Biology subjects

Li, S.

Publications and source records attributed to Li, S..

At least 1,081 records · Page 60Linked to original sources

bioRxiv2020

Exploring cell-specific miRNA regulation with single-cell miRNA-mRNA co-sequencing data

BackgroundExisting computational methods for studying miRNA regulation are mostly based on bulk miRNA and mRNA expression data. However, bulk data only allows the analysis of miRNA regulation regarding a group of cells, rather than the miRNA regulation unique to individual cells. Recent advance in single-cell miRNA-mRNA co-sequencing technology has opened a way for investigating miRNA regulation at single-cell level. However, as currently single-cell miRNA-mRNA co-sequencing data is just emerging and only available at small-scale, there is a strong need of novel methods to exploit existing single-cell data for the study of cell-specific miRNA regulation. ResultsIn this work, we propose a new method, CSmiR (Cell-Specific miRNA regulation) to use single-cell miRNA-mRNA co-sequencing data to identify miRNA regulatory networks at the resolution of individual cells. We apply CSmiR to the miRNA-mRNA co-sequencing data in 19 K562 single-cells to identify cell-specific miRNA-mRNA regulatory networks for understanding miRNA regulation in each K562 single-cell. By analyzing the obtained cell-specific miRNA-mRNA regulatory networks, we observe that the miRNA regulation in each K562 single-cell is unique. Moreover, we conduct detailed analysis on the cell-specific miRNA regulation associated with the miR-17/92 family as a case study. Finally, through exploring cell-cell similarity matrix characterized by cell-specific miRNA regulation, CSmiR provides a novel strategy for clustering single-cells to help understand cell-cell crosstalk. ConclusionsTo the best of our knowledge, CSmiR is the first method to explore miRNA regulation at a single-cell resolution level, and we believe that it can be a useful method to enhance the understanding of cell-specific miRNA regulation.

bioinformatics↗

bioRxiv2020

Understanding the phase separation characteristics of nucleocapsid protein provides a new therapeutic opportunity against SARS-CoV-2

The ongoing coronavirus disease 2019 (COVID-19) pandemic has raised an urgent need to develop effective therapeutics against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). As a potential antiviral drug target, the nucleocapsid (N) protein of SARS-CoV-2 functions as a viral RNA chaperone and plays vital and multifunctional roles during the life cycle of coronavirus1-3. In this study, we discovered that the N protein of SARS-CoV-2 undergoes liquid-liquid phase separation (LLPS) both in vitro and in vivo, which is further modulated by viral RNA. In addition, we found that, the core component of the RNA-dependent RNA polymerase (RdRp) of SARS-CoV-2, nsp12, preferentially partitions into the N protein condensates. Moreover, we revealed that, two small molecules, i.e., CVL218 and PJ34, can be used to intervene the N protein driven phase separation and loosen the compact structures of the condensates of the N-RNA-nsp12 complex of SARS-CoV-2. The discovery of the LLPS-mediated interplay between N protein and nsp12 and the corresponding modulating compounds illuminates a feasible way to improve the accessibility of antiviral drugs (e.g., remdesivir) to their targets (e.g., nsp12/RdRp), and thus may provide useful hints for further development of effective therapeutic strategies against SARS-CoV-2.

biophysics↗

bioRxiv2020

A Chinese hamster transcription start site atlas that enables targeted editing of CHO cells

Chinese hamster ovary (CHO) cells, with their human-compatible glycosylation and high protein titers, are the most widely used cells for producing biopharmaceuticals. Engineering gene expression in CHO is key to improving drug quality and affordability. However, engineering gene expression or activating silent genes requires accurate annotation of the underlying regulatory elements and transcription start sites (TSSs). Unfortunately, most TSSs in the Chinese hamster genome were computationally predicted and are frequently inaccurate. Here, we revised TSS annotations for 15,308 Chinese hamster genes and 4,478 non-coding RNAs based on experimental data from CHO-K1 cells and 10 hamster tissues. The experimental realignment and discovery of TSSs now expose previously hidden motifs, such as the TATA box. We further demonstrate, by targeting the glycosyltransferase gene Mgat3, how accurate annotations readily facilitate activating silent genes by CRISPRa to obtain more human-like glycosylation. Together, we envision our annotation and data will provide a rich resource for the CHO community, improve genome engineering efforts and aid comparative and evolutionary studies.

genomics↗

bioRxiv2020

Proteomic analysis of microtubule inner proteins (MIPs) in Rib72 null Tetrahymena cells reveals functional MIPs

Motile cilia and flagella are built from stable populations of doublet microtubules that comprise their axonemes. Their unique stability is brought about, at least in part, by a network of Microtubule Inner Proteins (MIPs) found in the lumen of their doublet microtubules. Rib72A and Rib72B were identified as microtubule inner proteins (MIPs) in the motile cilia of Tetrahymena thermophila. Loss of these proteins leads to ciliary defects and loss of multiple MIPs. We performed mass spectrometry coupled with proteomic analysis and bioinformatics to identify the MIPs lost in RIB72A/B knockout (KO) Tetrahymena cells. From this analysis we identified a number of candidate MIPs and pursued one, Fap115, for functional characterization. We find that loss of Fap115 results in disrupted cell swimming and aberrant ciliary beating. Cryo-electron tomography reveals that Fap115 localizes to MIP6a in the A-tubule of the doublet microtubules. Overall, our results highlight the complex relationship between MIPs, ciliary structure, and ciliary function.

molecular biology↗

bioRxiv2020

Tissue-specific accumulation profiles of phorbol esters in Jatropha curcas and gene induction in response to abiotic and biotic stresses

Jatropha curcas L. (J. curcas), a shrub plant of the Euphorbiaceae family, has received enormous attention as a promising biofuel plant for the production of biodiesel and medical potential in ethnopharmacology. However, the tumor-promoter toxin phorbol esters present in J. curcas raises concerns for health and environmental risk as its large-scale cultivation limits the use of meal obtained after oil extraction for animal feed. Here, we determined the variation of phorbol ester profiles and contents in eight J. curcas tissues by high performance liquid chromatography (HPLC) and found phorbol esters present in all parts of the plant except the seed shell. We showed tissue-specific patterns of accumulation of phorbol esters and associated terpenoids at the transcriptomic level with high transcript levels in reproductive and young tissues. Genes involved in the same module of terpenoids biosynthesis were positively correlated. We further present diverse abiotic and biotic stresses that had different effects on the accumulation of transcripts in terpenoids shared and branched terpenoid pathways in plant seedlings. The fine-tuning of terpenoids biosynthesis may link with ecological functions in plants under extreme environments and defense against pathogens.

plant biology↗

bioRxiv2020

Ribosomal protein S7 ubiquitination during ER stress in yeast is associated with selective mRNA translation and stress outcome

eIF2 phosphorylation-mediated translational regulation is crucial for global translation repression by various stresses, including the unfolded protein response (UPR). However, translational control during UPR has not been demonstrated in yeast. This study investigated ribosome ubiquitination-mediated translational controls during UPR. Tunicamycin-induced ER stress enhanced the levels of ubiquitination of the ribosomal proteins uS10, uS3 and eS7. Not4-mediated monoubiquitination of eS7A was required for resistance to tunicamycin, whereas E3 ligase Hel2-mediated ubiquitination of uS10 was not. Ribosome profiling showed that the monoubiquitination of eS7A was crucial for translational regulation, including the upregulation of the spliced form of HAC1 (HAC1i) mRNA and the downregulation of Histidine triad NucleoTide-binding 1 (HNT1) mRNA. Downregulation of the deubiquitinating enzyme complex Upb3-Bre5 increased the levels of ubiquitinated eS7A during UPR in an Ire1-independent manner. These findings suggest that the monoubiquitination of ribosomal protein eS7A plays a crucial role in translational controls during the ER stress response in yeast.

molecular biology↗

bioRxiv2020

Chronic in vivo STED nanoscopy uncovers multiple drivers of shape volatility in stable cortical spines

Excitatory synapses on dendritic spines of pyramidal neurons are considered a central memory locus. To foster both continuous adaption as well as the storage of long-term information, spines need to be plastic and stable at the same time. Here we advanced in vivo STED nanoscopy to superresolve distinct features of dendritic spines (head size, neck length and width) in mouse neocortex for up to one month. While LTP-dependent changes predict highly correlated modifications of spine geometry, we find both, uncorrelated dynamics, as well as correlated changes, indicating multiple independent drivers of spine remodeling. The magnitude of this remodeling suggests substantial fluctuations in synaptic strength, and is exaggerated in a mouse model of neurodegeneration. Despite this high degree of volatility, all spine features also exhibit persistent components that are maintained over long periods of time. Thus, at the nanoscale, stable dendritic spines exhibit a delicate balance of stability and volatility.

neuroscience↗

bioRxiv2020

Neonicotinoids stimulate H2-limited methane emission in Periplaneta americana through the regulation of gut bacterium community

Methane emitted by insects is considered to be an important source of atmospheric methane. Here we report the stimulation of methane emission in Periplaneta americana, an insect species with abundant methanogens, by neonicotinoids, insecticides widely used to control insect pests. The application of cycloxaprid (CYC) and imidacloprid (IMI) caused foregut expansion in P. americana, and increased the methane production and emission. Antibiotics could mostly eliminate the stimulatory effects. In P. americana gut, hydrogen levels increased and pH values decreased, which could be significantly explained by the gut bacterium community change. The proportion of several bacterium genera increased in guts following CYC treatment, and four genera from five with increased proportions could generate hydrogen at anaerobic conditions. Hydrogen is a central intermediate in methanogenesis. Gut methanogens could use the increased hydrogen to produce more methane, especially at acidic conditions. Following neonicotinoid applications, all increased methanogens in both foregut and hindgut used hydrogen as electron donor to produce methane. Besides, the up-regulation of mcrA, encoding the enzyme that catalyzes the final step of methanogenesis, suggested an enhanced methane production ability in present methanogens. In the termite Coptotermes chaohuensis, another methanogen-abundant insect species, hydrogen levels in gut and methane emission significantly increased after neonicotinoid treatment, which was similar to the results in P. americana. In summary, neonicotinoids changed bacterium community in P. americana gut to generate more hydrogen, which then stimulate gut methanogens to produce and emit more methane. The finding raised a new concern over neonicotinoid applications, and might be a potential environmental risk associated with global warming.

ecology↗

bioRxiv2020

Proneural genes define ground state rules to regulate neurogenic patterning and cortical folding

Transition from smooth, lissencephalic brains to highly-folded, gyrencephalic structures is associated with neuronal expansion and breaks in neurogenic symmetry. Here we show that Neurog2 and Ascl1 proneural genes regulate cortical progenitor cell differentiation through cross-repressive interactions to sustain neurogenic continuity in a lissencephalic rodent brain. Using in vivo lineage tracing, we found that Neurog2 and Ascl1 expression defines a lineage continuum of four progenitor pools, with double+ progenitors displaying several unique features (least lineage-restricted, complex gene regulatory network, G2 pausing). Strikingly, selective killing of double+ progenitors using split-Cre;Rosa-DTA transgenics breaks neurogenic symmetry by locally disrupting Notch signaling, leading to cortical folding. Finally, consistent with NEUROG2 and ASCL1 driving discontinuous neurogenesis and folding in gyrencephalic species, their transcripts are modular in folded macaque cortices and pseudo-folded human cerebral organoids. Neurog2/Ascl1 double+ progenitors are thus Notch-ligand expressing niche cells that control neurogenic periodicity to determine cortical gyrification. HIGHLIGHTSO_LINeurog2 and Ascl1 expression defines four distinct transitional progenitor states C_LIO_LIDouble+ NPCs are transcriptionally complex and mark a lineage branch point C_LIO_LIDouble+ NPCs control neurogenic patterning and cortical folding via Notch signaling C_LIO_LINeurog2 and Ascl1 expression is modular in folded and not lissencephalic cortices C_LI eTOC BLURBEmergence of a gyrencephalic cortex is associated with a break in neurogenic continuity across the cortical germinal zone. Han et al. identify a pool of unbiased neural progenitors at a lineage bifurcation point that co-express Neurog2 and Ascl1 and produce Notch ligands to control neurogenic periodicity and cortical folding.

developmental biology↗

bioRxiv2020

Mouse Single Pancreatic β Cell Transcriptomics Reveal Sexual Dimorphism of Transcriptomes and Identify Sex-dependent Type 2 Diabetes Altered Genes

Type 2 diabetes (T2D), characterized by malfunction of pancreatic {beta} cells, is affected by multiple cues including sex differences. Nevertheless, mechanisms of sex differences in type 2 diabetes susceptibility and pathogenesis remain unclear. Using single-cell RNA sequencing (scRNA-seq) technology, we showed that sexual dimorphism of transcriptome exists in mouse {beta} cells. Our analysis further revealed the existence of sex-dependent type 2 diabetes altered genes in high fat diet induced T2D model, suggesting divergences in pathological mechanisms of type 2 diabetes between sexes. Our results indicated that sex should be taken into consideration when treating diabetes, which was further validated by the sex-matched and sex-mismatched islet transplantation in mice. Compared to sex-matched transplants, sex-mismatched transplants showed downregulation of genes involved in the longevity regulating pathway in {beta} cells and led to impaired glucose tolerance in diabetic mice. Taken together, our findings could advance current understanding of type 2 diabetes pathogenesis with sexually dimorphic perspectives and provide new insights to the development of precision medicine.

bioinformatics↗

bioRxiv2020

BRD4-mediated repression of p53 is a target for combination therapy in AML

Acute Myeloid Leukemia (AML) is a typically-lethal molecularly heterogeneous disease, with few broad-spectrum therapeutic targets. Unusually, most AML retain wild-type TP53, encoding the pro-apoptotic tumor suppressor p53. MDM2 inhibitors (MDM2i), which activate wild-type p53, and BET inhibitors (BETi), targeting the BET-family co-activator BRD4, both show encouraging pre-clinical activity, but limited clinical activity as single agents. Here, we report synergistic toxicity of combined MDM2i and BETi towards AML cell lines, primary human blasts and mouse models, resulting from BETis ability to evict an unexpected repressive form of BRD4 from p53 target genes, and hence potentiate MDM2i-induced p53 activation. These results indicate that wild-type TP53 and a transcriptional repressor function of BRD4 together represent a potential broad-spectrum synthetic therapeutic vulnerability for AML.

cell biology↗

bioRxiv2020

Generation of glucocorticoid resistant SARS-CoV-2 T-cells for adoptive cell therapy

Adoptive cell therapy with viral-specific T cells has been successfully used to treat life-threatening viral infections, supporting the application of this approach against COVID-19. We expanded SARS-CoV-2 T-cells from the peripheral blood of COVID-19-recovered donors and non-exposed controls using different culture conditions. We observed that the choice of cytokines modulates the expansion, phenotype and hierarchy of antigenic recognition by SARS-CoV-2 T-cells. Culture with IL-2/4/7 but not other cytokine-driven conditions resulted in >1000 fold expansion in SARS-CoV-2 T-cells with a retained phenotype, function and hierarchy of antigenic recognition when compared to baseline (pre-expansion) samples. Expanded CTLs were directed against structural SARS-CoV-2 proteins, including the receptor-binding domain of Spike. SARS-CoV-2 T-cells could not be efficiently expanded from the peripheral blood of non-exposed controls. Since corticosteroids are used for the management of severe COVID-19, we developed an efficient strategy to inactivate the glucocorticoid receptor gene (NR3C1) in SARS-CoV-2 CTLs using CRISPR-Cas9 gene editing.

immunology↗

bioRxiv2020

In vivo and in vitro mechanistic characterization of a clinically relevant PolγA mutation

Mutations in POLG, encoding POL{gamma}A, the catalytic subunit of the mitochondrial DNA polymerase, cause a spectrum of disorders characterized by mtDNA instability. However, the molecular pathogenesis of POLG-related diseases is poorly understood and efficient treatments are missing. Here, we generated a POLGA449T/A449T mouse model, which reproduces the most common human recessive mutation of POLG, encoding the A467T change, and dissected the mechanisms underlying pathogenicity. We show that the A449T mutation impairs DNA binding and mtDNA synthesis activities of POL{gamma} in vivo and in vitro. Interestingly, the A467T mutation also strongly impairs interactions with POL{gamma}B, the homodimeric accessory subunit of holo-POL{gamma}. This allows the free POL{gamma}A to become a substrate for LONP1 protease degradation, leading to dramatically reduced levels of POL{gamma}A, which in turn exacerbates the molecular phenotypes of PolgA449T/A449T mice. Importantly, we validated this mechanism for other mutations affecting the interaction between the two POL{gamma} subunits. We suggest that LONP1 dependent degradation of POL{gamma}A can be exploited as a target for the development of future therapies.

genetics↗

bioRxiv2020

A key linear epitope for a potent neutralizing antibody to SARS-CoV-2 S-RBD

The spread of SARS-CoV-2 confers a serious threat to the public health without effective intervention strategies1-3. Its variant carrying mutated Spike (S) protein D614G (SD614G) has become the most prevalent form in the current global pandemic4,5. We have identified a large panel of potential neutralizing antibodies (NAbs) targeting the receptor-binding domain (RBD) of SARS-CoV-2 S6. Here, we focused on the top 20 potential NAbs for the mechanism study. Of them, the top 4 NAbs could individually neutralize both authentic SARS-CoV-2 and SD614G pseudovirus efficiently. Our epitope mapping revealed that 16/20 potent NAbs overlapped the same steric epitope. Excitingly, we found that one of these potent NAbs (58G6) exclusively bound to a linear epitope on S-RBD (termed as 58G6e), and the interaction of 58G6e and the recombinant ACE2 could be blocked by 58G6. We confirmed that 58G6e represented a key site of vulnerability on S-RBD and it could positively react with COVID-19 convalescent patients plasma. We are the first, as far as we know, to provide direct evidences of a linear epitope that can be recognized by a potent NAb against SARS-CoV-2 S-RBD. This study paves the way for the applications of these NAbs and the potential safe and effective vaccine design.

immunology↗

bioRxiv2020

Stanniocalcin 1a is a Ca2+-regulated switch controlling epithelial cell quiescence-proliferation balance and Ca2+ uptake

The mechanisms governing cell quiescence-proliferation balance are poorly defined. Using a zebrafish model, here we report that Stc1a, a glycoprotein known as a hypocalcemic hormone, not only inhibits epithelial calcium uptake but also functions as a Ca2+-regulated switch controlling epithelial cell quiescence-proliferation balance. Among the 4 stc genes, only the stc1a expression is [Ca2+]-dependent. Genetic deletion of stc1a, but not stc2b, resulted in elevated body Ca2+ contents, ectopic Ca2+ deposit, body swelling, and premature death. Reducing epithelial calcium channel Trpv6-mediated Ca2+ uptake alleviated these phenotypes. Loss of Stc1a also promoted quiescent epithelial cells to re-enter the cell cycle. This action was accompanied by local IGF signaling activation and increased expression in papp-aa, a zinc metalloproteinase degrading Igfbp5a. Genetic deletion of papp-aa or igfbp5a abolished the elevated epithelial cell reactivation in stc1a-/- mutants. Likewise, inhibition of IGF1 receptor, PI3 kinase, Akt, and Tor signaling abolished epithelial cell reactivation. These results reveal that Stc1a plays dual roles in regulating epithelial calcium uptake and cell quiescence-proliferation balance and implicate Trpv6 and Papp-aa-Igfbp5a-IGF signaling in these functions.

developmental biology↗

bioRxiv2020

Evaluation of Nanopore sequencing technology to differentiate Salmonella serotypes and serotype variants with the same or closely related antigenic formulae

Our previous study demonstrated that whole genome sequencing (WGS) data generated by Oxford Nanopore Technologies (ONT) can be used for rapid and accurate prediction of Salmnonella serotypes. However, one limitation is that established methods for WGS-based serotype prediction cannot differentiate certain serotypes and serotype variants with the same or closely related antigenic formulae. This study aimed to evaluate Nanopore sequencing and corresponding data analysis for differentiation of these serotypes and serotype variants, thus overcoming this limitation. Five workflows that combined different flow cells, library construction methods and basecaller models were evaluated and compared. The workflow that consisted of the R9 flow cell, rapid sequencing library construction kit and guppy basecaller with base modified model performed best for Single Nucleotide Polymorphism (SNP) analysis. With this workflow, as high as 99.98% matched the identity of the assembled genomes and only less than five high quality SNPs (hqSNPs) between ONT and Illumina sequencing data were achieved. SNP typing allowed differentiation of Choleraesuissensu stricto, Choleraesuis var. Kunzendorf, Choleraesuis var. Decatur, Paratyphi C, and Typhisuis that share the same antigenic formula 6,7:c:1,5. Prophage prediction further distinguished Orion var. 15+ and Orion var. 15+, 34+. Our study improves the readiness of ONT as a Salmonella subtyping and source tracking tool for food industry applications. HighlightsO_LISalmonella serotypes or serotype variants with the same antigenic formula were differentiated by SNP typing. C_LIO_LINanopore sequencing followed by phage prediction identified the Salmonella serotype variants caused by phage conversion. C_LIO_LIThe latest ONT technology is capable of high fidelity SNP typing of Salmonella. C_LI

microbiology↗

bioRxiv2020

Fast and inexpensive whole genome sequencing library preparation from intact yeast cells

Through the increase in the capacity of sequencing machines massively parallel sequencing of thousands of samples in a single run is now possible. With the improved throughput and resulting drop in the price of sequencing, the cost and time for preparation of sequencing libraries have become the major bottleneck in large-scale experiments. Methods using a hyperactive variant of the Tn5 transposase efficiently generate libraries starting from cDNA or genomic DNA in a few hours and are highly scalable. For genome sequencing, however, the time and effort spent on genomic DNA isolation limits the practicability of sequencing large numbers of samples. Here, we describe a highly scalable method for preparing high quality whole-genome sequencing libraries directly from yeast cultures in less than three hours at 34 cents per sample. We skip the rate-limiting step of genomic DNA extraction by directly tagmenting yeast spheroplasts and add a nucleosome release step prior to enrichment PCR to improve the evenness of genomic coverage. Resulting libraries do not show any GC-bias and are comparable in quality to libraries processed from genomic DNA with a commercially available Tn5-based kit. We use our protocol to investigate CRISPR/Cas9 on- and off-target edits and reliably detect edited variants and shared polymorphisms between strains. Our protocol enables rapid preparation of unbiased and high-quality, sequencing-ready indexed libraries for hundreds of yeast strains in a single day at a low price. By adjusting individual steps of our workflow we expect that our protocol can be adapted to other organisms.

genomics↗

bioRxiv2020

Two-stage Dynamic Deregulation of Metabolism Improves Process Robustness & Scalability in Engineered E. coli.

We report improved strain and bioprocess robustness as a result of the dynamic deregulation of central metabolism using two-stage dynamic control. Dynamic control is implemented using combinations of CRISPR interference and controlled proteolysis to reduce levels of central metabolic enzymes in the context of a standardized two-stage bioprocesses. Reducing the levels of key enzymes alters metabolite pools resulting in deregulation of the metabolic network. The deregulated network is more robust to environmental conditions improving process robustness, which in turn leads to predictable scalability from high throughput small scale screens to fully instrumented bioreactors as well as to pilot scale production. Additionally, as these two-stage bioprocesses are standardized, a need for traditional process optimization is minimized. Predictive high throughput approaches that translate to larger scales are critical for metabolic engineering programs to truly take advantage of the rapidly increasing throughput and decreasing costs of synthetic biology. In this work we demonstrate that the improved robustness of E. coli strains engineered for the improved scalability of the important industrial chemicals alanine, citramalate and xylitol, from microtiter plates to pilot reactors.

synthetic biology↗