Biology subjects

Liu, Q.

Publications and source records attributed to Liu, Q..

At least 649 records · Page 36Linked to original sources

bioRxiv2018

Subjugation of TGFβ Signaling by Human Papilloma Virus in Head and Neck Squamous Cell Carcinoma Shifts DNA Repair from Homologous Recombination to Alternative End-Joining

Purpose: Following cytotoxic therapy, 70% of patients with human papillomavirus (HPV) positive oropharyngeal head and neck squamous cell carcinoma (HNSCC) are alive at 5 years compared to 30% of those with similar HPV-negative cancer, which is thought to be due to dysregulation of DNA repair. Loss of transforming growth factor {beta} (TGF{beta}) signaling is a poorly studied consequence of HPV that could contribute to this phenotype.\n\nExperimental Design: Human HNSCC cell lines (n=9), patient-derived xenografts (n=9), tissue microarray (n=194), TCGA expression data and primary tumor specimens (n=10) were used to define the relationship between TGF{beta} competency, response to DNA damage, and type of DNA repair.\n\nResults: Analysis of HNSCC specimens in situ and in vitro showed that HPV associates with loss of TGF{beta} signaling that increases the response to radiation or cisplatin. TGF{beta} suppressed miR-182 that inhibited both BRCA1, necessary for homologous recombination repair, and FOXO3, which is required for ATM kinase activity. TGF{beta} signaling blockade by either HPV or inhibitors released this control, compromised HRR and increased response to PARP inhibition. Antagonizing miR-182 rescued the homologous recombination deficit in HPV+ cells. Loss of TGF{beta} signaling unexpectedly increased error-prone, alternative end-joining repair.\n\nConclusions: HPV-positive HNSCC cells are unresponsive to TGF{beta}. Abrogated TGF{beta} signaling compromises homologous recombination and shifts reliance on alt-EJ repair that provides a mechanistic basis for sensitivity to PARP inhibitors. The effect of HPV in HNSCC provides critical validation of TGF{beta}s role in DNA repair proficiency and further raises the translational potential of TGF{beta} inhibitors in cancer therapy.

cancer biology↗

bioRxiv2018

Mammalian CST averts replication failure by preventing G-quadruplex accumulation

Human CST (CTC1-STN1-TEN1) is an RPA-like complex that associates with G-rich single-strand DNA and helps resolve replication problems both at telomeres and genome-wide. We previously showed that CST binds and disrupts G-quadruplex (G4) DNA in vitro, suggesting that CST may prevent in vivo blocks to replication by resolving G4 structures. Here, we demonstrate that CST binds and unfolds G4 with similar efficiency to RPA. In cells, CST is recruited to telomeric and non-telomeric chromatin upon G4 stabilization. STN1 depletion increases G4 accumulation and slows bulk genomic DNA replication. At telomeres, combined STN1 depletion and G4 stabilization causes multi-telomere FISH signals and telomere loss, hallmarks of deficient telomere duplex replication. Strand-specific telomere FISH indicates preferential loss of C-strand DNA while analysis of BrdU uptake during leading and lagging-strand telomere replication shows preferential under-replication of lagging telomeres. Together these results indicate a block to Okazaki fragment synthesis. Overall, our findings indicate a novel role for CST in maintaining genome integrity through resolution of G4 structures both ahead of the replication fork and on the lagging strand template.

molecular biology↗

bioRxiv2018

Reliable Multiplex Sequencing with Rare Index Mis-Assignment on DNB-Based NGS Platform

BackgroundMassively-parallel-sequencing, coupled with sample multiplexing, has made genetic tests broadly affordable. However, intractable index mis-assignments (commonly exceeds 1%) were repeatedly reported on some widely used sequencing platforms.\n\nResultsHere, we investigated this quality issue on BGI sequencers using three library preparation methods: whole genome sequencing (WGS) with PCR, PCR-free WGS, and two-step targeted PCR. BGIs sequencers utilize a unique DNB technology which uses rolling circle replication for DNA-nanoball preparation; this linear amplification is PCR free and can avoid error accumulation. We demonstrated that single index mis-assignment from free indexed oligos occurs at a rate of one in 36 million reads, suggesting virtually no index hopping during DNB creation and arraying. Furthermore, the DNB-based NGS libraries have achieved an unprecedentedly low sample-to-sample mis-assignment rate of 0.0001% to 0.0004% under recommended procedures.\n\nConclusionsSingle indexing with DNB technology provides a simple but effective method for sensitive genetic assays with large sample numbers.

genomics↗

bioRxiv2018

The probiotic effectiveness in experimental colitis is correlated with gut microbiome and host genetic features

Current evidence to support extensive use of probiotics in inflammatory bowel disease is limited and factors contribute to the inconsistent effectiveness of clinical probiotic therapy are not completely known. Here, as a proof-of-concept, we utilized Bifidobacterium longum JDM 301, a widely used commercial probiotic strain in China, to study potential factors that may influence the beneficial effect of probiotics in experimental colitis. We found that the probiotic therapeutic effect was varied across individual mouse even with the same genetic background and consuming the same type of food. The different probiotic efficacy was highly correlated with different microbiome features in each mouse. Consumption of a diet rich in fat can change the host sensitivity to mucosal injury-induced colitis but did not change the host responsiveness to probiotic therapy. Finally, the host genetic factor TLR2 was required for a therapeutic effect of B. longum JDM 301. Together, our results suggest that personalized microbiome and genetic features may modify the probiotic therapeutic effect.

microbiology↗

bioRxiv2018

Quantitative assessment of cell population diversity in single-cell landscapes

Single-cell RNA-sequencing (scRNA-seq) has become a powerful tool for the systematic investigation of cellular diversity. As a number of computational tools have been developed to identify and visualize cell populations within a single scRNA-seq dataset, there is a need for methods to quantitatively and statistically define proportional shifts in cell population structures across datasets, such expansion or shrinkage, or emergence or disappearance of cell populations. Here we present sc-UniFrac, a framework to statistically quantify compositional diversity in cell populations between single-cell transcriptome landscapes. sc-UniFrac enables sensitive and robust quantification in simulated and experimental datasets in terms of both population identity and quantity. We have demonstrated the utility of sc-UniFrac in multiple applications, including assessment of biological and technical replicates, classification of tissue phenotypes, identification and definition of altered cell populations, and benchmarking batch correction tools. sc-UniFrac provides a framework for quantifying diversity or alterations in cell populations across conditions, and has broad utility for gaining insight on how cell populations respond to perturbations.

bioinformatics↗

bioRxiv2018

Cycloserine for Treatment of Multidrug-Resistant Tuberculosis in China: A Retrospective Observational Study

ObjectivesCycloserine is crucial in multidrug-resistant tuberculosis (MDR-TB) treatment. Although extensive research has been carried out on MDR-TB, most researchers have not treated cycloserine in much detail. Therefore, we evaluate the efficacy and safety of cycloserine and seek to clarify the role of cycloserine for treatment of simple MDR-TB, pre-extensively drug-resistant tuberculosis (pre-XDR-TB), and extensively drug-resistant tuberculosis (XDR-TB).\n\nPatients and methodsA retrospective observational study was performed in China. We determined the treatment outcome as the primary outcome for 144 cycloserine-treated and 181 cycloserine-nontreated patients according to the definitions of WHO. The proportion of patients with sputum-culture conversion and the frequency of adverse drug reactions related to cycloserine were assessed as well.\n\nResultsAmong 325 MDR-TB patients, 144 were treated with cycloserine and 100 (69.4%) out of 144 successfully completed treatment. Compared with patients in non-cycloserine group, the hazard ratio of any unfavorable treatment outcome was 0.53 (95%CI: 0.35-0.81, P=0.003). Culture conversion rate at the intensive phase was similar whether cycloserine was administered or not (P=0.703). Of the 144 patients treated with cycloserine, a total of 16 (11.1%) patients experienced side-effects related to cycloserine, including 7 patients who discontinued cycloserine permanently.\n\nConclusionsCycloserine could be an attractive agent to treat MDR-TB. Its safety profile warrants use in the most of MDR-TB cases. Cycloserine significantly improved the chance of favorable outcome for patients with simple MDR-TB but not pre-XDR-TB and XDR-TB. More aggressive regimens might be required for pre-XDR-TB or XDR-TB patients.

microbiology↗

bioRxiv2018

Hemolysin liberates bacterial outer membrane vesicles for cytosolic lipopolysaccharide sensing

Inflammatory caspase-11/4/5 recognize cytosolic LPS from invading Gram-negative bacteria and induce pyroptosis and cytokine release, forming rapid innate antibacterial defenses. Since extracellular or vacuole-constrained bacteria are thought to rarely access the cytoplasm, how their LPS are exposed to the cytosolic sensors is a critical event for pathogen recognition. Hemolysin is a pore-forming bacterial toxin, which was generally accepted to rupture cell membrane, leading to cell lysis. Whether and how hemolysin participates in non-canonical inflammasome signaling remains uncovered. Here, we show that hemolysin-overexpressed enterobacteria triggered significantly increased caspase-4 activation in human intestinal epithelial cells (IECs). Hemolysin promoted LPS cytosolic delivery from extracellular bacteria through dynamin-dependent endocytosis. Further, we revealed that hemolysin was largely associated with bacterial outer membrane vesicles (OMVs) and induced rupture of OMV-containing vacuoles, subsequently increasing LPS exposure to the cytosolic sensor. Accordingly, overexpression of hemolysin promoted caspase-11 dependent IL-18 secretion, gut inflammation, and enterocyte pyroptosis in orally-infected mice, which was associated with restricting bacterial colonization in vivo. Together, our work reveals a concept that hemolysin promotes noncanonical inflammasome activation via liberating OMVs for cytosolic LPS sensing, which offers insights into innate immune surveillance of dysregulated hemolysin via caspase-11/4 in intestinal antibacterial defenses.\n\nSignificanceSensing of lipopolysaccharide (LPS) in the cytosol triggers non-canonical inflammasome-mediated innate responses. Recent work revealed that bacterial outer membrane vesicles (OMVs) enables LPS to access the cytosol for extracellular bacteria. However, since intracellular OMVs are generally constrained in endosomes, how OMV-derived LPS gain access to the cytosol remains unknown. Here, we reported that hemolysin largely bound with OMVs and entered cells through dynamin-dependent endocytosis. Intracellular hemolysin significantly impaired OMVs-constrained vacuole integrity and increased OMV-derived LPS exposure to the cytosolic sensor, which promoted non-canonical inflammasome activation and restricted bacterial gut infections. This work reveals the role of hemolysin in promoting non-canonical inflammasome activation and alerting host immune recognition, which provides insights into the more sophisticated biological functions of hemolysin upon infection.

immunology↗

bioRxiv2018

The identification of an anti-thrombin molecule via the screening of semi-random DNA libraries

Thrombosis remains one of the leading causes of mortality and morbidity in the world. Thrombin is a key enzyme involved in the blood clotting processes, which can be intervened by low concentrations of Hirudin. The C-terminal dodecapeptide of Hirudin was capable of inhibiting thrombosis. This peptide has been partially randomized in this report, and the coding sequences have been expressed in yeast as chimerical peptides for secretion into the culture media. Two other semi-random modules have been processed likewise. The supernatant was subsequently tested for anti-thrombin activities. DNA sequencing indicated that the putative positive clone encoded a single serine residue followed by a stop codon. The Ninhydrin assay of the culture supernatant of the positive clone indicated a high content of amino acid. Electrospray Mass Spectrometry showed a distinct peak at 430.5 when the expression products from Pichia pastoris were examined, suggesting that the compound may be a dimannosylated serine, as yeast possesses glycosylation at serine residues. The observed effects of -Mannosidase treatments on the function of yeast induction products are consistent with this assumption. Partial randomization of peptides and proteins may accelerate directed evolution, yielding unprecedented number of variants for functional interrogation and drug development.

molecular biology↗

bioRxiv2018

NanoMod: a computational tool to detect DNA modifications using Nanopore long-read sequencing data

BackgroundRecent advances in single-molecule sequencing techniques, such as Nanopore sequencing, improved read length, increased sequencing throughput, and enabled direct detection of DNA modifications through the analysis of raw signals. These DNA modifications include naturally occurring modifications such as DNA methylations, as well as modifications that are introduced by DNA damage or through synthetic modifications to one of the four standard nucleotides.\n\nMethodsTo improve the performance of detecting DNA modifications, especially synthetically introduced modifications, we developed a novel computational tool called NanoMod. NanoMod takes raw signal data on a pair of DNA samples with and without modified bases, extracts signal intensities, performs base error correction based on a reference sequence, and then identifies bases with modifications by comparing the distribution of raw signals between two samples, while taking into account of the effects of neighboring bases on modified bases (\"neighborhood effects\").\n\nResultsWe evaluated NanoMod on simulation data sets, based on different types of modifications and different magnitudes of neighborhood effects, and found that NanoMod outperformed other methods in identifying known modified bases. Additionally, we demonstrated superior performance of NanoMod on an E. coli data set with 5mC (5-methylcytosine) modifications.\n\nConclusionsIn summary, NanoMod is a flexible tool to detect DNA modifications with single-base resolution from raw signals in Nanopore sequencing, and will greatly facilitate large-scale functional genomics experiments in the future that use modified nucleotides.

bioinformatics↗

bioRxiv2018

VirTect: a computational method for detecting virus species from RNA-Seq and its application in head and neck squamous cell carcinoma

Next generation sequencing (NGS) provides an opportunity to detect viral species from RNA-seq data on human tissues, but existing computational approaches do not perform optimally on clinical samples. We developed a bioinformatics method called VirTect for detecting viruses in neoplastic human tissues using RNA-seq data. Here, we used VirTect to analyze RNA-seq data from 363 HNSCC (head and neck squamous cell carcinoma) patients and identified 22 HPV-induced HNSCCs. These predictions were validated by manual review of pathology reports on histopathologic specimens. Compared to two existing prediction methods, VirusFinder and VirusSeq, VirTect demonstrated superior performance with many fewer false positives and false negatives. The majority of HPV carcinogenesis studies thus far have been performed on cervical cancer and generalized to HNSCC. Our results suggest that HPV-induced HNSCC involves unique mechanisms of carcinogenesis, so understanding these molecular mechanisms will have a significant impact on therapeutic approaches and outcomes. In summary, VirTect can be an effective solution for the detection of viruses with NGS data, and can facilitate the clinicopathologic characterization of various types of cancers with broad applications for oncology.\n\nSignificance StatementWe developed a new bioinformatics tool, and reported the new inside of HPV carcinogenesis mechanism in HPV-induced head and neck squamous cell carcinoma (HNSCC). This novel bioin-formatics tool and the new knowledge of HPV-induced HNSCC will facilitate the development of target therapies for treating HNSCC.

bioinformatics↗

bioRxiv2018

Loss of SDHB reprograms energy metabolisms and inhibits high fat diet induced metabolic syndromes

Mitochondrial respiratory complex II utilizes succinate, key substrate of the Krebs cycle, for oxidative phosphorylation, which is essential for glucose metabolism. Mutations of complex II cause cancers and mitochondrial diseases, raising a critical question of the (patho-)physiological functions. To address the fundamental role of complex II in systemic energy metabolism, we specifically knockout SDHB in mice liver, a key complex II subunit that tethers the catalytic SDHA subunit and transfers the electrons to ubiquinone, and found that SHDB deficiency abolishes the assembly of complex II without affecting other respiration complexes while largely retaining SDHA stability. SHDB ablation reprograms energy metabolism and hyperactivates the glycolysis, Krebs cycle and {beta}-oxidation pathways, leading to catastrophic energy deficit and early death. Strikingly, sucrose supplementation or high fat diet resumes both glucose and lipid metabolism and prevent early death. Also, SDHB deficient mice are completely resistant to high fat diet induced obesity. Our findings reveal that the unanticipated role of complex II orchestrating both lipid and glucose metabolisms, and suggest that SDHB is an ideal therapeutic target for combating obesity.

molecular biology↗

bioRxiv2018

Evidence of independent acquisition and adaption of ultra-small bacteria to human hosts across the highly diverse yet reduced genomes of the phylum Saccharibacteria

Recently, we discovered that a member of the Saccharibacteria/TM7 phylum (strain TM7x) isolated from the human oral cavity, has an ultra-small cell size (200-300nm), a highly reduced genome (705 Kbp) with limited de novo biosynthetic capabilities, and a very novel lifestyle as an obligate epibiont on the surface of another bacterium 1. There has been considerable interest in uncultivated phyla, particularly those that are now classified as the proposed candidate phyla radiation (CPR) reported to include 35 or more phyla and are estimated to make up nearly 15% of the domain Bacteria. Most members of the larger CPR group share genomic properties with Saccharibacteria including reduced genomes (<1Mbp) and lack of biosynthetic capabilities, yet to date, strain TM7x represents the only member of the CPR that has been cultivated and is one of only three CPR routinely detected in the human body. Through small subunit ribosomal RNA (SSU rRNA) gene surveys, members of the Saccharibacteria phylum are reported in many environments as well as within a diversity of host species and have been shown to increase dramatically in human oral and gut diseases. With a single copy of the 16S rRNA gene resolved on a few limited genomes, their absolute abundance is most often underestimated and their potential role in disease pathogenesis is therefore underappreciated. Despite being an obligate parasite dependent on other bacteria, six groups (G1-G6) are recognized using SSU rRNA gene phylogeny in the oral cavity alone. At present, only genomes from the G1 group, which includes related and remarkably syntenic environmental and human oral associated representatives1, have been uncovered to date. In this study we systematically captured the spectrum of known diversity in this phylum by reconstructing completely novel Class level genomes belonging to groups G3, G6 and G5 through cultivation enrichment and/or metagenomic binning from humans and mammalian rumen. Additional genomes for representatives of G1 were also obtained from modern oral plaque and ancient dental calculus. Comparative analysis revealed remarkable divergence in the host-associated members across this phylum. Within the human oral cavity alone, variation in as much as 70% of the genes from nearest oral clade (AAI 50%) as well as wide GC content variation is evident in these newly captured divergent members (G3, G5 and G6) with no environmental relatives. Comparative analyses suggest independent episodes of transmission of these TM7 groups into humans and convergent evolution of several key functions during adaptation within hosts. In addition, we provide evidence from in vivo collected samples that each of these major groups are ultra-small in size and are found attached to larger cells.

microbiology↗

bioRxiv2018

ClusterMine: a Knowledge-integrated Clustering Approach based on Expression Profiles of Gene Sets

MotivationClustering analysis is essential for understanding complex biological data. In widely used methods such as hierarchical clustering (HC) and consensus clustering (CC), expression profiles of all genes are often used to assess similarity between samples for clustering. These methods output sample clusters, but are not able to provide information about which gene sets (functions) contribute most to the clustering. So interpretability of their results is limited. We hypothesized that integrating prior knowledge of annotated biological processes would not only achieve satisfying clustering performance but also, more importantly, enable potential biological interpretation of clusters.\n\nResultsHere we report ClusterMine, a novel approach that identifies clusters by assessing functional similarity between samples through integrating known annotated gene sets, e.g., in Gene Ontology. In addition to outputting cluster membership of each sample as conventional approaches do, it outputs gene sets that are most likely to contribute to the clustering, a feature facilitating biological interpretation. Using three cancer datasets, two single cell RNA-sequencing based cell differentiation datasets, one cell cycle dataset and two datasets of cells of different tissue origins, we found that ClusterMine achieved similar or better clustering performance and that top-scored gene sets prioritized by ClusterMine are biologically relevant.\n\nImplementation and availabilityClusterMine is implemented as an R package and is freely available at: www.genemine.org/clustermine.php\n\nContactjxwang@csu.edu.cn\n\nSupplementary InformationSupplementary data are available at Bioinformatics online.

bioinformatics↗

bioRxiv2018

Combining high-throughput micro-CT-RGB phenotyping and genome-wide association study to dissect the genetic architecture of tiller growth in rice

Traditional phenotyping of rice tillers is time consuming and labor intensive and lags behind the rapid development of rice functional genomics. Thus, dynamic phenotyping of rice tiller traits at a high spatial resolution and high-throughput for large-scale rice accessions is urgently needed. In this study, we developed a high-throughput micro-CT-RGB (HCR) imaging system to non-destructively extract 730 traits from 234 rice accessions at 9 time points. We used these traits to predict the grain yield in the early growth stage, and 30% of the grain yield variance was explained by 2 tiller traits in the early growth stage. A total of 402 significantly associated loci were identified by GWAS, and dynamic and static genetic components were found across the nine time points. A major locus associated with tiller angle was detected at nine time points, which contained a major gene TAC1. Significant variants associated with tiller angle were enriched in the 3'-UTR of TAC1. Three haplotypes for the gene were found and tiller angles of rice accessions containing haplotype H3 were much smaller. Further, we found two loci contained associations with both vigor-related HCR traits and yield. The superior alleles would be beneficial for breeding of high yield and dense planting.\n\nHighlightCombining high-throughput micro-CT-RGB phenotyping facility and genome-wide association study to dissect the genetic architecture of rice tiller development by using the indica subpopulation.

genetics↗

bioRxiv2017

Unconsciously Implanted Visuoauditory Memory in the Presence of Cholecystokinin Retrieved in Behavioral Contexts

We investigated whether visuoauditory association can be artificially implanted in rodents and then retrieved in a behaviorally relevant context. Rats were trained to approach the left or right hole of a behavioral apparatus to retrieve a reward depending on the side of electrical stimulation of the auditory cortex (EAC) they received and mice were fear-conditioned to EAC. Next, an irrelevant visual stimulus (VS) was repeatedly paired with EAC in the presence of cholecystokinin (CCK) or with activation of terminals of entorhinal CCK neurons in the auditory cortex. In subsequent behavioral testing with VS, rats approached the hole associated with reward availability and mice showed a freezing response to the VS. A CCK antagonist blocked the establishment of visuoauditory association, whereas a CCK agonist rescued the deficit of association. Our findings provide a scientific foundation for \"memory implantation\" and indicate that CCK is the switching chemical for formation of visuoauditory association.

neuroscience↗

bioRxiv2017

NRT1.1-dependent NH4+ toxicity in Arabidopsis is associated with disturbed balance between NH4+ uptake and assimilation

A high concentration of a sole ammonium (NH4+) source in growth media is often toxic to plants. The nitrate transporter NRT1.1 is involved in plant NH4+ toxicity; however, its mechanism remains undefined. In this study, wild-type Arabidopsis (Col-0) and NRT1.1 mutants (chl1-1 and chl1-5) were grown hydroponically in NH4NO3 and (NH4)2SO4 media to evaluate NRT1.1 function in NH4+ stress responses. All plants grew normally in mixed N sources, but Col-0 displayed more chlorosis, and lower biomass and photosynthesis than the NRT1.1 mutants in the (NH4)2SO4 condition. Grafting experiments between Col-0 and chl1-5 further confirmed that NH4+ toxicity is NRT1.1-dependent. In (NH4)2SO4 medium, NRT1.1 facilitated the higher expression of NH4+ transporters, increasing NH4+ uptake. Additionally, glutamine synthetase (GS) and glutamate synthetase (GOGAT) in roots of Col-0 plants decreased and soluble sugar accumulated significantly, whereas pyruvate kinase (PK)-mediated glycolysis was not affected, all of which contributed to NH4+ accumulation. In contrast, the NRT1.1 mutants reduced NH4+ accumulation and enhanced NH4+ assimilation through glutamate dehydrogenase (GDH) and glutamate-oxaloacetate transamination (GOT) activity. In addition, the upregulation of genes involved in senescence in Col-0 plants treated with (NH4)2SO4 suggests that ethylene could be involved in NH4+ toxicity responses. Our results indicate that NH4+ toxicity is dependent on NRT1.1 in Arabidopsis, characterized by enhanced NH4+ accumulation and by perturbed NH4+ metabolism, which stimulated ethylene-induced plant senescence.\n\nHighlight: Nitrate transporter NRT1.1 enhances NH4+ accumulation, disturbs the NH4+ metabolism, and aggravates NH4+ toxicity in Arabidopsis when grown under sole NH4+ condition.

plant biology↗

bioRxiv2017

Hi-TOM: a platform for high-throughput tracking of mutations induced by CRISPR/Cas systems

The CRISPR/Cas system has been extensively applied to make precise genetic modifications in various organisms. Despite its importance and widespread use, large-scale mutation screening remains time-consuming, labour-intensive and costly. Here, we describe a cheap, practicable and high-throughput screening strategy that allows parallel screening of 96 x N (N denotes the number of targets) genome-modified sites. The strategy simplified and streamlined the process of next-generation sequencing (NGS) library construction by fixing the bridge sequences and barcoding primers. We also developed Hi-TOM (available at http://www.hi-tom.net/hi-tom/), an online tool to track the mutations with precise percentage. Analysis of the samples from rice, hexaploid wheat and human cells reveals that the Hi-TOM tool has high reliability and sensitivity in tracking various mutations, especially complex chimeric mutations that frequently induced by genome editing. Hi-TOM does not require specially design of barcode primers, cumbersome parameter configuration or additional data analysis. Thus, the streamlined NGS library construction and comprehensive result output make Hi-TOM particularly suitable for high-throughput identification of all types of mutations induced by CRISPR/Cas systems.

bioinformatics↗

bioRxiv2017

New Cell Fate Potentials and Switching Kinetics Uncovered in a Classic Bistable Genetic Switch

Bistable switches are common gene regulatory motifs directing two mutually exclusive gene expression states, and consequently distinct cell fates. Theoretical studies suggest that the simple circuitry of bistable switches is sufficient to encode more than two cell fates due to the non-equilibrium, heterogeneous cellular environment, allowing a high degree of adaptation and differentiation. However, new cell fates arising from a classic bistable switch without rewiring the circuitry have not been experimentally observed. By developing a new, dual single-molecule gene-expression reporting system in live E. coli cells, we investigated the expression dynamics of two mutually repressing transcription factors, CI and Cro, in the classic genetic switch of bacteriophage {lambda}. We found that in addition to the two expected high-Cro and high-CI production states, there existed two new ones, in which neither CI nor Cro was produced, or both CI and Cro were produced. We constructed the corresponding potential landscape and mapped the transition kinetics between the four production states, providing insight into possible state-switching rates and paths. These findings uncover new cell fate potentials beyond the classical picture of {lambda} switch, and open a new window to explore the genetic and environmental origins of the cell fate decision-making process in gene regulatory networks.

biophysics↗