Segregation of a M404V mutation of the p62/sequestosome 1 (p62/SQSTM1) gene with polyostotic Paget's disease of bone in an Italian family

Mutations of the p62/Sequestosome 1 gene (p62/SQSTM1) account for both sporadic and familial forms of Paget's disease of bone (PDB). We originally described a methionine-->valine substitution at codon 404 (M404V) of exon 8, in the ubiquitin protein-binding domain of p62/SQSTM1 gene in an Italian PDB patient. The collection of data from the patient's pedigree provided evidence for a familial form of PDB. Extension of the genetic analysis to other relatives in this family demonstrated segregation of the M404V mutation with the polyostotic PDB phenotype and provided the identification of six asymptomatic gene carriers. DNA for mutational analysis of the exon 8 coding sequence was obtained from 22 subjects, 4 PDB patients and 18 clinically unaffected members. Of the five clinically ascertained affected members of the family, four possessed the M404V mutation and exhibited the polyostotic form of PDB, except one patient with a single X-ray-assessed skeletal localization and one with a polyostotic disease who had died several years before the DNA analysis. By both reconstitution and mutational analysis of the pedigree, six unaffected subjects were shown to bear the M404V mutation, representing potential asymptomatic gene carriers whose circulating levels of alkaline phosphatase were recently assessed as still within the normal range. Taken together, these results support a genotype-phenotype correlation between the M404V mutation in the p62/SQSTM1 gene and a polyostotic form of PDB in this family. The high penetrance of the PDB trait in this family together with the study of the asymptomatic gene carriers will allow us to confirm the proposed genotype-phenotype correlation and to evaluate the potential use of mutational analysis of the p62/SQSTM1 gene in the early detection of relatives at risk for PDB.     

Effects of extremely low frequency electromagnetic fields on membrane-associated enzymes

The effects of extremely low frequency electromagnetic fields of 75 Hz were studied on different membrane-associated enzymes. Only the activities of three enzymes out of seven exposed to the field decreased approximately of about 54-61% with field amplitudes above a threshold of 73-151 microT depending on the enzyme. The same field had no effect on the activities of either integral membrane enzymes such as Ca,ATPase, Na/K,ATPase, and succinic dehydrogenase or peripheral membrane enzymes such as photoreceptor PDE. The decrease in enzymatic activity of the field-sensitive enzymes was independent of the time of permanence in the field and was completely reversible. When these enzymes were solubilized with Triton, no effect of the field was obtained on the enzymatic activity, suggesting the crucial role of the membrane in determining the conditions for enzyme inactivation. The role of the particular linkage of the field-sensitive enzymes to the membranes is also discussed.     

Dermal fibroblasts from pseudoxanthoma elasticum patients have raised MMP-2 degradative potential

Cultured fibroblasts from the dermis of normal subjects and of Pseudoxanthoma elasticum (PXE) patients were analysed for enzyme activity, protein and mRNA expression of metalloproteases (MMP-2, MMP-3, MMP-9, MT1-MMP) and of their specific inhibitors (TIMP-1, TIMP-2 and TIMP-3). MMP-3, MMP-9 and TIMP-3 mRNAs and proteins failed to be detected in both the medium and the cell layer of both controls and PXE patients. MMP-2 mRNA was significantly more expressed in PXE than in control cell lines, whereas MT1-MMP, TIMP-1 and TIMP-2 mRNAs appeared unchanged. MMP-2 was significantly higher in the cell extracts from PXE fibroblasts than in control cells, whereas differences were negligible in the cell medium. Data suggest that PXE fibroblasts have an increased proteolytic potential, and that MMP-2 may actively contribute to connective tissue alterations in this genetic disorder.     

Evaluating markers for selecting a patient's treatment

Selecting the best treatment for a patient's disease may be facilitated by evaluating clinical characteristics or biomarker measurements at diagnosis. We consider how to evaluate the potential impact of such measurements on treatment selection algorithms. For example, magnetic resonance neurographic imaging is potentially useful for deciding whether a patient should be treated surgically for Carpal Tunnel Syndrome or should receive less-invasive conservative therapy. We propose a graphical display, the selection impact (SI) curve that shows the population response rate as a function of treatment selection criteria based on the marker. The curve can be useful for choosing a treatment policy that incorporates information on the patient's marker value exceeding a threshold. The SI curve can be estimated using data from a comparative randomized trial conducted in the population as long as treatment assignment in the trial is independent of the predictive marker. Estimating the SI curve is therefore part of a post hoc analysis to determine whether the marker identifies patients that are more likely to benefit from one treatment over another. Nonparametric and parametric estimates of the SI curve are proposed in this article. Asymptotic distribution theory is used to evaluate the relative efficiencies of the estimators. Simulation studies show that inference is straightforward with realistic sample sizes. We illustrate the SI curve and statistical inference for it with data motivated by an ongoing trial of surgery versus conservative therapy for Carpal Tunnel Syndrome.     

Partially supervised learning using an EM-boosting algorithm

Training data in a supervised learning problem consist of the class label and its potential predictors for a set of observations. Constructing effective classifiers from training data is the goal of supervised learning. In biomedical sciences and other scientific applications, class labels may be subject to errors. We consider a setting where there are two classes but observations with labels corresponding to one of the classes may in fact be mislabeled. The application concerns the use of protein mass-spectrometry data to discriminate between serum samples from cancer and noncancer patients. The patients in the training set are classified on the basis of tissue biopsy. Although biopsy is 100% specific in the sense that a tissue that shows itself to have malignant cells is certainly cancer, it is less than 100% sensitive. Reference gold standards that are subject to this special type of misclassification due to imperfect diagnosis certainty arise in many fields. We consider the development of a supervised learning algorithm under these conditions and refer to it as partially supervised learning. Boosting is a supervised learning algorithm geared toward high-dimensional predictor data, such as those generated in protein mass-spectrometry. We propose a modification of the boosting algorithm for partially supervised learning. The proposal is to view the true class membership of the samples that are labeled with the error-prone class label as missing data, and apply an algorithm related to the EM algorithm for minimization of a loss function. To assess the usefulness of the proposed method, we artificially mislabeled a subset of samples and applied the original and EM-modified boosting (EM-Boost) algorithms for comparison. Notable improvements in misclassification rates are observed with EM-Boost.     

IS THE GROWTH RATE HYPOTHESIS APPLICABLE TO MICROALGAE?1

The growth rate hypothesis (GRH) asserts, from known biochemistry, that maintaining high growth rates requires high concentrations of ribosomes. Since ribosomes are rich in phosphorus (P), the GRH predicts a positive correlation between growth rate and P content; this correlation is observed in some organisms. We consider the application of the GRH to phytoplankton and identify several key problems that require further research before the hypothesis can be accepted for these organisms. There are severe methodological problems that confound interpretation of data for testing the GRH. These problems include the measurement of protein and nucleic acids (such that ratio of these components carries a high level of uncertainty), studies of steady‐state versus dynamic systems, and the presentation of data per cell (especially as cell size varies with growth rate limitations) and the calculation of growth rates. In addition, because of the short generation times and rapid responses of these organisms to perturbations, ribosome and RNA content is expected to vary in response to (de)repression of various systems; content may increase on application of growth‐limiting stress. Finally, that most phytoplankton accumulate P when not P stressed conflicts with the GRH. In consequence, the value of the GRH for any sort of predictive role in nature appears to be severely limited. We conclude that the GRH cannot be assumed to apply to phytoplankton taxa without first performing experimental tests under transient conditions.     

Limitations on microalgal growth at very low photon fluence rates: the role of energy slippage

The lower limits of photosynthetically useable radiation at which growth and photosynthesis can occur establish the lower boundaries for the extent of photolithotrophy in the biosphere. Photolithotrophic growth denotes the capacity to grow with photons as the sole energy input. Slippage in terms of photosynthetic energy conversion implies a less than theoretical stoichiometry of energy-transduction process(es) such as the dissipation of intermediates of O₂ evolution and of ATP synthesis (H⁺/e⁻ and H⁺/ATP ratios). Slippage is particularly important in limiting the growth of photolithotrophic organisms at very low photon fluence rates. We found that Dunaliella tertiolecta and Phaeodactylum tricornutum avoid such reductions in photon use efficiency by increasing the size and number of their photosynthetic units, respectively, and by altering Q_A reduction kinetics on the reducing side of PS II. P. tricornutum is also less susceptible to slippage in terms of the breakdown of intermediates in its O₂ evolution pathway than D. tertiolecta. Minimizing H⁺ leakage through the CF₀–CF₁ ATP synthetase (and other H⁺ porters) is also discussed briefly. In combination, strategies employed by P.␣tricornutum effectively allow it to grow and photosynthesize at lower rates of energy input than D. tertiolecta, consistent with our observations. Differences in the responses of the photosynthetic apparatus of these two marine microalgae are mechanistic and probably representative of evolutionary divergences associated with strategies for dealing with environmental perturbations.     

Effect of polyamines on in vitro anther culture of Citrus clementina Hort. ex Tan

The improvement of the induction rate in Citrus anther culture is important for taking practical advantage of the haploid potential in breeding. The influence of polyamines on anther culture of Citrus clementina, cv Nules, with particular attention to the free, soluble and insoluble-conjugated polyamine levels, has been investigated. Putrescine, spermidine and putrescine plus spermidine, were added to the standard induction medium. Before culture, spermidine was the most abundant among the free polyamines detected in anthers. The exogenous supply of either putrescine or spermidine, either independently or combined, effected greater uptake and accumulation of polyamines. The addition of 2 mM spermidine to the medium stimulated gametic embryogenesis in clementine Nules, whereas putrescine did not influence embryo production. Regenerants were mostly tri-haploids; a few doubled-haploids and no haploid plants were obtained.     

Pressure affects the structure and the dynamics of the D-galactose/D-glucose-binding protein from Escherichia coli by perturbing the C-terminal domain of the protein

The effect of the pressure on the structure and stability of the D-galactose/D-glucose binding protein from Escherichia coli in the absence (GGBP) and in the presence (GGBP/Glc) of glucose was studied by Fourier transform infrared (FT-IR) spectroscopy and molecular dynamic (MD) simulations. FT-IR spectroscopy experiments showed that the protein beta-structures are more resistant than alpha-helices structures to pressure value increases. In addition, the infrared data indicated that the binding of glucose stabilizes the protein structure against high pressure values, and the protein structure does not completely unfold up to pressure values close to 9000 bar. MD simulations allow a prediction of the most probable configuration of the protein, consistent with the increasing pressures on the two systems. The detailed analysis of the structures at molecular level confirms that, among secondary structures, alpha-helices are more sensitive than beta-structures to the destabilizing effect of high pressure and that glucose is able to preserve the structure of the protein in the complex. Moreover, the evidence of the different resistance of the two domains of this protein to high pressure is investigated and explained at a molecular level, indicating the importance of aromatic amino acid in protein stabilization.