Biology subjects

Shi, L.

Publications and source records attributed to Shi, L..

At least 253 records · Page 14Linked to original sources

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

Bimodal Regulation of the PRC2 Complex by USP7 Underlies Melanomagenesis

Although overexpression of EZH2, a catalytic subunit of the polycomb repressive complex 2 (PRC2), is an eminent feature of various cancers, the regulation of its abundance and function remains insufficiently understood. We report here that the PRC2 complex is physically associated with ubiquitin-specific protease USP7 in melanoma cells where USP7 acts to deubiquitinate and stabilize EZH2. Interestingly, we found that USP7-catalyzed H2BK120 deubiquitination is a prerequisite for chromatin loading of PRC2 thus H3K27 trimethylation. Genome-wide analysis of the transcriptional targets of the USP7/PRC2 complex identified a cohort of genes including FOXO1 that are involved in cell growth and proliferation. We demonstrated that the USP7/PRC2 complex drives melanoma cell proliferation and tumorigenesis in vitro and in vivo. We showed that the expression of both USP7 and EZH2 elevates during melanoma progression, corresponding to a diminished FOXO1 expression, and the level of the expression of USP7 and EZH2 strongly correlates with histological grades and prognosis of melanoma patients. These results reveal a dual role for USP7 in the regulation of the abundance and function of EZH2, supporting the pursuit of USP7 as a therapeutic target for melanoma.

cell biology↗

bioRxiv2019

Distinct antagonist-bound inactive states underlie the divergence in the structures of the dopamine D2 and D3 receptors

Understanding how crystal structures reflect the range of possible G protein-coupled receptor (GPCR) states is critical for rational drug discovery (RDD). Combining computational simulations with mutagenesis and binding studies, we find that the structure of the dopamine D2 receptor (D2R)/risperidone complex captures an inactive receptor conformation that accommodates some but not all antagonist scaffolds. Indeed, we find that eticlopride binds D2R in a configuration very similar to that seen in the D3R structure, in a pose that is incompatible with the D2R/risperidone structure. Moreover, our simulations reveal that extracellular loops 1 and 2 (EL1 and EL2) are highly dynamic, with spontaneous transitions of EL2 from the helical conformation in the D2R/risperidone structure to an extended conformation similar to that in the D3R/eticlopride structure. Our results highlight previously unappreciated conformational diversity and dynamics in the inactive state of a GPCR with potential functional implications. These findings are also of paramount importance for RDD as limiting a virtual screen to one state will miss relevant ligands.

biophysics↗

bioRxiv2019

Deconvolute individual genomes from metagenome sequences through short read clustering

MotivationMetagenome assembly from short next-generation sequencing data is a challenging process due to its large scale and computational complexity. Clustering short reads before assembly offers a unique opportunity for parallel downstream assembly of genomes with individualized optimization. However, current read clustering methods suffer either false negative (under-clustering) or false positive (over-clustering) problems.\n\nResultsBased on a previously developed scalable read clustering method on Apache Spark, SpaRC, that has very low false positives, here we extended its capability by adding a new method to further cluster small clusters. This method exploits statistics derived from multiple samples in a dataset to reduce the under-clustering problem. Using a synthetic dataset from mouse gut microbiomes we show that this method has the potential to cluster almost all of the reads from genomes with sufficient sequencing coverage. We also explored several clustering parameters that deferentially affect genomes with various sequencing coverage.\n\nAvailabilityhttps://bitbucket.org/berkeleylab/jgi-sparc/.\n\nContactzhongwang@lbl.gov

bioinformatics↗

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

Kindlin-3 Mutation in Mesenchymal Stem Cells Results in Enhanced Chondrogenesis

Identifying patient mutations driving skeletal development disorders has driven our understanding of bone development. Integrin adhesion deficiency disease is caused by a Kindlin-3 (fermitin family member 3) mutation and its inactivation results in bleeding disorders and osteopenia. In this study, we uncover a role for Kindlin-3 in the differentiation of bone marrow mesenchymal stem cells (BMSCs) down the chondrogenic lineage. Kindlin-3 expression increased with chondrogenic differentiation similar to RUNX2. BMSCs isolated from a Kindlin-3 deficient patient expressed chondrocyte markers including SOX9 under basal conditions, which were further enhanced with chondrogenic differentiation. Rescue of integrin activation by a constitutively activated {beta}3 integrin construct increased adhesion to multiple extracellular matrices and reduced SOX9 expression to basal levels. Growth plates from mice expressing a mutated Kindlin-3 with the integrin binding site ablated demonstrated alterations in chondrocyte maturation similar to that seen with the human Kindlin-3 deficient BMSCs. These findings suggest that Kindlin-3 expression mirrors RUNX2 during chondrogenesis.\n\nSUMMARYThis study by Kerr et al. describes a new role for Kindlin-3 in controlling early chondrocyte differentiation from mesenchymal stem cells and later hypertrophic differentiation of chondrocytes.

cell biology↗

bioRxiv2019

Comparative performance of the BGI and Illumina sequencing technology for single-cell RNA-sequencing

The libraries generated by high-throughput single cell RNA-sequencing platforms such as the Chromium from 10X Genomics require considerable amounts of sequencing, typically due to the large number of cells. The ability to use this data to address biological questions is directly impacted by the quality of the sequence data. Here we have compared the performance of the Illumina NextSeq 500 and NovaSeq 6000 against the BGI MGISEQ-2000 platform using identical Single Cell 3 libraries consisting of over 70,000 cells. Our results demonstrate a highly comparable performance between the NovaSeq 6000 and MGISEQ-2000 in sequencing quality, and cell, UMI, and gene detection. However, compared with the NextSeq 500, the MGISEQ-2000 platform performs consistently better, identifying more cells, genes, and UMIs at equalised read depth. We were able to call an additional 1,065,659 SNPs from sequence data generated by the BGI platform, enabling an additional 14% of cells to be assigned to the correct donor from a multiplexed library. However, both the NextSeq 500 and MGISEQ-2000 detected similar frequencies of gRNAs from a pooled CRISPR single cell screen. Our study provides a benchmark for high capacity sequencing platforms applied to high-throughput single cell RNA-seq libraries.

genomics↗

bioRxiv2019

Toll-like receptor 2 stimulation augments esophageal epithelial barrier integrity

BackgroundA key concept of the hygiene hypothesis is that the microbiome modulates both epithelial barrier integrity as well as host immune responses. Defective expression of tight junction complex proteins alters this homeostatic process, and plays a role in atopic disorders including eosinophilic esophagitis. We tested the hypothesis that Toll-like receptor 2 (TLR2) stimulation improves esophageal barrier function in a cell-intrinsic manner by upregulation of TJ-protein expression using an in vitro model of human epithelium. MethodsPattern recognition receptor expression was assessed in esophageal epithelial cells from patients with EoE and non-EoE control patients. Functional consequences of TLR2 stimulation were investigated using human esophageal EPC2-hTERT cells in the three-dimensional air-liquid interface culture (ALI) model to evaluate transepithelial electrical resistance (TEER) and FITC-Dextran permeability. Characterization of TLR2-stimulated ALI cultures was performed by histology, immunohistochemistry, western blotting and chromatin immunoprecipitation. ResultsTLR2 stimulation increased TEER (1.28 to 1.31-fold) and decreased paracellular permeability to FITC-Dextran. Notably, TLR2 stimulation-induced increases in TEER were abolished by treatment with anti-TLR2 blocking antibody. Tight junction complex proteins claudin 1 and zonula occludens 1 were increased following TLR2 stimulation, and chromatin immunoprecipitation analysis demonstrated significant increase in histone 4 acetyl binding at the CLDN1 enhancer and promoter following zymosan treatment, implying the occurrence of durable chromatin changes in the esophageal epithelium. ConclusionsOur findings reveal that the TLR2 pathway may play a regulatory role as a mechanism that maintains epithelial barrier homeostasis in the esophagus.

immunology↗

bioRxiv2019

Changes in fungal communities across a forest disturbance gradient

Deforestation has a substantial impact on above ground biodiversity, but the response of below ground soil fungi remains poorly understood. In a tropical montane rainforest in southwestern China, plots were established along a forest degradation gradient ranging from mature and regenerated forests to open land to examine the impacts of forest degradation and deforestation on ecosystem diversity and function. Here, we evaluate the changes in below ground fungal diversity and community composition using a metabarcoding approach. Soil saprotrophic fungal richness declined with increasing forest disturbance. For example, Penicillium spp. (Phosphorus (P) solubilizing fungi) dominated in mature forest, but were less abundant in regenerating forest and showed the lowest abundance in open land sites. Conversely, the abundance of facultative pathogenic fungi increased along the disturbance gradient. The decline in soil saprophytic fungi may be a direct result of forest disturbance or it may be associated with increased availability of soil phosphorus indirectly through an increase in soil pH. The increase in abundance of facultative pathogenic fungi may be related to reduce competition with saprotrophic fungi, changes in microclimate or increased spore rain. These results demonstrate a loss of dominant P solubilizing saprotrophic fungi along the disturbance gradient, indicated a change from soil P limitation in mature tropical forest to soil C limitation in deforested sites. The increased prevalence of pathogenic fungi may inhibit plant succession following deforestation. Overall, this research demonstrates that soil fungi can be used as a sensitive indicator for soil health to evaluate the consequences of forest disturbance.\n\nImportanceThe soil fungal functional group changes in response to forest disturbance indicated a close interaction between the above-ground plant community and the below-ground soil biological community. Soil saprotrophic fungi declined in relative abundance with increasing forest disturbance. At the same time, the relative abundance of facultative pathogenic fungi increased. The loss of saprotrophic fungal richness and abundance may have been a direct result of forest disturbance or an indirect result of changes in soil pH and soil P. Furthermore, the dominant P solubilizing saprotrophic fungi was replaced by diverse facultative pathogenic fungi, which have weaker C decomposition ability. These changes potentially indicate a shift from soil phosphate limitation to carbon limitation following deforestation. This study suggests that changes in fungal functional group composition can be used as an indicator of the effects of forest disturbance on soil carbon and nutrients.

ecology↗

bioRxiv2018

Chemogenetic ligands for translational neurotheranostics

Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) are a popular chemogenetic technology for manipulation of neuronal activity in uninstrumented awake animals with potential for precision medicine-based clinical theranostics. DREADD ligands developed to date are not appropriate for such translational applications. The prototypical DREADD agonist clozapine N-oxide (CNO) lacks brain entry and converts to clozapine. The second-generation DREADD agonist, Compound 21 (C21), was developed to overcome these limitations. We found that C21 has low brain penetrance, weak affinity, and low in vivo DREADD occupancy. To address these drawbacks, we developed two new DREADD agonists, JHU37152 and JHU37160, and the first dedicated positron emission tomography (PET) DREADD radiotracer, [18F]JHU37107. JHU37152 and JHU37160 exhibit high in vivo DREADD potency. [18F]JHU37107 combined with PET allows for DREADD detection in locally-targeted neurons and at their long-range projections, enabling for the first time, noninvasive and longitudinal neuronal projection mapping and potential for neurotheranostic applications.

neuroscience↗

bioRxiv2018

eIF2B activator prevents neurological defects caused by a chronic Integrated Stress Response

The Integrated Stress Response (ISR) attenuates the rate of protein synthesis while inducing expression of stress proteins in cells. Various insults activate kinases that phosphorylate the GTPase eIF2 leading to inhibition of its exchange factor eIF2B. Vanishing White Matter (VWM) is a neurological disease caused by eIF2B mutations that, like phosphorylated eIF2, reduce its activity. We show that introduction of a human VWM mutation into mice leads to persistent ISR induction in the central nervous system. ISR activation precedes myelin loss and development of motor deficits. Remarkably, long-term treatment with a novel eIF2B activator, 2BAct, prevents all measures of pathology and normalizes the transcriptome and proteome of VWM mice. 2BAct stimulates the remaining activity of mutant eIF2B complex in vivo, abrogating the maladaptive stress response. Thus, 2BAct-like molecules may provide a promising therapeutic approach for VWM and provide relief from chronic ISR induction in a variety of other disease contexts.

neuroscience↗

bioRxiv2018

Intraflagellar transport protein 74 is essential for mouse spermatogenesis and male fertility by regulating axonemal microtubule assembly in mice

IFT74 is a component of the core intraflagellar transport (IFT) complex, a bidirectional movement of large particles along the axoneme microtubules for cilia formation. In this study, we investigated its role in sperm flagella formation and discovered that mice deficiency in IFT74 in male germ cells were infertile associated with low sperm counts and immotile sperm. The few developed spermatozoa displayed misshaped heads and short tails. Transmission electron microscopy revealed abnormal flagellar axoneme in the seminiferous tubules where sperm are made. Clusters of unassembled microtubules were present in the spermatids. Testicular expression levels of IFT27, IFT57, IFT81, IFT88 and IFT140 were significantly reduced in the mutant mice, with the exception of IFT20 and IFT25. The levels of ODF2 and SPAG16L proteins were also not changed. However, the processed AKAP4 protein, a major component of the fibrous sheath, a unique structure of sperm tail, was significantly reduced. Our study demonstrates that IFT74 is essential for mouse sperm formation, probably through assembly of the core axoneme and fibrous sheath, and highlights a potential genetic factor (IFT74) that contributes to human infertility in men.

developmental biology↗

bioRxiv2018

A familial case of Syndactyly type IV due to a novel duplication of ~222.23 kb covering exons 2-17 of the LMBR1 gene: a case report

Syndactyly is one of the most frequent hereditary limb malformations with clinical and genetical complexity. Autosomal dominant Syndactyly type IV (SD4) is a very rare form of syndactyly, occurring as a result of heterozygous mutation in an SHH regulatory element (ZRS) that resides in intron 5 of the LMBR1 gene on chromosome 7q36.3. The SD4 is characterized by complete cutaneous syndactyly of all fingers, cup-shaped hands due to flexion of the fingers and accompanied by polydactyly. Here, we firstly reported a big Chinese family, manifesting cup-shaped hands consistent with SD4 and intrafamilial heterogeneity in clinical phenotype of tibial and fibulal shortening, triphalangeal thumb-polysyndactyly syndrome (TPTPS). Genetically, we identified a novel duplication of [~]222.23 kb covering exons 2-17 of the LMBR1 gene in this family by next generation sequencing. This case expands our new clinical understanding of SD4 phenotype.

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

Lsm12 mediates Polη deubiquitination to help Saccharomyces cerevisiae resist oxidative stress

In Saccharomyces cerevisiae, the Y-family DNA polymerase {eta} (Pol{eta}) regulates genome stability in response to different forms of environmental stress by translesion DNA synthesis. To elucidate the role of Pol{eta} in oxidative stress-induced DNA damage, we deleted or overexpressed the corresponding gene RAD30, and used transcriptome analysis to screen the potential genes associated with RAD30 to respond to DNA damage. Under 2 mM H2O2, deletion of RAD30 resulted in a 2.2-fold decrease in survival and a 2.8-fold increase in DNA damage, whereas overexpression of RAD30 increased survival and decreased DNA damage by 1.2- and 1.4-fold, respectively, compared with that of the wild-type strain. Transcriptome and phenotypic analysis identified Lsm12 as a main factor involved in oxidative stress-induced DNA damage. Deleting LSM12 caused growth defects while its overexpression enhanced cell growth under 2 mM H2O2. This effect was due to the physical interaction of Lsm12 with the UBZ domain of Pol{eta} to enhance Pol{eta} deubiquitination through Ubp3, and consequently promote Pol{eta} recruitment. Overall, these findings demonstrate that Lsm12 is a novel regulator mediating Pol{eta} deubiquitination to promote its recruitment under oxidative stress. Furthermore, this study provides a potential strategy to maintain the genome stability of industrial strains during fermentation.\n\nIMPORTANCEPol{eta} was shown to be critical for cell growth in the yeast Saccharomyces cerevisiae, and deletion of its corresponding gene RAD30 caused a severe growth defect under exposure to oxidative stress with 2 mM H2O2. Furthermore, we found that Lsm12 physically interacts with Pol{eta} and promotes Pol{eta} deubiquitination and recruitment. Overall, these findings indicate Lsm12 as a novel regulator mediating Pol{eta} deubiquitination that regulates its recruitment in response to DNA damage induced by oxidative stress.

cell biology↗

bioRxiv2018

CTC Marker CD117/c-kit Represents a Prostate Cancer Stem-Like Subpopulation Driving Progression, Migration, and TKI Resistance

Cancer stem-like cells (CSCs) are associated with cancer progression, metastasis, and recurrence, and may also represent a subset of circulating tumor cells (CTCs). In our prior study, CTCs in advanced prostate cancer patients were found to express CD117/c-kit in a liquid biopsy. Whether CD117 expression played an active or passive role in the aggressiveness and migration of these CTCs remained an open question. In this study, we show that CD117 expression in prostate cancer patients is associated with decreased overall and progression-free survival and that activation and phosphorylation of CD117 increases in prostate cancer patients with higher Gleason grades. To determine how CD117 expression and activation by its ligand stem cell factor (SCF, kit ligand, steel factor) alter prostate cancer aggressiveness, we used LNCaP-C4-2 and PC3-mm human prostate cancer cells, which contain a CD117+ subpopulation. We demonstrate that CD117+ cells display increased proliferation and migration. In prostaspheres, CD117 expression enhances sphere formation. In both 2D and 3D cultures, stemness marker gene expression is higher in CD117+ cells. Using xenograft limiting dilution assays and serial tumor initiation assays, we show that CD117+ cells represent a CSC population. Combined, these data indicate that CD117 expression potentially promotes tumor initiation and metastasis. Further, in cell lines, CD117 activation by SCF promotes faster proliferation and invasiveness, while blocking CD117 activation with tyrosine kinase inhibitors (TKIs) decreased progression in a context-dependent manner. We demonstrate that CD117 expression and activation drives prostate cancer aggressiveness through the CSC phenotype and TKI resistance.

cancer biology↗

bioRxiv2018

Optical Imaging of Metabolic Dynamics in Animals

Direct visualization of metabolic dynamics in living tissues with high spatial and temporal resolution is essential to understanding many biological processes. Here we introduce a platform that combines deuterium oxide (D2O) probing with stimulated Raman scattering microscopy (DO-SRS) to image in situ metabolic activities. Enzymatic incorporation of D2O-derived deuterium into macromolecules generates carbon-deuterium (C-D) bonds, which track biosynthesis in tissues and can be imaged by SRS in situ. Within the broad vibrational spectra of C-D bonds, we discovered lipid-, protein-, and DNA-specific Raman shifts and developed spectral unmixing methods to obtain C-D signals with macromolecular selectivity. DO-SRS enabled us to probe de novo lipogenesis in animals, image protein biosynthesis without tissue bias, and simultaneously visualize lipid and protein metabolism and reveal their different dynamics. DO-SRS, being noninvasive, universally applicable, and cost-effective, can be adapted to a broad range of biological systems to study development, tissue homeostasis, aging, and tumor heterogeneity.

biophysics↗

bioRxiv2018

Inducible expression of immediate early genes is regulated through dynamic chromatin association by NF45/ILF2 and NF90/ILF3

Immediate early gene (IEG) transcription is rapidly activated by diverse stimuli without requiring new protein synthesis. This transcriptional regulation is assumed to involve constitutively expressed nuclear factors that are targets of signaling cascades initiated at the cell membrane. NF45 and its heterodimeric partner NF90 are chromatin-interacting proteins that are constitutively expressed and localized predominantly in the nucleus. Previously, NF90 chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) in K562 erythroleukemia cells revealed its enriched association with chromatin at active promoters and strong enhancers. NF90 specifically occupied the promoters of IEGs. Here, ChIP in serum-starved HEK293 cells demonstrated that NF45 and NF90 pre-exist and specifically occupy the promoters of IEG transcription factors EGR1, FOS and JUN. Cellular stimulation with phorbol myristyl acetate increased NF90 occupancy, while decreasing NF45 occupancy at promoters of EGR1, FOS and JUN. In HEK293 cells stably transfected with doxycycline-inducible shRNA vectors targeting NF90 or NF45, doxycycline-mediated knockdown of NF90 or NF45 attenuated the inducible expression of EGR1, FOS, and JUN at the levels of mRNA and protein. NF90 and NF45 operate as constitutively-expressed transcriptional regulators of IEGs. Dynamic chromatin association of NF45 and NF90 at IEG promoters are observed upon stimulation, and NF45 and NF90 contribute to inducible expression of IEGs. NF45 and NF90 operate as chromatin regulators of the immediate early response.

cell biology↗