Biology subjects

Shi, J.

Publications and source records attributed to Shi, J..

At least 289 records · Page 16Linked to original sources

bioRxiv2020

The construction and application of a new 17-plex Y-STR system using universal fluorescent PCR

Y-chromosomal short tandem repeat (Y-STR) polymorphisms are useful in forensic identification, population genetics and human structures. However, the current Y-STR systems are limited in discriminating distant relatives in a family with a low discrimination power. Increasing the capacity of detecting Y chromosomal polymorphisms will drastically narrow down the matching number of genealogy populations or pedigrees. In this study, we developed a system containing 17 Y-STRs that are complementary to the current commercially available Y-STR kits. This system was constructed by multiplex PCR with expected sizes of 126-400 bp labeled by different fluorescence molecules (DYS715, DYS709, DYS716, DYS713 and DYS607 labeled by FAM; DYS718, DYS723, DYS708 and DYS714 labeled by JOE; DYS712, DYS717, DYS721 and DYS605 labeled by TAMRA; and DYS719, DYS726, DYS598 and DYS722 labeled by ROX). The system was extensively tested for sensitivity, male specificity, species specificity, mixture, population genetics and mutation rates following the Scientific Working Group on DNA Analysis Methods (SWGDAM) guidelines. The genetic data were obtained from eight populations with a total of 1260 individuals. Our results showed that all the 17 Y-STRs are human- and male-specific and include only one copy of the Y-chromosome. The 17 Y-STR system detects 143 alleles and has a high discrimination power (0.996031746). Mutation rates were different among the 17 Y-STRs, ranging from 0.30% to 3.03%. In conclusion, our study provides a robust, sensitive and cost-effective genotyping method for human identification, which will be beneficial for narrowing the search scope when applied to genealogy searching with the Y-STR DNA databank.

genetics↗

bioRxiv2020

Generation of multi-transgenic pigs using PiggyBac transposons co-expressing pectinase, xylanase, cellulase?β-1.3-1.4-glucanase and phytase

The current challenges facing the pork industry are to maximize feed efficiency and minimize fecal emissions. Unlike ruminants, pigs lack a number of digestive enzymes like pectinase, xylanase, cellulase, {beta}-1.3-1.4-glucanase and phytase to hydrolyze the cell walls of grains to release endocellular nutrients into their digestive tracts. Herein, we synthesized multiple cellulase and pectinase genes derived from lower organisms and then codon optimized these genes to be expressed in pigs. These genes were then cloned into our previously optimized XynB (xylanase)- EsAPPA (phytase) bicistronic construct. We then successfully generated transgenic pigs that expressed four enzymes (Pg7fn (pectinase), XynB (xylanase), EsAPPA (phytase) and TeEGI (cellulase and {beta}-glucanase)) using somatic cell cloning. Expression of these genes was parotid gland specific. Enzymatic assays using the saliva of these founders demonstrated high levels of phytase (2.0~3.4 U/mL) and xylanase (0.25~0.42 U/mL) activity, but low levels of pectinase (0.06~0.08 U/mL) activity. These multi-transgenic pigs are expected to contribute to enhance feed utilization and reduce environmental impact.

genetics↗

bioRxiv2020

Nonspecific membrane interactions can modulate BK channel activation

Large-conductance potassium (BK) channels are transmembrane (TM) proteins that can be synergistically and independently activated by membrane voltage and intracellular Ca2+. The only covalent connection between the cytosolic Ca2+ sensing domain and the TM pore and voltage sensing domains is a 15-residue "C-linker". To determine the linker's role in BK activation, we designed a series of linker sequence scrambling mutants to suppress potential complex interplay of specific interactions with the rest of the protein. The results revealed a surprising sensitivity of BK activation to the linker sequence. Combing atomistic simulations and further mutagenesis experiments, we demonstrated that nonspecific interactions of the linker with membrane alone could directly modulate BK activation. The C-linker thus plays more direct roles in mediating allosteric coupling between BK domains than previously assumed. Our results also suggest that covalent linkers could directly modulate TM protein function and should be considered an integral component of the sensing apparatus.

biophysics↗

bioRxiv2020

Macromolecular and biochemical changes of G0 fission yeast nuclei

When starved of nitrogen, fission yeast Schizosaccharomyces pombe cells enter a quiescent "G0" state with smaller nuclei and transcriptional repression. The genomics of S. pombe G0 cells has been well studied, but much of its nuclear cell biology remains unknown. Here we use confocal microscopy, immunoblots, and electron cryotomography to investigate the cytological, biochemical, and ultrastructural differences between S. pombe proliferating, G1-arrested, and G0 cell nuclei, with an emphasis on the histone acetylation, RNA polymerase II fates, and macromolecular complex packing. Compared to proliferating cells, G0 cells have lower levels of histone acetylation, nuclear RNA polymerase II, and active transcription. The G0 nucleus has similar macromolecular crowding yet fewer chromatin-associated multi-megadalton globular complexes. Induced histone hyperacetylation in G0 results in cells that have larger nuclei and therefore less compact chromatin. However, these histone-hyperacetylated G0 cells remain transcriptionally repressed with similar nuclear crowding. Canonical nucleosomes - those that resemble the crystal structure - are rare in proliferating, G1-arrested, and G0 cells. Our study therefore shows that extreme changes in nucleus physiology are possible without extreme reorganisation at the macromolecular level. SummaryWe use multiple cell-biological techniques to compare proliferating and quiescent G0 fission yeast cell nucleus transcription, histone acetylation, macromolecular packing.

cell biology↗

bioRxiv2020

The landscape of mouse epididymal cells defined by the single-cell RNA-Seq

Spermatozoa acquire their fertilizing ability and forward motility properties during epididymal transit. Although lots of attempts elucidating the functions of different cell types in epididymis, the composition of epididymal tubal and cell types are still largely unknown. Using single-cell RNA sequence, we analyzed the cell constitutions and their gene expression profiles of adult epididymis derived from caput, corpus and cauda epididymis with a total of 12,597 cells. This allowed us to elucidate the full range of gene expression changes during epididymis and derive region-specific gene expression signatures along the epididymis. A total of 7 cell populations were identified with all known constituent cells of mouse epididymis, as well as two novel cell types. Our analyses revealed a segment to segment variation of the same cell type in the three different part of epididymis and generated a reference dataset of epididymal cell gene expression. Focused analyses uncovered nine subtypes of principal cell. Two subtypes of principal cell, c0.3 and c.6 respectively, in our results supported with previous finding that they mainly located in the caput of mouse epididymis and play important roles during sperm maturation. We also showed unique gene expression signatures of each cell population and key pathways that may concert epididymal epithelial cell-sperm interactions. Overall, our single-cell RNA seq datasets of epididymis provide a comprehensive potential cell types and information-rich resource for the studies of epididymal composition, epididymal microenvironment regulation by the specific cell type, or contraceptive development, as well as a gene expression roadmap to be emulated in efforts to achieve sperm maturation regulation in the epididymis.

cell biology↗

bioRxiv2019

Personalized genealogical history inferred from biobank-scale IBD segments

When modern biobanks collect genotype information for a significant fraction of a population, dense genetic connections of a person can be traced using identity by descent (IBD) segments. These connections offer opportunities to characterize individuals in the context of the underlying populations. Here, we conducted an individual-centric analysis of IBDs among the UK Biobank participants that represent 0.7% of the UK population. On average, one UK individual shares IBDs over 5 cM with 14,000 UK Biobank participants, which we refer to as "cousins". Using these segments, approximately 80% of a persons genome can be reconstructed. Also, using changes of cousin counts sharing IBDs at different lengths, we identified a group, potentially British Jews, who has a distinct pattern of familial expansion history. Finally, using the enrichment of cousins in ones neighborhood, we identified regional variations of personal preference favoring living closer to ones extended families. In summary, our analysis revealed genetic makeup, personal genealogical history, and social behaviors at population scale, opening possibilities for further studies of individuals genetic connections in biobank data.

genetics↗

bioRxiv2019

Membrane dynamics promote sensitivity of epithelial cancer targets to pro-necrotic activity of Natural Killer cells

Natural Killer (NK) cells play an important role in cancer immunosurveillance and therapy. However, the target selectivity of NK cell activity is still poorly understood. Here we used live-cell reporters to unravel differential epithelial cancer target killing by primary human NK cells. We found highly variable fraction of killing by distinct NK cell cytotoxic modes that was not determined by NK ligand expression. Rather, epithelial plasma membrane dynamics driven by ROCK-mediated blebs and/or Rac1-mediated lamellipodia promoted necrotic mode in preference to apoptotic mode of killing. Inhibition of granzyme and key necroptosis regulators RIP1, RIP3 and MLKL significantly attenuated necrotic killing, revealing a novel NK cytotoxic pathway by granzyme-induced necroptosis that conferred target selectivity. Our results not only elucidate a new NK cell effector mechanism but also suggest tissue microenvironment and oncogenic signaling pathways that promote membrane dynamics, e.g., Rac1 and Rho/ROCK, could be exploited to enhance proinflammatory NK cell killing.

cell biology↗

bioRxiv2019

Allele-specific open chromatin in human iPSC neurons elucidates functional non-coding disease variants

Functional interpretation of noncoding disease variants, which likely regulate gene expression, has been challenging. Chromatin accessibility strongly influences gene expression during neurodevelopment; however, to what extent genetic variants can alter chromatin accessibility in the context of brain disorders/traits is unknown. Using human induced pluripotent stem cell (iPSC)-derived neurons as a neurodevelopmental model, we identified abundant open-chromatin regions absent in adult brain samples and thousands of genetic variants exhibiting allele-specific open-chromatin (ASoC). ASoC variants are overrepresented in brain enhancers, transcription-factor-binding sites, and quantitative-trait-loci associated with gene expression, histone modification, and DNA methylation. Notably, compared to open chromatin regions and other commonly used functional annotations, neuronal ASoC variants showed much stronger enrichments of risk variants for various brain disorders/traits. Our study provides the first snapshot of the neuronal ASoC landscape and a powerful framework for prioritizing functional disease variants.\n\nOne Sentence SummaryAllele-specific open chromatin informs functional disease variants

genetics↗

bioRxiv2019

A Hypomorphic Cystathionine β-Synthase Gene Contributes to Cavefish Eye Loss by Disrupting Optic Vasculature

Vestigial structures are key indicators of evolutionary descent but the mechanisms underlying their development are poorly understood. This study examines vestigial eye formation in the teleost Astyanax mexicanus, which consists of a sighted surface-dwelling morph and different populations of blind cave morphs. Cavefish embryos initially develop optic primordia but vestigial eyes are formed during larval development. Multiple genetic factors are involved in cavefish eye loss but none of the mutated genes have been identified. Here we identify cystathionine {beta}-synthase (cbsa), which encodes the key enzyme of the transsulfuration pathway, as a mutated gene responsible for eye degeneration in multiple cavefish populations. The inactivation of cbsa affects eye development by inducing accumulation of the transsulfuration intermediate homocysteine and defects in optic vasculature, including aneurysms and eye hemorrhages, leading to oxygen deficiency. Our findings suggest that localized modifications in the circulatory system and hypoxia had important roles in the evolution of vestigial eyes in blind cavefish.

developmental biology↗

bioRxiv2019

Two-stage electro-mechanical coupling of a Kv channel in voltage-dependent activation

In voltage-gated potassium (KV) channels, the voltage-sensing domain (VSD) undergoes sequential activation from the resting state to the intermediate state and activated state to trigger pore opening via electro-mechanical (E-M) coupling. However, the spatial and temporal details underlying E-M coupling remain elusive. Here, we leverage KV7.1s unique two open states associated with the VSD adopting the intermediate and activated conformations to report a two-stage E-M coupling mechanism in voltage-dependent gating of KV7.1 as triggered by VSD activations to the intermediate and then activated state. When the S4 segment transitions to the intermediate state, the hand-like C-terminus of the VSD-pore linker (S4-S5L) interacts with the pore in the same subunit. When S4 then proceeds on to the fully-activated state, the elbow-like hinge between S4 and S4-S5L engages with the pore to activate conductance. This two-stage \"hand-and-elbow\" gating mechanism elucidates distinct tissue-specific modulations, pharmacology, and disease pathogenesis of KV7.1, and likely applies to numerous KV channels.

biophysics↗

bioRxiv2019

Sphingomyelinase disables Piezo1 channel inactivation to enable sustained response to mechanical force

Piezo1 channels are determinants of vascular responses to fluid flow. They importantly provide sustained response to flow, but this property contrasts with the rapid inactivation that has become a hallmark of the channels in heterologous overexpression studies. Here we reveal a mechanism by which blood vessels disable inactivation to enable sustained physiological response. Creation of a molecular model of Piezo1 channel in defined lipid membranes suggested potential modulation by sphingomyelin and its product ceramide. Biological relevance was indicated by the observation that exogenous sphingomyelinase enhanced Piezo1-mediated Ca2+ entry in cultured endothelial cells. We therefore hypothesised that endogenous sphingomyelinase suppresses channel inactivation. Remarkably, in endothelium freshly-isolated from murine artery, neutral sphingomyelinase inhibitors or genetic disruption of sphingomyelin phosphodiesterase 3 (SMPD3) caused flow- and pressure-activated Piezo1 channels to become inactivating. SMPD3 retained its ability to disable inactivation in cell-free membrane patches, providing evidence for a membrane localised effect. The data suggest that inherent inactivation of Piezo1 channels is disabled by enzymatic control of lipid environment to enable physiological response to mechanical force.

physiology↗

bioRxiv2019

Computational reconstruction of the complete Piezo1 structure reveals a unique footprint and specific lipid interactions

Piezo1 is a critical mechanical sensor in many cells. It is activated by mechanical force thus allowing cells to sense the physical environment and respond to stress. Structural data have suggested that Piezo1 has a curved shape. Here, we use computational approaches to model, for the first time, the 3D structure of the full-length Piezo1 in an asymmetric membrane. A number of novel insights emerge: (i) Piezo1 creates a dome in the membrane with a trilobed topology that extends beyond the radius of the protein, (ii) Piezo1 changes the lipid environment in its vicinity via specific interactions with cholesterol and PIP2 molecules, (iii) changes in cholesterol concentration that change the membrane stiffness result in changes in the depth of the dome created by Piezo1, and iv) modelling of the N-terminal region that is missing from current structures modifies Piezo1 membrane footprint, suggesting the importance of this region in Piezo1 function.

biophysics↗

bioRxiv2019

Molecular mechanisms of fentanyl mediated β-arrestin biased signaling

The development of novel analgesics with improved safety profiles to combat the opioid epidemic represents a central question to G protein coupled receptor structural biology and pharmacology: What chemical features dictate G protein or {beta}-arrestin signaling? Here we use adaptively biased molecular dynamics simulations to determine how fentanyl, a potent {beta}-arrestin biased agonist, activates the -opioid receptor (OR). The resulting fentanyl-bound pose provides rational insight into a wealth of historical structure-activity-relationship on its chemical scaffold. We found that fentanyl and the synthetic opioid peptide DAMGO require M153 to induce {beta}-arrestin coupling, while M153 was dispensable for G protein coupling. We propose and validate a mechanism where the n-aniline ring of fentanyl mediates OR {beta}-arrestin through a novel M153 \"microswitch\" by synthesizing fentanyl-based derivatives that exhibit complete, clinically desirable, G protein biased coupling. Together, these results provide molecular insight into fentanyl mediated {beta}-arrestin biased signaling and a rational framework for further optimization of fentanyl-based analgesics with improved safety profiles.\n\nAuthor SummaryThe global opioid crisis has drawn significant attention to the risks associated with over-use of synthetic opioids. Despite the public attention, and perhaps in-line with the profit-based incentives of the pharmaceutical industry, there is no public structure of mu-opioid receptor bound to fentanyl or fentayl derivatives. A publicly available structure of the complex would allow open-source development of safer painkillers and synthetic antagonists. Current overdose antidotes, antagonists, require natural products in their synthesis which persists a sizable barrier to market and develop better antidotes. In this work we use advance molecular dynamics techniques to obtain the bound geometry of mu-opioid receptor with fentanyl (and derivatives). Based on our in-silico structure, we synthesized and tested novel compounds to validate our predicted structure. Herein we report the bound state of several dangerous fentanyl derivatives and introduce new derivatives with signaling profiles that may lead to lower risk of respiratory depression.

biophysics↗

bioRxiv2019

A novel xenograft model of human HCC in immunocompetent mouse

Hepatocellular carcinoma (HCC) is one of the most common malignant tumors that threaten human health; thus, the establishment of an animal model with clinical features similar to human liver cancer is of important practical significance. Taking advantage of the novel microcarrier-6, human HCC cells was injected into immunocompetent mice to establish a novel human HCC patient-derived xenograft (PDX) model. Primary HCC cells were isolated from fresh liver cancer tissues, which were subsequently co-cultured with microcarrier-6 to construct a three-dimensional tumor cell culture model in vitro. The HCC-microcarrier complex was implanted into mice by subcutaneous inoculation, and the tumor formation time, tumor formation rate, and pathological manifestation were recorded. Changes of immune parameters in mice were detected by flow cytometry. The success rate was 60% (6/10) in the establishment of liver cancer PDX mouse model, and the total tumor formation rate of the tumor-forming model is 80-100%. H&E staining and immunohistochemical experiments indicate that the model well retained the characteristics of the primary tumor. Interestingly, M2 macrophages in tumor-bearing mice increased significantly, and the levels of CD4+ T cells were significantly reduced. Through the application of the microcarrier-6 in immunocompetent mice, we successfully established a novel human HCC PDX model, which can be used to better study and further elucidate the occurrence and pathogenic mechanism of HCC.

cancer biology↗

bioRxiv2019

Cryo-ET analysis of budding yeast synaptonemal complexes in situ

In meiosis, cells undergo two sequential rounds of cell division, termed meiosis I and meiosis II. Textbook models of the meiosis I substage called pachytene show that nuclei have conspicuous 100-nm-wide, ladder-like synaptonemal complexes (SC), which form between homologous chromosomes. It remains unknown if cells have any other large, meiosis-specific nuclear structures. Here we present cryo-ET analysis of frozen-hydrated budding yeast cells before, during, and after pachytene. We found no evidence for the dense ladder-like structures expected of the SC or the ordered chromatin loops expected to project from their sides. Instead, we found large quantities of 12-nm-wide triple-helices that pack into crystalline bundles. These structures are present in meiotic cells, but not in interphase cells, so we call them meiotic triple helices (MTHs). MTHs are enriched in the nucleus but not enriched in the cytoplasm. Bundles of MTHs form at the same time as SCs in wild-type cells and also in mutant cells that are unable to form SCs. These results suggest that in yeast, SCs are not crystalline and that they coexist with large, previously unreported meiotic machines.

cell biology↗

bioRxiv2019

Crl activates transcription by stabilizing the active conformation of the master stress factor σS

{sigma}S is a master transcription initiation factor that protects bacterial cells from various harmful environmental stresses and antibiotic pressure. Although its mechanism remains unclear, it is known that full activation of {sigma}S-mediated transcription requires a {sigma}S-specific activator, Crl. In this study, we determined a 3.80 [A] cryo-EM structure of an E. coli transcription activation complex (E. coli Crl-TAC) comprising E. coli {sigma}S-RNAP holoenzyme, Crl, and a nucleic-acid scaffold. The structure reveals that Crl interacts with the domain 2 of {sigma}S ({sigma}S2), sharing no interaction with promoter DNA. Subsequent hydrogen-deuterium exchange mass spectrometry (HDX-MS) results indicate that Crl stabilizes key structural motifs of {sigma}S2 to promote the assembly of {sigma}S-RNAP holoenzyme and also to facilitate formation of the RNA polymerase-promoter DNA open complex (RPo). Our study demonstrates a unique DNA contact-independent mechanism of transcription activation, thereby defining a previously unrecognized mode of transcription activation in cells.

biochemistry↗

bioRxiv2019

CRISPR/Cas9-mediated integration of large transgene into pig potential safe harbor

Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (Cas9) is a precise genome manipulating tool which can produce targeted gene mutations in various cells and organisms. Although CRISPR/Cas9 can efficiently generate gene knock-out, the gene knock-in efficiency mediated by homology-directed repair (HDR) remains low, especially for large fragment integration. In this study, we established an efficient method for CRISPR/Cas9-mediated integration of large transgene cassette carrying salivary gland-expressing multiple digestion enzymes ({approx} 20 kbp) in CEP112 locus in pig fetal fibroblasts. Our results showed that using homologous donor with a short left arm and a long right arm yielded the best CRISPR/Cas9-mediated knock-in efficiency, and the targeting efficiency in potential safe harbor CEP112 locus are higher than ROSA26 locus. The CEP112 knock-in cell lines were used as nuclear donors for somatic cell nuclear transfer to create genetically modified pigs. We found that knock-in pig (705) successfully expressed three microbial enzymes ({beta}-glucanase, xylanase, and phytase) in salivary gland, suggesting potential safe harbor CEP112 locus supports exogenous genes expression by a tissue-specific promoter. In summary, we successfully targeted CEP112 locus using our optimal homology arm system for precise modification of pigs, and established a modified pig model for foreign digestion enzyme expression in saliva.

bioengineering↗

bioRxiv2019

Substrate Transport is Mediated not only by P-glycoprotein but also by Lipid Penetration

In association with large-scale conformational changes, the members of the ATP-binding cassette (ABC) transporter superfamily such as P-glycoprotein (P-gp) pump endogenous cytotoxic substances and exogenous drugs out of cells. Here, a series of nonequilibrium-driven molecular dynamics (MD) simulations are sophisticatedly combined to provide a generally effective access to quantitatively investigate such a complex biological process that has been posing a great challenge for experiments and computational simulations. Both common features and unique characteristics of multiple ligands (substrates or inhibitors) that are recognized by P-gps from mouse and human species are quantitatively explored, providing additional insights into experimentally suggested ligand transport pathways and summarizing the important roles of not only different P-gps but also lipids in regulating ligand transport. These findings reveal the molecular mechanism underlying the transport of ligands by P-gps from different species and emphasize the consideration of lipid effects on the future design of effective P-gp inhibitors.

biophysics↗