Flow cytometric measurement of cellular FLICE-inhibitory protein (c-FLIP) in ovarian carcinoma effusions

H. P. Dong, A. K. Ree Rosnes, A. J. Bock, A. Holth, V. A. Flørenes, C. G. Trope’, B. Risberg and B. Davidson Flow cytometric measurement of cellular FLICE‐inhibitory protein (c‐FLIP) in ovarian carcinoma effusions Objective: The objective of this study was to establish a flow cytometry assay for measuring c‐FLIP in serous effusions. In addition, we studied the clinical relevance in ovarian carcinoma effusions of this inhibitor protein in the death receptor signalling pathway of apoptosis. Methods: Two c‐FLIP antibodies were tested using Western blotting and the best performing one was used for titration of c‐FLIP expression in a panel of five cell lines, consisting of ovarian carcinoma, breast carcinoma and malignant mesothelioma. The concentration that provided the best signal‐to‐noise ratio was used for comparison of the performance of three fixation and permeabilization protocols. The best performing protocol was chosen for analysis of 69 ovarian carcinoma effusions. c‐FLIP expression was analysed for association with clinicopathological parameters and survival. Results: Rabbit polyclonal c‐FLIP by Abcam and the IntraStain kit by Dako performed best. c‐FLIP expression was detected in tumour cells in all 69 effusions (expression range 21–100%, median = 80%). No association was found between c‐FLIP expression and clinicopathological parameters, including chemoresponse and survival. However, an inverse correlation was found between c‐FLIP levels and expression of the previously studied apoptosis marker cleaved caspase‐3 (P = 0.029). Conclusions: An assay for measuring c‐FLIP in cytology specimens is presented. c‐FLIP is frequently expressed in ovarian carcinoma effusions, but its expression appears to be unrelated to disease aggressiveness.     

Comparison of different analysis techniques for the determination of muscle onset in individuals with patellofemoral pain syndrome

To understand patellofemoral pain syndrome (PFPS), recent studies have focused on assessing the onset in the vastus medialis and vastus lateralis to determine whether there is a delay between these muscles’ activation. However, the results of these studies are not in agreement, as some research shows that there is a delay in the VMO, while others do not show delay. It has been suggested that this discrepancies may be due to differences in the signal processing and analysis. For this reason, this study aimed to compare the three techniques used for onset determination – automatic detection, visual inspection and cross-correlation – and to verify whether these methods are able to detect PFPS. The surface electromyography evaluation procedure was conducted in 22 pain-free control individuals and 11 with PFPS diagnoses, during a stair climbing. The standard error of measurement (SEM) showed that cross-correlation presents the lower variation (2.56/3.27, control/PFPS) in relation to visual (3.77/10.19, control/PFPS) and automatic detection (43.23/51.98, control/PFPS, respectively). But when using the cross-correlation technique, we were not able to distinguish the groups (−6.56/−9.74 ms, control/PFPS, p = 0.15). Therefore, use of muscle onset may not be the best way to distinguish individuals with PFPS.     

Computing fragmentation trees from metabolite multiple mass spectrometry data

Since metabolites cannot be predicted from the genome sequence, high-throughput de novo identification of small molecules is highly sought. Mass spectrometry (MS) in combination with a fragmentation technique is commonly used for this task. Unfortunately, automated analysis of such data is in its infancy. Recently, fragmentation trees have been proposed as an analysis tool for such data. Additional fragmentation steps (MS(n)) reveal more information about the molecule. We propose to use MS(n) data for the computation of fragmentation trees, and present the Colorful Subtree Closure problem to formalize this task: There, we search for a colorful subtree inside a vertex-colored graph, such that the weight of the transitive closure of the subtree is maximal. We give several negative results regarding the tractability and approximability of this and related problems. We then present an exact dynamic programming algorithm, which is parameterized by the number of colors in the graph and is swift in practice. Evaluation of our method on a dataset of 45 reference compounds showed that the quality of constructed fragmentation trees is improved by using MS(n) instead of MS2 measurements.     

A new disruption vector (pDHO) to obtain heterothallic strains from both Saccharomyces cerevisiae and Saccharomyces pastorianus

Yeasts are responsible for several traits in fermented beverages, including wine and beer, and their genetic manipulation is often necessary to improve the quality of the fermentation product. Improvement of wild-type strains of Saccharomyces cerevisiae and Saccharomyces pastorianus is difficult due to their homothallic character and variable ploidy level. Homothallism is determined by the HO gene in S. cerevisiae and the Sc-HO gene in S. pastorianus. In this work, we describe the construction of an HO disruption vector (pDHO) containing an HO disruption cassette and discuss its use in generating heterothallic yeast strains from homothallic Saccharomyces species.     

A mechanism of carbapenem resistance due to a new insertion element (ISPa133) in Pseudomonas aeruginosa

This study explored the evolutionary mechanism by which the clinical isolate PA110514 yields the imipenemresistant derivative PA116136. Both isolates were examined by PFGE and SDS-PAGE, which led to the identification of a new insertion sequence, ISPa133. This element was shown to have distinct chromosomal locations in each of the original isolates that appeared to explain the differences in imipenem susceptibilty. In strain PA110514, ISPa133 is located 56 nucleotides upstream of the translational start codon, which has no effect on expression of the porin OprD. However, in strain PA116136 ISPa133 it is located in front of nucleotide 696 and, by interrupting the coding region, causes a loss of OprD expression, thus conferring imipenem resistance. In vitro experiments mimicking the natural conditions of selective pressure yielded imipenem-resistant strains in which ISPa133 similarly interrupted oprD. A mechanism is proposed whereby ISPa133 acts as a mobile switch, with its position in oprD depending on the degree of selective pressure exerted by imipenem.     

Methicillin-resistant Staphylococcus epidermidis carrying biofilm formation genes: detection of clinical isolates by multiplex PCR

Staphylococcus epidermidis is the most prevalent coagulase-negative Staphylococcus (CNS) and is a major cause of hospital bacteremia. Based on 18 reference strains and 149 Staphylococcus clinical strains, used in a novel multiplex PCR method, the aim of this study was to identify S. epidermidis with respect to the sequence of three genes: recN, which encodes a recombination/repair protein, mecA (methicillin resistance), and icaAB, which is involved in biofilm formation. Amplicons of 219 bp (S. epidermidis-recN gene), 154 bp (mecA gene), and 546 bp (icaAB genes) were obtained. Reliable results were achieved for 100% of the evaluated strains, suggesting that this new multiplex-PCR approach could be useful for the accurate identification of methicillin-resistant S. epidermidis with the potential to produce biofilm.     

Microbial community composition of anoxic marine sediments in the Bay of Cádiz (Spain)

The composition of the microbial community inhabiting the anoxic coastal sediments of the Bay of Cádiz (southern Spain) was investigated using a molecular approach consisting of PCR cloning and denaturing gradient gel electrophoresis (DGGE), based on 16S rRNA sequences. The total cell count was 1-5 × 10? cells/g sediment and, as determined by catalyzed reporter deposition-fluorescent in situ hybridization (CARD-FISH), the proportion of Bacteria to Archaea was about 70:30. The analysis of 16S-rRNA gene sequences revealed a wide spectrum of microorganisms, which could be grouped into 111 operational taxonomic units (OTUs). Many of the OTUs showed high phylogenetic similarity to microorganisms living in marine sediments of diverse geographic origin. The phylogenetic groups that were predominantly detected were Firmicutes, Deltaproteobacteria, and Gammaproteobacteria, accounting for 23, 15, and 14% of the clones, respectively. Diversity in the domain Archaea was significantly lower than in the domain Bacteria. The majority of the archaeal OTUs belonged to the Crenarchaeota phylum. Since most of the sequences could not be identified precisely at the genus/species level, the functional roles of the microorganisms in the ecosystem could not be inferred. However, seven OTUs affiliated with the Delta- and Epsilonproteobacteria were identified down to the genus level, with all of the identified genera known to occur in sulfate-rich marine environments.     

A novel marker for assessment of liver matrix remodeling: an enzyme-linked immunosorbent assay (ELISA) detecting a MMP generated type I collagen neo-epitope (C1M)

A competitive enzyme-linked immunosorbent assay (ELISA) for detection of a type I collagen fragment generated by matrix metalloproteinases (MMP) -2, -9 and -13, was developed (CO1-764 or C1M). The biomarker was evaluated in two preclinical rat models of liver fibrosis: bile duct ligation (BDL) and carbon tetra chloride (CCL4)-treated rats. The assay was further evaluated in a clinical study of prostate-, lung- and breast-cancer patients stratified according to skeletal metastases. A technically robust ELISA assay specific for a MMP-2, -9 and -13 neo-epitope was produced and seen to be statistically elevated in BDL rats compared to baseline levels as well as significantly elevated in CCL4 rats stratified according to the amount of total collagen in the livers. CO1-764 levels also correlated significantly with total liver collagen and type I collagen mRNA expression in the livers. Finally, the CO1-764 marker was not correlated with skeletal involvement or number of bone metastases. This ELISA has the potential to assess the degree of liver fibrosis in a non-invasive manner.     

Simultaneously targeting mitochondria and endoplasmic reticulum by photodynamic therapy induces apoptosis in human lymphoma cells

Photodynamic therapy (PDT) and photodetection with protoporphyrin IX (PpIX) precursors have widely been used in the diseases with abnormally proliferative cells, but the mechanism of the modality is not fully understood yet. In this study 70-95% of apoptotic cells after PDT with PpIX precursor, hexaminolevulinate (HAL) in two human lymphoma cell lines, Namalwa and Bjab, were confirmed by fluorescence microscopy, electron microscopy and flow cytometry. HAL-derived PpIX was mainly distributed in the mitochondria and endoplasmic reticulum (ER), both of which were initial targets after light exposure causing two major pathways simultaneously involved in the apoptotic induction. One was the mitochondrial pathway including the release of cytochrome c, cleavage of caspases-9/-3, poly(ADP-ribose) polymerase and DNA fragmentation factor. The other was the ER stress-mediated pathway triggering a transient increase in the cytosolic Ca(2+) level after photodamage to the ER calcium pump protein SERCA2. The released Ca(2+) further initiated the caspase-8 cleavage. The use of both extracellular Ca(2+) chelator EGTA and intracellular Ca(2+) chelator BAPTA-AM confirmed that such cytosolic Ca(2+) originated from the ER rather than extracellular Ca(2+)-containing medium. About 30% of the apoptosis was blocked with BAPTA-AM alone; while a complete inhibition of such apoptosis was achieved with a combination of the caspase-9 inhibitor Z-LEHD-FMK and caspase-8 inhibitor Z-IETD-FMK, thus quantifying each role of the mitochondrial and ER pathways.