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At least 19 recordsLinked to original sources

PubMed2025

A neural network model enables worm tracking in challenging conditions and increases signal-to-noise ratio in phenotypic screens.

High-resolution posture tracking of C. elegans has applications in genetics, neuroscience, and drug screening. While classic methods can reliably track isolated worms on uniform backgrounds, they fail when worms overlap, coil, or move in complex environments. Model-based tracking and deep learning approaches have addressed these issues to an extent, but there is still significant room for improvement in tracking crawling worms. Here we train a version of the DeepTangle algorithm developed for swimming worms using a combination of data derived from Tierpsy tracker and hand-annotated data for more difficult cases. DeepTangleCrawl (DTC) outperforms existing methods, reducing failure rates and producing more continuous, gap-free worm trajectories that are less likely to be interrupted by collisions between worms or self-intersecting postures (coils). We show that DTC enables the analysis of previously inaccessible behaviours and increases the signal-to-noise ratio in phenotypic screens, even for data that was specifically collected to be compatible with legacy trackers including low worm density and thin bacterial lawns. DTC broadens the applicability of high-throughput worm imaging to more complex behaviours that involve worm-worm interactions and more naturalistic environments including thicker bacterial lawns.

Caenorhabditis elegans↗

PubMed2025

The limitations of small molecule and genetic screening in phenotypic drug discovery.

Phenotypic screens carried out with functional genomics or small molecules have led to novel biological insights, revealed previously unknown targets for drug discovery programs, and provided starting points for the development of first-in-class therapies. Despite being valuable research tools, genetic and compound screening also have significant limitations. This perspective aims to shed a light on those limitations and provide mitigation strategies when available, with a goal of helping phenotypic screening practitioners gain an understanding of how and when to best utilize either approach.

Drug Discovery↗

PubMed2025

The critical role of PSAC channel in malaria parasite survival is driven home by phenotypic screening under relevant nutrient levels.

Spreading resistance to front-line treatments necessitate the search for new classes of antimalarials. Limitations of standard screening conditions lead us to develop an assay using culture media that more closely reflects nutrient levels in human serum to reveal new therapeutically relevant parasite pathways. Our approach was validated by testing 22k compounds followed by a full 750k compound screen and identified 29 chemotypes with higher activity in nutrient restricted media that were further characterized. Through a combination of chemo-genomics and innovative photocatalytic proximity labeling proteomics, we identified the target of two compounds as the CLAG3 component of the plasmodial surface anion channel (PSAC). Strikingly, every one of the other 29 chemotypes selected was also found to block PSAC activity, highlighting the importance of this nutrient channel for parasite survival under physiological conditions. The effect of PSAC inhibitors in the in vivo humanized mouse model was confirmed.

Animals↗

PubMed2026

CRISPR/Cas9 loss-of-function screen in a neuronal model of AP-4 deficiency identifies ATG9A trafficking modulators.

Biallelic loss-of-function variants in adaptor protein complex 4 (AP-4) disrupt trafficking of transmembrane proteins at the trans-Golgi network, including autophagy-related protein 9A (ATG9A), leading to childhood-onset hereditary spastic paraplegia (AP-4-HSP). AP-4-HSP is characterized by features of both a neurodevelopmental and a degenerative neurological disease. To investigate the molecular mechanisms underlying AP-4-HSP and identify potential therapeutic targets, we conducted an arrayed CRISPR/Cas9 loss-of-function screen of 8,478 genes, targeting the "druggable genome," in a human neuronal model of AP-4 deficiency. Through this phenotypic screen and subsequent experiments, key modulators of ATG9A trafficking were identified, and complementary pathway analyses provided insights into the regulatory landscape of ATG9A transport. Knockdown of ANPEP and NPM1 enhanced ATG9A availability outside the trans-Golgi network, suggesting that they regulate ATG9A localization. These findings deepen our understanding of ATG9A trafficking in the context of AP-4 deficiency and offer a framework for the development of targeted interventions for AP-4-HSP.

Humans↗

PubMed2026

Alpha-thalassaemia early eluting peak for alpha-thalassaemia --SEA carrier screening: a multicentre diagnostic comparison with immunochromatographic strip test and haemoglobin H inclusion test.

While high-performance liquid chromatography (HPLC) is well-established for β-thalassaemia and haemoglobinopathies, phenotypic screening for α0-thalassaemia has been limited. To address this limitation, we aimed to translate the discovery of the α-thalassemia early eluting peak (αEEP) in HPLC into clinical practice by comparing its diagnostic performance with other existing methods (haemoglobin H inclusion test [HbHi] and immunochromatographic strip test [ICT]) in a multicentre setting, and elucidating the nature of the αEEP by liquid chromatography-tandem mass spectrometry (LC-MS/MS). With a cohort of 820 genotyped patients, the αEEP showed superior diagnostic performance in detecting --SEA (sensitivity 99.6%, specificity 100%) compared with HbHi (sensitivity 95.8%, P = 0.006; specificity 97.3%, P < 0.001) and ICT (sensitivity 95.8%, P = 0.006; specificity 75.4%, P < 0.001). Both HbHi and ICT showed reduced sensitivity in β-thalassaemia carriers versus non-carriers. ICT showed reduced specificity when Hb F ≥ 1% compared with < 1%. The αEEP remained robust across all subgroups. LC-MS/MS revealed a strong association between the αEEP and embryonic ζ-globin chains (P < 0.001). The αEEP offered cost reductions of 98.6% over HbHi and 97.3% over ICT. Collectively, the αEEP is a highly reliable and cost-effective marker for detecting --SEA carriers, enabling a novel "all-in-one" HPLC screening strategy for --SEA, β-thalassaemia and haemoglobinopathies. Trial registration number: not applicable.

Humans↗

PubMed2025

Rethinking GWAS: how lessons from genetic screens and artificial intelligence could reveal biological mechanisms.

MOTIVATION: Modern single-cell omics data are key to unraveling the complex mechanisms underlying risk for complex diseases revealed by genome-wide association studies (GWAS). Phenotypic screens in model organisms have several important parallels to GWAS which the author explores in this essay. RESULTS: The author provides the historical context of such screens, comparing and contrasting similarities to association studies, and how these screens in model organisms can teach us what to look for. Then the author considers how the results of GWAS might be exhaustively interrogated to interpret the biological mechanisms underpinning disease processes. Finally, the author proposes a general framework for tackling this problem computationally, and explore the data, mechanisms, and technology (both existing and yet to be invented) that are necessary to complete the task. AVAILABILITY AND IMPLEMENTATION: There are no data or code associated with this article.

Genome-Wide Association Study↗

PubMed2026

Accelerate Your Science: Direct-to-Biology Strategies in Medicinal Chemistry.

Direct-to-biology (D2B) is a powerful strategy that accelerates early drug discovery. It enables compounds to be synthesized in miniaturized formats and evaluated directly as crude reaction mixtures. This bypasses the need for purification during the initial design-make-test cycle. Advances in robust synthetic methodologies, automation, reaction miniaturization, and biological screening have transformed D2B from a proof-of-concept approach into a versatile medicinal chemistry platform. This platform is applicable to fragment optimization, covalent ligands, macrocycles, proteolysis-targeting chimeras (PROTACs), molecular glues, and cellular phenotypic screening. This perspective focuses on the synthetic transformations, assay technologies, and platform implementations that drive modern D2B workflows. It emphasizes reaction robustness, assay compatibility, and practical implementation. Analysis of the current literature revealed that D2B is more governed by reaction reliability than synthetic diversity. Amide coupling and click chemistry dominate reported workflows, while more complex transformations remain underexplored. We discuss the complementary strengths and limitations of biochemical, biophysical, and cellular readouts, identify current bottlenecks in reaction scope and data management, and highlight emerging opportunities arising from reaction miniaturization, machine learning, automated experimentation, and advanced synthetic methodologies. Rather than replacing conventional medicinal chemistry, D2B fundamentally shifts experimental effort from purification toward early biological validation and is poised to become an integral component of future medicinal chemistry workflows.

Humans↗

PubMed2026

Identification of a putative RocS homolog through phenotypic profiling of uncharacterized essential genes in Streptococcus mutans.

Genome-wide viability catalogs produced by transposon sequencing (Tn-seq) and CRISPR interference (CRISPRi) have successfully mapped the essential genome of Streptococcus mutans . In this study, we combined predictive bioinformatics, conditional CRISPRi transcriptional silencing, transmission electron microscopy, transcriptomics, and genetic suppressor screens to investigate nine poorly characterized essential genes in S. mutans . From this screen, phenotypic and genetic analyses identified SMU_393 as a functional homolog of the pneumococcal chromosome segregation factor, RocS. Depletion of SMU_393 resulted in abnormal cell widening, hypersensitivity to DNA damage, and a significant subpopulation of anucleate cells. These phenotypes were bypassed by a spontaneous surface-exposed missense mutation ( dnaA Q197E ) within the AAA+ ATPase domain of the replication initiator. Together, this study refines annotations within the S. mutans essential genome and provides genetic insights into streptococcal chromosome segregation and cell cycle control.

Journal Article↗

PubMed2026

Chromosome-Scale Genome Analysis Reveals Locus-Specific Disruption of the Citrinin-Associated Region in a Furu-Derived Monascus ruber Strain BC20.

Monascus species are widely used in traditional fermented foods for pigment and flavor formation, but citrinin contamination remains a major safety concern that limits broader food applications. Therefore, this study aimed to evaluate the citrinin risk of a furu-derived Monascus ruber strain, BC20, by integrating phenotypic screening across food-relevant matrices with genome-resolved analysis. After 14 days of cultivation across eight matrices, including fungal media as well as dairy-, cereal-, and bran-based substrates, citrinin was not detected by immunoaffinity cleanup combined with HPLC-FLD (LOD, 4 μg/kg; LOQ, 12 μg/kg). To investigate the genetic basis of this phenotype, we generated a chromosome-scale genome assembly for BC20 and conducted comparative analyses across a total of 19 Monascus genomes. ANI analysis and phylogenomic inference consistently placed BC20 within the ruber-pilosus clade. Comparative synteny analysis showed that the citrinin-associated locus in BC20 no longer retained an intact cluster configuration but instead exhibited a remnant-locus architecture, and similar patterns were also observed in several related genomes from the same clade. By contrast, the monacolin K (mk) locus remained syntenically conserved in BC20, supporting locus-specific structural disturbance rather than assembly-derived pseudo-absence. Additionally, its antifungal susceptibility was determined. Overall, BC20 represents a M. ruber candidate strain with undetectable citrinin, and this study provides a practical analytical framework for citrinin risk screening in food-related Monascus isolates.

biosynthetic gene cluster↗

PubMed2026

Screening of Fermentative Strains for Reducing the Allergenicity of a Whey Protein-Soy Protein System and Genomic Characterization of the Selected Strain.

Dual-protein systems combining whey protein isolate (WPI) and soy protein isolate (SPI) offer complementary nutritional benefits but are limited by the presence of major allergens. Lactic acid bacteria (LAB) fermentation provides a promising strategy to mitigate this limitation. In this study, Lacticaseibacillus paracasei JM053, selected from 13 LAB strains based on phenotypic screening, significantly reduced the in vitro allergenicity of the dual-protein system, increasing the IgE-binding inhibition rate to 48.75%. Whole-genome sequencing and characterization of JM053 revealed a comprehensive proteolytic system, including the proline-specific peptidase genes pepX and pepQ, which may contribute to the degradation of allergenic peptide sequences. Combined with in silico bioinformatic analysis, potential cleavage sites within the linear epitopes of the dual-protein system were predicted based on the substrate specificity of the identified proteases, offering a testable hypothesis for the strain's mechanism of action. In addition, in vitro safety assessment and genomic analysis supported the safety potential, stress tolerance, and probiotic characteristics of JM053. Collectively, this study provides a valuable candidate strain for the development of hypoallergenic dual-protein products and offers preliminary genomic insights into LAB-mediated allergenicity reduction.

Lacticaseibacillus paracasei↗

PubMed2026

Evaluation of carbapenem inactivation method-based phenotypic assays for the detection of GES-type carbapenemases in Enterobacterales, Pseudomonas aeruginosa, and Acinetobacter baumannii.

UNLABELLED: Detection of GES-type carbapenemases remains challenging because of their low prevalence and frequently weak hydrolytic activity against carbapenems. Carbapenem inactivation method (CIM)-based assays are widely used as phenotypic screening tools for carbapenemase detection; however, their performance in large collections of GES producers has not been systematically evaluated. We assessed the performance of CIM, modified CIM (mCIM), and CIM-Tris in a diverse collection of GES-producing clinical isolates, including 110 Enterobacterales and 108 Pseudomonas aeruginosa, recovered from Spanish hospitals (2010-2024), and 10 Acinetobacter baumannii isolates, mostly obtained from a hospital in Egypt. Whole-genome sequencing was carried out for species confirmation and resistome analysis. Meropenem MICs were determined by broth microdilution. Overall, 92.1% of isolates were GES-carbapenemase producers (CP), whereas 7.9% expressed GES-type extended-spectrum β-lactamases (ESBLs). In Enterobacterales (predominantly carrying blaGES-6), mCIM improved sensitivity compared with CIM (63.6% vs 40.0%), although many isolates remained undetected due to low meropenem MICs (MIC50, 0.5 µg/mL). In CP-P. aeruginosa (mainly blaGES-5), CIM, mCIM, and CIM-Tris showed sensitivities of 89.1%, 94.6%, and 100%, respectively; however, CIM-Tris yielded false-positive results in 50% of non-CP isolates (mostly blaGES-1 producers). Meropenem MICs in P. aeruginosa were higher (MIC50, >32 µg/mL). In A. baumannii, CIM-Tris improved sensitivity compared with CIM (100% vs 25.0%). These findings indicate that CIM-based methods can detect GES-type carbapenemases, but performance varies according to bacterial species and GES variant, and reduced specificity may occur in isolates producing GES-type ESBLs. Complementary molecular testing may therefore be necessary to ensure accurate detection of GES-type carbapenemases in routine clinical laboratories. IMPORTANCE: GES-type carbapenemases represent an important but underrecognized diagnostic challenge due to their low global prevalence, heterogeneous hydrolytic activity, and the limited performance data available for routine phenotypic detection methods. Although CIM-based assays are widely implemented in clinical microbiology laboratories for carbapenemase screening, their performance against GES-producing organisms has not been comprehensively evaluated across different bacterial genera and GES variants. In this study, we evaluated the performance of CIM, modified CIM (mCIM), and CIM-Tris in a large multicenter collection of well-characterized GES-producing clinical isolates, including Enterobacterales, Pseudomonas aeruginosa, and Acinetobacter baumannii. Our findings demonstrate substantial variability in assay performance according to bacterial species and GES variant. Notably, mCIM improved sensitivity among Enterobacterales with low meropenem MICs, whereas CIM-Tris achieved excellent sensitivity in P. aeruginosa and A. baumannii but at the expense of reduced specificity in isolates producing GES-type ESBLs. To the best of our knowledge, this is the first study directly comparing multiple CIM-based approaches in such a large and taxonomically diverse collection of GES-producing isolates.

beta-Lactamases↗

PubMed2025

Genetic and phenotypic diversity of wine-associated Hanseniaspora species.

The genus Hanseniaspora includes apiculate yeasts commonly found in fruit- and fermentation-associated environments. Their genetic diversity and evolutionary adaptations remain largely unexplored despite their ecological and oenological significance. This study investigated the phylogenetic relationships, genome structure, selection patterns, and phenotypic diversity of Hanseniaspora species isolated primarily from Australian wine environments, focusing on Hanseniaspora uvarum, the most abundant non-Saccharomyces yeast in wine fermentation. A total of 151 isolates were sequenced, including long-read genomes for representatives of the main phylogenetic clades. Comparative genomics revealed ancestral chromosomal rearrangements between the slow-evolving lineage (SEL) and fast-evolving lineage (FEL) that could have contributed to their evolutionary split, as well as significant loss of genes associated with mRNA splicing, chromatid segregation and signal recognition particle protein targeting in the FEL. Pangenome analysis within H. uvarum identified extensive copy number variation, particularly in genes related to xenobiotic tolerance and nutrient transport. Investigation into the selective landscape following the FEL/SEL divergence identified diversifying selection in 229 genes in the FEL, with significant enrichment in genes within the lysine biosynthetic pathway. Furthermore, phenotypic screening of 116 isolates revealed substantial intraspecific diversity, with specific species exhibiting enhanced ethanol, osmotic, copper, SO₂, and cold tolerance.

Wine↗

PubMed1979

alpha 1-Antitrypsin deficiency and susceptibility to lung disease.

This subject concerns the complex interrelationship of a genetically determined protein deficiency, enzymes which are inhibited by that protein, environmental challenges such as cigarette smoke and industrial pollutants, and the occurrence of obstructive lung disease (Fig. 1). Unequivocal establishment of an etiological role for AAT deficiency, especially of intermediate degree, has proven to be difficult. Confounding variables such as enzyme concentration in PMN and PAMs, duration of exposure to potential environmental hazards, differences in laboratory methods utilized in measuring AAT and in studying pulmonary function all require investigation. The definitive study, incorporating all of these and other factors, has yet to be conducted. No single, clear-cut conclusion can be drawn from analysis of present studies. In those circumstances in which heterozygotes appear to be predisposed to COPD, phenotypic screening of the population at potential risk, such as industrial workers may be appropriate. Conversely, in conditions in which no association is demonstrated, such screening would not be justified. Perhaps, the best one can do is to suggest a "Scotch verdict"; that is, the issue of causation is not proven.

Animals↗

PubMed2024

Genome mining for new enediyne antibiotics.

Enediyne antibiotics epitomize nature's chemical creativity. They contain intricate molecular architectures that are coupled with potent biological activities involving double-stranded DNA scission. The recent explosion in microbial genome sequences has revealed a large reservoir of novel enediynes. However, while hundreds of enediyne biosynthetic gene clusters (BGCs) can be detected, less than two dozen natural products have been characterized to date as many clusters remain silent or sparingly expressed under standard laboratory growth conditions. This review focuses on four distinct strategies, which have recently enabled discoveries of novel enediynes: phenotypic screening from rare sources, biosynthetic manipulation, genomic signature-based PCR screening, and DNA-cleavage assays coupled with activation of silent BGCs via high-throughput elicitor screening. With an abundance of enediyne BGCs and emerging approaches for accessing them, new enediyne natural products and further insights into their biogenesis are imminent.

Enediynes↗

PubMed2026

TRIM21 induces selective autophagy of viruses and bacteria.

TRIM21 is an exceptionally versatile ubiquitin ligase that can be directed by antibodies to target oligomeric protein scaffolds, viral capsids, and proteopathic aggregates for intracellular degradation. How the cell degrades these typically resistant substrates remains poorly understood. To address this, we used TRIM21 viral restriction to create a genome-wide phenotypic screen for antibody-dependent capsid degradation. We identify an antimicrobial selective macroautophagy pathway in mammalian cells, which we term "antibody-directed xenophagy" (ADX). We show that this mechanism restricts structurally diverse pathogens, including adenovirus and Salmonella. Using quantitative microscopy, we demonstrate that TRIM21 rapidly intercepts antibody-pathogen complexes, leading to ubiquitin ligase activation. Following this, selective autophagy adaptors are recruited, and viral cargoes are delivered to lysosomes. This process reduces Salmonella pathology and bacterial tissue invasion in mice. We propose that TRIM21 evolved through competition with pathogens to induce autophagy of diverse and complex substrates, potentially explaining its versatility for targeted protein degradation.

TRIM21 Protein↗

PubMed2026

Canalesolide A, a Structurally Unique Polyhydroxy Macrolide from the Marine Cyanobacterium Okeania sp. with Potent Antitrypanosomal Activity.

The discovery of structurally novel natural products remains central to expanding biologically relevant chemical space, particularly within underexplored marine metabolite classes. Herein, we report the discovery and complete structural elucidation of canalesolide A, a new polyhydroxylated macrolide isolated from the marine cyanobacterium Okeania sp. The compound was identified through an integrated workflow combining phenotypic screening against Trypanosoma brucei and LC-MS/MS-based molecular networking, enabling rapid prioritization of bioactive fractions and dereplication of known metabolite families. Spectroscopic analysis revealed that canalesolide A belongs to the bastimolide-related class of macrolides but exhibits a distinct structural architecture. Its structure was established by integrating ultrahigh-resolution NMR spectroscopy, empirical configurational analysis of polyol systems, targeted model compound synthesis, and controlled chemical degradation and derivatization. This combined strategy resolved stereochemical motifs that were inaccessible by direct analysis of the intact macrolide alone, providing a transferable approach for assigning densely oxygenated marine macrolides. Genome mining identified the putative biosynthetic gene cluster and proposed biosynthetic pathway for a bastimolide-related macrolide. Canalesolide A displays potent, low nanomolar antitrypanosomal activity against human-infective subspecies of T. brucei with rapid elimination of parasites within 1 h at 1 μM. Although moderate mammalian cytotoxicity was observed, preliminary in vivo efficacy/toxicity studies in infected mice suggest a narrow therapeutic window highlighting the need for improved selectivity. This study expands the structural and biosynthetic diversity of polyhydroxylated macrolides and establishes a generalizable framework for resolving stereochemically complex natural products.

Macrolides↗

PubMed2025

FDA-approved drug repurposing in zebrafish identifies thyroid hormone and other compounds as potential antithrombotics.

Venous thromboembolism (VTE) is a highly prevalent medical condition with limited therapeutic options and an incomplete understanding of its acquired and inherited subtypes. The zebrafish is a model with the benefits of external development, fecundity, optical transparency, and hemostasis that demonstrates conservation with mammals. We utilized zebrafish as a phenotypic screening tool to identify novel therapeutic options for preventing VTE. A library of FDA-approved compounds was screened for suppression of acquired (elevated estrogen) and spontaneous (protein C deficiency) thrombosis. We found that thyroid hormone, receptor tyrosine kinase (RTK) inhibitors, and proton-pump inhibitors (PPIs) effectively modulated levels of thrombosis, particularly in the estrogen-induced model. These also showed a more favorable hemostatic profile than standard therapies, suggesting alternative mechanisms. Genome editing of thyroid hormone receptor proved that thyroid hormone action is on target. A retrospective electronic health record (EHR) analysis found that thyroid-hormone prescriptions in hormonal contraceptive users correlated with a higher VTE risk, potentially limiting direct repurposing but highlighting thyroid signaling as a pathway involved in estrogen-induced thrombosis. Together, these data identify several drug classes that can be tailored to specific subtypes of VTE and help elucidate distinct pathways driving thrombosis.

FDA-approved compounds↗

PubMed1979

Screening methods using sulfamethazine for determining acetylator phenotype.

Analysis of sulfamethazine (SMZ) kinetics in man has revealed complexities including wide intersubject variability. In our study, an attempt was made to assess the potential influence of changes in nonmetabolic parameters (absorption and urinary elimination rate constants) on the markers of acetylation capacity normally used in clinical screening procedures to determine phenotype. Seven normal subjects were classified as slow (SA) or fast acetylators (FA) according to their metabolic rate constant for SMZ (Km), plasma SMZ half-life, and percentage of N-acetyl SMZ in a 6-hr blood sample (PI6), a 5- to 6-hr urine collection (UI5--6), or a 6-hr total urine collection (UI6). Computer simulations were applied to baseline SMZ kinetic data from these subjects, varying nonmetabolic kinetic parameters over experimentally defined ranges singly, or in parallel with 1 or more of the other parameters. The simulations indicate that all the usual phenotyping procedures were sensitive to changes in absorption and urinary elimination rate constants. While these predictions require experimental confirmation, results show that the PI6 method is least sensitive to such changes, suggesting this method may minimize errors in phenotyping screening.

Absorption↗