FUSE: Improving the estimation and imputation of variant impacts in functional screening.

Deep mutational scanning enables high-throughput functional assessment of genetic variants. While phenotypic measurements from screening assays generally align with clinical outcomes, experimental noise may affect the accuracy of individual variant estimates. We developed the FUSE (functional substitution estimation) pipeline, which leverages measurements collectively within screening assays to improve the estimation of variant impacts. Drawing data from 115 published functional assays, FUSE assesses the mean functional effect per amino acid position and makes estimates for individual allelic variants. It enhances the correlation of variant functional effects from different assay platforms and increases the classification accuracy of missense variants in ClinVar across 29 genes (area under the receiver operating characteristic [ROC] curve [AUC] from 0.83 to 0.90). In UK Biobank patients with rare missense variants in BRCA1, LDLR, or TP53, FUSE improves the classification accuracy of associated phenotypes. FUSE can also impute variant effects for substitutions not experimentally screened. This approach improves accuracy and broadens the utility of data from functional screening.

Advancements in mammalian display technology for therapeutic antibody development and beyond: current landscape, challenges, and future prospects.

The evolving development landscape of biotherapeutics and their growing complexity from simple antibodies into bi- and multi-specific molecules necessitates sophisticated discovery and engineering platforms. This review focuses on mammalian display technology as a potential solution to the pressing challenges in biotherapeutic development. We provide a comparative analysis with established methodologies, highlighting key aspects of mammalian display technology, including genetic engineering, construction of display libraries, and its pivotal role in hit selection and/or developability engineering. The review delves into the mechanisms underpinning developability-driven selection via mammalian display and their broader implications. Applications beyond antibody discovery are also explored, alongside advancements towards function-first screening technologies, precision genome engineering and AI/ML-enhanced libraries, situating them in the context of mammalian display. Overall, the review provides a comprehensive overview of the current mammalian display technology landscape, underscores the expansive potential of the technology for biotherapeutic development, addresses the critical challenges for the full realisation of this potential, and examines advances in related disciplines that might impact the future application of mammalian display technologies.

Characterization of ATP-binding cassette transporters associated with emamectin benzoate tolerance: from the model insect Drosophila melanogaster to the agricultural pest Spodoptera frugiperda.

BACKGROUND: Multiple families of detoxification genes, including the increasingly recognized family of ATP-binding cassette (ABC) transporters, work together to influence the toxicity of synthetic insecticides and thus their resistance. Effective management of insecticide resistance requires identification of all toxicity-affecting members from each family of toxicity-related genes. RESULTS: Here, we used emamectin benzoate (EB), ABC transporters and Spodoptera frugiperda as a working case to test whether the strategy of 'from the model insect Drosophila melanogaster to agricultural pests' can identify all or most ABC transporter members related to EB tolerance in S. frugiperda. After confirming the involvement of ABC transporters in the toxicity of EB against fruit fly with the ABC inhibitor verapamil, four ABC transporter genes (DmCG3327, DmCG11147, DmCG4822, and DmCG7627) were found to be involved in EB tolerance using RNA interference-based family-wide functional screening. A combination of phylogenic analysis and a reciprocal TBLASTN search identified five S. frugiperda ABC transporter members as homologs (SfABCC4, SfABCG1, and SfABCG23) or one-way best hits (SfABCG4 and SfABCG20) of the four fly ABC genes. Real-time quantitative polymerase chain reaction (qPCR) analysis found that all five S. frugiperda ABC transporter genes were inducible by EB, and expressed in all the developmental stages and larval tissues, but with significant quantitative differences among stages and tissues. A cytotoxicity assay of ABC-overexpressing Sf9 cell lines showed that all the five S. frugiperda ABC transporter genes made Sf9 cells tolerant to EB. CONCLUSIONS: This study not only identifies nine ABC transporter genes related to EB tolerance from D. melanogaster (four genes) and S. frugiperda (five genes), but also demonstrates the utility and effectiveness of the 'model to pests' strategy to identify most toxicity-affecting members from a given family of toxicity-related genes. © 2024 Society of Chemical Industry.

Intrinsic and extrinsic variables impacting upper quarter Y-balance test scores in sporting cohorts: A systematic review.

INTRODUCTION: The upper quarter y-balance test (YBT-UQ) is a functional screening tool used to detect musculoskeletal injury risk, aid rehabilitation, and monitor dynamic function, strength and control, yet little is currently known about intrinsic and extrinsic factors that influence reach scores. OBJECTIVES: This systematic review aimed to determine if age, sex, or interventions influenced reach scores and whether between-limb differences were common in non-injured sporting populations, with a secondary aim to identify if sport impacted YBT-UQ reach. METHODS: Web of Science, PubMed, and SportDiscus were systematically searched from January 2012 to November 16, 2023, revealing twenty-three studies satisfying inclusion criteria of published in English between 2012 and 2023, healthy participants of any age including both males and females, athletic populations, YBT-UQ use to assess upper limb mobility/stability, report normalised reach scores, and peer-reviewed full-texts. Methodological quality was evaluated via National Institutes of Health (NIH) quality assessment tools for controlled interventions, observational cohort and cross-sectional designs, and pre-post with no control group. RESULTS: Age, sex, sport, and fatigue were influencing factors; greater reach scores were achieved in older athletes (i.e. >18 years), males, and in a well-rested state. Between-limb differences were not common in sporting populations; therefore, asymmetries may be useful for practitioners to aid injury risk identification. CONCLUSION: This is the first systematic review investigating YBT-UQ influencing factors and thereby provides context for clinicians regarding characteristics that impact reach scores in sporting populations, from which normative values could be determined and further aid clinical decisions or areas to improve regarding injury risk.

Discovery platforms for RNA therapeutics.

RNA therapeutics are emerging as a unique opportunity to drug currently "undruggable" molecules and diseases. While their advantages over conventional, small molecule drugs, their therapeutic implications and the tools for their effective in vivo delivery have been extensively reviewed, little attention has been so far paid to the technological platforms exploited for the discovery of RNA therapeutics. Here, we provide an overview of the existing platforms and ex vivo assays for RNA discovery, their advantages and disadvantages, as well as their main fields of application, with specific focus on RNA therapies that have reached either phase 3 or market approval.

Functional Screening of cDNA Expression Library for Novel Ctenophore Photoproteins.

The functional screening of cDNA libraries (or functional cloning) enables isolation of cDNA genes encoding novel proteins with unknown amino acid sequences. This approach is the only way to identify a protein sequence in the event of shortage of biological material for obtaining pure target protein in amounts sufficient to determine its primary structure, since sensitive functional test for a target protein is only required to successfully perform functional cloning. Commonly, bioluminescent proteins from representatives belonging to different taxa significantly differ in sequences due to independent origin of bioluminescent systems during evolution. Nonetheless, these proteins are frequently similar in functions and can use even the same substrate of bioluminescence reaction, allowing the use of the same functional test for screening. The cDNA genes encoding unknown light-emitting proteins can be identified during functional screening with high sensitivity, which is provided by modern light recording equipment making possible the detection of a very small amount of a target protein. Here, we present the protocols for isolation of full-size cDNA genes for the novel bioluminescent protein family of light-sensitive Ca2+-regulated photoproteins in the absence of any sequence information by functional screening of plasmid cDNA expression library. The protocols describe all the steps from gathering animals to isolation of individual E. coli colonies carrying full-size cDNA genes using photoprotein berovin from ctenophore Beroe abyssicola as an illustrative example.

A robust high-throughput functional screening assay for plant pathogen effectors using the TMV-GFP vector.

Uncovering the function of phytopathogen effectors is crucial for understanding mechanisms of pathogen pathogenicity and for improving our ability to protect plants from diseases. An increasing number of effectors have been predicted in various plant pathogens. Functional characterization of these effectors has become a major focus in the study of plant-pathogen interactions. In this study, we designed a novel screening system that combines the TMV (tobacco mosaic virus)-GFP vector and Agrobacterium-mediated transient expression in the model plant Nicotiana benthamiana. This system enables the rapid identification of effectors that interfere with plant immunity. The biological function of these effectors can be easily evaluated by observing the GFP fluorescence signal using a UV lamp within just a few days. To evaluate the TMV-GFP system, we initially tested it with well-described virulence and avirulence type III effectors from the bacterial pathogen Ralstonia solanacearum. After proving the accuracy and efficiency of the TMV-GFP system, we successfully screened a novel virulence effector, RipS1, using this approach. Furthermore, using the TMV-GFP system, we reproduced consistent results with previously known cytoplasmic effectors from a diverse array of pathogens. Additionally, we demonstrated the effectiveness of the TMV-GFP system in identifying apoplastic effectors. The easy operation, time-saving nature, broad effectiveness, and low technical requirements of the TMV-GFP system make it a promising approach for high-throughput screening of effectors with immune interference activity from various pathogens.

Interrogation of RNA-protein interaction dynamics in bacterial growth.

Characterising RNA-protein interaction dynamics is fundamental to understand how bacteria respond to their environment. In this study, we have analysed the dynamics of 91% of the Escherichia coli expressed proteome and the RNA-interaction properties of 271 RNA-binding proteins (RBPs) at different growth phases. We find that 68% of RBPs differentially bind RNA across growth phases and characterise 17 previously unannotated proteins as bacterial RBPs including YfiF, a ncRNA-binding protein. While these new RBPs are mostly present in Proteobacteria, two of them are orthologs of human mitochondrial proteins associated with rare metabolic disorders. Moreover, we reveal novel RBP functions for proteins such as the chaperone HtpG, a new stationary phase tRNA-binding protein. For the first time, the dynamics of the bacterial RBPome have been interrogated, showcasing how this approach can reveal the function of uncharacterised proteins and identify critical RNA-protein interactions for cell growth which could inform new antimicrobial therapies.

Recent advances in the culture-independent discovery of natural products using metagenomic approaches.

Natural products derived from bacterial sources have long been pivotal in the discovery of drug leads. However, the cultivation of only about 1% of bacteria in laboratory settings has left a significant portion of biosynthetic diversity hidden within the genomes of uncultured bacteria. Advances in sequencing technologies now enable the exploration of genetic material from these metagenomes through culture-independent methods. This approach involves extracting genetic sequences from environmental DNA and applying a hybrid methodology that combines functional screening, sequence tag-based homology screening, and bioinformatic-assisted chemical synthesis. Through this process, numerous valuable natural products have been identified and synthesized from previously uncharted metagenomic territories. This paper provides an overview of the recent advancements in the utilization of culture-independent techniques for the discovery of novel biosynthetic gene clusters and bioactive small molecules within metagenomic libraries.

Differences and relationship in functional movement screen (FMS™) scores and physical fitness in males and female semi-professional soccer players.

Background: Soccer is the world's most popular sport for both men and women. Tests of athletic and functional performance are commonly used to assess physical ability and set performance goals. The Functional Movement Screen (FMS™) is a widely used seven-test battery developed by practitioners to provide interpretable measure of movement quality. The main objective of the present study was twofold, to analyze the relationship between FMS™ results from male and female soccer players and to compare their physical performance in different tests. Methods: A total of twenty-eight semi-professional soccer players: fourteen male (age: 21.29 ± 1.64 years; weight: 70.66 ± 5.29 kg; height: 171.86 ± 5.35 cm; BMI: 20.90 ± 2.22 kg/m2) and fourteen females (age: 20.64 ± 1.98 years; weight: 63.44 ± 5.83 kg; height: 166.21 ± 12.18 cm; BMI: 23.02 ± 2.50 kg/m2) were recruited for this study. A paired sample t-test was used for determining differences as a repeated measures analysis. All the participants conducted the following tests: The Functional Movement Test (FMS™), 10-m linear sprint, 5-0-5 COD Test and Yo-Yo Intermittent Recovery Test-Level 1 (YYIRT Level 1). Results: A t-test with data from 505 COD (change of direction) test showed significant differences between groups, p = 0.001, d = 1.11, revealing faster times in male soccer players (2.50 ± 0.19) in respect with female soccer players (2.70 ± 0.17). Crucially, a t-test with data from FMS did not reveal significant differences between groups. Multiple regression for V02max revealed significant effects (r = 0.55, r2 = 0.30, adjusted r2 = 0.24, F = 5.21, p = 0.04 and standard error = 2.20). On the other hand, multiple regression for 10-m sprint showed significant effects (r = 0.58, r2 = 0.33, adjusted r2 = 0.28, F = 5.98, p = 0.03). The impact of these factors on the correlation between FMS™ scores and physical performance measures can vary among individuals. Discussion/Conclusion: This study demonstrates the necessity of utilizing and applying multiple field-based tests to evaluate the movement and capabilities of physical performance in sports. Crucially, consider individual variations and factors such as training background, fitness level, and sport-specific demands when interpreting the relationship between the FMS™ and physical performance in both sexes.

Rapid Identification of High-Temperature Responsive Genes Using Large-Scale Yeast Functional Screening System in Potato.

As the third largest global food crop, potato plays an important role in ensuring food security. However, it is particularly sensitive to high temperatures, which seriously inhibits its growth and development, thereby reducing yield and quality and severely limiting its planting area. Therefore, rapid, and high-throughput screening for high-temperature response genes is highly significant for analyzing potato high-temperature tolerance molecular mechanisms and cultivating new high-temperature-tolerant potato varieties. We screened genes that respond to high temperature by constructing a potato cDNA yeast library. After high-temperature treatment at 39 °C, the yeast library was subjected to high-throughput sequencing, and a total of 1931 heat resistance candidate genes were screened. Through GO and KEGG analysis, we found they were mainly enriched in "photosynthesis" and "response to stimuli" pathways. Subsequently, 12 randomly selected genes were validated under high temperature, drought, and salt stress using qRT-PCR. All genes were responsive to high temperature, and most were also induced by drought and salt stress. Among them, five genes ectopically expressed in yeast enhance yeast's tolerance to high temperatures. We provide numerous candidate genes for potato response to high temperature stress, laying the foundation for subsequent analysis of the molecular mechanism of potato response to high temperature.

Involvement of lncRNAs in cancer cells migration, invasion and metastasis: cytoskeleton and ECM crosstalk.

Cancer is the main cause of death worldwide and metastasis is a major cause of poor prognosis and cancer-associated mortality. Metastatic conversion of cancer cells is a multiplex process, including EMT through cytoskeleton remodeling and interaction with TME. Tens of thousands of putative lncRNAs have been identified, but the biological functions of most are still to be identified. However, lncRNAs have already emerged as key regulators of gene expression at transcriptional and post-transcriptional level to control gene expression in a spatio-temporal fashion. LncRNA-dependent mechanisms can control cell fates during development and their perturbed expression is associated with the onset and progression of many diseases including cancer. LncRNAs have been involved in each step of cancer cells metastasis through different modes of action. The investigation of lncRNAs different roles in cancer metastasis could possibly lead to the identification of new biomarkers and innovative cancer therapeutic options.

Temporal, Kinematic and Kinetic Variables Derived from a Wearable 3D Inertial Sensor to Estimate Muscle Power during the 5 Sit to Stand Test in Older Individuals: A Validation Study.

The 5-Sit-to-stand test (5STS) is widely used to estimate lower limb muscle power (MP). An Inertial Measurement Unit (IMU) could be used to obtain objective, accurate and automatic measures of lower limb MP. In 62 older adults (30 F, 66 ± 6 years) we compared (paired t-test, Pearson's correlation coefficient, and Bland-Altman analysis) IMU-based estimates of total trial time (totT), mean concentric time (McT), velocity (McV), force (McF), and MP against laboratory equipment (Lab). While significantly different, Lab vs. IMU measures of totT (8.97 ± 2.44 vs. 8.86 ± 2.45 s, p = 0.003), McV (0.35 ± 0.09 vs. 0.27 ± 0.10 m∙s-1, p < 0.001), McF (673.13 ± 146.43 vs. 653.41 ± 144.58 N, p < 0.001) and MP (233.00 ± 70.83 vs. 174.84 ± 71.16 W, p < 0.001) had a very large to extremely large correlation (r = 0.99, r = 0.93, and r = 0.97 r = 0.76 and r = 0.79, respectively, for totT, McT, McF, McV and MP). Bland-Altman analysis showed a small, significant bias and good precision for all the variables, but McT. A sensor-based 5STS evaluation appears to be a promising objective and digitalized measure of MP. This approach could offer a practical alternative to the gold standard methods used to measure MP.

Functional evaluation of epilepsy-associated KCNT1 variants in multiple cellular systems reveals a predominant gain of function impact on channel properties.

OBJECTIVE: Gain of function variants in the sodium-activated potassium channel KCNT1 have been associated with pediatric epilepsy disorders. Here, we systematically examine a spectrum of KCNT1 variants and establish their impact on channel function in multiple cellular systems. METHODS: KCNT1 variants identified from published reports and genetic screening of pediatric epilepsy patients were expressed in Xenopus oocytes and HEK cell lines. Variant impact on current magnitude, current-voltage relationships, and sodium ion modulation were examined. RESULTS: We determined basic properties of KCNT1 in Xenopus oocyte and HEK systems, including the role of extra- and intracellular sodium in regulating KCNT1 activity. The most common six KCNT1 variants demonstrated strong gain of function (GOF) effects on one or more channel properties. Analysis of 36 total variants identified phenotypic heterogeneity but a strong tendency for pathogenic variants to exert GOF effects on channel properties. By controlling intracellular sodium, we demonstrate that multiple pathogenic KCNT1 variants modulate channel voltage dependence by altering the sensitivity to sodium ions. SIGNIFICANCE: This study represents the largest systematic functional examination of KCNT1 variants to date. We both confirm previously reported GOF channel phenotypes and expand the number of variants with in vitro GOF effects. Our data provide further evidence that novel KCNT1 variants identified in epilepsy patients lead to disease through generalizable GOF mechanisms including increases in current magnitude and/or current-voltage relationships.

Euchromatic Histone Lysine Methyltransferase 2 Inhibition Enhances Carfilzomib Sensitivity and Overcomes Drug Resistance in Multiple Myeloma Cell Lines.

Proteasome inhibitors (PIs) are extensively used for the therapy of multiple myeloma. However, patients continuously relapse or are intrinsically resistant to this class of drugs. In addition, adverse toxic effects such as peripheral neuropathy and cardiotoxicity could arise. Here, to identify compounds that can increase the efficacy of PIs, we performed a functional screening using a library of small-molecule inhibitors covering key signaling pathways. Among the best synthetic lethal interactions, the euchromatic histone-lysine N-methyltransferase 2 (EHMT2) inhibitor UNC0642 displayed a cooperative effect with carfilzomib (CFZ) in numerous multiple myeloma (MM) cell lines, including drug-resistant models. In MM patients, EHMT2 expression correlated to worse overall and progression-free survival. Moreover, EHMT2 levels were significantly increased in bortezomib-resistant patients. We demonstrated that CFZ/UNC0642 combination exhibited a favorable cytotoxicity profile toward peripheral blood mononuclear cells and bone-marrow-derived stromal cells. To exclude off-target effects, we proved that UNC0642 treatment reduces EHMT2-related molecular markers and that an alternative EHMT2 inhibitor recapitulated the synergistic activity with CFZ. Finally, we showed that the combinatorial treatment significantly perturbs autophagy and the DNA damage repair pathways, suggesting a multi-layered mechanism of action. Overall, the present study demonstrates that EHMT2 inhibition could provide a valuable strategy to enhance PI sensitivity and overcome drug resistance in MM patients.

An Agrobacterium-Mediated Transient Expression Method for Functional Assay of Genes Promoting Disease in Monocots.

Agrobacterium-mediated transient expression (AMTE) has been widely used for high-throughput assays of gene function in diverse plant species. However, its application in monocots is still limited due to low expression efficiency. Here, by using histochemical staining and a quantitative fluorescence assay of ß-glucuronidase (GUS) gene expression, we investigated factors affecting the efficiency of AMTE on intact barley plants. We found prominent variation in GUS expression levels across diverse vectors commonly used for stable transformation and that the vector pCBEP produced the highest expression. Additionally, concurrent treatments of plants with one day of high humidity and two days of darkness following agro-infiltration also significantly increased GUS expression efficiency. We thus established an optimized method for efficient AMTE on barley and further demonstrated its efficiency on wheat and rice plants. We showed that this approach could produce enough proteins suitable for split-luciferase assays of protein-protein interactions on barley leaves. Moreover, we incorporated the AMTE protocol into the functional dissection of a complex biological process such as plant disease. Based on our previous research, we used the pCBEP vector to construct a full-length cDNA library of genes upregulated during the early stage of rice blast disease. A subsequent screen of the library by AMTE identified 15 candidate genes (out of ~2000 clones) promoting blast disease on barley plants. Four identified genes encode chloroplast-related proteins: OsNYC3, OsNUDX21, OsMRS2-9, and OsAk2. These genes were induced during rice blast disease; however, constitutive overexpression of these genes conferred enhanced disease susceptibility to Colletotrichum higginsianum in Arabidopsis. These observations highlight the power of the optimized AMTE approach on monocots as an effective tool for facilitating functional assays of genes mediating complex processes such as plant-microbe interactions.

Activation Profile of TAS2R2, the 26th Human Bitter Taste Receptor.

SCOPE: To avoid ingestion of potentially harmful substances, humans are equipped with about 25 bitter taste receptor genes (TAS2R) expressed in oral taste cells. Humans exhibit considerable variance in their bitter tasting abilities, which are associated with genetic polymorphisms in bitter taste receptor genes. One of these variant receptor genes, TAS2R2, is initially believed to represent a pseudogene. However, TAS2R2 exists in a putative functional variant within some populations and can therefore be considered as an additional functional bitter taste receptor. METHODS AND RESULTS: To learn more about the function of the experimentally neglected TAS2R2, a functional screening with 122 bitter compounds is performed. The study observes responses with eight of the 122 bitter substances and identifies the substance phenylbutazone as a unique activator of TAS2R2 among the family of TAS2Rs, thus filling one more gap in the array of cognate bitter substances. CONCLUSIONS: The comprehensive characterization of the receptive range of TAS2R2 allows the classification into the group of TAS2Rs with a medium number of bitter agonists. The variability of bitter taste and its potential influences on food choice in some human populations may be even higher than assumed.

Joint estimation and imputation of variant functional effects using high throughput assay data.

Deep mutational scanning assays enable the functional assessment of variants in high throughput. Phenotypic measurements from these assays are broadly concordant with clinical outcomes but are prone to noise at the individual variant level. We develop a framework to exploit related measurements within and across experimental assays to jointly estimate variant impact. Drawing from a large corpus of deep mutational scanning data, we collectively estimate the mean functional effect per AA residue position within each gene, normalize observed functional effects by substitution type, and make estimates for individual allelic variants with a pipeline called FUSE (Functional Substitution Estimation). FUSE improves the correlation of functional screening datasets covering the same variants, better separates estimated functional impacts for known pathogenic and benign variants (ClinVar BRCA1, p=2.24×10-51), and increases the number of variants for which predictions can be made (2,741 to 10,347) by inferring additional variant effects for substitutions not experimentally screened. For UK Biobank patients who carry a rare variant in TP53, FUSE significantly improves the separation of patients who develop cancer syndromes from those without cancer (p=1.77×10-6). These approaches promise to improve estimates of variant impact and broaden the utility of screening data generated from functional assays.

Identification and Characterization of a Novel Carboxylesterase Belonging to Family VIII with Promiscuous Acyltransferase Activity Toward Cyanidin-3-O-Glucoside from a Soil Metagenomic Library.

An alkaline esterase, designated as EstXT1, was identified through functional screening from a metagenomic library. Sequence analysis revealed that EstXT1 belonged to the family VIII carboxylesterases and contained a characteristic conserved S-x-x-K motif and a deduced catalytic triad Ser56-Lys59-Tyr165. EstXT1 exhibited the strongest activity toward methyl ferulate at pH 8.0 and temperature 55°C and retained over 80% of its original activity after incubation in the pH range of 7.0-10.6 buffers. Biochemical characterization of the recombinant enzyme showed that it was activated by Zn2+ and Co2+ metal ion, while inhibited by Cu2+ and CTAB. EstXT1 exhibited significant promiscuous acyltransferase activity preferred to the acylation of benzyl alcohol acceptor using short-chain pNP-esters (C2-C8) as acyl-donors. A structure-function analysis indicated that a WAG motif is essential to acyltransferase activity. This is the first report example that WAG motif plays a pivotal role in acyltransferase activity in family VIII carboxylesterases beside WGG motif. Further experiment indicated that EstXT1 successfully acylated cyanidin-3-O-glucoside in aqueous solution. The results from the current investigation provided new insights for the family VIII carboxylesterase and lay a foundation for the potential applications of EstXT1 in food and biotechnology fields.