Biology subjects

Gallagher, J.

Publications and source records attributed to Gallagher, J..

9 recordsLinked to original sources

bioRxiv2025

Engineering of CRISPR-Cas PAM recognition using deep learning of vast evolutionary data

CRISPR-Cas enzymes must recognize a protospacer-adjacent motif (PAM) to edit a genomic site, significantly limiting the range of targetable sequences in a genome. Machine learning-based protein engineering provides a powerful solution to efficiently generate Cas protein variants tailored to recognize specific PAMs. Here, we present Protein2PAM, an evolution-informed deep learning model trained on a dataset of over 45,000 CRISPR-Cas PAMs. Protein2PAM rapidly and accurately predicts PAM specificity directly from Cas proteins across Type I, II, and V CRISPR-Cas systems. Using in silico deep mutational scanning, we demonstrate that the model can identify residues critical for PAM recognition in Cas9 without utilizing structural information. As a proof of concept for protein engineering, we employ Protein2PAM to computationally evolve Nme1Cas9, generating variants with broadened PAM recognition and up to a 50-fold increase in PAM cleavage rates compared to the wild-type under in vitro conditions. This work represents the first successful application of machine learning to achieve customization of Cas enzymes for alternate PAM recognition, paving the way for personalized genome editing.

synthetic biology↗

bioRxiv2024

Comparing alpha-synuclein-interactomes between multiple systems atrophy and Parkinsons disease reveals unique and shared pathological features.

IntroductionPrimary synucleinopathies, such as Parkinsons disease (PD), Dementia with Lewy bodies (DLB), and multiple system atrophy (MSA), are neurodegenerative disorders with some shared clinical and pathological features. Aggregates of alpha-synuclein (syn) phosphorylated at serine 129 (PSER129) are the hallmark of synucleinopathies, which for PD/DLB are found predominantly in neurons (Neuronal cytoplasmic inclusions "NCIs"), but for MSA, aggregates are primarily found in oligodendroglia (Glial cytoplasmic inclusions "GCIs"). It remains unclear if the distinct pathological presentation of PD/DLB and MSA are manifestations of distinct or shared pathological processes. We hypothesize that the distinct synucleinopathies MSA and PD/DLB share common molecular features. MethodsUsing the in-situ proximity labeling technique biotinylation by antibody recognition (BAR), we compare aggregated syn-interactomes (BAR-PSER129) and total syn-interactomes (BAR-MJFR1) between MSA (n=5) and PD/DLB (n=10) in forebrain and midbrain structures. ResultsFor BAR-PSER129 and BAR-MJFR1 captures, syn was the most significantly enriched protein in PD/DLB and MSA. In PD/DLB, BAR-PSER129 identified 194 syn-aggregate-interacting proteins, while BAR-MJFR1 identified 245 syn interacting proteins. In contrast, in the MSA brain, only 38 and 175 proteins were identified for each capture, respectively. When comparing MSA and PD/DLB, a high overlap (59.5%) was observed between BAR-MJFR1 captured proteins, whereas less overlap (14.4%) was observed for BAR-PSER129. Direct comparison between MSA and PD/DLB revealed 79 PD/DLB-associated proteins and only three MSA-associated proteins (CBR1, CRYAB, and GFAP). Pathway enrichment analysis revealed PD/DLB interactions were dominated by vesicle/SNARE-associated pathways, in contrast to MSA, which strongly enriched for metabolic/catabolic, iron, and cellular oxidant detoxification pathways. A subnetwork of cytosolic antioxidant enzymes called peroxiredoxins drove cellular detoxification pathways in MSA. A common network of 26 proteins, including neuronal-specific proteins (e.g., SNYGR3) with HSPA8 at the core, was shared between MSA and DLB/PD. Extracellular exosome pathways were universally enriched regardless of disease or BAR target protein. ConclusionSynucleinopathies have divergent and convergent syn-aggregate interactions, indicating unique and shared pathogenic mechanisms. MSA uniquely involves oxidant detoxification processes in glial cells, while vesicular processes in neurons dominate PD/DLB. Shared interactions, specifically SNYGR3 (i.e., a neuronal protein), between MSA and PD/DLB suggest neuronal axons origin for both diseases. In conclusion, we provide syn aggregates protein interaction maps for two distinct synucleinopathies.

neuroscience↗

bioRxiv2024

Adapting protein language models for structure-conditioned design

Generative models for protein design trained on experimentally determined structures have proven useful for a variety of design tasks. However, such methods are limited by the quantity and diversity of structures used for training, which represent a small, biased fraction of protein space. Here, we describe proseLM, a method for protein sequence design based on adaptation of protein language models to incorporate structural and functional context. We show that proseLM benefits from the scaling trends of underlying language models, and that the addition of non-protein context - nucleic acids, ligands, and ions - improves recovery of native residues during design by 4-5% across model scales. These improvements are most pronounced for residues that directly interface with non-protein context, which are faithfully recovered at rates >70% by the most capable proseLM models. We experimentally validated proseLM by optimizing the editing efficiency of genome editors in human cells, achieving a 50% increase in base editing activity, and by redesigning therapeutic antibodies, resulting in a PD-1 binder with 2.2 nM affinity.

bioengineering↗

bioRxiv2024

Design of highly functional genome editors by modeling the universe of CRISPR-Cas sequences

Gene editing has the potential to solve fundamental challenges in agriculture, biotechnology, and human health. CRISPR-based gene editors derived from microbes, while powerful, often show significant functional tradeoffs when ported into non-native environments, such as human cells. Artificial intelligence (AI) enabled design provides a powerful alternative with potential to bypass evolutionary constraints and generate editors with optimal properties. Here, using large language models (LLMs) trained on biological diversity at scale, we demonstrate the first successful precision editing of the human genome with a programmable gene editor designed with AI. To achieve this goal, we curated a dataset of over one million CRISPR operons through systematic mining of 26 terabases of assembled genomes and meta-genomes. We demonstrate the capacity of our models by generating 4.8x the number of protein clusters across CRISPR-Cas families found in nature and tailoring single-guide RNA sequences for Cas9-like effector proteins. Several of the generated gene editors show comparable or improved activity and specificity relative to SpCas9, the prototypical gene editing effector, while being 400 mutations away in sequence. Finally, we demonstrate an AI-generated gene editor, denoted as OpenCRISPR-1, exhibits compatibility with base editing. We release OpenCRISPR-1 publicly to facilitate broad, ethical usage across research and commercial applications.

synthetic biology↗

bioRxiv2020

Cortical dynamics underlying social behavior in dominance hierarchy and spatial navigation

Rodents establish dominance hierarchy as a social ranking system in which one subject acts as dominant over all the other subordinate individuals. Dominance hierarchy regulates food access and mating opportunities, but little is known of its significance in collective behavior, for instance during navigation for foraging or migration. Here, we implemented a simplified goal-directed spatial navigation task in mice and found that the social context exerts significant influence on individual decision-making, even when efficient navigation rules leading to reward had been previously learned. Thus, decision-making and consequent task performance were strongly dependent on contingent social interactions arising during collective navigation, yet their influence on individual behavior was outlined by dominance hierarchy. Dominant animals did not behave as leaders during navigation; conversely, they were most sensitive to social context. Social ranking in turn was reflected in the neural activity and connectivity patterns of the prefrontal cortex and hippocampus, both in anesthetized and behaving mice. These results suggest that the interplay between contingent social interactions and dominance hierarchy can regulate behavioral performance, supported by the intrinsic matrix of coordinated activity in the hippocampal-prefrontal circuit. Significance StatementDecision-making is shaped by intrinsic features, such as memory-stored information, and external influences, such as social interactions, yet their interplay is not well understood. We studied decision-making during collective behavior and found that instead of prioritizing memory-based pertinent information, mice shifted their individual decisions according to contingent social interactions arising in the social context. Conversely, constitutive social interactions, such as dominance hierarchy, were relevant to outline the effect of the social environment on individual behavior. Our results suggest that intrinsic hippocampal-cortical activity and connectivity patterns define social interactions. Hence, intrinsic cortical dynamics underlie behavioral performance during social decision-making.

neuroscience↗

bioRxiv2019

Evaluating the efficacy of enzalutamide and the development of resistance in a preclinical mouse model of type-I endometrial carcinoma

Androgen Receptor (AR) signaling is a critical driver of hormone-dependent prostate cancer and has also been proposed to have biological activity in female hormone-dependent cancers, including type I endometrial carcinoma (EMC). In this study, we evaluated the preclinical efficacy of a third-generation AR antagonist, enzalutamide, in a genetic mouse model of EMC, Sprr2f-Cre;Ptenfl/fl. In this model, ablation of Pten in the uterine epithelium leads to localized and distant malignant disease as observed in human EMC. We hypothesized that administering enzalutamide through the diet would temporarily decrease the incidence of invasive and metastatic carcinoma, while prolonged administration would result in development of resistance and loss of efficacy. Short-term treatment with enzalutamide reduced overall tumor burden through increased apoptosis but failed to prevent progression of invasive and metastatic disease. These results suggest that AR signaling may have biphasic, oncogenic and tumor suppressive roles in EMC that are dependent on disease stage. Enzalutamide treatment increased Progesterone Receptor (PR) expression within both stromal and tumor cell compartments. Prolonged administration of enzalutamide decreased apoptosis, increased tumor burden and resulted in the clonal expansion of tumor cells expressing high levels of p53 protein, suggestive of acquired Trp53 mutations. In conclusion, we show that enzalutamide induces apoptosis in EMC but has limited efficacy overall as a single agent. Induction of PR, a negative regulator of endometrial proliferation, suggests that adding progestin therapy to enzalutamide administration may further decrease tumor burden and result in a prolonged response.

cancer biology↗

bioRxiv2019

Effects of MCHM on yeast metabolism

On January 2014 approximately 10,000 gallons of crude 4-Methylcyclohexanemethanol (MCHM) and propylene glycol phenol ether (PPH) were accidentally released into the Elk River, West Virginia, contaminating the tap water of around 300,000 residents. Crude MCHM is an industrial chemical used as flotation reagent to clean coal. At the time of the spill, MCHMs toxicological data were limited, an issue that have been addressed by different studies focused on understanding the immediate and long-term effects of MCHM on human health and the environment. Using S. cerevisiae as a model organism we study the effect of acute exposition to crude MCHM on metabolism. Yeasts were treated with MCHM 3.9 mM in YPD for 30 minutes. Polar and lipid metabolites were extracted from cells by a chloroform-methanol-water mixture. The extracts were then analyzed by direct injection ESI-MS and by GC-MS. The metabolomics analysis was complemented with flux balance analysis simulations done with genome-scale metabolic network models (GSMNM) of MCHM treated vs non-treated control. We integrated the effect of MCHM on yeast gene expression from RNA-Seq data within these GSMNM. 181 and 66 metabolites were identified by the ESI-MS and GC-MS procedures, respectively. From these 38 and 34 relevant metabolites were selected from ESI-MS and GC-MS respectively, for 72 unique compounds. MCHM induced amino acid accumulation, via its effects on amino acid metabolism, as well as a potential impairment of ribosome biogenesis. MCHM affects phospholipid biosynthesis and decrease the levels of ergosterol, with a potential impact in the biophysical properties of yeast cellular membranes. The FBA simulations were able to reproduce the deleterious effect of MCHM on cells growth and suggest that the effect of MCHM on ubiquinol:ferricytochrome c reductase reaction, caused by the under-expression of CYT1 gene, could be the driven force behind the observed effect on yeast metabolism and growth.

pharmacology and toxicology↗

bioRxiv2018

Of puzzles and pavements: a quantitative exploration of leaf epidermal cell shape

SummaryThe epidermal cells of leaves lend themselves readily to observation and display many shapes and types: tabular pavement cells, complex trichomes, and stomatal complexes1. Pavement cells from Zea mays (maize) and Arabidopsis thaliana (arabidopsis) both have highly undulate anticlinal walls and are held as representative of monocots and eudicots, respectively. In these two model species, we have a nuanced understanding of the molecular mechanisms that generate undulating pavement cell shape2-9. This model-system dominance has led to two common assumptions: first, that particular plant lineages are characterized by particular pavement cell shapes; and second, that undulatory pavement cell shapes are common enough to be model shapes. To test these assumptions, we quantified pavement cell shape in the leaves of 278 vascular plant taxa and assessed cell shape metrics across large taxonomic groups. We settled on two metrics that described cell shape diversity well in this dataset: aspect ratio (degree of cell elongation) and solidity (a proxy for margin undulation). We found that pavement cells in the monocots tended to have weakly undulating margins, pavement cells in ferns had strongly undulating margins, and pavement cells in the eudicots showed no particular degree of undulation. Indeed, we found that cells with strongly undulating margins, like those of arabidopsis and maize, were in the minority in seed plants. At the organ level, we found a trend towards cells with more undulating margins on the abaxial leaf surface vs. the adaxial surface. We also detected a correlation between cell and leaf aspect ratio: highly elongated leaves tended to have highly elongated cells (low aspect ratio), but not in the eudicots. This indicates that while plant anatomy and plant morphology can be connected, superficially similar leaves can develop through very different underlying growth dynamics (cell expansion and division patterns). This work reveals the striking diversity of pavement cell shapes across vascular plants, and lays the quantitative groundwork for testing hypotheses about pavement cell form and function.\n\n\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=139 SRC=\"FIGDIR/small/361717_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (83K):\norg.highwire.dtl.DTLVardef@1a571a8org.highwire.dtl.DTLVardef@c8cf7dorg.highwire.dtl.DTLVardef@1ae3090org.highwire.dtl.DTLVardef@f666ce_HPS_FORMAT_FIGEXP M_FIG C_FIG

plant biology↗

bioRxiv2018

Mitochondrial D-loop DNA analyses of Norway Lobster (Nephrops norvegicus) reveals genetic isolation between Atlantic and Mediterranean populations

Nephrops norvegicus is a commercially valuable demersal fisheries species. Relatively little is understood about this species population dynamics across its distribution with previous mitochondrial and microsatellite studies failing to identify significant population-level differentiation. In this study, sequence variation in the mitochondrial (mtDNA) D-loop was analysed from samples across the distribution range. Analysis of a 375bp fragment of the D-loop revealed significant genetic differentiation between samples from the northeast Atlantic and the East Mediterranean (FST = 0.107, P<0.001). Tau ({tau}), theta ({theta}0 and {theta}1) and Fus Fs values suggest the species spread between 10,500 to 19,000 ybp and subsequently expanded rapidly across the Atlantic.

genetics↗