) with 35.9% harboured genes capable of conferring resistance to 10 to 14 different drug classes. By in silico multi-locus sequence typing, majority of the MDR isolates belonged to 4 STs (ST4638, ST4479, ST4633 and ST4488). Thirty-four intact plasmids carrying MDR and virulence genes, and 130 intact prophages were identified from 17 complete E. albertii genomes. Ten plasmid replicon types were found to be significantly associated with MDR. The 130 intact prophages were clustered into 5 groups, with group 5 prophages harbouring more virulence genes. Our findings provided fundamental insights into the population structure, virulence variation and MDR of E. albertii. Impact statementE. albertii is newly recognized foodborne pathogen causing diarrhea. Elucidation of its genomic features is important for surveillance and control of E. albertii infections. In this work, 169 E. albertii genomes from difference sources and regions in China were collected and sequenced, which contributed to the currently limited genomic data pool of E. albertii. In combination with 312 publicly available genomes from 14 additional countries, the population structure of E. albertii was defined. The presence and subtypes of virulence genes in different lineages were significantly different, indicating potential pathogenicity variation. Additionally, the presence of multidrug resistance (MDR) genes was alarmingly high in the Chinese dominated lineages. MDR associated STs and plasmid subtypes were identified, which could be used as sentinels for MDR surveillance. Moreover, the subtypes of plasmids and prophages were distributed differently across lineages, and were found to contribute to the acquisition of virulence and MDR genes of E. albertii. Altogether, this work reveals the diversity of E. albertii and characterized its genomic features in unprecedented detail. Data SummaryAll newly sequenced data in this work were deposited in National Center for Biotechnology Information (NCBI) under the BioProject of PRJNA693666, including 6 complete genomes and raw reads of 164 E. albertii isolates.

genomics↗

bioRxiv2020

Glycocalyx-Mediated Vascular Dissemination of Circulating Tumor Cells

The glycocalyx on tumor cells has been recently identified as an important driver for cancer progression, possibly providing critical opportunities for treatment. Metastasis, in particular, is often the limiting step in the survival to cancer, yet our understanding of how tumor cells escape the vascular system to initiate metastatic sites remains limited. Using an in vitro model of the human microvasculature, we assess here the importance of the tumor and vascular glycocalyces during tumor cell extravasation. Through selective manipulation of individual components of the glycocalyx, we reveal a novel mechanism whereby tumor cells prepare an adhesive vascular niche by depositing components of the glycocalyx along the endothelium. Accumulated hyaluronic acid shed by tumor cells subsequently mediates adhesion to the endothelium via the glycoprotein CD44. Trans-endothelial migration and invasion into the stroma occurs through binding of the isoform CD44v to components of the sub-endothelial extra-cellular matrix. Targeting of the hyaluronic acid-CD44 glycocalyx complex results in significant reduction in the extravasation of tumor cells. These studies provide evidence of tumor cells repurposing the glycocalyx to promote adhesive interactions leading to cancer progression. Such glycocalyx-mediated mechanisms may be therapeutically targeted to hinder metastasis and improve patient survival.

cancer biology↗

bioRxiv2019

TLR2 signaling pathway combats Streptococcus uberis infection by inducing production of mitochondrial reactive oxygen species

Mastitis caused by Streptococcus uberis is a hazardous clinical disease in dairy animals. In this study, the role of Toll-like receptors (TLRs) and TLR-mediated signaling pathways in mastitis caused by S. uberis was investigated using mouse models and mammary epithelial cells (MECs). We used S. uberis to infect mammary glands of wild type, TLR2-/- and TLR4-/- mice and quantified the adaptor molecules in TLR signaling pathways, proinflammatory cytokines, tissue damage and bacterial count in mammary glands. When compared with TLR4 deficiency, TLR2 deficiency induced more severe pathological changes through myeloid differentiation primary response 88 (MyD88)-mediated signaling pathways during S. uberis infection. In MECs, TLR2 detected S. uberis infection and induced mitochondrial reactive oxygen species (mROS) to assist host control of secretion of inflammatory factors and elimination of intracellular S. uberis. Our results demonstrate that TLR2-mediated mROS have a significant effect on S. uberis-induced host defense responses in mammary glands as well as MECs. Author summaryS. uberis contributes significantly to global mastitis and remains a major obstacle for inflammation elimination due to its ability to form persistent infection in mammary tissue. The Toll-like receptor (TLR) family plays a significant role in identifying infections of intracellular bacteria and further triggering inflammatory reactions in immune cells. However, the detailed molecular mechanism by which TLR is regulated, and whether MECs, as the main cells in mammary gland, are tightly involved in these processes is poorly understood. Here, we used S. uberis to infect mammary glands of wild type, TLR2-/-, TLR4-/- mice and MECs to assess pathogenesis, proinflammatory cytokines, ROS as well as mROS levels during infection. We found that during S.uberis infection, it is TLR2 deficiency that induced more severe pathological changes through MyD88-mediated signaling pathways. In addition, our work demonstrates that mROS mediated by TLR2 has an important role in host defense response to combat S. uberis infection in mammary glands as well as MECs.

pathology↗

bioRxiv2019

Germline-encoded TCR-MHC contacts promote TCR V gene bias in umbilical cord blood T cell repertoire

T cells recognize antigens as peptides bound to major histocompatibility complex (MHC) proteins through T cell receptors (TCRs) on their surface. To recognize a wide range of pathogens, each individual possesses a substantial number of TCRs with an extremely high degree of variability. It remains controversial whether germline-encoded TCR repertoire is shaped by MHC polymorphism and, if so, what is the preference between MHC genetic variants and TCR V gene compatibility. To investigate the \"net\" genetic association between MHC variations and TRBV genes, we applied quantitative trait locus (QTL) mapping to test the associations between MHC polymorphism and TCR {beta} chain V (TRBV) genes usage using umbilical cord blood (UCB) samples of 201 Chinese newborns. We found TRBV gene and MHC loci that are predisposed to interact with one another differ from previous conclusions. The majority of MHC amino acid residues associated with the TRBV gene usage show spatial proximities in known structures of TCR-pMHC complexes. These results show for the first time that MHC variants bias TRBV gene usage in UCB of Chinese ancestry and indicate that germline-encoded contacts influence TCR-MHC interactions in intact T cell repertoires.

immunology↗

bioRxiv2018

Optical imaging of single protein size, charge, mobility, binding and conformational change

Protein analysis has relied on electrophoresis, mass spectroscopy and immunoassay, which separate, detect and identify proteins based on the size, charge, mobility and binding to antibodies. However, measuring these quantities at the single molecule level has not been possible. We tether a protein to a surface with a flexible polymer, drive the protein into mechanical oscillation with an alternating electric field, and image the protein oscillation with a near field imaging method, from which we determine the size, charge, mobility of the protein. We also measure binding of antibodies to single proteins and ligand binding-induced conformational changes in single proteins. This work provides new capabilities for protein analysis and disease biomarker detection at the single molecule level.

biophysics↗

bioRxiv2018

Investigating the conformational response of the Sortilin receptor upon binding endogenous peptide- and protein ligands by HDX-MS

Sortilin is a multifunctional transmembrane neuronal receptor involved in sorting of neurotrophic factors and apoptosis signalling. So far, structural characterization of Sortilin and its endogenous ligands has been limited to crystallographic studies of Sortilin in complex with the neuropeptide Neurotensin. Here, we use hydrogen/deuterium exchange mass spectrometry to investigate the conformational response of Sortilin to binding biological ligands including the peptides Neurotensin and the Sortilin propeptide and the proteins Progranulin and pro-Nerve growth factor-{beta}. The results show that the ligands employ two binding sites inside the cavity of the {beta}-propeller of Sortilin. However, ligands have distinct differences in their conformational impact on the receptor. Interestingly, the protein ligands induce conformational stabilization in a remote membrane-proximal domain, hinting at an unknown conformational link between the ligand binding region and this membrane-proximal region of Sortilin. Our findings improves our molecular understanding of Sortilin and how it mediates diverse ligand-dependent functions important in neurobiology.

biophysics↗

bioRxiv2018

Single-cell Transcriptomic Landscape of Nucleated Cells in Umbilical Cord Blood

Umbilical cord blood (UCB) transplant is a therapeutic option for both pediatric and adult patients with a variety of hematologic diseases such as several types of blood cancers, myeloproliferative disorders, genetic diseases, and metabolic disorders. However, the level of cellular heterogeneity and diversity of nucleated cells in the UCB has not yet been assessed in an unbiased and systemic fashion. In the current study, nucleated cells from UCB were subjected to single-cell RNA sequencing, a technology enabled simultaneous profiling of the gene expression signatures of thousands of cells, generating rich resources for further functional studies. Here, we report the transcriptomic maps of 19,052 UCB cells, covering 11 major cell types. Many of these cell types are comprised of distinct subpopulations, including distinct signatures in NK and NKT cell types in the UCB. Pseudotime ordering of nucleated red blood cells (NRBC) identifies wave-like activation and suppression of transcription regulators, leading to a polarized cellular state, which may reflect the NRBC maturation. Progenitor cells in the UBC also consist two subpopulations with divergent transcription programs activated, leading to specific cell-fate commitment. Collectively, we provide this comprehensive single-cell transcriptomic landscape and show that it can uncover previously unrecognized cell types, pathways and gene expression regulations that may contribute to the efficacy and outcome of UCB transplant, broadening the scope of research and clinical innovations.

genomics↗

bioRxiv2018

Identification and quantification of Lyme pathogen strains by deep sequencing of outer surface protein C (ospC) amplicons

Mixed infection of a single tick or host by Lyme disease spirochetes is common and a unique challenge for diagnosis, treatment, and surveillance of Lyme disease. Here we describe a novel protocol for differentiating Lyme strains based on deep sequencing of the hypervariable outer-surface protein C locus (ospC). Improving upon the traditional DNA-DNA hybridization method, the next-generation sequencing-based protocol is high-throughput, quantitative, and able to detect new pathogen strains. We applied the method to over one hundred infected Ixodes scapularis ticks collected from New York State, USA in 2015 and 2016. Analysis of strain distributions within individual ticks suggests an overabundance of multiple infections by five or more strains, inhibitory interactions among co-infecting strains, and presence of a new strain closely related to Borreliella bissettiae. A supporting bioinformatics pipeline has been developed. With the newly designed pair of universal ospC primers targeting intergenic sequences conserved among all known Lyme pathogens, the protocol could be used for culture-free identification and quantification of Lyme pathogens in wildlife and clinical specimens across the globe.

microbiology↗