Biology subjects

Lewis, B.

Publications and source records attributed to Lewis, B..

10 recordsLinked to original sources

bioRxiv2026

Antibody treatment targeting nitrated alpha-synuclein counteracts protein spreading pathology

-Synuclein nitration is a prominent post-translational modification in Parkinsons disease, but whether nitrated -synuclein merely reflects oxidative stress or actively contributes to pathology remains unclear. Here, we generated and characterized 6G6, an antibody selective for Tyr39-nitrated -synuclein, and tested whether targeting this modified -synuclein species affected pathology in different mouse models of -synuclein aggregation and spread. In two models of -synuclein overexpression targeting medullary vagal neurons, oxidative stress was induced by either exposure to the herbicide paraquat or transgenic heterozygous expression of the Gba1-L444P mutation. Both conditions were characterized by robust -synuclein spreading that was markedly counteracted by 6G6 administration. A third model consisted of an injection of -synuclein fibrils into the striatum of -synuclein-overexpressing mice. In this model, treatment with 6G6 protected against fibril-induced aggregate pathology and ensuing degeneration of nigral dopaminergic neurons. In a pilot human study, CSF levels of Tyr39-nitrated -synuclein were measured and found increased in Parkinson patients as compared to controls. These findings identify Tyr39-nitrated -synuclein as a pathogenic, therapeutically targetable -synuclein species linking oxidative/nitrative stress to PD pathological processes.

neuroscience↗

bioRxiv2026

Sub-second Extracellular Impedance Measurement of Epithelial Cell Monolayers using Step Excitations and Time-domain Analysis

Extracellular electrochemical impedance spectroscopy (EIS) is emerging as a powerful technique in in vitro epithelial research, offering quantitative insights into barrier integrity, morphology, and apical-basolateral polarity noninvasively through metrics such as transepithelial electrical resistance (TER/TEER), transepithelial capacitance (TEC), and membrane ratio (), respectively. However, due to the broad range of frequencies probed, EIS typically requires tens of seconds per measurement, limiting its ability to capture more rapid biological phenomena. We present Time-domain Epithelial Impedance Measurement (TEIM), a method for sub-second extracellular impedance measurements of epithelial cell monolayers based on step (Heaviside function) current excitation and time-domain analysis of the voltage transients, without the need for Fourier transforms. We experimentally demonstrate TEIM measuring TER, TEC, , and model-derived impedance spectrum at [~]0.3 s sampling rate, which represents a 100- fold improvement in time resolution compared with traditional EIS. The accuracy and precision of TEIM were benchmarked against EIS on both electrical circuits and epithelial cell monolayers of immortalized Human Bronchial Epithelial (16HBE) and Human Colorectal Adenocarcinoma (Caco-2) (n = 3 for each), and average percent errors for TER, TEC, and ranged from 0.17-3.55%, 1.13-8.96%, and 0.59-26.35%, respectively. Application of TEIM to monitor Caco-2 responses to saponin, a quick-acting pore-forming detergent, revealed smoothly gated double-exponential transient TER and TEC dynamics that were too rapid to be adequately captured previously using EIS. Overall, TEIM enables electrophysiology studies of rapid changes in epithelial cell culture models and possibly more complex in vitro models, holding promise for future applications in areas such as disease modeling, therapeutic development, and beyond.

bioengineering↗

bioRxiv2026

PTMOverlay: A Proteomic Tool to Visualize Post-Translational Modifications Across Evolution

Evolutionary conservation has been considered a hallmark of essential basic functions in cells. Therefore, the study of evolutionarily conserved post-translational modifications (PTMs) can provide insight into their role in protein function. In this context, mass spectrometry can identify and quantify thousands of PTM sites. However, a major bottleneck lies in analyzing the large amounts of data collected by the mass spectrometer. Here we address the need for a protein sequence alignment tool for multiple PTMs across several species. We developed a tool named PTMOverlay that takes peptide identification output files and overlays PTM sites onto multiple protein sequence alignments. Examining 31 bacteria isolates, we combined their protein sequences with select PTM types, including acetylation, phosphorylation, monomethylation, dimethylation, and trimethylation. The tool revealed a variety of conserved modification sites on the bacterial central carbon metabolism. Further structural analysis revealed possible interactions between methylated arginine and lysine residues with phosphothreonine/serine sites on the homodimer interface of enolase. Overall, this tool can parse large amounts of mass spectrometry data and allows for more informed and efficient selection of sites for future studies of protein function.

systems biology↗

bioRxiv2025

Spinning disk confocal microscopy with a 25 Megapixel Camera

Spinning disk confocal microscopy enables fast optical sectioning with low phototoxicity but is often inaccessible due to high hardware costs. We present a low-cost solution using a 25 megapixel machine vision CMOS camera (Sony IMX540, FLIR Blackfly S) and a custom-built spinning disk. The system uses a back-illuminated sensor with high quantum efficiency (69% at 525 nm) and low read noise (2.31 electrons). High-resolution images of Thy1-GFP mouse brain slices and H&E-stained rat testis verified performance across 3D tissue volumes. The custom disk, made with 18 {micro}m pinholes (180 {micro}m pitch) on a chrome photomask and mounted to an optical chopper motor, enables stable, near-telecentric imaging. Micro-Manager software integration allows synchronized control of all hardware, which demonstrates that affordable CMOS sensors can potentially replace sCMOS in spinning disk microscopy, offering an open-source, scalable solution for advanced imaging.

bioengineering↗

bioRxiv2025

Scouring the human Hsp70 network uncovers diverse chaperone safeguards buffering TDP-43 toxicity

Cytoplasmic aggregation and concomitant dysfunction of the prion-like, RNA-binding protein TDP-43 underpin several fatal neurodegenerative diseases, including amyotrophic lateral sclerosis. To elucidate endogenous defenses, we systematically scoured the entire human Hsp70 network for buffers of TDP-43 toxicity. We identify 30 J-domain proteins (2 DNAJAs, 10 DNAJBs, 18 DNAJCs), 6 Hsp70s, and 5 nucleotide-exchange factors that mitigate TDP-43 toxicity. Specific chaperones reduce TDP-43 aggregate burden and detoxify diverse synthetic or disease-linked TDP-43 variants. Sequence-activity mapping unveiled unexpected, modular mechanisms of chaperone-mediated protection. Typically, DNAJBs collaborate with Hsp70 to suppress TDP-43 toxicity, whereas DNAJCs act independently. In human cells, specific chaperones increase TDP-43 solubility and enhance viability under proteotoxic stress. Strikingly, spliceosome-associated DNAJC8 and DNAJC17 retain TDP-43 in the nucleus and promote liquid-phase behavior. Thus, we disambiguate a diverse chaperone arsenal embedded in the human proteostasis network that counters TDP-43 toxicity and illuminate mechanistic gateways for therapeutic intervention in TDP-43 proteinopathies.

biochemistry↗

bioRxiv2022

Identification of genomic alterations with clinical impact in canine splenic hemangiosarcoma

BackgroundCanine hemangiosarcoma (HSA) is an aggressive cancer of endothelial cells associated with short survival times. Understanding the genomic landscape of HSA is critical to developing more effective therapeutic strategies. ObjectivesTo determine the relationships between genomic and clinical features including treatment and outcome in canine splenic HSA. Animals109 dogs with primary splenic HSA treated by splenectomy that had tumor sequencing via the FidoCure(R) Precision Medicine Platform targeted sequencing panel. MethodsPatient signalment, weight, metastasis at diagnosis, treatment, and survival time were retrospectively evaluated. The incidence of genomic alterations in individual genes and their relationship to patient variables and outcome were assessed. ResultsSomatic mutations in TP53 (n = 45), NRAS (n = 20), and PIK3CA (n = 19) were most common. Survival was associated with metastases at diagnosis, germline variants in SETD2 and NOTCH1, and nominally with breed. Age at diagnosis was associated with NRAS mutations and breed. TP53 and PIK3CA mutations were found in larger dogs, germline SETD2 variants in smaller dogs. Doxorubicin (DOX) treatment did not significantly improve survival time, while targeted therapies had a significant early survival benefit. Conclusions and clinical importanceDOX treatment may provide limited clinical benefit for dogs with splenic HSA, while targeted therapy may provide early survival benefit. Genetic signatures associated with splenic HSA may be useful in guiding targeted therapy to improve outcomes. Germline variants, age, size, and breed may be useful prognostic factors and provide insight into the genomic landscape of the tumor.

genomics↗

bioRxiv2021

Shared hotspot mutations in spontaneously arising cancers position dog as an unparalleled comparative model for precision therapeutics

Naturally occurring canine cancers have remarkable similarities to their human counterparts. In order to determine whether these similarities occur at the molecular level, we investigated hotspot mutations in a variety of spontaneously arising canine cancers and found high concordance in oncogenic drivers between cancers in both species. These findings suggest that canines may present a powerful and complementary model for preclinical investigations for targeted cancer therapeutics. Through analysis of 708 client-owned dogs from 96 breeds (plus mixed breeds) with 23 common tumor types, we discovered mutations in 50 well-established oncogenes and tumor suppressors, and compared them to those reported in human cancers. TP53 is the most commonly mutated gene, detected in 30.81% of canine tumors overall and >40% in hemangiosarcoma and osteosarcoma. Canine tumors share mutational hotspots with human tumors in oncogenes including PIK3CA, KRAS, NRAS, BRAF, KIT and EGFR. Hotspot mutations with significant (P<0.0001) association to tumor type include NRAS G61R and PIK3CA H1047R in hemangiosarcoma, ERBB2 V659E in pulmonary carcinoma, and BRAF V588E in urothelial carcinoma. This work positions canines as excellent spontaneous models of human cancers that can help to investigate a wide spectrum of targeted therapies.

cancer biology↗

bioRxiv2020

Visualising G-quadruplex DNA dynamics in live cells by fluorescence lifetime imaging microscopy

Guanine rich regions of oligonucleotides fold into quadruple-stranded structures called G-quadruplexes (G4). Increasing evidence suggests that these G4 structures form in vivo and play a crucial role in cellular processes. However, their direct observation in live cells remains a challenge. Here we demonstrate that a fluorescent probe (DAOTA-M2) in conjunction with Fluorescence Lifetime Imaging Microscopy (FLIM) can identify G4 within nuclei of live and fixed cells. We present a new FLIM-based cellular assay to study the interaction of non-fluorescent small molecules with G4 and apply it to a wide range of drug candidates. We also demonstrate that DAOTA-M2 can be used to study G4 stability in live cells. Reduction of FancJ and RTEL1 expression in mammalian cells increases the DAOTA-M2 lifetime and therefore suggests an increased number of G4 in these cells, implying that FancJ and RTEL1 play a role in resolving G4 structures in cellulo.

biophysics↗

bioRxiv2017

Encoding And Decoding Dynamic Sensory Signals With Recurrent Neural Networks: An Application Of Conceptors To Birdsongs

In a constantly changing environment the brain has to make sense of dynamic patterns of sensory input. These patterns can refer to stimuli with a complex and hierarchical structure which has to be inferred from the neural activity of sensory areas in the brain. Such areas were found to be locally recurrently structured as well as hierarchically organized within a given sensory domain. While there is a great body of work identifying neural representations of various sensory stimuli at different hierarchical levels, less is known about the nature of these representations. In this work, we propose a model that describes a way to encode and decode sensory stimuli based on the activity patterns of multiple, recurrently connected neural populations with different receptive fields. We demonstrate the ability of our model to learn and recognize complex, dynamic stimuli using birdsongs as exemplary data. These birdsongs can be described by a 2-level hierarchical structure, i.e. as sequences of syllables. Our model matches this hierarchy by learning single syllables on a first level and sequences of these syllables on a top level. Model performance on recognition tasks is investigated for an increasing number of syllables or songs to recognize and compared to state-of-the-art machine learning approaches. Finally, we discuss the implications of our model for the understanding of sensory pattern processing in the brain. We conclude that the employed encoding and decoding mechanisms might capture general computational principles of how the brain extracts relevant information from the activity of recurrently connected neural populations.

neuroscience↗

bioRxiv2017

Demonstration of short-term plasticity in the dorsolateral prefrontal cortexwith theta burst stimulation: A TMS-EEG study

ObjectivesTo examine the effects of intermittent TBS (iTBS) and continuous TBS (cTBS) on cortical reactivity in the dorsolateral prefrontal cortex.\n\nMethods10 healthy participants were stimulated with either iTBS, cTBS or sham at F3 electrode. Single- and paired-pulse TMS and concurrent electroencephalography (EEG) were used to assess change in cortical reactivity and long-interval intracortical inhibition (LICI) via TMS-evoked potentials (TEPs) and TMS-evoked oscillations.\n\nResultsSignificant increases in N120 amplitudes (p < 0.01) were observed following iTBS over prefrontal cortex. Changes in TMS-evoked theta oscillations and LICI of theta oscillations were also observed following iTBS (increase) and cTBS (decrease). Change in LICI of theta oscillations correlated with change in N120 amplitude following TBS (r = -0.670, p = 0.001).\n\nConclusionsThis study provides preliminary evidence that TBS produces direct changes in cortical reactivity in the prefrontal cortex. Combining TBS with TMS-EEG may be a useful approach to optimise stimulation paradigms prior to the conduct of clinical trials.\n\nSignificanceTBS is able to modulate cortical reactivity and cortical inhibition in the prefrontal cortex.\n\nHighlightsO_LIEffects of iTBS and cTBS were studied in the DLPFC using TMS-EEG\nC_LIO_LIiTBS increased N120 amplitude, theta power and LICI of theta\nC_LIO_LIcTBS decreased theta power alone\nC_LI

neuroscience↗