bioRxiv : the preprint server for biology最新文献

{"title":"Design of Orthogonal Far-Red, Orange and Green Fluorophore-binding Proteins for Multiplex Imaging.","authors":"Long Tran, Shajesh Sharma, Steffen Klein, David Juergens, Justin Decarreau, Bingxu Liu, Yujia Wang, Asim K Bera, Alex Kang, Jon Woods, Emily Joyce, Dionne K Vafeados, Nicole Roullier, Wei Chen, Gyu Rie Lee, Julia Mahamid, Luke D Lavis, Linna An, David Baker","doi":"10.1101/2025.08.03.668343","DOIUrl":"10.1101/2025.08.03.668343","url":null,"abstract":"<p><p>Fluorescent proteins and small molecule dyes have complementary strengths for biological imaging: the former are genetically manipulatable enabling tagging of specific proteins and detection of protein interactions, while the latter have greater photostability and brightness but are difficult to target. To combine these strengths, we used de novo protein design to generate binders to three bright, stable, cell-permeable dyes spanning the visible spectrum: JF657 (far red), JF596 (orange-red) and JF494 (green). For each dye, we obtain nanomolar binders with weak or no binding to the other two dyes; the accuracy of the design approach is confirmed by a crystal structure of one binder which is very close to the design model. Fusion of the JF567, JF596 and JF494 binders to three different targets followed by staining with the three dyes simultaneously enables multiplex imaging. We further expand functionality by incorporating an active site carrying out nucleophilic aromatic substitution to form a covalent linkage with the dye, and developing split versions which reconstitute fluorescence at subcellular locations where both halves are present, enabling both protein-protein interaction detection and chemically induced dimerization with fluorescence reporting. Our designs combine the advantages of fluorescent proteins and small molecule dyes and should be broadly useful for cellular imaging.</p>","PeriodicalId":519960,"journal":{"name":"bioRxiv : the preprint server for biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-09-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12424662/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145067338","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"In Vitro Evidence to Support Amphotericin B and Flucytosine Combination Therapy for Talaromycosis.","authors":"Heera Natesan Sambath, Shawin Vitsupakorn, Kaushik Sreerama Reddy, Lottie Brown, Thu Nguyen Thi Mai, Matthew Burke, Jialin Liu, Emily Evans, Charles Giamberardino, John Perfect, Hoa Ngo Thi, Thuy Le","doi":"10.1101/2025.09.23.677804","DOIUrl":"10.1101/2025.09.23.677804","url":null,"abstract":"<p><strong>Background: </strong><i>Talaromyces marneffei</i> causes talaromycosis, a life-threatening fungal disease with limited treatment options. The standard treatment of amphotericin B (AmB) induction followed by itraconazole consolidation still results in 15% to 30% mortality. This study aimed to investigate the potential of AmB and flucytosine (5FC) combination therapy to enhance antifungal activity.</p><p><strong>Methods: </strong>The in vitro antifungal activity of AmB and 5FC alone and in combination against 60 <i>T. marneffei</i> clinical isolates was evaluated using a validated colorimetric antifungal susceptibility assay and the checkerboard method. The minimum inhibitory concentration (MIC) was defined as the lowest drug concentration inhibiting ≥ 95% fungal growth (MIC₉₅) for both AmB and 5FC. The combination effect between AmB and 5FC against <i>T. marneffei</i> was determined using fractional inhibitory concentration index. Combination effects were further tested using a time-kill assay.</p><p><strong>Results: </strong>The MIC₉₅ was 0.25 - 2 μg/mL (geometric mean [GM] 0.68 μg/mL) for AmB, and 0.03 - 0.5 μg/mL (GM 0.28 μg/mL) for 5FC. Full synergy was observed in 4 isolates (7%), and indifference was observed in the remaining 56 isolates (93%). The time-kill experiments revealed a concentration-dependent fungicidal activity of AmB, and concentration-independent fungistatic effect of 5FC. Synergy between AmB and 5FC was confirmed, showing greater than 2-log₁₀ reduction in colony forming units when used in combination. No antagonism was observed.</p><p><strong>Conclusions: </strong>Our study demonstrated in vitro evidence of synergistic activity between AmB and 5FC against <i>T. marneffei</i>, providing the evidence to support in vivo and clinical trial testing of AmB and 5FC combination therapy, and dosing reduction strategies of 5FC.</p><p><strong>Lay summary: </strong>This study demonstrated in vitro synergy between amphotericin B and flucytosine against <i>Talaromyces marneffei</i>, providing the proof of concept to test this antifungal combination in a clinical trial of talaromycosis.</p>","PeriodicalId":519960,"journal":{"name":"bioRxiv : the preprint server for biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-09-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12485967/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145215194","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Spatial isoform sequencing at sub-micrometer single-cell resolution reveals novel patterns of spatial isoform variability in brain cell types.","authors":"Lieke Michielsen, Andrey D Prjibelski, Careen Foord, Wen Hu, Julien Jarroux, Justine Hsu, Alexandru I Tomescu, Iman Hajirasouliha, Hagen U Tilgner","doi":"10.1101/2025.06.25.661563","DOIUrl":"10.1101/2025.06.25.661563","url":null,"abstract":"<p><p>Spatial long-read technologies are becoming increasingly common but lack nanometer- and therefore often single-cell resolution. This leaves the question unanswered of whether spatially variable isoforms represent spatial variability within one cell type or differences in cell-type composition between different regions. Here, we developed Spl-ISO-Seq2 (220nm spot size and 500nm resolution), and the accompanying software packages Spl-IsoQuant-2 and Spl-IsoFind, enabling long-read sequencing using 140 million barcodes compared to 80,000 previously. Applying this to the adult mouse brain, we compared spatial variability by examining (a) differential isoform abundance between known brain regions and (b) spatial isoform patterns that do not align with predefined regions. While the former revealed more spatial isoform differences, both approaches identified overlapping hits, e.g., <i>Rps24</i> in oligodendrocytes. For <i>Snap25,</i> previously known to exhibit spatial isoform variation, we now show that this variability occurs in excitatory neurons. The second approach also uncovered patterns not captured by predefined-region comparisons, e.g., <i>Tnnc1</i> in excitatory neurons. Furthermore, we show that a surprising number of spatial isoform signals is not driven by cell-type composition alone. Finally, we applied our software to public Visium HD 3' long-read data to demonstrate its applicability and strong reproducibility across protocols and biological replicates. Taken together, our experimental and analytical methods enrich spatial transcriptomics with a so-far elusive isoform view of spatial variation for individual cell types.</p>","PeriodicalId":519960,"journal":{"name":"bioRxiv : the preprint server for biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-09-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12262416/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144645009","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A novel Notch and WNT signaling mechanism contribute to paediatric DCM: a pathway to new therapeutics.","authors":"Obed O Nyarko, Ethan Rausch, Jared R H Goff, Anis Karimpour-Fard, Caitlyn S Conard, Laura Hernandez-Lagunas, McKenna P A Burns, Brisa Peña, Shelley D Miyamoto, Brian L Stauffer, Carmen C Sucharov","doi":"10.1101/2025.06.07.658396","DOIUrl":"10.1101/2025.06.07.658396","url":null,"abstract":"<p><strong>Background: </strong>Paediatric Idiopathic dilated cardiomyopathy (iDCM) is a life-threatening disease. The lack of disease-specific animal models limits our understanding of its mechanisms. We previously demonstrated that paediatric iDCM serum-circulating proteins promote pathologic remodeling <i>in vitro</i>, and that secreted frizzled related protein 1 (sFRP1) increases stiffness in cardiomyocytes. Here we investigated the mechanisms by which sFRP1 contributes to iDCM.</p><p><strong>Methods: </strong>The effect of sFRP1 in combination with isoproterenol (ISO) (to recapitulate the increase in circulating catecholamine observed in paediatric iDCM) was evaluated in neonatal rat ventricular myocytes (<i>in vitro</i>), and in neonatal rats through intraperitoneal injections (<i>in vivo</i>). Function and molecular mechanisms were investigated through echocardiography and next-generation-sequencing. Protein levels and localization were determined by Western blot. Tissue stiffness was measured by Atomic Force Microscopy. <i>In vitro</i> and <i>in vivo</i> data were compared to explanted human heart tissue.</p><p><strong>Results: </strong>We show that ISO+sFRP1 reactivates the fetal gene program <i>in vitro,</i> and promotes cardiac dysfunction, dilation and stiffness <i>in vivo</i>. Importantly, we show stiffness is also increased in paediatric iDCM hearts. We identified co-activation of Notch and WNT signaling in both ISO+sFRP1-treated rats and paediatric iDCM hearts. Mechanistically, <i>in vitro</i> inhibition of Notch or β-catenin prevented pathological remodeling, and Notch inhibition improved cardiac function, myocardial stiffness and ventricular dilation in ISO+sFRP1-treated rats.</p><p><strong>Conclusion: </strong>We identified alterations in Notch and WNT signaling in paediatric iDCM hearts and in our model. Notch inhibition abrogated pathologic changes <i>in vitro</i> and <i>in vivo</i>. These findings provide novel mechanistic insights and a potential therapeutic target for paediatric iDCM.</p>","PeriodicalId":519960,"journal":{"name":"bioRxiv : the preprint server for biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-09-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12157673/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144277361","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Dominant spinal muscular atrophy linked mutations in the cargo binding domain of BICD2 result in altered interactomes and dynein hyperactivity.","authors":"Hannah Neiswender, Jessica E Pride, Rajalakshmi Veeranan-Karmegam, Phylicia Allen, Grace Neiswender, Avneesh Prabakar, Caili Hao, Xingjun Fan, Graydon B Gonsalvez","doi":"10.1101/2025.05.13.653647","DOIUrl":"10.1101/2025.05.13.653647","url":null,"abstract":"<p><p>Cytoplasmic dynein-1 (dynein) is responsible for the transport of most cellular cargo towards the minus end of microtubules. Dynein activation requires the multi-subunit dynactin complex and an activating cargo adaptor. The adaptors serve to link dynein with cargo and to fully activate the motor. Mutations in one of these activating adaptors, Bicaudal-D2 (BICD2), are associated with a neurodegenerative disease called Spinal Muscular Atrophy with Lower Extremity Predominance (SMALED2). The molecular defect that underlies SMALED2 is largely unknown. In addition to interacting with dynein, BICD2 has also been shown to associate with KIF5B, a plus-end directed microtubule motor. We hypothesized that interactome changes associated with mutant versions of BICD2, and the resulting differences in cargo transport, might underlie the etiology of SMALED2. To test our hypothesis, we first defined the interactome of wild-type BICD2. This led to the identification of known BICD2 interacting proteins in addition to potentially novel cargo such as components of the HOPS complex, a six-subunit complex involved in endo-lysosomal trafficking. We next determined the interactome of three SMALED2 linked mutants in BICD2, two of which reside in the cargo binding domain. Interestingly, all three mutations resulted in BICD2-mediated dynein hyper-activation. Furthermore, all three mutants were associated with interactome changes. One of these mutants, BICD2_R747C, was deficient in binding to HOPS complex components and the nucleoporin RANBP2. In addition, this mutant also resulted in a gain of function interaction with GRAMD1A, a protein localized to the endoplasmic reticulum. This gain of function interaction resulted in mis-localization of GRAMD1A in BICD2_R747C expressing cells. Collectively, our results suggest that dynein hyperactivity, interactome changes, and cargo transport defects might contribute to the symptoms associated with SMALED2.</p>","PeriodicalId":519960,"journal":{"name":"bioRxiv : the preprint server for biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-09-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12132236/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144218245","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Genomic plasticity drives olfactory adaptation in a pest fly.","authors":"Qi Xue, Hany K M Dweck","doi":"10.1101/2025.09.18.677102","DOIUrl":"10.1101/2025.09.18.677102","url":null,"abstract":"<p><p>Preference shifts in insects are often driven by changes in the olfactory system, yet the underlying mechanisms remain unclear. The worldwide pest <i>Drosophila suzukii</i>, which oviposits in ripe rather than overripe fruits, provides a powerful model to study these mechanisms and their behavioral consequences. Here, we show that this shift is linked to functional remodeling in four olfactory receptor neurons: ab2B, ab3A, ab4B, and ab10A. While ab3A and ab10A exhibit tuning changes shared with the non-pest relative <i>D. biarmipes</i>, ab2B and ab4B display species-specific adaptations in <i>D. suzukii</i>. These changes result not only from receptor sequence divergence but also from novel innovations: receptor co-expression in ab3A and partitioned expression of Or67a paralogs in ab2B and ab10A. Together, these findings show how genomic plasticity in chemosensory gene families enables rapid sensory adaptation and niche transition.</p>","PeriodicalId":519960,"journal":{"name":"bioRxiv : the preprint server for biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-09-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12458127/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145153097","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Replication stress increases de novo CNVs across the malaria parasite genome.","authors":"Noah J Brown, Aleksander Luniewski, Xuanxuan Yu, Michelle D Warthan, Shiwei Liu, Julia Zulawinska, Syed Ahmad, Nadia Prasad, Molly Congdon, Webster Santos, Feifei Xiao, Jennifer L Guler","doi":"10.1101/2024.12.19.629492","DOIUrl":"10.1101/2024.12.19.629492","url":null,"abstract":"<p><p>Changes in the copy number of large genomic regions, termed copy number variations (CNVs), contribute to important phenotypes. CNVs are readily identified using conventional approaches when present in a large fraction of the cell population. However, CNVs in only a few genomes are often overlooked but important; if beneficial, a de novo CNV that arises in a single genome can expand during selection to create a population of cells with novel characteristics. While single cell methods for studying de novo CNVs are increasing, we continue to lack information about CNV dynamics in rapidly evolving microbial populations. Here, we investigated de novo CNVs in the genome of the Plasmodium parasite that causes human malaria. The highly AT-rich P. falciparum genome readily accumulates CNVs that facilitate rapid adaptation. We employed low-input genomics and specialized computational tools to evaluate the impact of sub-lethal stress on the de novo CNV rate. We observed a significant increase in genome-wide de novo CNVs following treatment with an antimalarial compound that inhibits replication. De novo CNVs encompassed genes from various cellular pathways participating in human infection. This snapshot of CNV dynamics emphasizes the connection between replication stress, DNA repair, and CNV generation in this important microbial pathogen.</p>","PeriodicalId":519960,"journal":{"name":"bioRxiv : the preprint server for biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-09-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11722320/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142974362","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Multiplex mapping of protein-protein interaction interfaces.","authors":"Jingxuan He, Ling Nan Zou, Vidhi Pareek, Stephen J Benkovic","doi":"10.1101/2025.01.28.635392","DOIUrl":"10.1101/2025.01.28.635392","url":null,"abstract":"<p><p>We describe peptide mapping through Split Antibiotic Resistance Complementation (SpARC-map), a method to identify the probable interface between two interacting proteins. Our method is based on in vivo affinity selection inside a bacterial host, and uses high throughput DNA sequencing to infer the probable protein-protein interaction (PPI) interfaces. SpARC-map uses only routine microbiology techniques, with no reliance on specialized instrumentation, dedicated reagents, or reconstituting protein complexes in vitro. SpARC-map can be tuned to detect PPIs over a broad range of affinities, multiplexed to probe multiple PPIs in parallel, and its nonspecific background can be precisely measured, enabling the sensitive detection of weak PPIs. Using SpARC-map, we recover known PPI interfaces in the p21-PCNA, p53-MDM2, and MYC-MAX complexes. We also use SpARC-map to probe the purinosome, the weakly bound complex of six purine biosynthetic enzymes, where no PPI interfaces are known. There, we identify interfaces that satisfy structural requirements for substrate channeling, as well as protein surfaces that participate in multiple distinct interactions, which we validate using site-specific photocrosslinking in live human cells. Finally, we show that SpARC-map results can impose stringent constraints on machine learning based structure prediction.</p>","PeriodicalId":519960,"journal":{"name":"bioRxiv : the preprint server for biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-09-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11838385/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143461777","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Mechanisms of Neural Representation and Segregation of Multiple Spatially Separated Visual Stimuli.","authors":"Steven Wiesner, Bikalpa Ghimire, Xin Huang","doi":"10.1101/2025.09.11.675659","DOIUrl":"10.1101/2025.09.11.675659","url":null,"abstract":"<p><p>Segregating objects from one another and the background is essential for scene understanding, object recognition, and visually guided action. In natural scenes, it is common to encounter spatially separated stimuli, such as distinct figure-ground regions, adjacent objects, and partial occlusions. Neurons in mid- and high-level visual cortex have large receptive fields (RFs) that often encompass multiple, spatially separated stimuli. It is unclear how neurons represent and segregate multiple stimuli within their RFs, and the role of spatial cues for such representation. To investigate these questions, we recorded neuronal responses in the middle temporal (MT) cortex of monkeys to spatially separated stimuli that moved simultaneously in two directions. We found that, across motion directions, response tuning to multiple stimuli was systematically biased toward the stimulus located at the more-preferred RF subregion of the neuron. The sign and magnitude of this spatial-location bias were correlated with the spatial preference of the neuron for single stimuli presented in isolation. We demonstrated that neuronal responses to multiple stimuli can be captured by an extended normalization model, which is a sum of the responses elicited by individual stimuli weighted by the spatial preference of the neuron. We also proposed a circuit implementation for the model. Our results indicate that visual neurons leverage spatial selectivity within their RFs to represent multiple spatially separated stimuli. The spatial-location bias in neuronal responses enables individual components of multiple stimuli to be represented by a population of neurons with different spatial preferences, providing a neural substrate for segregating multiple stimuli.</p>","PeriodicalId":519960,"journal":{"name":"bioRxiv : the preprint server for biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-09-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12439922/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145081823","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Massively parallel interrogation of the fitness of natural variants in ancient signaling pathways reveals pervasive local adaptation.","authors":"Jose Aguilar-Rodriguez, Jean C C Vila, Shi-An A Chen, Manuel Razo-Mejia, Olivia M Ghosh, Daniel F Jarosz, Hunter B Fraser, Dmitri A Petrov","doi":"10.1101/2024.10.30.621178","DOIUrl":"10.1101/2024.10.30.621178","url":null,"abstract":"<p><p>The nature of standing genetic variation remains a central debate in population genetics, with differing perspectives on whether common variants are almost always neutral as suggested by neutral and nearly neutral theories or whether they can commonly have large functional and fitness effects as proposed by the balance theory. We address this question by mapping the fitness effects of over 9,000 natural variants in the Ras/PKA and TOR/Sch9 pathways-key regulators of cell proliferation in eukaryotes-across four conditions in <i>Saccharomyces cerevisiae</i> . While most variants are neutral in our assay, ~3,500 exhibited significant fitness effects. These non-neutral variants tend to be missense and to affect conserved, more densely packed, and less solvent-exposed protein regions. While some of these non-neutral variants are younger and rarer, and more often found in heterozygous states-consistent with purifying selection-a substantial fraction is present at high frequencies in the population, which is expected under balancing selection. Indeed, we find that variants with a positive fitness effect in our laboratory measurement show strong signs of local adaptation as they tend to be found specifically in domesticated strains isolated from human-made environments. Our findings support the view that while many common variants might be effectively neutral, a significant proportion have locally adaptive functional consequences and are driven into a subset of the population by local positive selection. This study highlights the potential to combine high-throughput precision genome editing with fitness measurements to explore natural genetic variation on a pathway-wide scale, thereby bridging the gap between population genetics and functional genomics to understand the nature of evolutionary forces in the wild.</p>","PeriodicalId":519960,"journal":{"name":"bioRxiv : the preprint server for biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-09-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11565963/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142650057","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}