Biology subjects

Aharoni, A.

Publications and source records attributed to Aharoni, A..

At least 19 recordsLinked to original sources

bioRxiv2026

Spatially enforced engagement of PD-1⁺ T cells with AXL⁺ cells reprograms the tumor microenvironment and overcomes anti-PD-1 resistance

Background: AXL overexpression promotes tumor-cell plasticity, local immune suppression, and resistance to PD-1 blockade. Conventional anti-AXL and anti-PD-1 antibodies combinations act independently and do not ensure physical proximity between PD-1+ T cells and AXL+ cells within the immunosuppressive tumor microenvironment. Methods: To address this limitation, we developed a non-tandem, tetravalent IgG bispecific antibody designed to simultaneously engage PD-1 and AXL (BiTE-AXL/PD-1). Its antitumor activity was evaluated using in vitro functional assays and humanized and immunocompetent head and neck squamous cell carcinoma (HNSCC) models and compared with the combination of monospecific anti-AXL and anti-PD-1 antibodies. Spatial multiplex imaging was used to characterize treatment-associated changes in the tumor microenvironment of an anti-PD-1-resistant murine model. Results: BiTE-AXL/PD-1 enhanced T-cell activation, promoted AXL-dependent tumor-T-cell conjugation, and increased tumor-cell killing in vitro, outperforming the combination of the two monospecific antibodies. In humanized and immunocompetent HNSCC models, BiTE treatment significantly delayed tumor growth compared with the antibody combination and was well tolerated, with no overt toxicity. Spatial multiplex imaging revealed a shift from a fibroblast-dominated microenvironment characterized by exhausted CD8+ T cells toward dendritic-cell- and CD8+ T-cell-enriched niches associated with antitumor immune activity. Conclusion: BiTE-AXL/PD-1 promotes physical proximity between PD-1+ T cells and AXL+ cells, enhances antitumor immune responses, and remodels the immunosuppressive tumor microenvironment. These findings support PD-1/AXL bispecific targeting as a promising therapeutic strategy to overcome resistance to PD-1 blockade in HNSCC.

cancer biology↗

bioRxiv2026

Quinoa produces an insect molting hormone through a plant biosynthetic gene cluster

The steroid hormone 20-hydroxyecdysone (20E) controls molting and metamorphosis in arthropods, yet it also accumulates in plants, where its biosynthesis has remained unknown. We show that quinoa (Chenopodium quinoa) produces 20E through a seven-gene biosynthetic cluster encoding five cytochrome P450s, a fatty acid hydroxylase-like enzyme (FAH), and a 2-oxoglutarate-dependent dioxygenase (2OGD). Distinct mutant alleles, complementation crosses, and chemical rescue established FAH as essential for pathway entry, whereas natural accessions carrying a large cluster deletion lacked 20E and all detectable intermediates. Heterologous reconstruction in Nicotiana benthamiana demonstrated that the cluster is sufficient to convert lathosterol to 20E through an unusual FAH-catalyzed C6 oxidation and 2OGD-mediated C5 epimerization. Plants and arthropods therefore evolved distinct enzymatic routes to the same molecule. This pathway provides a blueprint for engineering 20E and defining its broader ecological functions.

plant biology↗

bioRxiv2026

Context-dependent determinants of CRISPR-Cas9 editing efficiency revealed through cross-species endogenous editing analysis

Accurate prediction of CRISPR-Cas9 guide RNA (gRNA) editing efficiency remains limited, particularly outside human systems, where models trained on exogenous human datasets show poor generalization. We analyzed Cas9 efficiency and repair outcomes using novel endogenous editing data from four human cell types, two tomato cell types, and cells from giant river prawn and black soldier fly. While integrating publicly available predictors via ensemble frameworks improved performance, our analysis revealed hundreds of novel features affecting activity. Crucially, dominant features related to sites competition for gRNA, and local geometric properties varied across systems, highlighting the strong context dependence of Cas9 efficiency and arguing against a universal model. Interestingly, codon usage bias-based features also emerged as informative predictors, as they are proxies for chromatin accessibility. In contrast, trends in repair outcomes remained conserved. This work provides essential resources for more generalizable CRISPR guide design.

synthetic biology↗

bioRxiv2026

Cruciform-forming AT/TA repeats are acted upon by structure selective endonucleases and Rad51 prior to repositioning to the nuclear periphery for repair

Structure-forming DNA repeats can pose a barrier to DNA replication and repair, creating chromosomal fragile sites. An AT/TA DNA repeat, derived from the Flex1 region of human common fragile site FRA16D, can form hairpin and cruciform structures which interfere with DNA replication. When inserted into the S. cerevisiae genome, the Flex1(AT)34 repeat stimulates chromosome deletions in a manner dependent on the Mus81-Mms4 nuclease and the SLX4 nuclease scaffold. It was previously found that hairpin forming CAG/CTG repeats move to the nuclear periphery to maintain genomic stability. Here, we show that a structure forming AT/TA repeat also relocalizes to the nuclear periphery in late S/G2 phase in a replication and length-dependent manner. In contrast to the CAG repeat, this shift in nuclear positioning is dependent on polySUMOylation and the activity of the Mus81-Mms4 nuclease. Processing by the Mre11 nuclease and Rad51-dependent strand exchange occurs prior to repositioning. Replication analysis indicates that the replisome likely bypasses the AT/TA repeat leaving behind a DNA structure that initiates relocation to the nuclear periphery. We conclude that AT/TA repeats form post-replicative DNA structures that are targeted for nuclease cleavage and require hairpin processing and repositioning to the nuclear periphery for homologous recombination-dependent repair.

genetics↗

bioRxiv2025

Replisome progression regulates R-loop mediated transcriptional repression

Maintaining cellular proliferation necessitates the synchronized activity of diverse molecular machineries operating in parallel on the genome. Wide-spread transcription and R-loop formation can interfere with genome duplication, causing transcription-replication conflicts (TRCs). Here, we use live-cell imaging for simultaneous monitoring of replication fork progression and transcription dynamics of an R-loop prone gene. While robust replisome progression through R-loops is observed in wild-type cells, it is impaired in RnaseH, Mph1 and Sen1 mutants. Intriguingly, we find that R-loop formation inhibits gene transcription, but this inhibition is reversed by replisome passage, demonstrating the dynamic crosstalk between replication, transcription and R-loops in a single cell cycle. Unexpectedly, we find that R-loops also have beneficial roles in reducing the density of RNAPII molecules on the gene, thereby preventing fork stalling upon high RNAPII occupancy. These findings illuminate that regulating R-loops and RNAPII density together is critical for preventing harmful TRCs.

cell biology↗

bioRxiv2025

Creation and Validation of a Proteome-Wide Yeast Library for Protein Detection and Analysis

A significant challenge in cell biology is to uncover the function of uncharacterized proteins. Surprisingly a quarter of the proteome is still poorly understood even in the most well studied model organisms. Systematic methodologies, including the use of tagged protein collections, have emerged as a powerful approach to address this gap. Despite the availability of proteome- wide collections featuring various fused proteins, the impact of tag size on protein function highlighted the need for using minimally disruptive tags for functional genomic studies. To rise to this challenge, we have created a proteome-wide collection of yeast strains in which proteins are N-terminally tagged with the Hemagglutinin (HA) epitope. The library leverages the compact size of the HA tag to minimize drawbacks associated with larger tags while enabling efficient functional analysis. We showcase the potential uses of our library for systematically evaluating protein size, abundance and localization using an in vivo labeling approach. Our characterization underscores the potential utility of a proteome-wide HA-tagged library in revealing novel aspects of cell biology, providing an additional powerful tool for functional genomics.

molecular biology↗

bioRxiv2024

Spec2Class: Accurate Prediction of Plant Secondary Metabolite Class using Deep Learning

Mass spectrometry (MS)-based data is commonly used in studying metabolism and natural products, but typically requires domain-specific skill and experience to analyze. Existing computational tools for non-targeted metabolite analysis (i.e., metabolomics) mostly rely on comparison to reference MS spectral libraries for metabolite identification, limiting the annotation of metabolites for which reference spectra do not exist. This is the case in plant secondary metabolites, where most spectral features remain unidentified. Here, we developed Spec2Class, a deep-learning algorithm for the identification and classification of plant secondary metabolites from liquid chromatography (LC)-MS/MS spectra. We used the in-house spectral library of 7973 plant metabolite chemical standards, alongside publicly available data, to train Spec2Class to classify LC-MS/MS spectra to 43 common plant secondary metabolite classes. Tested on held out sets, our algorithm achieved an overall accuracy of 73%, outperforming state-of-the-art classification. We further established a prediction certainty parameter to set a threshold for low-confidence results. Applying this threshold, we reached an accuracy of 93% on an unseen dataset. We show a high robustness of our prediction to noise and to the data acquisition method. Spec2Class is publicly available and is anticipated to facilitate metabolite identification and accelerate natural product discovery. Significance StatementUntargeted mass spectrometry (MS) is essential for natural product discovery but is limited by product identification, which is often manual and requires domain-specific skills. Spec2Class addresses this limitation by accurately classifying plant secondary metabolites from LC-MS/MS spectra without reliance on reference spectral libraries. Trained on a substantial dataset and using a prediction certainty threshold, it outperforms state-of-the-art algorithms with 93% accuracy. This tool demonstrates high robustness against noise and different data acquisition methods, promising to streamline metabolite identification and expedite natural product research. Spec2Class is open-source, publicly available, and easy to integrate into natural product discovery pipelines.

biochemistry↗

bioRxiv2023

A transient dermal niche and dual epidermal programs underlie sweat gland development

Eccrine glands are mammalian skin appendages indispensable for human thermoregulation. Like all skin-derived appendages, eccrine glands form from multipotent progenitors in the basal skin epidermis. It remains unclear how epidermal progenitors progressively specialize to specifically form eccrine glands, precluding efforts to regenerate these vital organs. Herein, we applied single nucleus transcriptomics to compare the expression content of wildtype, eccrine-forming mouse skin to that of mice harboring a skin-specific disruption of Engrailed 1 (En1), a transcription factor that promotes the formation of eccrine glands in both humans and mice. We identify two concurrent epidermal transcriptomes in the earliest eccrine anlagen: a predominant transcriptome that is shared with hair follicles, and a vastly underrepresented transcriptome that is En1-dependent and eccrine-specific. We demonstrate that differentiation of the eccrine anlage requires the induction of a transient and transcriptionally unique dermal niche that forms around each developing gland in humans and mice. Our study defines the transcriptional determinants underlying eccrine identity in the epidermis and uncovers the dermal niche required for eccrine developmental progression. By identifying these defining components of the eccrine developmental program, our findings set the stage for directed efforts to regenerate eccrine glands for comprehensive skin repair.

developmental biology↗

bioRxiv2023

Carotenoid retention during post-harvest storage of Capsicum annuum: the role of the fruit surface structure.

Degradation of carotenoids in food crops during post-harvest storage results in major economic and nutritional losses. In this study, a pepper (Capsicum annuum) panel for post-harvest carotenoid retention was studied to elucidate underlying mechanisms associated with this commercial trait of interest. Quantitative determination of carotenoid pigments and concurrent cellular analysis indicated that those pepper fruit with thicker lipid exocarp layers and smooth surfaces, following post-harvest drying and storage, possessed increased carotenoid retention. Total cutin monomer content increased in high carotenoid retention fruits and sub-epidermal cutin deposits were responsible for the difference in exocarp thickness. Cutin biosynthesis and cuticle precursor transport genes were differentially expressed between high and low carotenoid retention genotypes, and this supports the finding that fruit cuticle biosynthesis is associated with carotenoid retention. Carotenoids were located within cells embedded within the sub-epidermal cutin layer, and these carotenoids were protected from degradation due to the lack of permeability of the fruit surface to reactive oxygen species, and their precursors. The identification of a novel role for the pepper fruit surface in protecting against carotenoid degradation serves as an important discovery for the function of the fruit cuticle and provides an exploitable resource to enhance fruit quality. Highlight statementCarotenoid pigments in Chilli pepper confer post-harvest colour and nutritional quality. Analysis of diverse commercial genotypes indicates the involvement of the fruit surface in carotenoid retention

plant biology↗

bioRxiv2022

Arabidopsis thaliana induces multigenerational stress tolerance against biotic and abiotic stressors and memorization of host colonization in Bacillus subtilis

Bacillus subtilis is a beneficial bacterium that supports plant growth and protects it from bacterial, fungal, and viral infections. Here using a simplified system of B. subtilis, and Arabidopsis thaliana interactions, we found that history-dependent behavior is a potentially important manifestation of host colonization, worth classifying and quantifying. To study history-dependent adaptation to plant hosts, we develop a simple framework for measuring the physiological memory of B. subtilis following its interaction with Arabidopsis thaliana. We found that A. thaliana secretions reduce the lag time in pre-exposed bacteria compared with naive B. subtilis cells, even after their complete removal. Pre-exposed B. subtilis cells colonized plant roots more efficiently than naive bacteria, and were more resistant to biotic and abiotic stressors such as salicylic acid, and high salinity. Descendants of bacteria treated with plant secretions had an advantage in the competition against unexposed bacteria for root colonization. The effect of plant secretions was independent of their roles as nitrogen and carbon sources. Transcriptome analysis of both ancestors and descendants revealed that a specific set of plant-induced processes, among them c-di-AMP homeostasis, and the general stress response, maintain the signature of association with the plant in descendants of pre-exposed bacteria. Consistently, plant secretions compensated for the loss of c-di-AMP cyclases but required the general stress response and the master regulator Spo0A to exert their short and long-term effects. Overall, our work demonstrates that bacterial memory manifested by multigenerational reversible adaptation to plant hosts confirms an advantage to symbiotic bacteria during competition.

microbiology↗

bioRxiv2021

Fatty Alcohols, a Minor Component of the Tree Tobacco Surface Wax, Reduce Insect Herbivory

Despite decades of research resulting in a comprehensive understanding of epicuticular wax biosynthesis and metabolism, the function of these almost ubiquitous metabolites in plant-herbivore interactions remains unresolved. To develop a better understanding of this role, we investigated plant-herbivore interactions in four Nicotiana glauca (tree tobacco) genome edited mutants. This included [eceriferum1 (cer1), eceriferum3 (cer3), {beta}-ketoacyl-coA synthase6 (kcs6), and fatty acyl-coA reductase (far)] displaying a wide range of alkane and fatty alcohol abundances. Three interaction classes were examined: chewing herbivory with seven caterpillar and one snail species, phloem feeding with Myzus persicae (green peach aphid), and egg laying with Bemisia tabaci (sweet potato whitefly). We found that high wax load and alkane abundance did not reduce caterpillar or snail herbivory. However, fatty alcohol content was negatively correlated with caterpillar growth, suggesting a role in reducing insect herbivory despite its lower levels. Aphid reproduction and feeding activity were not correlated with wax load and composition but are potentially affected by altered cutin composition of cer1 mutants. When examining non-feeding activities, wax crystal morphology could explain the preference of B. tabaci to lay eggs on wildtype plants relative to cer1 and far mutants. Accordingly, the fatty alcohol wax component reduces caterpillar herbivory on the chemical level, but oviposition is increased when wax crystals are dense. The results suggest that this varied response between herbivore classes and species, at times displaying increased and at times reduced fitness in response to altered wax composition is in part a consequence of co-evolution that shaped the specific effects of different N. glauca metabolites such as anabasine and fatty alcohols in plant-herbivore interactions.

plant biology↗

bioRxiv2021

Cuticular Wax Composition is Essential for Plant Recovery Following Drought with Little Effect under Optimal Conditions

Despite decades of extensive study, the role of cuticular lipids in sustaining plant fitness is far from being understood. To answer this fundamental question, we employed genome editing in tree tobacco (Nicotiana glauca) plants and generated mutations in 16 different cuticular lipids-related genes. We chose tree tobacco due to the abundant, yet simply composed epicuticular waxes deposited on its surface. Five out of 9 different mutants that displayed a cuticular lipids-related phenotype were selected for in depth analysis. They had either reduced total wax load or complete deficiency in certain wax components. This led to substantial modification in surface wax crystal structure and to elevated cuticular water loss. Remarkably, under non-stressed conditions, mutant plants with altered wax composition did not display elevated transpiration or reduced growth. However, once exposed to drought, plants lacking alkanes were not able to strongly reduce their transpiration, leading to leaf death and impaired recovery upon resuscitation, and even to stem cracking, a phenomenon typically found in trees experiencing drought stress. In contrast, plants deficient in fatty alcohols exhibited an opposite response, having reduced cuticular water loss and rapid recovery following drought. This differential response was part of a larger trend, of no common phenotype connecting plants with a glossy appearance. We conclude that alkanes are essential under drought response and much less under normal non-stressed conditions, enabling plants to seal their cuticle upon stomatal closure, reducing leaf death and facilitating a speedy recovery.

plant biology↗

bioRxiv2021

ABA homeostasis and long-distance translocation is redundantly regulated by ABCG ABA importers

The effects of abscisic acid (ABA) on plant growth, development and response to the environment depend on local ABA concentrations. Here, we exploited a genome-scale amiRNA screen, targeting the Arabidopsis transportome, to show that ABA homeostasis is regulated by two previously unknown ABA transporters. ABCG17 and ABCG18 are localized to the plasma membranes of leaf mesophyll and stem cortex cells to redundantly promote ABA import, leading to conjugated inactive ABA sinks, thus restricting stomatal closure. ABCG17 and ABCG18 double knockdown revealed that the transporters encoded by these genes not only limit stomatal aperture size, conductance and transpiration while increasing water-use efficiency but also control ABA translocation from the shoot to the root to regulate lateral root emergence. The proposed ABCG17- and ABCG18-dependent ABA glucosyl ester shoot sink mechanism is restrained under abiotic stress conditions to further activate the ABA responses.

plant biology↗

bioRxiv2021

Inhibition of gibberellin accumulation by water deficiency promotes fast and long-term 'drought avoidance' responses in tomato

Plants reduce transpiration to avoid dehydration during drought episodes by stomatal closure and inhibition of canopy growth. While abscisic acid (ABA) has a primary role in drought avoidance, previous studies suggest that gibberellin (GA), might also be involved. Here we show in tomato (Solanum lycopersicum) that shortage of water inhibited the expression of the GA biosynthesis genes GA20 oxidase1 (GA20ox1) and GA20ox2 and induced the GA-deactivating gene GA2ox7 in leaves and guard cells, resulting in reduced bioactive GA levels. Drought regulation of GA metabolism was mediated by ABA-dependent and independent pathways, and by the transcription factor DEHYDRATION RESPONSIVE ELEMENT BINDING (DREB), TINY1. Mutations in GA20ox1 and GA20ox2 reduced water loss due to the smaller canopy area. On the other hand, loss of GA2ox7 did not affect leaf size, but attenuated stomatal response to water deficiency; during soil dehydration, ga2ox7 plants closed their stomata and reduced transpiration later than WT, suggesting that ga2ox7 stomata are hyposensitive to soil dehydration. Together, the results suggest that drought-induced GA deactivation in guard cells contributes to stomatal closure at the early stages of soil dehydration, whereas inhibition of GA synthesis in leaves promotes mainly the long-term reduction in canopy growth to reduce transpiration area.

molecular biology↗

bioRxiv2020

Key histone chaperones have distinct roles in replisome progression and genomic stability

Replication-coupled (RC) nucleosome assembly is an essential process in eukaryotic cells in order to maintain chromatin structure during DNA replication. The deposition of newly synthesized H3/H4 histones during DNA replication is facilitated by specialized histone chaperones. Although the contribution of these histone chaperones to genomic stability has been thoroughly investigated, their effect on replisome progression is much less understood. By exploiting a time-lapse microscopy system for monitoring DNA replication in individual live cells, we examined how mutations in key histone chaperones including CAC1, RTT106, RTT109 and ASF1, affect replication fork progression. Our experiments revealed that mutations in CAC1 or RTT106 that directly deposit histones on the DNA, slowdown replication fork progression. In contrast, analysis of cells mutated in the intermediary ASF1 or RTT109 histone chaperones revealed that replisome progression is not affected. We found that mutations in histone chaperones including ASF1 and RTT109 lead to extended G2/M duration, elevated number of RPA foci and in some cases, increased spontaneous mutation rate. Our research suggests that histone chaperones have distinct roles in enabling high replisome progression and maintaining genome stability during cell cycle progression. Author SummaryHistone chaperones (HC) play key roles in maintaining the chromatin structure during DNA replication in eukaryotic cells. Despite extensive studies on HCs, little is known regarding their importance for replication fork progression during S-phase. Here, we utilized a live-cell imaging approach to measure the progression rates of single replication forks in individual yeast cells mutated in key histone chaperones. Using this approach, we show that mutations in CAC1 or RTT106 HCs that directly deposit histones on the DNA lead to slowdown of replication fork progression. In contrast, mutations in ASF1 or RTT109 HCs that transfers H3/H4 to CAC1 or RTT106, do not affect replisome progression but lead to post replication defects. Our results reveal distinct functions of HCs in replication fork progression and maintaining genome stability.

cell biology↗

bioRxiv2020

The evolution of human SIRT1 for multi-specificity is enabled by active-site adaptation

Many enzymes that catalyze protein post-translational modifications (PTM) can specifically modify multiple target proteins. However, little is known regarding the molecular basis and evolution of multi-specificity in these enzymes. Here, we used bioinformatics and experimental approaches to investigate the molecular basis and evolution of multi-specificity in the sirtuin-1 (SIRT1) deacetylase. Guided by bioinformatics analysis of SIRT1 orthologues and substrates, we identified and examined key mutations that have occurred during the evolution of human SIRT1. We found that while these mutants maintain high activity toward conserved histone substrates they exhibit a dramatic loss of activity toward acetylated p53 protein that appeared only in Metazoa. These results demonstrate that active site substitutions in SIRT1 are essential to enable the inclusion of p53 substrate. Our results are consistent with a model in which promiscuous ancestral activity can provide an evolutionary starting point from which multispecificity toward a number of cellular substrates can develop.

biochemistry↗

bioRxiv2018

Chance and pleiotropy dominate genetic diversity in complex bacterial environments

How does environmental complexity affect the evolution of single genes? Here, we measured the effects of a set of mutants of Bacillus subtilis glutamate dehydrogenase across 19 different environments - from homogenous single cell populations in liquid media to heterogeneous biofilms, plant roots and soil communities. The effects of individual gene mutations on organismal fitness were highly reproducible in liquid cultures. Strikingly, however, 84% of the tested alleles showed opposing fitness effects under different growth conditions (environmental pleiotropy). In biofilms and soil samples, different alleles dominated in parallel replica experiments. Accordingly, we found that in these heterogeneous cell communities the fate of mutations was dictated by a combination of selection and drift. The latter relates to programmed prophage excisions that occurred along biofilm development. Overall, per individual condition, by the combined action of selection, pleiotropy and chance, a wide range of glutamate dehydrogenase mutations persisted and sometimes fixated. However, across longer periods and multiple environments, nearly all this diversity would be lost - indeed, considering all environments and conditions we have tested, wild-type is the fittest allele.

microbiology↗

bioRxiv2018

Bacterial virulence against an oceanic bloom-forming phytoplankter is mediated by algal DMSP

Emiliania huxleyi is a bloom forming microalga that impacts the global sulfur cycle by producing large amounts of dimethylsulfoniopropionate (DMSP) and its volatile metabolic product dimethyl sulfide (DMS). Top-down regulation of E. huxleyi blooms is attributed to viruses and grazers, however, the possible involvement of algicidal bacteria in bloom demise is still elusive. We isolated from a North Atlantic E. huxleyi bloom a Roseobacter strain, Sulfitobacter D7, which exhibited algicidal effects against E. huxleyi upon co-culturing. Both the alga and the bacterium were found to co-occur during a natural E. huxleyi bloom, therefore establishing this host-pathogen system as an attractive, ecologically relevant model for studying alga-bacterium interaction in the oceans. During interaction, Sulfitobacter D7 consumed and metabolized algal DMSP to produce high amounts of methanethiol, an alternative product of DMSP catabolism. We revealed a unique strain-specific response, in which E. huxleyi strains that exuded higher amounts of DMSP were more susceptible to Sulfitobacter D7 infection. Intriguingly, exogenous application of DMSP enhanced bacterial virulence and induced susceptibility in a resistant algal strain to the bacterial pathogen. This DMSP-dependent pathogenicity was highly specific as compared to supplementation of propionate and glycerol. We propose a novel function for DMSP, in addition to its central role in mutualistic interactions, as a mediator of bacterial virulence that may regulate E. huxleyi blooms.

microbiology↗