Biology subjects

Wang, K.

Publications and source records attributed to Wang, K..

At least 505 records · Page 28Linked to original sources

bioRxiv2019

A Chemical Probe of CARM1 Alters Epigenetic Plasticity against Breast Cancer Cell Invasion

CARM1 is a cancer-relevant protein arginine methyltransferase that regulates many aspects of transcription. Its pharmacological inhibition is a promising anti-cancer strategy. Here SKI-73 is presented as a CARM1 chemical probe with pro-drug properties. SKI-73 can rapidly penetrate cell membranes and then be processed into active inhibitors, which are retained intracellularly with 10-fold enrichment for days. These compounds were characterized for their potency, selectivity, modes of action, and on-target engagement. SKI-73 recapitulates the effect of CARM1 knockout against breast cancer cell invasion. Single-cell RNA-seq analysis revealed that the SKI-73-associated reduction of invasiveness act via altering epigenetic plasticity and suppressing the invasion-prone subpopulation. Interestingly, SKI-73 and CARM1 knockout alter the epigenetic plasticity with remarkable difference, arguing distinct modes of action between the small-molecule and genetic perturbation. We therefore discovered a CARM1-addiction mechanism of cancer metastasis and developed a chemical probe to target this process.

biochemistry↗

bioRxiv2019

Tracking human population structure through time from whole genome sequences

The genetic diversity of humans, like many species, has been shaped by a complex pattern of population separations followed by isolation and subsequent admixture. This pattern, reaching at least as far back as the appearance of our species in the paleontological record, has left its traces in our genomes. Reconstructing a populations history from these traces is a challenging problem. Here we present a novel approach based on the Multiple Sequentially Markovian Coalescent (MSMC) to analyse the population separation history. Our approach, called MSMC-IM, uses an improved implementation of the MSMC (MSMC2) to estimate coalescence rates within and across pairs of populations, and then fits a continuous Isolation-Migration model to these rates to obtain a time-dependent estimate of gene flow. We show, using simulations, that our method can identify complex demographic scenarios involving post-split admixture or archaic introgression. We apply MSMC-IM to whole genome sequences from 15 worldwide populations, tracking the process of human genetic diversification. We detect traces of extremely deep ancestry between some African populations, with around 1% of ancestry dating to divergences older than a million years ago.\n\nAuthor SummaryHuman demographic history is reflected in specific patterns of shared mutations between the genomes from different populations. Here we aim to unravel this pattern to infer population structure through time with a new approach, called MSMC-IM. Based on estimates of coalescence rates within and across populations, MSMC-IM fits a time-dependent migration model to the pairwise rate of coalescences. We implemented this approach as an extension to existing software (MSMC2), and tested it with simulations exhibiting different histories of admixture and gene flow. We then applied it to the genomes from 15 worldwide populations to reveal their pairwise separation history ranging from a few thousand up to several million years ago. Among other results, we find evidence for remarkably deep population structure in some African population pairs, suggesting that deep ancestry dating to one million years ago and older is still present in human populations in small amounts today.

evolutionary biology↗

bioRxiv2019

Morphine analgesia and mu opioid receptor signaling require programed death protein 1 (PD-1)

Opioids such as morphine produce analgesia via mu opioid receptor (MOR), but opioid receptor signaling is not fully understood. Here we report that morphine analgesia and MOR signaling require neuronal Programmed cell death protein-1 (PD-1). We found that morphine-induced antinociception following systemic or intrathecal injection was compromised in Pd1-/- mice. Morphine analgesia was also abrogated in wild-type mice after treatment with Nivolumab, a clinically used anti-PD-1 monoclonal antibody. Morphine produced analgesia by suppressing calcium currents in DRG neurons and excitatory synaptic transmission in spinal cord neurons, but strikingly, both actions were impaired by PD-1 blockade. In a mouse model of bone cancer, the antinociceptive action of systemic morphine was compromised in Pd1-/- mice. Finally, PD-L1 and morphine produce synergistic analgesia. Our findings demonstrate that PD-1 also acts as a neuro checkpoint inhibitor and mediates opioid-induced analgesia and MOR signaling in neurons.

neuroscience↗

bioRxiv2019

MicroRNA-transcriptome networks in whole blood and monocytes of women undergoing preterm labor

Preterm birth is attributed to neonatal morbidity as well as cognitive and physiological challenges. We have previously identified significant differences in mRNA expression in whole blood and monocytes, as well as differences in miRNA concentration in blood plasma, extracellular vesicles (EV) and EV-depleted plasma in women undergoing spontaneous preterm labor (sPTL). The goal of this analysis was to identify differences in miRNA expression within whole blood (WB) and peripheral monocytes (PM) from the same population of women undergoing sPTL compared to nonlaboring controls matched by gestational age. We performed single end small RNA sequencing in whole blood and peripheral monocytes from women undergoing sPTL with active contractions(24-34 weeks of gestation, N=15) matched for gestational age to healthy pregnant non laboring controls (>37 weeks gestation, N=30) who later delivered at term as a part of the Ontario Birth Study (Toronto, Ontario CA). We identified significant differences in expression of 16 miRNAs in PMs and 9 miRNAs in WB in women undergoing sPTL. In PMs, these miRNAs were predicted targets of 541 genes, including 28 previously associated with sPTL. In WB, miRNAs were predicted to target 303 genes, including 9 previously associated with sPTL. These genes were involved in a variety of immune pathways, including interleukin 2 signaling. This study is the first to identify changes in miRNA expression in WB and PMs of women undergoing sPTL. Our results shed light on potential mechanisms by which miRNAs may play a role in mediating systemic inflammatory response in pregnant women that deliver prematurely.

systems biology↗

bioRxiv2019

An Array of 60,000 Antibodies for Proteome-Scale Antibody Generation and Target Discovery

Antibodies are essential for elucidating the roles of genes decoded by genome sequencing. However, affordable technology for proteome-scale antibody generation does not exist. To address this, we developed the Proteome Epitope Tag Antibody Library (PETAL) and its array. PETAL consists of 62,208 mAbs against 15,199 peptides from diverse proteomes. PETAL harbors binders for a great multitude of proteins in nature due to antibody multispecificity, an intrinsic feature of an antibody. Distinctive combinations of 10,000-20,000 mAbs were found to target specific proteomes by array screening. Phenotype-specific mAb-target pairs were discovered for maize and zebrafish samples. Immunofluorescence and flow cytometry mAbs for human membrane proteins and ChIP-seq mAbs for transcription factors were identified from respective proteome-binding PETAL mAbs. Differential screening of cell surface proteomes of tumor and normal tissues discovered internalizing tumor antigens for antibody-drug conjugates. By discovering high affinity mAbs at a fraction of current time and cost, PETAL enables proteome-scale antibody generation and target discovery.

systems biology↗

bioRxiv2019

Detection of Base Analogs Incorporated During DNA Replication by Nanopore Sequencing

DNA synthesis is a fundamental requirement for cell proliferation and DNA repair, but no tools exist to identify the location, direction and speed of replication forks with base pair resolution. Mammalian cells have the ability to incorporate thymidine analogs along with the natural A, T, G and C bases during DNA synthesis, which allows for labelling of replicating or repaired DNA. Most sequencing platforms rely on base-pairing to identify the four canonical nucleotides, and are thus unable to distinguish them from these analogs. In contrast, the Oxford Nanopore Technologies (ONT) MinION infers nucleotide identity from changes in the ionic current as DNA strands are pulled through nanopores and can in principle differentiate noncanonical nucleotides from natural ones. Here, we demonstrate the use of the ONT MinION to detect 11 different thymidine analogs including CldU, BrdU, IdU, as well as, EdU alone or coupled to Biotin and other bulky adducts in synthetic DNA templates. We also show detection of IdU in the genome of mouse pluripotent stem cells. We find that different modifications generate variable shifts in ionic signals, providing a method of using analog combinations to identify the location and direction of DNA synthesis and repair at high resolution. We conclude that this novel method has the potential for single-base, genome-wide examination of DNA replication in stem cell differentiation or cell transformation.

genomics↗

bioRxiv2019

Optimal Scheduling of Bevacizumab and Pemetrexed/Cisplatin Dosing in Non-Small Cell Lung Cancer

Bevacizumab-pemetrexed/cisplatin (BEV-PEM/CIS) is a first line therapeutic for advanced non-squamous non-small cell lung cancer (NSCLC). Bevacizumab potentiates PEM/CIS cytotoxicity by inducing transient tumor vasculature normalization. BEV-PEM/CIS has a narrow therapeutic window. Therefore, it is an attractive target for administration schedule optimization. The present study leverages our previous work on BEV-PEM/CIS pharmacodynamic modeling in NSCLC-bearing mice to estimate the optimal gap in the scheduling of sequential BEV-PEM/CIS. We predicted the optimal gap in BEV-PEM/CIS dosing to be 2.0 days in mice and 1.2 days in humans. Our simulations suggest that the efficacy loss in scheduling BEV-PEM/CIS at too great of a gap is much less than the efficacy loss in scheduling BEV-PEM/CIS at too short of a gap.

pharmacology and toxicology↗

bioRxiv2019

Recruitment and disruption of value encoding during alcohol seeking

A critical area of inquiry in the neurobiology of alcohol abuse is the neural mechanisms by which cues gain the ability to elicit alcohol use. We previously showed that cue-evoked activity in rat ventral pallidum (VP) robustly encodes the value of cues trained under both Pavlovian and instrumental contingencies, despite a stronger relationship between cue-evoked responses and behavioral latency after instrumental training. Here, we assessed VP neural representations of cue value in rats trained with a Pavlovian conditioned stimulus (CS+) that predicted alcohol delivery, and in rats trained with an instrumental discriminative stimulus (DS) that predicted alcohol availability if the rat entered the reward port during the cue. We also examined the impact of alcohol exposure itself on the integrity of this type of signaling in rats trained with sucrose. Decoding of cue value based on VP firing was blunted for an alcohol CS+ versus an alcohol DS, as well as in comparison to a sucrose DS or CS+. Further, homecage alcohol exposure had opposing effects on VP encoding of cue value for a sucrose DS versus a sucrose CS+, enhancing decoding accuracy for the DS and reducing decoding accuracy for the CS+. These findings suggest that problem alcohol seeking may result from biased engagement of specific reward-related processes via changes in VP signaling.

neuroscience↗

bioRxiv2018

Mechanistic insights on metabolic dysfunction in PTSD: Role of glucocorticoid receptor sensitivity and energy deficit

PTSD is associated with metabolic comorbidities; however it is not clear how the neuroendocrine disturbances affect metabolism. To analyze this we employed a systems biological approach using an integrated mathematical model of metabolism, HPA axis and inflammation. We combined the metabolomics, neuroendocrine, clinical lab and cytokine data from combat-exposed veterans with and without PTSD, to characterize the differences in regulatory effects. We used the pattern of fold change in metabolites representing pathway level differences as reference for metabolic control analysis (MCA) using the model. MCA revealed parameters constituting the HPA axis, inflammation and GPCR pathway that yielded metabolic dysfunction consistent with PTSD. To support this, we performed causal analysis between regulatory components and the significantly different metabolites in our sample. Causal inference revealed that the changes in glucocorticoid receptor sensitivity were mechanistically associated with metabolic dysfunction and the effects were jointly mediated by insulin resistance, inflammation, oxidative stress and energy deficit.

systems biology↗

bioRxiv2018

Clonal seeds in hybrid rice using CRISPR/Cas9

Heterosis, the observation that first generation hybrids outcompete the parental lines, is widely used in increasing the productivity and yield of agricultural crops1,2. However, heterosis is lost in the following generations because of genetic segregation. In addition, the high cost of hybrid seed production hinders the application of heterosis in many crops. Clonal reproduction through seeds could be revolutionary for agriculture by allowing self-propagation of F1 hybrids3,4. Here we show that heterozygosity of F1 hybrid rice can be fixed and thus propagated without additional crossing. First, we showed that multiplex editing of three key meiotic genes5,6 in hybrid rice leads to the production of clonal diploid gametes and tetraploid seeds. Next, editing of the MATRILINEAL (MTL) gene that involved in fertilization7,8 results in the induction of haploid seeds in hybrid rice. By simultaneous editing of these four endogenous genes in hybrid rice using the CRISPR/Cas9 system, we obtained in one generation plants able to propagate clonally through seeds. This opens the possibility to fix heterozygosity of hybrid varieties in food crops.

plant biology↗

bioRxiv2018

Isolation and characterization of extracellular vesicles from Caenorhabditis elegans for multi-omic analysis.

Cells from bacteria to human release vesicles into their extracellular environment. These extracellular vesicles (EVs) contain multiple classesof molecules, including nucleic acids, proteins, and lipids. The isolation and analysis of EV cargos from mammalian cell culture and liquid biopsysamples has become a powerful approach for uncovering the messages that are packaged into these organelles. However, this approach has not been tenable in invertebrate model systems due to lack of sufficient amounts of pure EVs. Here we report a robust and reproducible procedure to isolateEVs from Caenorhabditis elegans with yields similar to those obtained from human cell culture. Through nanoparticle tracking, transmission electron microscopy, flow cytometry, mass spectrometry, RNAseq, and immunoaffinity analysis we provide the first ever detailed characterization of C. elegans EV composition and demonstrate that C. elegans EVs share fundamentally similar properties with their mammalian counterparts. These include vesicle size, enrichment for lipid rafts, and similar types of RNA and protein cargos. This ability of isolate pure EVs on ascale amenable to multiple types of downstream analyses permits, multi-omics characterization of EV cargos in an invertebrate model system.

cell biology↗

bioRxiv2018

Dietary lysozyme supplement alters serum biochemical makers and milk metabolite profile of sows via gut microbiota

Lysozyme is an important antimicrobial agent with promising future in replacing antibiotics in livestok production. The aim of current study was to determine variations in sows gut microbiota, serum immunity and breast milk metabolite profile mediated by lysozyme supplementation.Thirty-six pregnant sows were assigned to a control group without supplementation and two treatments with 0.5 g/kg and 1.0 g/kg lysozyme provided in formula feed for 21days. Microbiota analysis based on 16s RNA high-throughput sequencing and untargeted liquid chromatography tandem mass spectrometry were applied and combined in analysis. Serum biochemical indicators and immunoglobulins were also determined. Sows received 1.0kg/t lyszoyme treatment shown significant redution in microbial diversity. Spirochaetes, Euryarchaeota and Actinobacteria significantly increased while Firmicutes showed a remarkable reduction in 1.0kg/t treated group compared with control. Pyrimidine metabolism,Purine metabolism and Amino acid related enzymes were significantly upregulated in 1.0kg/t lysozyme treated group. The richness of gram-positive bacteria were significantly down-regulated by lysozyme treatments. Serum aspartate transaminase (AST) activity was significantly un-regulated. Serum IgM levels were significantly higher in the 1.0 kg/t group compared with control, while IgA levels was significantly lower in 1.0kg/t group. Over thirty metabolites from sows breast milk including L-Glutamine,creatine and L-Arginine were sigficantly altered by lysozyme treatment. There existed crucial correlations among gut microbiota, serum immunity and breast milk metabolites where lactobacillus and prevotella may play a key role in lysozyme mediated host-microbial interactions. Overall, lysozyme supplementation could effectively improve the composition, metabolic functions and phenotypes of sows gut microbiota and it also benefit sows with better immune status and breast milk composition.\n\nImportanceEnteric infections caused by pathogens have a significant negative effect on neonatal survival and animal health in swine production. The application of antibiotics in feeds at subtherapeutic levels could improve performance and overall health and is used extensively throughout the industry. However, abuse of antibiotics is contributing to the high level of drug resistance in microbial communities and rising concerns regarding human health. Here, we revealed that lysozyme supplementation could effectively improve the composition, metabolic functions and phenotypes of sows gut microbiota and it also benefit sows with better immune status and breast milk composition. These findings confirmed that lysozyme could be a suitable alternative to antibiotics in swine production.

microbiology↗

bioRxiv2018

Localization of balanced chromosome translocation breakpoints by long-read sequencing on the Oxford Nanopore platform

Structural variants (SVs) in genomes, including translocations, inversions, insertions, deletions and duplications, remain difficult to be detected reliably by traditional genomic technologies. In particular, balanced translocations and inversions cannot be detected by microarrays since they do not alter chromosome copy numbers; they cannot be reliably detected by short-read sequencing either, since many breakpoints are located within repetitive regions of the genome that are unmappable by short reads. However, the detection and the precise localization of breakpoints at the nucleotide level are important to study the genetic causes in patients carrying balanced translocations or inversions. Long-read sequencing techniques, such as the Oxford Nanopore Technology (ONT), may detect these SVs in a more direct, efficient and accurate manner. In this study, we applied whole-genome long-read sequencing on the Oxford Nanopore GridION sequencer to detect the breakpoints from 6 carriers of balanced translocations and one carrier of inversion, where SVs had initially been detected by karyotyping at the chromosome level. The results showed that all the balanced translocations were detected with [~]10X coverage and were consistent with the karyotyping results. PCR and Sanger sequencing confirmed 8 of the 14 breakpoints to single base resolution, yet other breakpoints cannot be refined to single-base due to their localization at highly repetitive regions or pericentromeric regions, or due to the possible presence of local deletions/duplications. Our results indicate that low-coverage whole-genome sequencing is an ideal tool for the precise localization of most translocation breakpoints and may provide haplotype information on the breakpoint-linked SNPs, which may be widely applied in SV detection, therapeutic monitoring, assisted reproduction technology (ART) and preimplantation genetic diagnosis (PGD).

genetics↗

bioRxiv2018

Genome-wide selection footprints and deleterious variations in young Asian allotetraploid rapeseed

Brassica napus (AACC, 2n=38), is an important oilseed crop grown worldwide. However, little is known about the population evolution of this species, the genomic difference between its major genetic clusters, such as European and Asian rapeseed, and impacts of historical large-sale introgression events in this young tetraploid. In this study, we reported the de novo assembly of the genome sequences of an Asian rapeseed (B. napus), Ningyou 7 and its four progenitors and carried out de novo assembly-based comparison, pedigree and population analysis with other available genomic data from diverse European and Asian cultivars. Our results showed that Asian rapeseed originally derived from European rapeseed, but it had subsequently significantly diverged, with rapid genome differentiation after intensive local breeding selection. The first historical introgression of B. rapa dramatically broadened the allelic pool of Asian B. napus, but decreased their deleterious variations. The secondary historical introgression of European rapeseed (canola-quality) has reshaped Asian rapeseed into two groups, accompanied by an increase in genetic load. This study demonstrates distinctive genomic footprints by recent intra- and inter-species introgression events for local adaptation, and provide novel insights for understanding the rapid genome evolution of a young allopolyploid crop.

genomics↗

bioRxiv2018

LinkedSV: Detection of mosaic structural variants from linked-read exome and genome sequencing data

Linked-read sequencing provides long-range information on short-read sequencing data by barcoding reads originating from the same DNA molecule, and can improve the detection and breakpoint identification for structural variants (SVs). We present LinkedSV for SV detection on linked-read sequencing data. LinkedSV considers barcode overlapping and enriched fragment endpoints as signals to detect large SVs, while it leverages read depth, paired-end signals and local assembly to detect small SVs. Benchmarking studies demonstrates that LinkedSV outperforms existing tools, especially on exome data and on somatic SVs with low variant allele frequencies. We demonstrate clinical cases where LinkedSV identifies disease causal SVs from linked-read exome sequencing data missed by conventional exome sequencing, and show examples where LinkedSV identifies SVs missed by high-coverage long-read sequencing. In summary, LinkedSV can detect SVs missed by conventional short-read and long-read sequencing approaches, and may resolve negative cases from clinical genome/exome sequencing studies.

bioinformatics↗

bioRxiv2018

scMerge: Integration of multiple single-cell transcriptomics datasets leveraging stable expression and pseudo-replication

Concerted examination of multiple collections of single cell RNA-Seq (scRNA-Seq) data promises further biological insights that cannot be uncovered with individual datasets. However, such integrative analyses are challenging and require sophisticated methodologies. To enable effective interrogation of multiple scRNA-Seq datasets, we have developed a novel algorithm, named scMerge, that removes unwanted variation by combining stably expressed genes and utilizing pseudo-replicates across datasets. Analysis of large collections of publicly available datasets demonstrates that scMerge performs well in multiple scenarios and enhances biological discovery, including inferring cell developmental trajectories.

bioinformatics↗

bioRxiv2018

UBE2G1 Governs the Destruction of Cereblon Neomorphic Substrates

The immunomodulatory drugs (IMiDs) thalidomide, lenalidomide, and pomalidomide as well as the novel cereblon modulating agents (CMs) including CC-122, CC-220 and cereblon-based proteolysis-targeting chimaeras (PROTACs) repurpose the Cul4-RBX1-DDB1-CRBN (CRL4CRBN) E3 ubiquitin ligase complex to induce the degradation of specific neomorphic substrates via polyubiquitination in conjunction with an E1 ubiquitin-activating enzyme and E2 ubiquitin-conjugating enzymes, which have until now remained elusive. Here we show that the ubiquitin-conjugating enzymes UBE2G1 and UBE2D3 cooperatively promote the polyubiquitination of CRL4CRBN neomorphic substrates in a cereblon- and CM-dependent manner via a sequential ubiquitination mechanism: UBE2D3 transforms the neomorphic substrates into mono-ubiquitinated forms, upon which UBE2G1 catalyzes K48-linked polyubiquitin chain extension. Blockade of UBE2G1 diminishes the ubiquitination and degradation of neomorphic substrates, and consequent antitumor activities elicited by all tested CMs. For example, UBE2G1 inactivation significantly attenuated the degradation of myeloma survival factors IKZF1 and IKZF3 induced by lenalidomide and pomalidomide, hence conferring drug resistance. UBE2G1-deficient myeloma cells, however, remained sensitive to a more potent IKZF1/3 degrader CC-220. Collectively, these findings suggest that loss of UBE2G1 activity might be a resistance mechanism to drugs that hijack the CRL4CRBN to eliminate disease-driving proteins, and that this resistance mechanism can be overcome by next-generation CMs that destroy the same targeted protein more effectively.

molecular biology↗

bioRxiv2018

Temporal Small RNA Expression Profiling Under Drought Reveals a Potential Regulatory Role of snoRNAs in Drought Responses of Maize

Small RNAs (sRNAs) are short noncoding RNAs that play roles in many biological processes, including drought responses in plants. However, how the expression of sRNAs dynamically changes with the gradual imposition of drought stress in plants is largely unknown. We generated time-series sRNA sequence data from maize seedlings under drought stress and under well-watered conditions at the same time points. Analyses of length, functional annotation, and abundance of 736,372 non-redundant sRNAs from both drought and well-watered data, as well as genome copy number and chromatin modifications at the corresponding genomic regions, revealed distinct patterns of abundance, genome organization, and chromatin modifications for different sRNA classes of sRNAs. The analysis identified 6,646 sRNAs whose regulation was altered in response to drought stress. Among drought-responsive sRNAs, 1,325 showed transient down-regulation by the seventh day, coinciding with visible symptoms of drought stress. The profiles revealed drought-responsive microRNAs, as well as other sRNAs that originated from ribosomal RNAs (rRNAs), splicing small nuclear RNAs, and small nucleolar RNAs (snoRNA). Expression profiles of their sRNA derivers indicated that snoRNAs might play a regulatory role through regulating stability of rRNAs and splicing small nuclear RNAs under drought condition.

genomics↗