Biology subjects

Shen, J.

Publications and source records attributed to Shen, J..

At least 181 records · Page 10Linked to original sources

bioRxiv2021

SHERRY2: A method for rapid and sensitive single cell RNA-seq

Prevalent single cell transcriptomic profiling (scRNA-seq) mechods are mainly based on synthesis and enrichment of full-length double-stranded complementary DNA. These approaches are challenging to generate accurate quantification of transcripts when their abundance is low or their full-length amplifications are difficult. Based on our previous finding that Tn5 transposase can directly cut-and-tag DNA/RNA hetero-duplexes, we present SHERRY2, a specifically optimized protocol for scRNA-seq without second strand cDNA synthesis. SHERRY2 is free of pre-amplification and eliminates the sequence-dependent bias. In comparison with other widely-used scRNA-seq methods, SHERRY2 exhibits significantly higher sensitivity and accuracy even for single nuclei. Besides, SHERRY2 is simple and robust, and can be easily scaled up to high-throughput experiments. When testing single lymphocytes and neuron nuclei, SHERRY2 not only obtained accurate countings of transcription factors and long non-coding RNAs, but also provided bias-free results that enriched genes in specific cellular components or functions, which outperformed other protocols. With a few thousand cells sequenced by SHERRY2, we confirmed expression and dynamics of Myc in different cell types of germinal centers, which were previously only revealed by gene-specific amplification methods. SHERRY2 is able to provide high sensitivity, high accuracy, and high throughput for those applications that require high number of genes identified in each cell. It can reveal the subtle transcriptomic difference between cells and facilitate important biological discoveries.

genomics↗

bioRxiv2021

Electron relay via diffusible and protein-bound flavin cofactor in human six-transmembrane epithelial antigen of the prostate (STEAP)

Six transmembrane epithelial antigen of the prostate (STEAP) is a family of membrane-embedded hemoproteins with four members, STEAP1-4, all of which have a transmembrane domain (TMD) that chelates a heme prosthetic group. STEAP2-4, but not STEAP1, have an intracellular oxidoreductase domain (OxRD) so that an electron transfer chain composed of NADPH, FAD, and heme is established to mediate electron transfer across cell membranes. However, it is not known whether STEAP1 can establish a physiologically relevant electron transfer chain. Here we show that reduced FAD binds to STEAP1 and enables reduction of the heme. We also show that a soluble cytochrome b5 reductase can dock on STEAP1 and serve as a surrogate OxRD to reduce the heme. These results provide the first evidence that STEAP1 can support a cross-membrane electron transfer chain. It is not clear whether FAD, which relays electrons from NADPH to heme and interacts with both OxRD and TMD, remains constantly bound to the STEAPs. We found that FAD reduced by STEAP2 can be utilized by STEAP1, supporting the hypothesis that FAD is diffusible rather than staying bound to STEAP2. We determined the structure of human STEAP2 in complex with NADP+ and FAD to an overall resolution of 3.2 [A] by cryo-electron microscopy. The structure of STEAP2 shows that the two cofactors bind similarly to those in the STEAP4 structure and thus a diffusible FAD is likely a general feature of the electron transfer mechanism in the STEAPs. The structure of STEAP2 also shows that its extracellular regions are less structured than those of STEAP4 or STEAP1, and further experiments show that STEAP2 reduces Fe3+-NTA with a rate significantly slower than STEAP1. These results establish a solid foundation for understanding the function and mechanisms of STEAP family of proteins.

biochemistry↗

bioRxiv2021

Fracture healing is delayed in the absence of gasdermin signaling

Amino-terminal fragments from proteolytically cleaved gasdermins (GSDMs) form plasma membrane pores that enable the secretion of interleukin-1{beta} (IL-1{beta}) and IL-18. Excessive GSDM- mediated pore formation can compromise the integrity of the plasma membrane thereby causing the lytic inflammatory cell death, pyroptosis. We found that GSDMD and GSDME were the only GSDMs that were readily expressed in bone microenvironment. Therefore, we tested the hypothesis that GSDMD and GSDME are implicated in fracture healing owing to their role in the obligatory inflammatory response following injury. We found that bone callus volume and biomechanical properties of injured bones were significantly reduced in mice lacking either GSDM compared with wild-type (WT) mice, indicating that fracture healing was compromised in mutant mice. However, compound loss of GSDMD and GSDME did not exacerbate the outcomes, suggesting shared actions of both GSDMs in fracture healing. Mechanistically, bone injury induced IL-1{beta} and IL-18 secretion in vivo, a response that was mimicked in vitro by bone debris and ATP, which function as inflammatory danger signals. Importantly, the secretion of these cytokines was attenuated in conditions of GSDMD deficiency. Finally, deletion of IL-1 receptor reproduced the phenotype of Gsdmd or Gsdme deficient mice, implying that inflammatory responses induced by the GSDM-IL-1 axis promote bone healing after fracture.

immunology↗

bioRxiv2021

Transmembrane helices mediate the formation of a stable ternary complex of cyt b5 reductase, cyt b5, and SCD1

Mammalian cytochrome b5 (cyt b5) and cytochrome b5 reductase (b5R) are electron carrier proteins required for many membrane-embedded oxidoreductases. Both cyt b5 and b5R have a cytosolic domain anchored to the membrane by a single transmembrane helix (TM). It is not clear if b5R, cyt b5 and their partner oxidoreductases assemble as binary or ternary complexes. Here we show that b5R and cyt b5 form a stable binary complex, and that b5R, cyt b5 and a membrane-embedded oxidoreductase, stearoyl-CoA desaturase 1 (SCD1) form a stable ternary complex. The formation of the complexes significantly enhances electron transfer rates, and that the single TM of cyt b5 and b5R mediated assembly of the complexes. These results reveal a novel functional role of TMs in cyt b5 and b5R and suggest that an electron transport chain composed of a stable ternary complex may be a general feature in oxidoreductases that require the participation of cyt b5 and b5R.

biochemistry↗

bioRxiv2021

Low frequency somatic copy number alterations in normal human lymphocytes revealed by large scale single-cell whole genome profiling

Genomic-scale somatic copy number alterations in healthy humans are difficult to investigate because of low occurrence rates and the structural variations stochastic natures. Using a Tn5-transposase assisted single-cell whole genome sequencing method, we sequenced over 20,000 single lymphocytes from 16 individuals. Then, with the scale increased to a few thousand single cells per individual, we found that about 7.5% of the cells had large-size copy number alterations. Trisomy 21 was the most prevalent aneuploid event among all autosomal copy number alterations, while monosomy X occurred most frequently in over-30-year-old females. In the monosomy X single cells from individuals with phased genomes and identified X-inactivation ratios in bulk, the inactive X Chromosomes were lost more often than were the active ones.

genomics↗

bioRxiv2021

Targeting a central feature of asthma using a cell type-selective IL-13-responsive enhancer

IL-13 is a central mediator of asthma1-3. Here, we used genome-wide approaches to characterize genes and regulatory elements modulated by IL-13 and other asthma-associated cytokines in airway epithelial cells and showed how they can be used for therapeutic purposes. Using bulk and single cell RNA-seq, we found distinctive responses to IL-13, IL-17, and interferons in human bronchial epithelial basal, ciliated, and secretory cells. H3K27ac ChIP-seq revealed that IL-13 had widespread effects on regulatory elements. Detailed characterization of an enhancer of SPDEF, a transcription factor required for pathologic mucin production, revealed that STAT6 and KLF5 binding sites cooperate to drive IL-13-dependent transcription selectively in secretory cells. Using this enhancer to drive CRISPRi and knockdown either SPDEF or the mucin MUC5AC showed the potential use of this approach for asthma therapeutics. This work identifies numerous genes and regulatory elements involved in cell type-selective cytokine responses and showcases their use for therapeutic purposes.

immunology↗

bioRxiv2021

Reference genomes of 545 silkworms enable high-throughput exploring genotype-phenotype relationships

The silkworm Bombyx mori is a domestic insect for silk production and a lepidopteran model. The currently available genomes limit a full understanding of its genetic and phenotypic diversity. Here we assembled long-read genomes of 545 domestic and wild silkworms and constructed a high-resolution pan-genome dataset. We found that the silkworm population harbors extremely variable genomes containing 7,308 new gene families, 4,260 (22%) core gene families, and 3,432,266 non-redundant SVs. We deciphered a series of causal genes and variants associated with domestication, breeding, and ecological adaptation traits, and experimentally validated two of those genes using CRISPR-Cas9 or RNA interference. This unprecedented large-scale genomic resource allows for high-throughput screening of interesting traits for functional genomic research and breeding improvement of silkworms and may serve as a guideline for traits decoding in other species.

genomics↗

bioRxiv2021

Impedance flow cytometry for viability analysis of Corynebacterium glutamicum

Corynebacterium glutamicum efficiently produces glutamate when growth is inhibited. Analyses of viability in this non-growing state requires time consuming plating and determination of colony forming units. We here establish impedance flow cytometry measurements to assess the viability of non-growing, glutamate producing C. glutamicum cultures within minutes.

microbiology↗

bioRxiv2021

Quantifying microbial associations of dissolved organic matter under global change

Microbes play a critical role in regulating the size, composition, and turnover of dissolved organic matter (DOM), which is one of the largest pools of carbon in aquatic ecosystems. Global change may alter DOM-microbe associations with implications for biogeochemical cycles, although disentangling these complex interactions remains a major challenge. Here we develop a framework called Energy-Diversity-Trait integrative Analysis (EDTiA) to examine the associations between DOM and bacteria along temperature and nutrient gradients in a manipulative field experiment on mountainsides in contrasting subarctic and subtropical climates. In both study regions, the chemical composition of DOM correlated with bacterial communities, and was primarily controlled by nutrients and to a lesser degree by temperature. At a molecular-level, DOM-bacteria associations depended strongly on the molecular traits of DOM, with negative associations indicative of decomposition as molecules are more biolabile. Using bipartite networks, we further demonstrated that negative associations were more specialized than positive associations indicative of DOM production. Nutrient enrichment promoted specialization of positive associations, but decreased specialization of negative associations particularly at warmer temperatures in subtropical climate. These global change drivers influenced specialization of negative associations most strongly via molecular traits, while both molecular traits and bacterial diversity similarly affected positive associations. Together, our framework provides a quantitative approach to understand DOM-microbe associations and wider carbon cycling across scales under global change.

ecology↗

bioRxiv2021

Visualizing the pH in Escherichia coli colonies via the sensor protein mCherryEA allows high-throughput screening of mutant libraries

Cytoplasmic pH is tightly regulated by diverse active mechanisms and interconnected regulatory processes in bacteria. Many processes and regulators underlying pH-homeostasis have been identified via phenotypic screening of strain libraries towards non-growth at low or high pH values. Direct screens with respect to changes of the internal pH in mutant strain collections are limited by laborious methods including fluorescent dyes or radioactive probes. Genetically encoded biosensors equip single organisms or strain libraries with an internal sensor molecule already during the generation of the strain. In this study, we used the pH-sensitive mCherry variant mCherryEA as ratiometric pH biosensor. We visualized the internal pH of E. coli colonies on agar plates by the use of a Gel-Doc imaging system. Combining this imaging technology with robot-assisted colony picking and spotting allowed us to screen and select mutants with altered internal pH values from a small transposon mutagenesis derived E. coli library. Identification of the TN- insertion sites in strains with altered internal pH levels revealed that the transposon was inserted into trkH (encoding a transmembrane protein of the potassium uptake system) or the rssB gene (encoding the anti-adaptor protein RssB which mediates the proteolytic degradation of the general stress response regulator RpoS), two genes known to be associated with pH-homeostasis and pH stress adaptation. This successful screening approach demonstrates that the pH- sensor based analysis of arrayed colonies on agar plates is a sensitive approach for the fast identification of genes involved in pH-homeostasis or pH stress adaptation in E. coli. ImportancePhenotypic screening of strain libraries on agar plates has become a versatile tool to understand gene functions and to optimize biotechnological platform organisms. Screening is supported by genetically encoded biosensors that allow to easily measure intracellular processes. For this purpose, transcription-factor-based biosensors have emerged as the sensor-type of choice. Here, the target stimulus initiates the activation of a response gene (e.g. a fluorescent protein) followed by transcription, translation and maturation. Due to this mechanistic principle, biosensor readouts are delayed and cannot report the actual intracellular state of the cell in real-time. To capture fast intracellular processes adequately, fluorescent reporter proteins are extensively applied. But these sensor-types are not utilized for phenotypic screenings so far. To take advantage of their properties, we here established an imaging method, which allows to apply a fast ratiometric sensor protein for assessing the internal pH of colonies in a high-thoughput manner.

microbiology↗

bioRxiv2021

DAB2IP inhibits p53 ubiquitin-mediated degradation by competitively binding to GRP75 and suppresses tumor malignancy in colon cancer

Increasing evidence has shown that DAB2IP acts as a tumor suppressor and plays an inhibition role in many tumors. However, the underlying mechanism is still uncertain. Our study shows that DAB2IP is positively associated with a better prognosis in colon cancer patients with wild-type TP53 expression. In vitro assay shows that DAB2IP elicits potent tumor-suppressive effects on inhibiting cell invasiveness, colony formation and promoting cell apoptosis in wild-type TP53 colon cancer cell lines. Subsequently, DAB2IP is demonstrated to up-regulate the stability of wild-type TP53 by inhibiting its degradation in a ubiquitin-proteasome-dependent manner. Using mass spectrometry profiling, we unveil that DAB2IP and p53 could both interact with the ubiquitin ligase-related protein, GRP75. Mechanistically, DAB2IP could competitively bind with GRP75, thus reducing GRP75-mediated p53 ubiquitination and degradation. Finally, animal experiments also reveal that DAB2IP inhibits the tumor progression in vivo. In conclusion, our study presents a novel function of DAB2IP in GRP75-driven wild-type p53 degradation, which provides a new insight in DAB2IP-induced tumor suppression and provides a novel molecular aspect of the p53 pathway.

cancer biology↗

bioRxiv2021

Reactivities of the Front Pocket N-Cap Cysteines in Human Kinases

Discovery of targeted covalent inhibitors directed at nucleophilic cysteines is attracting enormous interest. The front pocket (FP) N-cap cysteine has been the most popular site of covalent modification in kinases. Curiously, a long-standing hypothesis associates the N-cap position with cysteine hyper-reactivity; however, traditional computational methods suggest that the FP N-cap cysteines in all human kinases are predominantly unreactive at physiological pH. Here we applied a newly developed GPU-accelerated continuous constant pH molecular dynamics (CpHMD) tool to test the N-cap hypothesis and elucidate the cysteine reactivities. Simulations showed that the N-cap cysteines in BTK/BMX/TEC/ITK/TXK, JAK3, and MKK7 sample the reactive thiolate form to varying degrees at physiological pH; however, those in BLK and EGFR/ERBB2/ERBB4 which contain an Asp at the N-cap+3 position adopt the unreactive thiol form. The latter argues in favor of the base-assisted thiol-Michael addition mechanisms as suggested by the quantum mechanical calculations and experimental structure-function studies of EGFR inhibitors. Analysis revealed that the reactive N-cap cysteines are stabilized by hydrogen bond as well as electrostatic interactions, and in their absence a N-cap cysteine is unreactive due to desolvation. To test a corollary of the N-cap hypothesis, we also examined the reactivities of the FP N-cap+2 cysteines in JNK1/JNK2/JNK3 and CASK. Additionally, our simulations predicted the reactive cysteine and lysine locations in all 15 kinases. Our findings offer a systematic understanding of cysteine reactivities in kinases and demonstrate the predictive power and physical insights CpHMD can provide to guide the rational design of targeted covalent inhibitors.

biophysics↗

bioRxiv2021

Circularly permuted LOV domain as an engineering module for optogenetic tools

We re-engineered a commonly-used light-sensing protein, LOV domain, using a circular permutation strategy to allow photoswitchable control of the C-terminus of a peptide. We demonstrate that the use of circularly permuted LOV domain on its own or together with the original LOV could expand the engineering capabilities of optogenetic tools.

synthetic biology↗

bioRxiv2021

Mechanism and Kinetics of Fentanyl Dissociation from the mu-Opioid Receptor

Driven by illicit fentanyl, opioid related deaths have reached the highest level in 2020. Currently, an opioid over-dose is resuscitated by the use of naloxone, which competitively binds and antagonizes the {micro}-opioid receptor (mOR). Thus, knowledge of the residence times of opioids at mOR and the unbinding mechanisms is valuable for assessing the effectiveness of naloxone. In the present study we calculate the fentanyl-mOR dissociation time and elucidate the mechanism by applying an enhanced sampling molecular dynamics (MD) technique. Two sets of metadynamics simulations with different initial structures were performed while accounting for the protonation state of the conserved H2976.52, which has been suggested to modulate the ligand-mOR affinity and binding mode. Surprisingly, with the N{delta}-protonated H2976.52, fentanyl can descend as much as 10 [A] below the level of the conserved D1473.32 before escaping the receptor, and has a calculated residence time{tau} of 38 s. In contrast, with the N - and doubly protonated H2976.52, the calculated{tau} are 2.6 s and 0.9 s, respectively. Analysis suggests that formation of the piperidine-Hid297 hydrogen bond strengthens the hydrophobic contacts with the transmembrane helix (TM) 6, allowing fentanyl to explore a deep pocket. Considering the experimental{tau} of [~]4 min for fentanyl and the role of TM6 in mOR activation, we suggest that the deep insertion mechanism may be biologically relevant. The work paves the way for large-scale computational predictions of opioid dissociation rates to inform the regulatory decision. The profound role of the histidine protonation state found here may shift the paradigm in computational studies of ligand-receptor kinetics.

biophysics↗

bioRxiv2021

Sustained Oxygenation Accelerates Diabetic Wound Healing by Simultaneously Promoting Epithelialization and Angiogenesis, and Decreasing Tissue Inflammation

Non-healing diabetic wound is one of the most common complications for diabetic patients. Chronic hypoxia is among the prominent factors that delay the wound healing process. Therefore, sustained oxygenation to alleviate hypoxia is hypothesized to promote diabetic wound healing. Yet it cannot be achieved by current clinical approaches including hyperbaric oxygen therapy. Herein, we developed a sustained oxygenation system consisting of oxygen-release microspheres and a reactive oxygen species (ROS)-scavenging hydrogel. The hydrogel was used to capture the ROS that is elevated in the diabetic wounds, and that may be generated due to oxygen release. The sustainedly released oxygen augmented survival and migration of keratinocytes and dermal fibroblasts; promoted angiogenic growth factor expression, and angiogenesis in the diabetic wounds; and decreased M1 macrophage density. These effects led to a significant increase of wound closure rate. These findings reveal that sustained oxygenation alone without using drugs is capable of healing diabetic wounds.

bioengineering↗

bioRxiv2021

Single-cell analyses reveal a continuum of cell state and composition changes in the malignant transformation of polyps to colorectal cancer

To chart cell composition and cell state changes that occur during the transformation of healthy colon to precancerous adenomas to colorectal cancer (CRC), we generated 451,886 single-cell chromatin accessibility profiles and 208,557 single-cell transcriptomes from 48 polyps, 27 normal tissues, and 6 CRCs collected from patients with and without germline APC mutations. A large fraction of polyp and CRC cells exhibit a stem-like phenotype, and we define a continuum of epigenetic and transcriptional changes occurring in these stem-like cells as they progress from normal to CRC. Advanced polyps contain increasing numbers of stem-like cells, regulatory T-cells, and a subtype of FOX-regulated pre-cancer associated fibroblasts. In the cancerous state, we observe T-cell exhaustion, RUNX1-regulated cancer associated fibroblasts, and increasing accessibility associated with HNF4A motifs in epithelia. Methylation changes in sporadic CRC are strongly anti-correlated with accessibility changes along this continuum, further identifying regulatory markers for molecular staging of polyps.

genomics↗

bioRxiv2021

Scaling dictates the decoder structure

Despite fluctuations in embryo size within a species, the spatial gene expression pattern and hence the embryonic structure develop in proportion with embryo size, known as the scaling phenomenon. For morphogen induced patterning of gene expression, the positional information encoded in the morphogen profile is decoded by the downstream genetic network (the decoder). In this paper, we show that the requirement of scaling sets severe constraints on the geometric structure of the decoder, which in turn enables deduction of mutants behavior and extraction of regulation information without going into any molecular details. We demonstrate that the Drosophila gap gene system achieves scaling in the way consistent with our theory - the decoder geometry required by scaling correctly accounts for the observed gap gene expression pattern in nearly all maternal morphogen mutants. Furthermore, the regulation logic and the coding/decoding strategy of the gap gene system can also be revealed from the decoder geometry. Our work provides a general theoretical framework on a large class of problems where scaling output is induced by non-scaling input, as well as a unified understanding of scaling, mutants behavior and gene regulation for the Drosophila gap gene system.

developmental biology↗

bioRxiv2021

YAP1 induces invadopodia formation by transcriptionally activating TIAM1 through its enhancer in breast cancer

Yes-associated protein 1 (YAP1), a central component of the Hippo pathway, plays an important role in tumor metastasis; however, the mechanism remains to be elucidated. Here, we reported that YAP1 could induce invadopodia formation and promote tumor metastasis in breast cancer cells. We identified that TIAM1, the guanine nucleotide exchange factor, is a target of YAP1-TEAD4 complex. YAP1 promotes TEAD4 binding to the enhancer region of TIAM1, which activates TIAM1 expression and subsequently increases RAC1 activity. These findings reveal the functional role of Hippo signaling in invadopodia, and provide potential molecular targets for preventing tumor metastasis in breast cancer. SignificanceThrough regulating the enhancer region of TIAM1, YAP1 induces invadopodia formation and promotes tumor metastasis in breast cancer.

cancer biology↗