Revised assignment of room-temperature chlorophyll fluorescence emission bands in single living cells of Chlamydomonas reinhardtii

Room temperature (RT) microspectrofluorimetry in vivo of single cells has a great potential in photosynthesis studies. In order to get new information on RT chlorophyll fluorescence bands, we analyzed the spectra of Chlamydomonas reinhardtii mutants lacking fundamental proteins of the thylakoid membrane and spectra of photoinhibited WT cells. RT spectra of single living cells were characterized thorough derivative analyses and Gaussian deconvolution. The results obtained suggest that the dynamism in LHCII assembly could be sufficient to explain the variations in amplitudes of F680 (free LHCII), F694 (LHCII-PSII) and F702 (LHCII aggregates); F686 was assigned to the PSII core. Based on the revised assignments and on the variations observed, we discuss the meaning of the two fluorescence emission ratios F680/(F686 + F694) and F702/(F686 + F694), showing that these are sensitive parameters under moderate photoinhibition. In the most photoinhibited samples, the RT spectra tended to degenerate, showing characteristics of mutants that are partly depleted in PSII.     

Biomarkers in malignant mesothelioma: diagnostic and prognostic role of soluble mesothelin-related peptide

Soluble mesothelin-related peptide (SMRP) is a biomarker that has been proposed for differential diagnosis from pleural metastatic cancer, as well as prognosis and treatment monitoring of malignant pleural mesothelioma (MM). The aim of this study was to evaluate the role of SMRP in clinic management of MM. We assayed the SMRP concentrations in 354 subjects: 109 healthy volunteers with no history of exposure to asbestos, 26 patients with previous occupational asbestos exposure but who were free from pleural or parenchymal disease, 48 patients with asbestosis, 110 patients with pleural plaques, 25 patients with lung cancer, and 36 patients with MM. We also tested SMRP titers in 2 patients with MM at 5 different times of the disease, to evaluate the trend of the biomarker in the course of therapy. Our data confirm previous experiences with the use of SMRP as a diagnostic marker of MM. Low SMRP levels at diagnosis seem to have a positive prognostic significance.     

Oncosuppressor methylation: a possible key role in colon metastatic progression

K‐RAS and BRAF gene mutations are mandatory to set anti‐EGFR therapy in metastatic colorectal cancer (mCRC) patients. Due to the relationship of these mutations with tumor epigenotype, we hypothesized the potential role of oncosuppressor methylation of genes involved in K‐RAS/BRAF pathway (CDKN2A, RASSF1A, and RARbeta suppressor genes) in inhibiting EGFR signaling cascade. Primary tumor and synchronous liver metastatic tissues of 75 mCRC patients were characterized for promoter methylation by QMSP and for K‐RAS and BRAF mutations. RARbeta, RASSF1A, and CDKN2A genes were methylated in 82%, 35%, and 26% of primary tumors, respectively. RASSF1A resulted significantly more frequently methylated in liver metastasis than in primary site (P = 0.015), while RARbeta was significantly lower methylated in distant metastasis (P = 1.2 × 10^?6). As regards methylation content, RASSF1A methylation status was significantly higher in liver metastasis with respect to primary tumor (P = 0.000) underlying the role of this gene in liver metastatic progression. In our series K‐RAS and BRAF were mutated in 39% and 4% of cases, respectively. Methylation frequencies seemed to be unrelated to gene mutations; on the other hand, RASSF1A mean content methylation resulted significantly higher in liver than in primary tumor (288.78 vs. 56.23, respectively, P = 0.05) only in K‐RAS wild‐type cases sustaining a specific role of this gene in metastatic site thus supporting its function in strengthening the apoptotic role of K‐RAS. These evidences held the role of oncosuppressor methylation in both colon tumorigenesis and progression and suggested that epigenetic events should be taken into account when biological therapies in mCRC patients have to be set. J. Cell. Physiol. 226: 1934–1939, 2011. © 2010 Wiley‐Liss, Inc.     

The digital photobiology compendium: perspectives on a web-based teaching tool from learners and developers.

The DPC offers many benefits for learners, teachers and developers involved in creation of teaching materials in photobiology. Modifications and additions can be made relatively easily. Anonymous peer review of modules, allowing them to be cited as peer reviewed publications, is likely to encourage new submissions.     

Inflammation may modulate IL-6 and C-reactive protein gene expression in the adipose tissue: the role of IL-6 cell membrane receptor

Only few studies have been addressed to the presence and regulation of C-reactive protein (CRP) gene expression in different districts of adipose tissue, and no study has investigated the role of adipose tissue in presence of inflammation. Therefore, the aim of this study was to investigate the inflammatory involvement of either adipose tissue or adipose cells (adipocytes and stromal cells, respectively) in patients with chronic inflammatory disease, focusing on regional adipose tissue CRP gene expression. Eighteen patients with inflammatory disease and 14 healthy controls were enrolled. All subjects underwent specific surgical procedures. Inflamed and noninflamed patients provided samples of subcutaneous and/or omental adipose tissue. All samples were analyzed by RT-PCR and real-time PCR for specific gene expression. In addition, both adipocytes and stromal cells were studied by real-time PCR and immunoprecipitation to evaluate either gene or protein expression of CRP. Our results (real-time PCR) demonstrated a higher gene expression of CRP, IL-6, and both IL-6 membrane receptors in subcutaneous samples of inflamed patients than in healthy controls. Furthermore, in omental fragments of inflamed patients, an enhanced mRNA abundance of the same genes, compared with subcutaneous, was observed. The results obtained at cellular level did not provide evidence of any difference between adipocytes and stromal cell CRP gene expression, whereas immunoprecipitation demonstrated the presence of CRP in inflamed subjects. These results provide first-time evidence of the involvement of adipose tissue in the course of chronic inflammatory diseases, with a different degree of participation of the different adipose tissue districts.     

Discovery of a regular nesting area of loggerhead turtle <Emphasis Type="Italic">Caretta caretta</Emphasis> in southern Italy: a new perspective for national conservation

Loggerhead turtle Caretta caretta nesting in Italy had been reported to be limited to the Pelagian Islands and only sporadically elsewhere. As presence of loggerhead turtle nests had occasionally been reported (1988–1999) along about 200 km of the Ionian coast of Calabria, we carried out a project to assess the actual state of the nesting population between 2000 and 2004. We divided the coastline in two sectors (A: 52 km, and B: 146 km) that were monitored from mid-June to end of July for a total of n = 174 monitoring days and 1,813.6 km patrolled on foot with different intensities (extensive versus intensive). In sector B, through extensive monitoring we did not find any emergence tracks, but in sector A by intensive survey (2002–2004: one survey/3.64 days) we detected 3–8 nests/year. In total, 25 nests (both observed and reported), were recorded in our study area, and an assessment of a total of 15–16 nests/year was suggested. These figures, within the national scenario depicted from the review of known nesting events in the last 40 years (88 records concerning more than 143–144 nests), show that loggerhead turtle nesting has been underestimated in Italy, due to inadequate monitoring protocols, and that nesting is more frequent than expected (at least 30–40 nests/year). Conservation strategies in Italy should then focus not only on the reduction of mortality at sea, but also include large-scale actions to preserve scattered (but regular) nesting events.     

Phase studies of model biomembranes: Complex behavior of DSPC/DOPC/Cholesterol

We have undertaken a series of experiments to examine the behavior of individual components of cell membranes. Here we report an initial stage of these experiments, in which the properties of a chemically simple lipid mixture are carefully mapped onto a phase diagram. Four different experimental methods were used to establish the phase behavior of the 3-component mixture DSPC/DOPC/chol: (1) confocal fluorescence microscopy observation of giant unilamellar vesicles, GUVs; (2) FRET from perylene to C20:0-DiI; (3) fluorescence of dilute dyes C18:2-DiO and C20:0-DiI; and (4) wide angle X-ray diffraction. This particular 3-component mixture was chosen, in part, for a high level of immiscibility of the components in order to facilitate solving the phase behavior at all compositions. At 23 °C, a large fraction of the possible compositions for this mixture give rise to a solid phase. A region of 3-phase coexistence of {Lα + Lβ + Lo} was detected and defined based on a combination of fluorescence microscopy of GUVs, FRET, and dilute C20:0-DiI fluorescence. At very low cholesterol concentrations, the solid phase is the tilted-chain phase Lβ′. Most of the phase boundaries have been determined to be within a few percent of the composition. Measurements of the perturbations of the boundaries of this accurate phase diagram could serve as a means to understand the behaviors of a range of added lipids and proteins.     

A three-dimensional microfluidized liver system to assess hepatic drug metabolism and hepatotoxicity

In this study, we propose the design and fabrication of a liver system on a chip. We first chose the most suitable three-dimensional liver-like model between cell spheroids and microtissue precursors, both based on the use of hepatocellular carcinoma cells (HepG2) to provide proof-of-concept data. Spheroids displayed high cell density but low expression of the typical hepatic biomarkers, whereas microtissue precursors showed stable viability and function over the entire culture time. The two liver-like models were compared in terms of cell viability, function, metabolism, and the P-glycoprotein 1 (P-gp) transport-protein expression with the microtissue precursors showing the best performance. Thus, we cultured them into a microfluidic biochip featured with three parallel channels shaped to mimic the hepatic sinusoids. To assess the detoxification potential of the microtissue-loaded biochip we challenged it with a model molecule (ethanol) at different concentrations and time points. Ethanol cytotoxicity was detected by a noninvasive measurement of cell viability based on cell autofluorescence. As expected, a dose-dependent decrease of albumin and urea secretion was observed in the ethanol-treated samples. We believe that the described totally human-derived platform, suitable for integration into a multiorgan microfluidic system, can provide a consistent innovative platform for drug development and toxicity studies.