Osteogenic differentiation of skeletal muscle progenitor cells is activated by the DNA damage response.

Heterotopic ossification (HO) is a pathological condition characterized by the deposition of mineralized tissue in ectopic locations such as the skeletal muscle. The precise cellular origin and molecular mechanisms underlying HO are still debated. In our study we focus on the differentiation of mesoangioblasts (MABs), a population of multipotent skeletal muscle precursors. High-content screening for small molecules that perturb MAB differentiation decisions identified Idoxuridine (IdU), an antiviral and radiotherapy adjuvant, as a molecule that promotes MAB osteogenic differentiation while inhibiting myogenesis. IdU-dependent osteogenesis does not rely on the canonical BMP-2/SMADs osteogenic pathway. At pro-osteogenic conditions IdU induces a mild DNA Damage Response (DDR) that activates ATM and p38 eventually promoting the phosphorylation of the osteogenesis master regulator RUNX2. By interfering with this pathway IdU-induced osteogenesis is severely impaired. Overall, our study suggests that induction of the DDR promotes osteogenesis in muscle resident MABs thereby offering a new mechanism that may be involved in the ectopic deposition of mineralized tissue in the muscle.

Comprehensive experimental and computational analysis of binding energy hot spots at the NF-κB essential modulator/IKKß protein-protein interface.

We report a comprehensive analysis of binding energy hot spots at the protein-protein interaction (PPI) interface between nuclear factor kappa B (NF-κB) essential modulator (NEMO) and IκB kinase subunit ß (IKKß), an interaction that is critical for NF-κB pathway signaling, using experimental alanine scanning mutagenesis and also the FTMap method for computational fragment screening. The experimental results confirm that the previously identified NEMO binding domain (NBD) region of IKKß contains the highest concentration of hot-spot residues, the strongest of which are W739, W741, and L742 (ΔΔG = 4.3, 3.5, and 3.2 kcal/mol, respectively). The region occupied by these residues defines a potentially druggable binding site on NEMO that extends for ~16 Å to additionally include the regions that bind IKKß L737 and F734. NBD residues D738 and S740 are also important for binding but do not make direct contact with NEMO, instead likely acting to stabilize the active conformation of surrounding residues. We additionally found two previously unknown hot-spot regions centered on IKKß residues L708/V709 and L719/I723. The computational approach successfully identified all three hot-spot regions on IKKß. Moreover, the method was able to accurately quantify the energetic importance of all hot-spot residues involving direct contact with NEMO. Our results provide new information to guide the discovery of small-molecule inhibitors that target the NEMO/IKKß interaction. They additionally clarify the structural and energetic complementarity between "pocket-forming" and "pocket-occupying" hot-spot residues, and further validate computational fragment mapping as a method for identifying hot spots at PPI interfaces.

Foliar anatomical and morphological variation in Nothofagus pumilio seedlings under controlled irradiance and soil moisture levels.

Foliar anatomy and morphology are strongly related to physiological performance; therefore, phenotypic plasticity in leaves to variations in environmental conditions, such as irradiance and soil moisture availability, can be related to growth rate and survivorship, mainly during critical growth phases, such as establishment. The aim of this work was to analyze changes in the foliar internal anatomy (tissue proportions and cell dimensions) and external morphology (leaf length, width and area) of Nothofagus pumilio (Poepp. et Endl.) Krasser seedlings growing in a greenhouse under controlled irradiance (three levels) and soil moisture (two levels) during one growing season (measured three times), and to relate them to physiological traits. Three irradiance levels (4, 26 and 64% of the natural incident light) and two soil moisture levels (40 and 80% soil capacity) were evaluated during November, January and March. Internal foliar anatomy of seedlings was analyzed using digital photographs of histological cuttings, while leaf gross morphology was measured using digital calipers and image analysis software. Most internal anatomical variables presented significant differences under different irradiance levels during the growing season, but differences were not detected between soil moisture levels. Palisade parenchyma was the tissue most sensitive to irradiance levels, and high irradiance levels (64% natural incident light) produced greater values in most of the internal anatomical variables than lower irradiance levels (4-24% natural incident light). Complementarily, larger leaves were observed in medium and low irradiance levels, as well as under low soil moisture levels (40% soil capacity). The relationship of main results with some eco-physiological traits was discussed. Foliar internal anatomical and external morphological plasticity allows quick acclimation of seedlings to environmental changes (e.g., during harvesting). These results can be used to propose new forest practices that consider soil moisture and light availability changes to maintain high physiological performance of seedlings.

Latino Parents' and Adolescents' Perceptions of the Needs and Issues of Adolescents in Their Community.

Data from The Community Needs Assessment Survey were examined to understand the issues parents and adolescents felt were most important to address for the adolescents in their community. The sample of 1,784 Latino respondents consisted of 892 parent/adolescent dyads. Factor analyses found parents and adolescents identified and prioritized the same six factors: education and career planning, abuse and victimization, adolescent behavior problems, adolescent sexuality, socioeconomic stressors, and relationships. However, parent ratings for all factors were higher, indicating a higher level of concern. The article analyzes differences by age/generation (parents versus adolescents), by immigrant versus native status, and by type of dyad based on the latter (i.e., US born adolescent/US born parent, US born adolescent/immigrant parent, immigrant adolescent/immigrant parent). The authors discuss the complex interaction of these factors and the implications for practice and research.

Quantitative surface plasmon resonance imaging: a simple approach to automated angle scanning.

Here we present an automated angle-scanning surface plasmon resonance imaging (SPRi) instrument which provides multiplexed, quantitative reflectance data over a wide angular range. Angle-dependent artifacts, which arise from the simple optical setup, are corrected using software. This enables monitoring of significantly different surface coatings in many solvents, which would be outside the dynamic range of typical fixed-angle instruments. Operation in the visible to near-infrared range without the need for reconfiguration extends the instrument capabilities to increase sensitivity or to investigate the optical properties of surface films. This instrument provides maximum flexibility to study a wide range of systems with full exploitation of the quantitative capabilities of SPRi achieved by fitting data to the Fresnel model.

A generic approach for vertical integration of nanowires.

We report on the collective integration technology of vertically aligned nanowires (NWs). Si and ZnO NWs have been used in order to develop a generic technological process. Both mineral and organic planarizations of the as-grown nanowires have been achieved. Chemical vapour deposition (CVD) oxides, spin on glass (SOG), and polymer have been investigated as filling materials. Polishing and/or etching of the composite structures have been set up so as to obtain a suitable morphology for the top and bottom electrical contacts. Electrical and optical characterizations of the integrated NWs have been performed. Contacts ohmicity has been demonstrated and specific contact resistances have been reported. The photoconducting properties of polymer-integrated ZnO NWs have also been investigated in the UV-visible range through collective electrical contacts. A small increase of the resistivity in the ZnO NWs under sub-bandgap illumination has been observed and discussed. A comparison of the photoluminescence (PL) spectra at 300 K of the as-grown and SOG-integrated ZnO nanowires has shown no significant impact of the integration process on the crystal quality of the NWs.

Kinetic discrimination of sequence-specific DNA-drug binding measured by surface plasmon resonance imaging and comparison to solution-phase measurements.

We demonstrate the use of surface plasmon resonance (SPR) imaging for direct detection of small-molecule binding to surface-bound DNA probes. Using a carefully designed array surface, we quantitatively discriminate between the interactions of a model drug with different immobilized DNA binding sites. Specifically, we measure the association and dissociation intercalation rates of actinomycin-D (ACTD) to and from double-stranded 5'-TGCT-3' and 5'-GGCA-3' binding sites. The rates measured provide mechanistic information about the DNA-ACTD interaction; ACTD initially binds nonspecifically to DNA but exerts its activity by dissociating slowly from strong affinity sites. We observe a slow dissociation time of kd-1 = 3300 +/- 100 s for ACTD bound to the strong affinity site 5'-TGCT-3' and a much faster dissociation time (210 +/- 15 s) for ACTD bound weakly to the site 5'-GGCA-3'. These dissociation rates, which differ by an order of magnitude, determine the binding affinity for each site (8.8 x 10(6) and 1.0 x 10(6) M(-1), respectively). We assess the effect the surface environment has on these biosensor measurements by determining kinetic and thermodynamic constants for the same DNA-ACTD interactions in solution. The surface suppresses binding affinities approximately 4-fold for both binding sites. This suppression suggests a barrier to DNA-drug association; ACTD binding to duplex DNA is approximately 100 times slower on the surface than in solution.

Secondary structure effects on DNA hybridization kinetics: a solution versus surface comparison.

The hybridization kinetics for a series of designed 25mer probe-target pairs having varying degrees of secondary structure have been measured by UV absorbance and surface plasmon resonance (SPR) spectroscopy in solution and on the surface, respectively. Kinetic rate constants derived from the resultant data decrease with increasing probe and target secondary structure similarly in both solution and surface environments. Specifically, addition of three intramolecular base pairs in the probe and target structure slow hybridization by a factor of two. For individual strands containing four or more intramolecular base pairs, hybridization cannot be described by a traditional two-state model in solution-phase nor on the surface. Surface hybridization rates are also 20- to 40-fold slower than solution-phase rates for identical sequences and conditions. These quantitative findings may have implications for the design of better biosensors, particularly those using probes with deliberate secondary structure.

Effects of combined dietary supplementation on oxidative and inflammatory status in dyslipidemic subjects.

BACKGROUND AND AIM: Dyslipidemia is one of the main risk factors for atherosclerosis, usually the underlying cause of cardiovascular diseases which are the major cause of morbidity and mortality in developed countries. The aim of this study was to assess the effects and the advantages of a combined dietary supplementation with PUFA n-3, vitamin E, niacin and gamma-oryzanol on lipid profile, inflammatory status and oxidative balance. METHODS AND RESULTS: Fifty-seven dyslipidemic volunteers were randomly assigned to receive: placebo (group A, 19 subjects); PUFA n-3 and vitamin E (group B, 18 subjects); the same as B plus gamma-oryzanol and niacin (group C, 20 subjects). Lipid profile, reactive oxygen species (ROS), total antioxidant capacity (TAC), vitamin E, interleukin 1-beta (IL1-beta), tumor necrosis factor (TNF-alpha) and thromboxane B2 (TXB2) were determined at baseline (T0) and after four months (T1). All dyslipidemic subjects showed, at baseline, oxidative stress and, after four months, all biochemical markers improved significantly in groups treated with dietary supplementation. Particularly in group C all lipid patterns improved significantly. CONCLUSIONS: Our findings demonstrate that the strategy of combining different compounds, which protect each other and act together at different levels of the lipid chain production, improves lipid profile, inflammatory and oxidative status, allowing us to reduce the dose of each compound under the threshold of its side effects.

Quantitative angle-resolved SPR imaging of DNA-DNA and DNA-drug kinetics.

We demonstrate the quantitative characterization of DNA-DNA and DNA-drug interactions by angle-resolved surface plasmon resonance (SPR) imaging. Combining the angle-scanning capabilities of traditional SPR with the spatial definition capabilities of imaging, we directly measure DNA and drug surface coverages and kinetics simultaneously for multiple patterned spots. We find excellent agreement of DNA-DNA hybridization kinetics and thermodynamics measured by both the imaging system and traditional SPR. Instrument response and sensitivity is further demonstrated by successful measurement of association and dissociation kinetics of actinomycin-D binding to a low-density doubled-stranded DNA binding sequence. Without independent calibration, analysis of angle-resolved SPR imaging data yields 2.9 +/- 0.1 drugs per duplex at saturation coverage, consistent with all available duplex binding sites being occupied.

Sequence-dependent DNA immobilization: specific versus nonspecific contributions.

We present results of the first systematic study on in situ sequence-dependent kinetics for short single-strand oligonucleotide surface immobilization. By measuring film coverage for both thiolated and nonthiolated homo-oligomers as a function of adsorption time, we determine the relative contribution of specific thiol-surface and nonspecific DNA-surface interactions to the overall mechanism of DNA-thiol attachment to gold. We find that sequence-dependent nonspecific surface interactions play a significant role in DNA-thiol immobilization, influencing not only the kinetics but also the extent of oligomer adsorption. For example, sequences that initially form strong, rapid nonspecific contacts with the surface hinder long-time thiol adsorption (i.e., poly(dA)-thiol). In contrast, sequences with nucleotides that initially bind slowly and weakly to the surface (i.e., poly(dT)-thiol) do not obstruct further thiol adsorption, resulting in higher film coverage and Langmuir immobilization kinetics. This view of the DNA-thiol immobilization mechanism is further supported by sequence-dependent rinsing losses observed for thiolated DNA strands but not for analogous nonthiolated strands. Nonthiolated strands contact the surface strongly in a more horizontal orientation, whereas thiolated strands attain a more upright orientation that allows vertical DNA-DNA base-stacking. The results clearly illustrate the importance and interplay of competitive specific and nonspecific forces in forming DNA-thiol films. The specific coverage attained and the time dependence of the adsorption process depend on the prevailing sequence composition.

Effect of incremental doses of folate on homocysteine and metabolically related vitamin concentrations in nondiabetic patients on peritoneal dialysis.

The role of folate supplementation in reducing hyperhomocystinemia in patients on dialysis has been reported, but the optimal dose of folate is still unknown. The aim of the present study was to investigate whether greater than 5 mg/day folate supplementation provides any additional effect on plasma homocysteine (HCY) levels. The study was prospective, open, and had no control group. Of the 64 eligible nondiabetic patients on peritoneal dialysis with hyperhomocystinemia (>20 micromol/L), 56 were given oral folate (5 mg/day) for 3 months. When Hcy did not fall below 20 micromol/L, folate doses were increased by 5 mg every 3 months to up to 15 mg/day. With 5 mg/day supplementation, serum folate concentrations increased above the upper confidence limit in 23 patients and erythrocyte folate concentrations in 27 patients. Hcy levels decreased to less than 15 micromol/L in 6 cases and by more than 50% in 12 cases. Nineteen of the remaining patients were given 10 mg/day folate. After increasing the dose, serum and erythrocyte folate levels rose above the upper detection limit. In one patient, plasma Hcy concentrations decreased to less than 15 micromol/L. Ten patients were given 15 mg/day oral folate for an additional 3 months with no effect on homocystinemia. This study confirms that oral folate supplementation may improve hyperhomocystinemia even in patients on dialysis with normal serum or erythrocyte folate concentrations. In fact, serum and erythrocyte levels cannot predict the effect of supplementation on plasma Hcy levels. However, 5 mg/day folate supplementation normalized Hcy in 10% of cases and reduced Hcy levels in another 21%. Increasing the folate dose to greater than 5 mg/day had a minimal (10 mg/day) or no (15 mg/day) additional effect on Hcy concentrations. Despite the minimal effect of increasing folate doses, given the low cost, the absence of side effects, and the high cardiovascular risk for patients on peritoneal dialysis, a careful attempt to increase the dose of oral folate up to 10 mg/day might be suggested.

Hybridization of mismatched or partially matched DNA at surfaces.

We investigate how probe density influences hybridization for unlabeled target oligonucleotides that contain mismatched sequences or targets that access different binding locations on the immobilized probe. We find strong probe density effects influencing not only the efficiency of hybridization but also the kinetics of capture. Probe surfaces are used repeatedly, and the potentially large contributions of sample-to-sample variations in surface heterogeneity and nonspecific adsorption are addressed. Results of kinetic, equilibrium, and temperature-dependent studies, obtained using in-situ surface plasmon resonance (SPR) spectroscopy, show that hybridization for surface immobilized DNA is quite different from the well-studied solution-phase reaction. Surface hybridization depends strongly on the target sequence and probe density. Much of the data can be explained by the presence of steric crowding at high probe density; however, the behavior of mismatched sequences cannot be understood using standard models of hybridization even at the lowest density studied. In addition to unusual capture kinetics observed for the mismatched targets, we find that the binding isotherms can be fit only if a heterogeneous model is used. For mismatched targets, the Sips model adequately describes probe-target binding isotherms; for perfectly matched targets, the Langmuir model can be used.

[Resistance to antiretroviral drugs]. / La resistenza ai farmaci antiretrovirali.

Since the HIV eradication is an utopian goal the only hope we have is the appropriate follow up of the disease by maintaining the RNA viral load under control and the level of CD4+ sufficient for a minimum of immunocompetence guarantee. The international guideline for the management of HIV infected patients, annually updated, recently confirmed the importance of the GART (guided antiretroviral therapy) based on information derived from drug resistance laboratory tests. A general survey on the antiretroviral drugs, on the mutations involving drug resistance and on the tests for their detection could be interesting also for whom not specifically working on the field.

Genotyping in HIV drug resistance mutations: epidemiology in 145 patients.

The epidemiology of HIV-1 genomic mutations causing antiretroviral drug resistance, in 145 HIV-1 infected patients were obtained using a new sequencing technique. All patients were in a follow up treatment for at least 4 months with all drugs available in clinical practice in accordance to international guidelines. The percentage of the mutations were calculated both on the number of all participants and on the number of patients who received the drugs selecting for that specific resistance. It was possible then to evaluate patients who showed the mutation without receiving the drug. We consider this new sequencing method reliable and hopeful in clinical practice, giving the opportunity for a guided therapy for the single patient and in detecting and monitoring the antiretroviral drug resistance mutations in the pertinent geographic area following a periodic surveillance program.