Publications by authors named "Klaus Gerwert"

142 Publications

Shape decomposition algorithms for laser capture microdissection.

Algorithms Mol Biol 2021 Jul 8;16(1):15. Epub 2021 Jul 8.

Bioinformatics Group, Faculty of Biology and Biotechnology, Ruhr University Bochum, Bochum, Germany.

Background: In the context of biomarker discovery and molecular characterization of diseases, laser capture microdissection is a highly effective approach to extract disease-specific regions from complex, heterogeneous tissue samples. For the extraction to be successful, these regions have to satisfy certain constraints in size and shape and thus have to be decomposed into feasible fragments.

Results: We model this problem of constrained shape decomposition as the computation of optimal feasible decompositions of simple polygons. We use a skeleton-based approach and present an algorithmic framework that allows the implementation of various feasibility criteria as well as optimization goals. Motivated by our application, we consider different constraints and examine the resulting fragmentations. We evaluate our algorithm on lung tissue samples in comparison to a heuristic decomposition approach. Our method achieved a success rate of over 95% in the microdissection and tissue yield was increased by 10-30%.

Conclusion: We present a novel approach for constrained shape decomposition by demonstrating its advantages for the application in the microdissection of tissue samples. In comparison to the previous decomposition approach, the proposed method considerably increases the amount of successfully dissected tissue.
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http://dx.doi.org/10.1186/s13015-021-00193-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8265035PMC
July 2021

The Ras dimer structure.

Chem Sci 2021 May 4;12(23):8178-8189. Epub 2021 May 4.

Biospectroscopy, Center for Protein Diagnostics (PRODI), Ruhr University Bochum 44801 Bochum Germany

Oncogenic mutated Ras is a key player in cancer, but despite intense and expensive approaches its catalytic center seems undruggable. The Ras dimer interface is a possible alternative drug target. Dimerization at the membrane affects cell growth signal transduction. studies indicate that preventing dimerization of oncogenic mutated Ras inhibits uncontrolled cell growth. Conventional computational drug-screening approaches require a precise atomic dimer model as input to successfully access drug candidates. However, the proposed dimer structural models are controversial. Here, we provide a clear-cut experimentally validated N-Ras dimer structural model. We incorporated unnatural amino acids into Ras to enable the binding of labels at multiple positions click chemistry. This labeling allowed the determination of multiple distances of the membrane-bound Ras-dimer measured by fluorescence and electron paramagnetic resonance spectroscopy. In combination with protein-protein docking and biomolecular simulations, we identified key residues for dimerization. Site-directed mutations of these residues prevent dimer formation in our experiments, proving our dimer model to be correct. The presented dimer structure enables computational drug-screening studies exploiting the Ras dimer interface as an alternative drug target.
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http://dx.doi.org/10.1039/d1sc00957eDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8208300PMC
May 2021

The subcellular arrangement of alpha-synuclein proteoforms in the Parkinson's disease brain as revealed by multicolor STED microscopy.

Acta Neuropathol 2021 Jun 11. Epub 2021 Jun 11.

Department of Anatomy and Neurosciences, Clinical Neuroanatomy and Biobanking, Amsterdam Neuroscience, Amsterdam UMC, Location VU University Medical Center, O2 building, room 13 E11, De Boelelaan 1108, 1081 HZ, Amsterdam, The Netherlands.

Various post-translationally modified (PTM) proteoforms of alpha-synuclein (aSyn)-including C-terminally truncated (CTT) and Serine 129 phosphorylated (Ser129-p) aSyn-accumulate in Lewy bodies (LBs) in different regions of the Parkinson's disease (PD) brain. Insight into the distribution of these proteoforms within LBs and subcellular compartments may aid in understanding the orchestration of Lewy pathology in PD. We applied epitope-specific antibodies against CTT and Ser129-p aSyn proteoforms and different aSyn domains in immunohistochemical multiple labelings on post-mortem brain tissue from PD patients and non-neurological, aged controls, which were scanned using high-resolution 3D multicolor confocal and stimulated emission depletion (STED) microscopy. Our multiple labeling setup highlighted a consistent onion skin-type 3D architecture in mature nigral LBs in which an intricate and structured-appearing framework of Ser129-p aSyn and cytoskeletal elements encapsulates a core enriched in CTT aSyn species. By label-free CARS microscopy we found that enrichments of proteins and lipids were mainly localized to the central portion of nigral aSyn-immunopositive (aSyn+) inclusions. Outside LBs, we observed that 122CTT aSyn+ punctae localized at mitochondrial membranes in the cytoplasm of neurons in PD and control brains, suggesting a physiological role for 122CTT aSyn outside of LBs. In contrast, very limited to no Ser129-p aSyn immunoreactivity was observed in brains of non-neurological controls, while the alignment of Ser129-p aSyn in a neuronal cytoplasmic network was characteristic for brains with (incidental) LB disease. Interestingly, Ser129-p aSyn+ network profiles were not only observed in neurons containing LBs but also in neurons without LBs particularly in donors at early disease stage, pointing towards a possible subcellular pathological phenotype preceding LB formation. Together, our high-resolution and 3D multicolor microscopy observations in the post-mortem human brain provide insights into potential mechanisms underlying a regulated LB morphogenesis.
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http://dx.doi.org/10.1007/s00401-021-02329-9DOI Listing
June 2021

Quantum Cascade Laser-Based Infrared Imaging as a Label-Free and Automated Approach to Determine Mutations in Lung Adenocarcinoma.

Am J Pathol 2021 07 15;191(7):1269-1280. Epub 2021 May 15.

Center for Protein Diagnostics, Biospectroscopy, Germany; Department of Biophysics, Faculty of Biology and Biotechnology, Ruhr University Bochum, Bochum, Germany. Electronic address:

Therapeutic decisions in lung cancer critically depend on the determination of histologic types and oncogene mutations. Therefore, tumor samples are subjected to standard histologic and immunohistochemical analyses and examined for relevant mutations using comprehensive molecular diagnostics. In this study, an alternative diagnostic approach for automatic and label-free detection of mutations in lung adenocarcinoma tissue using quantum cascade laser-based infrared imaging is presented. For this purpose, a five-step supervised classification algorithm was developed, which was not only able to detect tissue types and tumor lesions, but also the tumor type and mutation status of adenocarcinomas. Tumor detection was verified on a data set of 214 patient samples with a specificity of 97% and a sensitivity of 95%. Furthermore, histology typing was verified on samples from 203 of the 214 patients with a specificity of 97% and a sensitivity of 94% for adenocarcinoma. The most frequently occurring mutations in adenocarcinoma (KRAS, EGFR, and TP53) were differentiated by this technique. Detection of mutations was verified in 60 patient samples from the data set with a sensitivity and specificity of 95% for each mutation. This demonstrates that quantum cascade laser infrared imaging can be used to analyze morphologic differences as well as molecular changes. Therefore, this single, one-step measurement provides comprehensive diagnostics of lung cancer histology types and most frequent mutations.
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http://dx.doi.org/10.1016/j.ajpath.2021.04.013DOI Listing
July 2021

Time-resolved spectroscopic and electrophysiological data reveal insights in the gating mechanism of anion channelrhodopsin.

Commun Biol 2021 05 14;4(1):578. Epub 2021 May 14.

Biospectroscopy, Center for Protein Diagnostics (PRODI), Ruhr University Bochum, Bochum, Germany.

Channelrhodopsins are widely used in optogenetic applications. High photocurrents and low current inactivation levels are desirable. Two parallel photocycles evoked by different retinal conformations cause cation-conducting channelrhodopsin-2 (CrChR2) inactivation: one with efficient conductivity; one with low conductivity. Given the longer half-life of the low conducting photocycle intermediates, which accumulate under continuous illumination, resulting in a largely reduced photocurrent. Here, we demonstrate that for channelrhodopsin-1 of the cryptophyte Guillardia theta (GtACR1), the highly conducting C = N-anti-photocycle was the sole operating cycle using time-resolved step-scan FTIR spectroscopy. The correlation between our spectroscopic measurements and previously reported electrophysiological data provides insights into molecular gating mechanisms and their role in the characteristic high photocurrents. The mechanistic importance of the central constriction site amino acid Glu-68 is also shown. We propose that canceling out the poorly conducting photocycle avoids the inactivation observed in CrChR2, and anticipate that this discovery will advance the development of optimized optogenetic tools.
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http://dx.doi.org/10.1038/s42003-021-02101-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8121809PMC
May 2021

Application of Label-Free Proteomics for Quantitative Analysis of Urothelial Carcinoma and Cystitis Tissue.

Methods Mol Biol 2021 ;2228:283-292

Medizinisches Proteom-Center (MPC) Medical Faculty, Ruhr-University Bochum, Bochum, Germany.

A label-free approach based on a highly reproducible and stable workflow allows for quantitative proteome analysis . Due to advantages compared to labeling methods, the label-free approach has the potential to measure unlimited samples from clinical specimen monitoring and comparing thousands of proteins. The presented label-free workflow includes a new sample preparation technique depending on automatic annotation and tissue isolation via FTIR-guided laser microdissection, in-solution digestion, LC-MS/MS analyses, data evaluation by means of Proteome Discoverer and Progenesis software, and verification of differential proteins. We successfully applied this workflow in a proteomics study analyzing human cystitis and high-grade urothelial carcinoma tissue regarding the identification of a diagnostic tissue biomarker. The differential analysis of only 1 mm of isolated tissue cells led to 74 significantly differentially abundant proteins.
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http://dx.doi.org/10.1007/978-1-0716-1024-4_20DOI Listing
June 2021

Genetic predisposition, Aβ misfolding in blood plasma, and Alzheimer's disease.

Transl Psychiatry 2021 05 1;11(1):261. Epub 2021 May 1.

Network Aging Research, Heidelberg University, Heidelberg, Germany.

Alzheimer's disease is highly heritable and characterized by amyloid plaques and tau tangles in the brain. The aim of this study was to investigate the association between genetic predisposition, Aβ misfolding in blood plasma, a unique marker of Alzheimer associated neuropathological changes, and Alzheimer's disease occurrence within 14 years. Within a German community-based cohort, two polygenic risk scores (clinical Alzheimer's disease and Aβ based) were calculated, APOE genotype was determined, and Aβ misfolding in blood plasma was measured by immuno-infrared sensor in 59 participants diagnosed with Alzheimer's disease during 14 years of follow-up and 581 participants without dementia diagnosis. Associations between each genetic marker and Aβ misfolding were assessed through logistic regression and the ability of each genetic marker and Aβ misfolding to predict Alzheimer's disease was determined. The Alzheimer's disease polygenic risk score and APOE ε4 presence were associated to Aβ misfolding (odds ratio, 95% confidence interval: per standard deviation increase of score: 1.25, 1.03-1.51; APOE ε4 presence: 1.61, 1.04-2.49). No association was evident for the Aβ polygenic risk score. All genetic markers were predictive of Alzheimer's disease diagnosis albeit much less so than Aβ misfolding (areas under the curve: Aβ polygenic risk score: 0.55; AD polygenic risk score: 0.59; APOE ε4: 0.63; Aβ misfolding: 0.84). Clinical Alzheimer's genetic risk was associated to early pathological changes (Aβ misfolding) measured in blood, however, predicted Alzheimer's disease less accurately than Aβ misfolding itself. Genetic predisposition may provide information regarding disease initiation, while Aβ misfolding could be important in clinical risk prediction.
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http://dx.doi.org/10.1038/s41398-021-01380-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8088439PMC
May 2021

Microsecond-Resolved Infrared Spectroscopy on Nonrepetitive Protein Reactions by Applying Caged Compounds and Quantum Cascade Laser Frequency Combs.

Anal Chem 2021 05 21;93(17):6779-6783. Epub 2021 Apr 21.

Competence Center for Biospectroscopy, Center for Protein Diagnostics (ProDi), Ruhr University Bochum, 44801 Bochum, Germany.

Infrared spectroscopy is ideally suited for the investigation of protein reactions at the atomic level. Many systems were investigated successfully by applying Fourier transform infrared (FTIR) spectroscopy. While rapid-scan FTIR spectroscopy is limited by time resolution (about 10 ms with 16 cm resolution), step-scan FTIR spectroscopy reaches a time resolution of about 10 ns but is limited to cyclic reactions that can be repeated hundreds of times under identical conditions. Consequently, FTIR with high time resolution was only possible with photoactivable proteins that undergo a photocycle. The huge number of nonrepetitive reactions, e.g., induced by caged compounds, were limited to the millisecond time domain. The advent of dual-comb quantum cascade laser now allows for a rapid reaction monitoring in the microsecond time domain. Here, we investigate the potential to apply such an instrument to the huge class of G-proteins. We compare caged-compound-induced reactions monitored by FTIR and dual-comb spectroscopy by applying the new technique to the α subunit of the inhibiting G protein and to the larger protein-protein complex of Gα with its cognate regulator of G-protein signaling (RGS). We observe good data quality with a 4 μs time resolution with a wavelength resolution comparable to FTIR. This is more than three orders of magnitude faster than any FTIR measurement on G-proteins in the literature. This study paves the way for infrared spectroscopic studies in the so far unresolvable microsecond time regime for nonrepetitive biological systems including all GTPases and ATPases.
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http://dx.doi.org/10.1021/acs.analchem.1c00666DOI Listing
May 2021

Aβ misfolding in blood plasma measured by immuno-infrared-sensor as an age-independent risk marker of Alzheimer's disease.

Alzheimers Dement (Amst) 2021 10;13(1):e12151. Epub 2021 Feb 10.

Division of Clinical Epidemiology and Aging Research German Cancer Research Center Heidelberg Germany.

Introduction: Determining potential risk factors of amyloid beta (Aβ) misfolding in blood, a risk marker for clinical Alzheimer's disease (AD), could have important implications for its utility in future research and clinical settings.

Methods: Participants aged 50 to 75 years attending a general health examination were recruited for a prospective community-based cohort study in Saarland, Germany, in 2000 to 2002. For these analyses, participants with available Aβ misfolding measurements and clinical AD information at 17-year follow-up were included ( = 444).

Results: Age did not show any association with Aβ misfolding in plasma; however, a strong association of both age and Aβ misfolding with the incidence of clinical AD was evident. Education and cardiovascular diseases were likewise not associated with Aβ misfolding.

Discussion: Structural measurement of Aβ misfolding in blood plasma is an age-independent risk marker of clinical AD among older adults, supporting that risk of clinical AD is already largely determined before older adulthood.
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http://dx.doi.org/10.1002/dad2.12151DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7875190PMC
February 2021

Amyloid-β misfolding as a plasma biomarker indicates risk for future clinical Alzheimer's disease in individuals with subjective cognitive decline.

Alzheimers Res Ther 2020 12 24;12(1):169. Epub 2020 Dec 24.

Competence Center for Biospectroscopy, Center for Protein Diagnostics (PRODI), Ruhr-University Bochum, Bochum, Germany.

Background: We evaluated Aβ misfolding in combination with Aβ ratio as a prognostic tool for future clinical progression to mild cognitive impairment (MCI) or dementia due to Alzheimer's disease (AD) in individuals with subjective cognitive decline (SCD).

Methods: Baseline plasma samples (n = 203) from SCD subjects in the SCIENCe project and Amsterdam Dementia Cohort (age 61 ± 9 years; 57% male, mean follow-up time 2.7 years) were analyzed using immuno-infrared-sensor technology. Within 6 years of follow-up, 22 (11%) individuals progressed to MCI or dementia due to AD. Sensor readout values > 1646 cm reflected normal Aβ folding; readouts at ≤ 1646 cm reflected low and at < 1644 cm high misfolding. We used Cox proportional hazard models to quantify Aβ misfolding as a prognostic biomarker for progression to MCI and dementia due to AD. The accuracy of the predicted development of MCI/AD was determined by time-dependent receiver operating characteristic (t-ROC) curve analyses that take individual follow-up and conversion times into account. Statistical models were adjusted for age, sex, and APOEε4 status. Additionally, plasma Aβ data measured by SIMOA were statistically analyzed and compared.

Results: All 22 patients who converted to MCI or AD-dementia within 6 years exhibited Aβ misfolding at baseline. Cox analyses revealed a hazard ratio (HR) of 19 (95% confidence interval [CI] 2.2-157.8) for future conversion of SCD subjects with high misfolding and of 11 (95% CI 1.0-110.1) for those with low misfolding. T-ROC curve analyses yielded an area under the curve (AUC) of 0.94 (95% CI 0.86-1.00; 6-year follow-up) for Aβ misfolding in an age, sex, and APOEε4 model. A similar model with plasma Aβ ratio yielded an AUC of 0.92 (95% CI, 0.82-1.00). The AUC increased to 0.99 (95% CI, 0.99-1.00) after inclusion of both Aβ misfolding and the Aβ ratio.

Conclusions: A panel of structure- and concentration-based plasma amyloid biomarkers may predict conversion to clinical MCI and dementia due to AD in cognitively unimpaired subjects. These plasma biomarkers provide a noninvasive and cost-effective alternative for screening early AD pathological changes. Follow-up studies and external validation in larger cohorts are in progress for further validation of our findings.
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http://dx.doi.org/10.1186/s13195-020-00738-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7761044PMC
December 2020

Label-free vibrational imaging of different Aβ plaque types in Alzheimer's disease reveals sequential events in plaque development.

Acta Neuropathol Commun 2020 12 11;8(1):222. Epub 2020 Dec 11.

Division of Biospectroscopy, Center for Protein Diagnostics (PRODI), Ruhr University Bochum, Bochum, Germany.

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.
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http://dx.doi.org/10.1186/s40478-020-01091-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7733282PMC
December 2020

A representation learning approach for recovering scatter-corrected spectra from Fourier-transform infrared spectra of tissue samples.

J Biophotonics 2021 03 27;14(3):e202000385. Epub 2020 Dec 27.

Center for Protein Diagnostics, Ruhr-University Bochum, Gesundheitscampus 4, Bochum, Germany.

Infrared spectra obtained from cell or tissue specimen have commonly been observed to involve a significant degree of scattering effects, often Mie scattering, which probably overshadows biochemically relevant spectral information by a nonlinear, nonadditive spectral component in Fourier transform infrared (FTIR) spectroscopic measurements. Correspondingly, many successful machine learning approaches for FTIR spectra have relied on preprocessing procedures that computationally remove the scattering components from an infrared spectrum. We propose an approach to approximate this complex preprocessing function using deep neural networks. As we demonstrate, the resulting model is not just several orders of magnitudes faster, which is important for real-time clinical applications, but also generalizes strongly across different tissue types. Using Bayesian machine learning approaches, our approach unveils model uncertainty that coincides with a band shift in the amide I region that occurs when scattering is removed computationally based on an established physical model. Furthermore, our proposed method overcomes the trade-off between computation time and the corrected spectrum being biased towards an artificial reference spectrum.
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http://dx.doi.org/10.1002/jbio.202000385DOI Listing
March 2021

TDP-43 as structure-based biomarker in amyotrophic lateral sclerosis.

Ann Clin Transl Neurol 2021 01 2;8(1):271-277. Epub 2020 Dec 2.

Center for Protein Diagnostics (ProDi), Ruhr University Bochum, Bochum, Germany.

Pathologic alterations of Transactivation response DNA-binding protein 43 kilo Dalton (TDP-43) are a major hallmark of amyotrophic lateral sclerosis (ALS). In this pilot study, we analyzed the secondary structure distribution of TDP-43 in cerebrospinal fluid of ALS patients (n = 36) compared to Parkinson´s disease patients (PD; n = 30) and further controls (Ctrl; n = 24) using the immuno-infrared sensor technology. ALS patients could be discriminated from PD and Ctrl with a sensitivity/specificity of 89 %/77 % and 89 %/83 %, respectively. Our findings demonstrate that TDP-43 misfolding measured by the immuno-infrared sensor technology has the potential to serve as a biomarker candidate for ALS.
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http://dx.doi.org/10.1002/acn3.51256DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7818221PMC
January 2021

Comparability of Raman Spectroscopic Configurations: A Large Scale Cross-Laboratory Study.

Anal Chem 2020 12 21;92(24):15745-15756. Epub 2020 Nov 21.

Mass Spectrometry Laboratory, MolSys Research Unit, University of Liege, Place du 20 Aoǔt 7, 4000 Liège, Belgium.

The variable configuration of Raman spectroscopic platforms is one of the major obstacles in establishing Raman spectroscopy as a valuable physicochemical method within real-world scenarios such as clinical diagnostics. For such real world applications like diagnostic classification, the models should ideally be usable to predict data from different setups. Whether it is done by training a rugged model with data from many setups or by a primary-replica strategy where models are developed on a 'primary' setup and the test data are generated on 'replicate' setups, this is only possible if the Raman spectra from different setups are consistent, reproducible, and comparable. However, Raman spectra can be highly sensitive to the measurement conditions, and they change from setup to setup even if the same samples are measured. Although increasingly recognized as an issue, the dependence of the Raman spectra on the instrumental configuration is far from being fully understood and great effort is needed to address the resulting spectral variations and to correct for them. To make the severity of the situation clear, we present a round robin experiment investigating the comparability of 35 Raman spectroscopic devices with different configurations in 15 institutes within seven European countries from the COST (European Cooperation in Science and Technology) action Raman4clinics. The experiment was developed in a fashion that allows various instrumental configurations ranging from highly confocal setups to fibre-optic based systems with different excitation wavelengths. We illustrate the spectral variations caused by the instrumental configurations from the perspectives of peak shifts, intensity variations, peak widths, and noise levels. We conclude this contribution with recommendations that may help to improve the inter-laboratory studies.
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http://dx.doi.org/10.1021/acs.analchem.0c02696DOI Listing
December 2020

The Effect of (-)-Epigallocatechin-3-Gallate on the Amyloid-β Secondary Structure.

Biophys J 2020 07 10;119(2):349-359. Epub 2020 Jun 10.

Department of Chemistry, Yale University, New Haven, Connecticut. Electronic address:

Amyloid-β (Aβ) is a macromolecular structure of great interest because its misfolding and aggregation, along with changes in the secondary structure, have been correlated with its toxicity in various neurodegenerative diseases. Small drug-like molecules can modulate the amyloid secondary structure and therefore have raised significant interest in applications to active and passive therapies targeting amyloids. In this study, we investigate the interactions of epigallocatechin-3-gallate (EGCG), found in green tea, with Aβ polypeptides, using a combination of in vitro immuno-infrared sensor measurements, docking, molecular dynamics simulations, and ab initio calculations. We find that the interactions of EGCG are dominated by only a few residues in the fibrils, including hydrophobic π-π interactions with aromatic rings of side chains and hydrophilic interactions with the backbone of Aβ, as confirmed by extended (1-μs-long) molecular dynamics simulations. Immuno-infrared sensor data are consistent with degradation of Aβ fibril induced by EGCG and inhibition of Aβ fibril and oligomer formation, as manifested by the recovery of the amide-I band of monomeric Aβ, which is red-shifted by 26 cm when compared to the amide-I band of the fibrillar form. The shift is rationalized by computations of the infrared spectra of Aβ42 model structures, suggesting that the conformational change involves interchain hydrogen bonds in the amyloid fibrils that are broken upon binding of EGCG.
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http://dx.doi.org/10.1016/j.bpj.2020.05.033DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7376235PMC
July 2020

Label-free, automated classification of microsatellite status in colorectal cancer by infrared imaging.

Sci Rep 2020 06 23;10(1):10161. Epub 2020 Jun 23.

Ruhr University Bochum, Center for Protein Diagnostics (ProDi), Biospectroscopy, Bochum, Germany.

Challenging histopathological diagnostics in cancer include microsatellite instability-high (MSI-H) colorectal cancer (CRC), which occurs in 15% of early-stage CRC and is caused by a deficiency in the mismatch repair system. The diagnosis of MSI-H cannot be reliably achieved by visual inspection of a hematoxylin and eosin stained thin section alone, but additionally requires subsequent molecular analysis. Time- and sample-intensive immunohistochemistry with subsequent fragment length analysis is used. The aim of the presented feasibility study is to test the ability of quantum cascade laser (QCL)-based infrared (IR) imaging as an alternative diagnostic tool for MSI-H in CRC. We analyzed samples from 100 patients with sporadic CRC UICC stage II and III. Forty samples were used to develop the random forest classifier and 60 samples to verify the results on an independent blinded dataset. Specifically, 100% sensitivity and 93% specificity were achieved based on the independent 30 MSI-H- and 30 microsatellite stable (MSS)-patient validation cohort. This showed that QCL-based IR imaging is able to distinguish between MSI-H and MSS for sporadic CRC - a question that goes beyond morphological features - based on the use of spatially resolved infrared spectra used as biomolecular fingerprints.
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http://dx.doi.org/10.1038/s41598-020-67052-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7311536PMC
June 2020

Grayscale representation of infrared microscopy images by extended multiplicative signal correction for registration with histological images.

J Biophotonics 2020 08 11;13(8):e201960223. Epub 2020 May 11.

Faculty of Science and Technology, Norwegian University of Life Sciences, Ås, Norway.

Fourier-transform infrared (FTIR) microspectroscopy is rounding the corner to become a label-free routine method for cancer diagnosis. In order to build infrared-spectral based classifiers, infrared images need to be registered with Hematoxylin and Eosin (H&E) stained histological images. While FTIR images have a deep spectral domain with thousands of channels carrying chemical and scatter information, the H&E images have only three color channels for each pixel and carry mainly morphological information. Therefore, image representations of infrared images are needed that match the morphological information in H&E images. In this paper, we propose a novel approach for representation of FTIR images based on extended multiplicative signal correction highlighting morphological features that showed to correlate well with morphological information in H&E images. Based on the obtained representations, we developed a strategy for global-to-local image registration for FTIR images and H&E stained histological images of parallel tissue sections.
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http://dx.doi.org/10.1002/jbio.201960223DOI Listing
August 2020

Toward noninvasive follow-up of low-risk bladder cancer - Rationale and concept of the UroFollow trial.

Urol Oncol 2020 12 18;38(12):886-895. Epub 2020 Mar 18.

Urologie 24, Nürnberg, Germany; Department of Urology, Friedrich-Alexander University, Erlangen, Germany. Electronic address:

Background: Follow-up recommendations for patients with nonmuscle invasive bladder cancer (NMIBC) are largely based upon expert opinion. A growing body of evidence suggests that current follow-up strategies for bladder cancer patients with low and intermediate risk represent overdiagnosis and may lead to overtreatment. The goal of this study is to explore the options of a noninvasive follow-up in patients with pTa G1-2/low-grade NMIBC.

Methods: The risks and options for a urine marker-guided, noninvasive follow-up of patients with pTa G1-2/low-grade NMIBC were defined and the study design for a prospective randomized trial (UroFollow) was developed based upon the current literature.

Results: The investigators postulated that follow-up of patients with pTa G1-2/low-grade NMIBC requires a high sensitivity of urinary tumor markers. However, data from prospective studies with prediagnostic urine samples are scarce, even for approved markers, and cross-sectional studies with symptomatic patients overestimate the sensitivity. So far, cell-based markers (e.g., uCyt+ and UroVysion) in urine appeared to have higher sensitivities and specificities in low-grade NMIBC than urine cytology and markers analyzing soluble tumor-associated antigens. Marker panels are more sensitive than single-marker approaches at the expense of a lower specificity. Given a prospective randomized comparison with a marker sensitivity of 80% compared to usual care with cystoscopy, the sample size calculation yielded that 62 to 185 patients under study per arm are needed depending on different recurrence rates.

Conclusions: Based upon these findings the UroFollow trial has been designed as a prospective randomized study comparing a noninvasive marker-based (UroVysion, NMP22, urine cytology, and ultrasound) follow-up with the current standard of care over a period of 3 years.
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http://dx.doi.org/10.1016/j.urolonc.2020.01.006DOI Listing
December 2020

Estimation of Protein-Ligand Unbinding Kinetics Using Non-Equilibrium Targeted Molecular Dynamics Simulations.

J Chem Inf Model 2019 12 22;59(12):5135-5147. Epub 2019 Nov 22.

Department of Biophysics , Ruhr-University Bochum , 44780 Bochum , Germany.

We here report on nonequilibrium targeted molecular dynamics simulations as a tool for the estimation of protein-ligand unbinding kinetics. Correlating simulations with experimental data from SPR kinetics measurements and X-ray crystallography on two small molecule compound libraries bound to the N-terminal domain of the chaperone Hsp90, we show that the mean nonequilibrium work computed in an ensemble of trajectories of enforced ligand unbinding is a promising predictor for ligand unbinding rates. We furthermore investigate the molecular basis determining unbinding rates within the compound libraries. We propose ligand conformational changes and protein-ligand nonbonded interactions to impact on unbinding rates. Ligands may remain longer at the protein if they exhibit strong electrostatic and/or van der Waals interactions with the target. In the case of ligands with a rigid chemical scaffold that exhibit longer residence times, transient electrostatic interactions with the protein appear to facilitate unbinding. Our results imply that understanding the unbinding pathway and the protein-ligand interactions along this path is crucial for the prediction of small molecule ligands with defined unbinding kinetics.
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http://dx.doi.org/10.1021/acs.jcim.9b00592DOI Listing
December 2019

Prediction of Alzheimer's disease diagnosis within 14 years through Aβ misfolding in blood plasma compared to APOE4 status, and other risk factors.

Alzheimers Dement 2020 02 6;16(2):283-291. Epub 2020 Jan 6.

Network Aging Research, Heidelberg University, Heidelberg, Germany.

Introduction: Alzheimer's disease (AD) has a long prodromal stage and identifying high-risk individuals is critical. We aimed to investigate the ability of Aβ misfolding in blood plasma, APOE4 status, and dementia risk factors to predict diagnosis of AD.

Methods: Within a community-based cohort, Aβ misfolding in plasma measured by immuno-infrared sensor and APOE genotype were determined at baseline in 770 participants followed over 14 years. Associations between Aβ misfolding, APOE4, and other predictors with clinical AD, vascular dementia, and mixed dementia diagnoses were assessed.

Results: Aβ misfolding was associated with a 23-fold increased odds of clinical AD diagnosis within 14 years. No association was observed with vascular dementia/mixed dementia diagnoses. APOE4-positive participants had a 2.4-fold increased odds of clinical AD diagnosis within 14 years.

Discussion: Aβ misfolding in blood plasma was a strong, specific risk prediction marker for clinical AD even many years before diagnosis in a community-based setting.
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http://dx.doi.org/10.1016/j.jalz.2019.08.189DOI Listing
February 2020

High-content screening Raman spectroscopy (HCS-RS) of panitumumab-exposed colorectal cancer cells.

Analyst 2019 Oct 18;144(20):6098-6107. Epub 2019 Sep 18.

Leibniz Institute of Photonics Technology, Albert Einstein str. 9, 07745 Jena, Germany.

Raman spectroscopy can provide the biomolecular fingerprint of a cell in a label-free manner. Although a variety of clinical and biomedical applications have been demonstrated, the method remains largely a niche technology. The two main problems are the complexity of data acquisition and the complexity of data analysis. Generally, Raman measurements are performed manually and require a substantial amount of time. This, on the other hand, frequently results in a low number of samples and hence with questionable statistical evaluation. Here, we propose an automated high content screening Raman spectroscopy (HCS-RS) platform, which can perform a series of experiments without human interaction, significantly increasing the number of measured samples and making the measurement more reliable. The automated image processing of bright field images in combination with automatic spectral acquisition of the molecular fingerprint of cells exposed to different physiological conditions enables label-free high content screening applications. The performance of the developed HCS-RS platform is demonstrated by investigating the effect of panitumumab on SW48 and SW480 colorectal cancer cells with wild-type and mutated K-RAS, respectively, in a series of concentrations. Our result indicates that the increased content of panitumumab prohibits the activation of the MAP kinase of the colorectal cancer cells with wild-type K-RAS strongly, whereas there is no significant effect on the K-RAS mutated cells. Moreover, the relative amount of the panitumumab content present in the cells is determined from the Raman spectral information, which could be beneficial for personalized patient treatment.
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http://dx.doi.org/10.1039/c9an01176eDOI Listing
October 2019

Fast and Noninvasive Diagnosis of Cervical Cancer by Coherent Anti-Stokes Raman Scattering.

Anal Chem 2019 11 17;91(21):13900-13906. Epub 2019 Sep 17.

ZyDoLab , 44137 Dortmund , Germany.

Cervical cancer is the fourth most common cancer in women worldwide, and early detection of its precancerous lesions can decrease mortality. Cytopathology, HPV testing, and histopathology are the most commonly used tools in clinical practice. However, these methods suffer from many limitations such as subjectivity, cost, and time. Therefore, there is an unmet clinical need to develop new noninvasive methods for the early detection of cervical cancer. Here, a novel noninvasive, fast, and label-free approach with high accuracy is presented using liquid-based cytology Pap smears. CARS and SHG/TPF imaging was performed at one wavenumber on the Pap smears from patients with specimens negative for intraepithelial lesions or malignancy (NILM), and low-grade (LSIL) and high-grade (HSIL) squamous intraepithelial lesions. The normal, LSIL, and HSIL cells were selected on the basis of the ratio of the nucleus to the cytoplasm and cell morphology. Raman spectral imaging of single cells from the same smears was also performed to provide integral biochemical information of cells. Deep convolutional neural networks (DCNNs) were trained independently with CARS, SHG/TPF, and Raman images, taking into account both morphotextural and spectral information. DCNNs based on CARS, SHG/TPF, or Raman images have discriminated between normal and cancerous Pap smears with 100% accuracy. These results demonstrate that CARS/SHG/TPF microscopy has a prospective use as a label-free imaging technique for the fast screening of a large number of cells in cytopathological samples.
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http://dx.doi.org/10.1021/acs.analchem.9b03395DOI Listing
November 2019

Lamprey Parapinopsin ("UVLamP"): a Bistable UV-Sensitive Optogenetic Switch for Ultrafast Control of GPCR Pathways.

Chembiochem 2020 03 30;21(5):612-617. Epub 2019 Oct 30.

Department of General Zoology and Neurobiology, Ruhr University Bochum, ND7/31, Universitätsstasse 150, 44780, Bochum, Germany.

Optogenetics uses light-sensitive proteins, so-called optogenetic tools, for highly precise spatiotemporal control of cellular states and signals. The major limitations of such tools include the overlap of excitation spectra, phototoxicity, and lack of sensitivity. The protein characterized in this study, the Japanese lamprey parapinopsin, which we named UVLamP, is a promising optogenetic tool to overcome these limitations. Using a hybrid strategy combining molecular, cellular, electrophysiological, and computational methods we elucidated a structural model of the dark state and probed the optogenetic potential of UVLamP. Interestingly, it is the first described bistable vertebrate opsin that has a charged amino acid interacting with the Schiff base in the dark state, that has no relevance for its photoreaction. UVLamP is a bistable UV-sensitive opsin that allows for precise and sustained optogenetic control of G protein-coupled receptor (GPCR) pathways and can be switched on, but more importantly also off within milliseconds via lowintensity short light pulses. UVLamP exhibits an extremely narrow excitation spectrum in the UV range allowing for sustained activation of the G pathway with a millisecond UV light pulse. Its sustained pathway activation can be switched off, surprisingly also with a millisecond blue light pulse, minimizing phototoxicity. Thus, UVLamP serves as a minimally invasive, narrow-bandwidth probe for controlling the G pathway, allowing for combinatorial use with multiple optogenetic tools or sensors. Because UVLamP activated G signals are generally inhibitory and decrease cellular activity, it has tremendous potential for health-related applications such as relieving pain, blocking seizures, and delaying neurodegeneration.
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http://dx.doi.org/10.1002/cbic.201900485DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7079062PMC
March 2020

Reversible Immuno-Infrared Sensor for the Detection of Alzheimer's Disease Related Biomarkers.

ACS Sens 2019 07 10;4(7):1851-1856. Epub 2019 Jul 10.

Department of Biophysics , Ruhr-Universität Bochum , Universitätsstrasse 150 , D-44801 Bochum , Germany.

The development of biosensors for medical purposes is a growing field. An immuno-infrared biosensor for the preclinical detection of Alzheimer's disease (AD) in body fluids was developed. The key element of this sensor is an ATR crystal with chemically modified surface to catch the biomarker out of the body fluid. So far, the immuno-infrared sensor can be used only once and requires time-consuming steps of sensor exchange, sensor cleaning, and novel surface functionalization. Here, we developed an immuno-infrared sensor providing a reusable surface and showcase its performance by the detection of the AD biomarker proteins Aβ and Tau in human cerebrospinal fluid (CSF). The sensor surface is covalently coated with the immunoglobulin binding proteins Protein A or Protein G. These were employed for noncovalent immobilization of antibodies and the subsequent immobilization and analysis of their antigens. The reversible antibody immobilization can be repeated several times with the same or different antibodies. Further, the more specific binding of the antibody via its Fc region instead of the conventional NHS coupling leads to a 3-4-fold higher antigen binding capacity of the antibody. Thus, the throughput, sensitivity, and automation capacity of the immuno-infrared biosensor are significantly increased as compared to former immuno-infrared assays. This immuno-sensor can be used with any antibody that binds to Protein A or Protein G.
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http://dx.doi.org/10.1021/acssensors.9b00631DOI Listing
July 2019

Lewy pathology in Parkinson's disease consists of crowded organelles and lipid membranes.

Nat Neurosci 2019 07 24;22(7):1099-1109. Epub 2019 Jun 24.

Roche Pharma Research and Early Development, Neuroscience, Ophthalmology, and Rare Diseases Discovery and Translational Area/Neuroscience Discovery, Roche Innovation Center Basel, Basel, Switzerland.

Parkinson's disease, the most common age-related movement disorder, is a progressive neurodegenerative disease with unclear etiology. Key neuropathological hallmarks are Lewy bodies and Lewy neurites: neuronal inclusions immunopositive for the protein α-synuclein. In-depth ultrastructural analysis of Lewy pathology is crucial to understanding pathogenesis of this disease. Using correlative light and electron microscopy and tomography on postmortem human brain tissue from Parkinson's disease brain donors, we identified α-synuclein immunopositive Lewy pathology and show a crowded environment of membranes therein, including vesicular structures and dysmorphic organelles. Filaments interspersed between the membranes and organelles were identifiable in many but not all α-synuclein inclusions. Crowding of organellar components was confirmed by stimulated emission depletion (STED)-based super-resolution microscopy, and high lipid content within α-synuclein immunopositive inclusions was corroborated by confocal imaging, Fourier-transform coherent anti-Stokes Raman scattering infrared imaging and lipidomics. Applying such correlative high-resolution imaging and biophysical approaches, we discovered an aggregated protein-lipid compartmentalization not previously described in the Parkinsons' disease brain.
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http://dx.doi.org/10.1038/s41593-019-0423-2DOI Listing
July 2019

Deep representation learning for domain adaptable classification of infrared spectral imaging data.

Bioinformatics 2020 01;36(1):287-294

Center for Protein Diagnostics (ProDi), 44801 Bochum, Germany.

Motivation: Applying infrared microscopy in the context of tissue diagnostics heavily relies on computationally preprocessing the infrared pixel spectra that constitute an infrared microscopic image. Existing approaches involve physical models, which are non-linear in nature and lead to classifiers that do not generalize well, e.g. across different types of tissue preparation. Furthermore, existing preprocessing approaches involve iterative procedures that are computationally demanding, so that computation time required for preprocessing does not keep pace with recent progress in infrared microscopes which can capture whole-slide images within minutes.

Results: We investigate the application of stacked contractive autoencoders as an unsupervised approach to preprocess infrared microscopic pixel spectra, followed by supervised fine-tuning to obtain neural networks that can reliably resolve tissue structure. To validate the robustness of the resulting classifier, we demonstrate that a network trained on embedded tissue can be transferred to classify fresh frozen tissue. The features obtained from unsupervised pretraining thus generalize across the large spectral differences between embedded and fresh frozen tissue, where under previous approaches separate classifiers had to be trained from scratch.

Availability And Implementation: Our implementation can be downloaded from https://github.com/arnrau/SCAE_IR_Spectral_Imaging.

Supplementary Information: Supplementary data are available at Bioinformatics online.
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http://dx.doi.org/10.1093/bioinformatics/btz505DOI Listing
January 2020

GTP Hydrolysis Without an Active Site Base: A Unifying Mechanism for Ras and Related GTPases.

J Am Chem Soc 2019 07 26;141(27):10684-10701. Epub 2019 Jun 26.

Department of Chemistry-BMC , Uppsala University , Box 576, S-751 23 Uppsala , Sweden.

GTP hydrolysis is a biologically crucial reaction, being involved in regulating almost all cellular processes. As a result, the enzymes that catalyze this reaction are among the most important drug targets. Despite their vital importance and decades of substantial research effort, the fundamental mechanism of enzyme-catalyzed GTP hydrolysis by GTPases remains highly controversial. Specifically, how do these regulatory proteins hydrolyze GTP without an obvious general base in the active site to activate the water molecule for nucleophilic attack? To answer this question, we perform empirical valence bond simulations of GTPase-catalyzed GTP hydrolysis, comparing solvent- and substrate-assisted pathways in three distinct GTPases, Ras, Rab, and the G subunit of a heterotrimeric G-protein, both in the presence and in the absence of the corresponding GTPase activating proteins. Our results demonstrate that a general base is not needed in the active site, as the preferred mechanism for GTP hydrolysis is a conserved solvent-assisted pathway. This pathway involves the rate-limiting nucleophilic attack of a water molecule, leading to a short-lived intermediate that tautomerizes to form HPO and GDP as the final products. Our fundamental biochemical insight into the enzymatic regulation of GTP hydrolysis not only resolves a decades-old mechanistic controversy but also has high relevance for drug discovery efforts. That is, revisiting the role of oncogenic mutants with respect to our mechanistic findings would pave the way for a new starting point to discover drugs for (so far) "undruggable" GTPases like Ras.
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http://dx.doi.org/10.1021/jacs.9b03193DOI Listing
July 2019

Comparison of the molecular properties of retinitis pigmentosa P23H and N15S amino acid replacements in rhodopsin.

PLoS One 2019 17;14(5):e0214639. Epub 2019 May 17.

Division of Biomedical Sciences, Medical School, University of Warwick, Coventry, United Kingdom.

Mutations in the RHO gene encoding for the visual pigment protein, rhodopsin, are among the most common cause of autosomal dominant retinitis pigmentosa (ADRP). Previous studies of ADRP mutations in different domains of rhodopsin have indicated that changes that lead to more instability in rhodopsin structure are responsible for more severe disease in patients. Here, we further test this hypothesis by comparing side-by-side and therefore quantitatively two RHO mutations, N15S and P23H, both located in the N-terminal intradiscal domain. The in vitro biochemical properties of these two rhodopsin proteins, expressed in stably transfected tetracycline-inducible HEK293S cells, their UV-visible absorption, their Fourier transform infrared, circular dichroism and Metarhodopsin II fluorescence spectroscopy properties were characterized. As compared to the severely impaired P23H molecular function, N15S is only slightly defective in structure and stability. We propose that the molecular basis for these structural differences lies in the greater distance of the N15 residue as compared to P23 with respect to the predicted rhodopsin folding core. As described previously for WT rhodopsin, addition of the cytoplasmic allosteric modulator chlorin e6 stabilizes especially the P23H protein, suggesting that chlorin e6 may be generally beneficial in the rescue of those ADRP rhodopsin proteins whose stability is affected by amino acid replacement.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0214639PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6524802PMC
January 2020
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