Biology subjects

Wu, T.

Publications and source records attributed to Wu, T..

At least 217 records · Page 12Linked to original sources

bioRxiv2020

DNA damage induced during mitosis undergoes DNA repair synthesis

Understanding the mitotic DNA damage response (DDR) is critical to our comprehension of cancer, premature aging and developmental disorders which are marked by DNA repair deficiencies. In this study we use a micro-focused-laser to induce DNA damage in selected mitotic chromosomes to study the subsequent repair response. Our findings demonstrate that (1) mitotic cells are capable of DNA repair as evidenced by DNA synthesis at damage sites, (2) Repair is attenuated when DNA-PKcs and ATM are simultaneously compromised, (3) Laser damage may permit the observation of previously undetected DDR proteins when damage is elicited by other methods in mitosis, and (4) Twenty five percent of mitotic DNA-damaged cells undergo a subsequent mitosis. Together these findings suggest that mitotic DDR is more complex than previously thought and may involve factors from multiple repair pathways that are better understood in interphase.

cell biology↗

bioRxiv2019

A portable and cost-effective microfluidic system for massively parallel single-cell transcriptome profiling

Single-cell technologies are becoming increasingly widespread and have been revolutionizing our understanding of cell identity, state, diversity and function. However, current platforms can be slow to apply to large-scale studies and resource-limited clinical arenas due to a variety of reasons including cost, infrastructure, sample quality and requirements. Here we report DNBelab C4 (C4), a negative pressure orchestrated, portable and cost-effective device that enables high-throughput single-cell transcriptional profiling. C4 system can efficiently allow discrimination of species-specific cells at high resolution and dissect tissue heterogeneity in different organs, such as murine lung and cerebral cortex. Finally, we show that the C4 system is comparable to existing platforms but has huge benefits in cost and portability and, as such, it will be of great interest for the wider scientific community.

genomics↗

bioRxiv2019

De novo assembly of a Tibetan genome and identification of novel structural variants associated with high altitude adaptation

Structural variants (SVs) may play important roles in human adaption to extreme environments such as high altitude but have been under-investigated. Here, combining long-read sequencing with multiple scaffolding techniques, we assembled a high-quality Tibetan genome (ZF1), with a contig N50 length of 24.57 mega-base pairs (Mb) and a scaffold N50 length of 58.80 Mb. The ZF1 assembly filled 80 remaining N-gaps (0.25 Mb in total length) in the reference human genome (GRCh38). Markedly, we detected 17,900 SVs, among which the ZF1-specific SVs are enriched in GTPase activity that is required for activation of the hypoxic pathway. Further population analysis uncovered a 163-bp intronic deletion in the MKL1 gene showing large divergence between highland Tibetans and lowland Han Chinese. This deletion is significantly associated with lower systolic pulmonary arterial pressure, one of the key adaptive physiological traits in Tibetans. Moreover, with the use of the high quality de novo assembly, we observed a much higher rate of genome-wide archaic hominid (Altai Neanderthal and Denisovan) shared non-reference sequences in ZF1 (1.32%-1.53%) compared to other East Asian genomes (0.70%-0.98%), reflecting a unique genomic composition of Tibetans. One such archaic-hominid shared sequence, a 662-bp intronic insertion in the SCUBE2 gene, is enriched and associated with better lung function (the FEV1/FVC ratio) in Tibetans. Collectively, we generated the first high-resolution Tibetan reference genome, and the identified SVs may serve as valuable resources for future evolutionary and medical studies.

genomics↗

bioRxiv2019

MicroRNA-378 suppressed osteogenesis of mesenchymal stem cells and impaired bone formation via inactivating Wnt/β-catenin signaling

MicroRNAs (miRNAs) have been reported to serve as silencers to repress gene expression at post-transcriptional level. Multiple miRNAs have been demonstrated to play important roles in osteogenesis. MiR-378, a conserved miRNA, was reported to mediate bone metabolism and influence bone development, but the detailed function and underlying mechanism remain obscure. In this study, the miR-378 transgenic (TG) mouse was developed to study the role of miR-378 in osteogenic differentiation as well as bone formation. The abnormal bone tissues and impaired bone quality were displayed in the miR-378 TG mice, and a delayed healing effect was observed during bone fracture of the miR-378 TG mice. The osteogenic differentiation of MSCs derived from this TG mouse was also inhibited. We also found that miR-378 mimics suppressed while anti-miR-378 promoted osteogenesis of human MSCs. Two Wnt family members Wnt6 and Wnt10a were identified as bona fide targets of miR-378, and their expression were decreased by this miRNA, which eventually induced the inactivation of Wnt/{beta}-catenin signaling. Finally, the sh-miR-378 modified MSCs were locally injected into the fracture sites in an established mouse fracture model. The results indicated that miR-378 inhibitor therapy could promote bone formation and stimulate healing process in vivo. In conclude, miR-378 suppressed osteogenesis and bone formation via inactivating Wnt/{beta}-catenin signaling, suggesting miR-378 may be a potential therapeutic target for bone diseases.

cell biology↗

bioRxiv2019

The succession pattern of bacterial diversity in compost using pig manure mixed with wood chips analyzed by 16S rRNA gene analysis

The pig manure mixed with wood chips and formed compost by means of fermentation. We found that the protease activity, organic matter content and ammonium nitrogen concentration were higher in the early stage of composting. Meanwhile, the urease activity was highest in the high temperature period. The carbon to nitrogen ratio of the compost decreased continuously with fermentation. The dynamic change in the composition of bacterial overtime in the compost of a 180 kg piles were explored using microbial diversity analysis. The results showed that the microbial species increased with the compost fermentation. At the early stage of composting, the phyla of Firmicutes and Actinomycetes were dominant. The microbes in the high temperature period were mainly composed of Firmicutes and Proteobacteria while the proportion of Bacteroides was increased during the cooling period. In the compost of maturity stage, the proportion of Chloroflexi increased, becoming dominant species with other microorganisms including Firmicutes, Proteobacteria, Bacteroides, Chloroflexi but not Actinomycetes. Bacteria involved in lignocellulose degradation, such as those of the Thermobifida, Cellvibrio, Mycobacterium, Streptomyces and Rhodococcus, were concentrated in the maturity stages of composting. Through correlation analysis, the environmental factors including organic matter, ammonium nitrogen and temperature were consistent with the succession of microbial including Rhodocyclaceae, Anaerolineaceae, Thiopseudomonas, Sinibacillus and Tepidimicrobium. The change of urease activity and carbon to nitrogen ratio corresponded to microbial communities, mainly containing Anaerolineaceae, Rhodocyclaceae, Luteimoas, Bacillaceae, Corynebacterium, Bacillus, Anaerococcus, Lactobacillus, Ignatzschineria, and Bacillaceae.

microbiology↗

bioRxiv2019

Phase separation of TAZ compartmentalizes the transcription machinery to promote gene expression

TAZ promotes cell proliferation, development, and tumorigenesis by regulating target gene transcription. However, how TAZ orchestrates the transcriptional responses remains poorly defined. Here we demonstrate that TAZ forms nuclear condensates via liquid-liquid phase separation to compartmentalize its DNA binding co-factor TEAD4, the transcription co-activators BRD4 and MED1 and the transcription elongation factor CDK9 for activation of gene expression. TAZ, but not its paralog YAP, forms phase-separated droplets in vitro and liquid-like nuclear condensates in vivo, and this ability is negatively regulated by Hippo signaling via LATS-mediated phosphorylation and mediated by the coiled-coil domain. Deletion of the TAZ coiled-coil domain or substitution with the YAP coiled-coil domain does not affect the interaction of TAZ with its partners, but prevents its phase separation and more importantly, its ability to induce target gene expression. Thus, our study identifies a novel mechanism for the transcriptional activation by TAZ and demonstrates for the first time that pathway-specific transcription factors also engage the phase separation mechanism for efficient transcription activation.

cell biology↗

bioRxiv2019

Sequencing and comparative analysis of three Chlorella genomes provide insights into strain-specific adaptation to wastewater

Microalgal Chlorella has been demonstrated to process wastewater efficiently from piggery industry, yet optimization through genetic engineering of such a bio-treatment is currently challenging, largely due to the limited data and knowledge in genomics. In this study, we first investigated the differential growth rates among three wastewater-processing Chlorella strains: Chlorella sorokiniana BD09, Chlorella sorokiniana BD08 and Chlorella sp. Dachan, and the previously published Chlorella sorokiniana UTEX 1602, showing us that BD09 maintains the best tolerance in synthetic wastewater. We then performed genome sequencing and analysis, resulting in a high-quality assembly for each genome with scaffold N50 >2 Mb and genomic completeness [≥] 91%, as well as genome annotation with 9,668, 10,240, 9,821 high-confidence gene models predicted for BD09, BD08, and Dachan, respectively. Comparative genomics study unravels that metabolic pathways, which are involved in nitrogen and phosphorus assimilation, were enriched in the faster-growing strains. We found that gene structural variation and genomic rearrangement might contribute to differential capabilities in wastewater tolerance among the strains, as indicated by gene copy number variation, domain reshuffling of orthologs involved, as well as a [~]1 Mb-length chromosomal inversion we observed in BD08 and Dachan. In addition, we speculated that an associated bacterium, Microbacterium chocolatum, which was identified within Dachan, play a possible role in synergizing nutrient removal. Our three newly sequenced Chlorella genomes provide a fundamental foundation to understand the molecular basis of abiotic stress tolerance in wastewater treatment, which is essential for future genetic engineering and strain improvement.

genomics↗

bioRxiv2019

Functional tunability from a distance: Rheostat positions influence allosteric coupling between two distant binding sites

For protein mutagenesis, a common expectation is that important positions will behave like on/off \"toggle\" switches (i.e., a few substitutions act like wildtype, most abolish function). However, there exists another class of important positions that manifests a wide range of functional outcomes upon substitution: \"rheostat\" positions. Previously, we evaluated rheostat positions located near the allosteric binding sites for inhibitor alanine (Ala) and activator fructose-1,6-bisphosphate (Fru-1,6-BP) in human liver pyruvate kinase. When substituted with multiple amino acids, many positions demonstrated moderate rheostat effects on allosteric coupling between effector binding and phosphoenolpyruvate (PEP) binding in the active site. Nonetheless, the combined outcomes of all positions sampled the full range of possible allosteric coupling (full tunability). However, that study only evaluated allosteric tunability of \"local\" positions, i.e., positions were located near the binding sites of the allosteric ligand being assessed. Here, we evaluated tunability of allosteric coupling when mutated sites were distant from the allosterically-coupled binding sites. Positions near the Ala binding site had rheostat outcomes on allosteric coupling between Fru-1,6-BP and PEP binding. In contrast, positions in the Fru-1,6-BP site exhibited modest effects on coupling between Ala and PEP binding. Analyzed in aggregate, both PEP/Ala and PEP/Fru-1,6-BP coupling were again fully tunable by amino acid substitutions at this limited set of distant positions. Furthermore, some positions exhibited rheostatic control over multiple parameters and others exhibited rheostatic effects on one parameter and toggle control over a second. These findings highlight challenges in efforts to both predict/interpret mutational outcomes and engineer functions into proteins.

biochemistry↗

bioRxiv2019

Do neutral protein positions really exist? A case study with allostery in human liver pyruvate kinase

Understanding how each residue position contributes to protein function has been a long-standing goal in protein science. Substitution studies have historically focused on conserved protein positions. However, substitutions of nonconserved positions can also modify function. Indeed, we recently identified nonconserved positions that have large substitution effects in human liver pyruvate kinase (hLPYK), including altered allosteric coupling. To facilitate a comparison of which characteristics determine when a nonconserved position does vs. does not contribute to function, the goal of the current work was to identify neutral positions in hLPYK. However, existing hLPYK data showed that three features commonly associated with neutral positions - high sequence entropy, high surface exposure, and alanine scanning - lacked the sensitivity needed to guide experimental studies. We used multiple evolutionary patterns identified in a sequence alignment of the PYK family to identify which positions were least patterned, reasoning that these were most likely to be neutral. Nine positions were tested with a total of 117 amino acid substitutions. Although exploring all potential functions is not feasible for any protein, five parameters associated with substrate/effector affinities and allosteric coupling were measured for hLPYK variants. For each position, the aggregate functional outcomes of all variants were used to quantify a "neutrality" score. Three positions showed perfect neutral scores for all five parameters. Furthermore, the nine positions showed larger neutral scores than 17 positions located near allosteric binding sites. Thus, our strategy successfully enriched the dataset for positions with neutral and modest substitutions.

biochemistry↗

bioRxiv2019

Simulation-based comparison of Biopharmaceutics Classification System and drug structure

BackgroundThe Biopharmaceutics Classification System (BCS), which classifies bioactive molecules based on solubility and permeability, is widely used to guide new drug development and drug formulation, as well as predict pharmacokinetics. Here we performed computer simulations to study correlations between a molecules structure and its BCS classification.\n\nMethodsA total of 411 small molecules were assigned to BCS categories based on published drug data, and their Pybel-FP4 fingerprints were extrapolated. The information gain(IG) of each fingerprint was calculated and its characteristic structure analyzed. IG was calculated using multiple thresholds, and results were verified using support vector machine prediction, while taking into account the dose coefficient(0-0.1, 0.1-1, or>1). Structural functional features common to fingerprints of compounds in each type of BCS class were determined using computer simulations.\n\nResultsBCS classes III and IV appear to share several structural and functional characteristics, including Secondary aliphaticamine, Michael_acceptor, Isothiourea, and Sulfonamide Sulfonic_derivatives.\n\nConclusionWe demonstrate that our approach can correlate characteristic fingerprints of small-molecule drugs with BCS classifications, which may help guide the development and optimization of new drugs.

molecular biology↗

bioRxiv2019

Cascaded-CNN: Deep Learning to Predict Protein Backbone Structure from High-Resolution Cryo-EM Density Maps

Cryo-electron microscopy (cryo-EM) has become a leading technology for determining protein structures. Recent advances in this field have allowed for atomic resolution. However, predicting the backbone trace of a protein has remained a challenge on all but the most pristine density maps (< 2.5[A] resolution). Here we introduce a deep learning model that uses a set of cascaded convolutional neural networks (CNNs) to predict C atoms along a proteins backbone structure. The cascaded-CNN (C-CNN) is a novel deep learning architecture comprised of multiple CNNs, each predicting a specific aspect of a proteins structure. This model predicts secondary structure elements (SSEs), backbone structure, and C atoms, combining the results of each to produce a complete prediction map. The cascaded-CNN is a semantic segmentation image classifier and was trained using thousands of simulated density maps. This method is largely automatic and only requires a recommended threshold value for each evaluated protein. A specialized tabu-search path walking algorithm was used to produce an initial backbone trace with C placements. A helix-refinement algorithm made further improvements to the -helix SSEs of the backbone trace. Finally, a novel quality assessment-based combinatorial algorithm was used to effectively map C traces to obtain full-atom protein structures. This method was tested on 50 experimental maps between 2.6[A] and 4.4[A] resolution. It outperformed several state-of-the-art prediction methods including RosettaES, MAINMAST, and a Phenix based method by producing the most complete prediction models, as measured by percentage of found C atoms. This method accurately predicted 88.5% (mean) of the C atoms within 3[A] of a proteins backbone structure surpassing the 66.8% mark achieved by the leading alternate method (Phenix based fully automatic method) on the same set of density maps. The C-CNN also achieved an average RMSD of 1.23[A] for all 50 experimental density maps which is similar to the Phenix based fully automatic method. The source code and demo of this research has been published at https://github.com/DrDongSi/Ca-Backbone-Prediction.

molecular biology↗

bioRxiv2019

Protein tertiary structure modeling driven by deep learning and contact distance prediction in CASP13

Prediction of residue-residue distance relationships (e.g. contacts) has become the key direction to advance protein tertiary structure prediction since 2014 CASP11 experiment, while deep learning has revolutionized the technology for contact and distance distribution prediction since its debut in 2012 CASP10 experiment. During 2018 CASP13 experiment, we enhanced our MULTICOM protein structure prediction system with three major components: contact distance prediction based on deep convolutional neural networks, contact distance-driven template-free (ab initio) modeling, and protein model ranking empowered by deep learning and contact prediction, in addition to an update of other components such as template library, sequence database, and alignment tools. Our experiment demonstrates that contact distance prediction and deep learning methods are the key reasons that MULTICOM was ranked 3rd out of all 98 predictors in both template-free and template-based protein structure modeling in CASP13. Deep convolutional neural network can utilize global information in pairwise residue-residue features such as co-evolution scores to substantially improve inter-residue contact distance prediction, which played a decisive role in correctly folding some free modeling and hard template-based modeling targets from scratch. Deep learning also successfully integrated 1D structural features, 2D contact information, and 3D structural quality scores to improve protein model quality assessment, where the contact prediction was demonstrated to consistently enhance ranking of protein models for the first time. The success of MULTICOM system in the CASP13 experiment clearly shows that protein contact distance prediction and model selection driven by powerful deep learning holds the key of solving protein structure prediction problem. However, there are still major challenges in accurately predicting protein contact distance when there are few homologous sequences to generate co-evolutionary signals, folding proteins from noisy contact distances, and ranking models of hard targets.

bioinformatics↗

bioRxiv2019

Common functional networks in the mouse brain revealed by multi-centre resting-state fMRI analysis

Preclinical applications of resting-state functional magnetic resonance imaging (rsfMRI) offer the possibility to non-invasively probe whole-brain network dynamics and to investigate the determinants of altered network signatures observed in human studies. Mouse rsfMRI has been increasingly adopted by numerous laboratories world-wide. Here we describe a multi-centre comparison of 17 mouse rsfMRI datasets via a common image processing and analysis pipeline. Despite prominent cross-laboratory differences in equipment and imaging procedures, we report the reproducible identification of several large-scale resting-state networks (RSN), including a murine default-mode network, in the majority of datasets. A combination of factors was associated with enhanced reproducibility in functional connectivity parameter estimation, including animal handling procedures and equipment performance. Our work describes a set of representative RSNs in the mouse brain and highlights key experimental parameters that can critically guide the design and analysis of future rodent rsfMRI investigations.

neuroscience↗

bioRxiv2019

A next generation approach to species delimitation reveals the role of hybridization in a cryptic species complex of corals

BackgroundOur ability to investigate processes shaping the evolutionary diversification of corals (Cnidaria: Anthozoa) is limited by a lack of understanding of species boundaries. Discerning species has been challenging due to a multitude of factors, including homoplasious and plastic morphological characters and the use of molecular markers that are either not informative or have not completely sorted. Hybridization can also blur species boundaries by leading to incongruence between morphology and genetics. We used traditional DNA barcoding and restriction-site associated DNA sequencing combined with coalescence-based and allele-frequency methods to elucidate species boundaries and simultaneously examine the potential role of hybridization in a speciose genus of octocoral, Sinularia.\n\nResultsSpecies delimitations using two widely used DNA barcode markers, mtMutS and 28S rDNA, were incongruent with one another and with the morphospecies identifications, likely due to incomplete lineage sorting. In contrast, 12 of the 15 morphospecies examined formed well-supported monophyletic clades in both concatenated RAxML phylogenies and SNAPP species trees of >6,000 RADSeq loci. DAPC and Structure analyses also supported morphospecies assignments, but indicated the potential for two additional cryptic species. Three morphologically distinct species pairs could not, however, be distinguished genetically. ABBA-BABA tests demonstrated significant admixture between some of those species, suggesting that hybridization may confound species delimitation in Sinularia.\n\nConclusionsA genomic approach can help to guide species delimitation while simultaneously elucidating the processes generating diversity in corals. Results support the hypothesis that hybridization is an important mechanism in the evolution of Anthozoa, including octocorals, and future research should examine the contribution of this mechanism in generating diversity across the coral tree of life.

evolutionary biology↗

bioRxiv2019

Dean flow assisted single cell and bead encapsulation for high performance single cell expression profiling

Single-cell RNA sequencing examines the transcriptome of individual cells and reveals the inter-cell transcription heterogeneity, playing a critical role in both scientific research and clinical applications. Recently, droplet microfluidics-based platform for expression profiling has been shown as a powerful tool to capture of the transcriptional information on single cell level. Despite the breakthrough this platform brought about, it required the simultaneous encapsulation of single cell and single barcoded bead, the incidence of which was very low. Suboptimal capturing efficiency limited the throughput of the Drop-seq platform. In this work, we leveraged the advance in inertial microfluidics-based cell sorting and designed a microfluidic chip for high efficiency cell-bead co-encapsulation, increasing the capturing rate by more than four folds. Specifically, we adopted spiral and serpentine channels and ordered cells/beads before the encapsulation region. We characterized the effect of cell concentration on the capturing rate and achieved a cell-bead co-capturing rate up to 3%. We tested this platform by co-encapsulating barcoded beads and human-mouse cell mixtures. The sequencing data distinguished the majority of human and mice expressions, with the doublet rate being as low as 5.8%, indicating that the simultaneous capturing of two or more cells in one droplet was minimal even when using high cell concentration. This chip design showed great potential in improving the efficiency for future single cell expression profiling.

bioengineering↗

bioRxiv2019

Identification of Factors Complicating Bioluminescence Imaging

In vivo bioluminescence imaging (BLI) has become a standard, non-invasive imaging modality for following gene expression or the fate of proteins and cells in living animals. Currently, bioluminescent reporters used in laboratories are mostly derivatives of two major luciferase families: ATP-dependent insect luciferases and ATP-independent marine luciferases. Inconsistent results have been reported for experiments using different bio-luminescent reporters and users are often confused when trying to choose an optimal bioluminescent reporter for a given research purpose. Herein, we re-examined inconsistency in several experimental settings and identified factors, such as ATP dependency, serum stability, and molecular size, which significantly affected BLI results. We expect this study will make the research community aware of these factors and facilitate more accurate interpretation of BLI data by considering the nature of each bioluminescent reporter.

biochemistry↗

bioRxiv2018

Whole blood transcriptome analysis in bipolar disorder reveals strong lithium effect

Bipolar disorder (BD) is a highly heritable mood disorder with complex genetic architecture and poorly understood etiology. We performed a whole blood transcriptome analysis in a BD case-control sample (Nsubjects = 480) by RNA sequencing. While we observed widespread differential gene expression patterns between affected and unaffected individuals, these effects were largely linked to lithium treatment at the time of blood draw (FDR < 0.05, Ngenes = 976) rather than BD diagnosis itself (FDR < 0.05, Ngenes = 6). These lithium-associated genes were enriched for cell signaling and immune response functional annotations, among others, and were associated with neutrophil cell-type proportions, which were elevated in lithium users. Neither genes with altered expression in cases nor in lithium users were enriched for BD, schizophrenia, and depression genetic risk based on information from genome-wide association studies, nor was gene expression associated with polygenic risk scores for BD. Our findings suggest that BD is associated with minimal changes in whole blood gene expression independent of medication use but underline the importance of accounting for medication use and cell type heterogeneity in psychiatric transcriptomic studies. The results of our study add to mounting evidence of lithium's cell signaling and immune-related mechanisms.

genomics↗

bioRxiv2018

Highly Structured Homolog Pairing Reflects Functional Organization of the Drosophila Genome

Trans-homolog interactions encompass potent regulatory functions, which have been studied extensively in Drosophila, where homologs are paired in somatic cells and pairing-dependent gene regulation, or transvection, is well-documented. Nevertheless, the structure of pairing and whether its functional impact is genome-wide have eluded analysis. Accordingly, we generated a diploid cell line from divergent parents and applied haplotype-resolved Hi-C, discovering that homologs pair relatively precisely genome-wide in addition to establishing trans-homolog domains and compartments. We also elucidated the structure of pairing with unprecedented detail, documenting significant variation across the genome. In particular, we characterized two forms: tight pairing, consisting of contiguous small domains, and loose pairing, consisting of single larger domains. Strikingly, active genomic regions (A-type compartments, active chromatin, expressed genes) correlated with tight pairing, suggesting that pairing has a functional role genome-wide. Finally, using RNAi and haplotype-resolved Hi-C, we show that disruption of pairing-promoting factors results in global changes in pairing.\n\nOne Sentence SummaryHaplotype-resolved Hi-C reveals structures of homolog pairing and global implications for gene activity in hybrid PnM cells.

genetics↗