Biology subjects

Xu, X.

Publications and source records attributed to Xu, X..

At least 757 records · Page 42Linked to original sources

bioRxiv2018

Cancer therapy with a CRISPR-assistant telomerase-activating gene expression system

Telomerase is silent in most normal somatic cells while active in 90% of tumor cells. Various telomerase activity inhibitors have been developed to treat cancer but all failed due to side effects. Here we acted oppositely to develop a cancer therapy named telomerase-activating gene expression (Tage) by utilizing the telomerase activity in tumor cells. By using CRISPR/Cas9 functions, the Tage system can effectively kill various cancer cells, including HepG2, HeLa, PANC-1, MDA-MB-453, A549, HT-29, SKOV-3, Hepa1-6, and RAW264.7, without effecting normal cells. By using homothallic switching endonuclease and adeno-associated virus, the Tage system realizes its in vivo application. The virus-loaded Tage system can significantly and specifically kill the cancer cells in mice by intravenous drug administration without side effects or toxicity.\n\nOne Sentence Summary: Killing cancer cells in body with a gene therapy missile detonated by telomerase.

cancer biology↗

bioRxiv2018

Engineering targeted deletions in the mitochondrial genome

Summary ParagraphMitochondria are a network of critical intracellular organelles with diverse functions ranging from energy production to cell signaling. The mitochondrial genome (mtDNA) consists of 37 genes that support oxidative phosphorylation and are prone to dysfunction that can lead to currently untreatable diseases. Further characterization of mtDNA gene function and creation of more accurate models of human disease will require the ability to engineer precise genomic sequence modifications. To date, mtDNA has been inaccessible to direct modification using traditional genome engineering tools due to unique DNA repair contexts in mitochondria1. Here, we report a new DNA modification process using sequence-specific transcription activator-like effector (TALE) proteins to manipulate mtDNA in vivo and in vitro for reverse genetics applications. First, we show mtDNA deletions can be induced in Danio rerio (zebrafish) using site-directed mitoTALE-nickases (mito-nickases). Using this approach, the protein-encoding mtDNA gene nd4 was deleted in injected zebrafish embryos. Furthermore, this DNA engineering system recreated a large deletion spanning from nd5 to atp8, which is commonly found in human diseases like Kearns-Sayre syndrome (KSS) and Pearson syndrome. Enrichment of mtDNA-deleted genomes was achieved using targeted mitoTALE-nucleases (mitoTALENs) by co-delivering both mito-nickases and mitoTALENs into zebrafish embryos. This combined approach yielded deletions in over 90% of injected animals, which were maintained through adulthood in various tissues. Subsequently, we confirmed that large, targeted deletions could be induced with this approach in human cells. In addition, we show that, when provided with a single nick on the mtDNA light strand, the binding of a terminal TALE protein alone at the intended recombination site is sufficient for deletion induction. This \"block and nick\" approach yielded engineered mitochondrial molecules with single nucleotide precision using two different targeted deletion sites. This precise seeding method to engineer mtDNA variants is a critical step for the exploration of mtDNA function and for creating new cellular and animal models of mitochondrial disease.

molecular biology↗

bioRxiv2018

Dectin-3 recognizes cryptococcal glucuronoxylomannan to initiate host defense against cryptococcosis

Cryptococcus neoformans and Cryptococcus gattii cause life-threatening meningoencephalitis and pneumonia in immunosuppressed and immunocompetent individuals. Given the structural differences of major polysaccharide glucuronoxylomannan (GXM) between C. neoformans and C. gattii, it remains unclear that how innate immune system recognizes GXM. Here, we report that C-type lectin receptor Dectin-3 (MCL encoded by Clec4d) is a direct receptor for GXMs from C. neoformans serotype AD (C.n-AD) and C. gattii serotype B (C.g-B). GXMs from C.n-AD and C.g-B activated both NF-{kappa}B and ERK pathways to induce the pro-inflammatory cytokine production, whereas it was completely abolished due to deficiency of Dectin-3 or its downstream adaptor protein CARD9. Upon pulmonary C.n-AD and C.g-B infection, Dectin-3- and CARD9-deficient mice were highly susceptible and showed augmented lung injury due to impairment of alveolar macrophage accumulation and killing activities. These results demonstrate that Dectin-3 contributes to host immunity against Cryptococcus infection through selectively recognizingGXM.

immunology↗

bioRxiv2018

A catalog of microbial genes from the bovine rumen reveals the determinants of herbivory

BackgroundThe rumen microbiota provides essential services to its host and, through its role in ruminant production, contributes to human nutrition and food security. A thorough knowledge of the genetic potential of rumen microbes will provide opportunities for improving the sustainability of ruminant production systems. The availability of gene reference catalogs from gut microbiomes has advanced the understanding of the role of the microbiota in health and disease in humans and other mammals. In this work, we established a catalog of reference prokaryote genes from the bovine rumen.\n\nResultsUsing deep metagenome sequencing we identified 13,825,880 non-redundant prokaryote genes from the bovine rumen. Compared to human, pig and mouse gut metagenome catalogs, the rumen is larger and richer in functions and microbial species associated with the degradation of plant cell wall material and production of methane. Genes encoding enzymes catalyzing the breakdown of plant polysaccharides showed a particularly high richness that is otherwise impossible to infer from available genomes or shallow metagenomics sequencing. The catalog expands by several folds the dataset of carbohydrate-degrading enzymes described in the rumen. Using an independent dataset from a group of 77 cattle fed 4 common dietary regimes, we found that only <0.1% of genes were shared by all animals, which contrast with a large overlap for functions, i.e. 63% for KEGG functions. Different diets induced differences in the relative abundance rather than the presence or absence of genes explaining the great adaptability of cattle to rapidly adjust to dietary changes.\n\nConclusionsThese data bring new insights into functions, carbohydrate-degrading enzymes and microbes of the rumen that is complementing the available information on microbial genomes. The catalog is a significant biological resource enabling deeper understanding of phenotypes and biological processes and will be expanded as new data is made available.

microbiology↗

bioRxiv2018

The critical role of ASD-related gene CNTNAP3 in regulating synaptic development and social behavior in mice

Accumulated genetic evidences indicate that the contactin associated protein-like (CNTNAP) family is implicated in autism spectrum disorders (ASD). In this study, we identified genetic mutations in the CNTNAP3 gene from Chinese Han ASD cohorts and Simons Simplex Collections. We found that CNTNAP3 interacted with synaptic adhesion proteins Neuroligin1 and Neuroligin2, as well as scaffolding proteins PSD95 and Gephyrin. Significantly, we found that CNTNAP3 played an opposite role in controlling the development of excitatory and inhibitory synapses in vitro and in vivo, in which ASD mutants exhibited loss-of-function effects. In this study, we showed that Cntnap3-null mice exhibited deficits in social interaction, spatial learning and prominent repetitive behaviors. These evidences elucidate the pivotal role of CNTNAP3 in synapse development and social behaviors, providing the mechanistic insights for ASD.

neuroscience↗

bioRxiv2018

Xist Intron 1 Repression by TALE Transcriptional Factor Improves Somatic Cell Reprogamming in Mice

Xist is the master regulator of X chromosome inactivation (XCI). In order to further understand the Xist locus in reprogramming of somatic cells to induced pluripotent stem cells (iPSCs) and in somatic cell nuclear transfer (SCNT), we tested transcription-factor-like effectors (TALE)-based designer transcriptional factors (dTFs), which were specific to numerous regions at the Xist locus. We report that the selected dTF repressor 6 (R6) binding the intron 1 of Xist, which did not affect Xist expression in mouse embryonic fibroblasts (MEFs), substantially improved the iPSC generation and the SCNT preimplantation embryo development. Conversely, the dTF activator targeting the same genomic region of R6 decreased iPSC formation, and blocked SCNT-embryo development. These results thus uncover the critical requirement for the Xist locus in epigenetic resetting, which is not directly related to Xist transcription. This may provide a unique route to improving the reprogramming.

developmental biology↗

bioRxiv2018

Microglia inhibition rescues developmental hypofrontality in a mouse model of mental illness

Cognitive deficits, core features of mental illness, largely result from dysfunction of prefrontal-hippocampal networks. This dysfunction emerges already during early development, before a detectable behavioral readout, yet the cellular elements controlling the abnormal maturation are still unknown. Combining in vivo electrophysiology and optogenetics with neuroanatomy and pharmacology in neonatal mice mimicking the dual genetic - environmental etiology of psychiatric disorders, we identified pyramidal neurons in layer II/III of the prefrontal cortex as key elements causing disorganized oscillatory entrainment of local circuits in beta-gamma frequencies. Their abnormal firing rate and timing result from sparser dendritic arborization and lower spine density. Pharmacological modulation of aberrantly hyper-mature microglia rescues morphological, synaptic and functional neuronal deficits and restores the early circuit function. Elucidation of the cellular substrate of developmental miswiring related to later cognitive deficits opens new perspectives for identification of neurobiological targets, amenable to therapies.\n\nHighlightsO_LIMice mimicking the etiology of mental illness have dysregulated prefrontal network\nC_LIO_LIStructural and synaptic deficits cause abnormal rate and timing of pyramidal firing\nC_LIO_LIWeaker activation of prefrontal circuits results from deficits of pyramidal neurons\nC_LIO_LIRescue of microglial function restores developing prefrontal circuits\nC_LI

neuroscience↗

bioRxiv2018

SlARF10, an auxin response factor, is required for chlorophyll and sugar accumulation during tomato fruit development

Tomato green fruits photosynthesis contributes to fruit growth and carbon economy. Tomato auxin response factor 10 (SlARF10) is one of the members of ARF family. Our results showed that SlARF10 locates in the nucleus and has no transcriptional activity. SlARF10 was expressed in various tomato tissues, but highly expressed in green fruit. Up-regulation of SlARF10 produced dark green phenotype of fruits, whereas down-regulation of SlARF10 had light green phenotype. Autofluorescence and chlorophyll content analysis confirmed the phenotypes, which indicated that SlARF10 plays an important role in chlorophyll accumulation in tomato fruits. Up-regulation of SlARF10 increased the photochemical potential in tomato leaves and fruits. Furthermore, the SlARF10 up-regulating lines displayed improved accumulation of starch in fruits, whereas SlARF10 suppressed lines had inhibited starch accumulation. Up-regulation of SlARF10 increased the expression of AGPases, the starch biosynthesis genes. SlARF10 up-regulating lines had increased accumulation of SlGLK1 and SlGLK2 transcripts in fruits. The promoter sequence of SlGLK1 gene had two conserved ARF binding sites. SlARF10 may regulate the expression of SlGLK1, thus controlling chlorophyll accumulation, photosynthesis rates and sugars synthesis in fruits. Our study provided more insight on the link between auxin signaling, chloroplastic activity and sugar metabolism during the development of tomato fruits.\n\nAbbreviations\n\nHighlightSlARF10 played an important role in the chlorophyll accumulation and photosynthesis in tomato fruits. SlARF10 was involved in starch accumulation by controlling the expression of starch synthesis related enzyme genes. SlARF10 may regulate the expression of SlGLK1, thus controlling chlorophyll accumulation, photosynthesis rates and sugars synthesis in tomato fruits.

plant biology↗

bioRxiv2018

The actin cytoskeleton of the mouse sperm flagellum is organized in a helical structure

Conception of a new mammalian organism is determined by the fusion of a sperm cell with an oocyte during fertilization. Motility is one of the features of sperm that allows them to succeed in fertilization, and their flagellum is essential for this function. Longitudinally, the flagellum divides into the midpiece, the principal piece, and the end piece. A precise cytoskeletal architecture of the sperm tail is key for the acquisition of fertilization competence. It has been proposed that the actin cytoskeleton plays essential roles in the regulation of sperm motility, however, actin organization in sperm remains elusive. In the present work, we found different types of actin structures in the sperm tail, using stochastic optical reconstruction microscopy (STORM). In the principal piece, actin is radially distributed between the axoneme and the plasma membrane. The actin-associated proteins spectrin and adducin are also found in these structures. Strikingly, polymerized actin in the midpiece forms a double-helix that accompanies mitochondria. Our findings illustrate a novel specialized structure of actin filaments in a mammalian cell.

cell biology↗

bioRxiv2018

Characterization of mannitol metabolism genes in Saccharina explains its key role in mannitol biosynthesis and evolutionary significance in Laminariales

As a unique photosynthetic product in brown algae, mannitol exhibits high synthesis and accumulation in Saccharina japonica. Mannitol acts as a carbon-storage compound and is an osmoprotectant, imparting increased tolerance to osmotic stress. However, the underlying biochemical and molecular mechanisms in macroalgae have not been studied. Analysis of genomic and transcriptomic data has shown that mannitol metabolism in S. japonica is a circular pathway composed of four steps. In this study, one S. japonica mannitol-1-phosphate dehydrogenase (M1PDH2) and two mannitol-1-phosphatase (M1Pase) proteins were recombinantly expressed to analysis enzyme biochemical properties. RNA sequencing and droplet digital polymerase chain reaction were used to analyze the gene expression patterns of mannitol metabolism in different generations, tissues, sexes, and abiotic stresses. Our findings revealed insights into the mannitol synthesis pathways in brown algae. All genes were constitutively expressed in all samples, allowing maintenance of basic mannitol anabolism and dynamic maintenance of the \"saccharide pool\" in vivo as the main storage and antistress mechanism. Enzyme assays confirmed that the recombinant proteins produced mannitol, with the specific activity of SjaM1Pase1 being 1.8-4831 times that of other algal enzymes. Combined with the transcriptional analysis, SjaM1Pase1 was shown to be the dominant gene of mannitol metabolism. Mannitol metabolism genes in multicellular filamentous (gametophyte) and large parenchyma thallus (sporophyte) generations had different expression levels and responded differently under environmental stresses (hyposaline and hyperthermia) in gametophytes and sporophytes. The considerable variation in enzyme characteristics and expression of mannitol synthesis genes suggest their important ecophysiological significance in the evolution of complex systems (filamentous and thallus) and environmental adaptation of Laminariales.

molecular biology↗

bioRxiv2018

The brown alga Saccharina japonica features distinct vanadium-dependent bromoperoxidases and iodoperoxidases

Marine algae have an extraordinary ability to absorb halogens which provide algae with an inorganic antioxidant impacting atmospheric chemistry. Although brown algal Laminariales species are the most efficient iodine accumulators among all living systems, and Saccharina japonica is the primary material used for iodine extraction, the functions and regulatory mechanisms of these species have not been fully documented. In this study, a functional genomics analysis of the algal vanadium-dependent haloperoxidase (vHPO) gene family was conducted; there genes can introduce halogen atoms into organic compounds. The comprehensive analyses regarding the bioinformatics and phylogenetics of novel genomic and transcriptomic sequencing data of 21 Rhodophyta and 19 Ochrophyta marine algal species revealed that brown algal vHPOs have two gene types, vanadium-dependent bromoperoxidase (vBPO) and vanadium-dependent iodoperoxidase (vIPO), with secondary endosymbiotic host origin. The enzyme activity of S. japonica vBPO and vIPO were verified for the first time and were quite stable in a wide range of temperature and pH values. However, the specific activity and optimal conditions were considerably different between vBPO and vIPO. The transcript expression analysis in different S. japonica tissues (including rhizoids), generations (sporophytes and gametophytes), sexes (male and female), and stress conditions (hyposaline and hyperthermia) also showed great differences between vBPOs and vIPOs. Most of the vBPOs were constitutively expressed with higher expression dose, which may be responsible for basal halogen metabolism. On the contrary, vIPOs mainly showed specific expression, which may be involved in tissue differentiation, generation differentiation, sex differentiation, and stress regulation. Comprehensive analysis of gene family evolution, enzyme biochemical characteristics, and complex transcriptional mechanisms were conducive to the environmental adaptation and sophisticated system evolution of Laminariales. The successful bromination of small-molecule compound substrate by SjavBPO provided high activity and efficient enzymatic tools for artificial synthesis of halogenated compounds.

molecular biology↗

bioRxiv2017

Uterine progesterone signaling is a target for metformin therapy in polycystic ovary syndrome

Impaired progesterone (P4) signaling is linked to endometrial dysfunction and infertility in women with polycystic ovary syndrome (PCOS). Here we report for the first time that elevated expression of progesterone receptor (PGR) isoforms A and B parallels increased estrogen receptor (ER) expression in PCOS-like rat uteri. The aberrant PGR-targeted gene expression in PCOS-like rats before and after implantation overlaps with dysregulated expression of Fkbp52 and Ncoa2, two genes that contribute to the development of uterine P4 resistance. In vivo and in vitro studies of the effects of metformin on the regulation of the uterine P4 signaling pathway under PCOS conditions showed that metformin directly inhibits the expression of PGR and ER along with the regulation of several genes that are targeted dependently or independently of PGR-mediated uterine implantation. Functionally, metformin treatment corrected the abnormal expression of cell-specific PGR and ER and some PGR-target genes in PCOS-like rats with implantation. Additionally, we documented how metformin contributes to the regulation of the PGR-associated MAPK/ERK/p38 signaling pathway in the PCOS-like rat uterus. Our data provide novel insights into how metformin therapy regulates uterine P4 signaling molecules under PCOS conditions.

physiology↗

bioRxiv2017

CRISPR-typing PCR (ctPCR), a new Cas9-based DNA detection method

This study develops a new method for detecting and typing target DNA based on Cas9 nuclease, which was named as ctPCR, representing Cas9/sgRNA- or CRISPR-typing PCR. The technique can detect and discriminate target DNA easily, rapidly, specifically, and sensitively. This technique detects target DNA in three steps: (1) amplifying target DNA with PCR by using a pair of universal primers (PCR1); (2) treating PCR1 products with a process referred to as CAT, representing Cas9 cutting, A tailing and T adaptor ligation; (3) amplifying the CAT-treated DNA with PCR by using a pair of general-specific primers (gs-primers) (PCR2). The technique was verified by detecting HPV16 and HPV18 L1 gene in 13 different high-risk human papillomavirus (HPV) subtypes. The technique was also detected two high-risk HPVs (HPV16 and HPV18) in cervical carcinoma cells (HeLa and SiHa) by detecting the L1 and E6/E7 genes, respectively. In this method, PCR1 was performed to determine if the detected DNA sample contained the target DNA (such as virus infection), while PCR2 was performed to discriminate which genotypic target DNA was present in the detected DNA sample (such as virus subtypes). With these proof-of-concept experiments, this study provides a new CRISPR-based DNA detection and typing method.

biochemistry↗

bioRxiv2017

Characterisation of the pathogenicity of strains of Pseudomonas syringae towards cherry and plum

Bacterial canker is a major disease of cherry and other stone fruits caused by several pathovars of Pseudomonas syringae. These are P.s pv. morsprunorum race 1 (Psm R1), P.s pv. morsprunorum race 2 (Psm R2) and P.s pv. syringae (Pss). Psm R1 and R2 were originally designated as races of the same pathovar, however phylogenetic analysis has revealed them to be distantly related. This study characterised the pathogenicity of P. syringae on cherry and plum, in the field and the laboratory. The field experiment identified variation in host cultivar susceptibility to the different pathogen clades. The cherry cultivar Merton Glory exhibited a broad resistance to all clades, whilst cultivar Van showed race-specific resistance. Psm R1 may be divided into a race structure with some strains pathogenic to both cherry and plum and others only pathogenic to plum. The results of laboratory-based pathogenicity tests were compared to results obtained on whole-trees. Only cut shoot inoculations were found to be sensitive enough to detect cultivar variation in susceptibility. Measuring population growth of bacteria in detached leaves reliably discriminated pathogens from non-pathogens. In addition, symptom appearance discriminated Psm races from non-pathogens which triggered a rapid hypersensitive response (HR). The pathogen Pss rapidly induced disease lesions and therefore may exhibit a more necrotrophic lifestyle than hemi-biotrophic Psm races. This in-depth study of pathogenic interactions, identification of host resistance and optimisation of laboratory assays, will provide a framework for future genetic dissection of virulence and host resistance mechanisms.

plant biology↗

bioRxiv2017

Assessment of the aging of the brown adipose tissue by 18F-FDG PET/CT imaging in the progeria mouse model LmnaG609G/G609G

Brown adipose tissue (BAT) is an important energy metabolic organ that is closely related to obesity, type 2 diabetes, and atherosclerosis. Aging is one of the most important determinants of BAT activity. In this study, we used 18F-FDG PET/CT imaging to assess the aging of the BAT in LmnaG609G/G609G mice. To evaluate the BAT activity, LmnaG609G/G609G and wild-type (WT) mice were injected with 18F-FDG, and PET/CT imaging was performed. The maximum standardized uptake value (SUVMax) of the BAT was measured and the target/nontarget (T/NT) values of BAT were calculated. The transcription and the protein expression levels of the uncoupling protein 1 (UCP1), beta3-adrenergic receptor ({beta}3-AR), and the PRdomain-containing16 (PRDM16), were measured by quantitative real-time polymerase chain reaction (RT-PCR) and Western blotting or immunohistochemical analysis. Apoptosis and cell senescence of the BAT, in WT and LmnaG609G/G609G mice, was detected by Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), and by CDKN2A/p16INK4a immunohistochemical staining, respectively. At 14 weeks of age, the BAT SUVMax and the expression levels of UCP1, {beta}3-AR and PRDM16 in LmnaG609G/G609G mice was significantly lower than that in WT mice. At the same time, the number of p16INK4a and TUNEL positively stained cells (%) increased in LmnaG609G/G609G mice. LmnaG609G/G609G mice are an ideal model for studying BAT aging. The aging characteristics and the aging mechanism of BAT in LmnaG609G/G609G mice can mimic normal BAT aging.

cell biology↗

bioRxiv2017

MapZ forms a stable ring structure that acts as a track for FtsZ treadmilling in Streptococcus mutans

Bacterial binary division requires the accurate placement of the division machinery. FtsZ, the vital component of the division machinery, can assemble into filaments and self-organize into a ring structure (Z-ring) at the proper site for cell division. Thus, understanding how bacteria control the spatiotemporal formation of the FtsZ ring is crucial for small molecule and nanoparticle antibacterial drug discovery. MapZ, a recently identified FtsZ regulator in Streptococcaceae, has been found to localize at the mid-cell and position the FtsZ ring. However, the mechanism is still unclear. Here, by using total internal reflection fluorescence microscopy, super-resolution imaging, and single molecule tracking, we investigated the mechanism by which MapZ regulates the FtsZ ring position. The results show that FtsZ exhibites dynamic treadmilling motion in S. mutans. Importantly, depletion of MapZ leads to an unconstrained movement of treadmilling FtsZ filaments and a shorter lifetime of the constricting FtsZ ring. Furthermore, by revealing that MapZ forms an immobile ring-like nanostructure at the division site, our study suggests that MapZ forms a stable ring that acts as a nanotrack to guide and restrict treadmilling FtsZ filaments in S. mutans, representing a novel way in which bacteria control the division.

microbiology↗

bioRxiv2017

Species and Cell-Type Properties of Classically Defined Human and Rodent Neurons and Glia

Determination of the molecular properties of genetically targeted cell types has led to fundamental insights into mouse brain function and dysfunction. Here, we report an efficient strategy for precise exploration of gene expression events in specific cell types in a broad range of species, including postmortem human brain. We demonstrate that classically defined, homologous neuronal and glial cell types differ between rodent and human by the expression of hundreds of orthologous, cell specific genes. Confirmation that these genes are differentially active was obtained using epigenetic mapping and immunofluorescence localization. Studies of sixteen human postmortem brains revealed cell-specific molecular responses to aging, and the induction of a shared, robust response to an unknown external event experienced by three donors. Our data establish a comprehensive approach for analysis of unique molecular events associated with specific circuits and cell types in a wide variety of human conditions.

neuroscience↗

bioRxiv2017

Sharing genetic admixture and diversity of public biomedical datasets

Genetic ancestry and admixture are critical co-factors to study phenotype-genotype associations using cohorts of human subjects. Most publically available molecular datasets - genomes, exomes or transcriptomes - are however missing this information or only share self-reported ancestry. This represents a limitation to identify and re-purpose datasets to investigate the contribution of race and ethnicity to diseases and traits. we propose an analytical framework to enrich the meta-data from publically available cohorts with admixture information and a resulting diversity score at continental resolution, calculated directly from the data. We illustrate the utility and versatility of the framework using The Cancer Genome Atlas datasets indexed and searched through the DataMed Data Discovery Index. Data repositories or data contributors can use this framework to provide, as metadata, admixture for controlled access datasets, minimizing the work involved in requesting a dataset that may ultimately prove inadequate for a researchers purpose. With the increasingly global scale of human genetics research, research on disease risk and susceptibility would benefit greatly from the adequate estimation and sharing of admixture data following a framework such as the one presented.

genetics↗