Single-cell analysis of yeast, mammalian cells, and fungal spores with a microfluidic pressure-driven chip-based system.

BACKGROUND: Cytomics aims at understanding the function of cellular systems by analysis of single cells. Recently, there has been a growing interest in single cell measurements being performed in microfluidic systems. These systems promise to integrate staining, measurement, and analysis in a single system. One important aspect is the limitation of allowable cell sizes due to microfluidic channel dimensions. Here we want to demonstrate the broad applicability of microfluidic chip technology for the analysis of many different cell types. METHODS: We have developed a microfluidic chip and measurement system that allows flow cytometric analysis of fluorescently stained cells from different organisms. In this setup, the cells are moved by pressure-driven flow inside a network of microfluidic channels and are analyzed individually by fluorescence detection. RESULTS: We have successfully applied the system to develop a methodology to detect viable and dead cells in yeast cell populations. Also, we have measured short interfering RNA (siRNA) mediated silencing of protein expression in mammalian cells. In addition, we have characterized the infection state of Magnaportae grisea fungal spores. CONCLUSIONS: Results obtained with the microfluidic system demonstrate a broad applicability of microfluidic flow cytometry to measurement of various cell types.     

2,4-dinitrophenylhydrazine carbonyl assay in metal-catalysed protein glycoxidation

Using an experimental in vitro glycation model, long-term incubations of bovine serum albumin with glucose (fructose) resulted in a significant increase in protein content of 2,4-dinitrophenylhydrazine (DNPH)-reactive carbonyl groups, which could be strongly inhibited by anaerobiosis and metal chelation. The pattern of yields of the protein-bound DNPH was not in accordance with that of the sugar-derived carbonyls determined as the ketoamine Amadori product. In spite of the fact that the contribution of the final advanced glycation end-products to the total DNPH-reactivity of glycation-altered protein remains unclear, the present results stress the need of oxidative steps in formation of most of the DNPH-reactive carbonyl compounds generated by glycation. The results provide evidence that, in protein glycoxidation, the DNPH assay is selective enough to discriminate between protein-bound carbonyls produced by metal-catalysed oxidations and those formed in the early glycation steps.     

Early fetal like slow Na+ current in heart cells of cardiomyopathic hamster

Using the whole-cell voltage-clamp technique, early embryonic tetrodotoxin (TTX) and Mn2⁺-insensitive slow Na⁺ current was detected in 10-22 week old fetal human heart cells as well as in 1-day-old and young cardiomyopathic hamster myocytes. This slow Na⁺ current in both heart cell preparations has the same kinetics and pharmacology. This type of slow Na⁺ current was absent in heart cells of newborn and young normal hamsters and became less present in myocytes of 19 and 22 week old human heart myocytes. Our results demonstrate that the slow Na⁺ channel does exist in early fetal human life and this type of channel continues to be functional after birth in myocytes of the hereditary cardiomyopathic hamster.     

Oxidative modification of serum albumin in an experimental glycation model of diabetes mellitus in vitro: effect of the pyridoindole antioxidant stobadine

Under conditions of an experimental in vitro glycation model, the pyridoindole antioxidant stobadine significantly inhibited glycation-related fluorescence changes of bovine serum albumin as well as the yield of 2,4-dinitrophenylhydrazine-reactive carbonyls with an efficacy comparable to that of the reference antioxidants Trolox C and 2-keto-4-methiolbutyric acid, and more efficiently than did aminoguanidine. Since stobadine did not affect the covalent binding of glucose, the protective effect may be explained by the ability of the drug to eliminate free radical intermediates of glyco-oxidation reactions, operative after the preceding glycation steps.     

Caspases in yeast apoptosis-like death: facts and artefacts

Various findings suggest that programmed cell death (PCD) is induced in yeast as a response to the impact of a deleterious environment and/or an intracellular defect. Moreover, the specifically localized PCD within multicellular colonies seems to be important for the safe degradation of cell subpopulations to simple compounds that can be used as nutrients by healthy survivors occurring in propitious colony areas, being thus important for proper development and survival of the yeast population. In spite of this, the question remains whether yeast dies by real apoptosis, i.e. death involving caspases, or by other kinds of PCD. A large group of mammalian caspases includes those that are responsible for monitoring of the stimulus and initiating the dying process, as well as those involved in the execution of death. Additionally, paracaspases and metacaspases, that share some homology with real caspases, but possibly differ in substrate specificity, have been identified in plants, fungi, Dictyostelium and metazoa. In yeast, one homologue of caspases, metacaspase Mca1p/Yca1p, has been identified so far, although there are several indications of the presence of other caspase-like activities in yeast. In this minireview, we summarize various data on the possible involvement of Mca1p and other caspase-like activities in yeast PCD.     

Gallbladder carcinoma cells in cerebrospinal fluid as the first manifestation of a tumor. A case report

BACKGROUND: Meningeal carcinomatosis (MC) rarely occurs as the first evidence of a tumor. In such cases cytology of the cerebrospinal fluid is crucial to the diagnosis. The most frequent primary MCs are lung and breast cancers. MC from a gallbladder carcinoma is uncommon. CASE: A 58-year-old woman presented with paroxysmal headaches, seizures and coma. Analysis of the cerebrospinal fluid revealed carcinoma cells and a low protein concentration. Only postmortem examination discovered gallbladder adenocarcinoma to be the source of the tumor cells. CONCLUSION: A case with the onset of MC secondary to rare mucinous adenocarcinoma of the gallbladder is presented. Cytology of the cerebrospinal fluid was the only examination that uncovered malignancy. Nine similar cases were found in the literature. Low cerebrospinal fluid protein seems to be of diagnostic value.     

Tissue Specific Electrochemical Fingerprinting

Background

Proteomics and metalloproteomics are rapidly developing interdisciplinary fields providing enormous amounts of data to be classified, evaluated and interpreted. Approaches offered by bioinformatics and also by biostatistical data analysis and treatment are therefore of extreme interest. Numerous methods are now available as commercial or open source tools for data processing and modelling ready to support the analysis of various datasets. The analysis of scientific data remains a big challenge, because each new task sets its specific requirements and constraints that call for the design of a targeted data pre-processing approach.

Methodology/Principal Findings

This study proposes a mathematical approach for evaluating and classifying datasets obtained by electrochemical analysis of metallothionein in rat 9 tissues (brain, heart, kidney, eye, spleen, gonad, blood, liver and femoral muscle). Tissue extracts were heated and then analysed using the differential pulse voltammetry Brdicka reaction. The voltammograms were subsequently processed. Classification models were designed making separate use of two groups of attributes, namely attributes describing local extremes, and derived attributes resulting from the level = 5 wavelet transform.

Conclusions/Significance

On the basis of our results, we were able to construct a decision tree that makes it possible to distinguish among electrochemical analysis data resulting from measurements of all the considered tissues. In other words, we found a way to classify an unknown rat tissue based on electrochemical analysis of the metallothionein in this tissue.