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

1The brains of Parkinson's 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 FRET-Based FLIM Method to Probe Membrane-Induced Alpha-Synuclein Aggregation in Neurons.

1Parkinson's 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 Förster 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.

Site-specific seeding of Lewy pathology induces distinct pre-motor cellular and dendritic vulnerabilities in the cortex.

Circuit-based biomarkers distinguishing the gradual progression of Lewy pathology across synucleinopathies remain unknown. Here, we show that seeding of α-synuclein preformed fibrils in mouse dorsal striatum and motor cortex leads to distinct prodromal-phase cortical dysfunction across months. Our findings reveal that while both seeding sites had increased cortical pathology and hyperexcitability, distinct differences in electrophysiological and cellular ensemble patterns were crucial in distinguishing pathology spread between the two seeding sites.

Real-Time Visualization of HIV-1 RNA Detection Using Loop-Mediated Isothermal Amplification-Enabled Particle Diffusometry.

Isothermal nucleic acid amplification tests, NAATs, such as reverse transcription-loop-mediated isothermal amplification (RT-LAMP), offer promising capabilities to perform real-time semiquantitative detection of viral pathogens. These tests provide rapid results, utilize simple instrumentation for single-temperature reactions, support efficient user workflows, and are suitable for field use. Herein, we present a novel and robust method for real-time monitoring of HIV-1 RNA RT-LAMP utilizing a novel implementation of particle diffusometry (PD), a diffusivity quantification technique using fluorescent particles, to quantify viral concentration in nuclease-free water.

Proteins clump: Mechanics and transport during neurodegeneration.

This summary of recent contributions in the Biophysical Journal from 2020 to 2023 highlights new mechanistic insights into key biomechanical and biophysical aspects of neurodegeneration. Neurodegeneration encompasses complex diseases characterized by the progressive loss of neuronal function, often linked to protein accumulation and aggregation. Several factors, including mechanical properties and structural composition of brain tissue, formation of proteinaceous condensates within cells, and protein transport between cells, impact the loss of neural function.

Biomolecular Interaction Analysis Quantification with a Low-Volume Microfluidic Chip and Particle Diffusometry.

Microfluidic techniques are widely applied in biomolecular analysis and disease diagnostic assays. While the volume of the sample that is directly used in such assays is often only femto-to microliters, the "dead volume" of solutions supplied in syringes and tubing can be much larger, even up to milliliters, increasing overall reagent use and making analysis significantly more expensive. To reduce the difficulty and cost, we designed a new chip using a low volume solution for analysis and applied it to obtain real-time data for protein-protein interaction measurements.

Two-phase Porous Media Flow Model Based on the Incompressible Navier-Stokes Equation.

Two-phase porous media flow is important in many applications from drug delivery to groundwater diffusion and oil recovery and is of particular interest to biomedical diagnostic test developers using cellulose and nitrocellulose membranes with limited fluid sample volumes. This work presents a new two-phase porous media flow model based on the incompressible Navier-Stokes equation. The model aims to address the limitations of existing methods by incorporating a partial saturation distribution in porous media to account for limited fluid volumes.

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. While it is intuitive that the ECM remodels in concert, little is known regarding how matrix composition and organization change during development.

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 (CaMKIIα).

Immobilization of azide-functionalized proteins to micro- and nanoparticles directly from cell lysate.

Immobilization of proteins and enzymes on solid supports has been utilized in a variety of applications, from improved protein stability on supported catalysts in industrial processes to fabrication of biosensors, biochips, and microdevices. A critical requirement for these applications is facile yet stable covalent conjugation between the immobilized and fully active protein and the solid support to produce stable, highly bio-active conjugates. Here, we report functionalization of solid surfaces (gold nanoparticles and magnetic beads) with bio-active proteins using site-specific and biorthogonal labeling and azide-alkyne cycloaddition, a click chemistry.

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 Alzheimer's 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 the overall NFT burden. Various immunobased analytical methods, including Western blotting and ELISA, have been used to quantify pT181-tau in human biofluids.

Mechanistic Computational Modeling of Implantable, Bioresorbable Drug Release Systems.

Implantable, bioresorbable drug delivery systems offer an alternative to current drug administration techniques; allowing for patient-tailored drug dosage, while also increasing patient compliance. Mechanistic mathematical modeling allows for the acceleration of the design of the release systems, and for prediction of physical anomalies that are not intuitive and may otherwise elude discovery. This study investigates short-term drug release as a function of water-mediated polymer phase inversion into a solid depot within hours to days, as well as long-term hydrolysis-mediated degradation and erosion of the implant over the next few weeks.

An Integrative Biology Approach to Quantify the Biodistribution of Azidohomoalanine .

Background: 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. We have previously demonstrated that labeling the murine proteome is feasible via injection of azidohomoalanine (Aha), an ncAA and methionine (Met) analog, without the need for Met depletion.

Quantifying Brownian motion in the presence of simple shear flow with particle diffusometry.

Particle diffusometry, a technology derived from particle image velocimetry, quantifies the Brownian motion of particles suspended in a quiescent solution by computing the diffusion coefficient. Particle diffusometry has been used for pathogen detection by measuring the change in solution viscosity due to amplified DNA from a specific gene target. However, particle diffusometry fails to calculate accurate measurements at elevated temperatures and fluid flow.

Measurement of Protein-Protein Interaction Dynamics Using Microfluidics and Particle Diffusometry.

The measurement and optimization of protein-protein interactions are critical in the design of biotherapeutics, biomolecular sensing elements, and functional protein-based biomaterials among other biomolecular sciences and engineering. Current gold standard assays require specifically designed core facilities, equipment, and expertise to implement the measurement, making it inconvenient for most labs unless implemented routinely. We developed a new method aiming at measuring protein binding kinetics based on microfluidics and particle diffusometry (PD), which only needs very general lab equipment, including a fluorescence microscope, a syringe pump, and a simple microchannel fabricated on a glass slide.

PD-LAMP smartphone detection of SARS-CoV-2 on chip.

In 2019 the COVID-19 pandemic, caused by SARS-CoV-2, demonstrated the urgent need for rapid, reliable, and portable diagnostics. The COVID-19 pandemic was declared in January 2020 and surges of the outbreak continue to reoccur. It is clear that early identification of infected individuals, especially asymptomatic carriers, plays a huge role in preventing the spread of the disease.

Towards the use of a smartphone imaging-based tool for point-of-care detection of asymptomatic low-density malaria parasitaemia.

Background: Globally, there are over 200 million cases of malaria annually and over 400,000 deaths. Early and accurate detection of low-density parasitaemia and asymptomatic individuals is key to achieving the World Health Organization (WHO) 2030 sustainable development goals of reducing malaria-related deaths by 90% and eradication in 35 countries. Current rapid diagnostic tests are neither sensitive nor specific enough to detect the low parasite concentrations in the blood of asymptomatic individuals.

A smartphone-based particle diffusometry platform for sub-attomolar detection of Vibrio cholerae in environmental water.

Each year, 3.4 million people die from waterborne diseases worldwide. Development of a rapid and portable platform for detecting and monitoring waterborne pathogens would significantly aid in reducing the incidence and spread of infectious diseases.

The Dynamic Relationship of Breast Cancer Cells and Fibroblasts in Fibronectin Accumulation at Primary and Metastatic Tumor Sites.

In breast cancer (BC), tissue stiffening via fibronectin (FN) and collagen accumulation is associated with advanced disease progression at both the primary tumor and metastatic sites. Here, we evaluate FN production in 15 BC cell lines, representing a variety of subtypes, phenotypes, metastatic potentials, and chemotherapeutic sensitivities. We demonstrate that intracellular and soluble FN is initially lost during tumorigenic transformation but is rescued in all lines with epithelial-mesenchymal plasticity (EMP).

Perlecan Knockdown Significantly Alters Extracellular Matrix Composition and Organization During Cartilage Development.

Perlecan is a critical proteoglycan found in the extracellular matrix (ECM) of cartilage. In healthy cartilage, perlecan regulates cartilage biomechanics and we previously demonstrated perlecan deficiency leads to reduced cellular and ECM stiffness This change in mechanics may lead to the early onset osteoarthritis seen in disorders resulting from perlecan knockdown such as Schwartz-Jampel syndrome (SJS). To identify how perlecan knockdown affects the material properties of developing cartilage, we used imaging and liquid chromatography-tandem mass spectrometry (LC-MS/MS) to study the ECM in a murine model of SJS, Perlecan knockdown led to defective pericellular matrix formation, whereas the abundance of bulk ECM proteins, including many collagens, increased.

Fluorescent Probes for Monitoring Serine Ubiquitination.

In a radical departure from the classical E1-E2-E3 three-enzyme mediated ubiquitination of eukaryotes, the recently described bacterial enzymes of the SidE family of effectors utilize NAD to ligate ubiquitin onto target substrate proteins. This outcome is achieved via a two-step mechanism involving (1) ADP ribosylation of ubiquitin followed by (2) phosphotransfer to a target serine residue. Here, using fluorescent NAD analogues as well as synthetic substrate mimics, we have developed continuous assays enabling real-time monitoring of both steps of this mechanism.

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.

Dynamics of Non-Canonical Amino Acid-Labeled Intra- and Extracellular Proteins in the Developing Mouse.

Introduction: Mapping protein synthesis and turnover during development will provide insight into functional tissue assembly; however, quantitative characterization has been hindered by a lack of tools. To address this gap, we previously demonstrated murine embryos can be labeled with the non-canonical amino acid azidohomoalanine (Aha), which enables the enrichment and identification of newly synthesized proteins. Using this technique, we now show how protein turnover varies as a function of both time and cellular compartment during murine development.

Protein Labeling and Bioconjugation Using N-Myristoyltransferase.

Methods that allow for labeling of proteins cotranslationally within protein expression systems have had wide-ranging applications in health, engineering, and medicine. Bioorthogonal chemistries that allow for conjugation of proteins or biomolecules of interest to substrates (fluorophores, gold nanoparticles, polymers, etc.) in living cells without prior enrichment or purification have likewise enabled advances in technology to study and engineer cellular and biomolecular systems.

Non-canonical amino acid labeling in proteomics and biotechnology.

Metabolic labeling of proteins with non-canonical amino acids (ncAAs) provides unique bioorthogonal chemical groups during de novo synthesis by taking advantage of both endogenous and heterologous protein synthesis machineries. Labeled proteins can then be selectively conjugated to fluorophores, affinity reagents, peptides, polymers, nanoparticles or surfaces for a wide variety of downstream applications in proteomics and biotechnology. In this review, we focus on techniques in which proteins are residue- and site-specifically labeled with ncAAs containing bioorthogonal handles.