Publications by authors named "Fatih Demir"

31 Publications

MANTI: Automated Annotation of Protein N-Termini for Rapid Interpretation of N-Terminome Data Sets.

Anal Chem 2021 04 17;93(13):5596-5605. Epub 2021 Mar 17.

Central Institute for Engineering, Electronics and Analytics, ZEA-3, Forschungszentrum Jülich GmbH, 52425 Jülich, Germany.

Site-specific proteolytic processing is an important, irreversible post-translational protein modification with implications in many diseases. Enrichment of protein N-terminal peptides followed by mass spectrometry-based identification and quantification enables proteome-wide characterization of proteolytic processes and protease substrates but is challenged by the lack of specific annotation tools. A common problem is, for example, ambiguous matches of identified peptides to multiple protein entries in the databases used for identification. We developed MaxQuant Advanced N-termini Interpreter (MANTI), a standalone Perl software with an optional graphical user interface that validates and annotates N-terminal peptides identified by database searches with the popular MaxQuant software package by integrating information from multiple data sources. MANTI utilizes diverse annotation information in a multistep decision process to assign a conservative preferred protein entry for each N-terminal peptide, enabling automated classification according to the likely origin and determines significant changes in N-terminal peptide abundance. Auxiliary R scripts included in the software package summarize and visualize key aspects of the data. To showcase the utility of MANTI, we generated two large-scale TAILS N-terminome data sets from two different animal models of chemically and genetically induced kidney disease, puromycin adenonucleoside-treated rats (PAN), and heterozygous Wilms Tumor protein 1 mice (WT1). MANTI enabled rapid validation and autonomous annotation of >10 000 identified terminal peptides, revealing novel proteolytic proteoforms in 905 and 644 proteins, respectively. Quantitative analysis indicated that proteolytic activities with similar sequence specificity are involved in the pathogenesis of kidney injury and proteinuria in both models, whereas coagulation processes and complement activation were specifically induced after chemical injury.
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http://dx.doi.org/10.1021/acs.analchem.1c00310DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8027985PMC
April 2021

Host apoplastic cysteine protease activity is suppressed during the mutualistic association of Lolium perenne and Epichloë festucae.

J Exp Bot 2021 Apr;72(9):3410-3426

Institute for Plant Sciences, University of Cologne, Cologne, Germany.

Plants secrete various defence-related proteins into the apoplast, including proteases. Papain-like cysteine proteases (PLCPs) are central components of the plant immune system. To overcome plant immunity and successfully colonize their hosts, several plant pathogens secrete effector proteins inhibiting plant PLCPs. We hypothesized that not only pathogens, but also mutualistic microorganisms interfere with PLCP-meditated plant defences to maintain endophytic colonization with their hosts. Epichloë festucae forms mutualistic associations with cool season grasses and produces a range of secondary metabolites that protect the host against herbivores. In this study, we performed a genome-wide identification of Lolium perenne PLCPs, analysed their evolutionary relationship, and classified them into nine PLCP subfamilies. Using activity-based protein profiling, we identified four active PLCPs in the apoplast of L. perenne leaves that are inhibited during endophyte interactions. We characterized the L. perenne cystatin LpCys1 for its inhibitory capacity against ryegrass PLCPs. LpCys1 abundance is not altered during the mutualistic interaction and it mainly inhibits LpCP2. However, since the activity of other L. perenne PLCPs is not sensitive to LpCys1, we propose that additional inhibitors, likely of fungal origin, are involved in the suppression of apoplastic PLCPs during E. festucae infection.
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http://dx.doi.org/10.1093/jxb/erab088DOI Listing
April 2021

DeepCoroNet: A deep LSTM approach for automated detection of COVID-19 cases from chest X-ray images.

Authors:
Fatih Demir

Appl Soft Comput 2021 May 8;103:107160. Epub 2021 Feb 8.

Firat University, Technology Faculty, Electrical-Electronics Engineering Department, Elazig, Turkey.

The new coronavirus, known as COVID-19, first emerged in Wuhan, China, and since then has been transmitted to the whole world. Around 34 million people have been infected with COVID-19 virus so far, and nearly 1 million have died as a result of the virus. Resource shortages such as test kits and ventilator have arisen in many countries as the number of cases have increased beyond the control. Therefore, it has become very important to develop deep learning-based applications that automatically detect COVID-19 cases using chest X-ray images to assist specialists and radiologists in diagnosis. In this study, we propose a new approach based on deep LSTM model to automatically identify COVID-19 cases from X-ray images. Contrary to the transfer learning and deep feature extraction approaches, the deep LSTM model is an architecture, which is learned from scratch. Besides, the Sobel gradient and marker-controlled watershed segmentation operations are applied to raw images for increasing the performance of proposed model in the pre-processing stage. The experimental studies were performed on a combined public dataset constituted by gathering COVID-19, pneumonia and normal (healthy) chest X-ray images. The dataset was randomly separated into two sections as training and testing data. For training and testing, these separations were performed with the rates of 80%-20%, 70%-30% and 60%-40%, respectively. The best performance was achieved with 80% training and 20% testing rate. Moreover, the success rate was 100% for all performance criteria, which composed of accuracy, sensitivity, specificity and F-score. Consequently, the proposed model with pre-processing images ensured promising results on a small dataset compared to big data. Generally, the proposed model can significantly improve the present radiology based approaches and it can be very useful application for radiologists and specialists to help them in detection, quantity determination and tracing of COVID-19 cases throughout the pandemic.
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http://dx.doi.org/10.1016/j.asoc.2021.107160DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7868740PMC
May 2021

Abundance of metalloprotease FtsH12 modulates chloroplast development in Arabidopsis thaliana.

J Exp Bot 2021 Apr;72(9):3455-3473

Department of Chemistry, Umeå University, Umeå, Sweden.

The ATP-dependent metalloprotease FtsH12 (filamentation temperature sensitive protein H 12) has been suggested to participate in a heteromeric motor complex, driving protein translocation into the chloroplast. FtsH12 was immuno-detected in proplastids, seedlings, leaves, and roots. Expression of Myc-tagged FtsH12 under its native promotor allowed identification of FtsHi1, 2, 4, and 5, and plastidic NAD-malate dehydrogenase, five of the six interaction partners in the suggested import motor complex. Arabidopsis thaliana mutant seedlings with reduced FTSH12 abundance exhibited pale cotyledons and small, deformed chloroplasts with altered thylakoid structure. Mature plants retained these chloroplast defects, resulting in slightly variegated leaves and lower chlorophyll content. Label-free proteomics revealed strong changes in the proteome composition of FTSH12 knock-down seedlings, reflecting impaired plastid development. The composition of the translocon on the inner chloroplast membrane (TIC) protein import complex was altered, with coordinated reduction of the FtsH12-FtsHi complex subunits and accumulation of the 1 MDa TIC complex subunits TIC56, TIC214 and TIC22-III. FTSH12 overexpressor lines showed no obvious phenotype, but still displayed distinct differences in their proteome. N-terminome analyses further demonstrated normal proteolytic maturation of plastid-imported proteins irrespective of FTSH12 abundance. Together, our data suggest that FtsH12 has highest impact during seedling development; its abundance alters the plastid import machinery and impairs chloroplast development.
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http://dx.doi.org/10.1093/jxb/eraa550DOI Listing
April 2021

Comparison of the Effects of Four Treatment Techniques Commonly Used in Ureteral Stone Treatment on Patients' Daily Physical Functioning: An Observational Randomized-Controlled Study.

J Endourol 2021 Jan 21;35(1):8-13. Epub 2020 Oct 21.

Department of Urology, Erciyes University, Kayseri, Turkey.

To investigate the effect of four different techniques used in the treatment of ureteral stones on patients' daily physical functioning (PF) and quality of life (QoL). Patients who underwent ureterorenoscopy (URS)-with or without Double-J stenting (DJS)-and extracorporeal shock wave lithotripsy (SWL) were divided into four groups: Group I: SWL ( = 29), Group II: URS ( = 43), Group III: URS +4.8F DJS ( = 39), Group IV: URS +6F DJS ( = 42), and Group V: Control ( = 30). Short Form-36 (SF-36) was administered to each participant both preoperatively and 14 days after operation. Based on the SF-36 results, the changes in patients' PF and QoL were evaluated. Ureteral stone treatment was performed in 202 patients. Of these, 153 patients who underwent an effective SWL or URS procedure in the first attempt were included in the study. Success rates in the first session were 53.7% (29/54) and 83.8% (124/148) for SWL and URS, respectively ( < 0.001). All the four groups were similar with regard to age, gender, body mass index, stone size, preoperative PF, and QoL. However, although postoperative PF, role limitations due to physical health, and energy/fatigue scores were similar in Group I, III, and IV, they were significantly higher in Group II. No major complication associated with SWL or URS occurred in any patient. However, in Group 2, DJS was inserted in three (7.7%) patients in the early postoperative period (within the first 48 hours) due to renal colic attacks secondary to ureterovesical junction mucosal edema. URS without DJS seems to be the most advantageous technique in the treatment of ureteral stones in terms of daily PF and QoL. However, it should be noted that patients undergoing URS may require postoperative emergency stenting, although rarely.
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http://dx.doi.org/10.1089/end.2020.0659DOI Listing
January 2021

Structural and functional studies of legumain beta reveal isoform specific mechanisms of activation and substrate recognition.

J Biol Chem 2020 09 21;295(37):13047-13064. Epub 2020 Jul 21.

Department of Biosciences, University of Salzburg, Salzburg, Austria. Electronic address:

The vacuolar cysteine protease legumain plays important functions in seed maturation and plant programmed cell death. Because of their dual protease and ligase activity, plant legumains have become of particular biotechnological interest, for the synthesis of cyclic peptides for drug design or for protein engineering. However, the molecular mechanisms behind their dual protease and ligase activities are still poorly understood, limiting their applications. Here, we present the crystal structure of legumain isoform β (AtLEGβ) in its zymogen state. Combining structural and biochemical experiments, we show for the first time that plant legumains encode distinct, isoform-specific activation mechanisms. Whereas the autocatalytic activation of isoform γ (AtLEGγ) is controlled by the latency-conferring dimer state, the activation of the monomeric AtLEGβ is concentration independent. Additionally, in AtLEGβ the plant-characteristic two-chain intermediate state is stabilized by hydrophobic rather than ionic interactions, as in AtLEGγ, resulting in significantly different pH stability profiles. The crystal structure of AtLEGβ revealed unrestricted nonprime substrate binding pockets, consistent with the broad substrate specificity, as determined by degradomic assays. Further to its protease activity, we show that AtLEGβ exhibits a true peptide ligase activity. Whereas cleavage-dependent transpeptidase activity has been reported for other plant legumains, AtLEGβ is the first example of a plant legumain capable of linking free termini. The discovery of these isoform-specific differences will allow us to identify and rationally design efficient ligases with application in biotechnology and drug development.
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http://dx.doi.org/10.1074/jbc.RA120.014478DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7489914PMC
September 2020

Histopathological and Clinical Features as Prognostic Factors of Atypical Meningiomas.

Turk Neurosurg 2020 Jun 23. Epub 2020 Jun 23.

Selcuk University, School of Medicine, Department of Pathology, Konya, Turkey.

Aim: To analyze the correlation of clinicopathologic prognostic parameters with atypical meningiomas (AMs) and recurrence development as well as progression-free survival (PFS).

Material And Methods: The neuropathology archive and hospital records of 75 patients with AM who underwent surgery in our institution between 2010 and 2019 were retrospectively reviewed. The pathological revision was performed according to the 2016 World Health Organization (WHO) criteria. Other clinicopathological parameters, such as age, gender, tumor location, preoperative tumor size, degree of resection, Psammoma body, nuclear atypia, main histological pattern, Ki67 labeling index (LI), radiotherapy, and dura and bone invasion, were also analyzed. Statistically, univariate and multivariate analyses were assessed to determine their potential impact on recurrence-related prognostic factors.

Results: Recurrence occurred in 20 patients. The mean PFS and follow-up time were 38.9 and 44.8 months, respectively. In univariate analysis, clinical and pathological features such as age of ≤55 years, female sex, skull base tumor location, larger preoperative tumor size, increased mitotic count, small cells, hypercellularity, sheeting, necrosis, and dura and bone invasion were remarkable in patients with recurrence, but were not statistically significant. In multivariate analysis, increased mitotic activity and brain invasion either considered alone or combined were significantly associated with PFS. Nuclear atypia was also not associated with both tumor recurrence and PFS. However, clinical features did not significantly influence the PFS.

Conclusion: This study found that recurrence could not be predicted by the presence of any of the clinicopathological features of AMs. We believe that molecular variables determined through routine neuropathological analysis will be needed in the future.
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http://dx.doi.org/10.5137/1019-5149.JTN.31161-20.1DOI Listing
June 2020

The Mouse Heart Mitochondria N Terminome Provides Insights into ClpXP-Mediated Proteolysis.

Mol Cell Proteomics 2020 08 28;19(8):1330-1345. Epub 2020 May 28.

Cologne Excellence Cluster on Cellular Stress Responses in Aging Associated Diseases (CECAD), Cologne, Germany, Medical Faculty and University Hospital, University of Cologne, Cologne, Germany; Central Institute for Engineering, Electronics and Analytics, ZEA-3, Forschungszentrum Jülich, Germany; Institute for Biochemistry, Faculty of Mathematics and Natural Sciences, University of Cologne, Cologne, Germany. Electronic address:

The mammalian mitochondrial proteome consists of more than 1100 annotated proteins and their proteostasis is regulated by only a few ATP-dependent protease complexes. Technical advances in protein mass spectrometry allowed for detailed description of the mitoproteome from different species and tissues and their changes under specific conditions. However, protease-substrate relations within mitochondria are still poorly understood. Here, we combined Terminal Amine Isotope Labeling of Substrates (TAILS) N termini profiling of heart mitochondria proteomes isolated from wild type and mice with a classical substrate-trapping screen using FLAG-tagged proteolytically active and inactive CLPP variants to identify new ClpXP substrates in mammalian mitochondria. Using TAILS, we identified N termini of more than 200 mitochondrial proteins. Expected N termini confirmed sequence determinants for mitochondrial targeting signal (MTS) cleavage and subsequent N-terminal processing after import, but the majority were protease-generated neo-N termini mapping to positions within the proteins. Quantitative comparison revealed widespread changes in protein processing patterns, including both strong increases or decreases in the abundance of specific neo-N termini, as well as an overall increase in the abundance of protease-generated neo-N termini in CLPP-deficient mitochondria that indicated altered mitochondrial proteostasis. Based on the combination of altered processing patterns, protein accumulation and stabilization in CLPP-deficient mice and interaction with CLPP, we identified OAT, HSPA9 and POLDIP2 and as novel bona fide ClpXP substrates. Finally, we propose that ClpXP participates in the cooperative degradation of UQCRC1. Together, our data provide the first landscape of the heart mitochondria N terminome and give further insights into regulatory and assisted proteolysis mediated by ClpXP.
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http://dx.doi.org/10.1074/mcp.RA120.002082DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8014998PMC
August 2020

ExteNDing Proteome Coverage with Legumain as a Highly Specific Digestion Protease.

Anal Chem 2020 02 31;92(4):2961-2971. Epub 2020 Jan 31.

Central Institute for Engineering, Electronics and Analytics, ZEA-3 , Forschungszentrum Jülich , 52428 Jülich , Germany.

Bottom-up mass spectrometry-based proteomics utilizes proteolytic enzymes with well characterized specificities to generate peptides amenable for identification by high-throughput tandem mass spectrometry. Trypsin, which cuts specifically after the basic residues lysine and arginine, is the predominant enzyme used for proteome digestion, although proteases with alternative specificities are required to detect sequences that are not accessible after tryptic digest. Here, we show that the human cysteine protease legumain exhibits a strict substrate specificity for cleavage after asparagine and aspartic acid residues during in-solution digestions of proteomes extracted from , mouse embryonic fibroblast cell cultures, and leaves. Generating peptides highly complementary in sequence, yet similar in their biophysical properties, legumain (as compared to trypsin or GluC) enabled complementary proteome and protein sequence coverage. Importantly, legumain further enabled the identification and enrichment of protein N-termini not accessible in GluC- or trypsin-digested samples. Legumain cannot cleave after glycosylated Asn residues, which enabled the robust identification and orthogonal validation of N-glycosylation sites based on alternating sequential sample treatments with legumain and PNGaseF and vice versa. Taken together, we demonstrate that legumain is a practical, efficient protease for extending the proteome and sequence coverage achieved with trypsin, with unique possibilities for the characterization of post-translational modification sites.
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http://dx.doi.org/10.1021/acs.analchem.9b03604DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7075662PMC
February 2020

Convolutional neural networks based efficient approach for classification of lung diseases.

Health Inf Sci Syst 2020 Dec 23;8(1). Epub 2019 Dec 23.

2Discipline of ECE, IIITDM, Jabalpur, India.

Treatment of lung diseases, which are the third most common cause of death in the world, is of great importance in the medical field. Many studies using lung sounds recorded with stethoscope have been conducted in the literature in order to diagnose the lung diseases with artificial intelligence-compatible devices and to assist the experts in their diagnosis. In this paper, ICBHI 2017 database which includes different sample frequencies, noise and background sounds was used for the classification of lung sounds. The lung sound signals were initially converted to spectrogram images by using time-frequency method. The short time Fourier transform (STFT) method was considered as time-frequency transformation. Two deep learning based approaches were used for lung sound classification. In the first approach, a pre-trained deep convolutional neural networks (CNN) model was used for feature extraction and a support vector machine (SVM) classifier was used in classification of the lung sounds. In the second approach, the pre-trained deep CNN model was fine-tuned (transfer learning) via spectrogram images for lung sound classification. The accuracies of the proposed methods were tested by using the ten-fold cross validation. The accuracies for the first and second proposed methods were 65.5% and 63.09%, respectively. The obtained accuracies were then compared with some of the existing results and it was seen that obtained scores were better than the other results.
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http://dx.doi.org/10.1007/s13755-019-0091-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6928168PMC
December 2020

The determination of the optimum conditions upon the leaching performance of calcined magnesite.

Heliyon 2019 Nov 26;5(11):e02830. Epub 2019 Nov 26.

Department of Chemical Engineering, Ataturk University, 25240, Erzurum, Turkey.

The aim of the study is to examine the reaction of calcined magnesite in the citric acid media, and after that it is determination of optimal conditions about the leaching performance of calcined magnesite by an experimental plan of Taguchi Statistical Method with L (4). The parameters such as particle size, reaction temperature, acid concentration, solid-liquid ratio and reaction period are tested. In this work, the optimal combination of the parameters that achieves robustness against noise factors is found as: 50 °C, 45 min, -319 μm, 0.125 g/mL, and 2M, respectively. Extraction efficiency of the magnesium from calcined magnesite is 98.86 %.
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http://dx.doi.org/10.1016/j.heliyon.2019.e02830DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6888728PMC
November 2019

Sensitive Determination of Proteolytic Proteoforms in Limited Microscale Proteome Samples.

Mol Cell Proteomics 2019 11 30;18(11):2335-2347. Epub 2019 Aug 30.

Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, Canada; Michael Cuccione Childhood Cancer Research Program, BC Children's Hospital, Vancouver, Canada. Electronic address:

Protein N termini unambiguously identify truncated, alternatively translated or modified proteoforms with distinct functions and reveal perturbations in disease. Selective enrichment of N-terminal peptides is necessary to achieve proteome-wide coverage for unbiased identification of site-specific regulatory proteolytic processing and protease substrates. However, many proteolytic processes are strictly confined in time and space and therefore can only be analyzed in minute samples that provide insufficient starting material for current enrichment protocols. Here we present High-efficiency Undecanal-based N Termini EnRichment (HUNTER), a robust, sensitive and scalable method for the analysis of previously inaccessible microscale samples. HUNTER achieved identification of >1000 N termini from as little as 2 μg raw HeLa cell lysate. Broad applicability is demonstrated by the first N-terminome analysis of sorted human primary immune cells and enriched mitochondrial fractions from pediatric cancer patients, as well as protease substrate identification from individual wild type and Vacuolar Processing Enzyme-deficient mutant seedlings. We further implemented the workflow on a liquid handling system and demonstrate the feasibility of clinical degradomics by automated processing of liquid biopsies from pediatric cancer patients.
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http://dx.doi.org/10.1074/mcp.TIR119.001560DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6823850PMC
November 2019

Towards the classification of heart sounds based on convolutional deep neural network.

Health Inf Sci Syst 2019 Dec 7;7(1):16. Epub 2019 Aug 7.

3Department of Electrical and Electronics Engineering, Faculty of Engineering, Bolu Abant Izzet Baysal University, 14280 Bolu, Turkey.

Background And Objective: Heart sound contains various important quantities that help early detection of heart diseases. Many methods have been proposed so far where various signal-processing techniques have been used on heart sounds for heart disease detection.

Methods: In this paper, a methodology is introduced for heart disease detection based on heart sounds. The proposed method employs three successive stages, such as spectrogram generation, deep feature extraction, and classification. In the spectrogram generation stage, the heart sounds are converted to spectrogram images by using time-frequency transformation.

Results: The deep features are extracted from three different pre-trained convolutional neural network models such as AlexNet, VGG16, and VGG19. Support vector machine classifier is used in the third stage of the proposed method. The proposed method is evaluated on two datasets, which are taken from The Classifying Heart Sounds Challenge.

Conclusions: The obtained results are compared with some of the existing methods. The comparisons show that the proposed method outperformed.
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http://dx.doi.org/10.1007/s13755-019-0078-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6684704PMC
December 2019

Cranially migrated lumbar intervertebral disc herniations: A multicenter analysis with long-term outcome.

J Craniovertebr Junction Spine 2019 Jan-Mar;10(1):57-63

Department of Neurosurgery, School of Medicine, Firat University, Elazig, Turkey.

Objective: Risk factors of cranial migration were investigated in patients with lumbar disc herniation (LDH) that migrated in the cranial direction and the long-term outcomes are discussed in this study.

Materials And Methods: Patients who underwent surgery for LDH at four different centers between 2012 and 2017 were studied. Extraligamentous discs were located in the lateral part of the posterior longitudinal ligament (PLL) within the spinal canal of the axial plane, and subligamentous discs were located under the PLL. The extent of cranial migration was calculated as a percentage of the height of the migrated corpus. Based on the extent of cranial migration, partial hemilaminectomy or hemilaminectomy was performed at different rates in each patient and the amount of laminectomy performed was recorded. During surgery, all free fragments were attempted to be removed. The appropriate technique was decided intraoperatively, and the surgery was performed on an individual patient basis.

Results: Of 1289 patients who underwent surgery for LDH, 654 (50.73%) had caudal migration, 576 (44.68%) had migration at the level of the disc, and 59 (4.57%) had cranial migration. Analysis of 59 patients with cranial migration according to the localization of the disc fragment revealed that 31 had extraligamentous and 28 had subligamentous fragments ( = 0.024).

Conclusions: Extraligamentous intervertebral disc fragments migrate more cranially than subligamentous intervertebral fragments. The anatomy of the PLL that varies along the corpus is the main reason for the weakness of the resistance of the disc material to the dorsolateral region, direction of discrete force vectors, and orientation of the disc fragment due to torsional vertebral movements.
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http://dx.doi.org/10.4103/jcvjs.JCVJS_15_19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6469315PMC
April 2019

A fungal substrate mimicking molecule suppresses plant immunity via an inter-kingdom conserved motif.

Nat Commun 2019 04 5;10(1):1576. Epub 2019 Apr 5.

Botanical Institute and Cluster of Excellence on Plant Sciences, University of Cologne, Cologne, D-50674, Germany.

Ustilago maydis is a biotrophic fungus causing corn smut disease in maize. The secreted effector protein Pit2 is an inhibitor of papain-like cysteine proteases (PLCPs) essential for virulence. Pit2 inhibitory function relies on a conserved 14 amino acids motif (PID14). Here we show that synthetic PID14 peptides act more efficiently as PLCP inhibitors than the full-length Pit2 effector. Mass spectrometry shows processing of Pit2 by maize PLCPs, which releases an inhibitory core motif from the PID14 sequence. Mutational analysis demonstrates that two conserved residues are essential for Pit2 function. We propose that the Pit2 effector functions as a substrate mimicking molecule: Pit2 is a suitable substrate for apoplastic PLCPs and its processing releases the embedded inhibitor peptide, which in turn blocks PLCPs to modulate host immunity. Remarkably, the PID14 core motif is present in several plant associated fungi and bacteria, indicating the existence of a conserved microbial inhibitor of proteases (cMIP).
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http://dx.doi.org/10.1038/s41467-019-09472-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6450895PMC
April 2019

Sirtilins - the new old members of the vitamin K-dependent coagulation factor family.

J Thromb Haemost 2019 03 13;17(3):470-481. Epub 2019 Feb 13.

Department of Biosciences, University of Salzburg, Salzburg, Austria.

Essentials Blood coagulation is driven by vitamin K (VK)-dependent proteases. We have identified and characterized 'sirtilin' as an additional VK-dependent protease. Sirtilins emerged early in the evolution of the coagulation system of vertebrates. Ubiquitous occurrence might indicate an important functional role of sirtilins. SUMMARY: Background Vitamin K (VK)-dependent proteases are major players in blood coagulation, including both the initiation and the regulation of the cascade. Five different members of this protease family have been described, comprising the following coagulation factors: factor VII, FIX, FX, protein C (PC), and prothrombin (FII). FVII, FIX, FX and PC share a typical domain architecture, with an N-terminal γ-carboxyglutamate (Gla) domain, two epidermal growth factor-like (EGF) domains, and a C-terminal trypsin-like serine protease (SP) domain. Objectives We have identified uncharacterized proteins in snake genomes showing the typical Gla-EGF1-EGF2-SP domain architecture but relatively low sequence conservation compared to known VK-dependent proteases. On the basis of sequence analysis, we hypothesized that these proteins are functional members of the VK-dependent protease family. Methods/results Using phylogenetic analyses, we confirmed the so-called 'sirtilins' as an additional VK-dependent protease class. These proteases were found in several vertebrates, including jawless fish, cartilaginous fish, bony fish, reptiles, birds, and marsupials, but not in other mammals. The recombinant zymogen form of Thamnophis sirtalis sirtilin was produced by in vitro renaturation, and was activated with human activated FXI. The activated form of sirtilin proteolytically cleaved peptide and protein substrates, including prothrombin. Mass spectrometry-based substrate profiling of sirtilin revealed a narrower sequence specificity than those of FIX and FX. Conclusions The ubiquitous occurrence of sirtilins in many vertebrate classes might indicate an important functional role. Understanding the detailed functions of sirtilins might contribute to a deeper understanding of the evolution and function of the vertebrate coagulation system.
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http://dx.doi.org/10.1111/jth.14384DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6850207PMC
March 2019

The proteome microenvironment determines the protective effect of preconditioning in cisplatin-induced acute kidney injury.

Kidney Int 2019 02 3;95(2):333-349. Epub 2018 Dec 3.

Department II of Internal Medicine, University of Cologne, Cologne, Germany; Center for Molecular Medicine, University of Cologne, Cologne, Germany; Cologne Excellence Cluster on Cellular Stress Responses in Aging Associated Diseases (CECAD), University of Cologne, Cologne, Germany; Systems Biology of Ageing Cologne (Sybacol), University of Cologne, Cologne, Germany. Electronic address:

Acute kidney injury (AKI) leads to significant morbidity and mortality; unfortunately, strategies to prevent or treat AKI are lacking. In recent years, several preconditioning protocols have been shown to be effective in inducing organ protection in rodent models. Here, we characterized two of these interventions-caloric restriction and hypoxic preconditioning-in a mouse model of cisplatin-induced AKI and investigated the underlying mechanisms by acquisition of multi-layered omic data (transcriptome, proteome, N-degradome) and functional parameters in the same animals. Both preconditioning protocols markedly ameliorated cisplatin-induced loss of kidney function, and caloric restriction also induced lipid synthesis. Bioinformatic analysis revealed mRNA-independent proteome alterations affecting the extracellular space, mitochondria, and transporters. Interestingly, our analyses revealed a strong dissociation of protein and RNA expression after cisplatin treatment that showed a strong correlation with the degree of damage. N-degradomic analysis revealed that most posttranscriptional changes were determined by arginine-specific proteolytic processing. This included a characteristic cisplatin-activated complement signature that was prevented by preconditioning. Amyloid and acute-phase proteins within the cortical parenchyma showed a similar response. Extensive analysis of disease-associated molecular patterns suggested that transcription-independent deposition of amyloid P-component serum protein may be a key component in the microenvironmental contribution to kidney damage. This proof-of-principle study provides new insights into the pathogenesis of cisplatin-induced AKI and the molecular mechanisms underlying organ protection by correlating phenotypic and multi-layered omics data.
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http://dx.doi.org/10.1016/j.kint.2018.08.037DOI Listing
February 2019

Low Level Texture Features for Snore Sound Discrimination.

Annu Int Conf IEEE Eng Med Biol Soc 2018 Jul;2018:413-416

Snoring is often associated with serious health risks such as obstructive sleep apnea and heart disease and may require targeted surgical interventions. In this regard, research into automatically and unobtrusively analysing the site of blockages that cause snore sounds is growing in popularity. Herein, we investigate the use of low level image texture features in classification of four specific types of snore sounds. Specifically, we explore histogram of local binary patterns (LBP) in dense grid of rectangular regions and histogram of oriented gradients (HOG) extracted from colour spectrograms for snore sound characterisation. Support vector machines with homogeneous mapping are used in the classification stage of the proposed method. Various experimental works are carried out with both LBP and HOG descriptors on the INTERSPEECH ComParE 2017 snoring sub-challenge dataset. Results presented indicate that LBP descriptors are better than the HOG descriptors in snore type detection and fusion of the LBP and HOG descriptors produces stronger results than either individual descriptor. Further, when compared to the challenge baseline and state-of-the-art deep spectrum features, our approach achieved relative percentage increases in unweighted average recall of 23.1% and 8.3% respectively.
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http://dx.doi.org/10.1109/EMBC.2018.8512459DOI Listing
July 2018

Heterologous expression and characterization of a novel serine protease from Daphnia magna: A possible role in susceptibility to toxic cyanobacteria.

Aquat Toxicol 2018 Dec 26;205:140-147. Epub 2018 Sep 26.

Department of Biochemistry, University of Cologne, 50674 Cologne, Germany.

Mass developments of toxin-producing cyanobacteria are frequently observed in freshwater ecosystems due to eutrophication and global warming. These mass developments can partly be attributed to cyanobacterial toxins, such as protease inhibitors (PIs), which inhibit digestive serine proteases of Daphnia, the major herbivore of phytoplankton and cyanobacteria. To date, mechanisms of this inhibition in the gut of the crustacean Daphnia magna are not known. Here, we characterize a single serine protease, chymotrypsin 448 (CT448), which is present in the gut of the crustacean D. magna. Sequence alignments with human serine proteases revealed that CT448 has a putative N-terminal pro-peptide which is extended compared to the mammalian homologs and within this pro-peptide two N-linked glycosylation motifs were found. CT448 was heterologously expressed in Sf21 insect cells using a baculovirus expression system for optimized protein production and secretion into the medium. The protein was purified via a one-step affinity chromatography, which resulted in a protein yield of 3.45 mg/l medium. The inactive precursor (zymogen) could be activated by tryptic digestion. This is the first example of a recombinant expression of an active crustacean serine protease, which functions in the gut of Daphnia. Proteomic identification of protease cleavage sites (PICS) and hydrolysation of various synthetic substrates showed that CT448 is a chymotrypsin-like elastase. In this study, we confirm that CT448 is a target of cyanobacterial protease inhibitors. Local evolutionary modifications of CT448 might render this proteolytic enzyme less susceptible against cyanobacterial secondary metabolites and might improve the fitness of Daphnia during cyanobacterial blooms.
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http://dx.doi.org/10.1016/j.aquatox.2018.09.013DOI Listing
December 2018

Benign metastasising leiomyoma with lymph node metastases in a patient with endometrial cancer after caesarean section.

J Obstet Gynaecol 2019 Apr 26;39(3):420-421. Epub 2018 Sep 26.

a Department of Obstetrics and Gynecology, Division of Gynecologic Oncology , Selcuk University Medical School , Konya , Turkey.

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http://dx.doi.org/10.1080/01443615.2018.1468743DOI Listing
April 2019

Human Autoantibodies against the AMPA Receptor Subunit GluA2 Induce Receptor Reorganization and Memory Dysfunction.

Neuron 2018 10 23;100(1):91-105.e9. Epub 2018 Aug 23.

Hans-Berger Department of Neurology, Jena University Hospital, Am Klinikum 1, 07747 Jena, Germany; Center for Sepsis Control and Care (CSCC), Jena University Hospital, Am Klinikum 1, 07747 Jena, Germany. Electronic address:

AMPA receptors are essential for fast excitatory transmission in the CNS. Autoantibodies to AMPA receptors have been identified in humans with autoimmune encephalitis and severe defects of hippocampal function. Here, combining electrophysiology and high-resolution imaging with neuronal culture preparations and passive-transfer models in wild-type and GluA1-knockout mice, we analyze how specific human autoantibodies against the AMPA receptor subunit GluA2 affect receptor function and composition, synaptic transmission, and plasticity. Anti-GluA2 antibodies induce receptor internalization and a reduction of synaptic GluA2-containing AMPARs followed by compensatory ryanodine receptor-dependent incorporation of synaptic non-GluA2 AMPARs. Furthermore, application of human pathogenic anti-GluA2 antibodies to mice impairs long-term synaptic plasticity in vitro and affects learning and memory in vivo. Our results identify a specific immune-neuronal rearrangement of AMPA receptor subunits, providing a framework to explain disease symptoms.
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http://dx.doi.org/10.1016/j.neuron.2018.07.048DOI Listing
October 2018

Calcified fibrous pseudotumor with Castleman disease.

Autops Case Rep 2018 Jul-Sep;8(3):e2018033. Epub 2018 Jul 30.

Selçuk University, Faculty of Medicine, Department of Pediatric Hematology and Oncology. Konya, Turkey.

Simultaneous calcified fibrous pseudotumor (CFT) and Castleman disease (CD) is an extremely rare association. CD is an uncommon lymphoproliferative disease that can arise in various sites of the body, while CFT is a rare type of benign fibrous lesion that frequently affects children and young adults, occurring as solitary or multiple lesions throughout the human body. Both entities are rare and exhibit typical and diverse histomorphological features. We report the case of a 15-year-old female patient, who, at the age of 13 had a biopsy performed at an external medical center; however, after 4 months the lesion had regrown. This lesion was removed with a surgical operation; however, it regrew 2 years later and was removed a third time. The results of the latter two biopsies were the same: CFT accompanying CD. The histologic examination of the excised lymph node and the surrounding tissue showed hyalinized fibrous tissue containing dystrophic and psammomatous calcification. In this case, the hyaline vascular type of CD was found to be intertwined with a CFT, which hampered the differentiation of whether both entities emerged within the lymph node or if the CFT developed from the soft tissue and then involved the lymph node. Future studies involving larger case series will provide a more precise insight, which should serve to resolve the current uncertainty.
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http://dx.doi.org/10.4322/acr.2018.033DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6066268PMC
July 2018

NMDAR encephalitis: passive transfer from man to mouse by a recombinant antibody.

Ann Clin Transl Neurol 2017 11 3;4(11):768-783. Epub 2017 Oct 3.

Department of Neurology Medical Faculty Heinrich Heine University Düsseldorf Düsseldorf Germany.

Objective: Autoimmune encephalitis is most frequently associated with anti-NMDAR autoantibodies. Their pathogenic relevance has been suggested by passive transfer of patients' cerebrospinal fluid (CSF) in mice in vivo. We aimed to analyze the intrathecal plasma cell repertoire, identify autoantibody-producing clones, and characterize their antibody signatures in recombinant form.

Methods: Patients with recent onset typical anti-NMDAR encephalitis were subjected to flow cytometry analysis of the peripheral and intrathecal immune response before, during, and after immunotherapy. Recombinant human monoclonal antibodies (rhuMab) were cloned and expressed from matching immunoglobulin heavy- (IgH) and light-chain (IgL) amplicons of clonally expanded intrathecal plasma cells (cePc) and tested for their pathogenic relevance.

Results: Intrathecal accumulation of B and plasma cells corresponded to the clinical course. The presence of cePc with hypermutated antigen receptors indicated an antigen-driven intrathecal immune response. Consistently, a single recombinant human GluN1-specific monoclonal antibody, rebuilt from intrathecal cePc, was sufficient to reproduce NMDAR epitope specificity in vitro. After intraventricular infusion in mice, it accumulated in the hippocampus, decreased synaptic NMDAR density, and caused severe reversible memory impairment, a key pathogenic feature of the human disease, in vivo.

Interpretation: A CNS-specific humoral immune response is present in anti-NMDAR encephalitis specifically targeting the GluN1 subunit of the NMDAR. Using reverse genetics, we recovered the typical intrathecal antibody signature in recombinant form, and proved its pathogenic relevance by passive transfer of disease symptoms from man to mouse, providing the critical link between intrathecal immune response and the pathogenesis of anti-NMDAR encephalitis as a humorally mediated autoimmune disease.
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http://dx.doi.org/10.1002/acn3.444DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5682115PMC
November 2017

N-Degradomic Analysis Reveals a Proteolytic Network Processing the Podocyte Cytoskeleton.

J Am Soc Nephrol 2017 Oct 19;28(10):2867-2878. Epub 2017 Jul 19.

Department II of Internal Medicine,

Regulated intracellular proteostasis, controlled in part by proteolysis, is essential in maintaining the integrity of podocytes and the glomerular filtration barrier of the kidney. We applied a novel proteomics technology that enables proteome-wide identification, mapping, and quantification of protein N-termini to comprehensively characterize cleaved podocyte proteins in the glomerulus We found evidence that defined proteolytic cleavage results in various proteoforms of important podocyte proteins, including those of podocin, nephrin, neph1, -actinin-4, and vimentin. Quantitative mapping of N-termini demonstrated perturbation of protease action during podocyte injury , including diminished proteolysis of -actinin-4. Differentially regulated protease substrates comprised cytoskeletal proteins as well as intermediate filaments. Determination of preferential protease motifs during podocyte damage indicated activation of caspase proteases and inhibition of arginine-specific proteases. Several proteolytic processes were clearly site-specific, were conserved across species, and could be confirmed by differential migration behavior of protein fragments in gel electrophoresis. Some of the proteolytic changes discovered also occurred in two models of podocyte damage (WT1 heterozygous knockout mice and puromycin aminonucleoside-treated rats). Thus, we provide direct and systems-level evidence that the slit diaphragm and podocyte cytoskeleton are regulated targets of proteolytic modification, which is altered upon podocyte damage.
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http://dx.doi.org/10.1681/ASN.2016101119DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5619959PMC
October 2017

Quantitative proteomics in plant protease substrate identification.

New Phytol 2018 05 11;218(3):936-943. Epub 2017 May 11.

ZEA-3 Analytics, Central Institute for Engineering, Electronics and Analytics, Forschungszentrum Jülich, Wilhelm-Johnen-Str., Jülich, 52425, Germany.

Contents Summary 936 I. Introduction 936 II. The quest for plant protease substrates - proteomics to the rescue? 937 III. Quantitative proteome comparison reveals candidate substrates 938 IV. Dynamic metabolic stable isotope labeling to measure protein turnover in vivo 938 V. Terminomics - large-scale identification of protease cleavage sites 939 VI. Substrate or not substrate, that is the question 940 VII. Concluding remarks 941 Acknowledgements 941 References 941 SUMMARY: Proteolysis is a central regulatory mechanism of protein homeostasis and protein function that affects all aspects of plant life. Higher plants encode for hundreds of proteases, but their physiological substrates and hence their molecular functions remain mostly unknown. Current quantitative mass spectrometry-based proteomics enables unbiased large-scale interrogation of the proteome and its modifications. Here we provide an overview of proteomics techniques that allow profiling of changes in protein abundance, measurement of proteome turnover rates, identification of protease cleavage sites in vivo and in vitro and determination of protease sequence specificity. We discuss how these techniques can help to reveal protease substrates and determine plant protease function, illustrated by recent studies on selected plant proteases.
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http://dx.doi.org/10.1111/nph.14587DOI Listing
May 2018

Profiling of Protein N-Termini and Their Modifications in Complex Samples.

Methods Mol Biol 2017 ;1574:35-50

Central Institute for Engineering, Electronics and Analytics, ZEA-3, Forschungszentrum Jülich, Wilhelm-Johnen-Str, 52425, Jülich, Germany.

Protein N termini are a unique window to the functional state of the proteome, revealing translation initiation sites, co-translation truncation and modification, posttranslational maturation, and further proteolytic processing into different proteoforms with distinct functions. As a direct readout of proteolytic activity, protein N termini further reveal proteolytic regulation of diverse biological processes and provide a route to determine specific substrates and hence the physiological functions for any protease of interest. Here, we describe our current protocol of the successful Terminal Amine Isotope Labeling of Substrates (TAILS) technique, which enriches protein N-terminal peptides from complex proteome samples by negative selection. Genome-encoded N termini, protease-generated neo-N termini, and endogenously modified N termini are all enriched simultaneously. Subsequent mass spectrometric analysis therefore profiles all protein N termini and their modifications present in a complex sample in a single experiment. We further provide a detailed protocol for the TAILS-compatible proteome preparation from plant material and discuss specific considerations for N terminome data analysis and annotation.
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http://dx.doi.org/10.1007/978-1-4939-6850-3_4DOI Listing
February 2018

Arabidopsis nanodomain-delimited ABA signaling pathway regulates the anion channel SLAH3.

Proc Natl Acad Sci U S A 2013 May 29;110(20):8296-301. Epub 2013 Apr 29.

Institute for Molecular Plant Physiology and Biophysics, University of Wuerzburg, 97082 Wuerzburg, Germany.

The phytohormone abscisic acid (ABA) plays a key role in the plant response to drought stress. Hence, ABA-dependent gene transcription and ion transport is regulated by a variety of protein kinases and phosphatases. However, the nature of the membrane-delimited ABA signal transduction steps remains largely unknown. To gain insight into plasma membrane-bound ABA signaling, we identified sterol-dependent proteins associated with detergent resistant membranes from Arabidopsis thaliana mesophyll cells. Among those, we detected the central ABA signaling phosphatase ABI1 (abscisic-acid insensitive 1) and the calcium-dependent protein kinase 21 (CPK21). Using fluorescence microscopy, we found these proteins to localize in membrane nanodomains, as observed by colocalization with the nanodomain marker remorin Arabidopsis thaliana remorin 1.3 (AtRem 1.3). After transient coexpression, CPK21 interacted with SLAH3 [slow anion channel 1 (SLAC1) homolog 3] and activated this anion channel. Upon CPK21 stimulation, SLAH3 exhibited the hallmark properties of S-type anion channels. Coexpression of SLAH3/CPK21 with ABI1, however, prevented proper nanodomain localization of the SLAH3/CPK21 protein complex, and as a result anion channel activation failed. FRET studies revealed enhanced interaction of SLAH3 and CPK21 within the plasma membrane in response to ABA and thus confirmed our initial observations. Interestingly, the ABA-induced SLAH3/CPK21 interaction was modulated by ABI1 and the ABA receptor RCAR1/PYL9 [regulatory components of ABA receptor 1/PYR1 (pyrabactin resistance 1)-like protein 9]. We therefore propose that ABA signaling via inhibition of ABI1 modulates the apparent association of a signaling and transport complex within membrane domains that is necessary for phosphorylation and activation of the S-type anion channel SLAH3 by CPK21.
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http://dx.doi.org/10.1073/pnas.1211667110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3657796PMC
May 2013

Darbepoetin α ameliorates neuronal damage in a rat model of acute ethanol intoxication.

Int J Neurosci 2013 Feb 19;123(2):99-103. Epub 2012 Nov 19.

T.C. Ministry of Health Haydarpasa Numune Research and Training Hospital, Internal Medicine Clinic, Hemodialysis Unit, Istanbul, Turkey.

Objective: Acute ethanol intoxication has been shown to cause oxidative damage in many organ systems including the brain. Erythropoietin has antioxidant effects and prevents neuronal damage in the animal model of ischemic brain injury. In this study, we aimed to investigate the effects of darbepoetin alpha, an analog of erythropoietin with a longer half-life and higher in vivo activity, on ethanol-induced acute brain injury.

Methods: Forty-eight Wistar albino rats were allocated to four groups. The first group received ethanol treatment (E), the second group was treated with ethanol and darbepoetin (ED), the third group received only saline treatment (S), and the fourth group received both saline and darbepoetin treatment (SD). Plasma S100-β and neuron-specific enolase (NSE) levels were measured. Histopathological evaluation of the brains was performed.

Results: The plasma S100-β and NSE levels were significantly lower in group ED compared with group E. In group E, we have observed focal red-neuron formation at the granular layer of the dentate gyrus. We did not observe any histopathological changes in the other groups (ED, S, and SD).

Conclusion: Our findings suggest that darbepoetin alpha has neuroprotective effect in acute ethanol intoxication, possibly through its antioxidant effect.
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http://dx.doi.org/10.3109/00207454.2012.738732DOI Listing
February 2013