Population analysis of a commercial Saccharomyces cerevisiae wine yeast in a batch culture by electric particle analysis, light diffraction and flow cytometry

Data from electric particle analysis, light diffraction and flow cytometry analysis provide information on changes in cell morphology. Here, we report analyses of Saccharomyces cerevisiae populations growing in a batch culture using these techniques. The size distributions were determined by electric particle analysis and by light diffraction in order to compare their outcomes. Flow cytometry parameters forward (related to cell size) and side (related to cell granularity) scatter were also determined to complement this information. These distributions of yeast properties were analysed statistically and by a complexity index. The cell size of Saccharomyces at the lag phase was smaller than that at the beginning of the exponential phase, whereas during the stationary phase, the cell size converged with the values observed during the lag phase. These experimental techniques, when used together, allow us to distinguish among and characterize the cell size, cell granularity and the structure of the yeast population through the three growth phases. Flow cytometry patterns are better than light diffraction and electric particle analysis in showing the existence of subpopulations during the different phases, especially during the stationary phase. The use of a complexity index in this context helped to differentiate these phases and confirmed the yeast cell heterogeneity.     

Doxorubicin induces mitochondrial permeability transition and contractile dysfunction in the human myocardium

In human atrial trabeculae, we examined the effects of doxorubicin on the isometric force of contraction, mitochondrial respiration, membrane potential and calcium retention capacity. Compared with untreated controls, doxorubicin induced contractile dysfunction and depression of mitochondrial respiration. Mitochondria isolated from doxorubicin-treated human atrial trabeculae displayed reduced transmembrane potential and calcium retention capacity. Cyclosporine A, a mitochondrial membrane transition pore opening blocker, prevented mitochondrial dysfunction and impaired contractile performance induced by doxorubicin. The study suggests that a mitochondrial membrane transition pore opening is involved in the development of doxorubicin cardiotoxicity in human hearts.     

Role of permissive neuraminidase mutations in influenza A/Brisbane/59/2007-like (H1N1) viruses

Neuraminidase (NA) mutations conferring resistance to NA inhibitors were believed to compromise influenza virus fitness. Unexpectedly, an oseltamivir-resistant A/Brisbane/59/2007 (Bris07)-like H1N1 H275Y NA variant emerged in 2007 and completely replaced the wild-type (WT) strain in 2008-2009. The NA of such variant contained additional NA changes (R222Q, V234M and D344N) that potentially counteracted the detrimental effect of the H275Y mutation on viral fitness. Here, we rescued a recombinant Bris07-like WT virus and 4 NA mutants/revertants (H275Y, H275Y/Q222R, H275Y/M234V and H275Y/N344D) and characterized them in vitro and in ferrets. A fluorometric-based NA assay was used to determine Vmax and Km values. Replicative capacities were evaluated by yield assays in ST6Gal1-MDCK cells. Recombinant NA proteins were expressed in 293T cells and surface NA activity was determined. Infectivity and contact transmission experiments were evaluated for the WT, H275Y and H275Y/Q222R recombinants in ferrets. The H275Y mutation did not significantly alter Km and Vmax values compared to WT. The H275Y/N344D mutant had a reduced affinity (Km of 50 vs 12 μM) whereas the H275Y/M234V mutant had a reduced activity (22 vs 28 U/sec). In contrast, the H275Y/Q222R mutant showed a significant decrease of both affinity (40 μM) and activity (7 U/sec). The WT, H275Y, H275Y/M234V and H275Y/N344D recombinants had comparable replicative capacities contrasting with H275Y/Q222R mutant whose viral titers were significantly reduced. All studied mutations reduced the cell surface NA activity compared to WT with the maximum reduction being obtained for the H275Y/Q222R mutant. Comparable infectivity and transmissibility were seen between the WT and the H275Y mutant in ferrets whereas the H275Y/Q222R mutant was associated with significantly lower lung viral titers. In conclusion, the Q222R reversion mutation compromised Bris07-like H1N1 virus in vitro and in vivo. Thus, the R222Q NA mutation present in the WT virus may have facilitated the emergence of NAI-resistant Bris07 variants.     

Quantitative analysis of NIM811, a cyclophilin inhibitor, in human dried blood spots using liquid chromatography-tandem mass spectrometry

A high-performance liquid chromatography-tandem mass spectrometric (LC-MS/MS) method has been developed and validated for the quantitative analysis of NIM811, a cyclophilin inhibitor, in human dried blood spot (DBS) samples, which were produced by spotting 20 μl whole blood onto FTA cards. A 3mm disc was cut from the DBS samples and extracted using methanol, followed by liquid-liquid extraction with MTBE. The reconstituted extracts were chromatographed using a Halo C(18) column and gradient elution for MS/MS detection. The possible impact of hematocrit, blood sample volume and punching location on DBS sampling was investigated. The results showed that blood sample volume or punching location has no impact on assay performance, but the presence of a high hematocrit resulted in significantly increased analyte concentrations measured from the high QC samples. The current method was fully validated over the range of 10.0-5000 ng/ml with correlation coefficients (r(2)) for three validation batches equal to or better than 0.991. The accuracy and precision (CV) at the LLOQ were -0.7 to 6.0% bias of the nominal value (10.0 ng/ml) and 10.2-2.3%, respectively. For the balance of QC samples (20.0, 50.0, 750, 1500 and 3750 ng/ml), the precision (CV) ranged from 3.2 to 11.7% and from 5.6 to 10.2%, respectively, for the intra-day and inter-day evaluations. The accuracy ranged from -6.8 to 8.5% and -0.2% to 2.7% bias, respectively, for the intra-day and inter-day batches. NIM811 is stable in the DBS samples for at least 24h at room temperature and 4h at 60°C. Interestingly, the long term stability (LTS) assessment showed that the stability of the analyte is better when the DBS samples were stored at a lower storage temperature (e.g. ≤ -60°C) compared to storage at room temperature. This is probably due to the interaction of the additives and/or other materials (e.g. cellulose, etc) on the DBS card with NIM811, a cyclic peptide. The current methodology has been applied to determine the NIM811 levels in DBS samples prepared from a clinical study.     

Identification of a Lysine Residue Important for the Catalytic Activity of Yeast Farnesyl Diphosphate Synthase

The Saccharomyces cerevisiae ERG20 gene (encoding farnesyl diphosphate synthase) has been subjected to a set of mutations at the catalytic site, at position K254 to determine the in vivo impact. The mutated strains have been shown to exhibit various growth rates, sterol profiles and monoterpenol producing capacities. The results obtained suggest that K at position 254 helps to stabilize one of the three Mg²⁺ forming a bridge between the enzyme and DMAPP, and demonstrate that destabilizing two of the three Mg²⁺ ions, by introducing a double mutation at positions K197 and K254, results in a loss of FPPS activity and a lethal phenotype.     

CAERUS: predicting CAncER oUtcomeS using relationship between protein structural information, protein networks, gene expression data, and mutation data

Carcinogenesis is a complex process with multiple genetic and environmental factors contributing to the development of one or more tumors. Understanding the underlying mechanism of this process and identifying related markers to assess the outcome of this process would lead to more directed treatment and thus significantly reduce the mortality rate of cancers. Recently, molecular diagnostics and prognostics based on the identification of patterns within gene expression profiles in the context of protein interaction networks were reported. However, the predictive performances of these approaches were limited. In this study we propose a novel integrated approach, named CAERUS, for the identification of gene signatures to predict cancer outcomes based on the domain interaction network in human proteome. We first developed a model to score each protein by quantifying the domain connections to its interacting partners and the somatic mutations present in the domain. We then defined proteins as gene signatures if their scores were above a preset threshold. Next, for each gene signature, we quantified the correlation of the expression levels between this gene signature and its neighboring proteins. The results of the quantification in each patient were then used to predict cancer outcome by a modified naïve Bayes classifier. In this study we achieved a favorable accuracy of 88.3%, sensitivity of 87.2%, and specificity of 88.9% on a set of well-documented gene expression profiles of 253 consecutive breast cancer patients with different outcomes. We also compiled a list of cancer-associated gene signatures and domains, which provided testable hypotheses for further experimental investigation. Our approach proved successful on different independent breast cancer data sets as well as an ovarian cancer data set. This study constitutes the first predictive method to classify cancer outcomes based on the relationship between the domain organization and protein network.     

Pyridoxine supply in human development

Vitamin B(6) has an important role in the function of the human nervous system. Experimental data are not generally available on the role in human development, but significant conclusions may be made from studies of the effect of disorders of B(6) vitamer metabolism. Vitamin B(6) comprises seven compounds - pyridoxal, pyridoxine, pyridoxamine and their respective 5' phosphates. The common active form in human tissue is the 5'-phosphate form of pyridoxal (PLP) most of which is found in muscle bound to phosphorylase. Like many vitamins, B(6) can function both as a co-enzyme and as a chaperone. Pyridoxal-5'-phosphate is the metabolically active form and is involved in 100 enzymatic reactions including carbohydrate, amino acid, and fatty acid metabolism. There is evidence that in some situations B(6) vitamers can function as antioxidants. The fetus is dependent on the placenta for supply of vitamin B(6) and the demand correlates with amino acid metabolism. Few reports are available on the role of B(6) in embryogenesis. Studies of human disorders where B(6) metabolism is blocked show a major role in neurotransmitter function with secondary cerebral and cerebellar hypoplasia. Pyridoxine potentiates vitamin A teratogenicity and an excess leads to peripheral nerve cell degeneration. The key role of vitamin B(6) in the developing human is in metabolism, especially of the neurotransmitters.     

Oceanographic anomalies and sea‐level rise drive mangroves inland in the Pacific coast of Mexico

Question: Although mangrove forests are generally regarded as highly threatened, some studies have shown that mangrove canopies in the Pacific coast of Mexico have been increasing in recent decades. We investigated the possible causes driving this reported mangrove expansion. Location: The mangrove lagoons of Magdalena Bay in Baja California, Mexico. Methods: We used 50‐year‐old aerial photographs and 24‐year‐old satellite images to compare long‐term vegetation change, surveyed a coastal vegetation transect to analyse flooding levels, compiled six decades of tidal and oceanographic information, as well as hurricane data to analyse changes in storm frequency or sea‐level conditions, and used isotopic analysis to date the age of trees along the gradient. Results: A significant increase in mangrove cover has occurred in backwaters of the lagoons during the last 40 years, and especially during the El Niño anomalies of the 1980s and 1990s, while at the same time the mangrove fringe has been receding. Conclusions: The observed change can be attributed to the combined action of the warm surface waters of El Niño events and sea‐level rise. Jointly, these two effects are sufficient to flood large areas of previously non‐flooded salt flats, dispersing mangrove seedlings inland. The inland expansion of mangroves, however, does not ease conservation concerns, as it is the seaward fringes, and not the inland margins, that provide the most valuable environmental services for fisheries and coastal protection.