Biology subjects

Pak, A.

Publications and source records attributed to Pak, A..

9 recordsLinked to original sources

bioRxiv2026

C19 trityl and silyl andrographolide analogs exhibit in vitro anticancer activity in murine cancer models

Andrographolide, a labdane diterpenoid isolated from A. paniculata has been investigated for activity in a broad panel of biological contexts, including its anti-inflammatory, antiviral, and anticancer properties. Previously, A-ring analogs of andrographolide bearing silyl and trityl ethers at C19 have demonstrated improved in vitro anticancer activity in human cancer cell lines, with putative activity through the Wnt1/{beta}-catenin signaling axis. In order to investigate the activity of this series of C19 andrographolide analogs, alongside a cyclic A-ring acetonide analog, as anticancer agents in murine cancer cell models, we evaluated antiproliferative activity of these compounds in B16F10 melanoma, CT26 colorectal adenocarcinoma, Neuro2a neuroblastoma, and 4T1 mammary ductal carcinoma cell lines, and found that most compounds exhibited potent micromolar level anticancer activity. Among these, all trityl ethers exhibited nearly indistinguishable IC50 values of 4-20 M, and generally exhibited greater potency compared to C19 silyl ether analogs. This anticancer activity was further validated through apoptosis flow cytometry, wherein we concluded that our compounds induced apoptosis to a greater extent than the parent compound, andrographolide. Further, we examine the role of modulation of canonical Wnt1/{beta}-catenin signaling in a series of co-delivery assays with CHIR-99021, a selective GSK-3{beta} kinase inhibitor, and MSAB, a selective {beta}-catenin degrader. Finally, we demonstrate that a selection of silyl ether analogs of andrographolide elicit cell cycle arrest by flow cytometry in a manner that is decoupled to in vitro potency trends. Altogether, we report the first in vitro evaluation of trityl and silyl ether analogs of andrographolide within murine adherent cancer cells.

cancer biology↗

bioRxiv2026

Thermoplastic polyurethane as a base material for soft, high-density neural interfaces

Polymers used as substrate and encapsulation for implantable devices are typically deposited layer-by-layer, leaving polymer-polymer interfaces that adhere poorly and become a critical failure point. Thermoplastic polyurethane (TPU) elastomers combine four properties not found together in any other candidate: decades of clinical use in long-term implants and availability in medical grade, a stiffness well below that of polyimide and parylene C, compatibility with microfabrication, and thermoplastic self-fusion, which removes the polymer-polymer interface entirely. This work introduces TPU as a base material for soft, high-density neural interfaces and demonstrates one route to realizing them. Gold is patterned on a sacrificial copper sheet by maskless lithography and transferred into TPU by vacuum lamination, giving conformally coated tracks down to 15 um at 30 um pitch within a single, merged polymer body. An additional electroplating step produces a nanostructured pinecone gold surface that increases the electrochemically active area of the electrodes, approximately doubling the charge injection capacity. Daisy-chain silicon chips were also embedded within the soft polymer body. The films transmit up to 88% at 470 nm, a wavelength commonly used for optogenetics. TPU showed no cytotoxicity towards differentiated SH-SY5Y cells and only a mild tissue response after 9.5 months of subcutaneous implantation in rats.

bioengineering↗

bioRxiv2026

Active locomotion predictively rescues head direction attractor dynamics in head-fixed mice

Head direction (HD) cells in the anterodorsal thalamic nuclei form the brains internal compass, and are often modeled as a ring attractor maintaining azimuth coding by leveraging continuous visual and inertial sensory input. Here, we test how the common experimental preparation of head-fixed animals alters this code. Complete head-fixation that creates vestibular conflict disrupts both unit and population encoding of head direction, while selectively constraining head-on-body movements either in real or virtual reality uniquely impairs HD population activity. More specifically, attractor dynamics is altered in head-restrained mice during periods of immobility, but remarkably recover several hundred milliseconds prior to locomotion onset. The rescue preceding movement onset suggests that an efference copy or prediction of a re-afferent signal is necessary to maintain HD network activity during head restraint. A computational model recapitulates these effects by perturbing lateral connectivity among HD neurons. More generally, the results indicate that the HD network is a context- and state-dependent predictive estimator, stabilized by forthcoming self-motion signals. The classic ring-attractor models should be revised to integrate context-dependent dynamics with prospective motor signals, offering a more complete account of how the brains compass remains stable across both naturalistic and constrained conditions.

neuroscience↗

bioRxiv2025

Transcriptional responses to proteotoxic stressors are profoundly diverse and tissue-specific

Cells counteract proteotoxic conditions by launching transcriptional stress responses. While synthesis of Heat shock proteins (HSPs) upon acute stress is well-characterized, how distinct proteotoxic conditions reshape the transcriptome remains poorly understood. Here, we analyse polyA+ RNA expression under heat shock, HSP90 inhibition, and polyglutamine (polyQ) aggregation. We find fundamentally distinct transcriptional responses to proteotoxic stressors, and a systemic deficiency of mice under chronic stress to launch acute responses. While heat shock and HSP90 inhibition induce chaperones, polyQ aggregation increases RNAs linked to transcription repression, chromatin remodeling, and autophagy. Analysing wildtype and Huntingtons Disease (HD) mice reveals tissue-specific transcriptional adaptations to polyQ, including repressed cell-type specific functions and altered energy metabolism. Despite profound reprogramming, remarkably few RNAs are consistently induced (Acy3, Abdh1, Tmc3) or reduced (Fos) across HD brain regions. These results emphasize cellular background in disease manifestation, and support energy metabolism and detoxifying enzymes as therapeutic targets in late-stage HD. Moreover, the systemic deficiency of chronically stressed mice to launch responses challenges strategies that rely on induced transcription. Altogether, we characterize transcription signatures to proteotoxic stresses, identify key trans-activators driving proteotoxic stress responses, provide an interactive gene-by-gene viewer of global changes, and delineate tissue-specific transcription programs in HD mice.

cell biology↗

bioRxiv2025

Deep cerebellar tFUS engages cortical circuits via convergent local and sensory-driven mechanisms

Transcranial focused ultrasound (tFUS) enables non-invasive access to deep brain structures with high spatial precision, but its mechanisms of action remain elusive and under active investigation. The leading hypothesis posits that tFUS exerts its neuromodulatory effects through direct activation of neurons, while others attribute effects to indirect activation of auditory pathways. Here, we investigated the neuromodulatory effects of tFUS on the dentato- thalamo-cortical (DTC) pathway by targeting either the lateral cerebellar nucleus (LCN) or auditory cortex (AUD) in anesthetized rats. Electroencephalography (EEG) recordings from bilateral motor cortices and the contralateral auditory cortex revealed that tFUS reliably evoked cortical potentials, whereas sham stimulation produced no responses. Both LCN and AUD stimulation activated the auditory cortex, suggesting auditory pathway involvements. However, only LCN stimulation elicited early and significant event-related potentials and gamma-band activity in the contralateral motor cortex, consistent with DTC pathway engagement. We propose that LCN-targeted stimulation engages the DTC pathway through modulation of neuronal excitability, while concurrent auditory inputs account for global cortical activation. These results are consistent with a hybrid mechanism in which tFUS modulates neuronal dynamics, converging both direct and indirect components depending on the stimulation site. Proposed framework reconciles competing views of tFUS as either a direct or indirect modulator and clarifies how ultrasound can differentially influence cortical circuits depending on the stimulation target.

neuroscience↗

bioRxiv2025

Improved T cell surfaceomics by depleting intracellularly labelled dead cells

Although the plasma membrane (PM) is among the most biologically important and therapeutically targeted cellular compartments, it is among the most challenging to faithfully capture using proteomic approaches. The quality of quantitative surfaceomics data depends heavily on the effectiveness of the cell surface enrichment used during sample preparation. Enrichment improves sensitivity for low abundance PM proteins and ensures that the changes detected reflect PM expression changes rather than whole cell changes. Cell surface biotinylation with PM-impermeable, amine-reactive reagents is a facile, accessible, and unbiased approach to enrich PM proteins. For unclear reasons however, it results in unexpectedly high contamination with intracellular proteins, reducing its utility. We report that biotinylating human cells with amine-reactive reagents intracellularly labels a small but reproducible population of non-viable cells. Although these dead cells represent only 5{+/-}2% of the total, we find that in T cell preparations the dead cells account for 90% of labelled proteins. Depleting Annexin V positive dead T cells post-labelling removes [~]99% of the intracellularly labelled cells, resulting in markedly improved PM identifications, peptide counts, and iBAQ intensities. Correspondingly, we found substantial depletion of intracellular proteins, particular of nuclear origin. Overall, the cumulative intensity of PM proteins increased from 4% to 55.8% with dead cell depletion. Finally, we demonstrate that immature ER/Golgi glycoforms of CD11a and CD18 are selectively removed by dead-cell depletion. We conclude that high intracellular labelling of non-viable cells is the major source of intracellular protein contaminants in amine-reactive surface enrichment methods and can be reduced by dead-cell depletion post-labelling, improving both sensitivity and accuracy of plasma membrane proteomics.

biochemistry↗

bioRxiv2024

Glycolytic flux controls retinal progenitor cell differentiation via regulating Wnt signaling

Metabolic pathways are remodeled in response to energy and other homeostatic demands and are dynamically regulated during embryonic development, suggesting a role in guiding cellular differentiation. Here, we show that glycolytic flux is required and sufficient to bias multipotent retinal progenitor cells (RPCs) to acquire a rod photoreceptor fate in the murine retina. In RPC-specific Phosphatase and tensin homolog conditional knock-out (Pten-cKO) and RPC-specific conditional gain-of-function of dominant active PFKB3 (cytoPFKB3) mice, glycolytic gene expression and activity are elevated, correlating with precocious rod photoreceptor differentiation and outer segment maturation. Conversely, glycolytic inhibition in retinal explants suppresses RPC proliferation and photoreceptor differentiation, achieved either with 2-deoxy-d-glucose, a competitive inhibitor of glucose metabolism, by lowering media pH, which disables PKM2, a rate-limiting enzyme, or by inhibiting lactate/H+ symporters, which lowers intracellular pH. Mechanistically, we show that Wnt signaling, the top-upregulated pathway in Pten-cKO retinas, is a glycolysis-dependent pathway. Pharmacological and genetic perturbation of Wnt signaling by knocking-out Ctnnb1, encoding {beta}-catenin, phenocopies glycolytic inhibition, suppressing RPC proliferation, photoreceptor differentiation and outer segment maturation. Thus, developmental rewiring of glycolytic flux modulates Wnt signaling to drive rod photoreceptor differentiation and maturation, an instructive role that may be exploited therapeutically for cell replacement strategies. IMPACT STATEMENTTransgenic and pharmacological approaches reveal developmental elevations in glycolytic flux have an instructive role in promoting rod photoreceptor differentiation and maturation via activation of Wnt signaling.

developmental biology↗

bioRxiv2020

A helical assembly of human ESCRT-I scaffolds reverse-topology membrane scission

The ESCRT complexes drive membrane scission in HIV-1 release, autophagosome closure, MVB biogenesis, cytokinesis, and other cell processes. ESCRT-I is the most upstream complex and bridges the system to HIV-1 Gag in virus release. The crystal structure of the headpiece of human ESCRT-I comprising TSG101:VPS28:VPS37B:MVB12A was determined, revealing an ESCRT-I helical assembly with a 12 molecule repeat. Electron microscopy confirmed that ESCRT-I subcomplexes form helical filaments in solution. Mutation of VPS28 helical interface residues blocks filament formation in vitro and autophagosome closure and HIV-1 release in human cells. Coarse grained simulations of ESCRT assembly at HIV-1 budding sites suggest that formation of a 12-membered ring of ESCRT-I molecules is a geometry-dependent checkpoint during late stages of Gag assembly and HIV-1 budding, and templates ESCRT-III assembly for membrane scission. These data show that ESCRT-I is not merely a bridging adaptor, but has an essential scaffolding and mechanical role in its own right.

biophysics↗

bioRxiv2020

Differential processing and habituation in distinct spatial frequency channels in V1 of a mouse model of fragile X syndrome

Extraction of both common and unique features across different visual inputs is crucial for animal survival. Regularities in the visual input lead to learning of the general principles governing an environment, whereas unique features are important for novelty detection. Low and high spatial frequencies (SF) represent two different channels of visual perception, which may be playing different roles in the processing of global pattern and local details. Alterations in the processing of these different SF channels may lead to impaired visual perception. Excessive detail-oriented processing and reduced habituation to sensory stimuli are some of the hallmarks of altered sensory perception in autism. However, the underlying neural mechanisms of these impairments are not understood. To gain insight into the pathophysiology of these impairments, we investigated the low and high SF channels in V1 of Fmr1 KO mice, the mouse model of Fragile X syndrome (FX). We first provide behavioral evidence for reduced habituation of both pupillary baseline and surprise responses in FX mice. Using silicon probe recordings, we demonstrate excessive processing of high SF stimuli in the late stages of visual responses in V1 of FX mice. We then show a reduced adaptation during a visual oddball paradigm in neurons preferring low but not high SF. Overall, our findings suggest that altered processing in distinct SF channels might contribute to altered visual perception and learning in FX and autism.

neuroscience↗