AFM provides very high resolution (fractions of a nm) topographical images of single molecules, live cells, recombinant proteins (i.e. amyoid, tau), or tissues. Measurement outputs also include elasticity, adhesion, chemical forces and molecular binding sites under physiological conditions. The JPK NanoWizard 4a AFM system is housed at the Byrd Microscopy Core (link) and is used by various lab, including Blair.

Analysis of cognition, mood, anxiety and depression, locomotion, and other behavioral outputs related to a wide variety of normal aging and pathological processes in mice and rats. These studies are conducted at the Byrd murine behavior core.

Circular dichroism spectroscopy

Circular dichroism is one of the major spectroscopic techniques for low resolution structural characterization of purified proteins, analysis of their conformational stability, and investigation of structural changes in proteins associated with their interactions with partners. This technique allows analyzing protein secondary and tertiary structure. The Jasco spectropolarimeter (Jasco-815) is used for these studies by Uversky. 

Dynamic light scattering

Protein aggregation is accompanied by the formation of particles whose size increases over time that leads to the increase in light scattering. Dynamic light scattering provides an opportunity to determine the size (hydrodynamic radius) and size distribution of particles, polymers, and biopolymers from 0.5 nm 1000 nm. The corresponding analysis is conducted by automated DynaPro Plate Reader (Wyatt Technology) equipped with the DynaPro Plate Reader Temperature Control unit (Wyatt Technology), which permits the Peltier-driven temperature regulation for the plate reader from 4°C - 70°C with an ambient temperature of 24°C. These studies are conducted by Uversky.

Fluorescence spectroscopy

Fluorescence spectroscopy is used for the analysis of the fluorescent properties of biological molecules. Since fluorescent properties of the fluorophore depend on its environment, fluorescence can be used for low resolution structural characterization of purified proteins, analysis of their conformational stability, investigation of structural changes in proteins associated with their interactions with partners, as well as it represents a unique tool to follow formation of amyloid fibrils. The Jasco Spectrofluorometer (FP-8300) is used for these studies by Uversky.

The C. Elegans (worm) model is a simple but elegant and powerful tool to study multiple facets of neurodegeneration and aging. We use genetically modified C. elegans to identify genetic modifiers of protein pathologies, such as amyloid, tau, and TDP-43. It is also used to study the general aging process and longevity. These studies are conducted by Jinwal.

Confocal microscopy, the ‘bread and butter’ of virtually all research investigations, is an optical imaging technique that offers shallow depth of field, elimination of out-of-focus glare, and ability to collect serial optical sections from thick specimens. The Byrd Microscopy Core houses 3 laser-scanning confocal units including Olympus FV10i, ZeissLSM 880, and NikonC2+ Ti-E.

Drosophila melanogaster, commonly known as the fruit fly, is a powerful genetic model system to study neurodegeneration, neurogenesis, axonal guidance and brain development. The Bhat lab specializes in the use of drosophila model systems.

Electrophysiology is the study of the electrical properties of biological cells and tissues. Such electrical properties are governed by neuronal synapses in the brain, which strengthen and weaken in response to increases or decreases in their activity – synaptic plasticity. As memories are thought to be represented by vast networks of synaptic connections in the brain, changes in synaptic plasticity correlate with learning and memory as well as neurodegeneration. Electrophysiological studies are conducted at the Byrd electrophysiology core and the Parent lab.

Viral vectors such as Adeno-associated virus (AAV) and lentivirus are used to express genes of interest in specified areas of the brain in rodent models, which facilitates the study of pathogenesis, neurodegeneration, and proof-of-concept therapeutics. AAV does not induce a significant immune response, which makes AAV quite attractive for gene therapy. The Nash lab specializes in AAV technology, and many Byrd labs use AAV and/or lentiviral gene expression tools.

Fluorescence reporter proteins/probes are coupled with live-cell confocal imaging to study many biological processes, including protein trafficking, axonal transport, mitochondrial membrane potential, mitochondrial fusion, calcium levels, protein turnover, etc. Techniques such as photoactivation, fluorescence resonance transfer (FRET), and ratiometric imaging are employed for a variety of different applications. These studies are conducted using sophisticated instruments at the Byrd Microscopy Core. These studies are conducted by multiple labs, including Thinakaran, Blair, Parent, Heckmann, and Dharap.

The ZEISS Axio Scan.Z1 instrument, a component of the Byrd Microscopy Core (link), is a powerful automated slide scanner with high throughput (100 whole slides) brightfield and fluorescence imaging capabilities used to image and quantitate brain tissue sections. The Li-Cor Odessey CLx imager is used to quantify immunoblots, in-cell westerns, microarrays, and tissues using near infrared (NIR) fluorescence, providing 6 logs of linear dynamic range. The Byrd also houses 2 Protein Simple Wes instruments, which allows investigators to separate and analyze proteins by size with pg-level sensitivity and provides quantitative data with up to 6 logs of linear dynamic range. All Byrd labs use these instruments.

Mitochondria, which are powerhouses of all cells, not only produce the vast majority of cellular energy (ATP) but also execute the cell death program. Mitochondrial dysfunction is common to all neurodegenerative diseases. We utilize the Seahorse XFe96 Analyzer coupled with pharmacological tools to measure mitochondrial respiration in real time in live cells, isolated mitochondria, or ex vivo tissues. These studies are conducted by multiple labs, including Heckmann.

Investigators utilize a variety of genetically modified mice to model proteinopathies such as amyloid, tau, TDP-43, and a-synuclein, which resemble those in human patient brains. Animal models are used to test genetic modifiers of pathology as well as potential therapeutic agents.

The multiphoton confocal microscopy allows investigators to image tissues (i.e. brain) at a subcellular resolution in live animals and ex vivo tissues using near infrared (NIR) pulse lasers coupled with optogenetic fluorescent proteins or fluorescent probes. The Nikon A1R HD multiphoton confocal system is housed at the Byrd Microscopy Core.

Use of the QuantStudioTM 12K Flex Real-Time PCR System together with OpenArray technology (housed in the Bickford lab) allows investigators to assess changes in the expression of 600 genes simultaneously from RNA isolated from healthy versus diseased cells and tissues.

Proteomics is the large-scale study of proteins often using mass spectrometry to identify protein fragments from complex biological samples. These studies are used to discover both the identity of proteins and change in protein levels from diseased versus healthy tissues, leading to discovery of novel disease-relevant biomarkers.

X-ray crystallography

X-ray crystallography is the main experimental technique used to determined three-dimensional structures of proteins, using X-ray diffraction by protein crystals. The high resolution structural information provided by X-ray crystallography is used to understand protein function and to engineer novel inhibitors. USF is a member of the SER-CAT beamline at the Advanced Photon Source (APS) of Argonne National Laboratory, a national synchrotron-radiation light source. This gives us regular access to one of the best X-ray facilities in the world and allows us to do data collection remotely through robotic systems. These studies are conducted by the Chen lab.  

Molecular docking  

Molecular docking uses computational programs to simulate molecular interactions between proteins and small molecule ligands. These calculations provide valuable insights into how proteins bind to their substrates and regulators, and offer an effective tool to screen compound libraries in silico and to design new small molecule inhibitors. Using the high performance scientific computing server at USF, we can virtually screen millions of commercially available compounds against proteins of interest and identify potential inhibitors. Subsequently, molecular docking can also be employed to evaluate modifications of lead compounds to enhance their activities. These studies are conducted by the Chen lab.