14 research outputs found
Confocal Raman Microscopy, Second Edition Jan Toporski, Thomas Dieing, and Olaf Hollricher (Eds.), Springer, New York, 2018. 596 pp. ISBN: 978-3319753782
Structural Dynamics of <i>Ec</i>DOS Heme Domain Revealed by Time-Resolved Ultraviolet Resonance Raman Spectroscopy
Protein dynamics on the subnanosecond to microsecond time scale was investigated for the isolated heme domain of a gas sensor protein, <i>Ec</i>DOS, with time-resolved ultraviolet resonance Raman (UVRR) spectroscopy. Rapid structural changes (<0.5 ns) due to CO dissociation and nanosecond structural relaxation following geminate recombination of CO were observed through a Raman band of Trp53 located near the heme. Microsecond transient UVRR spectra showed several phases of intensity changes in both Trp and Tyr bands. In hundreds of nanoseconds after CO photodissociation, the W18, W16, and W3 bands of Trp residues and Y8a band of Tyr residues decreased in intensity and were followed by the intensity recovery of Tyr band in 50 μs and of Trp bands in hundreds microseconds. This observation demonstrates that a change in the heme ligation triggers conformational changes in the protein moiety through heme side chains
Resonance Raman Spectroscopy Reveals the Origin of an Intermediate Wavelength Form in Photoactive Yellow Protein †
High-content screening Raman spectroscopy (HCS-RS) of panitumumab-exposed colorectal cancer cells
Label-Free Raman Spectroscopic Imaging Monitors the Integral Physiologically Relevant Drug Responses in Cancer Cells
Predictions about the cellular efficacy
of drugs tested <i>in vitro</i> are usually based on the
measured responses of
a few proteins or signal transduction pathways. However, cellular
proteins are highly coupled in networks, and observations of single
proteins may not adequately reflect the <i>in vivo</i> cellular
response to drugs. This might explain some large discrepancies between <i>in vitro</i> drug studies and drug responses observed in patients.
We present a novel <i>in vitro</i> marker-free approach
that enables detection of cellular responses to a drug. We use Raman
spectral imaging to measure the effect of the epidermal growth factor
receptor (EGFR) inhibitor panitumumab on cell lines expressing wild-type
Kirsten-Ras (K-Ras) and oncogenic K-Ras mutations. Oncogenic K-Ras
mutation blocks the response to anti-EGFR therapy in patients, but
this effect is not readily observed <i>in vitro</i>. The
Raman studies detect large panitumumab-induced differences <i>in vitro</i> in cells harboring wild-type K-Ras as seen in A
in red but not in cells with K-Ras mutations as seen in B; these studies
reflect the observed patient outcomes. However, the effect is not
observed when extracellular-signal-regulated kinase phosphorylation
is monitored. The Raman spectra show for cells with wild-type K-Ras
alterations based on the responses to panitumumab. The subcellular
component with the largest spectral response to panitumumab was lipid
droplets, but this effect was not observed when cells harbored K-Ras
mutations. This study develops a noninvasive, label-free, <i>in vitro</i> vibrational spectroscopic test to determine the
integral physiologically relevant drug response in cell lines. This
approach opens a new field of patient-centered drug testing that could
deliver superior patient therapies
Label-free vibrational imaging of different Aβ plaque types in Alzheimer’s disease reveals sequential events in plaque development
The neuropathology of Alzheimer’s disease (AD) is characterized by hyperphosphorylated tau neurofibrillary tangles (NFTs) and amyloid-beta (Aβ) plaques. Aβ plaques are hypothesized to follow a development sequence starting with diffuse plaques, which evolve into more compact plaques and finally mature into the classic cored plaque type. A better molecular understanding of Aβ pathology is crucial, as the role of Aβ plaques in AD pathogenesis is under debate. Here, we studied the deposition and fibrillation of Aβ in different plaque types with label-free infrared and Raman imaging. Fourier-transform infrared (FTIR) and Raman imaging was performed on native snap-frozen brain tissue sections from AD cases and non-demented control cases. Subsequently, the scanned tissue was stained against Aβ and annotated for the different plaque types by an AD neuropathology expert. In total, 160 plaques (68 diffuse, 32 compact, and 60 classic cored plaques) were imaged with FTIR and the results of selected plaques were verified with Raman imaging. In diffuse plaques, we detect evidence of short antiparallel β-sheets, suggesting the presence of Aβ oligomers. Aβ fibrillation significantly increases alongside the proposed plaque development sequence. In classic cored plaques, we spatially resolve cores containing predominantly large parallel β-sheets, indicating Aβ fibrils. Combining label-free vibrational imaging and immunohistochemistry on brain tissue samples of AD and non-demented cases provides novel insight into the spatial distribution of the Aβ conformations in different plaque types. This way, we reconstruct the development process of Aβ plaques in human brain tissue, provide insight into Aβ fibrillation in the brain, and support the plaque development hypothesis
Noninvasive Diagnosis of High-Grade Urothelial Carcinoma in Urine by Raman Spectral Imaging
The current gold
standard for the diagnosis of bladder cancer is
cystoscopy, which is invasive and painful for patients. Therefore,
noninvasive urine cytology is usually used in the clinic as an adjunct
to cystoscopy; however, it suffers from low sensitivity. Here, a novel
noninvasive, label-free approach with high sensitivity for use with
urine is presented. Coherent anti-Stokes Raman scattering imaging
of urine sediments was used in the first step for fast preselection
of urothelial cells, where high-grade urothelial cancer cells are
characterized by a large nucleus-to-cytoplasm ratio. In the second
step, Raman spectral imaging of urothelial cells was performed. A
supervised classifier was implemented to automatically differentiate
normal and cancerous urothelial cells with 100% accuracy. In addition,
the Raman spectra not only indicated the morphological changes that
are identified by cytology with hematoxylin and eosin staining but
also provided molecular resolution through the use of specific marker
bands. The respective Raman marker bands directly show a decrease
in the level of glycogen and an increase in the levels of fatty acids
in cancer cells as compared to controls. These results pave the way
for “spectral” cytology of urine using Raman microspectroscopy
Label-free vibrational imaging of different A plaque types in Alzheimer's disease reveals sequential events in plaque development
The neuropathology of Alzheimer's disease (AD) is characterized by hyperphosphorylated tau neurofibrillary tangles (NFTs) and amyloid-beta (A) plaques. A plaques are hypothesized to follow a development sequence starting with diffuse plaques, which evolve into more compact plaques and finally mature into the classic cored plaque type. A better molecular understanding of A pathology is crucial, as the role of A plaques in AD pathogenesis is under debate. Here, we studied the deposition and fibrillation of A in different plaque types with label-free infrared and Raman imaging. Fourier-transform infrared (FTIR) and Raman imaging was performed on native snap-frozen brain tissue sections from AD cases and non-demented control cases. Subsequently, the scanned tissue was stained against A and annotated for the different plaque types by an AD neuropathology expert. In total, 160 plaques (68 diffuse, 32 compact, and 60 classic cored plaques) were imaged with FTIR and the results of selected plaques were verified with Raman imaging. In diffuse plaques, we detect evidence of short antiparallel -sheets, suggesting the presence of A oligomers. A fibrillation significantly increases alongside the proposed plaque development sequence. In classic cored plaques, we spatially resolve cores containing predominantly large parallel -sheets, indicating A fibrils. Combining label-free vibrational imaging and immunohistochemistry on brain tissue samples of AD and non-demented cases provides novel insight into the spatial distribution of the A conformations in different plaque types. This way, we reconstruct the development process of A plaques in human brain tissue, provide insight into A fibrillation in the brain, and support the plaque development hypothesis
Detailed structural orchestration of Lewy pathology in Parkinson's disease as revealed by 3D multicolor STED microscopy
Post-translational modifications of alpha-synuclein (aSyn), in particular phosphorylation at Serine 129 (Ser129-p) and truncation of its C-terminus (CTT), have been implicated in Parkinson's disease (PD) pathophysiology. Although great interest has emerged for these species as potential biomarkers and therapeutic targets in PD, little is known about their (sub)cellular manifestation in the human brain. In this study, we mapped distribution patterns of Ser129-p and CTT aSyn in neurons with and without Lewy pathology. The combination of highly selective antibodies with multicolor STED microscopy allowed detailed phenotyping of subcellular neuropathology in PD. Nigral Lewy Bodies revealed an onion skin-like 3D architecture, with a framework of Ser129-p aSyn and neurofilaments encapsulating CTT and membrane-associated aSyn epitopes. Based on the identification of subcellular pathological phenotypes in this study, we present a novel hypothesis for maturation stages of Lewy pathology and provide evidence for a key role for Ser129-p aSyn in Lewy inclusion formation