Biology subjects

Du, X.

Publications and source records attributed to Du, X..

At least 127 records · Page 7Linked to original sources

bioRxiv2020

Parallel Movement-suppressing Striatal Output Pathways

The classic basal ganglia circuit model asserts a complete segregation of the two striatal output pathways. Empirical data argue that, in addition to indirect-pathway striatal projection neurons (iSPNs), direct-pathway striatal projection neurons (dSPNs) innervate the external globus pallidus (GPe). However, the functions of the latter were not known. In this study, we interrogated the organization principles of striatopallidal projections and their roles in full-body movement in mice (both males and females). In contrast to the canonical motor-promoting response of dSPNs in the dorsomedial striatum (DMSdSPNs), optogenetic stimulation of dSPNs in the dorsolateral striatum (DLSdSPNs) suppressed locomotion. Circuit analyses revealed that dSPNs selectively target Npas1+ neurons in the GPe. In a chronic 6-hydroxydopamine lesion model of Parkinsons disease, the dSPN-Npas1+ projection was dramatically strengthened. As DLSdSPN-Npas1+ projection suppresses movement, the enhancement of this projection represents a circuit mechanism for the hypokinetic symptoms of Parkinsons disease that has not been previously considered. In sum, our results suggest that dSPN input to the GPe is a critical circuit component that is involved in the regulation of movement in both healthy and parkinsonian states. Significance statementIn the classic basal ganglia model, the striatum is described as a divergent structure--it controls motor and adaptive functions through two segregated, opposing output streams. However, the experimental results that show the projection from direct-pathway neurons to the external pallidum have been largely ignored. Here, we showed that this striatopallidal sub-pathway targets a select subset of neurons in the external pallidum and is motor-suppressing. We found that this sub-pathway undergoes changes in a Parkinsons disease model. In particular, our results suggest that the increase in strength of this sub-pathway contributes to the slowness or reduced movements observed in Parkinsons disease.

neuroscience↗

bioRxiv2020

Dissociable Roles of Pallidal Neuron Subtypes in Regulating Motor Patterns

We have previously established that PV+ neurons and Npas1+ neurons are distinct neuron classes in the GPe-- they have different topographical, electrophysiological, circuit, and functional properties. Aside from Foxp2+ neurons, which are a unique subclass within the Npas1+ class, we lack driver lines that effectively capture other GPe neuron subclasses. In this study, we examined the utility of Kcng4-Cre, Npr3-Cre, and Npy2r-Cre mouse lines (both males and females) for the delineation of GPe neuron subtypes. By using these novel driver lines, we have provided the most exhaustive investigation of electrophysiological studies of GPe neuron subtypes to date. Corroborating our prior studies, GPe neurons can be divided into two statistically distinct clusters that map onto PV+ and Npas1+ classes. By combining optogenetics and machine learning-based tracking, we showed that optogenetic perturbation of GPe neuron subtypes generated unique behavioral structures. Our findings further highlighted the dissociable roles of GPe neurons in regulating movement and anxiety-like behavior. We concluded that Npr3+ neurons and Kcng4+ neurons are distinct subclasses of Npas1+ neurons and PV+ neurons, respectively. Finally, by examining local collateral connectivity, we inferred the circuit mechanisms involved in the motor patterns observed with optogenetic perturbations. In summary, by identifying mouse lines that allow for manipulations of GPe neuron subtypes, we created new opportunities for interrogations of cellular and circuit substrates that can be important for motor function and dysfunction. Significance statementWithin the basal ganglia, the external globus pallidus (GPe) has long been recognized for its involvement in motor control. However, we lacked an understanding of precisely how movement is controlled at the GPe level as a result of its cellular complexity. In this study, by using transgenic and cell-specific approaches, we showed that genetically-defined GPe neuron subtypes have distinct roles in regulating motor patterns. In addition, the in vivo contributions of these neuron subtypes are in part shaped by the local, inhibitory connections within the GPe. In sum, we have established the foundation for future investigations of motor function and disease pathophysiology.

neuroscience↗

bioRxiv2020

Robust Benchmark Structural Variant Calls of An Asian Using the State-of-Art Long Fragment Sequencing Technologies

The importance of structural variants (SVs) on phenotypes and human diseases is now recognized. Although a variety of SV detection platforms and strategies that vary in sensitivity and specificity have been developed, few benchmarking procedures are available to confidently assess their performances in biological and clinical research. To facilitate the validation and application of those approaches, our work established an Asian reference material comprising identified benchmark regions and high-confidence SV calls. We established a high-confidence SV callset with 8,938 SVs in an EBV immortalized B lymphocyte line, by integrating four alignment-based SV callers [from 109x PacBio continuous long read (CLR), 22x PacBio circular consensus sequencing (CCS) reads, 104x Oxford Nanopore long reads, and 114x optical mapping platform (Bionano)] and one de novo assembly-based SV caller using CCS reads. A total of 544 randomly selected SVs were validated by PCR and Sanger sequencing, proofing the robustness of our SV calls. Combining trio-binning based haplotype assemblies, we established an SV benchmark for identification of false negatives and false positives by constructing the continuous high confident regions (CHCRs), which cover 1.46Gb and 6,882 SVs supported by at least one diploid haplotype assembly. Establishing high-confidence SV calls for a benchmark sample that has been characterized by multiple technologies provides a valuable resource for investigating SVs in human biology, disease, and clinical diagnosis.

genomics↗

bioRxiv2020

Restoration of β-globin expression with optimally designed lentiviral vector for β-thalassemia treatment in Chinese patients

{beta}-thalassemia is one of the most prevalent genetic diseases worldwide. The current treatment for {beta}-thalassemia is allogeneic hematopoietic stem cell transplantation (HSCT), which is limited due to lack of matched donors. Gene therapy has been developed as an alternative therapeutic option for transfusion-ependent {beta}-thalassemia (TDT). However, successful gene therapy for {beta}-thalassemia patients in China has not been reported. Here we present the results of preclinical studies of an optimally designed LV named LentiHBBT87Q in hematopoietic stem cells (HSCs) derived from Chinese TDT patients. LentiHBBT87Q was selected from a series of LVs with optimized backbone and de novo cloning strategy. It contains an exogenous T87Q {beta}-globin gene (HBBT87Q) driven by a specific reconstituted locus control region (rLCR) and efficiently express HBB mRNA and HBB protein in erythroblasts derived from cord blood (CB) HSCs. To facilitate clinical transformation, we manufactured clinical grade LentiHBBT87Q (cLentiHBBT87Q) and optimized its transduction procedure. Importantly, transduction of cLentiHBBT87Qrestored expression of HBB monomer and adult hemoglobin (HbA) tetramer to relatively normal level in erythroblasts from bone marrow (BM) HSCs of Chinese TDT patients, that carry the most common mutation types and cover various genotypes, including {beta}0/{beta}0. Furthermore, viral integration sites (VIS) of cLentiHBBT87Q were similar to other LVs safely used in previous clinical trials and the associated risk of tumorigenesis was not observed in cLentiHBBT87Q transduced HSCs through comprehensive analysis. Taken together, we have engineered the cLentiHBBT87Q that can restore {beta}-globin expression in the HSCs-derived erythroblasts of Chinese TDT patients with minimal risk on tumorigenesis, providing a favorable starting point for future clinical application.

cell biology↗

bioRxiv2020

Parabrachial neuron types categorically encode thermoregulation variables during heat defense

Heat defense is crucial for survival and fitness, and its dysregulation may result in deaths due to poor management. Transmission of thermosensory signals into hypothalamic thermoregulation centers represent a key layer of regulation in heat defense. However, the mechanism by which these signals are transmitted into the hypothalamus remains poorly understood. Here, we reveal that glutamatergic prodynorphin and cholecystokinin neuron populations in the lateral parabrachial (LPB) are progressively recruited to defend elevated body temperature. These two nonoverlapping neuron types form circuitries with downstream preoptic hypothalamic neurons to inhibit BAT thermogenesis and activate tail vasodilation, respectively. Both circuitries are selectively activated by warm temperatures and are required for fever limiting. The prodynorphin circuitry is further required for regulation of energy expenditure and weight homeostasis. Thus, these findings establish that the genetic and functional specificity of heat defense neurons occurs as early as in the LPB and uncover categorical neuron types for encoding two heat defense variables, which may provide targets for treating thermoregulation disorders.

neuroscience↗

bioRxiv2020

The therapeutic effects of intratumoral injection of IL12IL2GMCSF fusion protein on canine tumors

Regulating the immune system through tumor immunotherapy to defeat tumors is currently one of the most popular methods of tumor treatment. We previously found that the combinations of IL12, IL2 and GMCSF has superior antitumor activities. In this study, IL12IL2GMCSF fusion protein was produced from 293 cells transduced by expression lentiviral vector. IL12IL2GMCSF fusion protein was injected into canine tumors, and the efficacy was monitored to evaluate the potential of tumor immunotherapy. In 14 canines injected with IL12IL2GMCSF protein, 6 had a complete response (CR), 7 had a partial response (PR), and 1 had stable disease (SD). IL12IL2GMCSF fusion protein has great anti-tumor potential. Intratumoral injection of IL12IL2GMCSF can effectively treat tumors without severe adverse effects. It provides a promising immune therapeutics in canine cancers.

cancer biology↗

bioRxiv2020

Haplotype-Resolved Assembly for Synthetic Long Reads Using a Trio-Binning Strategy

The accuracy and completeness of genome haplotyping are crucial for characterizing the relationship between human disease susceptibility and genetic variations, especially for the heterozygous variations. However, most of current variations are unphased genotypes, and the construction of long-range haplotypes remains challenging. We introduced a de novo haplotype-resolved assembly tool, HAST that exports two haplotypes of a diploid species for synthetic long reads with trio binning. It generates parental distinguishing k-mer libraries, partitions reads from the offspring according to the unique markers, and individually assembles them to resolve the haplotyping problem. Based on the stLFR co-barcoding data of an Asian as well as his parental massive parallel sequencing data, we utilized HAST to recover both haplotypes with a scaffold N50 of >11 Mb and an assembly accuracy of 99.99995% (Q63). The complete and accurate employment of long-range haplotyping information provided sub-chromosome level phase blocks (N50 [~]13 Mb) with 99.6% precision and 94.1% recall on average. We suggest that the accurate and efficient approach accomplishes the regeneration of the haplotype chromosomes with trio binning, thus promoting the determination of haplotype phase, the heterosis of crossbreeding, and the formation of autopolyploid and allopolyploid.

bioinformatics↗

bioRxiv2020

Chromosome-level genome assembly of the African pike, Hepsetus odoe

The order Characiformes is one of the largest components of the freshwater teleost fauna inhabiting exclusively in South America and Africa with great ecological and economical significance. Yet, quite limited genomic resources are available to study this group and their transatlantic vicariance. In this study we present a chromosome-level genome assembly of the African pike (Hepsetus odoe), a representative member of the African Characiformes. To this end, we generated 119, 11, and 67 Gb reads using the single tube long fragment read (stLFR), Oxford Nanopore, and Hi-C sequencing technologies, respectively. We obtained an 862.1 Mb genome assembly with the contig and scaffold N50 of 347.4 kb and 25.8 Mb, respectively. Hi-C sequencing produced 29 chromosomes with 742.5 Mb, representing 86.1% of the genome. 24,314 protein-coding genes were predicted and 23,999 (98.7%) genes were functionally annotated. The chromosomal-scale genome assembly will be useful for functional and evolutionary studies of the African pike and promote the study of Characiformes speciation and evolution.

genomics↗

bioRxiv2020

Once delayed non-invasive remote ischemic preconditioning protects against early stroke by modulating neuroinflammatory responses in rats

Once delayed non-invasive remote ischemic preconditioning (RIPC) has been proven to provide endogenous protection against injury induced by ischemia-reperfusion in the central nervous system. However, for thus ischemic preconditioning method, it is still unclear how long this protection can maintain and what the underlying mechanism is. In this study, we tested the hypothesis that once delayed non-invasive RIPC protects brain injury at short reperfusion time. The rat was stimulated by transient middle cerebral artery occlusion (MCAo) for 90 min, and subsequent reperfusion was performed at 6 h, 72 h and 7 days after MCAo. RIPC was conducted in both hind limbs 24 h before MCAo for 3 cycles (10 min ischemia/ 10 min reperfusion). The infarct size was measured by 2, 3, 5-triphenyl-2H-tetrazolium chloride (TTC) staining and Cresyl violet (CV) staining. The mRNA and protein levels of inflammatory cytokines in the brain were measured by real-time RT-PCR and ELISA. The results showed that once delayed non-invasive RIPC reduced the infarct size, improved neurological functions and behavioral performance at 6 and 72 h post-stroke. There was no change by reperfusion at 7 d after MCAo. RIPC reduced the levels of TNF, IL-1{beta} and IL-6 in the brain at 72 h post stroke. It also reduced the levels of TNF and IL-1{beta} when reperfusion at 6 h after MCAo. Our results strongly supported that once delayed non-invasive RIPC protects against stroke as a non-invasive neuroprotective strategy, which maintained for both short and middle term ischemic reperfusion time. The protective effect is mediated by the modulation of inflammatory response in the ischemic brain.

neuroscience↗

bioRxiv2020

Structural basis for the inhibition of COVID-19 virus main protease by carmofur, an antineoplastic drug

The antineoplastic drug Carmofur was shown to inhibit SARS-CoV-2 main protease (Mpro). Here the X-ray crystal structure of Mpro in complex with Carmofur reveals that the carbonyl reactive group of Carmofur is covalently bound to catalytic Cys145, whereas its fatty acid tail occupies the hydrophobic S2 subsite. Carmofur inhibits viral replication in cells (EC50 = 24.30 M) and it is a promising lead compound to develop new antiviral treatment for COVID-19.

biochemistry↗

bioRxiv2020

Adolescent rats show estrous cycle-mediated sex differences in extinction of conditioned fear

Anxiety disorders are more prevalent in females than males, and frequently emerge during adolescence. Despite this, preclinical research commonly focuses on adult males. Here we use Pavlovian fear conditioning and extinction in adolescent male and female rats to understand sex- and age-dependent processes relevant to anxiety disorders. In experiment 1, 35-day-old male and female rats were exposed to 6 pairings of a conditioned stimulus (CS, a tone) with an aversive unconditioned stimulus (US, a footshock). The next day they were extinguished in a contextually distinct chamber, via 60 presentations of the CS without the US. Extinction recall was tested 24 hours later in the extinction context. Estrous phase was monitored by cytology on vaginal smears taken 1 hour after each behavioral session. In experiment 2, male and female rats were given sham surgery or gonadectomy at 21 days of age. They were then trained and tested as for experiment 1. We observed that females in proestrus or met/diestrus during extinction showed delayed extinction and impaired extinction recall the next day compared to males. Ovariectomy enhanced extinction for female rats, but orchidectomy delayed extinction for males. Plasma analyses showed that met/di/proestrus phases were associated with high estradiol levels. These findings suggest that high plasma estradiol levels impair extinction for adolescent females. While these results contradict what is observed in adult animals, they are consistent with the prevalence of anxiety disorders observed in females. Our findings have important implications for understanding and treating anxiety in adolescents, particularly where treatment involves extinction-based therapies.

animal behavior and cognition↗

bioRxiv2020

The effect of a novel powerful ABA mimic on the improvement of color in grapes and its mechanism

Pigment content is an important quality attribute in the grape industry, and anthocyanins are the major fruit pigments. iso-PhABA is a novel and excellent ABA analog capable of the antimetabolic inactivation of ABA. In this study, we found that iso-PhABA improved the coloration of grape berries more obviously than ABA at concentrations of 2 mg/L and 5 mg/L in two grape varieties in China. iso-PhABA treatment enhanced the anthocyanin content in the two grape varieties; specifically, the anthocyanin and delphinidin contents increased in both the Jufeng and Xiahei varieties. An enzymatic activity test showed that iso-PhABA significantly promoted four key enzyme activities catalyzing anthocyanin biosynthesis. We also determined the affinity between iso-PhABA and ABA receptors using ABA as a control. The results indicated that iso-PhABA had significantly to moderately higher affinities for some ABA receptors, including PYR1, PYL2, PYL1, PYL3 and PYL10, which resulted in higher inhibition of the PP2C HAB1 in the presence of iso-PhABA than in the presence of ABA. iso-PhABA treatment increased the content of soluble sugars and grape yield without any apparent accompanying adverse effects on the quality of the grapes.

biochemistry↗

bioRxiv2020

Exploring the Pharmacological Mechanism of Bezoar on Cerebral Ischemic Stroke using a Network Pharmacology Approach

BackgroundIschemic stroke is a common clinical disease limited by its time window for treatment. In addition to its high mortality rate, only one treatment currently exists for ischemic stroke. Nonetheless, traditional Chinese medicine is often used as a reatment for the disease. Bezoar is a multi-functional drug that has been demonstrated to be effective for the treatment of ischemic stroke. However, its mechanism of action is yet to be fully elucidated. Based on network pharmacology, we explored the potential mechanism of action of bezoar. Symmap and literature data mining methods were used to obtain the target of bezoar. The mechanism of bezoar for the treatment of ischemic stroke was identified and ischemic stroke-related targets were retrieved using DrugBank, Online Mendelian Inheritance in Man, and Therapeutic Target Database. Protein-protein interaction networks were constructed using the Cytoscape plugin, BisoGenet, and analysed by topological methods. Gene Ontology and Kyoto Encyclopaedia of Genes and Genomes pathway enrichment were carried out via the Database for Annotation, Visualization, and Integrated Discovery server. We obtained 48 potential targets and 3 signalling pathways, including mitogen-activated protein kinase, hypoxia-inducible factor-1, and tumour necrosis factor signalling pathways. The mechanism of action of bezoar on ischemic stroke involves multiple targets and signalling pathways. Our research provides a network pharmacology framework for future Chinese medicinal research.

neuroscience↗

bioRxiv2020

Structure-based drug design, virtual screening and high-throughput screening rapidly identify antiviral leads targeting COVID-19

A new coronavirus (CoV) identified as COVID-19 virus is the etiological agent responsible for the 2019-2020 viral pneumonia outbreak that commenced in Wuhan1-4. Currently there is no targeted therapeutics and effective treatment options remain very limited. In order to rapidly discover lead compounds for clinical use, we initiated a program of combined structure-assisted drug design, virtual drug screening and high-throughput screening to identify new drug leads that target the COVID-19 virus main protease (Mpro). Mpro is a key CoV enzyme, which plays a pivotal role in mediating viral replication and transcription, making it an attractive drug target for this virus5,6. Here, we identified a mechanism-based inhibitor, N3, by computer-aided drug design and subsequently determined the crystal structure of COVID-19 virus Mpro in complex with this compound. Next, through a combination of structure-based virtual and high-throughput screening, we assayed over 10,000 compounds including approved drugs, drug candidates in clinical trials, and other pharmacologically active compounds as inhibitors of Mpro. Six of these inhibit Mpro with IC50 values ranging from 0.67 to 21.4 M. Ebselen also exhibited promising antiviral activity in cell-based assays. Our results demonstrate the efficacy of this screening strategy, which can lead to the rapid discovery of drug leads with clinical potential in response to new infectious diseases where no specific drugs or vaccines are available.

biochemistry↗

bioRxiv2020

Silicon modulates multi-layered defense against powdery mildew in Arabidopsis

Silicon (Si) has been widely employed in agriculture to enhance resistance against pathogens in many crop plants. However, the underlying molecular mechanisms of Si-mediated resistance remain elusive. In this study, the Arabidopsis-powdery mildew pathosystem was employed to investigate possible defense mechanisms engaged for Si-mediated resistance. Because Arabidopsis lacks efficient Si transporters and thus is a low Si-accumulator, two heterologous Si influx transporters (from barley and muskmelon) were individually expressed in wild-type Arabidopsis Col-0 and a panel of mutants defective in different immune signaling pathways. Results from infection tests showed that while very low leaf Si content slightly induced salicylic acid (SA)-dependent resistance, high Si promoted PAD4-dependent but EDS1- and SA-independent resistance against the adapted powdery mildew isolate Golovinomyces cichoracearum UCSC1. Intriguingly, our results also showed that high Si could largely reboot non-host resistance in an immune-compromised eds1/pad4/sid2 triple mutant background against a non-adapted powdery mildew isolate G. cichoracearum UMSG1. Taken together, our results suggest that assimilated Si modulates distinct, multi-layered defense mechanisms to enhance plant resistance against adapted and no-adapted powdery mildew pathogens, possibly via synergistic interaction with defense-induced callose

plant biology↗

bioRxiv2020

Characterization of a novel NDM-5-harboring plasmid from a carbapenem-resistant Escherichia coli isolate from China

A carbapenem-resistant Escherichia coli (sequence type 5415) strain was isolated from a male patient. S1-PFGE and Southern blotting showed that the blaNDM-5 gene was located on a novel 66-kb IncFII [F2:A-:B-] plasmid. Conjugation assays revealed that the blaNDM-5-bearing plasmid was self-transferrable. Genomic sequencing and comparative analysis suggested that plasmid p2947-NDM5 likely originated from a combination of an IncFII-type backbone and the blaNDM-5 flanking genetic elements. This study highlights the genetic complexity of an NDM-5 carbapenemase and the urgent need for continuous active monitoring. IMPORTANCECarbapenem resistance due to the NDM enzyme has become a serious clinical problem in medical care and public health. We here identified a blaNDM-5-positive E. coli strain ST5414. The blaNDM-5 gene was located on a novel self-transferrable IncFII type plasmid p2947-NDM5. Genomic sequencing and comparative analysis suggested that plasmid p2947-NDM5 likely originated from a combination of an IncFII-type backbone and the blaNDM-5 flanking genetic elements. The genetic context of NDM-5 is highly similar to that of animal origins, and we speculate that the blaNDM-harboring IncFII plasmid has a potential risk of being transmitted from animals to humans and should be paid more attention. Our findings highlight the threat of NDM-5 carbapenemase circulation and the urgency of stringent surveillance and control measures.

genomics↗

bioRxiv2019

Junctophilin-4 is essential for signalling at plasma membrane-endoplasmic reticulum junctions in sensory neurons

Junctions of endoplasmic reticulum and plasma membrane (ER-PM junctions) serve as signaling hubs in prokaryotic cells. ER-PM junctions are present in peripheral sensory neurons and are necessary for pro-inflammatory G protein coupled receptor signalling and for inflammatory pain generation. Yet, the principles of ER-PM junctions assembly and maintenance, as well as their role in inflammatory signaling in sensory neurons are only beginning to emerge. Here we discovered that a member of the junctophilin family of proteins, JPH4, is abundantly expressed in rat dorsal root ganglion (DRG) neurons and is necessary for the formation of store operated Ca2+ entry (SOCE) complex at the ER-PM junctions in response to the G-protein induced ER Ca2+ store depletion. Furthermore, we demonstrate a key role of the JPH4 and ER Ca2+ stores in the maintenance of inflammatory pain. Indeed, knockdown of JPH4 expression in DRG in vivo significantly reduced the duration of pain produced by inflammatory mediator bradykinin. Since the ER supplies Ca2+ for the excitatory action of multiple inflammatory mediators, we suggest that junctional Ca2+ signalling maintained by JPH4 is an important contributor to the inflammatory pain mechanisms.

neuroscience↗

bioRxiv2019

The genome of Mekong tiger perch (Datnioides undecimradiatus) provides insights into the phylogenic position of Lobotiformes and biological conservation

Mekong tiger perch (Datnioides undecimradiatus) is one ornamental fish and a vulnerable species, which belongs to order Lobotiformes. Here, we report a [~]595 Mb D. undecimradiatus genome, which is the first whole genome sequence in the order Lobotiformes. Based on this genome, the phylogenetic tree analysis suggested that Lobotiformes and Sciaenidae are closer than Tetraodontiformes, resolving a long-time dispute. We depicted the pigment synthesis pathway in Mekong tiger perch and result confirmed that this pathway had evolved from the shared whole genome duplication. We also estimated the demographic history of Mekong tiger perch, showing the effective population size suffered a continuous reduction possibly related to the contraction of immune-related genes. Our study provided a reference genome resource for the Lobotiformes, as well as insights into the phylogeny of Eupercaria and biological conservation.

genomics↗