Long-term expandable mouse and human-induced nephron progenitor cells enable kidney organoid maturation and modeling of plasticity and disease.

Nephron progenitor cells (NPCs) self-renew and differentiate into nephrons, the functional units of the kidney. Here, manipulation of p38 and YAP activity allowed for long-term clonal expansion of primary mouse and human NPCs and induced NPCs (iNPCs) from human pluripotent stem cells (hPSCs). Molecular analyses demonstrated that cultured iNPCs closely resemble primary human NPCs. iNPCs generated nephron organoids with minimal off-target cell types and enhanced maturation of podocytes relative to published human kidney organoid protocols. Surprisingly, the NPC culture medium uncovered plasticity in human podocyte programs, enabling podocyte reprogramming to an NPC-like state. Scalability and ease of genome editing facilitated genome-wide CRISPR screening in NPC culture, uncovering genes associated with kidney development and disease. Further, NPC-directed modeling of autosomal-dominant polycystic kidney disease (ADPKD) identified a small-molecule inhibitor of cystogenesis. These findings highlight a broad application for the reported iNPC platform in the study of kidney development, disease, plasticity, and regeneration.

Digital neuropsychological measures by defense automated neurocognitive assessment: reference values and clinical correlates.

Introduction: Although the growth of digital tools for cognitive health assessment, there's a lack of known reference values and clinical implications for these digital methods. This study aims to establish reference values for digital neuropsychological measures obtained through the smartphone-based cognitive assessment application, Defense Automated Neurocognitive Assessment (DANA), and to identify clinical risk factors associated with these measures. Methods: The sample included 932 cognitively intact participants from the Framingham Heart Study, who completed at least one DANA task. Participants were stratified into subgroups based on sex and three age groups. Reference values were established for digital cognitive assessments within each age group, divided by sex, at the 2.5th, 25th, 50th, 75th, and 97.5th percentile thresholds. To validate these values, 57 cognitively intact participants from Boston University Alzheimer's Disease Research Center were included. Associations between 19 clinical risk factors and these digital neuropsychological measures were examined by a backward elimination strategy. Results: Age- and sex-specific reference values were generated for three DANA tasks. Participants below 60 had median response times for the Go-No-Go task of 796 ms (men) and 823 ms (women), with age-related increases in both sexes. Validation cohort results mostly aligned with these references. Different tasks showed unique clinical correlations. For instance, response time in the Code Substitution task correlated positively with total cholesterol and diabetes, but negatively with high-density lipoprotein and low-density lipoprotein cholesterol levels, and triglycerides. Discussion: This study established and validated reference values for digital neuropsychological measures of DANA in cognitively intact white participants, potentially improving their use in future clinical studies and practice.

Intrinsic targeting of host RNA by Cas13 constrains its utility.

Cas13 can be used for the knockdown, editing, imaging or detection of RNA and for RNA-based gene therapy. Here by using RNA immunoprecipitation sequencing, transcriptome profiling, biochemical analysis, high-throughput screening and machine learning, we show that Cas13 can intrinsically target host RNA in mammalian cells through previously unappreciated mechanisms. Different from its known cis/trans RNA-cleavage activity, Cas13 can also cleave host RNA via mechanisms that are transcript-specific, independent of the sequence of CRISPR RNA and dynamically dependent on the conformational state of Cas13, as we show for several Cas13-family effectors encoded in one-vector and two-vector lentiviral systems. Moreover, host genes involved in viral processes and whose transcripts are intrinsically targeted by Cas13 contribute to constraining the lentiviral delivery and expression of Cas13. Our findings offer guidance for the appropriate use of lentiviral Cas13 systems and highlight the need for caution regarding intrinsic RNA targeting in Cas13-based applications.

USP10 strikes down ß-catenin by dual-wielding deubiquitinase activity and phase separation potential.

Wnt/ß-catenin signaling is a conserved pathway crucially governing development, homeostasis, and oncogenesis. Discoveries of its regulators hold great values in both basic and translational research. Through screening, we identified a deubiquitinase, USP10, as a critical modulator of ß-catenin. Mechanistically, USP10 binds to key scaffold Axin1 via conserved motifs and stabilizes Axin1 through K48-linked deubiquitination. Surprisingly, USP10 physically tethers Axin1 and ß-catenin and promotes the phase separation for ß-catenin suppression regardless of the enzymatic activity. Function-wise, USP10 enzymatic activity preferably regulates embryonic development and both the enzymatic activity and physical function jointly control intestinal homeostasis by antagonizing ß-catenin. In colorectal cancer, USP10 substantially represses cancer growth mainly through physical promotion of phase separation and correlates with Wnt/ß-catenin magnitude clinically. Collectively, we discovered USP10 functioning in multiple biological processes against ß-catenin and unearthed the enzyme-dependent and -independent "dual-regulating" mechanism. These two functions of USP10 work in parallel and are context dependent.

A deep learning-based drug repurposing screening and validation for anti-SARS-CoV-2 compounds by targeting the cell entry mechanism.

The recent outbreak of Corona Virus Disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been a severe threat to the global public health and economy, however, effective drugs to treat COVID-19 are still lacking. Here, we employ a deep learning-based drug repositioning strategy to systematically screen potential anti-SARS-CoV-2 drug candidates that target the cell entry mechanism of SARS-CoV-2 virus from 2635 FDA-approved drugs and 1062 active ingredients from Traditional Chinese Medicine herbs. In silico molecular docking analysis validates the interactions between the top compounds and host receptors or viral spike proteins. Using a SARS-CoV-2 pseudovirus system, we further identify several drug candidates including Fostamatinib, Linagliptin, Lysergol and Sophoridine that can effectively block the cell entry of SARS-CoV-2 variants into human lung cells even at a nanomolar scale. These efforts not only illuminate the feasibility of applying deep learning-based drug repositioning for antiviral agents by targeting a specified mechanism, but also provide a valuable resource of promising drug candidates or lead compounds to treat COVID-19.

Modeling kidney development, disease, and plasticity with clonal expandable nephron progenitor cells and nephron organoids.

Nephron progenitor cells (NPCs) self-renew and differentiate into nephrons, the functional units of the kidney. Here we report manipulation of p38 and YAP activity creates a synthetic niche that allows the long-term clonal expansion of primary mouse and human NPCs, and induced NPCs (iNPCs) from human pluripotent stem cells. Cultured iNPCs resemble closely primary human NPCs, generating nephron organoids with abundant distal convoluted tubule cells, which are not observed in published kidney organoids. The synthetic niche reprograms differentiated nephron cells into NPC state, recapitulating the plasticity of developing nephron in vivo. Scalability and ease of genome-editing in the cultured NPCs allow for genome-wide CRISPR screening, identifying novel genes associated with kidney development and disease. A rapid, efficient, and scalable organoid model for polycystic kidney disease was derived directly from genome-edited NPCs, and validated in drug screen. These technological platforms have broad applications to kidney development, disease, plasticity, and regeneration.

Modeling CRISPR-Cas13d on-target and off-target effects using machine learning approaches.

A major challenge in the application of the CRISPR-Cas13d system is to accurately predict its guide-dependent on-target and off-target effect. Here, we perform CRISPR-Cas13d proliferation screens and design a deep learning model, named DeepCas13, to predict the on-target activity from guide sequences and secondary structures. DeepCas13 outperforms existing methods to predict the efficiency of guides targeting both protein-coding and non-coding RNAs. Guides targeting non-essential genes display off-target viability effects, which are closely related to their on-target efficiencies. Choosing proper negative control guides during normalization mitigates the associated false positives in proliferation screens. We apply DeepCas13 to the guides targeting lncRNAs, and identify lncRNAs that affect cell viability and proliferation in multiple cell lines. The higher prediction accuracy of DeepCas13 over existing methods is extensively confirmed via a secondary CRISPR-Cas13d screen and quantitative RT-PCR experiments. DeepCas13 is freely accessible via http://deepcas13.weililab.org .

The Study of Alternative Splicing Events in Human Induced Pluripotent Stem Cells From a Down's Syndrome Patient.

Down's syndrome (DS) is one of the most commonly known disorders with multiple congenital disabilities. Besides severe cognitive impairment and intellectual disability, individuals with DS also exhibit additional phenotypes of variable penetrance and severity, with one or more comorbid conditions, including Alzheimer's disease, congenital heart disease, or leukemia. Various vital genes and regulatory networks had been studied to reveal the pathogenesis of the disease. Nevertheless, very few studies have examined alternative splicing. Alternative splicing (AS) is a regulatory mechanism of gene expression when making one multi-exon protein-coding gene produce more than one unique mature mRNA. We employed the GeneChip Human Transcriptome Array 2.0 (HTA 2.0) for the global gene analysis with hiPSCs from DS and healthy individuals. Examining differentially expressed genes (DEGs) in these groups and focusing on specific transcripts with AS, 466 up-regulated and 722 down-regulated genes with AS events were identified. These genes were significantly enriched in biological processes, such as cell adhesion, cardiac muscle contraction, and immune response, through gene ontology (GO) analysis of DEGs. Candidate genes, such as FN1 were further explored for potentially playing a key role in DS. This study provides important insights into the potential role that AS plays in DS.

Lycopene Aggravates Acute Gastric Injury Induced by Ethanol.

Lycopene is an important natural red pigment with strong singlet oxygen and peroxide free radical quenching ability. Ethanol directly destroys the epithelial cells of gastric mucosa, causing oxidative damage and inflammation. To evaluate the effect of lycopene on the ethanol induced gastric injury, 112 adult male Kunming mice were randomly divided into normal control, lycopene control, gastric injury control, omeprazole (20 mg/kg) positive control, and lycopene experimental groups (at doses of 10, 50, 100, and 150 mg/kg body weight) in this study. The general and pathological evaluation, gastric secretion, as well as the levels of antioxidant and inflammatory factors were detected. In lycopene experimental groups, the amount of gastric juice were lower than that in the gastric injury control group; the levels of T-SOD, and the levels of MDA and inflammatory factors (MMP-9 and MCP-1) decreased. However, general and pathological evaluation of gastric tissues revealed that lycopene (especially at high doses) could aggravate acute gastric mucosal injury induced by ethanol. Therefore, lycopene (especially at high doses) aggravates acute gastric mucosal injury caused by ethanol, but this was not due to oxidative stress or inflammatory factors. In lycopene control group, the levels of MTL, T-SOD, and NO increased, but the levels of ALT and AST decreased, indicating that lycopene has a protective effect on the stomach and liver when ethanol wasn't taken. It reminds us that, when alcohol is consumed in large quantities, consumption of lycopene products should be carefully considered.

A chemical-enhanced system for CRISPR-Based nucleic acid detection.

The CRISPR-based nucleic acid detection systems have shown great potential for point-of-care testing of viral pathogens, especially in the context of COVID-19 pandemic. Here we optimize several key parameters of reaction chemistry and develop a Chemical Enhanced CRISPR Detection system for nucleic acid (termed CECRID). For the Cas12a/Cas13a-based signal detection phase, we determine buffer conditions and substrate range for optimal detection performance, and reveal a crucial role of bovine serum albumin in enhancing trans-cleavage activity of Cas12a/Cas13a effectors. By comparing several chemical additives, we find that addition of L-proline can secure or enhance Cas12a/Cas13a detection capability. For isothermal amplification phase with typical LAMP and RPA methods, inclusion of L-proline can also enhance specific target amplification as determined by CRISPR detection. Using SARS-CoV-2 pseudovirus, we demonstrate CECRID has enhanced detection sensitivity over chemical additive-null method with either fluorescence or lateral flow strip readout. Thus, CECRID provides an improved detection power and system robustness, and helps to develop enhanced reagent formula or test kit towards practical application of CRISPR-based diagnostics.

An in silico drug repositioning workflow for host-based antivirals.

Drug repositioning represents a cost- and time-efficient strategy for drug development. Artificial intelligence-based algorithms have been applied in drug repositioning by predicting drug-target interactions in an efficient and high throughput manner. Here, we present a workflow of in silico drug repositioning for host-based antivirals using specially defined targets, a refined list of drug candidates, and an easily implemented computational framework. The workflow described here can also apply to more general purposes, especially when given a user-defined druggable target gene set. For complete details on the use and execution of this protocol, please refer to Li et al. (2021).

Untargeted Global Metabolomic Analysis Reveals the Mechanism of Tripropylamine-Enhanced Lycopene Accumulation in Blakeslea trispora.

We previously determined that the cyclase inhibitor tripropylamine (TPA) significantly enhances lycopene accumulation in Blakeslea trispora. To elucidate the mechanism of TPA-enhanced lycopene accumulation, the untargeted metabolome of B. trispora treated with TPA was analyzed by UHPLC-Q-TOF/MS. Forty-two differential metabolites were identified, of which 15 significantly differential metabolites meeting the following parameters were screened: variable importance for the projection > 1, P < 0.05, and fold change > 1.5. The down-regulated metabolites were mainly cyclic dipeptides, bacteriostatic compounds, and lipids, while the up-regulated metabolites were mainly unsaturated fatty acid. Furthermore, the bacteriostatic ability was poor, the extracellular and intracellular pH levels were high, and hyphae with vesicles were swollen locally in B. trispora after treatment with TPA. Our data suggest that the TPA enhances lycopene accumulation not only by inhibiting the cyclization of ß-carotene but also by down-regulating cyclic dipeptides for quorum sensing; up-regulating unsaturated fatty acids, 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphoethanolamine, and 4-hydroxybenzoate and down-regulating choline, resulting in locally swelling mycelium with vacuoles; and down-regulating bacteriostatic metabolites for metabolic flux redistribution.

A computational framework of host-based drug repositioning for broad-spectrum antivirals against RNA viruses.

RNA viruses are responsible for many zoonotic diseases that post great challenges for public health. Effective therapeutics against these viral infections remain limited. Here, we deployed a computational framework for host-based drug repositioning to predict potential antiviral drugs from 2,352 approved drugs and 1,062 natural compounds embedded in herbs of traditional Chinese medicine. By systematically interrogating public genetic screening data, we comprehensively cataloged host dependency genes (HDGs) that are indispensable for successful viral infection corresponding to 10 families and 29 species of RNA viruses. We then utilized these HDGs as potential drug targets and interrogated extensive drug-target interactions through database retrieval, literature mining, and de novo prediction using artificial intelligence-based algorithms. Repurposed drugs or natural compounds were proposed against many viral pathogens such as coronaviruses including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), flaviviruses, and influenza viruses. This study helps to prioritize promising drug candidates for in-depth evaluation against these virus-related diseases.

A Fully Conjugated 3D Covalent Organic Framework Exhibiting Band-like Transport with Ultrahigh Electron Mobility.

Although π-conjugated two dimensional (2D) covalent organic frameworks (COFs) have been extensively reported, developing fully π-conjugated 3D COFs is still an extremely difficult problem due to the lack of fully π-conjugated 3D linkers. We synthesize a fully conjugated 3D COF (BUCT-COF-1) by designing a saddle-shaped building block of aldehyde-substituted cyclooctatetrathiophene (COThP)-CHO. As a consequence of the fully conjugated 3D network, BUCT-COF-1 demonstrates ultrahigh Hall electron mobility up to ≈3.0 cm2 V-1 s-1 at room temperature, which is one order of magnitude higher than the current π-conjugated 2D COFs. Temperature-dependent conductivity measurements reveal that the charge carriers in BUCT- COF-1 exhibit the band-like transport mechanism, which is entirely different from the hopping transport phenomena observed in common organic materials. The findings indicate that fully conjugated 3D COFs can achieve electron delocalization and charge-transport pathways within the whole 3D skeleton, which may open up a new frontier in the design of organic semiconducting materials.

CRISP-view: a database of functional genetic screens spanning multiple phenotypes.

High-throughput genetic screening based on CRISPR/Cas9 or RNA-interference (RNAi) enables the exploration of genes associated with the phenotype of interest on a large scale. The rapid accumulation of public available genetic screening data provides a wealth of knowledge about genotype-to-phenotype relationships and a valuable resource for the systematic analysis of gene functions. Here we present CRISP-view, a comprehensive database of CRISPR/Cas9 and RNAi screening datasets that span multiple phenotypes, including in vitro and in vivo cell proliferation and viability, response to cancer immunotherapy, virus response, protein expression, etc. By 22 September 2020, CRISP-view has collected 10 321 human samples and 825 mouse samples from 167 papers. All the datasets have been curated, annotated, and processed by a standard MAGeCK-VISPR analysis pipeline with quality control (QC) metrics. We also developed a user-friendly webserver to visualize, explore, and search these datasets. The webserver is freely available at http://crispview.weililab.org.

Improving Cancer Immunotherapy with CRISPR-Based Technology.

The rapidly evolving field of immunotherapy has attracted great attention in the field of cancer research and already revolutionized the clinical practice standard for treating cancer. Genetically engineered T cells expressing either T cell receptors or chimeric antigen receptors represent novel treatment modalities and are considered powerful weapons to fight cancer. The immune checkpoint blockade, which harnesses the negative control signaling behind the anti-tumor immune response with therapeutic antibodies by blocking cytotoxic T lymphocyte-associated protein 4 or the programmed cell death 1 pathways are another mainstream direction for cancer immunotherapy. In addition to cytotoxic T cells, other immune cell types such as nature killer cells and macrophages also possess the ability to eradicate cancer cells, which may serve as the basis to develop novel cancer immunotherapies. The advent of cutting-edge genome editing technology, especially clustered regularly interspaced palindromic repeats (CRISPR)-based tools, has greatly expedited many biomedical research areas, including cancer immunology and immunotherapy. In this review, the contribution of current CRISPR techniques to basic and translational cancer immunology research is discussed, and the future for cancer immunotherapy in the age of CRISPR is predicted.

CDKL3 promotes osteosarcoma progression by activating Akt/PKB.

Osteosarcoma (OS) is a primary malignant bone neoplasm with high frequencies of tumor metastasis and recurrence. Although the Akt/PKB signaling pathway is known to play key roles in tumorigenesis, the roles of cyclin-dependent kinase-like 3 (CDKL3) in OS progression remain largely elusive. We have demonstrated the high expression levels of CDKL3 in OS human specimens and comprehensively investigated the role of CDKL3 in promoting OS progression both in vitro and in vivo. We found that CDKL3 regulates Akt activation and its downstream effects, including cell growth and autophagy. The up-regulation of CDKL3 in OS specimens appeared to be associated with Akt activation and shorter overall patient survival (P = 0.003). Our findings identify CDKL3 as a critical regulator that stimulates OS progression by enhancing Akt activation. CDKL3 represents both a biomarker for OS prognosis, and a potential therapeutic target in precision medicine by targeting CDKL3 to treat Akt hyper-activated OS.

Tau Pathology Promotes the Reorganization of the Extracellular Matrix and Inhibits the Formation of Perineuronal Nets by Regulating the Expression and the Distribution of Hyaluronic Acid Synthases.

Hyaluronic acid (HA) is the backbone of the extracellular matrix (ECM) and provides biochemical and physical support to aggrecan-based perineuronal nets (PNNs), which are associated with the selective vulnerability of neurons in Alzheimer's disease (AD). Here, we showed that HA synthases (HASs), including Has1, Has2, and Has3, were widely expressed in murine central nervous system. All types of HASs were localized to cell bodies of neurons; only Has1 existed in the membranes of neural axons. By using TauP301S transgenic (Tg) mouse model, we found that the axonal-localization of Has1 was abolished in TauP301S overexpressed mouse brain, and the redistribution of Has1 was also observed in human AD brains, suggesting that the localization of Has1 is dependent on intact microtubules which are regulated partially by the phosphorylation and dephosphorylation cycles of tau proteins. Furthermore, Has1 was reduced and Has3 was increased in TauP301S Tg mouse brain, resulting in the upregulation of shorter-chain HA in the ECM. These findings suggest that by abolishing the axonal-localization of Has1 and promoting the expression of Has3 and the synthesis of shorter-chain HA, the tau pathology breaks the balance of ECM components, promotes the reorganization of the ECM, and inhibits the formation of PNNs in the hippocampus, and then regulates neuronal plasticity during the progression of AD.