Biology subjects

Cheng, Y.

Publications and source records attributed to Cheng, Y..

At least 289 records · Page 16Linked to original sources

bioRxiv2019

Dosage effect of multiple genes accounts for multisystem disorder of myotonic dystrophy type 1

Multisystem manifestations in myotonic dystrophy type 1 (DM1) may be due to dosage reduction in multiple genes induced by aberrant expansion of CTG repeats in DMPK, including Dmpk and its neighboring genes (Six5 or Dmwd) and downstream Mbnl1. However, the direct evidences are lack. Here, we develop a new strategy to generate mice carrying multigene mutations in one step by injection of haploid embryonic stem cells with mutant Dmpk, Six5 and Mbnl1 into oocytes. The triple heterozygous mutant mice exhibit adult-onset DM1 phenotypes. With the additional mutation in Dmwd, quadruple heterozygous mutant mice recapitulate many major manifestations in congenital DM1. Moreover, muscle stem cells in both models display reduced stemness. Our results suggest that the complex symptoms of DM1 result from the reduced gene dosage of multiple genes.

genetics↗

bioRxiv2019

General and robust covalently linked graphene oxide affinity grids for high-resolution cryo-EM

Despite their great potential to facilitate rapid preparation of quite impure samples, affinity grids have not yet been widely employed in single particle cryo-EM. Here, we chemically functionalize graphene oxide coated grids and use a highly specific covalent affinity tag system. Importantly, our polyethylene glycol spacer keeps particles away from the air-water interface and graphene oxide surface, protecting them from denaturation or aggregation and permits high-resolution reconstructions of small particles.

biophysics↗

bioRxiv2019

eIF3 promotes early translation elongation to ensure mitochondrial homeostasis and skeletal muscle health

eIF3 is a multi-subunit complex with numerous functions in canonical translation initiation. eIF3 was also found to interact with 40S and 60S ribosomal proteins and translation elongation factors, but a direct involvement in translation elongation has never been demonstrated. We found that eIF3 deficiency reduced early ribosomal elongation speed between codons 25 and 75 on a set of [~]2,700 mRNAs encoding proteins associated with mitochondrial and membrane functions, resulting in defective synthesis of their encoded proteins. To promote elongation, eIF3 interacts with 80S ribosomes translating the first [~]60 codons and serves to recruit protein quality control factors, functions required for normal mitochondrial physiology. Accordingly, eIF3e+/- mice accumulate defective mitochondria in skeletal muscle and show a progressive decline in muscle strength. Hence, eIF3 interacts with 80S ribosomes to enhance, at the level of early elongation, the synthesis of proteins with membrane-associated functions, an activity that is critical for mitochondrial physiology and muscle health.

cell biology↗

bioRxiv2019

An All-to-All Approach to the Identification of Sequence-Specific Readers for Epigenetic DNA Modifications on Cytosine

The authors have withdrawn their manuscript whilst they perform additional experiments to test some of their conclusions further. Therefore, the authors do not wish this work to be cited as reference for the project. If you have any questions, please contact the corresponding author.Competing Interest StatementThe authors have declared no competing interest.

systems biology↗

bioRxiv2019

Combinatorial morphogenetic and mechanical cues to mimic bone development for defect repair

Endochondral ossification during long bone development and natural fracture healing initiates by mesenchymal cell condensation and is directed by local morphogen signals and mechanical cues. Here, we aimed to mimic these developmental conditions for regeneration of large bone defects. We hypothesized that engineered human mesenchymal stem cell (hMSC) condensations with in situ presentation of transforming growth factor-{beta}1 (TGF-{beta}1) and/or bone morphogenetic protein-2 (BMP-2) from encapsulated microparticles would promote endochondral regeneration of critical-sized rat femoral bone defects in a manner dependent on the in vivo mechanical environment. Mesenchymal condensations induced bone formation dependent on morphogen presentation, with dual BMP-2 + TGF-{beta}1 fully restoring mechanical bone function by week 12. In vivo ambulatory mechanical loading, initiated at week 4 by delayed unlocking of compliant fixation plates, significantly enhanced the bone formation rate in the four weeks after load initiation in the dual morphogen group. In vitro, local presentation of either BMP-2 alone or BMP-2 + TGF-{beta}1 initiated endochondral lineage commitment of mesenchymal condensations, inducing both chondrogenic and osteogenic gene expression through SMAD3 and SMAD5 signaling. In vivo, however, endochondral cartilage formation was evident only in the BMP-2 + TGF-{beta}1 group and was enhanced by mechanical loading. The degree of bone formation was comparable to BMP-2 soaked on collagen but without the ectopic bone formation that limits the clinical efficacy of BMP-2/collagen. In contrast, mechanical loading had no effect on autograft-mediated repair. Together, this study demonstrates a biomimetic template for recapitulating developmental morphogenic and mechanical cues in vivo for tissue engineering. One Sentence SummaryMimicking aspects of the cellular, biochemical, and mechanical environment during early limb development, chondrogenically-primed human mesenchymal stem cell condensations promoted functional healing of critical-sized femoral defects via endochondral ossification, and healing rate and extent was a function of the in vivo mechanical environment.

bioengineering↗

bioRxiv2019

Electron cryo-microscopy structures of remodeler-nucleosome intermediates suggest allosteric control through the nucleosome.

The SNF2h remodeler slides nucleosomes most efficiently as a dimer, yet how the two protomers avoid a tug-of-war is unclear. Furthermore, SNF2h couples histone octamer deformation to nucleosome sliding, but the underlying structural basis remains unknown. Here we present cryo-EM structures of SNF2h-nucleosome complexes with ADP-BeFx that capture two reaction intermediates. In one structure, histone residues near the dyad and in the H2A-H2B acidic patch, distal to the active SNF2h protomer, are disordered. The disordered acidic patch is expected to inhibit the second SNF2h promoter, while disorder near the dyad is expected to promote DNA translocation. The other structure doesnt show octamer deformation, but surprisingly shows a 2bp translocation. FRET studies indicate that ADP-BeFx predisposes SNF2h-nucleosome complexes for an elemental translocation step. We propose a model for allosteric control through the nucleosome, where one SNF2h protomer promotes asymmetric octamer deformation to inhibit the second protomer, while stimulating directional DNA translocation. One sentence summaryCryo-EM structures capture different conformational states of chromatin remodeler-nucleosome complexes.

biophysics↗

bioRxiv2018

A multi-site MicroLED Optrode Array for Neural Interfacing

We present an electrically addressable optrode array capable of delivering light to 181 sites in the brain, each providing sufficient light to optogenetically excite hundreds of neurons in vivo, developed with the aim to allow behavioural studies in large mammals. The device is a glass microneedle array directly integrated with a custom fabricated microLED device, which delivers light to 100 needle tips and 81 interstitial surface sites, giving 2-level optogenetic excitation of neurons in vivo. Light delivery and thermal properties are evaluated, with the device capable of peak irradiances > 80 mW/mm2 per needle at 50 ms pulse widths with tissue temperature increase less than 1 {degrees}C. Future designs are explored through optical and thermal modelling and benchmarked against the current device.

neuroscience↗

bioRxiv2018

The research progress of p53 tumour suppressor activity controlled by Numb in triple-negative breast cancer

Numb is known as a cell fate determinant as it determines the direction of cell differentiation by asymmetrically partitioning at mitosis. It has been shown to be a tumor suppressor, and there is frequent loss of Numb expression in breast cancer. Numb enters in a tricomplex with p53 and the E3 ubiquitin ligase HDM2 (also known as MDM2), thereby preventing ubiquitination and degradation of p53. The aim of this study was to investigate the expression and migration of Numb, HDM2 and p53 proteins in the membrane, cytoplasmic and nuclear fractions of MCF-10A cells and MDA-MB-231 cells. We extracted the cell fractions to detect changes of these three protein levels after re-expression of NUMB in the basal-like triple-negative cell line MDA-MB-231 and knocking down NUMB in the normal mammary epithelial cell line MCF-10A. Our results show that Numb protein can migrate from cytoplasm to nucleus of the MDA-MB-231 cells. Numb protein regulates p53 levels in nucleus of MCF-10A cells and MDA-MB-231 cells, and the Numb levels is positively correlated with p53 levels. We have a finding on HDM2 protein that after knocking down NUMB in MCF-10A cells, it was remarkably reduced in the membrane fraction of NUMB knockdown cells, but its mRNA levels was significantly increased. We also examined the Numb expression in 125 patients with triple-negative breast cancer, 61(48.8%) displayed deficient or reduced expression of Numb. The percent of Ki67>14% in retained Numb group was significantly lower than that in the reduced and deficient Numb group (86.00% vs. 98.40%, P=0.0171).

molecular biology↗

bioRxiv2018

Structural insight into TRPV5 channel function and modulation

TRPV5 (transient receptor potential vanilloid) is a unique calcium-selective TRP channel that is essential for calcium homeostasis. TRPV5 and its close homologue TRPV6 do not exhibit thermosensitivity or ligand-dependent activation, unlike other TRPV channels, but are constitutively opened at physiological membrane potentials. Here, we report high resolution electron cryo-microscopy (cryo-EM) structures of truncated and full length TRPV5 in lipid nanodisc, as well as a TRPV5 W583A mutant structure and a complex structure of TRPV5 with calmodulin (CaM). These structures highlight and explain functional differences between the thermosensitive and calcium-selective TRPV channels. An extended S1-S2 linker folds on top of the channel that might shield it from modulation by extracellular factors. Resident lipid densities in the homologous vanilloid pocket are different from those previously found in TRPV1, supporting a comparatively more rigid architecture of TRPV5. A ring of tryptophan residues (W583) at the bottom of the pore coordinates a density and mutation of W583 resultes in opening of the lower gate. Moreover, we provide structural insight into the calcium-dependent channel inactivation and propose a flexible stoichiometry for TRPV5 and CaM binding.

biophysics↗

bioRxiv2018

Human-like NSG mouse glycoproteins sialylation pattern changes the phenotype of human lymphocytes and sensitivity to HIV-1 infection

BackgroundThe use of immunodeficient mice transplanted with human hematopoietic stem cells is an accepted approach to study human-specific infectious diseases, like HIV-1, and to investigate multiple aspects of human immune system development. However, mouse and human are different in sialylation patterns of proteins due to evolutionary mutations of the CMP-N-acetylneuraminic acid hydroxylase (CMAH) gene that prevent formation of N-glycolylneuraminic acid from N-acetylneuraminic acid. How changes of mouse glycoproteins chemistry will affect phenotype and function of transplanted human hematopoietic stem cells and mature human immune cells in the course of HIV-1 infection is not known.\n\nResultsWe mutated mouse CMAH on the most widely human cells transplantation strain NOD/scid-IL2R{gamma} c-/- (NSG) mouse background using the CRISPR/Cas9 system. The new strain provides a better environment for human immune cells. Transplantation of human hematopoietic stem cells leads to broad B cells repertoire, higher sensitivity to HIV-1 infection, and enhanced proliferation of transplanted peripheral blood lymphocytes. The mice showed low effects on the clearance of human immunoglobulins and enhanced transduction efficiency of recombinant adeno-associated viral vector rAAV2/DJ8.\n\nConclusionNSG-cmah-/- mice expand the mouse models suitable for human cells transplantation and this new model has advantages in generating a human B cell repertoire. This strain is suitable to study different aspects of the human immune system development, might provide advantages in patient-derived tissue and cell transplantation, and could allow studies of viral vectors and infectious agents that are sensitive to human-like sialylation of mouse glycoproteins.

immunology↗

bioRxiv2018

The structural basis for activation of voltage sensor domains in an ion channel TPC1

Voltage sensing domains (VSDs) couple changes in transmembrane electrical potential to conformational changes that regulate ion conductance through a central channel. Positively charged amino acids inside each sensor cooperatively respond to changes in voltage. Our previous structure of a TPC1 channel captured the first example of a resting-state VSD in an intact ion channel. To generate an activated state VSD in the same channel we removed the luminal inhibitory Ca2+-binding site (Cai2+), that shifts voltage-dependent opening to more negative voltage and activation at 0 mV. Cryo-EM reveals two coexisting structures of the VSD, an intermediate state 1 that partially closes access to the cytoplasmic side, but remains occluded on the luminal side and an intermediate activated state 2 in which the cytoplasmic solvent access to the gating charges closes, while luminal access partially opens. Activation can be thought of as moving a hydrophobic insulating region of the VSD from the external side, to an alternate grouping on the internal side. This effectively moves the gating charges from the inside potential to that of the outside. Activation also requires binding of Ca2+ to a cytoplasmic site (Caa2+). An X-ray structure with Caa2+ removed and a near-atomic resolution cryo-EM structure with Cai2+ removed define how dramatic conformational changes in the cytoplasmic domains may communicate with the VSD during activation. Together four structures provide a basis for understanding the voltage dependent transition from resting to activated state, the tuning of VSD by thermodynamic stability, and this channels requirement of cytoplasmic Ca2+-ions for activation.

biophysics↗

bioRxiv2018

MICU1 modulates MCU ion selectivity and tolerance to manganese stress

The mitochondrial calcium uniporter is a highly selective ion channel composed of species-and tissue-specific structural and regulatory subunits. However, the contribution of each component to uniporter-mediated activity still remains unclear. Here, we employ an evolutionary and synthetic biology approach to investigate the functional inter-dependence between the pore-forming subunit MCU and the EF-hand protein MICU1. Using phylogenetic profiling and genetic complementation analyses, we show that MCU and MICU1 constitute the minimal eukaryotic unit of the uniporter, pointing towards a strong selective pressure behind their co-occurrence. Heterologous reconstitution of MCU-mediated and MICU1-gated mitochondrial calcium entry in vivo in yeast cells demonstrates that MICU1 per se is essential to protect yeast from MCU-dependent manganese cytotoxicity. Accordingly, MICU1 deletion significantly sensitizes human HEK-293 cells to manganese-induced stress. Our study identifies a critical role of MICU1 in the regulation of MCU ion selectivity, with potential implications for patients with MICU1 deficiency.

cell biology↗

bioRxiv2018

Population dynamics and transcriptomic responses of Pseudomonas aeruginosa in a complex laboratory microbial community

Pseudomonas aeruginosa is one of the dominant species when it co-exists with many other bacterial species in diverse environments. To understand its physiology and interactions with co-existing bacterial species in different conditions, we established physiologically reproducible eighteen-species communities, and found that P. aeruginosa became the dominant species in mixed-species biofilm community but not in the planktonic community. P. aeruginosa H1 type VI secretion system was highly induced in the mixed-species biofilm community compare to its mono-species biofilm, which was further demonstrated to play a key role for P. aeruginosa to gain fitness over other bacterial species. In addition, the type IV pili and Psl exopolysaccharide were shown to be required for P. aeruginosa to compete with other bacterial species in the biofilm community. Our study showed that the physiology of P. aeruginosa is strongly affected by interspecies interactions, and both biofilm determinants and H1 type VI secretion system contribute to P. aeruginosa fitness over other species in complex biofilm communities.\n\nImportancePseudomonas aeruginosa usually coexists with different bacterial species in natural environment. However, systematic comparative characterization of P. aeruginosa in complex microbial communities with its mono-species communities is lacking. We constructed mixed-species planktonic and biofilm communities consisting P. aeruginosa and seventeen other bacterial species to study the physiology and interaction of P. aeruginosa in complex multiple-species community. A single molecule detection platform, NanoString nCounter(R) 16S rRNA array, was used to shown that P. aeruginosa can become the dominant species in the biofilm communities while not in the planktonic communities. Comparative transcriptomic analysis and fluorescence-based quantification further revealed that P. aeruginosa H1 type VI secretion system and biofilm determinants are both required for its fitness in mixed-species biofilm communities.

microbiology↗

bioRxiv2018

Structural basis for cholesterol transport-like activity of the Hedgehog receptor Patched

Hedgehog protein signals mediate tissue patterning and maintenance via binding to and inactivation of their common receptor Patched, a twelve-transmembrane protein that otherwise would suppress activity of the seven-transmembrane protein, Smoothened. Loss of Patched function, the most common cause of basal cell carcinoma, permits unregulated activation of Smoothened and of the Hedgehog pathway. A cryo-EM structure of the Patched protein reveals striking transmembrane domain similarities to prokaryotic RND transporters. The extracellular domain mediates association of Patched monomers in an unusual dimeric architecture that implies curvature in the associated membrane. A central conduit with cholesterol-like contents courses through the extracellular domain and resembles that used by other RND proteins to transport substrates, suggesting Patched activity in cholesterol transport. Patched expression indeed reduces cholesterol activity in the inner leaflet of the plasma membrane, in a manner antagonized by Hedgehog stimulation and with implications for regulation of Smoothened.

biophysics↗

bioRxiv2018

Extracellular Vesicles promote host immunity during an M. tuberculosis infection through RNA Sensing

Extracellular vesicles (EVs) have been shown to carry microbial components and function in the host defense against infections. In this study, we demonstrate that Mycobacterium tuberculosis (M.tb) RNA is delivered into macrophage-derived EVs through an M.tb SecA2-dependent pathway, and that EVs released from M.tb-infected macrophages stimulate a host RIG-I/MAVS/TBK1/IRF3 RNA sensing pathway, leading to type I interferon production in recipient cells. These EVs also promote, in a RIG-I/MAVS-dependent manner, the maturation of M.tb-containing phagosomes through a noncanonical LC3 modification, leading to increased bacterial killing. Moreover, treatment of M.tb-infected macrophages or mice with a combination of moxifloxacin and EVs, isolated from M.tb-infected macrophages, significantly lowered bacterial burden relative to either treatment alone. We propose that EVs, which are preferentially removed by macrophages in vivo, may be developed in combination with effective antibiotics as a novel approach to treat drug-resistant TB.

microbiology↗

bioRxiv2018

A simple and robust procedure for preparing graphene-oxide cryo-EM grids

Graphene oxide (GO) sheets have been used successfully as a supporting substrate film in several recent cryogenic electron-microscopy (cryo-EM) studies of challenging biological macromolecules. However, difficulties in preparing GO-covered holey carbon EM grids have limited its widespread use. Here, we report a simple and robust method for covering holey carbon EM grids with GO sheets and demonstrate that these grids are suitable for high-resolution single particle cryo-EM. GO substrates adhere macromolecules, allowing cryo-EM grid preparation with lower specimen concentrations and providing partial protection from the air-water interface. Additionally, the signal from images of the GO lattice beneath the frozen-hydrated specimen can be discerned in many motion-corrected micrographs, providing a high-resolution fiducial for evaluating beam-induced motion correction.

biophysics↗

bioRxiv2018

Reverse-engineering flow-cytometry gating strategies for phenotypic labelling and high-performance cell sorting

MotivationRecent flow and mass cytometers generate 1,000,000 single cell datasets of dimensions 20 to 40. Many tools facilitate the discovery of new cell populations associated with diseases or physiology. These discoveries require the identification of new gating strategies, but gating strategies become exponentially harder to optimize when dimensionality increases. To facilitate this step we developed Hypergate, an algorithm which given a cell population of interest identifies a gating strategy optimized for high yield and purity.\n\nResultsHypergate achieves higher yield and purity than human experts, Support Vector Machines and Random-Forests on public datasets. We use it to revisit some established gating strategies for the identification of Innate lymphoid cells, which identifies concise and efficient strategies that allow gating these cells with fewer parameters but higher yield and purity than the current standards. For phenotypic description, Hypergates outputs are consistent with fields knowledge and sparser than those from a competing method.\n\nAvailability and ImplementationHypergate is implemented in R and available at http://github.com/ebecht/hypergate under an Open Source Initiative-compliant licence.

bioinformatics↗

bioRxiv2018

Efficient differentiation of vascular endothelial cells from dermal-derived mesenchymal stem cells induced by endothelial cell lines conditioned medium

ObjectiveTo directionally-differentiate dermis-derived mesenchymal stem cells (DMSCs) into vascular endothelial cells (VECs) in vitro, providing an experimental basis for studies on the pathogenesis and treatment of vascular diseases.\n\nMethodsAfter separation by adherent culture, VEC line supernatant, vascular endothelial growth factor (VEGF), bone morphogenetic protein-4 and hypoxia were used for the differentiation of VECs from DMSCs. The cell type was authenticated by flow cytometry, matrigel angiogenesis assay in vitro, and immunofluorescent staining during differentiation. The VEGF concentration was investigated by enzyme-linked immunosorbent assay.\n\nResultsAfter 28 days of differentiation, the cell surface marker CD31 was significantly positive (80%-90%) by flow cytometry in the VEC line-conditioned culture, which was significantly higher than in the other groups. Differentiated DMSCs had the ability to ingest Dil-ac-LDL and vascularize in the conditioned culture, but not in the other groups. In the VEC line supernatant, the concentration of VEGF was very low. The VEGF concentration changed along with the differentiation into VECs in the medium of the conditioned culture group.\n\nConclusionVEC line supernatant can induce the differentiation of DMSCs into VECs, possibly through the pathway except VEGF.

cell biology↗