Biology subjects

Zhang, W.

Publications and source records attributed to Zhang, W..

At least 631 records · Page 35Linked to original sources

bioRxiv2022

A Community Challenge to Predict Clinical Outcomes After Immune Checkpoint Blockade in Non-Small Cell Lung Cancer

PurposePredictive biomarkers of immune checkpoint inhibitors (ICIs) efficacy are currently lacking for non-small cell lung cancer (NSCLC). Here, we describe the results from the Anti-PD-1 Response Prediction DREAM Challenge, a crowdsourced initiative that enabled the assessment of predictive models by using data from two randomized controlled clinical trials (RCTs) of ICIs in first-line metastatic NSCLC. MethodsParticipants developed and trained models using public resources. These were evaluated with data from the CheckMate 026 trial (NCT02041533), according to the model-to-data paradigm to maintain patient confidentiality. The generalizability of the models with the best predictive performance was assessed using data from the CheckMate 227 trial (NCT02477826). Both trials were phase III RCTs with a chemotherapy control arm, which supported the differentiation between predictive and prognostic models. Isolated model containers were evaluated using a bespoke strategy that considered the challenges of handling transcriptome data from clinical trials. ResultsA total of 59 teams participated, with 417 models submitted. Multiple predictive models, as opposed to a prognostic model, were generated for predicting overall survival, progression-free survival, and progressive disease status with ICIs. Variables within the models submitted by participants included tumor mutational burden (TMB), programmed death ligand 1 (PD-L1) expression, and gene-expression-based signatures. The bestperforming models showed improved predictive power over reference variables, including TMB or PD-L1. ConclusionThis DREAM Challenge is the first successful attempt to use protected phase III clinical data for a crowdsourced effort towards generating predictive models for ICIs clinical outcomes and could serve as a blueprint for similar efforts in other tumor types and disease states, setting a benchmark for future studies aiming to identify biomarkers predictive of ICIs efficacy. Context summaryO_ST_ABSKey objectiveC_ST_ABSNot all patients with non-small cell lung cancer (NSCLC) eligible for immune checkpoint inhibitor (ICIs) respond to treatment, but accurate predictive biomarkers of ICIs clinical outcomes are currently lacking. This crowdsourced initiative enabled the robust assessment of predictive models using data from two randomized clinical trials of first-line ICI in metastatic NSCLC. Knowledge generatedModels submitted indicate that a combination of programmed death ligand 1 (PD-L1), tumor mutational burden (TMB), and immune gene signatures might be able to identify patients more likely to respond to ICIs. TMB and PD-L1 seemed important to predict progression-free survival and overall survival. Mechanisms including apoptosis, T-cell crosstalk, and adaptive immune resistance appeared essential to predict response. Relevance

cancer biology↗

bioRxiv2022

Ring-finger protein 34 facilitates nervous necrosis virus evading antiviral innate immunity by targeting TBK1 and IRF3 for ubiquitination and degradation

Ubiquitination, as one of the most prevalent posttranslational modifications of proteins, enables a tight control on host immune responses. Many viruses hijack the host ubiquitin system to regulate host antiviral responses for their survival. Here, we found that fish pathogen nervous necrosis virus (NNV) recruited an E3 ubiquitin ligase ring finger protein 34 (RNF34) to inhibit RLRs-mediated interferons (IFN) response via ubiquitinating TBK1 and IRF3. Ectopic expression of RNF34 greatly enhances NNV replication and prevents IFN production, while deficiency of RNF34 led to the opposite effect. Furthermore, RNF34 targets TBK1 and IRF3 via its RING domain. Of note, the interactions between RNF34 and TBK1 or IRF3 were conserved in different fish species. Mechanically, RNF34 promote K27-linked ubiquitination and degradation of TBK1 and IRF3, which in turn diminishing TBK1-induced translocation of IRF3 from cytoplasm to nucleus. Ultimately, NNV capsid protein (CP) was found directly bind with RNF34 and this interaction was conserved in different fishes, and CP induced TBK1 and IRF3 degradation and IFN suppression was depended on RNF34. Our finding demonstrated a novel mechanism by which NNV CP evaded host innate immunity via RNF34, and provided a potential drug target for the control of NNV infection. Author SummaryUbiquitination plays an essential role in the regulation of innate immune responses to pathogens. NNV, a kind of RNA virus, is the causal agent of a highly destructive disease in a variety of marine and freshwater fish. Previous study reported NNV could hijack the ubiquitin system to manipulate the hosts immune responses, however, how NNV utilizes ubiquitination to facilitate its own replication is not well understood. Here, we identified a novel distinct role of E3 ubiquitin ligase RNF34 as an IFN antagonist to promote NNV infection. Nervous necrosis virus capsid protein utilized RNF34 to target TBK1 and IRF3 for K27 and K48-linked ubiquitination degradation. Importantly, the interactions between RNF34 and CP, TBK1 or IRF3 are conserved in different fishes, suggesting it is a general immune evasion strategy exploited by NNV to target the IFN response via RNF34.

immunology↗

bioRxiv2022

JMJD3 is a Key Regulator of Dopamine Synthesis in Ventral Tegmental Area Dopamine Neurons.

Chronic pain is associated with midbrain dopamine levels. The molecules and mechanisms modulating this association remain to be elucidated. By using conditional knockout mice, we report that JMJD3 deficiency in midbrain dopamine neurons causes prolonged mechanical hyperalgesia regardless of sex and age. Both genetic defect and pharmaceutical inhibition of JMJD3 decrease dopamine level in midbrain and striatum, resulting from reduced tyrosine hydroxylase expression in midbrain dopamine neurons. Furthermore, epigenetic experiments reveal that JMJD3 is indispensable for the transcription of tyrosine hydroxylase through direct and indirect manners. These findings suggest JMJD3 and midbrain dopamine neurons as novel players and pharmaceutical targets for chronic pain regulation.

neuroscience↗

bioRxiv2022

A tRNA Modification-based strategy for Identifying amiNo acid Overproducers (AMINO)

Amino acids have a multi-billion-dollar market with rising demand, prompting the development of high-performance microbial factories. However, a general screening strategy applicable to all proteinogenic and non-proteinogenic amino acids is still lacking. Modification of the critical structure of tRNA could decrease the aminoacylation level of tRNA catalyzed by aminoacyl-tRNA synthetases. Involved in a two-substrate sequential reaction, amino acids with increased concentration could elevate the reduced aminoacylation rate caused by specific tRNA modification. Here, we developed a selection system for overproducers of specific amino acids using corresponding engineered tRNAs and reporter genes. As a proof-of-concept, overproducers of five amino acids such as L-tryptophan were screened out by growth-based and/or fluorescence-activated cell sorting (FACS)-based screening from random mutation libraries of Escherichia coli and Corynebacterium glutamicum, respectively. This study provided a universal strategy that could be applied to screen overproducers of proteinogenic and non-proteinogenic amino acids in amber-stop-codon-recoded or non-recoded hosts.

synthetic biology↗

bioRxiv2022

ppGpp is Present in and Functions to Regulate Sleep in Drosophila

Sleep is essential for animals, and receives inputs from circadian, homeostasis, and environment, yet the mechanisms of sleep regulation remain elusive. Discovery of molecules in living systems and demonstration of their functional roles are pivotal in furthering our understanding of the molecular basis of biology. Here we report that ppGpp (guanosine-5-diphosphate, 3-diphosphate), a molecule that has been detected in prokaryotes for more than five decades, is present in Drosophila, and plays an important role in regulation of sleep and SISL (starvation induced sleep loss). ppGpp is detected in germ-free Drosophila and hydrolyzed by an enzyme encoded by the mesh1 gene in Drosophila. Nighttime sleep and SISL were defected in mesh1 mutant flies, and rescued by expression of wildtype Mesh1, but not the enzymatically defective mutant Mesh1E66A. Ectopic expression of RelA, the E. coli synthetase for ppGpp, phenocopied mesh1 knockout mutants, whereas overexpression of Mesh1 resulted in the opposite phenotypes, supporting that ppGpp is both necessary and sufficient in sleep regulation. A chemoconnectomic screen followed by genetic intersection experiments implicate the Dilp2 neurons in the pars intercerebralis (PI) brain region as the site of ppGpp function. Our results have thus supported that ppGpp is present in animals after long lag since its discovery in bacteria, and revealed a physiological role of ppGpp in sleep regulation for the first time.

genetics↗

bioRxiv2022

Human Early Syncytiotrophoblasts Are Highly Susceptible to SARS-CoV-2 Infection

The ongoing and devastating pandemic of coronavirus disease 2019 (COVID-19) has led to a global public health crisis. COVID-19 is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and can potentially pose a serious risk to maternal and neonatal health. Cases of abnormal pregnancy and vertical transmission of SARS-CoV-2 from mother to foetus have been reported but no firm conclusions are drawn. Trophoblasts are the major constituents of the placenta to protect and nourish the developing foetus. However, direct in vivo investigation of trophoblasts susceptibility to SARS-CoV-2 and of COVID-19 and pregnancy is challenging. Here we report that human early syncytiotrophoblasts (eSTBs) are highly susceptible to SARS-CoV-2 infection in an angiotensin-converting enzyme 2 (ACE2)-dependent manner. From human expanded potential stem cells (hEPSCs), we derived bona fide trophoblast stem cells (TSCs) that resembled those originated from the blastocyst and the placenta in generating functional syncytiotrophoblasts (STBs) and extravillus trophoblasts (EVTs) and in low expression of HLA-A/B and amniotic epithelial (AME) cell signature. The EPSC-TSCs and their derivative trophoblasts including trophoblast organoids could be infected by SARS-CoV-2. Remarkably, eSTBs expressed high levels of ACE2 and produced substantially higher amounts of virion than Vero E6 cells which are widely used in SARS-CoV-2 research and vaccine production. These findings provide experimental evidence for the clinical observations that opportunistic SARS-CoV-2 infection during pregnancy can occur. At low concentrations, two well characterized antivirals, remdesivir and GC376, effectively eliminated infection of eSTBs by SARS-CoV-2 and middle east respiratory syndrome-related coronavirus (MERS-CoV), and rescued their developmental arrest caused by the virus infection. Several human cell lines have been used in coronavirus research. However, they suffer from genetic and/or innate immune defects and have some of the long-standing technical challenges such as cell transfection and genetic manipulation. In contrast, hEPSCs are normal human stem cells that are robust in culture, genetically stable and permit efficient gene-editing. They can produce and supply large amounts of physiologically relevant normal and genome-edited human cells such as eSTBs for isolation, propagation and production of coronaviruses for basic research, antivirus drug tests and safety evaluation.

cell biology↗

bioRxiv2022

KBPRNA: A novel method integrating bulk RNA-seq data and LINCS-L1000 gene signatures to predict kinase activity based on machine learning

BackgroundKinases are a type of enzymes which can transfer phosphate groups from high-energy and phosphate-donating molecules to specific substrates. Kinase activities could be utilized to be represented as specific biomarkers of specific cancer types. Nowadays novel algorithms have already been developed to compute kinase activities from phosphorylated proteomics data. However, phosphorylated proteomics sequencing could be costly expensive and need valuable samples. Moreover,not methods which could achieve kinase activities from bulk RNA-sequence data have been developed. Here we propose KBPRNA, a general computational framework for extracting specific kinase activities from bulk RNA-sequencing data in cancer samples. KBPRNA also achieves better performance in predicting kinase activities from bulk RNA-sequence data under cancer conditions benchmarking against other models. ResultsIn this study, we used LINCS-L1000 dataset which was used to be reported as efficient gene signatures in defining bulk RNA-seq data as input dataset of KBPRNA. Also, we utilized eXtreme Gradient Boosting (XGboost) as the main algorithm to extract valuable information to predict kinase activities. This model outperforms other methods such as linear regression and random forest in predicting kinase activities from bulk RNA-seq data. KBPRNA integrated tissue samples coming from breast invasive carcinoma, hepatocellular carcinoma, lung squamous cell carcinoma, Glioblastoma multiforme and Uterine Corpus Endometrial Carcinoma. It was found that KBPRNA achieved good performance with an average R score above threshold of 0.5 in kinase activity prediction. ConclusionsModel training and testing process showed that KBPRNA outperformed other machine learning methods in predicting kinase activities coming from various cancer types tissue samples. This model could be utilized to approximate basic kinase activities and link it with specific biological functions, which in further promoted the progress of cancer identification and prognosis.

bioinformatics↗

bioRxiv2022

A stable reference human transcriptome and proteome as a standard for reproducible omics experiments

In recent years, the development of high-throughput omics technology has greatly promoted the development of biomedicine. However, the poor reproducibility of omics techniques limits its application. It is necessary to use standard reference materials of complex RNAs or proteins to test and calibrate the accuracy and reproducibility of omics workflows. However, the transcriptome and proteome of most cell lines shift during culturing, which limits their applicability to serve as standard samples. In this study, we demonstrated that the human hepatocellular cell line MHCC97H has a very stable transcriptome (R2=0.966-0.995) and proteome (R2=0.934-0.976 for DDA, R2=0.942-0.986 for DIA) after 9 subculturing generations, which allows this stable standard sample to be stably produced on an industrial scale for several decades. Moreover, this stability was maintained across labs and platforms. In sum, our results justified a omics standard reference material and reference datasets for transcriptomic and proteomics research. This helps to further standardize the workflow and data quality of omics techniques and thus promotes the application of omics technology in precision medicine.

cell biology↗

bioRxiv2022

Spatial Localization Ability of Planarians Identified Through the Light Maze Paradigm

Spatial localization ability is crucial for free-living animals to fit the environment. As shown by previous studies, planarians can be conditioned to discriminate directions. However, due to their simplicity and primitiveness, they had never been considered to have true spatial localization ability to retrieve locations of objects and places in the environment. Here, we introduce a light maze training paradigm to demonstrate that a planarian worm can navigate to a former recognized place from the start point, even if the worm is transferred into a newly produced maze. This finding identifies the spatial localization ability of planarians for the first time, which provides clues for the evolution of spatial learning. Since the planarians have a primitive brain with simple structures, this paradigm can also provide a simplified model for a detailed investigation of spatial learning.

animal behavior and cognition↗

bioRxiv2022

Personalized Deep Learning based Source Imaging Framework Improves the Imaging of Epileptic Sources from MEG Interictal Spikes

Electromagnetic source imaging (ESI) has been widely used to image brain activities for research and clinical applications from MEG and EEG. It is a challenging task due to the ill-posedness of the problem and the complexity of modeling the underlying brain dynamics. Deep learning has gained attention in the ESI field for its ability to model complex distributions and has successfully demonstrated improved imaging performance for ESI. In this work, we investigated the capability of imaging epileptic sources from MEG interictal spikes using deep learning-based source imaging framework (DeepSIF). A generic DeepSIF model was first trained with a generic head model using a template MRI. A fine-tuning procedure was proposed to introduce personalized head model information into the neural network for a personalized DeepSIF model. Two models were evaluated and compared in extensive computer simulations. The MEG-DeepSIF approach was further rigorously validated for imaging epileptogenic regions from interictal spike recordings in focal epilepsy patients. We demonstrated that DeepSIF can be successfully applied to MEG recordings and the additional fine-tuning step for personalized DeepSIF can alleviate the impact of head model variations and further improve the performance significantly. In a cohort of 29 drug-resistant focal epilepsy patients, the personalized DeepSIF model provided a sublobar concordance of 93%, sublobar sensitivity of 77% and specificity of 99%, respectively. When compared to the seizure-onset-zone defined by intracranial recordings, the localization error is 15.78 {+/-} 5.54 mm; and when compared with resection volume in seizure free patients, the spatial dispersion is 8.19 {+/-} 8.14 mm. DeepSIF enables an accurate and robust imaging of spatiotemporal brain dynamics from MEG recordings, suggesting its unique value to neuroscience research and clinical applications.

bioengineering↗

bioRxiv2022

A pan-cancer interrogation of intronic polyadenylation and its association with cancer characteristics

mRNA cleavage and polyadenylation is an integral 2-step process in the generation of protein-encoding mRNA or noncoding transcripts. More than 60% of human genes have multiple polyadenylation sites either in the 3 untranslated region (3UTR-APA) or in the intronic/exonic region, resulting in expression of isoforms with alternative polyadenylation (APA) under different physiologic conditions. The 3UTR-APAs have been extensively studied, but the biology of intronic polyadenylations (IPA) remain largely unexplored. Here we characterized the IPA profiles of 9,679 patient samples across 32 cancer types from the Cancer Genome Atlas (TCGA) cohort. Overall, we identified 22,260 detectable IPA sites; 9,014 (40.5%) occurred in all 32 cancer types and 11,676 (52.5%) occurred in 2 to 31 cancer types. By comparing tumors and their paired normal tissues, we identified 180 to 4,645 dysregulated IPAs in 132 to 2,249 genes in each of 690 patient tumors from 22 cancer types that showed consistent patterns within individual cancer types. Furthermore, across all cancer types, IPA isoforms and their gene regulation showed consistent pan-cancer patterns, and cancer types with similar histologic features were clustered at higher levels of hierarchy. We selected 2,741 genes that were consistently regulated by IPAs across cancer types, including 1,834 pan-cancer tumor-enriched and 907 tumor-depleted IPA genes. Pan-cancer tumor-enriched IPA genes were amply represented in the functional pathways such as cilium assembly and DNA damage repair. Expression of IPA isoforms in DNA damage repair genes was associated with tumor mutation burdens. Expression of IPA isoforms of tumor-enriched IPA genes was also associated with patient characteristics (e.g., sex, race, cancer stages, and subtypes) in cancer-specific and feature-specific manners. Importantly, IPA isoform expression for some genes could be a more accurate prognostic marker than gene expression (summary of all possible isoforms). In summary, our study reveals the roles and the clinical relevance of tumor-associated IPAs in cancer.

bioinformatics↗

bioRxiv2022

Normalized Semi-Covariance Co-Efficiency Analysis of Spike Proteins from SARS-CoV-2 variant Omicron and Other Coronaviruses for their Infectivity and Virulence

Spectrum-based Mass-Charge modeling is increasingly used in biological analysis. To explain statistical phenomenon with positive and negative fluctuations of amino acid charges in spike protein sequences from Omicron and other coronaviruses, we propose calculation-based Mass-Charge modeling, a normalized derivation algorithm with exact Excel and MATLAB tool involving separate quadrant extension to normalized covariance, which is still compatible with Pearson covariance co-efficiency. The number of amino acids, molecular weight, isoelectric point, amino acid composition, charged residues, mass-charge ratio, hydropathicity of the proteins were taken into consideration in the analyses, and the relative peak and dip of the average with spike protein sequences based on hydrophobic mass to isoelectric charges of amino acids were also examined. The analyses with the algorithm provide more clear insights leading to revealing underline evolving trends of the viral proteins. Spike proteins from SARS-CoV-2 variants, seasonal and murine coronaviruses were taken as representative examples in this study. The analyses demonstrate that the Mass-Charge covariance co-efficiency can distinguish subtle differences between biological properties of spike proteins and correlate well with viral infectivity and virulence.

bioinformatics↗

bioRxiv2022

Membrane-remodeling protein ESCRT-III homologs incarnate the evolution and morphogenesis of multicellular magnetotactic bacteria

Endosomal sorting complex required transport (ESCRT) III proteins are essential for membrane remodeling and repair across all domains of life. Eukaryotic ESCRT-III and the cyanobacterial homologs PspA and Vipp1/Imm30 remodel membrane into vesicles, rings, filaments and tubular rods structures. Here our microscopy analysis showed that multicellular bacteria, referred to as magnetoglobules, possess multiple compartments including magnetosome organelles, polyphosphate granules, vesicles, rings, tubular rods, filaments and MVB-like structures. Therefore, membrane remodeling protein PspA might be required for the formation of these compartments, and contribute to the morphogenesis and evolution of multicellularity. To assess these hypotheses, we sequenced nine genomes of magnetoglobules and found a significant genome expansion compared to unicellular magnetotactic bacteria. Moreover, PspA was ubiquitous in magnetoglobules and formed a distinct clade on the tree of eubacterial and archaeal ESCRT-III. The phylogenetic feature suggested the evolution of magnetoglobules from a unicellular ancestor of deltaproteobacterium. Hetero-expression of ellipsoidal magnetoglobule pspA2 gene alone in Escherichia coli resulted in intracellular membrane aggregation. GFP fusion labeling revealed polar location of PspA2 in rod-shaped unicells and regular interval location in filamentous cells. Cryo-electron tomography analysis showed filament bundle, membrane sacculus, vesicles and MVB-like structure in the cells expressing PspA2. Moreover, electron-dense area with a similar distribution as GFP-PspA2 foci in filamentous cells changed the inward orientation of the septum, which might interfere with the cell division. Collectively, these results show the membrane remodeling function of magnetoglobule PspA proteins, which may contribute to morphogenesis and the evolution of multicellularity of magnetotactic bacteria.

microbiology↗

bioRxiv2022

Cryo-EM structures of amyloid-beta filaments with the Arctic mutation (E22G) from human and mouse brains

The Arctic mutation, encoding E693G in the amyloid precursor protein (APP) gene [E22G in amyloid-{beta} (A{beta})], causes dominantly inherited Alzheimers disease. Here we report the high-resolution cryo-EM structures of A{beta} filaments from the frontal cortex of a previously described case (A{beta}PParc1) with the Arctic mutation. Most filaments consist of two pairs of non-identical protofilaments that comprise residues V12-V40 (human Arctic fold A) and E11-G37 (human Arctic fold B). They have a substructure (residues F20-G37) in common with the folds of type I and type II A{beta}42. When compared to the structures of wild-type A{beta}42 filaments, there are subtle conformational changes in the human Arctic folds, because of the lack of a side chain at G22, which may strengthen hydrogen bonding between mutant A{beta} molecules and promote filament formation. A minority of A{beta}42 filaments of type II was also present, as were tau paired helical filaments. In addition, we report the cryo-EM structures of A{beta} filaments with the Arctic mutation from mouse knock-in line AppNL-G-F. Most filaments are made of two identical mutant protofilaments that extend from D1-G37 (murine Arctic fold). In a minority of filaments, two dimeric folds pack against each other in an anti-parallel fashion. The murine Arctic fold differs from the human Arctic folds, but shares some substructure.

neuroscience↗

bioRxiv2022

Adenovirus type 34 and HVR1-deleted Adenovirus type 5 do not bind to PF4: clearing the path towards vectors without thrombosis risk

The adenoviral vector based AstraZeneca and Janssen COVID vaccines have been associated with rare cases of thrombosis, a condition which depends on adenovirus binding to the blood protein Platelet Factor 4 (PF4). In order to identify adenoviruses with low or absent affinity for PF4, we screened dozens of types from various adenovirus species, and Adenovirus type 5 (Ad5) derived vectors carrying genetic or chemical modifications of different hexon hyper-variable regions (HVR). For this purpose, we established an armamentarium of techniques including ELISA-qPCR and Aggregate Pull-Down (APD), which enabled fast and sensitive assessments of virus-protein interactions. Unlike most tested serotypes, Ad34 did not bind to PF4. Likewise, the deletion or shielding of the HVR1 loop of Ad5 seemingly ablated its PF4 binding. Therefore, we showed that PF4 binds to adenovirus hexon through interactions dependent on HVR1, and identified vectors that may avoid or decrease the risk of thrombosis and represent safer candidates for vaccine or gene therapy vector development.

microbiology↗

bioRxiv2022

Integrated Network Pharmacology and Experimental Analysis Unveil Modulation of EGFR/MAPK Signaling Cascades in Acute Cerebral Ischemia-Reperfusion Injury by Qing-Tong-Hua-Yu Decoction

ObjectiveBased on network pharmacology, the response of Qing-tong-hua-yu Decoction (QTHY) to the regulation of EGFR/MAPK signaling cascade in cerebral ischemia-reperfusion injury was discussed and the possible mechanism of the protective effect of QTHY on the cerebral ischemia-reperfusion injury was studied. MethodsA compound-target disease-function-pathway network was established and analyzed based on the network pharmacology approach used in Chinese medicine. The correlation, which is between effect of the components of QTHY Decoction against CI/RI with EGFR/MAPK signalling cascade response, was observed. And then the degree of neurological deficits in each group was assessed after cerebral ischemia for 2 hours and reperfusion for 3 hours, 24 hours, 3 days and 7 days. Expression levels of EGFR and p44/42MAPK in ischemic brain tissue at different time points in various groups of rats were tested by Western bolt (WB), real-time quantitative PCR (RT-qPCR) and immunohistochemistry (IHC). ResultsNetwork pharmacology analysis revealed that QTHY-mediated treatment involved 439 key targets, in which the effect of QTHY groups against CI/RI was associated with EGFR/MAPK signaling cascade. QTHY treatment reduced neurological deficit scores and improved ischemic changes in rats. In addition, QTHY promoted EGFR and p44/42MAPK expression in the SVZ through the EGFR/MAPK signaling cascade, with varying degrees of improvement at different time points. ConclusionQTHY can better improve cerebral ischemia injury in CI / RI rats and exert the neuroprotective effect of cerebral ischemia-reperfusion injury. This may be related to the potential of QTHY to activate the EGFR / MAPK signaling cascade, which is consistent with the results of network pharmacology analysis.

neuroscience↗

bioRxiv2022

Instantaneous visual genotyping and facile site-specific transgenesis via CRISPR-Cas9 and phiC31 integrase

The zebrafish Danio rerio has become a popular model in functional genomics and genetic disease studies. However, when a zebrafish mutant line must be propagated as heterozygotes due to homozygous lethality, using standard genotyping methods to identify a population of homozygous mutant embryos is time-consuming and sometimes impractical due to downstream applications such as large-scale chemical screens. Here, we introduce TICIT, Targeted Integration by CRISPR-Cas9 and Integrase Technologies, which utilizes the site-specific DNA recombinase - phiC31 integrase - to insert fluorescent markers into CRISPR-Cas9-generated mutant alleles. It allows instantaneous determination of a zebrafishs genotype simply by examining its color. This technique, which relies on first knocking in a 39-basepair phiC31 landing site via CRISPR-Cas9, enables researchers to insert large DNA fragments at the same genomic location repeatedly and with high precision and efficiency. We demonstrated that TICIT could also be used to create reporter fish driven by an endogenous promoter. Additionally, we created a landing site located in the tyrosinase gene that could support transgene expression in a broad spectrum of tissue and cell types, acting as a putative safe harbor locus. Hence, TICIT can yield predictable and reproducible transgene expression, facilitate diverse applications in zebrafish, and may be applicable to cells in culture and other model organisms.

bioengineering↗

bioRxiv2022

Development of Scaffold-free 3D Cholangiocyte Organoids to Study the Progression of Primary Sclerosing Cholangitis

Organoids are novel in vitro models to study intercellular crosstalk between the different types of cells in the pathophysiology of disease. To better understand the underlying mechanisms driving the progression of primary sclerosing cholangitis (PSC), we developed scaffold-free multi-cellular 3D cholangiocyte organoids (3D-CHO) using primary liver cell lines derived from normal and PSC patients. Human liver samples from healthy donors and late-stage PSC patients were used to isolate primary cholangiocytes (EPCAM+/CK-19+), liver endothelial cells (LECs, CD31+), and hepatic stellate cells (HSCs, CD31-/CD68-/Desmin+/Vitamin A+). 3D-CHOs were formed using cholangiocytes:HSCs:LECs and kept viable for up to 1 month. Isolated primary cell lines and 3D-CHOs were further characterized by immunofluorescence (IF), qRT-PCR, and transmission electron microscopy. Gene expressions for cholangiocytes (SOX9, CFTR, EpCAM, AE, SCT, SCTR), fibrosis (ACTA2, COL1A1, DESMIN, TGF{beta}1), angiogenesis (PECAM, VEGF, CDH5, vWF), and inflammation (IL-6, TNF-) confirmed PSC phenotypes of 3D-CHOs. Since cholangiocytes develop a neuroendocrine phenotype and express neuromodulators, confocal-IF demonstrated that neurokinin-1 receptor (NK-1R, expressed by cholangiocytes and upregulated in PSC), was localized within CK-19+ cholangiocytes. Moreover, 3D-CHOs from PSC patients confirmed PSC phenotypes with upregulated NK-1R, tachykinin precursor 1, and downregulated membrane metalloendopeptidase. Our viable scaffold-free multiple-cell 3D-CHOs showed superiority as an in vitro model in mimicking PSC in vivo phenotypes compared to 2D cell culture, which can be used in PSC disease-related research.

bioengineering↗