Biology subjects

Kinzer-Ursem, T. L.

Publications and source records attributed to Kinzer-Ursem, T. L..

9 recordsLinked to original sources

bioRxiv2025

Antibody-Initiated Loop-Mediated Isothermal Nucleic Acid Amplification (ai-LAMP) as a New Biosensor for Antigen Detection

Highly sensitive protein detection is critical in research and healthcare diagnostics but remains limited to resource intensive environments. In contrast, lateral flow immunoassay (LFIA)-based diagnostics are popular point-of-care tests due to their simplicity and user-friendly format but lack sensitivity for detecting the low concentrations of proteins that are present early in infections and among asymptomatic individuals. To overcome these limitations, we developed a novel biosensor platform utilizing antibody-initiated loop-mediated isothermal amplification (ai-LAMP) with an LFIA readout for protein detection. This platform integrates protein-specific antibody-antigen binding with the robust signal amplification of LAMP. By conjugating pairs of antibodies to overlapping DNA strands, the presence of a target protein brings the DNA strands into proximity, completing a DNA target to directly initiate the LAMP reaction without any additional ligation step. This approach facilitates the rapid detection of low concentration proteins with a clear visual readout and can be performed in 3 steps from sample to answer. Using ai-LAMP we detected HIV-1 p24 on commercially available LFIAs at a limit of detection (LOD) of 20 fM (0.53 pg/mL), demonstrating 46x improvement over existing HIV p24 LFIAs. The use of ai-LAMP eliminates the need for sophisticated laboratory equipment to detect protein targets in low concentrations, paving a new way for rapid and accessible biomarker detection in clinical and research settings. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=139 SRC="FIGDIR/small/678293v2_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@5b587forg.highwire.dtl.DTLVardef@10f3587org.highwire.dtl.DTLVardef@1833eeborg.highwire.dtl.DTLVardef@197d1bf_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2024

A FRET-Based FLIM Method to Probe Membrane-Induced Alpha-Synuclein Aggregation in Neurons

1Parkinsons disease (PD) involves the aggregation of the protein alpha-synuclein, a process promoted by interactions with intracellular membranes. To study this phenomenon in neurons for the first time, we developed a fluorescence lifetime imaging (FLIM) method using Forster resonance energy transfer and self-quenching reporters, analyzed with a custom-built FLIM microscope. This method offers insights into aggregate formation in PD and can be broadly applied to probe protein-membrane interactions in neurons.

neuroscience↗

bioRxiv2024

Mechanisms of Alpha-Synuclein Seeded Aggregation in Neurons Revealed by Fluorescence Lifetime Imaging

1The brains of Parkinsons disease (PD) patients are characterized by the presence of Lewy body inclusions enriched with fibrillar forms of the presynaptic protein alpha-synuclein (aSyn). Despite related evidence that Lewy pathology spreads across different brain regions as the disease progresses, the underlying mechanism hence the fundamental cause of PD progression is unknown. The propagation of aSyn pathology is thought to potentially occur through the release of aSyn aggregates from diseased neurons, their uptake by neighboring healthy neurons via endocytosis, and subsequent seeding of native aSyn aggregation in the cytosol. A critical aspect of this process is believed to involve the escape of internalized ag-gregates from the endolysosomal compartment, though direct evidence of this mechanism in cultured neuron models remains lacking. In this study, we utilize a custom-built, time-gated fluorescence lifetime imaging microscope (FLIM) to investigate the progression of seeded ag-gregation over time in live cortical neurons. By establishing fluorescence lifetime sensitivity to aSyn aggregation level, we are able to monitor the proteins aggregation state. Through a FLIM analysis of neurons expressing aSyn-mVenus and exposed to aSyn preformed fibrils labeled with the acid-responsive dye pHrodo, we reveal the proteins aggregation state in both the cytosol and the endolysosomal compartment. The results indicate that aSyn seeds undergo partial disassembly prior to escaping the endocytic pathway, and that this escape is closely linked to the aggregation of cytosolic aSyn. In certain neurons, monomeric aSyn is found to translocate from the cytosol into the endolysosomal compartment, where it appar-ently forms aggregates in proximity to retained seeds. Additional analyses reveals zones of neuritic aSyn aggregates that overlaps with regions of microtubule disruption. Collectively, these findings enhance our understanding of aSyn pathology propagation in PD and other synucleinopathies, motivate additional experiments along these lines, and offer a path to guide the development of disease-modifying therapies.

neuroscience↗

bioRxiv2023

Effects of Neighboring Phosphorylation Events on the Affinities of pT181-Tau Antibodies

A tau variant phosphorylated on threonine 181 (pT181-tau) has been widely investigated as a potential Alzheimers disease (AD) biomarker in cerebrospinal fluid (CSF) and blood. pT181-tau is present in neurofibrillary tangles (NFTs) of AD brains, and CSF levels of pT181-tau correlate with overall NFT burden. Various immuno-based analytical methods, including Western blotting and ELISA, have been used to quantify pT181-tau in human biofluids. The reliability of these methods depends on the affinity and binding specificity of the antibodies used to measure pT181-tau levels. Although both of these properties could in principle be affected by phosphorylation within or near the antibodys cognate antigen, such effects have not been extensively studied. Here, we developed a biolayer interferometry assay to determine the degree to which the affinity of pT181-tau antibodies is altered by the phosphorylation of serine or threonine residues near the target epitope. Our results revealed that phosphorylation near T181 negatively affected the binding of pT181-tau antibodies to their cognate antigen to varying degrees. In particular, two of three antibodies tested showed a complete loss of affinity for the pT181 target when S184 or S185 was phosphorylated. These findings highlight the importance of selecting antibodies that have been thoroughly characterized in terms of affinity and binding specificity, addressing the potential disruptive effects of post-translational modifications in the epitope region, to ensure accurate biomarker quantitation.

neuroscience↗

bioRxiv2023

Methods Optimization for the Expression and Purification of Human Calcium Calmodulin-Dependent Protein Kinase II Alpha

Calcium/calmodulin-dependent protein kinase II (CaMKII) is a complex multifunctional kinase that is highly expressed in central nervous tissues and plays a key regulatory role in the calcium signaling pathway. Despite over 30 years of recombinant expression and characterization studies, CaMKII continues to be investigated for its impact on signaling cooperativity and its ability to bind multiple substrates through its multimeric hub domain. Here we compare and optimize protocols for the generation of full-length wild-type human calcium/calmodulin-dependent protein kinase II alpha (CaMKII). Side-by-side comparison of expression and purification in both insect and bacterial systems shows that the insect expression method provides superior yields of the desired autoinhibited CaMKII holoenzymes. Utilizing baculovirus insect expression system tools, our results demonstrate a high yield method to produce homogenous, monodisperse CaMKII in its autoinhibited state suitable for biophysical analysis. Advantages and disadvantages of these two expression systems (baculovirus insect cell versus Escherichia coli expression) are discussed, as well as purification optimizations to maximize the enrichment of full-length CaMKII.

biochemistry↗

bioRxiv2021

AN INTEGRATIVE BIOLOGY APPROACH TO QUANTIFY THE BIODISTRIBUTION OF AZIDOHOMOALANINE IN VIVO

Identification and quantitation of newly synthesized proteins (NSPs) are critical to understanding protein dynamics in development and disease. Probing the nascent proteome can be achieved using non-canonical amino acids (ncAAs) to selectively label the NSPs utilizing endogenous translation machinery, which can then be quantitated with mass spectrometry. Since its conception, ncAA labeling has been applied to study many in vitro systems and more recently the in vivo proteomes of complex organisms such as rodents. In vivo labeling is typically achieved by introducing ncAAs into diet, which requires extended labeling times. We have previously demonstrated that labeling the murine proteome is feasible via injection of azidohomoalanine (Aha), a ncAA and methionine (Met) analog, without the need for Met depletion. With the ability to isolate NSPs without applying stress from dietary changes, Aha labeling can address biological questions wherein temporal protein dynamics are significant. However, accessing this temporal resolution requires a more complete understanding of Aha distribution kinetics in tissues. Furthermore, studies of physiological effects of ncAA administration have been limited to gross observation of animal appearance. To address these gaps, we created a deterministic, compartmental model of the biokinetic transport and incorporation of Aha in mice. Parameters were informed from literature and experimentally. Model results demonstrate the ability to predict Aha distribution and labeling under a variety of dosing paradigms and confirms the use of the model as a tool for design of future studies. To establish the suitability of the method for in vivo studies, we investigated the impact of Aha administration on normal physiology by analyzing the plasma metabolome following Aha injection. We show that Aha administration does not significantly perturb cellular functions as reflected by an unchanged plasma metabolome compared to non-injected controls. Author SummaryAs the machinery of life, proteins play a key role in dynamic processes within an organism. As such, the response of the proteome to perturbation is increasingly becoming a critical component of biological and medical studies. Dysregulation of protein mechanisms following exposure to experimental treatment conditions can implicate physiological mechanisms of health and disease, elucidate toxin/drug response, and highlight potential targets for novel therapies. Traditionally, these questions have been probed by studying perturbations in total proteins following an experimental treatment. However, the proteome is expansive and noisy, often an early response can be indiscernible against the background of unperturbed proteins. Here, we apply a technique to selectively label newly synthesized proteins, which enables capturing early changes in protein behavior. We utilize an amino acid analog that naturally incorporates into proteins, and investigate the tissue distribution, protein labeling efficiency, and potential physiological impact of this analog in mice. Our results demonstrate that we can reproducibly predict protein labeling and that the administration of this analog does not significantly alter in vivo physiology over the course of our experimental study. We further present a computational model that can be used to guide future experiments utilizing this technique to study proteomic responses to stimuli.

bioengineering↗

bioRxiv2020

Extracellular matrix protein composition dynamically changes during murine forelimb development

The extracellular matrix (ECM) is an integral part of multicellular organisms, connecting different cell layers and tissue types. During morphogenesis and growth, tissues undergo substantial reorganization involving cellular proliferation, migration, and differentiation. While it is intuitive that the ECM remodels in concert, little is known regarding how matrix composition and organization change during development. We utilized tissue fractionation and mass spectrometry to define ECM protein (matrisome) dynamics during murine forelimb development and resolved significant differences in ECM composition as a function of development, disease and tissue type. Additionally, we used bioorthogonal non-canonical amino acid tagging (BONCAT) to label newly synthesized ECM within the developing forelimb. We demonstrate the feasibility of using BONCAT to enrich for newly synthesized matrisome components and identified differences in ECM synthesis between morphogenesis and growth. This resource will guide future research investigating the role of the matrisome during complex tissue development.

developmental biology↗

bioRxiv2019

A multi-state model of the CaMKII dodecamer suggests a role for calmodulin in maintenance of autophosphorylation

Ca2+/calmodulin-dependent protein kinase II (CaMKII) accounts for up to 2 percent of all brain protein and is essential to memory function. CaMKII activity is known to regulate dynamic shifts in the size and signaling strength of neuronal connections, a process known as synaptic plasticity. Increasingly, computational models are used to explore synaptic plasticity and the mechanisms regulating CaMKII activity. Conventional modeling approaches may exclude biophysical detail due to the impractical number of state combinations that arise when explicitly monitoring the conformational changes, ligand binding, and phosphorylation events that occur on each of the CaMKII holoenzymes twelve subunits. To manage the combinatorial explosion without necessitating bias or loss in biological accuracy, we use a specialized syntax in the software MCell to create a rule-based model of the twelve-subunit CaMKII holoenzyme. Here we validate the rule-based model against previous measures of CaMKII activity and investigate molecular mechanisms of CaMKII regulation. Specifically, we explore how Ca2+/CaM-binding may both stabilize CaMKII subunit activation and regulate maintenance of CaMKII autophosphorylation. Noting that Ca2+/CaM and protein phosphatases bind CaMKII at nearby or overlapping sites, we compare model scenarios in which Ca2+/CaM and protein phosphatase do or do not structurally exclude each others binding to CaMKII. Our results suggest a functional mechanism for the so-called \"CaM trapping\" phenomenon, such that Ca2+/CaM structurally excludes phosphatase binding and thereby prolongs CaMKII autophosphorylation. We conclude that structural protection of autophosphorylated CaMKII by Ca2+/CaM may be an important mechanism for regulation of synaptic plasticity.\n\nAuthor summaryIn the hippocampus, the dynamic fluctuation in size and strength of neuronal connections is thought to underlie learning and memory processes. These fluctuations, called synaptic plasticity, are in-part regulated by the protein calcium/calmodulin-dependent kinase II (CaMKII). During synaptic plasticity, CaMKII becomes activated in the presence of calcium ions (Ca2+) and calmodulin (CaM), allowing it to interact enzymatically with downstream binding partners. Interestingly, activated CaMKII can phosphorylate itself, resulting in state changes that allow CaMKII to be functionally active independent of Ca2+/CaM. Phosphorylation of CaMKII at Thr-286/287 has been shown to be a critical component of learning and memory. To explore the molecular mechanisms that regulate the activity of CaMKII holoenzymes, we use a rule-based approach that reduces computational complexity normally associated with representing the wide variety of functional states that a CaMKII holoenzyme can adopt. Using this approach we observe regulatory mechanisms that might be obscured by reductive approaches. Our results newly suggest that CaMKII phosphorylation at Thr-286/287 is stabilized by a mechanism in which CaM structurally excludes phosphatase binding at that site.

neuroscience↗

bioRxiv2017

Measuring Biotherapeutic Viscosity and Degradation On-Chip with Particle Diffusometry

In absence of efficient ways to test drug stability and efficacy, pharmaceuticals that have been stored outside of set temperature conditions are destroyed, often at great cost. This is especially problematic for biotherapeutics, which are highly sensitive to temperature fluctuations. Current platforms for assessing the stability of protein-based biotherapeutics in high throughput and in low volumes are unavailable outside of research and development laboratories and are not efficient for use in production, quality control, distribution, or clinical settings. In these alternative environments, microanalysis platforms could provide significant advantages for the characterization of biotherapeutic degradation. Here we present particle diffusometry (PD), a new technique to study degradation of biotherapeutic solutions. PD uses a simple microfluidic chip and microscope setup to calculate the Brownian motion of particles in a quiescent solution using a variation of particle image velocimetry (PIV) fundamentals. We show that PD can be used to measure the viscosity of protein solutions to discriminate intact protein from degraded samples as well as to determine the change in viscosity as a function of therapeutic concentration. PD viscosity analysis is applied to two particularly important biotherapeutic preparations: insulin, a commonly used protein for diabetic patients, and monoclonal antibodies which are an emerging class of biotherapeutics used to treat a variety of diseases such as autoimmune disorders and cancer. PD-based characterization of solution viscosity is a new tool for biotherapeutic analysis, and owing to its easy setup could readily be implemented at key points of the pharmaceutical delivery chain and in clinical settings.

bioengineering↗