Alterations in the Proteome and Phosphoproteome Profiles of Rat Hippocampus after Six Months of Morphine Withdrawal: Comparison with the Forebrain Cortex

The knowledge about proteome changes proceeding during protracted opioid withdrawal is lacking. Therefore, the aim of this work was to analyze the spectrum of altered proteins in the rat hippocampus in comparison with the forebrain cortex after 6-month morphine withdrawal. We utilized 2D electrophoretic workflow (Pro-Q® Diamond staining and Colloidal Coomassie Blue staining) which was preceded by label-free quantification (MaxLFQ). The phosphoproteomic analysis revealed six significantly altered hippocampal (Calm1, Ywhaz, Tuba1b, Stip1, Pgk1, and Aldoa) and three cortical proteins (Tubb2a, Tuba1a, and Actb). The impact of 6-month morphine withdrawal on the changes in the proteomic profiles was higher in the hippocampus—14 proteins, only three proteins were detected in the forebrain cortex. Gene Ontology (GO) enrichment analysis of differentially expressed hippocampal proteins revealed the most enriched terms related to metabolic changes, cytoskeleton organization and response to oxidative stress. There is increasing evidence that energy metabolism plays an important role in opioid addiction. However, the way how morphine treatment and withdrawal alter energy metabolism is not fully understood. Our results indicate that the rat hippocampus is more susceptible to changes in proteome and phosphoproteome profiles induced by 6-month morphine withdrawal than is the forebrain cortex.

A comparative study of two methods to determine acrosome integrity of frozen-thawed boar sperm

Coomassie blue staining has been reported as an effective and inexpensive method for evaluating the acrosome integrity of spermatozoa, though to date its use to evaluate cryopreserved boar sperm has not been reported. Moreover, there is no information concerning the agreement between Coomassie blue staining and fluorescein isothiocyanate conjugated peanut agglutinin and ethidium homodimer (FITC-PNA/EthD-1) methods for assessing sperm acrosome integrity for any species. The current study was performed to determine the efficacy and agreement between Coomassie blue and FITC-PNA/EthD-1 staining methods for evaluating the acrosome integrity of frozen-thawed boar sperm. A total of 25 semen samples were cryopreserved using lactose-egg yolk-based extender and loaded into 0.5 PVC-French straws. Sperm motility and motion characteristics were determined using a computer-assisted sperm analysis system. Sperm viability and plasma membrane integrity were evaluated using the SYBR-14/EthD-1 and hypo-osmotic swelling test, respectively. Acrosome integrity of frozen-thawed boar sperm was evaluated using both FITC-PNA/EthD-1 and Coomassie blue staining to assess the association between sperm acrosome integrity and agreement between these two methods. The average percent acrosome integrity of frozen-thawed boar sperm as determined by FITC-PNA/ EthD-1 and Coomassie blue staining was 48.8 ± 12.6% and 52.6 ± 13.6%, respectively (P>0.05). Interestingly, Coomassie blue staining found a correlation between sperm viability and acrosome integrity (r=0.609, P=0.002), while FITC-PNA/EthD-1 staining did not (P>0.05). However, the acrosome integrity of frozen-thawed boar sperm evaluated by FITC-PNA/ EthD-1 and Coomassie blue staining was significantly correlated (r=0.448, P=0.025, n=25). The Bland-Altman plot determined that this agreement was acceptable. In conclusion, the acrosome integrity of the frozen-thawed boar sperm assessed via Coomassie blue staining was significantly correlated with that obtained via the FITC-PNA/EthD-1 staining method, and the two methods showed good agreement. Moreover, the significant association between the acrosome integrity of frozen-thawed boar sperm determined by Coomassie blue staining with other sperm quality parameters indicates that this is an effective method for assessing the acrosome integrity of frozen-thawed sperm in pigs.

Microscopic and chemical characterization of PVC tube used for dialysis lines: A new approach

Polyvinylchloride is universally agreed upon to be the material of choice for tubings and for containers for medical application. Many alterations of the chemical/physical surface conditions, mainly due to an altered extrusion process, could influence its biocompatibility by promoting platelet aggregation. Biocompatibility and safety of the medical device must be preserved, also monitoring the migration of additives within polyvinylchloride during the diffusion process. A large variety of methods are used to verify the correct composition and extrusion of polyvinylchloride but, generally, they need long experimental time and are expensive. The aim of the study is to propose a simple, economic and rapid approach based on Fourier transform-infrared spectroscopy and Coomassie Blue staining. The method has been used to detect chemical and morphological defects caused by an altered extrusion process on 20/75 polyvinylchloride tubings in a blind test. This approach positively identified altered samples in 80% of the cases. The suggested approach represents a reliable and versatile method to detect and monitor surface defects by an easy, inexpensive and reproducible method.

The Helicobacter pylori Urease Virulence Factor Is Required for the Induction of Hypoxia-Induced Factor-1α in Gastric Cells

Chronic Helicobacter pylori infection increases the risk of gastric cancer and induction of hypoxia-induced factor (HIF), which is frequently associated with the development and progression of several types of cancer. We recently showed that H. pylori activation of the PI3K-AKT-mTOR pathway in gastric cells increased HIF-1α expression. Here, we identified the H. pylori virulence factor responsible for HIF-1α induction. A mutant of the H. pylori 84-183 strain was identified with reduced ability to induce HIF-1α. Coomassie blue staining of extracts from these bacteria separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) revealed poor expression of urease subunits that correlated with reduced urease activity. This finding was confirmed in the 26695 strain, where urease mutants were unable to induce HIF-1α expression. Of note, HIF-1α induction was also observed in the presence of the urease inhibitor acetohydroxamic acid at concentrations (of 20 mM) that abrogated urease activity in bacterial culture supernatants, suggesting that enzymatic activity of the urease is not required for HIF-1α induction. Finally, the pre-incubation of the human gastric adenocarcinoma cell line AGS with blocking antibodies against Toll-like receptor-2 (TLR2), but not TLR4, prevented HIF-1α induction. In summary, these results reveal a hitherto unexpected role for the urease protein in HIF-1α induction via TLR2 activation following H. pylori infection of gastric cells.

Profil Protein Trypanosoma evansi dari Daerah Geografis Berbeda di Indonesia Tahun 2012-2014 dengan Sodium Dodecil Sulphate Polyacrylamide Gel Electrophoresis (TRYPANOSOMA EVANSI PROTEIN PROFILE OF DIFFERENT GEOGRAPHICAL AREAS ORIGIN IN INDONESIA

Surra outbreak in 2012 has led to more than 1,700 animals have died in the province of East Nusa Tenggara (NTT) Indonesia. Surra case sporadically continues throughout the year in various areas, especially Kalimantan, Banten as well as other areas. Some reports reveal differences in protein profiles among multiple isolates of T. evansi. Therefore the purpose of this research were to find out the protein profile of each isolate T. evansi in Indonesia and the possible biological differences among them. Eleven isolates originating from the province of East Nusa Tenggara, South Kalimantan and Central Kalimantan, Banten, Lampung and Bengkulu has been isolated and purified Using DEAE. Trypanosoma isolate were frezeethawing repeatedly to obtain soluble protein. Furthermore, soluble protein is treated with heating or without heating and then each was run on SDS PAGE with Coomassie Blue staining. The protein profiles of all isolates were compared each other. The results showed that eleven isolates of T. evansi in Indonesia has a very diverse protein profile. Then for the purposes of development of diagnostic kit can be used whole lysate cell (WCL) as stock antigen in serological test process.

Proteomic Approach for Extracting Cytoplasmic Proteins from Streptococcus sanguinis using Mass Spectrometry

Streptococcus sanguinis is a commensal and early colonizer of oral cavity as well as an opportunistic pathogen of infectious endocarditis. Extracting the soluble proteome of this bacterium provides deep insights about the physiological dynamic changes under different growth and stress conditions, thus defining “proteomic signatures” as targets for therapeutic intervention. In this protocol, we describe an experimentally verified approach to extract maximal cytoplasmic proteins from Streptococcus sanguinis SK36 strain. A combination of procedures was adopted that broke the thick cell wall barrier and minimized denaturation of the intracellular proteome, using optimized buffers and a sonication step. Extracted proteome was quantitated using Pierce BCA Protein Quantitation assay and protein bands were macroscopically assessed by Coomassie Blue staining. Finally, a high resolution detection of the extracted proteins was conducted through Synapt G2Si mass spectrometer, followed by label-free relative quantification via Progenesis QI. In conclusion, this pipeline for proteomic extraction and analysis of soluble proteins provides a fundamental tool in deciphering the biological complexity of Streptococcus sanguinis.

Abstract 432: Endothelial Cells Express miR-33a-5p and Release Extracellular Vesicles Containing miR-33a-5p

Background: The microRNA miR-33a-5p increases cellular cholesterol by posttranscriptional silencing of genes involved in cholesterol efflux. Previous studies showed that miR-33a-5p inhibition protects against atherosclerosis. Atheroprotective effects of miR-33a-5p inhibition are attributed to actions in hepatocytes that raise plasma HDL and actions in macrophages that enhance cholesterol efflux. We hypothesized that miR-33a-5p is also expressed in primary arterial endothelial cells (EC) and that miR-33a-5p might contribute to atherosclerosis by promoting cholesterol accumulation in EC. We also hypothesized that EC could release miR-33a-5p in extracellular vesicles (EV; including exosomes and microvesicles) that transferred miR-33a-5p to neighboring vascular cells such as SMC and macrophages, inhibiting cholesterol efflux, promoting lipid accumulation in these cells, and accelerating atherosclerosis. Methods: We cultured bovine aortic endothelial cells (BAEC) in serum-free medium, collected the medium, and isolated EV by ultracentrifugation. To test whether we had isolated EV, we compared EV and BAEC lysates by SDS-PAGE, Coomassie blue staining, and immunoblotting. We measured EV size with nanoparticle tracking analysis and imaged the particles with transmission electron microscopy. We extracted total RNA from BAEC and EV, measured RNA size using BioAnalyzer, and detected miR-33a-5p expression using RT-PCR and restriction digestion as well as qRT-PCR. Results: Coomassie blue staining revealed large differences between BAEC and EV lysates. The exosome marker CD9 was present at higher levels in EV lysate compared to BAEC lysate. Most of the EV were in the size range of exosomes, and appeared as typical lipid membrane-bound spheres by electron microscopy. EV contained primarily small RNA (~25-200 nucleotides). miR-33a-5p was detected both in BAEC lysates and in detergent-soluble EV. Conclusions: Primary arterial EC express miR-33a-5p and release EV rich in small RNA, including miR-33a-5p. Manipulating miR-33a-5p expression in EC, for example with a miR-33a-5p antagomiR, may be an effective therapeutic approach for increasing cholesterol efflux from multiple vessel wall cell types and preventing/reversing atherosclerosis.

The effect of cultureware surfaces on functional and structural components of differentiated 3T3-L1 preadipocytes

Experiments using cultured primary cells or cell lines are a routine in vitro approach used across multiple biological disciplines, However, the structural and functional influences of various cultureware materials on cultured cells is not clearly understood. Surface treatments of cultureware have proven to have profound effects on cell viability and proliferation. In this study, we investigated the impact of polystyrene and fluorocarbon cultureware dishes on the proteomic profile of differentiated 3T3-L1 preadipocytes. After expansion and differentiation of cells on appropriate cultureware dishes, cell lysates were separated using two-dimensional gel electrophoresis and proteins were visualized with Coomassie blue staining. Spots with the highest differential expression between the two culture conditions were subsequently analyzed using matrix-assisted laser desorption/ionization mass spectrometry and the identified proteins were subjected to pathway analysis. We observed that 43% of all spots were differentially expressed depending on the cultureware. Pathway analysis revealed that glucose metabolism, mitochondrial structure and cell differentiation, represented by 14-3-3 protein-mediated signaling and the mitochondrial inner membrane organizing system (MINOS), were significantly affected by cultureware material. These results indicate that cultureware material can have a profound effect on key adipocyte functional pathways. These effects modifications of the cells should be reflected in the design of in vitro experiments and interpretation of their results.

Protein profile of the seminal plasma of collared peccaries (Pecari tajacu Linnaeus, 1758)

This study was conducted to characterize the major proteins of the peccary seminal plasma, based on the semen samples collected from nine adult and reproductively sound animals. Our approach included the use of two-dimensional electrophoresis followed by Coomassie blue staining and analysis of polypeptide maps with PDQuest Software (Bio-Rad). Proteins were identified by tandem mass spectrometry (LC–MS/MS). We detected 179 protein spots per gel and 98 spots were identified by mass spectrometry, corresponding to 23 different proteins. The combined intensity of those spots accounted for 56.2±6% of the intensities of all spots and 60.9% of the intensities of spots presented in every protein map. Protein spots identified as clusterin represented 19.7±8.3% of the integrated optical densities of all spots detected in the seminal plasma maps. There was a negative association (r=−0.87;P<0.05) between the intensity of a clusterin spot and the percentage of sperm with functional membrane. Spermadhesin porcine seminal plasma protein 1 and bodhesin 2 comprised 5.4±1.9 and 8.8±3.9% of the total intensity of all spots respectively. Many proteins appeared in a polymorphic pattern, such as clusterin (27 spots), epididymal secretory glutathione peroxidase (ten spots), inter-α-trypsin inhibitor (12 spots), and IgG-binding protein (ten spots), among others. In conclusion, we presently describe the major seminal plasma proteome of the peccary, which exhibits a distinct high expression of clusterin isoforms. Knowledge of wild species reproductive biology is crucial for an understanding of their survival strategies and adaptation in a changing environment.