Biology subjects

Parikh, V.

Publications and source records attributed to Parikh, V..

17 recordsLinked to original sources

bioRxiv2026

Decoding the Allosteric Paradox: A Dual Framework Integrating AI Cofolding Models with Landscape-Guided Interpretable AI Framework of Ligand-Protein Binding

Artificial intelligence (AI) has transformed prediction of protein structure and biomolecular interactions, yet modeling of allosteric regulation remains a persistent and unresolved challenge. We develop a dual explainable AI framework that systematically interrogates AI Co-Folding models AlphaFold3, Protenix, Boltz-2, Chai-1, and DynamicBind on rigorously stratified datasets of orthosteric and allosteric ligand-protein complexes. While all AI models excel in accurate modeling of orthosteric ligand binding, a universal and architecture-independent collapse emerges in prediction of allosteric complexes. The biophysical logic for this dichotomy is unveiled through physics-based lens of the energy landscape theory and local frustration analysis. Orthosteric binding creates dominant energetic funnels via ligand-induced minimal frustration quenching, while allosteric sites preserve neutral frustration landscapes in both apo and holo protein states. The findings show that conformational heterogeneity and evolutionary plasticity encoded in allosteric binding landscapes may conceal the recurrent recognition patterns AI models are trained to detect. By linking prediction outcomes to frustration landscapes, this study recasts AI shortcomings in allosteric ligand binding as diagnostic indicators of fundamental biophysical constraints, establishing a physics-informed framework that turns the allosteric blind spot into mechanistic insight for next-generation landscape-aware predictive tools.

biophysics↗

bioRxiv2025

Neutral Frustration Landscape Architecture of SARS-CoV-2 Spike -Antibody Interfaces Shapes Immune Evasion Mechanisms for Ultrapotent Neutralizing Antibodies and Determines Pathways of Viral Adaptation: Insights from Integrative Computational Approach

The relentless evolution of SARS-CoV-2 underscores the urgent need to decipher the molecular principles that enable certain antibodies to maintain exceptional breadth and resilience against immune escape. In this study, we employ a multi-pronged computational framework integrating structural analysis, conformational dynamics, mutational scanning, MM-GBSA binding energetics, and conformational and mutational frustration profiling to dissect the mechanisms of ultrapotent neutralization by a cohort of broadly reactive Class 1 antibodies (BD55-1205, 19-77, ZCP4C9, ZCP3B4) and the Class 4/1 antibody ADG20. We reveal a unifying biophysical architecture: these antibodies bind via rigid, pre-configured interfaces that distribute binding energy across extensive epitopes through numerous suboptimal yet synergistic interactions, predominantly with backbone atoms and conserved side chains. This distributed redundancy enables tolerance to mutations at key sites like F456L or A475V without catastrophic loss of affinity. Mutational scanning identifies a hierarchical hotspot organization where primary hotspots (e.g., H505, Y501, Y489, Y421) which overlap with ACE2-contact residues and incur high fitness costs upon mutation are buffered by secondary hotspots (e.g., F456, L455) that are more permissive to variation. MM-GBSA energy decomposition confirms that van der Waals-driven hydrophobic packing dominates binding, with primary hotspots contributing disproportionately to affinity, while electrostatic networks provide auxiliary stabilization that mitigates mutational effects. Critically, both conformational and mutational frustration analyses demonstrate that immune escape hotspots reside in neutral-frustration landscape that permit mutational exploration without destabilizing the RBD, explaining the repeated emergence of convergent mutations across lineages. Our results establish that broad neutralization arises not from ultra-high-affinity anchors, but rather from strategic energy distribution across rigid, evolutionarily informed interfaces. By linking distributed binding, neutral frustration landscapes, and viral fitness constraints, this framework provides a predictive blueprint for designing next-generation therapeutics and vaccines capable of withstanding viral evolution.

biophysics↗

bioRxiv2025

Deciphering the Mechanistic Continuum of Broadly Neutralizing Class 4 Antibodies Targeting Conserved Cryptic Epitopes of the SARS-CoV-2 Spike Protein : Operating at the Intersection of Binding, Allostery and Immune Escape

Understanding atomistic basis of multi-layer mechanisms employed by broadly reactive neutralizing antibodies of SARS-CoV-2 spike protein without directly blocking receptor engagement remains an important challenge in coronavirus immunology. Class 4 antibodies represent an intriguing case: they target a deeply conserved, cryptic epitope on the receptor-binding domain yet exhibit variable neutralization potency across subgroups F1 (CR3022, EY6A, COVA1-16), F2 (DH1047), and F3 (S2X259). In this study, we employed a multi-modal computational approach combining atomistic and coarse-grained simulations, mutational scanning of binding interfaces, binding free energy calculations, and allosteric modeling using dynamic network analysis to map the allosteric landscapes and binding hotspots of these antibodies. Through this approach, our data revealed that distal binding can influence ACE2 engagement and immune escape traits through the confluence of direct interfacial interactions and allosteric effects. We found that group F1 antibodies can operate via classic allostery by modulating flexibility in the receptor binding domain loop regions and indirectly interfering with ACE2 binding using long-range effects. Group F2 antibody DH1047 represents an intermediate mechanism, engaging residues T376, R408, V503, and Y508 hotspots which are both critical for ACE2 binding and under immune pressure. Mutational scanning and rigorous binding free energy calculations highlight the synergy between hydrophobic and electrostatic interactions, while dynamic network modeling reveals a shift toward localized communication pathways connecting the cryptic site to the ACE2 interface. Our results demonstrate how group F3 antibody S2X259 achieves efficient synergistic mechanism through confluence of direct competition with ACE2 and localized allosteric effects leading to stabilization of the spike protein at the cost of increased escape vulnerability. Dynamic network analysis identifies a shared "allosteric ring" embedded in the core of the receptor binding domain and serving a conserved structural scaffold mediating long-range signal propagation with antibody-specific extensions propagating toward the ACE2 interface. The findings of this study support a modular model of antibody-induced allostery where neutralization strategies evolve via refinement of peripheral network connections, rather than complete redesign of the epitope itself. Taken together, this study establishes a robust computational framework for atomistic understanding of mechanisms underlying neutralization activity and immune escape for class 4 antibodies which harness conformational dynamics, binding energetics, and allosteric modulation to influence viral entry. The findings highlight the modular evolution of neutralization strategies, where progressive refinement of peripheral interactions enhances potency but increases susceptibility to immune pressure.

biophysics↗

bioRxiv2025

Exploring Dynamic Modulation of Binding, Allostery and Immune Resistance in the SARS-CoV-2 Spike Complexes with Classes of Antibodies Targeting Cryptic Binding Sites: Antibody-Specific Augmentations of Conserved Allosteric Architecture Can Influence Evolution of Viral Escape

The ongoing evolution of SARS-CoV-2 variants has underscored the need to understand not only the structural basis of antibody recognition but also the dynamic and allosteric mechanisms that could underlie complexity of broad and escape-resistant neutralization. In this study, we employed a multi-scale approach integrating structural analysis, hierarchical molecular simulations, mutational scanning and network-based allosteric modeling to dissect how Class 4 antibodies (represented by S2X35, 25F9, and SA55) and Class 5 antibodies (represented by S2H97, WRAIR-2063 and WRAIR-2134) can modulate conformational behavior, binding energetics, allosteric interactions and immune escape patterns of the SARS-CoV-2 spike protein. Using hierarchical simulations of the antibody complexes with the spike protein and ensemble-based mutational scanning of binding interactions we showed that these antibodies through targeting conserved cryptic sites can exert allosteric effects that influence global conformational dynamics in the RBD functional regions. The ensemble-based mutational scanning of binding interactions revealed an excellent agreement with experimentally derived deep mutational scanning (DMS) data accurately recapitulating the known binding hotspots and escape mutations across all studied antibodies. The predicted destabilization values in functional sites are consistent with experimentally observed reductions in antibody binding affinity and immune escape profiles demonstrating that computational models can robustly reproduce and forecast mutation-induced immune escape trends. Using dynamic network modeling we characterized the antibody-induced changes in residue interaction networks and long-range interactions. The results revealed that class 4 antibodies can exhibit distinct patterns of allosteric influence despite targeting overlapping regions, while class 5 antibodies elicit consistently dense and broadly distributed allosteric networks and long-range stabilization of the RBD conformations. Dynamic network analysis identifies a conserved allosteric network core that mediates long-range interactions and incudes antibody specific allosteric extensions that connect the binding interface hotspots with allosteric hubs. This study suggests that mechanisms of binding and immune escape for classes of antibodies targeting cryptic binding sites may be determined by confluence of multiple factors including high-affinity binding and long-range allosteric effects that modulate RBD adaptability and propagation of dynamic constraints which can influence mechanisms of viral evolution and escape.

biophysics↗

bioRxiv2025

Nucleosome-mediated conformational switches in micro-eccDNAs

Extrachromosomal-circular DNA (eccDNA) are circular-DNA elements that are implicated in cellular processes such as genomic diversification and instability, along with other genomic mechanisms. However, their structure and chromatinization state is poorly understood. Here we identified a 358-bp circular micro-eccDNA molecule derived from the human Titin gene, which was selected for this study based on its size and sequence characteristics, and used atomistic molecular dynamics simulations to study its interaction with nucleosomes and corresponding topological state. We show the presence or absence of bound nucleosomes provides a topological switch between intact and denatured DNA.

biophysics↗

bioRxiv2025

Conformational Landscaping and Dynamic Mutational Profiling of Binding Interactions and Immune Escape for Broadly Neutralizing Class I Antibodies with SARS-CoV-2 Spike Protein: Distributed Binding Hotspot Networks Underlie Mechanism of Viral Resistance Against Existing Variants

The rapid evolution of SARS-CoV-2 has underscored the need for a detailed understanding of antibody binding mechanisms to combat immune evasion by emerging variants. In this study, we investigated the interactions between Class I neutralizing antibodies BD55-1205, BD-604, OMI-42, P5S-1H1, and P5S-2B10 and the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein using multiscale modeling which combined coarse-grained simulations and atomistic reconstruction of conformational landscapes together with mutational scanning of the binding interfaces, dynamic profiling of binding and immune escape using molecular mechanics generalized Born surface area (MM-GBSA) analysis. A central theme emerging from this work is the critical role of epitope breadth and interaction diversity in determining an antibody resilience to mutations. BD55-1205 antibody exemplifies the advantages of broad epitope coverage and distributed hotspot mechanisms. By engaging an extensive network of residues across the RBD, BD55-1205 minimizes its dependence on individual side-chain conformations, allowing it to maintain robust binding even when key residues are mutated. This adaptability is particularly evident in its tolerance to mutations at positions such as L455 and F456, which severely compromise other antibodies. The ability of BD55-1205 to sustain cumulative interactions underscores the importance of targeting diverse epitopes through multiple interaction mechanisms, a strategy that enhances resistance to immune evasion while maintaining functional integrity. In contrast, BD-604 and OMI-42, with localized binding mechanisms, are more vulnerable to escape mutations at critical positions such as L455, F456, and A475. P5S-1H1 and P5S-2B10 exhibit intermediate behavior, balancing specificity and adaptability but lacking the robustness of BD55-1205. Mutational scanning identified key residues Y421, Y489, and F456 as critical hotspots for RBD stability and antibody binding, highlighting their dual role in viral fitness and immune evasion. The computational predictions generated through mutational scanning and MM-GBSA analysis demonstrate excellent agreement with experimental data on average antibody escape scores. This study underscores the diversity of binding mechanisms employed by different antibodies and molecular basis for high affinity and excellent neutralization activity of the latest generation of antibodies.

biophysics↗

bioRxiv2025

A Conserved Architecture of Allosteric Communications and Regulatory Hotspots in the KRAS Complexes with Diverse Binding Proteins Controls Mechanisms of Effector Mimicry and Allosteric Modulation : Atomistic Revelations from Molecular Simulations and Mutational Scanning of Binding Energetics and Allostery

KRAS is a pivotal oncoprotein that regulates cell proliferation and survival through interactions with downstream effectors such as RAF1. Despite significant advances in understanding KRAS biology, the structural and dynamic mechanisms of KRAS allostery remain unresolved. In this study, we employ microsecond molecular dynamics simulations, mutational scanning and binding free energy calculations together with dynamic network modeling to dissect how engineered DARPin proteins K27, K55, K13, and K1 engage KRAS through diverse molecular mechanisms ranging from effector mimicry to conformational restriction and allosteric modulation. Mutational scanning across all four DARPin systems identifies a core set of evolutionarily constrained residues that function as universal hotspots in KRAS recognition. KRAS residues I36, Y40, M67, and H95 consistently emerge as critical contributors to binding stability. Binding free energy computations show that despite similar binding modes K27 relies heavily on electrostatic contributions from major binding hotspots while K55 exploits a dense hydrophobic cluster enhancing its effector-mimetic signature. Allosteric binders K13 and K19, by contrast, stabilize a KRAS-specific pocket introducing new hinges and bottlenecks that rewire the communication architecture of KRAS without full immobilization. Network-based analysis reveals a strikingly consistent theme: despite their distinct mechanisms of recognition, all systems engage a unifying allosteric architecture that spans multiple functional motifs. This architecture is not only preserved across complexes but also mirrors the intrinsic communication framework of KRAS itself, where specific residues function as central hubs transmitting conformational changes across the protein. By integrating dynamic profiling, energetic mapping, and network-modeling our study provides a multi-scale mechanistic roadmap for targeting KRAS, revealing how engineered proteins can exploit both conserved motifs and isoform-specific features to enable precision modulation of KRAS signaling in oncogenic contexts.

biophysics↗

bioRxiv2025

Exploring Diverse Binding Mechanisms of Broadly Neutralizing Antibodies S309, S304, CYFN-1006 and VIR-7229 Targeting SARS-CoV-2 Spike Omicron Variants: Integrative Computational Modeling Reveals Balance of Evolutionary and Dynamic Adaptability in Shaping Molecular Determinants of Immune Escape

Evolution of SARS-CoV-2 has led to the emergence of variants with increased immune evasion capabilities, posing significant challenges to antibody-based therapeutics and vaccines. The cross-neutralization activity of antibodies against Omicron variants is governed by a complex and delicate interplay of multiple energetic factors and interaction contributions. In this study, we conducted a comprehensive analysis of the interactions between the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein and four neutralizing antibodies S309, S304, CYFN1006, and VIR-7229. Using integrative computational modeling that combined all-atom molecular dynamics (MD) simulations, mutational scanning, and MM-GBSA binding free energy calculations, we elucidated the structural, energetic, and dynamic determinants of antibody binding. Our findings reveal distinct dynamic binding mechanisms and evolutionary adaptation driving broad neutralization effect of these antibodies. We show that S309 targets conserved residues near the ACE2 interface, leveraging synergistic van der Waals and electrostatic interactions, while S304 focuses on fewer but sensitive residues, making it more susceptible to escape mutations. The analysis of CYFN-1006.1 and CYFN-1006.2 antibody binding highlights broad epitope coverage with critical anchors at T345, K440, and T346, enhancing its efficacy against variants carrying the K356T mutation which caused escape from S309 binding. Our analysis of broadly potent VIR-7229 antibody binding to XBB.1.5 and EG.5 Omicron variants emphasized a large and structurally complex epitope, demonstrating certain adaptability and compensatory effects to F456L and L455S mutations. Mutational profiling identified key residues crucial for antibody binding, including T345, P337, and R346 for S309, and T385 and K386 for S304, underscoring their roles as evolutionary "weak spots" that balance viral fitness and immune evasion. The results of this energetic analysis demonstrate a good agreement between the predicted binding hotspots and critical mutations with respect to the latest experiments on average antibody escape scores. The results of this study dissect distinct energetic mechanisms of binding and importance of targeting conserved residues and diverse epitopes to counteract viral resistance. Broad-spectrum antibodies CYFN1006 and VIR-7229 maintain efficacy across multiple variants and achieve neutralization by targeting convergent evolution hotspots while enabling tolerance to mutations in these positions through structural adaptability and compensatory interactions at the binding interface. The results of this study underscore the diversity of binding mechanisms employed by different antibodies and molecular basis for high affinity and excellent neutralization activity of the latest generation of antibodies.

biophysics↗

bioRxiv2025

Atomistic Profiling of KRAS Interactions with Monobodies and Affimer Proteins Through Ensemble-Based Mutational Scanning Unveils Conserved Residue Networks Linking Cryptic Pockets and Regulating Mechanisms of Binding, Specificity and Allostery

KRAS, a historically "undruggable" oncogenic driver, has eluded targeted therapies due to its lack of accessible binding pockets in its active state. This study investigates the conformational dynamics, binding mechanisms, and allosteric communication networks of KRAS in complexes with monobodies (12D1, 12D5) and affimer proteins (K6, K3, K69) to characterize the binding and allosteric mechanisms and hotspots of KRAS binding. Through molecular dynamics simulations, mutational scanning, binding free energy analysis and network-based analyses, we identified conserved allosteric hotspots that serve as critical nodes for long-range communication in KRAS. Key residues in {beta}-strand 4 (F78, L80, F82), -helix 3 (I93, H95, Y96), {beta}-strand 5 (V114, N116), and -helix 5 (Y157, L159, R164) consistently emerged as hotspots across diverse binding partners, forming contiguous networks linking functional regions of KRAS. Notably, {beta}-strand 4 acts as a central hub for propagating conformational changes, while the cryptic allosteric pocket centered around H95/Y96 positions targeted by clinically approved inhibitors was identified as a universal hotspot for both binding and allostery. The study also reveals the interplay between structural rigidity and functional flexibility, where stabilization of one region induces compensatory flexibility in others, reflecting KRASs adaptability to perturbations. We found that monobodies stabilize the switch II region of KRAS, disrupting coupling between switch I and II regions and leading to enhanced mobility in switch I of KRAS. Similarly, affimer K3 leverages the 3-helix as a hinge point to amplify its effects on KRAS dynamics. Mutational scanning and binding free energy analysis highlighted the energetic drivers of KRAS interactions. revealing key hotspot residues, including H95 and Y96 in the 3 helix, as major contributors to binding affinity and selectivity. Network analysis identified {beta}-strand 4 as a central hub for propagating conformational changes, linking distant functional sites. The predicted allosteric hotspots strongly aligned with experimental data, validating the robustness of the computational approach. Despite distinct binding interfaces, shared hotspots highlight a conserved allosteric infrastructure, reinforcing their universal importance in KRAS signaling. The results of this study can inform rational design of small-molecule inhibitors that mimic the effects of monobodies and affimer proteins, challenging the "undruggable" reputation of KRAS.

biophysics↗

bioRxiv2024

Quantitative Characterization and Prediction of the Binding Determinants and Immune Escape Hotspots for Groups of Broadly Neutralizing Antibodies Against Omicron Variants: Atomistic Modeling of the SARS-CoV-2 Spike Complexes with Antibodies

The growing body of experimental and computational studies suggested that the cross-neutralization antibody activity against Omicron variants may be driven by balance and tradeoff of multiple energetic factors and interaction contributions of the evolving escape hotspots involved in antigenic drift and convergent evolution. However, the dynamic and energetic details quantifying the balance and contribution of these factors, particularly the balancing nature of specific interactions formed by antibodies with the epitope residues remain scarcely characterized. In this study, we performed molecular dynamics simulations, ensemble-based deep mutational scanning of SARS-CoV-2 spike residues and binding free energy computations for two distinct groups of broadly neutralizing antibodies : E1 group (BD55-3152, BD55-3546 and BD5-5840) and F3 group (BD55-3372, BD55-4637 and BD55-5514). Using these approaches, we examine the energetic determinants by which broadly potent antibodies can largely evade immune resistance. Our analysis revealed the emergence of a small number of immune escape positions for E1 group antibodies that correspond to R346 and K444 positions in which the strong van der Waals and interactions act synchronously leading to the large binding contribution. According to our results, E1 and F3 groups of Abs effectively exploit binding hotspot clusters of hydrophobic sites critical for spike functions along with selective complementary targeting of positively charged sites that are important for ACE2 binding. Together with targeting conserved epitopes, these groups of antibodies can lead to the expanded neutralization breadth and resilience to antigenic shift associated with viral evolution. The results of this study and the energetic analysis demonstrate excellent qualitative agreement between the predicted binding hotspots and critical mutations with respect to the latest experiments on average antibody escape scores. We argue that E1 and F3 groups of antibodies targeting binding epitopes may leverage strong hydrophobic interactions with the binding epitope hotspots critical for the spike stability and ACE2 binding, while escape mutations tend to emerge in sites associated with synergistically strong hydrophobic and electrostatic interactions.

biophysics↗

bioRxiv2024

Assessing Structures and Conformational Ensembles of Apo and Holo Protein States Using Randomized Alanine Sequence Scanning Combined with Shallow Subsampling in AlphaFold2 : Insights and Lessons from Predictions of Functional Allosteric Conformations

Proteins often exist in multiple conformational states, influenced by the binding of ligands or substrates. The study of these states, particularly the apo (unbound) and holo (ligand-bound) forms, is crucial for understanding protein function, dynamics, and interactions. In the current study, we use AlphaFold2 that combines randomized alanine sequence masking with shallow multiple sequence alignment subsampling to expand the conformational diversity of the predicted structural ensembles and capture conformational changes between apo and holo protein forms. Using several well-established datasets of structurally diverse apo-holo protein pairs, the proposed approach enables robust predictions of apo and holo structures and conformational ensembles, while also displaying notably similar dynamics distributions. These observations are consistent with the view that the intrinsic dynamics of allosteric proteins is defined by the structural topology of the fold and favors conserved conformational motions driven by soft modes. Our findings support the notion that AlphaFold2 approaches can yield reasonable accuracy in predicting minor conformational adjustments between apo and holo states, especially for proteins with moderate localized changes upon ligand binding. However, for large, hinge-like domain movements, AlphaFold2 tends to predict the most stable domain orientation which is typically the apo form rather than the full range of functional conformations characteristic of the holo ensemble. These results indicate that robust modeling of functional protein states may require more accurate characterization of flexible regions in functional conformations and detection of high energy conformations. By incorporating a wider variety of protein structures in training datasets including both apo and holo forms, the model can learn to recognize and predict the structural changes that occur upon ligand binding.

biophysics↗

bioRxiv2023

Sugar-feeding by invasive mosquito species on ornamental and wild plants

Feeding on plant-derived sugars is an essential component of mosquito biology that affects key aspects of their lives such as survival, metabolism, and reproduction. Mosquitoes locate plants to feed on using olfactory and visual cues. Aedes aegypti and Aedes albopictus are two invasive mosquito species to the US, and are vectors of diseases such as dengue fever, chikungunya, and Zika. These species live in heavily populated, urban areas, where they have a high accessibility to human hosts as well as to plants in backyards and town landscapes. Therefore, it is important to understand what plants may attract or repel mosquitoes to inform citizens and municipal authorities accordingly. Here, we analyzed Ae. aegypti and Ae. albopictus sugar-feeding behavior with eleven different commonly planted ornamental plant species. We then assessed feeding activity using the anthrone method and identified volatile composition of plant headspace using gas-chromatography mass-spectroscopy. Finally, we determined the sugar-feeding activity of field caught mosquitoes using the plant DNA barcoding technique and compared these results with the eleven ornamental species tested in the laboratory. The potential for application to disease vector control is also discussed.

ecology↗

bioRxiv2022

MODELING POLARITY SITE MOVEMENT IN YEAST VIA CALCIUM-MEDIATED REGULATION OF GAP ACTIVITY

Symmetry-breaking polarization is a process that occurs in many cells. In budding yeast cells specifically, this polarization is possible due to the presence of a positive feedback loop, which allows for one and only one front to develop. Furthermore, in mating yeast cells the detection of pheromones plays a crucial role in the cells development of a polarity site; however, due to the microscopic size of yeast cells, there is often error in the initial polarity sites accuracy. As a result, yeast cells can correct their polarity site by allowing the GTP-Cdc42 cluster to "wander". This is typically considered as the indecisive phase of a cell. Recent studies have identified that cytosolic Ca2+ bursts, caused by sexual pheromones, increase the activity of GTPase-activating protein (GAP). This project tests this from a modeling perspective by adding a module of code to an already established positive feedback model that allows periodic spikes of GAP activity to occur. The model was successfully able to simulate the effects of calcium-mediated GAP spikes by portraying oscillating polarity sites and "wandering" polarity sites, using specific parameters based on biochemical data.

systems biology↗

bioRxiv2022

Neuro-immune modulation of cholinergic signaling in an addiction vulnerability trait

Sign-tracking (ST) describes the propensity to approach and contact a Pavlovian reward cue. By contrast, goal-trackers (GTs) respond to such a cue by retrieving the reward. These behaviors index the presence of opponent cognitive-motivational traits, with STs exhibiting attentional control deficits, behavior dominated by incentive motivational processes, and vulnerability for addictive drug taking. Attentional control deficits in STs were previously attributed to attenuated cholinergic signaling, resulting from deficient translocation of intracellular choline transporters (CHTs) into synaptosomal plasma membrane. Here we investigated a post-translational modification of CHTs - poly-ubiquitination - and tested the hypothesis that elevated cytokine signaling in STs contributes to CHT modification. We demonstrated that intracellular CHTs, but not plasma membrane CHTs, are highly ubiquitinated in male and female sign-tracking rats when compared with GTs. Moreover, levels of cytokines measured in cortex and striatum, but not spleen, were higher in STs than in GTs. Activation of the innate immune system by systemic administration of the bacterial endotoxin lipopolysaccharide (LPS) elevated ubiquitinated CHT levels in cortex and striatum of GTs only, suggesting ceiling effects in STs. In spleen, LPS increased levels of most cytokines in both phenotypes. In cortex, LPS particularly robustly increased levels of the chemokines CCL2 and CXCL10. Phenotype-specific increases were restricted to GTs, again suggesting ceiling effects in STs. These results indicate that interactions between elevated brain immune modulator signaling and CHT regulation are essential components of the neuronal underpinnings of the addiction vulnerability trait indexed by sign-tracking. Significance StatementSign-tracking rats (STs) have emerged as a fruitful animal model for determining the neuro-behavioral foundations, including the cholinergic-attentional control deficits, which heighten the risk for the manifestation of addictive behaviors. The results from the present experiments suggest that, in STs, elevated levels of neuro-immune signaling interact with a post-translational modification of choline transporters that yields a relatively low transporter capacity. Furthermore, the effects of activation of the innate immune system, by administrating the endotoxin lipopolysaccharide, mechanistically supported such an interaction. The results extend research on the neuronal vulnerabilities for addictive behaviors to the role of neuro-immune signaling and suggests a new role of neuro-immune modulators in influencing complex cognitive-motivational traits.

neuroscience↗

bioRxiv2022

Computational Pipeline to Identify Gene signatures that Define Cancer Subtypes

MotivationThe heterogeneous nature of cancers with multiple subtypes makes them challenging to treat. However, multi-omics data can be used to identify new therapeutic targets and we established a computational strategy to improve data mining. ResultsUsing our approach we identified genes and pathways specific to cancer subtypes that can serve as biomarkers and therapeutic targets. Using a TCGA breast cancer dataset we applied the ExtraTreesClassifier dimensionality reduction along with logistic regression to select a subset of genes for model training. Applying hyperparameter tuning, increased the model accuracy up to 92%. Finally, we identified 20 significant genes using differential expression. These targetable genes are associated with various cellular processes that impact cancer progression. We then applied our approach to a glioma dataset and again identified subtype specific targetable genes. ConclusionOur research indicates a broader applicability of our strategy to identify specific cancer subtypes and targetable pathways for various cancers.

bioinformatics↗

bioRxiv2021

Disrupted choline clearance and sustained acetylcholine release in vivo by a common choline transporter coding variant associated with poor attentional control in humans

Transport of choline via the neuronal high-affinity choline transporter (CHT; SLC5A7) is essential for cholinergic terminals to synthesize and release acetylcholine (ACh). In humans, we previously demonstrated an association between a common CHT coding substitution (rs1013940; Ile89Val) and reduced attentional control as well as attenuated frontal cortex activation. Here, we used a CRISPR/Cas9 approach to generate mice expressing the I89V substitution and assessed, in vivo, CHT-mediated choline transport, and ACh release. Relative to wild type (WT) mice, CHT-mediated clearance of choline in mice expressing one or two Val89 alleles was reduced by over 80% cortex and by over 50% in striatum. Choline clearance in CHT Val89 mice was further reduced by neuronal inactivation. Deficits in ACh release, 5 and 10 min after repeated depolarization at a low, behaviorally relevant frequency, support an attenuated reloading capacity of cholinergic neurons in mutant mice. The density of CHTs in total synaptosomal lysates and neuronal plasma-membrane-enriched fractions was not impacted by the Val89 variant, indicating a selective impact on CHT function. When challenged with a visual disruptor to reveal attentional control mechanisms, Val89 mice failed to adopt a more conservative response bias. Structural modeling revealed that Val89 may attenuate choline transport by altering conformational changes of CHT that support normal transport rates. Our findings support the view that diminished, sustained cholinergic signaling capacity underlies perturbed attentional performance in individuals expressing CHT Val89. The CHT Val89 mouse serves as a valuable model to study heritable risk for cognitive disorders arising from cholinergic dysfunction. Significance StatementAcetylcholine (ACh) signaling depends on the functional capacity of the neuronal choline transporter (CHT). Previous research demonstrated that humans expressing the common CHT coding variant Val89 exhibit attentional vulnerabilities and attenuated fronto-cortical activation during attention. Here, we find that mice engineered to express the Val89 variant exhibit reduced CHT-mediated choline clearance and a diminished capacity to sustain ACh release. Additionally, Val89 mice lack cognitive flexibility in response to an attentional challenge. These findings provide a mechanistic and cognitive framework for interpreting the attentional phenotype associated with the human Val89 variant and establish a model that permits a more invasive interrogation of CNS effects as well as the development of therapeutic strategies for those, including Val89 carriers, with presynaptic cholinergic perturbations.

neuroscience↗

bioRxiv2019

Repetitive mild concussion in subjects with a vulnerable cholinergic system: lasting cholinergic-attentional impairments in CHT+/- mice.

Previous research emphasized the impact of traumatic brain injury on cholinergic systems and associated cognitive functions. Here we addressed the converse question: Because of the available evidence indicating cognitive and neuronal vulnerabilities in humans expressing low-capacity cholinergic systems or with declining cholinergic systems, do injuries cause more severe cognitive decline in such subjects, and what cholinergic mechanisms contribute to such a vulnerability? Using mice heterozygous for the choline transporter (CHT+/- mice) as a model for a limited cholinergic capacity, we investigated the cognitive and neuronal consequences of repeated, mild concussion injuries (rmCc). Following five rmCc, and compared with WT mice, CHT+/- mice exhibited severe and lasting impairments in sustained attention performance, consistent with effects of cholinergic losses on attention. However, rmCc did not affect the integrity of neuronal cell bodies and did not alter the density of cortical synapses. As a cellular mechanism potentially responsible for the attentional impairment in CHT+/- mice, we found that rmCc nearly completely attenuated performance-associated, CHT-mediated choline transport. These results predict that subjects with an already vulnerable cholinergic system will experience severe and lasting cognitive-cholinergic effects following even relatively mild injuries. If confirmed in humans, such subjects may be excluded from, or receive special protection against, activities involving injury risk. Moreover, the treatment and long-term outcome of traumatic brain injuries may benefit from determining the status of cholinergic systems and associated cognitive functions.

neuroscience↗