Biology subjects

Peshkin, L.

Publications and source records attributed to Peshkin, L..

At least 19 recordsLinked to original sources

bioRxiv2026

Protein accumulation upstream of Cdk1 activation regulates the competence and the timing of meiosis resumption

The capacity to resume meiosis is progressively acquired during oogenesis and is ultimately restricted to fully grown oocytes. Meiotic resumption is triggered by hormonal stimulation and requires activation of Cdk1, the universal driver of M-phase entry. Cdk1 activation occurs in two steps: an initial activation of Cdk1, followed by an amplification phase that drives cell cycle re-entry. The first step depends on the accumulation of proteins that promote Cdk1 activation, while the second step involves a regulatory network of kinases and phosphatases. Using TMT-based quantitative proteomics, we reveal that growing oocytes first acquire the ability to regulate protein homeostasis in response to hormonal stimulation, and only later gain the competence to amplify initial Cdk1 activity and enter meiosis. Notably, protein accumulation, occurring independently of Cdk1 activation, is controlled by both translational and non-translational mechanisms. Together, our findings show that the molecular competence to trigger Cdk1 activation is acquired in a stepwise manner during oocyte growth. The earliest regulatory layer is the acquisition of the ability to respond to hormonal stimulation by accumulating proteins that are required for efficient Cdk1 activation and meiotic resumption.

Developmental Biology↗

bioRxiv2026

Whole-Embryo 3D Quantification Reveals Conserved Topological Design and Scaling of Germ Layers in Xenopus

How embryos with markedly different absolute sizes and cell numbers establish comparable tissue organization during development remains a fundamental question in developmental biology. To address this question, we compared two closely related Xenopus species that differ substantially in embryonic size, Xenopus laevis and Xenopus tropicalis. We generated a whole-embryo quantitative 3D atlas of cell allocation, spatial organization, and mitotic dynamics at key time points between gastrulation to tailbud stages. Using tissue clearing, and 3D imaging we tracked single-nucleus coordinates across developmental milestones to resolve how body plans adapt to organismal scale. We show that embryonic scaling is not achieved through simple proportional changes in cell number. Instead, the smaller X. tropicalis embryo is characterized by a distinct high-density tissue organization associated with a persistently higher mitotic index (~1.4-fold higher than in X. laevis at both gastrula and tailbud stages). Across development, this is accompanied by a near-doubling of cell number in X. tropicalis without a proportional increase in embryo volume. We quantify tissue organization and find species-specific cellular architectures during gastrulation that largely converge by the tailbud stage. At this stage, homologous tissues display broadly similar structural profiles despite persistent differences in embryo size, cell number, and density. Together, our findings reveal that closely related vertebrate embryos can follow distinct cellular organization trajectories while converging toward comparable tissue architecture, providing a quantitative framework for understanding robust body plan formation across divergent physical scales.

Developmental Biology↗

bioRxiv2026

A kinome inhibitor screen implicates adhesion and growth factor signaling in cellular recovery after caspase activation

Apoptosis is a common form of regulated cell death and requires cysteine-aspartic proteases called effector caspases. Caspase activation triggers positive feedback, leading to the idea that apoptosis is irreversible. However, we and others have demonstrated that cancer cells can survive effector caspase activation and become more aggressive and drug-resistant as a result. Despite the profound implications of apoptotic reversal, also known as anastasis, for both regenerative medicine and cancer therapy, the molecular pathways that enable cells to survive executioner caspase activation remain largely unmapped. To systematically dissect this phenomenon, we developed a quantitative screening platform that combines inducible caspase activation with kinome-wide pharmacological profiling. This approach uniquely allowed us to: (1) identify pharmacological modulators of post-caspase survival, (2) identify specific kinases regulating post-caspase survival, and (3) distinguish general toxicity from anastasis effects. This approach implicated regulators of cell adhesion and the cytoskeleton, consistent with the known rounding of apoptotic cells and respreading during recovery. Growth factor signaling also emerged from the analysis. In addition to its expected effects on unstressed cells, fetal bovine serum markedly increased anastasis. Some growth factor combinations were more effective than individual ones at recapitulating the serum effect. Similarly, pleiotropic kinase inhibitors were generally more effective than selective ones. Nevertheless, selective Rho kinase inhibition significantly enhanced anastasis whereas Akt inhibition impaired it, suggesting that these kinases serve as central nodes that integrate multiple upstream inputs. Beyond identifying specific kinase targets, our work provides a framework for anti-anastasis therapies that could prevent cancer cell recovery after chemotherapy.

cell biology↗

bioRxiv2025

Using regularized regression and biological covariation to impute missing values in quantitative proteomics

Proteomics studies analyzing many samples typically generate datasets with missing values where many protein abundances are only quantified in a subset of assayed conditions. While multiplexing with isobaric tags can address this by combining multiple samples into a single injection, missing values are unavoidable when the sample count exceeds the number of available isobaric tags (currently >35). Such missing data complicates the interpretation of large-scale studies across diverse experimental conditions. Here, we introduce a method to impute missing values of relative protein abundance by leveraging measurements from other proteins in the dataset through regularized regression. Our technique, which is applicable to diverse datasets including different cell lines, animals, or biochemical perturbations, capitalizes on the hitherto overlooked biological covariation among protein abundance changes. Our analysis of eight published proteomics datasets reveals a robust imputation capability, achieving a median R2 of 0.55 to 0.8 between imputed and measured data. We demonstrate a similar imputation efficacy in multiple measurement modalities: TMT, DIA, label free, and TMT phosphoproteomics. When examining regression coefficients that were pivotal for accurate data imputation we found that those often mirror known biology. We propose that previously overlooked biological covariation might lead to the generation of novel hypotheses and ultimately advance our understanding of systems level protein organization.

biochemistry↗

bioRxiv2025

Normalized Raman Imaging for Studies of Tissue Physiology of the Kidney

Conventional histological relies on fixation, embedding, sectioning, and staining methods that distort cellular architecture, extract lipids, and introduces variability, limiting reproducibility. We present Normalized Stimulated Raman Imaging (NoRI) that enables quantitative, label-free protein and lipid measurements at high-spatial resolution. NoRI computationally corrects protein, lipid, and water Raman signals, allowing quantitative biomass measurements while preserving tissue architecture, facilitating analysis by convolutional neural networks. Applied to mouse kidney, NoRI accurately classified tubule types (F1-[harmonic mean of precision and recall against manual annotation]=0.93), anatomical regions (F1=0.91), and biological sex (F1=0.97), revealing greater cytoplasmic lipid (+6.9mg/mL; p=0.028) and nuclear protein (+26.3mg/mL; p<0.001) in female tubules. In acute kidney injury, NoRI captured dynamic lipid and protein organization and quantified brush border remodelling and lipid droplet changes over 25 days. Spatial lipid quantification was central for feature classification in AKI (F1=1.0). These results establish NoRI as a reproducible, quantitative method for diagnostics, histopathology, and feature discovery.

physiology↗

bioRxiv2025

Innovations in spinal cord cell type heterogeneity across vertebrate evolution

Vertebrates display remarkable diversity of sensorimotor behaviors, each adapted to distinct ecological and survival demands. This diversity raises fundamental questions about the evolutionary origin of motor control: do conserved spinal circuits underlie these behaviors, and how have they diverged across species. Recent studies detail spinal cell-type architecture in mammals but comparable, high-resolution atlases of the non-mammalian spinal cord are lacking. Here, we compare spinal cord cell types between fish, frogs, mice and humans, spanning [~]450 million years of evolution. Across species, we define highly conserved programs of cell type specification that segregate spinal neurons into nearly identical cardinal classes during development. This contrasts with adult stages, when spinal cell-type composition selectively diverges for excitatory neuron subpopulations. Using spatial transcriptomics, we localize this species divergence to the superficial, dorsal spinal cord, where variant neuropeptide expression defines mammalian-specific cell types. The most dorsal spinal cord thus emerges as a recently evolved hub for sensory integration in mammals, a neospinal cord analogous to the neocortex.

neuroscience↗

bioRxiv2025

High doses of electromagnetic radiation pre- and post- fertilization produce different developmental outcomes in Xenopus laevis.

We examined the effects of X-ray irradiation on Xenopus laevis, focusing on pre- and post-fertilization exposure. We applied X-ray doses of 10, 50, 100, 250, and 500 Gy. Fifty percent of the 360 eggs irradiated at 250 Gy failed to fertilize, while fertilized eggs developed normally until the gastrula stage. Doses ranging from 10 to 250 Gy caused developmental anomalies. High mortality rates were observed at doses of 100 to 500 Gy. Post-fertilization irradiation at 50 to 100 Gy resulted in 100% lethality, while exposure to 10 Gy led to only 13% lethality, although both exposure levels produced similar types of developmental anomalies compared to pre-fertilization irradiation. This study highlights how the timing and intensity of exposure critically affect embryo viability, especially during the sensitive stages of fertilization and gastrulation. We establish the necessary and sufficient dosage to further investigate the molecular mechanisms of X-ray damage to DNA and protein. HighlightHigh doses of electromagnetic radiation pre- and post-fertilization produce different developmental outcomes in Xenopus laevis.

pharmacology and toxicology↗

bioRxiv2025

Efficient Techniques for Comprehensive Tissue Sampling in Adult Xenopus

Xenopus has long been a pivotal model organism for investigating vertebrate development and disease, offering deep insights into cellular processes and gene function. Despite the wealth of information on embryonic Xenopu,s there remains a significant gap in standardized methods for adult tissue sampling, especially for modern approaches like quantitative proteomics. This study introduces a comprehensive protocol for rapid, precise, and efficient sampling of multiple tissues in adult Xenopu.sThe protocol addresses challenges associated with the subtle anatomical differences compared to other anurans, ensuring reproducibility even for those with limited experience in frog dissection. This protocol is optimized for high- quality biochemical analyses by prioritizing sample freshness. We are facilitating the rapid collection of up to 18 tissues within an hour. Additionally, the methods apply to perfused and unperfused conditions, providing flexibility for a range of experimental needs. This work not only fills a critical methodological gap for Xenopus laevisand tropicalisbut also serves as a valuable resource for researchers adapting techniques to similar amphibian models, thereby enhancing the scope and reliability of comparative biological and evolutionary studies. SummaryThis is part one of a comprehensive Xenopus sampling protocol. The tissues sampled are the heart ventricle, arterial trunk, left liver lobe, gallbladder, lung, pancreas, spleen, larynx, esophagus, stomach, intestines, testes, fat bodies, oviduct, paired kidneys, sciatic plexus, skin, thymus, and whole eye.

zoology↗

bioRxiv2024

An epigenetic clock for Xenopus tropicalis reveals age-associated sites enriched in H4K20me3-marked heterochromatin

DNA methylation clocks have been widely used for accurate age prediction, but most studies have been carried out on mammals. Here we present a clock for the aquatic frog Xenopus tropicalis, a widely used model organism in developmental biology and genomics. The clock, trained on targeted bisulfite sequencing data, achieved a cross-validation median absolute error of 0.70 years. To construct the clock, we collected DNA methylation data from 192 frogs at genomic regions containing CpG sites previously shown to have age-associated methylation in Xenopus. We found that CpG sites with methylation levels significantly increasing or decreasing with age are enriched in heterochromatic regions marked with H4K20me3 and H3K9me3. CpG sites with positively age-associated methylation levels are enriched in bivalent chromatin and gene bodies with H3K36me3, and tend to be proximal to lowly expressed genes. These epigenetic features of aging are similar to those found in mammals, suggesting evolutionary conservation of epigenetic aging mechanisms. Our clock enables future aging biology experiments that leverage the unique properties of amphibians.

genomics↗

bioRxiv2024

Decoding protein phosphorylation during oocyte meiotic divisions using phosphoproteomics

Oocyte meiotic divisions represent a critical process in sexual reproduction, as a diploid non-dividing oocyte is transformed into a haploid fertilizable egg, as a prelude for the subsequent embryonic divisions and differentiation. Although cell differentiation and proliferation are governed by transcription, oocyte maturation and early embryonic divisions depend entirely on changes in protein abundance and post- translational modifications. Here, we analyze the abundance and phosphorylation of proteins during Xenopus oocyte meiotic maturation. We reveal significant shifts in protein stability, related to spindle assembly, DNA replication and RNA-binding. Our analysis pinpoints broad changes in phosphorylation correlating with key cytological meiotic milestones, noteworthy changes in membrane trafficking, nuclear envelope disassembly and modifications in microtubule dynamics. Additionally, specific phosphorylation events target regulators of protein translation, Cdk1 and the Mos/MAPK pathway, thereby providing insight into the dynamics of Cdk1 activity, as related to the meiotic cell cycle. This study sheds light on the orchestration of protein dynamics and phosphorylation events during oocyte meiotic divisions, providing a rich resource for understanding the molecular pathways orchestrating meiotic progression in the frog, and most likely applicable to other vertebrate species.

developmental biology↗

bioRxiv2024

Genome-wide DNA Methylation Patterns in Daphnia magna Reveal Targeting to 5' Exons

Studying DNA methylation in Daphnia magna, a model organism in ecological and evolutionary research, offers valuable insights into pharmaceutical toxicity and behavioral ethology. In this study, we characterized DNA methyltransferases and mapped DNA methylation across the D. magna genome using whole-genome bisulfite sequencing. Our analysis revealed a highly expressed, nonfunctional de novo methyltransferase (DNMT3.1) alongside lowly expressed functional de novo methyltransferase DNMT3.2 and maintenance methyltransferase DNMT1. D. magna displays overall low DNA methylation, targeting primarily CpG dinucleotides. Methylation is sparse at promoters but elevated in the first exons downstream of transcription start sites, with these exons showing hypermethylation relative to adjacent introns. In contrast to prior studies, we observed minimal age-related changes in DNA methylation patterns that were not sufficiently robust to build an accurate epigenetic clock. These findings expand our understanding of the epigenetic landscape in D. magna.

systems biology↗

bioRxiv2024

Synergy Between TNFα and Proteostatic Stress Drives Cell Death and Guard Immunity

The production and sensing of type I interferons (IFN-I) are critical for antiviral defense, yet most virus-infected cells do not produce IFN-I or upregulate IFN-stimulated genes. Using quantitative proteomics and global protein synthesis measurements, we show that productive viral infection globally down-regulates protein synthesis, restricting the IFN response. Guard immunity, which responds to disruptions in essential cellular processes, might compensate for the lack of IFN-I response by rapidly killing infected cells. However, non-pathological stressors can also disrupt proteostasis, making it unclear how cells decide to trigger guard immunity. We hypothesized that TNF, produced by macrophages, provides a contextual signal allowing specificity. Using live-cell fluorescence microscopy and mathematical modeling, we showed that TNF synergizes with the rapid decay of the anti-apoptotic protein c-FLIP to induce cell death and prevent viral spread. Our findings demonstrate that TNF contextualizes proteostasis loss as non-sterile, enabling the activation of guard immunity to counteract viral infection.

immunology↗

bioRxiv2024

Single Cell Transcriptome Defines Cell Type Repertoire of Adult Daphnia magna.

Detailed knowledge of transcriptional responses to environmental and developmental cues is impossible without single cell (SC) resolution data. We performed two SC RNAseq experiments surveying transcriptional profiles of females and males of D. magna, a freshwater plankton crustacean which is both a classic and emerging new model for eco-physiology, toxicology, and evolutionary genomics. We were able to identify over 30 distinct cell types about half of which could be functionally annotated. First, we identified ovaries- and testis-related cell types by focusing on female- and male-specific clusters. Second, we compared markers between SC clusters and bulk RNAseq data on transcriptional profiles of early embryos, circulating hemocytes, midgut, heads (containing brain, eyes, muscles and hepatic caeca), antennae II, and carapace. Finally, we compared transcriptional profiles of Daphnia cell clusters with orthologous markers of 250+ cell types annotated in Drosophila cell atlas. This allowed us to recognize striated muscle cells, gut enterocytes, cuticular cells, as well as 5 different neuron types, including photoreceptors and 3 ovaries-related clusters, one of which tentatively identified as the germ line cells. One well-defined cluster showed a significant enrichment in markers of both hemocytes and fat body of Drosophila, but not with bulk RNAseq data from circulating hemocytes, allowing us to hypothesize the existence of non-circulating, fat body-associated population of hemocytes in Daphnia. On the other hand, the circulating hemocytes express numerous cuticular proteins suggesting their role, in addition to macrophagy, in wound repair. At the same time numerous cell types remain unidentified, including those that map to FCA groups ambiguously or are characterized by Daphnia-specific markers with no clear orthology in the fruitfly. Likewise, many known or presumed cell types or tissues in Daphnia have not been identified to SC clusters. A detailed in-situ hybridization study would be necessary to match not yet annotated SC clusters to functional cell groups. HighlightsO_LIFirst single-cell transcriptomic atlas for Daphnia magna, identifies > 30 distinct cell types. C_LIO_LINovel cell type representing circulating hemocytes may play a role in cuticle regeneration. C_LIO_LIEvidence for non-circulating hemocyte-like cells associated with the fat body in Daphnia. C_LIO_LICuticle/epithelial cells expressing photoreceptors, suggesting light-sensing capabilities. C_LIO_LISubfunctionalization of divergent paralogs across cell types for ecological versatility. C_LI

cell biology↗

bioRxiv2024

Short lifespan is one's fate, long lifespan is one's achievement: lessons from Daphnia

Studies of longevity and senescence rely on baseline life expectancy of reference genotypes measured in standardized conditions such as food level and group size. Variation in baseline lifespan data across labs and protocols and among genotypes can make longevity intervention studies difficult to compare, particularly when GxE interactions exist. Furthermore, extending the lifespan of a short-lived genotype or of any genotype under suboptimal conditions may be of a lesser theoretical and translational value than extending the maximal possible lifespan. Daphnia is rapidly becoming a model organism of choice for longevity research complementing data obtained on traditional models. In this study we report baseline longevity of several genotypes (parthenogenetic clones) of a long-lived species D. magna under a variety of laboratory protocols, aiming to document the highest possible lifespan, factors reducing it, and physiological parameters that change with age and correlate with longevity. Combining data from 25 different experiments across two labs we report strong differences among clones of different geographic origin, moderate effects of group size and medium composition on longevity, and strong GxE with respect to food level. Specifically, short-lived clones that tend to originate from small intermittent habitats show little or no caloric restriction (CR) longevity extension, while long-lived ones expand their lifespan even further when maintained at 25% of the ad libitum food. We find no evidence of any trade-offs between longevity and fecundity across clones or correlations with age-specific feeding rate. We find that in the short-lived, CR non-responsive clones show little correlation between longevity and two measures of lipid peroxidation (LPO: lipid hydroperoxides and MDA abundance). In contrast, the long-lived, CR-responsive clones show a positive longevity correlation with lipid hydroperoxide abundance at any age, and a negative correlation with MDA concentration measured at about median lifespan. This indicates differences among genotypes in longevity-related accumulation of LPO targets, efficiency of detoxification of LPO products, and/or their effects on longevity. Our observations support the hypothesis that a long lifespan can be affected by food availability and levels of oxidative damage, while genetically determined short lifespan remains short regardless. We suggest a set of condition and genotypes to be used as a reference for longevity studies in Daphnia.

ecology↗

bioRxiv2024

A revised single-cell transcriptomic atlas of Xenopus embryo reveals new differentiation dynamics.

This paper introduces an updated single-cell atlas for pivotal developmental stages in Xenopus, encompassing gastrulation, neurulation, and early tailbud. Notably surpassing its predecessor, the new atlas enhances gene mapping, read counts, and gene/cell type nomenclature. Leveraging the latest Xenopus tropicalis genome version, alongside advanced alignment pipelines and machine learning for cell type assignment, this release maintains consistency with previous cell type annotations while rectifying nomenclature issues. Employing an unbiased approach for cell type assignment proves especially apt for embryonic contexts, given the considerable number of non-terminally differentiated cell types. An alternative cell type attribution here adopts a fuzzy, non-deterministic stance, capturing the transient nature of early embryo progenitor cells by presenting an ensemble of types in superposition. The value of the new resource is emphasized through numerous examples, with a focus on previously unexplored germ cell populations where we uncover novel transcription onset features. Offering interactive exploration via a user-friendly web portal and facilitating complete data downloads, this atlas serves as a comprehensive and accessible reference.

developmental biology↗

bioRxiv2023

On standardization of controls in lifespan studies

The search for interventions to slow down and even reverse aging is a burgeoning field. The literature cites hundreds of supposedly beneficial pharmacological and genetic interventions in model organisms: mice, rats, flies and worms, where research into physiology is routinely accompanied by lifespan data. Naturally the negative results are more frequent, yet scientifically quite valuable if analyzed systematically. Yet, there is a strong "discovery bias", i.e. results of interventions which turn out not to be beneficial remain unpublished. Theoretically, all lifespan data is ripe for re-analysis: we could contrast the molecular targets and pathways across studies and help focus the further search for interventions. Alas, the results of most longevity studies are difficult to compare. This is in part because there are no clear, universally accepted standards for conducting such experiments or even for reporting such data. The situation is worsened by the fact that the authors often do not describe experimental conditions completely. As a result, works on longevity make up a set of precedents, each of which might be interesting in its own right, yet incoherent and incomparable. Here we point out specific issues and propose solutions for quality control by checking both inter- and intra-study consistency of lifespan data.

systems biology↗

bioRxiv2023

Effective Rapid Blood Perfusion in Xenopus

Xenopus has been a powerful model organism for understanding vertebrate development and disease for over a hundred years. Here we define a rapid blood perfusion protocol in Xenopus aimed at a consistent and drastic reduction of blood across tissues. Perfusion is done by inserting a needle directly into the ventricle and pumping heparin in PBS through the vascular system. The whole procedure should take about 10 minutes per frog. Blood is dominated by a few highly abundant proteins and cell types which create numerous issues by masking most other molecules and cell types of interest. Reproducible characterization of adult Xenopus tissues with quantitative proteomics and single cell transcriptomics will gain from applying this protocol prior to organ dissections defined in companion papers. The procedure is aimed at standardization of practice across the animals of different gender, age and Xenopus species, specifically X.laevis and X.tropicalis. SUMMARYAn effective rapid blood perfusion protocol to prepare tissue samples for transcriptomics and proteomics studies.

physiology↗

bioRxiv2022

Epigenetic profiling and incidence of disrupted development point to gastrulation as aging ground zero in Xenopus laevis

Recent studies suggest the existence of a natural rejuvenation event during early embryonic development of mice, followed by epigenetic aging. Here, by profiling embryonic DNA methylation in the African clawed frog, Xenopus laevis, we found that the epigenetic entropy basepoint maps to the gastrulation stage of embryogenesis and corresponds to a rapid increase in embryo transcript abundance. We further developed a frog aging clock, revealing that this species epigenetically ages. Application of this clock to developmental stages identified a decrease in epigenetic age during early embryogenesis, with the minimal age reached around gastrulation. By examining individual developmental trajectories of 6,457 embryos, we found that this stage is also accompanied by a higher incidence of disrupted development. Taken together, our data point to gastrulation as a critical stage for aging and natural rejuvenation, characterized by the lowest epigenetic age, increased mortality, nadir of DNA methylation entropy and rapid increase in embryo transcript abundance, defining aging ground zero as the basepoint where rejuvenation ends and the aging process begins.

genetics↗