Proteomic investigation of neural stem cell to oligodendrocyte precursor cell differentiation reveals phosphorylation-dependent Dclk1 processing.

Oligodendrocytes are generated via a two-step mechanism from pluripotent neural stem cells (NSCs): after differentiation of NSCs to oligodendrocyte precursor/NG2 cells (OPCs), they further develop into mature oligodendrocytes. The first step of this differentiation process is only incompletely understood. In this study, we utilized the neurosphere assay to investigate NSC to OPC differentiation in a time course-dependent manner by mass spectrometry-based (phospho-) proteomics. We identify doublecortin-like kinase 1 (Dclk1) as one of the most prominently regulated proteins in both datasets, and show that it undergoes a gradual transition between its short/long isoform during NSC to OPC differentiation. This is regulated by phosphorylation of its SP-rich region, resulting in inhibition of proteolytic Dclk1 long cleavage, and therefore Dclk1 short generation. Through interactome analyses of different Dclk1 isoforms by proximity biotinylation, we characterize their individual putative interaction partners and substrates. All data are available via ProteomeXchange with identifier PXD040652.

Effect of modulating glutamate signaling on myelinating oligodendrocytes and their development-A study in the zebrafish model.

Myelination is crucial for the development and maintenance of axonal integrity, especially fast axonal action potential conduction. There is increasing evidence that glutamate signaling and release through neuronal activity modulates the myelination process. In this study, we examine the effect of manipulating glutamate signaling on myelination of oligodendrocyte (OL) lineage cells and their development in zebrafish (zf). We use the "intensity-based glutamate-sensing fluorescent reporter" (iGluSnFR) in the zf model (both sexes) to address the hypothesis that glutamate is implicated in regulation of myelinating OLs. Our results show that glial iGluSnFR expression significantly reduces OL lineage cell number and the expression of myelin markers in larvae (zfl) and adult brains. The specific glutamate receptor agonist, L-AP4, rescues this iGluSnFR effect by significantly increasing the expression of the myelin-related genes, plp1b and mbpa, and enhances myelination in L-AP4-injected zfl compared to controls. Furthermore, we demonstrate that degrading glutamate using Glutamat-Pyruvate Transaminase (GPT) or the blockade of glutamate reuptake by L-trans-pyrrolidine-2,4-dicarboxylate (PDC) significantly decreases myelin-related genes and drastically declines myelination in brain ventricle-injected zfl. Moreover, we found that myelin-specific ClaudinK (CldnK) and 36K protein expression is significantly decreased in iGluSnFR-expressing zfl and adult brains compared to controls. Taken together, this study confirms that glutamate signaling is directly required for the preservation of myelinating OLs and for the myelination process itself. These findings further suggest that glutamate signaling may provide novel targets to therapeutically boost remyelination in several demyelinating diseases of the CNS.

Pathogenic variants in GNPTAB and GNPTG encoding distinct subunits of GlcNAc-1-phosphotransferase differentially impact bone resorption in patients with mucolipidosis type II and III.

PURPOSE: Pathogenic variants in GNPTAB and GNPTG, encoding different subunits of GlcNAc-1-phosphotransferase, cause mucolipidosis (ML) II, MLIII alpha/beta, and MLIII gamma. This study aimed to investigate the cellular and molecular bases underlying skeletal abnormalities in patients with MLII and MLIII. METHODS: We analyzed bone biopsies from patients with MLIII alpha/beta or MLIII gamma by undecalcified histology and histomorphometry. The skeletal status of Gnptgko and Gnptab-deficient mice was determined and complemented by biochemical analysis of primary Gnptgko bone cells. The clinical relevance of the mouse data was underscored by systematic urinary collagen crosslinks quantification in patients with MLII, MLIII alpha/beta, and MLIII gamma. RESULTS: The analysis of iliac crest biopsies revealed that bone remodeling is impaired in patients with GNPTAB-associated MLIII alpha/beta but not with GNPTG-associated MLIII gamma. Opposed to Gnptab-deficient mice, skeletal remodeling is not affected in Gnptgko mice. Most importantly, patients with variants in GNPTAB but not in GNPTG exhibited increased bone resorption. CONCLUSION: The gene-specific impact on bone remodeling in human individuals and in mice proposes distinct molecular functions of the GlcNAc-1-phosphotransferase subunits in bone cells. We therefore appeal for the necessity to classify MLIII based on genetic in addition to clinical criteria to ensure appropriate therapy.

Lysosomal Proteome and Secretome Analysis Identifies Missorted Enzymes and Their Nondegraded Substrates in Mucolipidosis III Mouse Cells.

Targeting of soluble lysosomal enzymes requires mannose 6-phosphate (M6P) signals whose formation is initiated by the hexameric N-acetylglucosamine (GlcNAc)-1-phosphotransferase complex (α2ß2γ2). Upon proteolytic cleavage by site-1 protease, the α/ß-subunit precursor is catalytically activated but the functions of γ-subunits (Gnptg) in M6P modification of lysosomal enzymes are unknown. To investigate this, we analyzed the Gnptg expression in mouse tissues, primary cultured cells, and in Gnptg reporter mice in vivo, and found high amounts in the brain, eye, kidney, femur, vertebra and fibroblasts. Consecutively we performed comprehensive quantitative lysosomal proteome and M6P secretome analysis in fibroblasts of wild-type and Gnptgko mice mimicking the lysosomal storage disorder mucolipidosis III. Although the cleavage of the α/ß-precursor was not affected by Gnptg deficiency, the GlcNAc-1-phosphotransferase activity was significantly reduced. We purified lysosomes and identified 29 soluble lysosomal proteins by SILAC-based mass spectrometry exhibiting differential abundance in Gnptgko fibroblasts which was confirmed by Western blotting and enzymatic activity analysis for selected proteins. A subset of these lysosomal enzymes show also reduced M6P modifications, fail to reach lysosomes and are secreted, among them α-l-fucosidase and arylsulfatase B. Low levels of these enzymes correlate with the accumulation of non-degraded fucose-containing glycostructures and sulfated glycosaminoglycans in Gnptgko lysosomes. Incubation of Gnptgko fibroblasts with arylsulfatase B partially rescued glycosaminoglycan storage. Combinatorial treatments with other here identified missorted enzymes of this degradation pathway might further correct glycosaminoglycan accumulation and will provide a useful basis to reveal mechanisms of selective, Gnptg-dependent formation of M6P residues on lysosomal proteins.

Identification of Poly(ethylene glycol) and Poly(ethylene glycol)-Based Detergents Using Peptide Search Engines.

Poly(ethylene glycol) (PEG) is one of the most common polymer contaminations in mass spectrometry (MS) samples. At present, the detection of PEG and other polymers relies largely on manual inspection of raw data, which is laborious and frequently difficult due to sample complexity and retention characteristics of polymer species in reversed-phase chromatography. We developed a new strategy for the automated identification of PEG molecules from tandem mass spectrometry (MS/MS) data using protein identification algorithms in combination with a database containing "PEG-proteins". Through definition of variable modifications, we extend the approach for the identification of commonly used PEG-based detergents. We exemplify the identification of different types of polymers by static nanoelectrospray tandem mass spectrometry (nanoESI-MS/MS) analysis of pure detergent solutions and data analysis using Mascot. Analysis of liquid chromatography-tandem mass spectrometry (LC-MS/MS) runs of a PEG-contaminated sample by Mascot identified 806 PEG spectra originating from four PEG species using a defined set of modifications covering PEG and common PEG-based detergents. Further characterization of the sample for unidentified PEG species using error-tolerant and mass-tolerant searches resulted in identification of 3409 and 3187 PEG-related MS/MS spectra, respectively. We further demonstrate the applicability of the strategy for Protein Pilot and MaxQuant.

The apoptotic impact of nisin as a potent bacteriocin on the colon cancer cells.

Nisin is a polycyclic peptide containing 34 amino acids produced by Lactococcus lactis during fermentation. Recently, researchers considered nisin as an anticancer peptide. Herein, the authors aim to evaluate the nisin effects on the apoptosis stimulation in the colon cancer cell line. The SW480 cells were exposed to discrepant concentrations of nisin and the cell viability as well as the expression of bcl-2 and bax genes and proteins were surveyed by the MTT assay, Real-Time PCR and western blotting method, respectively. Furthermore, the Ethidium bromide/Acridine orange staining was performed to visualize apoptotic cells. 4000, 3000, 2500 and 2000 µg/ml of nisin led to significant anti-proliferative impact and augmentation apoptotic index (bax/bcl-2 ratio) both at mRNA and protein levels (p < 0.05). Furthermore, the apoptotic impacts were demonstrated after Ethidium bromide/Acridine orange (EB/AO) staining to have a dose dependent manner. Our outcome suggested that nisin could induce apoptosis via intrinsic pathways and lead to cancerous cell death.

Bilosomes as Nanocarriers for the Drug and Vaccine Delivery against Gastrointestinal Infections: Opportunities and Challenges.

The gastrointestinal tract (GIT) environment has an intricate and complex nature, limiting drugs' stability, oral bioavailability, and adsorption. Additionally, due to the drugs' toxicity and side effects, renders are continuously seeking novel delivery systems. Lipid-based drug delivery vesicles have shown various loading capacities and high stability levels within the GIT. Indeed, most vesicular platforms fail to efficiently deliver drugs toward this route. Notably, the stability of vesicular constructs is different based on the different ingredients added. A low GIT stability of liposomes and niosomes and a low loading capacity of exosomes in drug delivery have been described in the literature. Bilosomes are nonionic, amphiphilic, flexible surfactant vehicles that contain bile salts for the improvement of drug and vaccine delivery. The bilosomes' stability and plasticity in the GIT facilitate the efficient carriage of drugs (such as antimicrobial, antiparasitic, and antifungal drugs), vaccines, and bioactive compounds to treat infectious agents. Considering the intricate and harsh nature of the GIT, bilosomal formulations of oral substances have a remarkably enhanced delivery efficiency, overcoming these conditions. This review aimed to evaluate the potential of bilosomes as drug delivery platforms for antimicrobial, antiviral, antifungal, and antiparasitic GIT-associated drugs and vaccines.

Ranitidine induces inhibition and structural changes in sucrase.

Ranitidine is an antagonist of histamine-2 (H(2)) receptor. It is employed to treat peptic ulcer and other conditions in which gastric acidity must be reduced. Sucrase is a hydrolytic enzyme that catalyzes the breakdown of sucrose to its monomer content. A liquid of yeast sucrase was developed for treatment of congenital sucrase-isomaltase deficiency (CSID) in human. In this study, the effect of ranitidine on yeast sucrase activity was investigated. Our results showed that ranitidine binds to sucrase and inhibits the enzyme in a noncompetitive manner. The K(i) and IC(50) values were measured to be about 2.3 and 2.2 mM, respectively. Fluorescence measurement showed conformational changes after binding of ranitidine to the enzyme. The fluorescence spectra showed that ranitidine could bind to both free enzyme and enzyme-substrate complex, which was accompanied with reduction of emission intensity and red shift production.

Identification of Unexpected Protein Modifications by Mass Spectrometry-Based Proteomics.

Peptide identification relies in the majority of mass spectrometry-based proteomics experiments on matching of experimental data against peptide and fragment ion masses derived from in silico digests of protein databases. One of the main drawbacks of this approach is that modifications have to be defined for database searching and therefore no unexpected modifications can be identified in a standard setup. Consequently, in many bottom-up proteomics experiments, unexpected modifications are not identified, even if high-quality fragment ion spectra of the modified peptides were acquired. It is therefore often not straightforward to identify unexpected modifications. In this protocol, we describe a stepwise procedure to identify unexpected modifications at peptides using the database search algorithm Mascot. The workflow includes parallel searches for the identification of known modifications at unexpected amino acids, error tolerant searches for modifications unexpected in the sample but known to the community, and mass tolerant searches for entirely unknown modifications. Furthermore, we suggest a follow-up strategy consisting of (1) verification of identified modifications in the initial dataset and (2) targeted experiments using synthetic peptides.

Motor Oil Classification Using Color Histograms and Pattern Recognition Techniques.

Motor oil classification is important for quality control and the identification of oil adulteration. In this work, we propose a simple, rapid, inexpensive and nondestructive approach based on image analysis and pattern recognition techniques for the classification of nine different types of motor oils according to their corresponding color histograms. For this, we applied color histogram in different color spaces such as red green blue (RGB), grayscale, and hue saturation intensity (HSI) in order to extract features that can help with the classification procedure. These color histograms and their combinations were used as input for model development and then were statistically evaluated by using linear discriminant analysis (LDA), quadratic discriminant analysis (QDA), and support vector machine (SVM) techniques. Here, two common solutions for solving a multiclass classification problem were applied: (1) transformation to binary classification problem using a one-against-all (OAA) approach and (2) extension from binary classifiers to a single globally optimized multilabel classification model. In the OAA strategy, LDA, QDA, and SVM reached up to 97% in terms of accuracy, sensitivity, and specificity for both the training and test sets. In extension from binary case, despite good performances by the SVM classification model, QDA and LDA provided better results up to 92% for RGB-grayscale-HSI color histograms and up to 93% for the HSI color map, respectively. In order to reduce the numbers of independent variables for modeling, a principle component analysis algorithm was used. Our results suggest that the proposed method is promising for the identification and classification of different types of motor oils.

CSEO - the Cigarette Smoke Exposure Ontology.

BACKGROUND: In the past years, significant progress has been made to develop and use experimental settings for extensive data collection on tobacco smoke exposure and tobacco smoke exposure-associated diseases. Due to the growing number of such data, there is a need for domain-specific standard ontologies to facilitate the integration of tobacco exposure data. RESULTS: The CSEO (version 1.0) is composed of 20091 concepts. The ontology in its current form is able to capture a wide range of cigarette smoke exposure concepts within the knowledge domain of exposure science with a reasonable sensitivity and specificity. Moreover, it showed a promising performance when used to answer domain expert questions. The CSEO complies with standard upper-level ontologies and is freely accessible to the scientific community through a dedicated wiki at https://publicwiki-01.fraunhofer.de/CSEO-Wiki/index.php/Main_Page. CONCLUSIONS: The CSEO has potential to become a widely used standard within the academic and industrial community. Mainly because of the emerging need of systems toxicology to controlled vocabularies and also the lack of suitable ontologies for this domain, the CSEO prepares the ground for integrative systems-based research in the exposure science.

Inhibition of renal alkaline phosphatase by cimetidine.

Alkaline phosphatase (ALP) belongs to hydrolase group of enzymes. It is responsible for removing phosphate groups from many types of molecules, including nucleotides and proteins. Cimetidine (trade name Tagamet) is an antagonist of histamine H2-receptor that inhibits the production of gastric acid. Cimetidine is used for the treatment of gastrointestinal diseases. In this study the inhibitory effect of cimetidine on mouse renal ALP activity was investigated. Our results showed that cimetidine can inhibit ALP by uncompetitive inhibition. In the absence of inhibitor the V(max) and K(m) of the enzyme were found to be 13.7 mmol/mg prot.min and 0.25 mM, respectively. Both the Vmax and Km of the enzyme decreased with increasing cimetidine concentrations (0- 1.2 mM). The Ki and IC(50) of cimetidine were determined to be about 0.5 mM and 0.52 mM, respectively.