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Genome-wide screening is a potent approach for comprehensively understanding the molecular mechanisms of biological phenomena. However, despite its widespread use in the past decades across various biological targets, its application to biochemical reactions with temporal and reversible biological outputs remains a formidable challenge. To uncover the molecular machinery underlying various biochemical reactions, we have recently developed the revival screening method, which combines flow cytometry-based cell sorting with library reconstruction from collected cells. Our refinements to the traditional genome-wide screening technique have proven successful in revealing the molecular machinery of biochemical reactions of interest. In this article, we elucidate the technical basis of revival screening, focusing on its application to CRISPR-Cas9 single guide RNA (sgRNA) library screening. Finally, we also discuss the future of genome-wide screening while describing recent achievements from in vitro and in vivo screening.

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BACKGROUND: New drugs to tackle the next pathway or mutation fueling cancer are constantly proposed, but 97% of them are doomed to fail in clinical trials, largely because they are identified by cellular or in silico screens that cannot predict their in vivo effect. METHODS: We screened an Adeno-Associated Vector secretome library (> 1000 clones) directly in vivo in a mouse model of cancer and validated the therapeutic effect of the first hit, EMID2, in both orthotopic and genetic models of lung and pancreatic cancer. RESULTS: EMID2 overexpression inhibited both tumor growth and metastatic dissemination, consistent with prolonged survival of patients with high levels of EMID2 expression in the most aggressive human cancers. Mechanistically, EMID2 inhibited TGFß maturation and activation of cancer-associated fibroblasts, resulting in more elastic ECM and reduced levels of YAP in the nuclei of cancer cells. CONCLUSION: This is the first in vivo screening, precisely designed to identify proteins able to interfere with cancer cell invasiveness. EMID2 was selected as the most potent protein, in line with the emerging relevance of the tumor extracellular matrix in controlling cancer cell invasiveness and dissemination, which kills most of cancer patients.

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Plant-based diets (PBDs) are renowned for managing and developing bioactive chemical inhibitors to combat obesity, a well-known global public health concern. There are currently no published research studies examining the effects of food plant mucilage dietary supplements on animal models of obesity induced by high-fat diets (HFD). The present research investigated the anti-obesity properties of the culinary plant Pedalium murex L. mucilage (PMM) in obese albino male rats models fed HFD. PMM's HR-LCMS phytochemical profiling and in silico evaluation of anti-obesity and drug-likeness using Schrodinger's Glide, QikProp, and GROMACS modules were also investigated. In vivo, anti-obesity model animal rat's daily dietary intake, common blood biochemical parameters, and histological examination of the liver and kidney tissues for the development of macrovesicular and microvesicular steatosis were all performed. Among the 46 Phytochemicals profiled, 7(14)-Bisabolene-2, 3, 10,11tetrol, Moschamine, and N-Feruloyltyramine show prominent anti-obesity activity and drug-like characteristics in silico. Rats given PMM showed significantly lower serum levels of total cholesterol (TC), low-density lipoprotein (LDL), and triglycerides (TGs), increased levels of high-density lipoprotein (HDL), as well as macro-and microvesicular steatosis, lobular inflammation of the liver and kidney tissues. This suggests that PMM is an effective natural anti-obesity therapeutic ingredient or dietary supplement with a high concentration of anti-obesity phytochemicals that mainly satisfies the needs for such natural anti-obesity medicine or a supplement.Communicated by Ramaswamy H. Sarma.

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The development of α-glucosidase inhibitors is essential for the prevention of type II diabetes. Previous research has investigated in vitro inhibition using isolated α-glucosidase, which may not accurately reflect physical processes. The method presented in this study aims to establish a rapid and inexpensive in vivo method to study the inhibition of α-glucosidase activity using Drosophila as a model organism. This method can be used to calculate the IC50 value of compounds of interest for inhibition of α-glucosidase activity. The method established in this study can be used for in vivo screening of anti-diabetic compounds. •A rapid and inexpensive in vivo method to study the inhibition of α-glucosidase activity.•This method can be used to calculate the IC50 value of compounds of interest for inhibition of α-glucosidase activity.•This is a useful method for in vivo screening of anti-diabetic compounds.

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Somatic mutations in nonmalignant tissues accumulate with age and injury, but whether these mutations are adaptive on the cellular or organismal levels is unclear. To interrogate genes in human metabolic disease, we performed lineage tracing in mice harboring somatic mosaicism subjected to nonalcoholic steatohepatitis (NASH). Proof-of-concept studies with mosaic loss of Mboat7, a membrane lipid acyltransferase, showed that increased steatosis accelerated clonal disappearance. Next, we induced pooled mosaicism in 63 known NASH genes, allowing us to trace mutant clones side by side. This in vivo tracing platform, which we coined MOSAICS, selected for mutations that ameliorate lipotoxicity, including mutant genes identified in human NASH. To prioritize new genes, additional screening of 472 candidates identified 23 somatic perturbations that promoted clonal expansion. In validation studies, liver-wide deletion of Tbx3, Bcl6, or Smyd2 resulted in protection against hepatic steatosis. Selection for clonal fitness in mouse and human livers identifies pathways that regulate metabolic disease.

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To develop safe subcutaneous formulations and minimize the risk of local irritation, it is essential to optimize the composition of active pharmaceutical ingredients and excipients. Depending on the physicochemical properties of the active pharmaceutical ingredient, additional excipients may be required to improve the stability and solubility of the active pharmaceutical ingredient. However, some of these excipients may not have been previously used in injectable drugs. Owing to the lack of safety data for such excipients, especially those used in subcutaneous dosing, it is important to evaluate their potential for local irritation during the early stages of formulation development. We evaluated the tolerability of 44 formulations with 24 candidate novel excipients, such as surfactants, polymers, and lipids, in a single subcutaneous dose in rats. Excipient formulations were administered as single bolus subcutaneous injections with an injection volume of 1 mL. The injection sites were observed for 2 days, and macroscopic and microscopic examinations were conducted. Local tolerability was evaluated on the basis of severity, incidence, and pathophysiology of each finding. Formulations that caused tissue degeneration or necrosis, which is indicative of tissue injury, were determined to be irritative and poorly tolerated. A single-dose subcutaneous screening study in rats was considered effective in ranking the safety of candidate excipients during the formulation optimization phase.

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Bacteria in biofilm formations are up to 1000 times less susceptible to antibiotics than their planktonic counterparts. Recognition of the role of biofilms in ∼80% of chronic infections, their contribution to bacterial tolerance and development of antimicrobial resistance, and thus the search for compounds with antibiofilm properties, has increased greatly in recent years. The need for robust experimental methods is therefore critical but currently undermined by inappropriate controls when dimethyl-sulfoxide (DMSO) is used to enhance test compound solubility. DMSO is known to have a limited effect on planktonic growth, but emerging data indicates that the solvent can affect biofilm formation even at low concentrations. Here, we present both a literature review on the application of DMSO in in vitro antibiofilm studies, as well as a series of experiments and Bayesian hormetic dose-response modelling to define the effects of DMSO alone and in combination with standard antibiotics using two clinically important biofilm-forming bacteria. DMSO has been used in 76 published studies to solubilise a wide variety of synthesised and natural products, including plant extracts, isolated secondary metabolites, modified lead molecules and proteins, in in vitro antibiofilm assays. DMSO solvent concentrations to which biofilms were exposed ranged between <1 and 100% but unfortunately, 35% of articles did not specify the DMSO concentrations used, 50% of articles did not include solvent controls and, of those that did, 26% did not specify control concentrations, 47% did not report or discuss control data, and 53% omitted media controls. In a further 12 studies, DMSO is used as a biofilm treatment, demonstrating the antibiofilm properties of this solvent at higher concentrations. We provide evidence that DMSO (between 0.03 and 25%) significantly inhibits biofilm formation in Pseudomonas aeruginosa, but not Streptococcus pneumoniae, and acts synergistically with standard antibiotics at very low concentrations (<1%). Interestingly, intermediate concentrations of DMSO (∼6%) strongly promote the growth of P. aeruginosa biofilms. As the research community strives to identify bioactive antimicrobial compounds, there is a need for increased scientific rigour when using DMSO as a solvent in antibiofilm assays.

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In prokaryotes, transcription factors (TFs) are of uttermost importance for the regulation of gene expression. However, the majority of TFs are not characterized today, which hampers both the understanding of fundamental processes and the development of TF-based applications, such as biosensors, used in metabolic engineering, synthetic biology, diagnostics, etc. One way of analyzing TFs is through in vivo screening, enabling the study of TF-promoter interactions, ligand inducibility, and ligand specificity in a high-throughput fashion. Here, an approach is described for the selection and cloning of TF-promoter pairs, the development of a reporter system, and the measurement and analysis of fluorescent reporter assays. Furthermore, the importance of a suitable inducible plasmid system is illustrated together with prospective adaptations to modify a reporter system's output signal. The given approach can be used for the investigation of native, heterologous, or even artificially created TFs in Escherichia coli, and can be extended toward use in other microorganisms.

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Synthetic riboswitches are a promising tool for conditional gene expression. In vitro selected aptamers used as binding domains for the design of RNA-based switches have to exhibit excellent binding affinity as well as ligand binding-induced structural changes. Selection via Capture-SELEX favors the enrichment of aptamers which exhibit both characteristics. For the Capture-SELEX, an RNA pool is used that gets immobilized onto a capture oligonucleotide by hybridization. Addition of the ligand frees the aptamers by their binding to the ligand, resulting in the release from the capture oligonucleotide through structural changes. These sequences get reverse transcribed, PCR amplified, and used for the following selection rounds. In this publication, we present a detailed protocol for Capture-SELEX, followed by screening in yeast to identify aptamers suitable for the design of synthetic riboswitches.

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Due to the emerging rise of multi-drug resistant bacteria, the discovery of novel antibiotics is of high scientific interest. Through their high chemodiversity of bioactive secondary metabolites, cyanobacteria have proven to be promising microorganisms for the discovery of antibacterial compounds. These aspects make appropriate antibacterial screening approaches for cyanobacteria crucial. Up to date, screenings are mostly carried out using a phenotypic methodology, consisting of cyanobacterial cultivation, extraction, and inhibitory assays. However, the parameters of these methods highly vary within the literature. Therefore, the common choices of parameters and inhibitory assays are summarized in this review. Nevertheless, less frequently used method variants are highlighted, which lead to hits from antimicrobial compounds. In addition to the considerations of phenotypic methods, this study provides an overview of developments in the genome-based screening area, be it in vivo using PCR technique or in silico using the recent genome-mining method. Though, up to date, these techniques are not applied as much as phenotypic screening.

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Cardiac arrhythmia, or irregular heart rhythm, is associated with morbidity and mortality and is described as one of the most important future public health challenges. Therefore, developing new models of cardiac arrhythmia is critical for understanding disease mechanisms, determining genetic underpinnings, and developing new therapeutic strategies. In the last few decades, the zebrafish has emerged as an attractive model to reproduce in vivo human cardiac pathologies, including arrhythmias. Here, we highlight the contribution of zebrafish to the field and discuss the available cardiac arrhythmia models. Further, we outline techniques to assess potential heart rhythm defects in larval and adult zebrafish. As genetic tools in zebrafish continue to bloom, this model will be crucial for functional genomics studies and to develop personalized anti-arrhythmic therapies.

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This paper focuses on preliminary in vitro and in vivo testing of new bivalent folate-targeted PEGylated doxorubicin (DOX) made by modular chemo-enzymatic processes (FA2-dPEG-DOX2). A unique feature is the use of monodisperse PEG (dPEG). The modular approach with enzyme catalysis ensures exclusive γ-conjugation of folic acid, full conversion and selectivity, and no metal catalyst residues. Flow cytometry analysis showed that at 10 µM concentration, both free DOX and FA2-dPEG-DOX2 would be taken up by 99.9% of triple-negative breast cancer cells in 2 h. Intratumoral injection to mice seemed to delay tumor growth more than intravenous delivery. The mouse health status, food, water consumption, and behavior remained unchanged during the observation.

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Restrictions on the use of antibiotics in the poultry industry stimulate the development of alternative nutritional solutions to maintain or improve poultry health. This requires more insight in the modulatory effects of feed additives on the immune system and microbiota composition. Compounds known to influence the innate immune system and microbiota composition were selected and screened in vitro, in ovo, and in vivo. Among all compounds, 57 enhanced NK cell activation, 56 increased phagocytosis, and 22 increased NO production of the macrophage cell line HD11 in vitro. Based on these results, availability and regulatory status, six compounds were selected for further analysis. None of these compounds showed negative effects on growth, hatchability, and feed conversion in in ovo and in vivo studies. Based on the most interesting numerical results and highest future potential feasibility, two compounds were analyzed further. Administration of glucose oligosaccharide and long-chain glucomannan in vivo both enhanced activation of intraepithelial NK cells and led to increased relative abundance of lactic acid bacteria (LAB) amongst ileum and ceca microbiota after seven days of supplementation. Positive correlations between NK cell subsets and activation, and relative abundance of LAB suggest the involvement of microbiota in the modulation of the function of intraepithelial NK cells. This study identifies glucose oligosaccharide and long-chain glucomannan supplementation as effective nutritional strategies to modulate the intestinal microbiota composition and strengthen the intraepithelial innate immune system.

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Isosteviol, an ent-beyerane diterpenoid, has been repeatedly reported to possess potent cardioprotective activity. With the aim of discovering new cardioprotective derivatives from isosteviol, 47 compounds, including 40 new ones, were synthesized and evaluated in vivo using the easy-handling and efficient zebrafish model. The structure-activity relationship of this type of compounds was thus discussed. Of these compounds, new derivative 15d exhibited the most pronounced efficacy in vivo. Our results indicated that 15d could effectively prevent the doxorubicin-induced morphological distortions and cardiac dysfunction in zebrafish. Its cardioprotective activity is much better than that of isosteviol, and Levosimendan in zebrafish model. The molecular mechanism underlying in H9c2 cells indicated that 15d protected cardiomyocyte death and damage through inhibiting the reactive oxygen species overproduction, restoring the mitochondrial membrane potential and maintaining morphology of mitochondrial. Thus, 15d merits further development as a potential cardioprotective clinical trial candidate. The present study is a successful example to combine synthesis, structure-activity relationship study and in vivo screening to effectively discover new cardioprotective agents from isosteviol.

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DNA barcoding allows the quantitative, biomarker-free tracking of individual cell populations in mixed/heterogeneous cell pools. Here, we describe a multiplexed in vivo screening platform based on DNA barcoding technology to interrogate compound libraries for their effect on metastatic seeding in vivo. We apply next-generation sequencing (NGS) technology to quantitatively analyze high-throughput compound screening in mice. Up to 96 compounds and controls can be screened for their effect on metastatic ability in a single mouse.

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Biological aging, and the diseases of aging, occur in a complex in vivo environment, driven by multiple interacting processes. A convergence of recently developed technologies has enabled in vivo pooled screening: direct administration of a library of different perturbations to a living animal, with a subsequent readout that distinguishes the identity of each perturbation and its effect on individual cells within the animal. Such screens hold promise for efficiently applying functional genomics to aging processes in the full richness of the in vivo setting. In this review, we describe the technologies behind in vivo pooled screening, including a range of options for delivery, perturbation and readout methods, and outline their potential application to aging and age-related disease. We then suggest how in vivo pooled screening, together with emerging innovations in each of its technological underpinnings, could be extended to shed light on key open questions in aging biology, including the mechanisms and limits of epigenetic reprogramming and identifying cellular mediators of systemic signals in aging.

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[This corrects the article DOI: 10.3389/fragi.2021.714926.].

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The continual introduction of new chemicals into the market necessitates fast, efficient testing strategies for evaluating their toxicity. Ideally, these high-throughput screening (HTS) methods should capture the entirety of biological complexity while minimizing reliance on expensive resources that are required to assess diverse phenotypic endpoints. In recent years, the zebrafish (Danio rerio) has become a preferred vertebrate model to conduct rapid in vivo toxicity tests. Previously, using HTS data on 1060 chemicals tested as part of the ToxCast program, we showed that early, 24 h post-fertilization (hpf), behavioral responses of zebrafish embryos are predictive of later, 120 h post-fertilization, adverse developmental endpoints-indicating that embryonic behavior is a useful endpoint related to observable morphological effects. Here, our goal was to assess the contributions (i.e., information gain) from multiple phenotypic data streams and propose a framework for efficient identification of chemical hazards. We systematically swept through analysis parameters for data on 24 hpf behavior, 120 hpf behavior, and 120 hpf morphology to optimize settings for each of these assays. We evaluated the concordance of data from behavioral assays with that from morphology. We found that combining information from behavioral and mortality assessments captures early signals of potential chemical hazards, obviating the need to evaluate a comprehensive suite of morphological endpoints in initial screens for toxicity. We have demonstrated that such a screening strategy is useful for detecting compounds that elicit adverse morphological responses, in addition to identifying hazardous compounds that do not disrupt the underlying morphology. The application of this design for rapid preliminary toxicity screening will accelerate chemical testing and aid in prioritizing chemicals for risk assessment.

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Ehrlichia chaffeensis, a tick-transmitted obligate intracellular rickettsial agent, causes human monocytic ehrlichiosis. In recent reports, we described substantial advances in developing random and targeted gene disruption methods to investigate the functions of E. chaffeensis genes. We reported earlier that the Himar1 transposon-based random mutagenesis is a valuable tool in defining E. chaffeensis genes critical for its persistent growth in vivo in reservoir and incidental hosts. The method also aided in extending studies focused on vaccine development and immunity. Here, we describe the generation and mapping of 55 new mutations. To define the critical nature of the bacterial genes, infection experiments were carried out in the canine host with pools of mutant organisms. Infection evaluation in the physiologically relevant host by molecular assays and by xenodiagnoses allowed the identification of many proteins critical for the pathogen's persistent in vivo growth. Genes encoding proteins involved in biotin biosynthesis, protein synthesis and fatty acid biosynthesis, DNA repair, electron transfer, and a component of a multidrug resistance (MDR) efflux pump were concluded to be essential for the pathogen's in vivo growth. Three known immunodominant membrane proteins, i.e., two 28-kDa outer membrane proteins (P28/OMP) and a 120-kDa surface protein, were also recognized as necessary for the pathogen's obligate intracellular life cycle. The discovery of many E. chaffeensis proteins crucial for its continuous in vivo growth will serve as a major resource for investigations aimed at defining pathogenesis and developing novel therapeutics for this and related pathogens of the rickettsial family Anaplasmataceae.

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Identifying miRNA target genes is difficult, and delineating which targets are the most biologically important is even more difficult. We devised a novel strategy to test the phenotypic impact of individual microRNA-target interactions by disrupting each predicted miRNA-binding site by CRISPR-Cas9 genome editing in C. elegans We developed a multiplexed negative selection screening approach in which edited loci are deep sequenced, and candidate sites are prioritized based on apparent selection pressure against mutations that disrupt miRNA binding. Importantly, our screen was conducted in vivo on mutant animals, allowing us to interrogate organism-level phenotypes. We used this approach to screen for phenotypic targets of the essential mir-35-42 family. By generating 1130 novel 3'UTR alleles across all predicted targets, we identified egl-1 as a phenotypic target whose derepression partially phenocopies the mir-35-42 mutant phenotype by inducing embryonic lethality and low fecundity. These phenotypes can be rescued by compensatory CRISPR mutations that retarget mir-35 to the mutant egl-1 3'UTR. This study demonstrates that the application of in vivo whole organismal CRISPR screening has great potential to accelerate the discovery of phenotypic negative regulatory elements in the noncoding genome.

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