Biology subjects

Zhao, N.

Publications and source records attributed to Zhao, N..

At least 91 records · Page 5Linked to original sources

bioRxiv2021

TREM2-H157Y Increases Soluble TREM2 Production and Reduces Amyloid Pathology

The p.H157Y variant of TREM2 (Triggering Receptor Expressed on Myeloid Cells 2) has been reported to increase Alzheimers disease (AD) risk. This mutation in the extracellular domain of TREM2 localizes at the cleavage site, leading to enhanced shedding. Here, we generated a novel Trem2 H157Y knock-in mouse model to investigate how this H157Y mutation impacts TREM2 proteolytic processing, synaptic function, and AD-related amyloid pathology. Consistent with previous in vitro findings, TREM2-H157Y increases the amount of soluble TREM2 (sTREM2) in the cortex and serum of mutant mice compared to the wild type controls. Interestingly, the Trem2 H157Y variant enhances synaptic plasticity without affecting microglial density and morphology. In the presence of amyloid pathology, TREM2-H157Y surprisingly accelerates A{beta} clearance and reduces amyloid burden and microgliosis. Taken together, our findings support a beneficial effect of the Trem2 H157Y mutation in synaptic function and in mitigating amyloid pathology. Considering the genetic association of TREM2 p.H157Y with AD, we speculate TREM2-H157Y might increase AD risk through an amyloid-independent pathway, as such its effects on tauopathy and neurodegeneration merit further investigation.

neuroscience↗

bioRxiv2021

Batch effects removal for microbiome data via conditional quantile regression (ConQuR)

Batch effects in microbiome data arise from differential processing of specimens and can lead to spurious findings and obscure true signals. Most existing strategies for mitigating batch effects rely on approaches designed for genomic analysis, failing to address the zero-inflated and over-dispersed microbiome data. Strategies tailored for microbiome data are restricted to association testing, failing to allow other analytic goals such as visualization. We develop the Conditional Quantile Regression (ConQuR) approach to remove microbiome batch effects using a two-part quantile regression model. It is a fundamental advancement in the field because it is the first comprehensive method that accommodates the complex distributions of microbial read counts, and it generates batch-removed zero-inflated read counts that can be used in and benefit all usual subsequent analyses. We apply ConQuR to real microbiome data sets and demonstrate its state-of-the-art performance in removing batch effects while preserving or even amplifying the signals of interest.

bioinformatics↗

bioRxiv2021

Generation and diversification of recombinant monoclonal antibodies for studying mitosis

Antibodies are indispensable tools used for a large number of applications in both foundational and translational bioscience research; however, there are drawbacks to using traditional antibodies generated in animals. These include a lack of standardization leading to problems with reproducibility, high costs of antibodies purchased from commercial sources, and ethical concerns regarding the large number of animals used to generate antibodies. To address these issues, we have developed practical methodologies and tools for generating low-cost, high-yield preparations of recombinant monoclonal antibodies and antibody fragments directed to protein epitopes from primary sequences. We describe these methods here, as well as approaches to diversify monoclonal antibodies, including customization of antibody species specificity, generation of genetically encoded small antibody fragments, and conversion of single chain antibody fragments (e.g. scFv) into full-length, bivalent antibodies. This study focuses on antibodies directed to epitopes important for mitotic cell division; however, the methods and reagents described here are applicable to antibodies and antibody fragments for use in any field.

cell biology↗

bioRxiv2021

Three-dimensional microenvironment regulates gene expression, function, and tight junction dynamics of iPSC-derived blood-brain barrier microvessels

The blood-brain barrier (BBB) plays a pivotal role in brain health and disease. In the BBB, brain microvascular endothelial cells (BMECs) are connected by tight junctions which regulate paracellular transport, and express specialized transporter systems which regulate transcellular transport. However, existing in vitro models of the BBB display variable physiological accuracy across a wide range of characteristics including gene/protein expression and barrier function. Here, we use an isogenic family of fluorescently-labeled iPSC-derived BMEC-like cells (iBMECs) and brain pericyte-like cells (iPCs) within two-dimensional confluent monolayers (2D) and three-dimensional (3D) tissue-engineered microvessels to explore how 3D microenvironment regulates gene expression and function of the in vitro BBB. We show that 3D microenvironment (shear stress, cell-ECM interactions, and cylindrical geometry) increases BBB phenotype and endothelial identity, and alters angiogenic and cytokine responses in synergy with pericyte co-culture. Tissue-engineered microvessels incorporating junction-labeled iBMECs enable study of the real-time dynamics of tight junctions during homeostasis and in response to physical and chemical perturbations.

bioengineering↗

bioRxiv2021

TREM2 interacts with TDP-43 and mediates microglial neuroprotection against TDP-43-related neurodegeneration

Triggering receptor expressed on myeloid cell 2 (TREM2) is a surface receptor that, in the central nervous system, is exclusively expressed on microglia. TREM2 variants have been linked to increased risk for neurodegenerative diseases, but the functional effects of microglial TREM2 remain largely unknown. To this end, we investigated TAR-DNA binding protein 43 kDa (TDP-43)-related neurodegenerative disease via viral-mediated expression of human TDP-43 protein (hTDP-43) in neonatal and adult mice or inducible expression of hTDP43 with defective nuclear localization signals in transgenic mice. We found that TREM2 deficiency impaired microglia phagocytic clearance of pathological TDP-43, and enhanced neuronal damage and motor function impairments. Mass cytometry analysis revealed that hTDP-43 induced a TREM2-dependent subpopulation of microglia with high CD11c expression and higher phagocytic ability. Using mass spectrometry and surface plasmon resonance analysis, we further demonstrated an interaction between TDP-43 and TREM2, in vitro and in vivo, in hTDP-43-expressing transgenic mouse brains. We computationally identified the region within hTDP-43 that interacts with TREM2 and observed the potential interaction in ALS patient tissues. Our data reveal the novel interaction between TREM2 and TDP-43, highlighting that TDP-43 is a possible ligand for microglial TREM2 and the interaction mediates neuroprotection of microglial TREM2 in TDP-43-related neurodegeneration.

neuroscience↗

bioRxiv2021

TMT-based proteomic analysis of the antibiotic effects of ShangKeHuangShui against Straphylococcus aureus.

Post-traumatic infection is a serious orthopedic trauma complication commonly caused by Staphylococcus aureus (SAU). ShangKeHuangShui, also known as Yellow Aqueous Concentrate of Traumatology Herbs (YACTH), is prepared from six Chinese herbal medicines, which has been used for decades in our hospital to prevent post-traumatic infection. In the present study, we investigated the in vitro antibacterial effects and underlying mechanism of YACTH against SAU. YACTH exhibited significant antibacterial activity against SAU with the minimum inhibitory concentration (MIC) of 8.625 mg/mL. Proteomic analysis based on Tandem mass tag (TMT) showed different protein expression levels in SAU under the YACTH and control conditions. Compared to the control group, the expression level of 490 proteins of YACTH treated group significantly changed (>1.2 fold, P <0.05). Biological informatics analysis showed that these differential proteins were widely involved in a variety of biological processes and multiple metabolic pathways. We then selected 26 target proteins to further conduct Parallel reaction monitoring (PRM). The results revealed that the 9 down-regulated proteins were mainly involved in RNA polymerase, oxidative phosphorylation, ABC transporter and glycolysis; whereas, the 17 up-regulated proteins are related to RNA degradation, mismatch repair and ribosome. In conclusion, YACTH, which contains the water-soluble components of six Chinese herbs, is first reported to exhibit antibacterial activity against SAU in vitro. The results provide novel insights into the antibacterial mechanisms of YACTH on protein networks, which may help us find new potential antibiotic targets.

microbiology↗

bioRxiv2021

After traumatic brain injury oligodendrocytes regain a plastic phenotype and can become astrocytes

After acute brain injuries various response cascades are evoked that direct the formation of the glial scar. Here, we report that acute lesions associated with a disruption of the blood-brain barrier trigger a re-programming within the oligodendrocyte lineage. In PLP-DsRed1/GFAP-EGFP and PLP-EGFPmem/GFAP-mRFP1 transgenic mice with cortical injuries, we transiently found PLP transgene-labelled cells with activated GFAP promoter activity adjacent to the lesion site. We termed them AO cells, based on their concomitant activity of astro- and oligodendroglial genes. By fate mapping using PLP- and GFAP-split Cre complementation and NG2-CreERT2 mice we observed that major portions of AO cells surprisingly differentiated into astrocytes. Using repeated long-term in vivo two-photon laser-scanning microscopy (2P-LSM) we followed oligodendrocytes after injury. We observed their conversion into astrocytes via the AO cell stage with silencing of the PLP promoter and simultaneous activation of the GFAP promoter. In addition, we provide evidence that this oligodendrocyte-to-astrocyte conversion depends on local cues. At the lesion site higher expression levels of various glial differentiation factors were detected. And indeed, local injection of IL-6 promoted the formation of AO cells. In summary, our findings highlight the plastic potential of oligodendrocytes in acute brain trauma. An altered environmental milieu affects gene expression programs of mature oligodendrocytes and induces a plastic differentiation stage with astrogliogenic potential via transitional AO cells.

neuroscience↗

bioRxiv2021

CUB domain containing protein 1 (CDPC1) is a target for radioligand therapy in castration resistant prostate cancer

PurposeRadioligand therapy (RLT) is relatively unexplored in metastatic castration resistant prostate cancer (mCRPC), with much of the focus having been on bone seeking radionuclides and PSMA-directed RLT. Herein, we evaluated if CUB domain containing protein 1 (CDCP1) can be exploited to treat mCRPC with RLT, particularly for subsets like small cell neuroendocrine prostate cancer (SCNC) that would not be expected to respond to current options. Experimental DesignCDCP1 mRNA levels were evaluated in the RNA-seq data from 119 recent mCRPC biopsies. Protein expression was assessed in twelve SCNC and adenocarcinoma patient derived xenografts. Saturation binding assays were performed with 4A06, a recombinant human antibody that targets the CDCP1 ectodomain. The feasibility of imaging and treating mCRPC in vivo was tested with 89Zr-4A06 and 177Lu-4A06. ResultsCDCP1 mRNA expression was observed in over 90% of mCRPC biopsies, including SCNC and in adenocarcinoma with low FOLH1 (PSMA) levels. A modest anticorrelation was observed between CDCP1 and PTEN. Overall survival was not significantly different based on CDCP1 mRNA levels, regardless of PTEN status. Full length and/or cleaved CDCP1 was expressed in ten of twelve PDX samples. Bmax values of ~22,000 and ~6,200 fmol/mg were calculated for two human prostate cancer cell lines. Five prostate cancer models were readily detected in vivo with 89Zr-4A06. 177Lu-4A06 significantly suppressed the growth of DU145 tumors compared to control. ConclusionsThe antitumor data and the overexpression of CDCP1 reported herein provide the first evidence promoting CDCP1 directed RLT as a treatment strategy for mCRPC. Statement of Translational RelevanceNew targets for RLT are needed to address the subset of mCRPC that cannot be treated with bone seeking radionuclides or PSMA directed RLT. We report herein the first data credentialing CDCP1 as a target for mCRPC, in both adenocarcinoma and neuroendocrine subtypes. Combined with low expression in normal human tissues, these data provide a compelling scientific rationale for testing CDCP1 directed RLT clinically in mCRPC patients alone or in combination with other systemic therapies.

cancer biology↗

bioRxiv2021

Visualizing looping of two endogenous genomic loci using synthetic zinc-finger proteins with anti-FLAG and anti-HA frankenbodies in living cells

In eukaryotic nuclei, chromatin loops mediated through cohesin are critical structures that regulate gene expression and DNA replication. Here we demonstrate a new method to visualize endogenous genomic loci using synthetic zinc-finger proteins harboring repeat epitope tags (ZF probes) for signal amplification via binding of tag-specific intracellular antibodies, or frankenbodies, fused with fluorescent proteins. We achieve this in two steps. First, we develop an anti-FLAG frankenbody that can bind FLAG-tagged proteins in diverse live-cell environments. The anti-FLAG frankenbody complements the anti-HA frankenbody, enabling two-color signal amplification from FLAG and HA-tagged proteins. Second, we develop a pair of cell-permeable ZF probes that specifically bind two endogenous chromatin loci predicted to be involved in chromatin looping. By coupling our anti-FLAG and anti-HA frankenbodies with FLAG- and HA-tagged ZF probes, we simultaneously visualize the dynamics of the two loci in single living cells. This reveals close association between the two loci in the majority of cells, but the loci markedly separate upon the triggered degradation of the cohesin subunit RAD21. Our ability to image two endogenous genomic loci simultaneously in single living cells provides a proof-of-principle that ZF probes coupled with frankenbodies are useful new tools for exploring genome dynamics in multiple colors.

cell biology↗

bioRxiv2021

A Drosophila Toolkit for Imaging of HA-tagged Proteins Unveiled a Block in Autophagy Flux in the Last Instar Larval Fat Body

For in vivo functional analysis of a protein of interest (POI), multiple transgenic strains with POI harboring different tags are needed but generation of these strains is still labor-intensive work. To overcome this, we developed a versatile Drosophila toolkit with a genetically encoded single-chain variable fragment for the HA epitope tag: "HA Frankenbody". This system allows various analyses of HA-tagged POI in live tissues by simply crossing an HA Frankenbody fly with an HA-tagged POI fly. Strikingly, the GFP-mCherry tandem fluorescent-tagged HA Frankenbody revealed a block in autophagic flux and an accumulation of enlarged autolysosomes in the last instar larval and prepupal fat body. Autophagy was dispensable for the swelling of lysosomes, indicating that lysosomal activity is downregulated at this stage. Furthermore, forced activation of lysosomes by fat body-targeted overexpression of Mitf, the single MiTF/TFE family gene in Drosophila, suppressed the lysosomal swelling and resulted in pupal lethality. Collectively, we propose that downregulated lysosomal function in the fat body plays a role in the metamorphosis of Drosophila.

cell biology↗

bioRxiv2021

Non-canonical Wnt/Ror2 signaling status regulates cell-matrix crosstalk to prompt directional tumor cell invasion and dissemination in breast cancer

Bidirectional cell-extracellular matrix (ECM) interactions represent fundamental exchanges during tumor progression. We demonstrate the noncanonical Wnt receptor, Ror2, regulates tumor cell-driven matrix remodeling in models of breast cancer. Wnt/Ror2 loss-of-function triggers tumor cell invasion, accompanied by changes in actin cytoskeleton, adhesion, and collagen crosslinking gene expression programs. E-cadherin levels decline upon Ror2 depletion, and spatially, we pinpoint the upregulation and redistribution of 5 and {beta}3 integrins together with the production of fibronectin in areas of invasion. Wnt/{beta}-catenin-dependent and Wnt/Ror2 alternative Wnt signaling appear to regulate distinct functions for tumor cells regarding their ability to modify cell-ECM exchanges during invasion. Furthermore, blocking either integrin or focal adhesion kinase (FAK), a downstream mediator of integrin-mediated signal transduction, abrogates the enhanced migration observed upon Ror2 loss. These results reveal a critical function for the alternative Wnt receptor, Ror2, as a determinant of reciprocal communication between tumor cells and ECM during cancer invasion and metastasis.

cancer biology↗

bioRxiv2021

Impaired bidirectional communication between interneurons and oligodendrocyte precursor cells affects cognitive behavior

Cortical neural circuits are complex but very precise networks of balanced excitation and inhibition (E/I). Yet, the molecular and cellular mechanisms that form the E/I balance are just beginning to emerge. Here, using conditional GABAB receptor-deficient mice we identified a GABA/TNF-related cytokine (TNFSF12)-mediated bidirectional communication pathway between Parvalbumin-positive (PV+) fast spiking interneurons and oligodendrocyte precursor cells (OPCs) that determines the density and function of interneurons in the developing medial prefrontal cortex (mPFC). Interruption of the GABAergic signaling to OPCs resulted in reduced myelination and hypoactivity of interneurons, strong changes of cortical network activities and impaired cognitive behavior. In conclusion, glial transmitter receptors are pivotal elements in finetuning distinct brain functions.

neuroscience↗

bioRxiv2021

Imaging translational control by Argonaute with single-molecule resolution in live cells

A major challenge to our understanding of translational control has been deconvolving the individual impact specific regulatory factors have on the complex dynamics of mRNA translation. MicroRNAs (miRNAs), for example, guide Argonaute and associated proteins to target mRNAs, where they direct gene silencing in multiple ways that are not well understood. To better deconvolve these dynamics, we have developed technology to directly visualize and quantify the impact of human Argonaute2 (Ago2) on the translation and subcellular localization of individual reporter mRNAs in living cells. We show that our combined translation and Ago2 tethering system reflects endogenous miRNA-mediated gene silencing. Using the system, we find that Ago2 association leads to progressive silencing of translation at individual mRNA. The timescale of silencing was similar to that of translation, consistent with a role for Ago2 in blocking translation initiation, leading to ribosome runoff. At early time points, we observed occasional brief bursts of translational activity at Ago2-tethered mRNAs undergoing silencing, suggesting that translational repression may initially be reversible. At later time points, Ago2-tethered mRNAs cluster and coalesce with endogenous P-bodies, where a translationally silent state is maintained. These results provide a framework for exploring miRNA-mediated gene regulation in live cells at the single-molecule level. Furthermore, our tethering-based, single-molecule reporter system will likely have wide-ranging application in studying general RNA-protein interactions.

molecular biology↗

bioRxiv2021

Extracellular vesicle molecular signatures characterize metastatic dynamicity in ovarian cancer

Late-stage diagnosis of ovarian cancer drastically lowers 5-year survival rate from 90% to 30%. Early screening tools that use non-invasive sampling methods combined with high specificity and sensitivity can significantly increase survival. Emerging research employing blood-based screening tools have shown promise in non-invasive detection of cancer. Our findings in this study show the potential of a small extracellular vesicle (sEV)-derived signature as a non-invasive longitudinal screening tool in ovarian cancer. We identified a 7-gene panel in these sEVs that overlapped with an established tissue-derived metastatic ovarian carcinoma signature. We found the 7-gene panel to be differentially expressed with tumor development and metastatic spread. While there were quantifiable changes in genes from the 7-gene panel in plasma-derived sEVs from ovarian cancer patients, we were unable to establish a definitive signature due to low sample number. The most notable finding was a significant change in the ascites-derived sEV gene signature that overlapped with that of the plasma-derived sEV signature at varying stages of disease progression. Taken together our findings show that differential expression of metastatic genes derived from circulating sEVs present a minimally invasive screening tool for ovarian cancer detection and longitudinal monitoring of molecular changes associated with progression and metastatic spread.

cancer biology↗

bioRxiv2021

Establishment and Resilience of Transplanted Gut Microbiota in Aged Mice

Fecal microbiota transplantation (FMT), a procedure in which fecal material is transferred from a donor to a recipient, has been increasingly used as a treatment to restore healthy gut microbiota. There is a substantial difference in the composition of gut microbiota between young and aged hosts, but little is known about whether age matching between the FMT donor and recipient affects microbiota restoration and long-term maintenance. In the present investigation, we aimed to study the establishment and resilience of transplanted gut microbiota in aged recipients. We treated naturally aged mice (20 months old) with a broad-spectrum antibiotic cocktail and monitored the restoration of gut microbiota over 8 weeks. The diversity of gut microbiota in aged mice failed to reach the baseline level via spontaneous recovery; in contrast, FMT from either (age-)matched or unmatched donors facilitated the recovery of gut microbiota diversity. The microbiota transplanted from different donors successfully established in the aged recipients and had long-term effects on the gene expression profiles of the host colon. Finally, we evaluated the long-term maintenance of transplanted microbiota via intentional disruption of gut homeostasis. We found that lack of age matching between FMT donors and recipients may decrease the resilience of transplanted gut microbiota against colonic inflammation. The results from our study systematically examining the effects of FMT on the gut homeostasis of aged hosts suggest that the compatibility between donors and recipients should be taken into account when implementing FMT.

microbiology↗

bioRxiv2020

Topoisomerase 1 inhibition therapy protects against SARS-CoV-2-induced inflammation and death in animal models.

The ongoing pandemic caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is currently affecting millions of lives worldwide. Large retrospective studies indicate that an elevated level of inflammatory cytokines and pro-inflammatory factors are associated with both increased disease severity and mortality. Here, using multidimensional epigenetic, transcriptional, in vitro and in vivo analyses, we report that Topoisomerase 1 (Top1) inhibition suppresses lethal inflammation induced by SARS-CoV-2. Therapeutic treatment with two doses of Topotecan (TPT), a FDA-approved Top1 inhibitor, suppresses infection-induced inflammation in hamsters. TPT treatment as late as four days post-infection reduces morbidity and rescues mortality in a transgenic mouse model. These results support the potential of Top1 inhibition as an effective host-directed therapy against severe SARS-CoV-2 infection. TPT and its derivatives are inexpensive clinical-grade inhibitors available in most countries. Clinical trials are needed to evaluate the efficacy of repurposing Top1 inhibitors for COVID-19 in humans.

microbiology↗

bioRxiv2020

F-Seq2: improving the feature density based peak caller with dynamic statistics

Genomic and epigenomic features are captured at a genome-wide level by using high-throughput sequencing technologies. Peak calling is one of the first essential steps in analyzing these features by delineating regions such as open chromatin regions and transcription factor binding sites. Our original peak calling software, F-Seq, has been widely used and shown to be the most sensitive and accurate peak caller for DNase I hypersensitive sites sequencing (DNase-seq) data. However, F-Seq lacks support for user-input control dataset nor reporting test statistics, limiting its ability to capture systematic and experimental biases and accurately estimate background distributions. Here we present an improved version, F-Seq2, which combined the power of kernel density estimation and a dynamic "continuous" Poisson distribution to robustly account for local biases and solve ties when ranking candidate peaks. In F-score and motif distance analysis, we demonstrated the superior performance of F-Seq2 than other competing peak callers used by the ENCODE Consortium on simulated and real ATAC-seq and ChIP-seq datasets. The output of F-Seq2 is suitable for irreproducible discovery rate (IDR) analysis as the test statistics calculated for individual candidate summit and ties are robustly solved.

bioinformatics↗

bioRxiv2020

Impact of temporal pH fluctuations on the coexistence of nasal bacteria

To manipulate nasal microbiota for respiratory health, we need to better understand how this microbial community is assembled and maintained. Previous work has demonstrated that the pH in the nasal passage experiences temporal fluctuations. Yet, the impact of such pH fluctuations on nasal microbiota is not fully understood. Here, we examine how temporal fluctuations in pH might affect the coexistence of nasal bacteria. We take advantage of the cultivability of nasal bacteria to experimentally assess their responses to pH. Based on experimentally observed responses, we formulate a mathematical model to numerically investigate the impact of temporal pH fluctuations on species coexistence. Through extensive numerical simulations, we find that the composition of nasal communities is robust against pH fluctuations. Our results suggest that nasal microbiota could be more robust than expected against environmental fluctuations.

ecology↗