Assessment of Blood Contamination in Biological Fluids Using MALDI-TOF MS

Biological fluid sample collection often includes the risk of blood contamination that may alter the proteomic profile of biological fluid. In proteomics studies, exclusion of contaminated samples is usually based on visual inspection and counting of red blood cells in the sample; analysis of specific blood derived proteins is less used. To fill the gap, we developed a fast and sensitive method for ascertainment of blood contamination in crude biological fluids, based on specific blood-derived protein, hemoglobin detection by MALDI-TOF MS. The MALDI-TOF MS based method allows detection of trace hemoglobin with the detection limit of 0.12 nM. UV-spectrometry, which was used as reference method, was found to be less sensitive. The main advantages of the presented method are that it is fast, effective, sensitive, requires very small sample amount and can be applied for detection of blood contamination in various biological fluids collected for proteomics studies. Method applicability was tested on human cerebrospinal and follicular fluid, which proteomes generally do not contain hemoglobin, however, which possess high risk for blood contamination. Present method successfully detected the blood contamination in 12 % of cerebrospinal fluid and 24 % of follicular fluid samples. High percentage of contaminated samples accentuates the need for initial inspection of proteomic samples to avoid incorrect results from blood proteome overlap.     

Plant plasma membrane‐resident receptors: Surveillance for infections and coordination for growth and development

As sessile organisms, plants are exposed to pathogen invasions and environmental fluctuations. To overcome the challenges of their surroundings, plants acquire the potential to sense endogenous and exogenous cues, resulting in their adaptability. Hence, plants have evolved a large collection of plasma membrane-resident receptors, including RECEPTOR-LIKE KINASEs(RLKs) and RECEPTOR-LIKE PROTEINs(RLPs) to perceive those signals and regulate plant growth,development, and immunity. The ability of RLKs and RLPs to recognize distinct ligands relies on diverse categories of extracellular domains evolved. Co-regulatory receptors are often required to associate with RLKs and RLPs to facilitate cellular signal transduction. RECEPTOR-LIKE CYTOPLASMIC KINASEs(RLCKs) also associate with the complex, bifurcating the signal to key signaling hubs, such as MITOGEN-ACTIVATED PROTEIN KINASE(MAPK) cascades, to regulate diverse biological processes. Here, we discuss recent knowledge advances in understanding the roles of RLKs and RLPs in plant growth, development, and immunity, and their connection with co-regulatory receptors, leading to activation of diverse intracellular signaling pathways.     

Structure Analysis of Proteins and Peptides by Difference Circular Dichroism Spectroscopy

The Protein Journal - Difference circular dichroism (CD) spectroscopy was used here to characterize changes in structure of flexible peptides upon altering their environments. Environmental changes...     

Methylene Blue Modulates Transendothelial Migration of Peripheral Blood Cells

Vasoplegia is a severe complication after cardiac surgery. Within the last years the administration of nitric oxide synthase inhibitor methylene blue (MB) became a new therapeutic strategy. Our aim was to investigate the role of MB on transendothelial migration of circulating blood cells, the potential role of cyclic cGMP, eNOS and iNOS in this process, and the influence of MB on endothelial cell apoptosis. Human vascular endothelial cells (HuMEC-1) were treated for 30 minutes or 2 hours with different concentrations of MB. Inflammation was mimicked by LPS stimulation prior and after MB. Transmigration of PBMCs and T-Lymphocytes through the treated endothelial cells was investigated. The influence of MB upon the different subsets of PBMCs (Granulocytes, T- and B-Lymphocytes, and Monocytes) was assessed after transmigration by means of flow-cytometry. The effect of MB on cell apoptosis was evaluated using Annexin-V and Propidium Iodide stainings. Analyses of the expression of cyclic cGMP, eNOS and iNOS were performed by means of RT-PCR and Western Blot. Results were analyzed using unpaired Students T-test. Analysis of endothelial cell apoptosis by MB indicated a dose-dependent increase of apoptotic cells. We observed time- and dose-dependent effects of MB on transendothelial migration of PBMCs. The prophylactic administration of MB led to an increase of transendothelial migration of PBMCs but not Jurkat cells. Furthermore, HuMEC-1 secretion of cGMP correlated with iNOS expression after MB administration but not with eNOS expression. Expression of these molecules was reduced after MB administration at protein level. This study clearly reveals that endothelial response to MB is dose- and especially time-dependent. MB shows different effects on circulating blood cell-subtypes, and modifies the release patterns of eNOS, iNOS, and cGMP. The transendothelial migration is modulated after treatment with MB. Furthermore, MB provokes apoptosis of endothelial cells in a dose/time-dependent manner.     

C2ORF40 suppresses breast cancer cell proliferation and invasion through modulating expression of M phase cell cycle genes

Recently, it has been suggested that C2ORF40 is a candidate tumor suppressor gene in breast cancer. However, the mechanism for reduced expression of C2ORF40 and its functional role in breast cancers remain unclear. Here we show that C2ORF40 is frequently silenced in human primary breast cancers and cell lines through promoter hypermethylation. C2ORF40 mRNA level is significantly associated with patient disease-free survival and distant cancer metastasis. Overexpression of C2ORF40 inhibits breast cancer cell proliferation, migration and invasion. By contrast, silencing C2ORF40 expression promotes these biological phenotypes. Bioinformatics and FACS analysis reveal C2ORF40 functions at G2/M phase by downregulation of mitotic genes expression, including UBE2C. Our results suggest that C2ORF40 acts as a tumor suppressor gene in breast cancer pathogenesis and progression and is a candidate prognostic marker for this disease.