Untargeted Metabolomics Analysis using LC-MSQTOF for Metabolite Profile Comparison between Patients with Myofascial Pain of Upper Trapezius Muscle versus Controls

Objective: This study aims to identify different biomarkers of Myofascial pain syndrome (MPS) using untargeted metabolomics screening. Materials and Methods: In a case-control study, serum samples from MPS patients (n = 19) and healthy controls (n = 10) were analyzed using reverse-phase liquid chromatography and mass spectrometry quadrupole time-of-flight (MS-QTOF). The resulted raw data was processed with Progenesis QI data analysis software. The HMBD database was used to identify the metabolites based on their fold change (>1.2), variable importance plot (>1) with P < 0.05. MetaboAnalyst 5.0 was used to generate metabolic network analysis for all identified metabolites. Results: The MPS group reported significantly higher pain on visual analog scale when compared with control while most of the other routine blood chemical profiles were not different. Twenty-seven metabolites were analyzed and identified with untargeted metabolomics analysis which could distinguish MPS patients from healthy controls. Inosine and chenodeoxycholic acid were abundant in the MPS group, whereas the others were low. Metabolites were divided into three categories: lipids, nucleotides, and organic compounds. Possible MPS metabolites included lysoSM (sphingomyelin), lysoPC (lysophosphatidylcholine), lysoPE (lysophosphatidylethanolamine), triglyceride, and inosine. Conclusion: These metabolite profiles, including glycerophospholipids mechanism and purine metabolism, indicate that the inflammatory process might be related to the mechanisms of MPS. A larger sample size, a different trigger point location, and modifications in therapy afterward should all be further explored

Evolution of mirror-image pain in temporomandibular joint osteoarthritis mouse model

Mirror-image pain is a kind of pain that occurs on the contralateral side, but its pathogenesis remains unclear.&nbsp; Objective: To develop an osteoarthritis mouse model for investigating mirror-image pain through observing nocifensive behaviors, histological changes, and nociceptive activity at days 3, 7, 14, 21, and 28 after the chemical induction of unilateral temporomandibular joint (TMJ) osteoarthritis. Methodology: We randomly divided 6-week-old mice into sham and complete Freund adjuvant groups. To induce nocifensive behaviors, we applied 0.04 g of von Frey filament, 10 psi of air puff, and cold acetone on both sides of whisker pads at different days. The histology of TMJ on both sides was observed by hematoxylin/eosin staining and microcomputed tomography scanning. Furthermore, the nociceptive activity was evaluated using the phosphorylated cyclic AMP response element binding protein (pCREB) and a microglia marker at different days in the trigeminal subnucleus caudalis. Results: Nocifensive behaviors against mechanical and temperature stimuli on the contralateral side became stronger than the baseline on day 28, in agreement with the elevation of the pCREB and the microglia marker in the trigeminal subnucleus caudalis. Thus, hypernociception on the contralateral side occurred at day 28. Conclusions: Clearly, the TMJ model with unilateral osteoarthritis exhibited mirror-image pain. Therefore, this model is useful in investigating the pathogenesis of pain and in developing treatments

Bacopa monnieri extract increases rat coronary flow and protects against myocardial ischemia/reperfusion injury

Background: This study explored Bacopa monnieri, a medicinal Ayurvedic herb, as a cardioprotectant against ischemia/reperfusion injury using cardiac function and coronary flow as end-points. Methods: In normal isolated rat hearts, coronary flow, left ventricular developed pressure, heart rate, and functional recovery were measured using the Langendorff preparation. Hearts were perfused with either (i) Krebs-Henseleit (normal) solution, (control), or with 30, 100 μg/ml B. monnieri ethanolic extract (30 min), or (ii) with normal solution or extract for 10 min preceding no-perfusion ischemia (30 min) followed by reperfusion (30 min) with normal solution. Infarct volumes were measured by triphenyltetrazolium staining. L-type Ca2+-currents (ICa, L) were measured by whole-cell patching in HL-1 cells, a mouse atrial cardiomyocyte cell line. Cytotoxicity of B. monnieri was assessed in rat isolated ventricular myocytes by trypan blue exclusion. Results: In normally perfused hearts, B. monnieri increased coronary flow by 63 ± 13% (30 μg/ml) and 216 ± 21% (100 μg/ml), compared to control (5 ± 3%) (n = 8–10, p < 0.001). B. monnieri treatment preceding ischemia/reperfusion improved left ventricular developed pressure by 84 ± 10% (30 μg/ml), 82 ± 10% (100 μg/ml) and 52 ± 6% (control) compared to pre- ischemia/reperfusion. Similarly, functional recovery showed a sustained increase. Moreover, B. monnieri (100 μg/ml) reduced the percentage of infarct size from 51 ± 2% (control) to 25 ± 2% (n = 6-8, p < 0.0001). B. monnieri (100 μg/ml) reduced ICa, L by 63 ± 4% in HL-1 cells. Ventricular myocyte survival decreased at higher concentrations (50–1000 μg/ml) B. monnieri. Conclusions: B. monnieri improves myocardial function following ischemia/reperfusion injury through recovery of coronary blood flow, contractile force and decrease in infarct size. Thus this may lead to a novel cardioprotectant strategy

Role of Doc2β in regulated exocytosis of large dense-core vesicles in bovine adrenal chromaffin cells

Doc2ß is a member of the double C2-domain (Doc2) protein family, a novel family of proteins having two C2 domains. Doc2ß is highly enriched in brain where it is associated with synaptic vesicles. However, Doc2ß is also expressed in nonneuronal tissues. Like its brain-specific counterpart Doc2a, Doc2ß interacts with Ca²⁺, phospholipids, Muncl3 and Muncl8, all of which are important elements for regulated exocytosis. While the significant role of Doc2a in regulated exocytosis was confirmed in PC 12 cells, the role of Doc2p in regulated exocytosis is still unresolved. Given the high homology, the complementary distribution in brain, the similarities in Ca²⁺-dependent phospholipid binding and interaction with other synaptic proteins between Doc2α and Doc2ß, it is hypothesised that Doc2ß performs a similar function in regulated exocytosis as Doc2α. The aim of this thesis is to study the possible roles of Doc2ß in regulated exocytosis and to elucidate the mechanisms of action.The adrenal chromaffin cell has been a popular model cell for the study of exocytosis. This cell contains catecholamine packaged in large dense-core vesicles (LDCVs) that undergo Ca²⁺-dependent exocytosis based on molecular machinery similar to that of synaptic transmission. Heterologous protein expression was achieved by using a Semliki Forest Virus (SFV) vector system, yielding high efficiency expression and minimal interference of membrane trafficking. Secretion was studied in both cell-population and single-cell levels. At the cell-population level, secretion of catecholamine was detected by using spectrofluorimetry. High resolution monitoring of secretion was achieved by using membrane capacitance measurements and electrochemical detection of catecholamine.Expression of Doc2ß fusion with green fluorescent protein (Doc2ß-EGFP) in bovine adrenal chromaffin cell showed both cytoplasmic and nuclear localisation. Punctate cytoplasmic fluorescence was identified in some cells which indicated the association with some intracytoplasmic compartments. Deletion of the amino terminal of Doc2ß (C2AB-EGFP) did not change the fluorescence distribution pattern. The expression of Doc2ß did not affect calcium currents and intracellular calcium level. Secretion induced by 50 mM KC1 depolarisation in intact cells or 1 μM free Ca²⁺ in ß-escin-permeabilised cells was not different among cells expressing green fluorescent protein, cells expressing wild-type Doc2ß and cells expressing Nterminal deletion of Doc2ß (C2AB). Secretion induced by whole-cell dialysis with solution containing 10 pM free calcium was also not different among those groups. The fusion-competent vesicle pool was not significantly changed by the expression of Doc2ß or C2AB. Application of phorbol ester similarly increased the fusioncompetent vesicle pool and catecholamine secretion in all cell groups. The results from this study indicate that Doc2ß is not essential for regulated exocytosis of LCDVs in bovine adrenal chromaffin cells

Effects of Ceftriaxone on Oxidative Stress and Inflammation in a Rat Model of Chronic Cerebral Hypoperfusion

International audienceCeftriaxone (CTX) exerts a neuroprotective effect by decreasing glutamate excitotoxicity. We further studied the underlying mechanisms and effects of CTX early post-treatment on behavior in a cerebral hypoperfusion rats. The rats’ common carotid arteries (2VO) were permanently ligated. CTX was treated after ischemia. Biochemical studies were performed to assess antioxidative stress and inflammation. Behavioral and histological studies were then tested on the ninth week after vessel ligation. The 2VO rats showed learning and memory deficits as well as working memory impairments without any motor weakness. The treatment with CTX was found to attenuate white matter damage, MDA production, and interleukin 1 beta and tumor necrosis factor alpha production, mainly in the hippocampal area. Moreover, CTX treatment could increase the expression of glia and the glial glutamate transporters, and the neuronal glutamate transporter. Taken together, our data indicate the neuroprotective mechanisms of CTX involving the upregulation of glutamate transporters’ expression. This increased expression contributes to a reduction in glutamate excitotoxicity and oxidative stress as well as pro-inflammatory cytokine production, thus resulting in the protection of neurons and tissue from further damage. The present study highlights the mechanism of the effect of CTX treatment and of the underlying ischemia-induced neuronal damage

Efficacy of Semliki Forest virus transduction of bovine adrenal chromaffin cells:an analysis of heterologous protein targeting and distribution

Проведено аналіз існуючих методів та підходів до побудови сучасних захищених інформаційно-комунікаційних систем та мереж в комерційних банках. Визначено переваги та недоліки методів, що розглядаються. Запропоновано підхід та методику модернізації існуючих мере