Biology subjects

Pak, V.

Publications and source records attributed to Pak, V..

6 recordsLinked to original sources

bioRxiv2026

Universal oligo adapters for high-efficiency DNA-barcoded antibody panel generation

DNA-barcoded antibodies are central to a broad range of spatial and dissociative assays and applications, including multiplexed imaging, single-cell profiling, and proximity detection. Direct modification of primary antibodies with defined DNA barcodes enables flexible panel design for multiplexed labeling of proteins. However, conventional antibody-oligonucleotide conjugation methods are inefficient, low-throughput, and prone to batch variability, limiting the reliable generation of orthogonally barcoded antibody panels. These challenges are particularly acute during initial panel development, where lengthy protocols, conjugation failures, large antibody input requirements, and the need for custom antibody formulations increase experimental cost and effort. Site-specific conjugation strategies based on antibody Fc-domain binders offer a promising alternative. We streamlined this foundational approach to establish its compatibility with multiplexed imaging in cells and tissues; however, generating a full barcoded library is still resource-intensive. This is because every unique DNA barcode must first be chemically linked to a separate binder before it can be attached to an antibody. To overcome the prominent bottleneck of rapidly and reliably generating DNA-barcoded antibody panels, we introduce the universal oligo adapter (UnO) strategy. UnO fundamentally changes the workflow from barcode-specific conjugation to universal barcode conversion. We build on the established photoreactive protein G binder and combine it with a universal oligonucleotide carrying a second ultrafast photocrosslinking group, 3-cyanovinylcarbazole (cnvK). This creates a dual-functional adapter: one photoreactive group enables covalent attachment to the antibody, while the cnvK-containing universal oligo simultaneously captures a user-defined DNA barcode through hybridization and UV crosslinking in a single step. Rather than preparing separate conjugation reactions for dozens of barcodes, UnO acts as a single reagent that covalently couples any desired barcode onto small quantities of off-the-shelf primary antibodies in minutes. We validate the generalizability and modularity of this approach across subcellular Immuno-SABER and tissue-based CODEX workflows for multiplex immunostaining. By converting antibody barcoding into a modular, one-step nucleic-acid adapter workflow, UnO reduces the cost, time, and complexity of generating DNA-barcoded antibody panels and provides an efficient, accessible solution to a central bottleneck in DNA-enabled multiplexed protein detection.

bioengineering↗

bioRxiv2026

ProPER: Programmable, multiplexed detection of molecular proximities and RNA life-cycle stages in situ

Proximity detection methods facilitate contextual analysis of biomolecules by reporting their microenvironment, spatial organization and molecular interactions. To expand these detection capabilities towards highly sensitive, quantitative, and multiplexed interaction mapping, we introduce Proximity Primer Exchange Reaction (ProPER). ProPER is a novel ligation-free proximity extension and controlled DNA amplification strategy that enables spatially resolved in situ detection of molecular proximities. In ProPER, extension of DNA barcodes into linear concatemers is made conditional on the spatial proximity of primer and hairpin pairs split across two targets, thereby enabling efficient coincidence or proximity detection with isothermal signal amplification for high-sensitivity imaging. The method is readily integrated with mainstream in situ assays such as immunofluorescence, FISH and metabolic labeling, and supports high-resolution visualization of diverse molecular modalities, including RNA-RNA or protein-protein interactions, even at dense labeling sites. Leveraging pre-validated orthogonal sequence pairs, ProPER allows simultaneous multiplexed detection of molecular proximities within the same cell and on the same target molecule. We applied multiplexed ProPER to resolve the life-cycle stages of individual RNA molecules. Quantitative tracking of transcriptional, splicing and translational states enabled construction of a kinetic model that identifies key regulatory steps driving differential expression kinetics of two inflammatory response genes. Overall, we establish ProPER as a versatile and efficient framework for in situ multiplexed proximity detection, overcoming key limitations of existing approaches. The programmable DNA encoding scheme enables advanced capabilities, such as proximity cascades and multivalent interaction detection, and provides a basis for integrating molecular proximity measurements into future combinatorial barcoding or spatial omics workflows.

molecular biology↗

bioRxiv2025

Molecular and neuropathological determinants of neuronal dysfunction in Alzheimer's disease

The biological basis of neuronal excitatory/inhibitory (E/I) imbalance in Alzheimers disease (AD) remains unclear. Using a comprehensive cohort with ante-mortem functional neuroimaging and post-mortem molecular data from the same participants, we mapped individual, whole-brain E/I imbalances through biophysical modeling. E/I ratios in regions supporting higher-order cognitive functions were significantly associated with cognitive performance and decline, with mediation by global neuropathological burden. We also observed a significant inverted U-shaped relationship between E/I ratios and neurofibrillary tangle severity, peaking at the limbic stage (Braak III-IV) in 14 brain areas, including the bilateral hippocampus and superior frontal gyrus. In addition, we identified 89 genes and 101 proteins that predict regional E/I ratios, with pathways related to synaptic signaling and immune response overrepresented. The generalizability of these molecular predictors was confirmed in two independent cohorts, achieving good classification performance for neuropathology severity and AD dementia. Lastly, the estimated E/I imbalances in AD aligned with whole-brain distributions of microglia and oligodendrocyte precursor cells, suggesting that spatial cellular organization contributes to vulnerability to neuronal dysfunction. Overall, this study provides critical insights into the cellular, molecular, and neuropathological signatures of circuit-level dysfunction in AD.

neuroscience↗

bioRxiv2025

Whole-Brain Cell-Cell Interaction Axes Explaining Tissue Vulnerability Across the Neurodegenerative Spectrum

Disrupted cell-cell communication represents a fundamental mechanism underlying neurodegeneration, yet how specific intercellular signaling patterns relate to regional brain vulnerability remains poorly understood. Here, we map whole-brain intercellular interaction networks and their spatial correspondence with tissue damage across 13 neurodegenerative conditions, including early- and late-onset Alzheimers disease, presenilin-1 mutations, clinical and pathological subtypes of frontotemporal lobar degeneration, Parkinsons disease, dementia with Lewy bodies, and amyotrophic lateral sclerosis. By integrating multiregional single-nucleus and bulk RNA-seq data with curated cell-cell interaction databases and structural MRI, we reconstruct over 1,000 whole-brain maps of ligand-receptor interactions and quantify their associations with regional atrophy patterns. Multivariate analysis identifies three dominant axes of intercellular communication that explain regional vulnerability to neurodegeneration. Notably, the first axis involves neuron-astrocyte-microglia interactions, explaining atrophy patterns shared by frontotemporal lobar degeneration and Alzheimers disease subtypes. Two complementary axes involving neurons, endothelial cells, and astrocytes explain patterns specific to mutations in PS1 and Parkinsons disease. Importantly, validation in an independent post-mortem cohort (N = 375) confirms that late-onset Alzheimers disease-associated cell-cell interactions predict observed frontal cortex atrophy. These results establish a systematic framework linking local intercellular communication networks to spatial patterns of neurodegeneration, revealing both shared and disease-specific molecular pathways that drive regional brain vulnerability and identifying cellular interaction targets for precision therapeutic interventions.

neuroscience↗

bioRxiv2023

Distinctive Whole-brain Cell-Types Strongly Predict Tissue Damage Patterns in Eleven Neurodegenerative Disorders

For over a century, brain research narrative has mainly centered on neuron cells. Accordingly, most neurodegenerative studies focus on neuronal dysfunction and their selective vulnerability, while we lack comprehensive analyses of other major cell types contribution. By unifying spatial gene expression, structural MRI, and cell deconvolution, here we describe how the human brain distribution of canonical cell types extensively predicts tissue damage in thirteen neurodegenerative conditions, including early-and late-onset Alzheimers disease, Parkinsons disease, dementia with Lewy bodies, amyotrophic lateral sclerosis, mutations in presenilin-1, and three clinical variants of frontotemporal lobar degeneration (behavioural variant, semantic and non-fluent primary progressive aphasia) along with associated 3-repeat and 4-repeat tauopathies and TDP43 proteinopathies types A and C. We reconstructed comprehensive whole-brain reference maps of cellular abundance for six major cell types and identified characteristic axes of spatial overlapping with atrophy. Our results support the strong mediating role of non-neuronal cells, primarily microglia and astrocytes, in spatial vulnerability to tissue loss in neurodegeneration, with distinct and shared across-disorders pathomechanisms. These observations provide critical insights into the multicellular pathophysiology underlying spatiotemporal advance in neurodegeneration. Notably, they also emphasize the need to exceed the current neuro-centric view of brain diseases, supporting the imperative for cell-specific therapeutic targets in neurodegeneration.

neuroscience↗

bioRxiv2023

Thermogenetic control of Ca2+ levels in cells and tissues

Virtually all major processes in cells and tissues are regulated by calcium ions (Ca2+). Understanding the influence of Ca2+ on cell function requires technologies that allow for non-invasive manipulation of intracellular calcium levels including the formation of calcium patterns, ideally in a way that is expandable to intact organisms. The currently existing tools for optical and optogenetic Ca2+ manipulation are limited with respect to response time, and tissue penetration depth. Here we present Genetically Encoded Calcium Controller (GECCO), a system for thermogenetic Ca2+ manipulation based on snake TRP channels optically controlled by infrared illumination. GECCO is functional in animal and plant cells and allows studying how cells decode different profiles of Ca2+ signals. GECCO enabled the shaping of insulin release from {beta}-cells, the identification of drugs that potentiate Ca2+-induced insulin release, and the generation of synthetic Ca2+ signatures in plants.

synthetic biology↗