Posttranslational modifications of proteins are key features in the identification of CSF biomarkers of multiple sclerosis.

BACKGROUND: Multiple sclerosis is an inflammatory and degenerative disease of the central nervous system (CNS) characterized by demyelination and concomitant axonal loss. The lack of a single specific test, and the similarity to other inflammatory diseases of the central nervous system, makes it difficult to have a clear diagnosis of multiple sclerosis. Therefore, laboratory tests that allows a clear and definite diagnosis, as well as to predict the different clinical courses of the disease are of utmost importance. Herein, we compared the cerebrospinal fluid (CSF) proteome of patients with multiple sclerosis (in the relapse-remitting phase of the disease) and other diseases of the CNS (inflammatory and non-inflammatory) aiming at identifying reliable biomarkers of multiple sclerosis. METHODS: CSF samples from the discovery group were resolved by 2D-gel electrophoresis followed by identification of the protein spots by mass spectrometry. The results were analyzed using univariate (Student's t test) and multivariate (Hierarchical Cluster Analysis, Principal Component Analysis, Linear Discriminant Analysis) statistical and numerical techniques, to identify a set of protein spots that were differentially expressed in CSF samples from patients with multiple sclerosis when compared with other two groups. Validation of the results was performed in samples from a different set of patients using quantitative (e.g., ELISA) and semi-quantitative (e.g., Western Blot) experimental approaches. RESULTS: Analysis of the 2D-gels showed 13 protein spots that were differentially expressed in the three groups of patients: Alpha-1-antichymotrypsin, Prostaglandin-H2-isomerase, Retinol binding protein 4, Transthyretin (TTR), Apolipoprotein E, Gelsolin, Angiotensinogen, Agrin, Serum albumin, Myosin-15, Apolipoprotein B-100 and EF-hand calcium-binding domain-containing protein. ELISA experiments allowed validating part of the results obtained in the proteomics analysis and showed that some of the alterations in the CSF proteome are also mirrored in serum samples from multiple sclerosis patients. CSF of multiple sclerosis patients was characterized by TTR oligomerization, thus highlighting the importance of analyzing posttranslational modifications of the proteome in the identification of novel biomarkers of the disease. CONCLUSIONS: The model built based on the results obtained upon analysis of the 2D-gels and in the validation phase attained an accuracy of about 80% in distinguishing multiple sclerosis patients and the other two groups.

Extracellular Electrophysiological Measurements of Cooperative Signals in Astrocytes Populations.

Astrocytes are neuroglial cells that exhibit functional electrical properties sensitive to neuronal activity and capable of modulating neurotransmission. Thus, electrophysiological recordings of astroglial activity are very attractive to study the dynamics of glial signaling. This contribution reports on the use of ultra-sensitive planar electrodes combined with low noise and low frequency amplifiers that enable the detection of extracellular signals produced by primary cultures of astrocytes isolated from mouse cerebral cortex. Recorded activity is characterized by spontaneous bursts comprised of discrete signals with pronounced changes on the signal rate and amplitude. Weak and sporadic signals become synchronized and evolve with time to higher amplitude signals with a quasi-periodic behavior, revealing a cooperative signaling process. The methodology presented herewith enables the study of ionic fluctuations of population of cells, complementing the single cells observation by calcium imaging as well as by patch-clamp techniques.

Ultrasensitive gold micro-structured electrodes enabling the detection of extra-cellular long-lasting potentials in astrocytes populations.

Ultra-sensitive electrodes for extracellular recordings were fabricated and electrically characterized. A signal detection limit defined by a noise level of 0.3-0.4 µV for a bandwidth of 12.5 Hz was achieved. To obtain this high sensitivity, large area (4 mm2) electrodes were used. The electrode surface is also micro-structured with an array of gold mushroom-like shapes to further enhance the active area. In comparison with a flat gold surface, the micro-structured surface increases the capacitance of the electrode/electrolyte interface by 54%. The electrode low impedance and low noise enable the detection of weak and low frequency quasi-periodic signals produced by astrocytes populations that thus far had remained inaccessible using conventional extracellular electrodes. Signals with 5 µV in amplitude and lasting for 5-10 s were measured, with a peak-to-peak signal-to-noise ratio of 16. The electrodes and the methodology developed here can be used as an ultrasensitive electrophysiological tool to reveal the synchronization dynamics of ultra-slow ionic signalling between non-electrogenic cells.

Neck Flexor and Extensor Muscle Endurance in Subclinical Neck Pain: Intrarater Reliability, Standard Error of Measurement, Minimal Detectable Change, and Comparison With Asymptomatic Participants in a University Student Population.

OBJECTIVE: The aims of this study were to assess intrarater reliability and to calculate the standard error of measurement (SEM) and minimal detectable change (MDC) for deep neck flexor and neck extensor muscle endurance tests, and compare the results between individuals with and without subclinical neck pain. METHODS: Participants were students of the University of Aveiro reporting subclinical neck pain and asymptomatic participants matched for sex and age to the neck pain group. Data on endurance capacity of the deep neck flexors and neck extensors were collected by a blinded assessor using the deep neck flexor endurance test and the extensor endurance test, respectively. Intraclass correlation coefficients (ICCs), SEM, and MDC were calculated for measurements taken within a session by the same assessor. Differences between groups for endurance capacity were investigated using a Mann-Whitney U test. RESULTS: The deep neck flexor endurance test (ICC = 0.71; SEM = 6.91 seconds; MDC = 19.15 seconds) and neck extensor endurance test (ICC = 0.73; SEM = 9.84 minutes; MDC = 2.34 minutes) are reliable. No significant differences were found between participants with and without neck pain for both tests of muscle endurance (P > .05). CONCLUSION: The endurance capacity of the deep neck flexors and neck extensors can be reliably measured in participants with subclinical neck pain. However, the wide SEM and MDC might limit the sensitivity of these tests.

Unraveling Mesenchymal Stem Cells' Dynamic Secretome Through Nontargeted Proteomics Profiling.

The modulatory and regenerative potential shown by the use of MSC secretomes has emphasized the importance of their proteomics profiling. Proteomic analysis, initially focused on the targeted analysis of some candidate proteins or the identification of the secreted proteins, has been changing to an untargeted profiling also based on the quantitative evaluation of the secreted proteins.The study of the secretome can be accomplished through several different proteomics-based approaches; however this analysis must overcome one key challenge of secretome analysis: the low amount of secreted proteins and usually their high dilution.In this chapter, a general workflow for the untargeted proteomic profile of MSC's secretome is presented, in combination with a comprehensive description of the major techniques/procedures that can be used. Special focus is given to the main procedures to obtain the secreted proteins, from secretome concentration by ultrafiltration to protein precipitation. Lastly, different proteomics-based approaches are presented, emphasizing alternative digestion techniques and available mass spectrometry-based quantitative methods.

Nitric oxide from inflammatory origin impairs neural stem cell proliferation by inhibiting epidermal growth factor receptor signaling.

Neuroinflammation is characterized by activation of microglial cells, followed by production of nitric oxide (NO), which may have different outcomes on neurogenesis, favoring or inhibiting this process. In the present study, we investigated how the inflammatory mediator NO can affect proliferation of neural stem cells (NSCs), and explored possible mechanisms underlying this effect. We investigated which mechanisms are involved in the regulation of NSC proliferation following treatment with an inflammatory stimulus (lipopolysaccharide plus IFN-γ), using a culture system of subventricular zone (SVZ)-derived NSCs mixed with microglia cells obtained from wild-type mice (iNOS(+/+)) or from iNOS knockout mice (iNOS(-/-)). We found an impairment of NSC cell proliferation in iNOS(+/+) mixed cultures, which was not observed in iNOS(-/-) mixed cultures. Furthermore, the increased release of NO by activated iNOS(+/+) microglial cells decreased the activation of the ERK/MAPK signaling pathway, which was concomitant with an enhanced nitration of the EGF receptor. Preventing nitrogen reactive species formation with MnTBAP, a scavenger of peroxynitrite (ONOO(-)), or using the ONOO(-) degradation catalyst FeTMPyP, cell proliferation and ERK signaling were restored to basal levels in iNOS(+/+) mixed cultures. Moreover, exposure to the NO donor NOC-18 (100 µM), for 48 h, inhibited SVZ-derived NSC proliferation. Regarding the antiproliferative effect of NO, we found that NOC-18 caused the impairment of signaling through the ERK/MAPK pathway, which may be related to increased nitration of the EGF receptor in NSC. Using MnTBAP nitration was prevented, maintaining ERK signaling, rescuing NSC proliferation. We show that NO from inflammatory origin leads to a decreased function of the EGF receptor, which compromised proliferation of NSC. We also demonstrated that NO-mediated nitration of the EGF receptor caused a decrease in its phosphorylation, thus preventing regular proliferation signaling through the ERK/MAPK pathway.