Biology subjects

Xu, M.

Publications and source records attributed to Xu, M..

At least 181 records · Page 10Linked to original sources

bioRxiv2022

Quantification of adulterated fox-derived components in meat Products by Drop Digital Polymerase Chain Reaction

There are still large research gaps in the testing standards for non-edible meat-derived components at present. Here we report a novel approach to quantification of adulterated fox-derived components in meat products by drop digital polymerase chain reaction(ddPCR). It was better to identify the adulteration that has been added either inadvertently or deliberately during the process by designing single primers on housekeeping genes. In this paper, the fox meat was used as the experimental materials and the relationship between sample quality and copy number was established by extracting DNA and using DNA concentration as an intermediary. The results of ddPCR showed that both the relationships between meat weight and DNA concentration and between DNA concentration and DNA copy number (C) were nearly linear within the dynamic range. The DNA concentration was utilized as an intermediate value to establish the formulae about the relationship between the original meat weight and DNA copy number: Mfox=0.05C+2.7. The methods feasibility was validated using artificial adulteration of different proportions The establishment of this method provides technical support for relevant regulatory authorities to monitor the adulteration of fox meat.

molecular biology↗

bioRxiv2022

Toxicity of microplastics and plastic additive co-exposure in liver Disse organoids from healthy donors and patient-derived induced pluripotent stem cells

The ubiquitous microplastics (MPs) and plastic additives in the environment usually form complexes, enter human blood circulation, and increase the risk of steatohepatitis. The liver Disse space plays a vital role in corresponding hepatic pathological processes. However, due to the limited understanding of the regulatory cues in multilineage maturation, the generation of large-scale Disse-like organoids (DOs) mirroring the comprehensive toxicity responses of MPs is challenging. Here, using human-induced pluripotent stem cells (hiPSCs), we biofabricated healthy donors and patient-derived DOs containing hepatocytes, endothelial cells, and hepatic stellate cells, resembling the features of Disse space. These organoids revealed that polystyrene MPs preferentially entered endothelial cells and then dispersed throughout the organoids, similar to reported studies in zebrafish. Co-exposure to MPs and tetrabromobisphenol A (TBBPA), a common plastic additive, showed enhanced accumulation of contamination in the organoids. We also biofabricated alcoholic liver disease (ALD) patient-derived DOs representing the specific disease transcriptional profiles. We found that co-exposure to MPs and TBBPA at environmental-related dosages significantly elevated the pathological transcriptional expression and biochemical profiles in patient-derived DOs but not in healthy organoids, suggesting that both hereditary factors and pollutants contribute to susceptibility to environmental toxicants. This study exemplified the value of biofabricated hiPSC-derived organoids in environmental toxicology and offered a powerful strategy for personalized toxicology evaluation.

bioengineering↗

bioRxiv2022

FRIZZLED 2 regulates limb development by mediating both β-catenin-dependent and independent Wnt signaling pathways

Human Robinow Syndrome and omodysplasia, characterized by skeletal limb and craniofacial defects, are associated with mutations in the Wnt receptor FZD2. However, as FZD2 can activate both canonical and non-canonical Wnt pathways, its precise functions and mechanisms of action in limb development are unclear. To address these questions, we generated mice harboring a single nucleotide insertion in the Dishevelled-interacting domain of Fzd2 (Fzd2em1Smill), causing a frameshift mutation similar to the effects of human syndromic FZD2 mutations. Fzd2em1Smill mutant mice had shortened limbs resembling those of Robinow Syndrome and omodysplasia patients. Fzd2em1Smill mutant embryos displayed decreased canonical Wnt signaling in developing limb mesenchyme and disruption of digit chondrocyte elongation and orientation, which is controlled by the WNT5A/PCP pathway. In line with this, we found that tissue-specific disruption of Fzd2 function in limb mesenchyme caused formation of shortened bone elements and was associated with deficiency in both Wnt/{beta}-catenin and WNT5A/PCP signaling. These findings indicate that FZD2 controls limb development by mediating both canonical and non-canonical Wnt pathways and reveal causality of pathogenic FZD2 mutations in Robinow Syndrome and omodysplasia patients. Summary statementHuman FZD2 mutations are associated with limb defects; using genetic mouse models we revealed causality of these mutations and showed that they disrupt both canonical and non-canonical Wnt signaling.

developmental biology↗

bioRxiv2022

Zinc Fingers and Homeoboxes 2 is Required for Diethylnitrosamine-induced Liver Tumor Formation in C57BL/6 Mice

Liver cancer, comprised mainly of hepatocellular carcinoma (HCC), is the third leading cause of cancer deaths worldwide and increasing in Western countries. We previously identified the transcription factor Zinc Fingers and Homeoboxes 2 (Zhx2) as a regulator of hepatic gene expression, and many Zhx2 target genes are dysregulated in HCC. Here, we investigate HCC in Zhx2-deficient mice using the Diethylnitrosamine (DEN)-induced liver tumor model. Our study using whole-body Zhx2 knock-out (Zhx2KO) mice revealed the complete absence of liver tumors 9 and 10 months after DEN exposure.Analysis soon after DEN treatment showed no differences in expression of the DEN bioactivating enzyme CYP2E1 and DNA polymerase delta 2, or in the numbers of {gamma}H2AX foci between Zhx2KO and wild-type (Zhx2wt) mice. The absence of Zhx2, therefore, did not alter DEN bioactivation or DNA damage. Zhx2KO livers showed fewer positive foci for Ki67 staining and reduced IL-6 and AKT2 expression compared to Zhx2wt livers, suggesting that Zhx2 loss reduces liver cell proliferation and may account for reduced tumor formation. Tumors were reduced but not absent in DEN-treated liver-specific Zhx2 knock-out mice, suggesting that Zhx2 acts in both hepatocytes and non-parenchymal cells to inhibit tumor formation. Analysis of data from The Cancer Genome Atlas and Clinical Proteomic Tumor Consortium indicated that ZHX2 mRNA and protein levels were significantly higher in HCC patients and associated with clinical pathological parameters. ConclusionsIn contrast to previous studies in human hepatoma cell lines and other HCC mouse models showing that Zhx2 acts as a tumor suppressor, our data indicate that Zhx2 acts as an oncogene in the DEN-induced HCC model and is consistent with the higher ZHX2 expression in HCC patients.

cancer biology↗

bioRxiv2022

Systematic characterization of chromodomain proteins revealsan H3K9me1/2 reader regulating aging in C. elegans

The chromatin organization modifier domain (chromodomain) is an evolutionally conserved motif across eukaryotic species. The chromodomain mainly functions as a histone methyl-lysine reader to modulate gene expression, chromatin spatial conformation and genome stability. Mutations or aberrant expression of chromodomain proteins can result in cancer and other human diseases. Here, we systematically tagged chromodomain proteins with green fluorescent protein (GFP) using CRISPR/Cas9 technology in C. elegans. By combining ChIP-seq analysis and imaging, we delineated a comprehensive expression and functional map of chromodomain proteins. We then conducted a candidate-based RNAi screening and identified factors that regulate the expression and subcellular localization of the chromodomain proteins. Specifically, we revealed a new H3K9me1/2 reader, CEC-5, both by in vitro biochemistry and in vivo ChIP assays. MET-2, an H3K9me1/2 writer, is required for CEC-5 association with heterochromatin. Both MET-2 and CEC-5 are required for the normal lifespan of C. elegans. Furthermore, a forward genetic screening identified a conserved Arginine124 of CEC-5s chromodomain, which was essential for CEC-5s association with chromatin and life span regulation. Thus, our work will serve as a reference to explore chromodomain functions and regulation in C. elegans and allow potential applications in aging-related human diseases.

molecular biology↗

bioRxiv2022

Fat Extract Modulates Calcium Signaling and Protects Against Hyperactive Osteoclastogenesis in Bone Remodeling with Antioxidant Capacity

Osteoclasts are cells which are primarily involved in bone remodeling and osteolytic bone diseases. Hyperactive osteoclastogenesis leads to pathological bone loss and microarchitectural deterioration, particularly in postmenopausal osteoporosis. However, given the limitations of current first-line osteoclast inhibitors, there is an urgent need for a novel antiresorptive agent with higher efficiency and fewer side effects. Cell-free fat extract (CEFFE) is the liquid fraction obtained from human adipose tissues, which are enriched with a variety of cytokines and growth factors. This study aims to explore its pharmaceutical effect on hyperactive osteoclast formation in vivo and in vitro. CEFFE exhibits excellent potentials to attenuate osteoclast-associated bone loss in an ovariectomy (OVX) mouse model and to inhibit RANKL-induced osteoclastogenesis in primary bone marrow-derived monocytes. Furthermore, the cationic protein fraction of CEFFE (CEFFE-Cation) is identified as the main inhibitory component in osteoclast formation assay. Excessive reactive oxygen species (ROS) production is the main cause of osteoclast overactivation. According to LC-MS/MS analysis, the CEFFE-Cation fraction mainly consists of various antioxidant enzymes, extracellular matrix, and secreted cytokines, which endow it with a superior antioxidant capacity. Ca2+ signaling contributes to osteoclast maturation. In addition to scavenging ROS, CEFFE-Cation is also capable of mitigating Ca2+ oscillation, calcineurin activation, and subsequent NFATc1 nuclear translocation during osteoclastogenesis. Overall, this study elucidates the promising translational potential of CEFFE as a next-generation personalized antiresorptive agent for osteolytic bone disease treatment.

pathology↗

bioRxiv2022

An engineered xCas12i with high activity, high specificity and broad PAM range

The type-V CRISPR effector Cas12i, with its smaller size, short crRNA guiding, and self-processing features, is a potentially versatile genome editing tool. By screening Cas12i proteins from a metagenomic database, we identified a natural variant with high activity in mammalian cells, named as xCas12i. We further engineered the PAM-interacting, REC, and RuvC domains for enhanced cleavage activity and specificity. This variant, named as high-fidelity Cas12Max, exhibited robust genome editing activity and minimal off-target activity with a broad 5-TN recognition profile. With the fusion of deaminase TadA8e and further optimization of xCas12i, the base editor dCas12i-Tad8e also showed the high editing efficiency. This study provides highly efficient and specific tools for gene therapy.

bioengineering↗

bioRxiv2022

A "cooperative collaboration" between HAND2 and MYCN shapes neuroblastoma cell identity

Noradrenergic neuroblastoma (NB) is characterized by a core transcriptional regulatory circuitry (CRC) comprised of transcription factors (TFs) such as PHOX2B, HAND2 and GATA3, which form a network with MYCN. Physiological levels of MYCN mainly binds to promoters but aberrantly upregulated MYCN in NB also binds to enhancers. How MYCN invades enhancers, and what role the CRC TFs play in this process is unknown. Here we find that HAND2 assists MYCN to invade enhancers through an "indirect cooperative" TF-DNA binding mechanism. HAND2 cooperates with MYCN to compete with nucleosomes and regulate global gene transcription. The treatment of NB with an Aurora A kinase inhibitor plus HDAC inhibitor potently downregulates both MYCN and the CRC TFs and suppresses MYCN-amplified NB tumor growth. Our study identifies a "cooperative collaboration" among MYCN and CRC TFs and the simultaneous targeting these TFs is an effective way to treat this aggressive pediatric tumor.

cancer biology↗

bioRxiv2022

Dynamic tripartite construct of interregional engram circuits underlies forgetting of extinction memory

Fear extinction allows for adaptive control of learned fear responses but often fails, resulting in a renewal or spontaneous recovery of the extinguished fear, i.e., forgetting of the extinction memory readily occurs. Using an activity-dependent neuronal labeling strategy, we demonstrate that engram neurons for fear extinction memory are dynamically positioned in the medial prefrontal cortex (mPFC), basolateral amygdala (BLA), and ventral hippocampus (vHPC), which constitute an engram construct in the term of directional engram synaptic connectivity from the BLA or vHPC to mPFC, but not that in the opposite direction, for retrieval of extinction memory. Fear renewal or spontaneous recovery switches the extinction engram construct from an accessible to inaccessible state, whereas additional extinction learning or optogenetic induction of long-term potentiation restores the directional engram connectivity and prevents the return of fear. Thus, the plasticity of engram construct underlies forgetting of extinction memory.

neuroscience↗

bioRxiv2022

Comprehensive multiomic profiling of somatic mutations in malformations of cortical development

Malformations of cortical development (MCD) are neurological conditions displaying focal disruption of cortical architecture and cellular organization arising during embryogenesis, largely from somatic mosaic mutations. Identifying the genetic causes of MCD has been a challenge, as mutations remain at low allelic fractions in brain tissue resected to treat epilepsy. Here, we report a genetic atlas from 317 brain resections, identifying 69 mutated genes through intensive profiling of somatic mutations, combining whole-exome and targeted-amplicon sequencing with functional validation and single-cell sequencing. Genotype-phenotype correlation analysis elucidated specific MCD gene sets associating distinct pathophysiological and clinical phenotypes. The unique spatiotemporal expression patterns identified by comparing single-nucleus transcriptional sequences of mutated genes in control and patient brains implicate critical roles in excitatory neurogenic pools during brain development, and in promoting neuronal hyperexcitability after birth.

genetics↗

bioRxiv2022

A toolkit of highly selective and sensitive genetically encoded neuropeptide sensors

Neuropeptides are key signaling molecules in the endocrine and nervous systems that regulate many critical physiological processes, including energy balance, sleep and circadian rhythms, stress, and social behaviors. Understanding the functions of neuropeptides in vivo requires the ability to monitor their dynamics with high specificity, sensitivity, and spatiotemporal resolution; however, this has been hindered by the lack of direct, sensitive and non-invasive tools. Here, we developed a series of GRAB (G protein-coupled receptor activation-based) sensors for detecting somatostatin (SST), cholecystokinin (CCK), corticotropin-releasing factor (CRF), neuropeptide Y (NPY), neurotensin (NTS), and vasoactive intestinal peptide (VIP). These fluorescent sensors utilize the corresponding GPCRs as the neuropeptide-sensing module with the insertion of a circular-permutated GFP as the optical reporter. This design detects the binding of specific neuropeptides at nanomolar concentration with a robust increase in fluorescence. We used these GRAB neuropeptide sensors to measure the spatiotemporal dynamics of endogenous SST release in isolated pancreatic islets and to detect the release of both CCK and CRF in acute brain slices. Moreover, we detect endogenous CRF release induced by stressful experiences in vivo using fiber photometry and 2-photon imaging in mice. Together, these new sensors establish a robust toolkit for studying the release, function, and regulation of neuropeptides under both physiological and pathophysiological conditions.

neuroscience↗

bioRxiv2022

The lectin LecB induces patches with basolateral characteristics at the apical membrane to promote Pseudomonas aeruginosa host cell invasion

The opportunistic bacterium Pseudomonas aeruginosa can infect mucosal tissues of the human body. To persist at the mucosal barrier, this highly adaptable pathogen has evolved many strategies, including invasion of host cells. Here, we show that the P. aeruginosa lectin LecB binds and cross-links fucosylated receptors at the apical plasma membrane of epithelial cells. This triggers a signaling cascade via Src kinases and PI3K leading to the formation of patches enriched with the basolateral marker PIP3 at the apical plasma membrane. This identifies LecB as causative bacterial factor for activating this well-known host cell response that is elicited upon apical binding of P. aeruginosa. Downstream of PI3K, Rac1 is activated to cause actin rearrangement and the outgrowth of protrusions at the apical plasma membrane. LecB-triggered PI3K activation also results in aberrant recruitment of caveolin-1 to the apical domain. In addition, we reveal a positive feedback loop between PI3K activation and apical caveolin-1 recruitment, which provides a mechanistic explanation for the previously observed implication of caveolin-1 in P. aeruginosa host cell invasion. Interestingly, LecB treatment also reversibly removes primary cilia. To directly prove the role of LecB for bacterial uptake, we coated bacteria-sized beads with LecB, which drastically enhanced their endocytosis. Furthermore, LecB deletion and LecB inhibition with L-fucose diminished the invasion efficiency of P. aeruginosa bacteria. Taken together, our study identifies LecB as missing link that can explain how PI3K signaling and caveolin-1 recruitment are triggered to facilitate invasion of epithelial cells from the apical side by P. aeruginosa. ImportanceAn intriguing feature of the bacterium P. aeruginosa is its ability to colonize highly diverse niches. P. aeruginosa can, besides biofilm formation, also enter and proliferate within epithelial host cells. Moreover, research during recent years has shown that P. aeruginosa possesses many different mechanisms to invade host cells. In this study we identify LecB as novel invasion factor. In particular, we show that LecB activates PI3K signaling, which is connected via a positive feedback loop to apical caveolin-1 recruitment, and leads to actin rearrangement at the apical plasma membrane. This provides a unifying explanation for the previously reported implication of PI3K and caveolin-1 in P. aeruginosa host cell invasion. In addition, our study adds a further function to the remarkable repertoire of the lectin LecB, which is all brought about by the capability of LecB to recognize fucosylated glycans on many different niche-specific host cell receptors.

microbiology↗

bioRxiv2022

Influence of residual microbial nucleic acid in instruments for hip joint arthroplasty on false-positive results of metagenomic sequencing

The purpose of this study was to analyze the effect of residual microbial nucleic acid in instruments for hip joint arthroplasty on false-positive sequencing results. Samples were taken from 3 different acetabular reamer for hip arthroplasty in 7 different hospitals. The whole process was strictly aseptic, metagenomic next-generation sequencing (mNGS) was performed according to standard operating procedures. The sterility of instruments was confirmed by culture method. The sequencing results of specimens from different hospitals were compared to analyze the difference of background bacteria. Bioinformatics analysis and visualization were presented through R language. A total of 26 samples were processed by mNGS, including 24 instrument swab samples, 1 blank swab control, and 1 blank water control. 254,314,707 reads were sequenced in all samples. The results showed that 1.13% of Clean Reads can be matched to pathogenic microorganism genomes, of which bacterial sequences account for 87.48%, fungal sequences account for 11.18%, parasite sequences account for 1.26%, and virus sequences account for 0.06%. The results of PCA (Principal Component Analysis) demonstrated that the distribution of bacteria on the surface of instruments was significantly different between medical institutions. Through the Venn diagram, it was found that 465 species of bacteria in all region hospitals, Liaocheng Peoples Hospital had a maximum of 340 species of bacteria, followed by Guanxian County Peoples Hospital with 169 species. The clustering heat map illustrated that the distribution of bacterial groups in three different instrument samples in the same hospital was basically the same, and the bacterial genera varied significantly among hospitals. The residual microbial nucleic acid fragments are mainly bacterial DNA and represent differences in different medical institutions, The establishment of independent background bacterial libraries in different medical institutions can effectively improve the accuracy of mNGS diagnosis and help to exclude background microorganisms interference.

genomics↗

bioRxiv2022

GPCR-IFP: A Web Server for Analyzing G-Protein Coupled Receptor-Ligand Fingerprint Interaction from Experimental Structures

G-protein coupled receptors (GPCRs) are interesting targets for a broad range of drugs. The comprehensions of the interactions mode between GPCRs and their ligands remains to be resolved. Here, we present GPCR-IFP, a web server which drives to gather experimental information supports a better understanding of ligand-receptor interaction and the chemical space. Despite the vast chemical library used for drug discovery, the structures of ligands for a particular receptor share some common molecular features. Thus, we foraged to collect all experimental structures of GPCRs and analyzed protein-ligand interactions which resulted in important unique patterns for both agonist and antagonist. All those results can be analyzed and visualized in GPCR-IFP, a friendly-used webserver.

bioinformatics↗

bioRxiv2022

ILnc: Prioritizing Long Non-coding RNAs for Pan-cancer Analysis of Immune Cell Infiltration

The distribution and extent of immune cell infiltration into solid tumors play pivotal roles in cancer immunology and therapy. Here we introduced an immune long non-coding RNA (lncRNA) signature-based method (ILnc), for estimating the abundance of 14 immune cell types from lncRNA transcriptome data. Performance evaluation through pure immune cell data shows that our lncRNA signature sets can be more accurate than protein-coding gene signatures. We found that lncRNA signatures are significantly enriched to immune functions and pathways, such as immune response and T cell activation. In addition, the expression of these lncRNAs is significantly correlated with expression of marker genes in corresponding immune cells. Application of ILnc in 33 cancer types provides a global view of immune infiltration across cancers and we found that the abundance of most immune cells is significantly associated with patient clinical signatures. Finally, we identified six immune subtypes spanning cancer tissue types which were characterized by differences in immune cell infiltration, homologous recombination deficiency (HRD), expression of immune checkpoint genes, and prognosis. Altogether, these results demonstrate that ILnc is a powerful and exhibits broad utility for cancer researchers to estimate tumor immune infiltration, which will be a valuable tool for precise classification and clinical prediction.

bioinformatics↗

bioRxiv2022

A high-performance genetically encoded fluorescent indicator for in vivo cAMP imaging

cAMP is a key second messenger that regulates diverse cellular functions including neural plasticity. However, the spatiotemporal dynamics of intracellular cAMP in intact organisms are largely unknown due to low sensitivity and/or brightness of current genetically encoded fluorescent cAMP indicators. Here, we report the development of the new circularly permuted GFP (cpGFP)-based cAMP indicator G-Flamp1, which exhibits a large fluorescence increase (a maximum {Delta}F/F0 of 1100% in HEK293T cells), relatively high brightness, appropriate affinity (a Kd of 2.17 {micro}M) and fast response kinetics (an association and dissociation half-time of 0.20 s and 0.087 s, respectively). Furthermore, the crystal structure of the cAMP-bound G-Flamp1 reveals one linker connecting the cAMP-binding domain to cpGFP adopts a distorted {beta}-strand conformation that may serve as a fluorescence modulation switch. We demonstrate that G-Flamp1 enables sensitive monitoring of endogenous cAMP signals in brain regions that are implicated in learning and motor control in living organisms such as fruit flies and mice.

molecular biology↗

bioRxiv2022

Heat Shock Factor 1 (HSF1) specifically potentiates c-MYC-mediated transcription independently of the canonical heat-shock response

Despite its pivotal roles in biology, how the transcriptional activity of c-MYC is attuned quantitatively remain poorly defined. Here, we show that heat shock factor 1 (HSF1), the master transcriptional regulator of the heat-shock, or proteotoxic stress, response, acts as a key modifier of the c-MYC-mediated transcription. HSF1 deficiency diminishes c-MYC DNA binding and dampens its transcriptional activity genome-widely. Mechanistically, c-MYC, MAX, and HSF1 assemble into a transcription factor complex on genomic DNAs and, surprisingly, the DNA binding of HSF1 is dispensable. Instead, HSF1 physically recruits the histone acetyltransferase GCN5, thereby promoting histone acetylation and augmenting c-MYC transcriptional activity. Thus, our studies reveal that HSF1 specifically potentiates the c-MYC-mediated transcription, distinct from its role in the canonical heat-shock response. Importantly, this mechanism of action engenders two distinct c-MYC activation states, primary and advanced, which may be important to accommodate diverse physiological and pathological conditions.

cancer biology↗

bioRxiv2022

A Suite of TMPRSS2 Assays for Screening Drug Repurposing Candidates as Potential Treatments of COVID-19

SARS-CoV-2 is the causative viral pathogen driving the COVID-19 pandemic that prompted an immediate global response to the development of vaccines and antiviral therapeutics. For antiviral therapeutics, drug repurposing allowed for rapid movement of existing clinical candidates and therapies into human clinical trials to be tested as COVID-19 therapies. One effective antiviral treatment strategy used early in symptom onset is to prevent viral entry. SARS-CoV-2 enters ACE2-expressing cells when the receptor-binding domain of the spike protein on the surface of SARS-CoV-2 binds to ACE2 followed by cleavage at two cut sites on the spike protein. TMPRSS2 has a protease domain capable of cleaving the two cut sites; therefore, a molecule capable of inhibiting the protease activity of TMPRSS2 could be a valuable antiviral therapy. Initially, we used a fluorogenic high-throughput screening assay for the biochemical screening of 6030 compounds in NCATS annotated libraries. Then, we developed an orthogonal biochemical assay that uses mass spectrometry detection of product formation to ensure that hits from the primary screen are not assay artifacts from the fluorescent detection of product formation. Finally, we assessed the hits from the biochemical screening in a cell-based SARS-CoV-2 pseudotyped particle entry assay. Of the six molecules advanced for further studies, two are approved drugs in Japan (camostat and nafamostat), two have entered clinical trials (PCI-27483 and otamixaban), while the other two molecules are peptidomimetic inhibitors of TMPRSS2 taken from the literature that have not advanced into clinical trials (compounds 92 and 114). This work demonstrates a suite of assays for the discovery and development of new inhibitors of TMPRSS2.

biochemistry↗