Biology subjects

Chen, W.

Publications and source records attributed to Chen, W..

At least 541 records · Page 30Linked to original sources

bioRxiv2019

Anlotinib overcomes multiple drug resistant of the colorectal cancer cells via inactivating PI3K/AKTpathway

BackgroundAnlotinib is a multi-tyrosine kinase inhibitor that has been reported to have activity against colorectal cancer. However, the functional mechanisms whereby anlotinib mediates against deadly drug-resistant colorectal cancer (CRC) has not been fully described-specifically, the potential mechanisms that inhibit proliferation and induce apoptosis remain largely unknown. MethodsMTT assays were used to detect cell viability and calculate the resistance index. Colony formation was used to evaluate the proliferation of resistant cells. DAPI staining was used to detect cell apoptosis morphologically. Annexin V-FITC with PI staining was used to detect early and late-stage apoptosis of cells. Cell cycle distribution was determined by Flow cytometry. Transwell assays were performed to examine the ability of migration and invasion. Cyclin D1 Survivin, CDK4, Bcl-2, Bax and changes of PI3K/AKT pathway were detected by Western blotting. Compared as a single agent or combined with anlotinib or LY294002, PI3K inhibitor (LY294002) was used to verify whether it inhibited drug-resistant CRC cells by lowering PI3K/AKT. ResultsHCT-8/5-FU cells showed multiple drug resistance. Drug resistance index of 5-FU, ADM and DDP were 390.27, 2.55 and 4.57, respectively. Anlotinib was shown to inhibit cell viability on HCT-8/5-FU and HCT-8 cells for 24 h and 48 h in a dosage- and time-dependent pattern. Compared with 48 h, intervened with anlotinib (0 M, 10 M, 20 M and 40 M) for 24 h, the HCT-8/5-FU cells were sensitive to anlotinib, and their sensitivity was greater than that of the parent cell line (HCT-8) at 24 h. Further, anlotinib inhibited the number of cloned cells significantly and had a significant inhibitory effect on cell cycle, mainly by blocking G1 transferring to S phase. Moreover, anlotinib could down-regulate the expression of survivin, cyclin D1, CDK4, caspase-3, Bcl-2, MMP-2, vimentin, MMP-9, and N-cadherin, while up-regulating cleaved-caspase-3, Bax and E-cadherin. Anlotinib inhibited the activity of the PI3K/AKT pathway and induced apoptosis in HCT-8/5-FU cells. Using LY294002, a specific PI3K inhibitor, our experiment found anlotinib can inhibit drug-resistant CRC cells by reducing PI3K and p-AKT activity-induce apoptosis. ConclusionsAnlotinib inhibited the proliferation, metastasis and induced apoptosis of HCT-8 / 5-FU cells; and the mechanism could be that anlotinib overcomes multiple drug resistant of the colorectal cancer cells via inactivating PI3K/AKT pathway.

pharmacology and toxicology↗

bioRxiv2019

LARP7 is a BRCA1 ubiquitinase substrate and regulates genome stability and tumorigenesis

Attenuated DNA repair leads to genomic instability and tumorigenesis. BRCA1/BARD1 are the best known tumor suppressors that promote homology recombination (HR) and arrest cell cycle at G2/M checkpoint. As E3 ubiquitin ligases, their ubiquitinase activity has been known to involve in the HR and tumor suppression, but the mechanism remains ambiguous. Here, we demonstrated upon genotoxic stress, BRCA1 together with BARD1 catalyzed the K48 ployubiquitination on LARP7, a 7SK RNA binding protein known to control RNAPII pausing, and thereby degraded it through 26S ubiquitin-proteasome pathway. Depleting LARP7 suppressed the expression of CDK1 complex, arrested cell at G2/M DNA damage checkpoint and reduced BRCA2 phosphorylation which thereby facilitated RAD51 recruitment to damaged DNA to enhance HR. Importantly, LARP7 depletion observed in breast patients lead to the chemoradiotherapy resistance both in vitro and in vivo. Together, this study unveils a mechanism by which BRCA1/BARD1 utilizes their E3 ligase activity to control HR and cell cycle, and highlights LARP7 as a potential target for cancer prevention and therapy.\n\nHighlightsO_LIDNA damage response downregulates LARP7 through BRCA1/BARD1\nC_LIO_LIBRCA1/BARD1 catalyzes the K48 polyubiquitination on LARP7\nC_LIO_LILARP7 promotes G2/M cell cycle transition and tumorigenesis via CDK1 complex\nC_LIO_LILARP7 disputes homology-directed repair that leads to tumor therapy resistance\nC_LI

molecular biology↗

bioRxiv2019

Optimizing a human fecal assay that elicits bacterial swarming

A distinct property of many bacteria is swarming: swift movement across a surface through flagella propulsion. Early research indicates that bacterial swarming can be a protective host response to intestinal inflammation. Central to the further study of bacterial swarming in human health is an effective and replicable assay for swarming that can accommodate complex material, such as fecal matter. To date, nearly all swarming assays described in the literature are specific for bacteria grown in culture, most often Pseudomonas. In this paper, we describe a protocol for discerning swarming of bacteria from frozen human fecal samples. Moreover, we tested 4 variables that may influence the effectiveness of the assay: the method by which frozen samples were thawed, the concentration of agar used in the Lysogenic broth (LB) agar plate, the volume of LB agar poured in the plate, and the volume of sample inoculated. We found that while the type of thaw and volume of LB agar had little to no effect on swarming, greater concentrations of agar were negatively correlated with swarming and greater volumes of the sample were positively correlated with swarming.

microbiology↗

bioRxiv2019

Role of Proinsulin Self-Association in Mutant INS gene-induced Diabetes of Youth

Abnormal interactions between misfolded mutant and wild-type (WT) proinsulin in the endoplasmic reticulum (ER) drive the molecular pathogenesis of Mutant-INS-gene induced Diabetes of Youth (MIDY). How these abnormal interactions are initiated remains unknown. Normally, proinsulin-WT dimerizes in the ER. Here, we suggest that the normal proinsulin-proinsulin contact surface, involving the B-chain, contributes to dominant-negative effects of misfolded MIDY mutants. Specifically, we find that proinsulin Tyr-B16, which is a key residue in normal proinsulin dimerization, helps confer dominant-negative behavior of MIDY mutant proinsulin-C(A7)Y. Substitutions of Tyr-B16 with ether Ala, Asp, or Pro in proinsulin-C(A7)Y each decrease the abnormal interactions between the MIDY mutant and proinsulin-WT, rescuing proinsulin-WT export, limiting ER stress, and increasing insulin production in {beta}-cells and human islets. This study reveals the first evidence indicating that noncovalent proinsulin-proinsulin contact initiates dominant-negative behavior of misfolded proinsulin, pointing to a novel therapeutic target to enhance bystander proinsulin export for increased insulin production.

cell biology↗

bioRxiv2019

Principles of 3D Nucleus Organization and Epigenetic Regulation in Diploid Genome Revealed by Multi-omic Data from Hybrid Mouse

Most mammalian genomes are diploid and previous studies have extensively investigated the average epigenetic profiles of homologous chromosomes. Here we use hybrid mice to distinguish the epigenetic status and three-dimensional organization of homologous chromosomes. We generated Hi-C, ChIP-seq and RNA-seq datasets from CD4 T cells of B6, Cast and hybrid mice, respectively, and systematically analyzed the 3D nucleus organization and epigenetic regulation. Our data indicate that the inter-chromosomal interaction patterns between homologous chromosomes are similar and the similarity is highly correlated with their allelic co-expression levels. Construction of 3D nucleus based on allele-specific interaction frequency revealed symmetric positioning of homologous chromosomes in the 3D nuclear space. The inter-chromosomal interactions at centromeres are significantly weaker than those at telomeres, indicating positioning of centromeres toward the inside of chromosome territories and telomeres toward the surface of chromosome territories. The majority A|B compartments or topologically associated domains (TADs) are consistent between B6 and Cast. We found 58% of the haploids in hybrids maintain their parental compartment status at B6/Cast divergent compartments due to cis-effect. About 95% of the trans-effected B6/Cast divergent compartments converge to same compartment status potentially due to a shared cellular environment. We found the differentially expressed genes between the two haploids in hybrid were associated with either genetic associated cis-effects or epigenetic associated trans-effects. The widespread epigenetic differences between B6 and Cast suggest that epigenetic changes may be major contributors to differences between B6 and Cast. Our data revealed symmetrical positioning of homologous chromosomes in 3D nucleus and enhanced our understanding of allele-specific epigenetic regulation.

genomics↗

bioRxiv2019

LncRNA-UCA1 inhibits the astrocyte activation in the temporal lobe epilepsy via regulating JAK/STAT signaling pathway

This article aimed to reveal the mechanism of Urothelial cancer associated 1 (UCA1) regulated astrocyte activation in temporal lobe epilepsy (TLE) rats via JAK/STAT signaling pathway. A model of TLE was established based on rats via kainic acid (KA) injection. All rats were divided into sham group, KA group, normal control (NC) + KA group and UCA1 + KA group. The Morris water maze was used to test the learning and memory ability of rats, and the expression of UCA1 in hippocampus was determined by qRT-PCR. Surviving neurons were counted by Nissl staining, and expression of glial cells glial fibrillary acidic protein, p-JAK1, and p-STAT and glutamate/aspartate transporter (GLAST) was analyzed by immunofluorescence and Western blot. A rat model of TLE was established by intraperitoneal injection of KA. QRT-PCR and fluorescence study showed that UCA1 inhibited astrocyte activation in hippocampus of epileptic rats. Meanwhile, the MWM analysis indicated that UCA1 improved the learning and memory in epilepsy rats. Moreover, the Nissl staining showed that UCA1 might has protective effect on neuronal injury induced by KA injection. Furthermore, the immunofluorescence and Western blot analysis revealed that the overexpression of UCA1 inhibited KA-induced abnormal elevation of GLAST, astrocyte activation of JAK/STAT signaling pathway, as well as hippocampus of epilepsy rats. UCA1 inhibited hippocampal astrocyte activation and GLAST expression in TLE rats via regulating JAK/STAT signaling, and improved the adverse reactions caused by epilepsy.

neuroscience↗

bioRxiv2019

GLUT1 inhibition blocks growth of RB1-positive Triple Negative Breast Cancer

Triple negative breast cancer (TNBC) is a deadly form of breast cancer due to the development of resistance to chemotherapy affecting over 30% of patients. New therapeutics and companion biomarkers are urgently needed. Recognizing the elevated expression of glucose transporter 1 (GLUT1, encoded by SLC2A1) and associated metabolic dependencies in TNBC, we investigated the vulnerability of TNBC cell lines and patient-derived samples to GLUT1 inhibition. We report that genetic or pharmacological inhibition of GLUT1 with BAY-876 impairs the growth of a subset of TNBC cells displaying high glycolytic and lower oxidative phosphorylation (OXPHOS) rates. Pathway enrichment analysis of gene expression data implicates E2F Targets pathway activity as a surrogate of OXPHOS activity. Furthermore, the protein levels of retinoblastoma tumor suppressor (RB1) are strongly correlated with the degree of sensitivity to GLUT1 inhibition in TNBC, where RB1-negative cells are insensitive to GLUT1 inhibition. Collectively, our results highlight a strong and targetable RB1-GLUT1 metabolic axis in TNBC and warrant clinical evaluation of GLUT1 inhibition in TNBC patients stratified according to RB1 protein expression levels.

cancer biology↗

bioRxiv2019

Bacterial Hyperswarming as a Protective Response to Intestinal Stress

Bacterial swarming, a collective movement on a surface, has rarely been associated with human pathophysiology. Here, we report for the first time that bacterial swarmers are associated with protection against intestinal inflammation. We show that bacterial swarmers are highly predictive of intestinal stress in mice and humans. We isolated a novel Enterobacter swarming strain, SM3, from mouse feces. SM3 and other known commensal swarmers contrast to their respective swarming-deficient, but swimming-competent isogenic strains abrogated intestinal inflammation in mice. Treatment of colitic mice with SM3, but not its mutants, enriched beneficial fecal anaerobes belonging to the family, Bacteroidales S24-7. We observed SM3 swarming associated pathways in the in vivo fecal metatranscriptomes. In vitro growth of S24-7 was enriched in presence of SM3 or its mutants conjecturing that bacterial swarming in vivo might influence SM3s access to S24-7 in the intestines. Overall, our work identifies a new paradigm in which intestinal stress allows for the emergence of swarming bacteria, which can counterintuitively heal intestinal inflammation.

microbiology↗

bioRxiv2019

Germline RNA helicases couple RNA binding to P granule assembly at nuclear periphery

P granules are phase-separated liquid droplets that play important roles in the maintenance of the germ cell fate in C. elegans. The localization and formation of P granules are highly dynamic, but mechanisms that regulate such processes remain poorly understood. Here we show that germline RNA helicases (GLHs) control the formation and disassembly of germ granules through their binding and release of RNAs, respectively. In addition, the FGG repeats in the GLHs promote the formation of germ granules at the perinucleus. Proteomic analyses of a mutation that traps RNA-bound GLH-1 complex revealed transient interactions of GLH-1 with several Argonautes and RNA binding proteins. Finally, we found that defects in perinuclear P granule formation correlate with the fertility defects observed in various GLH mutants. Together, our results highlight the versatile roles of RNA helicases in controlling the formation of liquid droplets in space and time.

cell biology↗

bioRxiv2019

FRA1 modulates cortical microtubule localization of CMU proteins

Construction of the cell wall demands harmonized deposition of cellulose and matrix polysaccharides. Cortical microtubules orient the deposition of cellulose by guiding the trajectory of plasma membrane-embedded cellulose synthase complexes. Vesicles containing matrix polysaccharides are thought to be transported by the FRA1 kinesin to facilitate their secretion along cortical microtubules. The cortical microtubule cytoskeleton thus provides a platform to coordinate the delivery of cellulose and matrix polysaccharides, but the underlying molecular mechanisms remain unknown. Here, we show that the tail region of the FRA1 kinesin physically interacts with CMU proteins which are important for the microtubule-dependent guidance of cellulose synthase complexes. Interaction with CMUs did not affect microtubule binding or motility of the FRA1 kinesin but had an opposing effect on the cortical microtubule localization of CMU1 and CMU2 proteins, thus regulating the lateral stability of cortical microtubules. Phosphorylation of the FRA1 tail region by CKL6 inhibited binding to CMUs and consequently reversed the extent of cortical microtubule decoration by CMU1 and CMU2. Genetic experiments demonstrated the significance of this interaction to the growth and reproduction of Arabidopsis thaliana plants. We propose that modulation of CMUs microtubule localization by FRA1 provides a mechanism to control the coordinated deposition of cellulose and matrix polysaccharides.

plant biology↗

bioRxiv2019

Single-cell transcriptomic landscape of cardiac neural crest cell derivatives during embryonic and neonatal development

RationaleCardiac neural crest cells (CNCCs) contribute greatly to cardiovascular development. A thorough understanding of the cell lineages, transcriptomic states and regulatory networks of CNCC derivatives during normal development is essential for deciphering the pathogenesis of CNCC-associated congenital anomalies. However, the transcriptomic landscape of CNCC derivatives during development has not yet been examined at a single-cell resolution.\n\nObjectiveWe sought to systematically characterize the cell lineages, define the developmental chronology and elucidate the transcriptomic dynamics of CNCC derivatives during embryonic and neonatal development.\n\nMethods and ResultsWe performed single-cell transcriptomic sequencing of 34,131 CNCC-derived cells in mouse hearts from eight developmental stages between E10.5 and P7. Through single-cell analyses and single-molecule fluorescence in situ hybridization, we confirmed the presence of CNCC-derived mural cells. Furthermore, we found the transition from CNCC-derived pericytes to microvascular smooth muscle cells, and identified the genes that were significantly regulated during this transition through pseudo-temporal analysis. CNCC-derived neurons first appeared at E10.5, which was earlier than previously recognized. In addition, the CNCC derivatives switched from a proliferative to a quiescent state with the progression of development. Gradual loss of the neural crest molecular signature with development was also observed in the CNCC derivatives. Our data suggested that many CNCC-derivatives had already committed or differentiated to a specific lineage when migrating to the heart. Finally, we characterized some previously unknown subpopulations of CNCC derivatives during development. For example, we found that Penk+ cells, which were mainly localized in outflow tract cushions, were all derived from CNCCs.\n\nConclusionsOur study provides novel insights into the cell lineages, molecular signatures, developmental chronology and state change dynamics of CNCC derivatives during embryonic and neonatal development. Our dataset constitutes a valuable resource that will facilitate future efforts in exploring the role of CNCC derivatives in development and disease.

developmental biology↗

bioRxiv2019

Suppression of membranous LRP5 recycling, WNT/β-catenin signaling, and colorectal tumorigenesis by 15-LOX-1 peroxidation of PI3P_linoleic acid

Aberrant Wnt/{beta}-catenin activation is a major driver of colorectal cancer (CRC), which is typically initiated by APC mutations. Additional modifiable factors beyond APC mutations have been recognized to be important for further potentiation of aberrant {beta}-catenin activation to promote colorectal tumorigenesis. These factors have yet to be clearly identified. Western-type diets are increasingly enriched in linoleic acid (LA). LA-enriched diet however promotes chemically-induced colorectal tumorigenesis in rodent models. Furthermore, the main metabolizing enzyme of LA, 15-lipoxygenase-1 (15-LOX-1), is transcriptionally silenced in CRC. Whether LA and 15-LOX-1 affect Wnt/{beta}-catenin signaling to modulate colorectal tumorigenesis is poorly understood. Herein, we report that high dietary LA promoted colorectal tumorigenesis in mice with intestinally targeted APC mutation (Apc{Delta}580) by upregulating a Wnt receptor, LRP5 expression, and {beta}-catenin activation. 15-LOX-1 transgenic expression in intestinal epithelial cells suppressed LRP5 expression, {beta}-catenin activation and subsequently CRC in these mice. In particular, 15-LOX-1 peroxidation of LA in phosphatidylinositol-3-phosphates (PI3P_LA) into PI3P_13-HODE decreased PI3P binding to SNX17and LRP5, which inhibited LRP5 recycling from endosomes to the plasma membrane, thereby leading to an increase of LRP5 lysosomal degradation. Our findings demonstrate for the first time that 15-LOX-1 metabolism of LA in PI3P to regulate LRP5 membrane abundance is a modifiable factor of Wnt/{beta}-catenin aberrant signaling that could be potentially therapeutically targeted to suppress colorectal tumorigenesis and progression.

cancer biology↗

bioRxiv2019

Modeling and treating GRIN2A developmental and epileptic encephalopathy in mice

NMDA receptors (NMDAR) play crucial roles in excitatory synaptic transmission. Rare variants of GRIN2A, which encodes the GluN2A NMDAR subunit, are associated with several intractable neurodevelopmental disorders, including developmental and epileptic encephalopathy (DEE). A de novo missense variant, p.Ser644Gly (c.1930A>G), was identified in a child with DEE, and Grin2a knockin mice were generated to model and extend understanding of this intractable childhood disease. Homozygous and heterozygous mutant mice exhibit altered hippocampal morphology at two weeks of age, and homozygotes exhibit lethal tonic-clonic seizures in the third week. Heterozygous adult mice display a variety of distinct features, including resistance to electrically induced partial seizures, as well as hyperactivity and repetitive and reduced anxiety behaviors. Multielectrode recordings of mutant neuronal networks reveal hyperexcitability and altered bursting and synchronicity. When expressed in heterologous cells, mutant receptors exhibit enhanced NMDAR agonist potency and slow deactivation following rapid removal of glutamate, as occurs at synapses. Consistent with these observations, NMDAR-mediated synaptic currents in hippocampal slices from mutant mice show a prolonged deactivation time course. Standard antiepileptic drug monotherapy was ineffective in the patient, but combined treatment of NMDAR antagonists with antiepileptic drugs substantially reduced the seizure burden albeit without appreciable developmental improvement. Chronic treatment of homozygous mutant mouse pups with NMDAR antagonists delayed the onset of lethal seizures but did not prevent them. These studies illustrate the power of modeling severe neurodevelopmental seizure disorders using multiple experimental modalities and suggest their extended utility in identifying and evaluating new therapies.

neuroscience↗

bioRxiv2019

Auxin-induced nanoclustering of membrane signaling complexes underlies cell polarity establishment in Arabidopsis

Cell polarity is fundamental to the development of both eukaryotic and prokaryotic organisms, yet the mechanism of its establishment remains poorly understood. Here we show that signal-activated nanoclustering of membrane proteins and a cytoskeleton-based feedback loop provide an important mechanism for the establishment of cell polarity. The phytohormone auxin promoted sterol-dependent nanoclustering of cell surface transmembrane receptor-like kinase 1 (TMK1) to initiate cell polarity during the morphogenesis of Arabidopsis puzzle piece-shaped leaf pavement cells (PC). Auxin-triggered nanoclustering of TMK1 stabilized flotillin-associated ordered nanodomains, which were essential for auxin-mediated formation of ROP6 GTPase nanoclusters that act downstream TMK1 to promote cortical microtubule ordering. Mathematical modeling further demonstrated the essential role of this auxin-mediated stabilization of TMK1 and ROP6 nanoclusters, and predicted the additional requirement of ROP6-dependent cortical microtubules for further stabilization of TMK1-sterol nanodomains and the polarization of PC. This prediction was experimentally validated by genetic and biochemical data. Our studies reveal a new paradigm for polarity establishment: A diffusive signal triggers cell polarization by activating cell surface receptor-mediated lateral segregation of signaling components and a cytoskeleton-mediated positive feedback loop of nanodomain stabilization.\n\nHighlightsO_LISterols are required for cell polarity in Arabidopsis leaf epidermal cells\nC_LIO_LIAuxin promotes lipid ordering and polar distribution of ordered lipid nanodomains at the plasma membrane (PM)\nC_LIO_LIAuxin stabilizes sterol-dependent nanoclustering of transmembrane kinase (TMK1), a PM auxin signal transducer\nC_LIO_LIAuxin-induced TMK1 nanoclustering is required but insufficient for cell polarization\nC_LIO_LIMicrotubule-based feedback stabilization of the auxin-induced TMK1 nanodomains can generate cell polarity\nC_LI

cell biology↗

bioRxiv2019

Genetic and molecular mechanism for distinct clinical phenotypes conveyed by allelic truncating mutations implicated in FBN1

The molecular and genetic mechanisms by which different single nucleotide variant (SNV) alleles in specific genes, or at the same genetic locus, bring about distinct disease phenotypes often remain unclear. Allelic truncating mutations of fibrillin-1(FBN1) cause either classical Marfan syndrome (MFS) or a more severe phenotype associated with Marfanoid-progeroid-lipodystrophy syndrome (MPLS). A total of three Marfan syndrome/Marfanoid patients (2 singletons and 1 parent-offspring trio) were recruited. Targeted next-generation sequencing was performed on all the participants. We analyzed the molecular diagnosis, patient clinical features, and the potential molecular mechanism involved in the MPLS subject in our cohort. We investigated a small cohort, consisting of two classical MFS and one MPLS patient from China, whose clinical presentation included scoliosis potentially requiring surgical intervention. We provide evidence that most nonsense and frameshift mutations lead to FBN1 null alleles due to mutant mRNA transcript degradation. In contrast, the more severe disease phenotype, MPLS, is caused by mutant mRNAs that are predicted to escape the nonsense mediated decay (NMD) surveillance pathway, making a mutant protein that exerts a dominant negative interference effect to FBN1 thus generating a gain-of-function (GoF) rather than a loss-of-function (LoF) allele as in MFS. Overall, we provide direct evidence that a dominant negative interaction of FBN1 potentially explains the distinct clinical phenotype in MPLS patients through genetic and functional analysis of the first Chinese patient with MPLS. Moreover, our study expands the mutation spectrum of FBN1 and highlights the potential molecular mechanism for MPLS patients.

genetics↗

bioRxiv2019

Comparative investigation into formycin A and pyrazofurin A biosynthesis reveals branch pathways for the construction of C-nucleoside scaffolds

Formycin A (FOR-A) and pyrazofurin A (PRF-A) are purine-related C-nucleoside antibiotics, in which ribose and a pyrazole-derived base are linked by a C-glycosidic bond, however, the logic underlying the biosynthesis of these molecules has remained largely unexplored. Here, we report the discovery of the pathways for FOR-A and PRF-A biosynthesis from diverse actinobacteria, and demonstrate that their biosynthesis is initiated by a lysine N6-monooxygenase. Moreover, we show that the forT and prfE (individually related to FOR-A and PRF-A biosynthesis) mutants are correspondingly capable of accumulating the unexpected pyrazole-related intermediates, compound 11 and 9a. We also decipher the enzymatic basis of ForT/PrfE for the C-glycosidic bond formation in FOR-A/PRF-A biosynthesis. To our knowledge, ForT/PrfE represents the first example of {beta}-RFA-P ({beta}-ribofuranosyl-aminobenzene 5-phosphate) synthase-like enzymes governing C-nucleoside scaffold construction in natural product biosynthesis. These data establish a foundation for combinatorial biosynthesis of related purine nucleoside antibiotics, and also open the way for target-directed genome mining of PRF-A/FOR-A related antibiotics.\n\nIMPORTANCEFormycin A (FOR-A) and pyrazofurin A (PRF-A) are well known for their unusual chemical structures and remarkable biological activities. Actually, deciphering FOR-A/PRF-A biosynthesis will not only expand biochemical repertoire for novel enzymatic reactions, but also permit the target-oriented genome mining of FOR-A/PRF-A related C-nucleoside antibiotics.

biochemistry↗

bioRxiv2019

Spatial and temporal control of CRISPR/Cas9-mediated gene editing delivered via a light-triggered liposome system

The CRISPR-Cas9 and related systems offer a unique genome editing tool allowing facile and efficient introduction of heritable and locus-specific sequence modifications in the genome. Despite its molecular precision, temporal and spatial control of gene editing with CRISPR-Cas9 system is very limited. We developed a light-sensitive liposome delivery system that offers a high degree of spatial and temporal control of gene editing with CRISPR/Cas9 system. We demonstrated its high transfection efficiency, by assessing the targeted knockout of eGFP gene in human HEK293 cells (52.8% knockout). We further validated our results at a single-cell resolution using an in vivo eGFP reporter system in zebrafish (77% knockout). To the best of our knowledge we reported the first proof-of-concept of spatio-temporal control of CRISPR/Cas9 by using light-triggered liposomes in both in vitro and in vivo environment.

bioengineering↗

bioRxiv2019

Identification of porcine RUNX1 as an LPS-dependent gene expression regulator in PBMCs by Super deepSAGE sequencing of multiple tissues

Genome-wide identification of gene expression regulators may facilitate our understanding of the transcriptome constructed by gene expression profiling experiment. These regulators may be selected as targets for genetic manipulations in farm animals. In this study, we developed a gene expression profile of 76,000+ unique transcripts for 224 porcine samples from 28 normal tissues collected from 32 animals using Super deepSAGE (serial analysis of gene expression by deep sequencing) technology. Excellent sequencing depth has been achieved for each multiplexed library, and principal component analysis showed that duplicated samples from the same tissues cluster together, demonstrating the high quality of the Super deepSAGE data. Comparison with previous research indicated that our results not only have excellent reproducibility but also have greatly extended the coverage of the sample types as well as the number of genes. Clustering analysis discovered ten groups of genes showing distinct expression patterns among those samples. Binding motif over representative analysis identified 41 regulators responsible for the regulation of these gene clusters. Finally, we demonstrate a potential application of this dataset to infectious and immune research by identifying an LPS-dependent transcription factor, runt-related transcription factor 1 (RUNX1), in peripheral blood mononuclear cells (PBMCs). The selected genes are specifically responsible for the transcription of toll-like receptor 2 (TLR2), lymphocyte-specific protein tyrosine kinase (LCK), vav1 oncogene (VAV1), and other 32 genes. These genes belong to the T and B cell signaling pathways, making them potential novel targets for the diagnostic and therapy of bacterial infections and other immune disorders.

genomics↗