Expression of soluble and functional full-length human matrix metalloproteinase-2 in Escherichia coli

Characterization of the matrix metalloproteinase-2 (MMP-2) substrates and understanding of its function remain difficult because up to date preparations containing minor amounts of other eukaryotic proteins that are co-purified with MMP-2 are still used. In this work, the expression of a soluble and functional full-length recombinant human MMP-2 (rhMMP-2) in the cytoplasm of Escherichia coli is reported, and the purification of this metalloproteinase is described. Culture of this bacterium at 18 degrees C culminated in maintenance of the soluble and functional rhMMP-2 in the soluble fraction of the E. coli lysate and its purification by affinity with gelatin-sepharose yielded approximately 0.12 mg/L of medium. Western Blotting and zymographic analysis revealed that the most abundant form was the 72-kDa MMP-2, but some gelatinolytic bands corresponding to proteins with lower molecular weight were also detected. The obtained rhMMP-2 was demonstrated to be functional in a gelatinolytic fluorimetric assay, suggesting that the purified rhMMP-2 was correctly folded. The method described here involves fewer steps, is less expensive, and is less prone to contamination with other proteinases and MMP inhibitors as compared to expression of rhMMP-2 in eukaryotic tissue culture. This protocol will facilitate the use of the full-length rhMMP-2 expressed in bacteria and will certainly help researchers to acquire new knowledge about the substrates and biological activities of this important proteinase. (C) 2011 Elsevier B.V. All rights reserved.

From Sample Processing to Quantification: A Full Electrochemical Approach for Neutral Analyte Derivatization, Capillary Electrophoresis Separation, and Contactless Conductivity Detection

A thin-layer electrochemical flow cell coupled to capillary electrophoresis with contactless conductivity detection (EC-CE-(CD)-D-4) was applied for the first time to the derivatization and quantification of neutral species using aliphatic alcohols as model compounds. The simultaneous electrooxidation of four alcohols (ethanol, 1-propanol, 1-butanol, and 1-pentanol) to the corresponding carboxylates was carried out on a platinum working electrode in acid medium. The derivatization step required 1 min at 1.6 V vs. Ag/AgCl under stopped flow conditions, which was preceded by a 10 s activation at 0 V. The solution close to the electrode surface was then hydrodynamically injected into the capillary, and a 2.5 min electrophoretic separation was carried out. The fully automated flow system operated at a frequency of 12 analyses per hour. Simultaneous determination of the four alcohols presented detection limits of about 5 x 10(-5) mol As a practical application with a complex matrix, ethanol concentrations were determined in diluted pale lager beer and in nonalcoholic beer. No statistically significant difference was observed between the EC-CE-(CD)-D-4 and gas chromatography with flame ionization detection (GC-FID) results for these samples. The derivatization efficiency remained constant over several hours of continuous operation with lager beer samples (n = 40).

Treatment of Nuclear-Donor Cells or Cloned Zygotes with Chromatin-Modifying Agents Increases Histone Acetylation But Does Not Improve Full-Term Development of Cloned Cattle

Although somatic cell nuclear transfer (SCNT) is a promising tool, its potential use is hampered by the high mortality rates during the development to term of cloned offspring. Abnormal epigenetic reprogramming of donor nuclei after SCNT is thought to be the main cause of this low efficiency. We hypothesized that chromatin-modifying agents (CMAs) targeting chromatin acetylation and DNA methylation could alter the chromatin configuration and turn them more amenable to reprogramming. Thus, bovine fibroblasts were treated with 5-aza-2'-deoxycytidine (AZA) plus trichostatin (TSA) or hydralazine (HH) plus valproic acid (VPA) whereas, in another trial, cloned bovine zygotes were treated with TSA. The treatment of fibroblasts with either AZA + TSA or HH + VPA increased histone acetylation, but did not affect the level of DNA methylation. However, treatment with HH + VPA decreased cellular viability and proliferation. The use of these cells as nuclear donors showed no positive effect on pre- and postimplantation development. Regarding the treatment of cloned zygotes with TSA, treated one-cell embryos showed an increase in the acetylation patterns, but not in the level of DNA methylation. Moreover, this treatment revealed no positive effect on pre- and postimplantation development. This work provides evidence the treatment of either nuclear donor cells or cloned zygotes with CMAs has no positive effect on pre- and postimplantation development of cloned cattle.

Constitutive models for biodegradable thermoplastic ropes for ligament repair

The concept behind a biodegradable ligament device is to temporarily replace the biomechanical functions of the ruptured ligament, while it progressively regenerates its capacities. However, there is a lack of methods to predict the mechanical behaviour evolution of the biodegradable devices during degradation, which is an important aspect of the project. In this work, a hyper elastic constitutive model will be used to predict the mechanical behaviour of a biodegradable rope made of aliphatic polyesters. A numerical approach using ABAQUS is presented, where the material parameters of the model proposal are automatically updated in correspondence to the degradation time, by means of a script in PYTHON. In this method we also use a User Material subroutine (UMAT) to apply a failure criterion base on the strength that decreases according to a first order differential equation. The parameterization of the material model proposal for different degradation times were achieved by fitting the theoretical curves with the experimental data of tensile tests on fibres. To model all the rope behaviour we had considered one step of homogenisation considering the fibres architectures in an elementary volume. (C) 2012 Elsevier Ltd. All rights reserved.

New Isomers in the Full Seniority Scheme of Neutron-Rich Lead Isotopes: The Role of Effective Three-Body Forces

The neutron-rich lead isotopes, up to Pb-216, have been studied for the first time, exploiting the fragmentation of a primary uranium beam at the FRS-RISING setup at GSI. The observed isomeric states exhibit electromagnetic transition strengths which deviate from state-of-the-art shell-model calculations. It is shown that their complete description demands the introduction of effective three-body interactions and two-body transition operators in the conventional neutron valence space beyond Pb-208.

The full Fisher matrix for galaxy surveys

Starting from the Fisher matrix for counts in cells, we derive the full Fisher matrix for surveys of multiple tracers of large-scale structure. The key step is the classical approximation, which allows us to write the inverse of the covariance of the galaxy counts in terms of the naive matrix inverse of the covariance in a mixed position-space and Fourier-space basis. We then compute the Fisher matrix for the power spectrum in bins of the 3D wavenumber , the Fisher matrix for functions of position (or redshift z) such as the linear bias of the tracers and/or the growth function and the cross-terms of the Fisher matrix that expresses the correlations between estimations of the power spectrum and estimations of the bias. When the bias and growth function are fully specified, and the Fourier-space bins are large enough that the covariance between them can be neglected, the Fisher matrix for the power spectrum reduces to the widely used result that was first derived by Feldman, Kaiser & Peacock. Assuming isotropy, a fully analytical calculation of the Fisher matrix in the classical approximation can be performed in the case of a constant-density, volume-limited survey.

Effects of Working Full-Time and Studying in the Evening Hours Among Young Apprentices and Trainees

Objective: This research aims to assess apprentices' and trainees' work conditions, psychosocial factors at work, as well as health symptoms after joining the labor force. Background: Despite the fact that there are over 3.5 million young working students in Brazil, this increasing rate brings with it difficult working conditions such as work pressure, heavy workloads, and lack of safety training. Method: This study was carried out in a nongovernmental organization (NGO) with 40 young members of a first job program in the city of Sao Paulo, Brazil. They filled out a comprehensive questionnaire focused on sociodemographic variables, working conditions, and health symptoms. Individual and collective semi-structured interviews were conducted. Empirical data analysis was performed using analysis of content. Results: The majority of participants mentioned difficulties in dealing with the pressure and their share of responsibilities at work. Body pains, headaches, sleep deprivation during the workweek, and frequent colds were mentioned. Lack of appropriate task and safety training contributed to the occurrence of work injuries. Conclusion: Having a full-time job during the day coupled with evening high school attendance may jeopardize these people's health and future. Application: This study can make a contribution to the revision and implementation of work training programs for adolescents. It can also help in the creation of more sensible policies regarding youth employment.

Niobium Effects on the Recrystallization Behavior on a Full Austenitic 15Cr-15Ni Stainless Steel Investigated Using Adaptive Neural Networks (ANN)

A new series of austenitic stainless steels-Nb stabilized, without Mo additions, non-susceptible to delta ferrite formation and devoid of intemetallic phases (sigma and chi), without deformation induced martensite is being developed, aiming at high temperature applications as well as for corrosive environments. The base steel composition is a 15Cr-15Ni with normal additions of Nb of 0.5, 1.0 and 2 wt%. Mechanical properties, oxidation and corrosion resistance already have been invetigated in previous papers. In this paper, the effects of Nb on the SFE, strain hardening and recrystallization resistance are evaluated with the help of Adaptive Neural Networks (ANN).

A familial case with interstitial 2q36 deletion: Variable phenotypic expression in full and mosaic state

Submicroscopic chromosomal anomalies play an important role in the etiology of craniofacial malformations, including midline facial defects with hypertelorism (MFDH). MFDH is a common feature combination in several conditions, of which Frontonasal Dysplasia is the most frequently encountered manifestation; in most cases the etiology remains unknown. We identified a parent to child transmission of a 6.2 Mb interstitial deletion of chromosome region 2q36.1q36.3 by array-CGH and confirmed by FISH and microsatellite analysis. The patient and her mother both presented an MFDH phenotype although the phenotype in the mother was much milder than her daughter. Inspection of haplotype segregation within the family of 2q36.1 region suggests that the deletion arose on a chromosome derived from the maternal grandfather. Evidences based on FISH, microsatellite and array-CGH analysis point to a high frequency mosaicism for presence of a deleted region 2q36 occurring in blood of the mother. The frequency of mosaicism in other tissues could not be determined. We here suggest that the milder phenotype observed in the proband's mother can be explained by the mosaic state of the deletion. This most likely arose by an early embryonic deletion in the maternal embryo resulting in both gonadal and somatic mosaicism of two cell lines, with and without the deleted chromosome. The occurrence of gonadal mosaicism increases the recurrence risk significantly and is often either underestimated or not even taken into account in genetic counseling where new mutation is suspected. (C) 2012 Elsevier Masson SAS. All rights reserved.

Development and optimization of biodegradable films based on achira flour

The influence of glycerol concentration (C-g), process temperature (T-p), drying temperature (T-s), and relative humidity (RH) on the properties of achira flour films was initially assessed. The optimized process conditions were C-g of 17g glycerol/100g flour, T-p of 90 degrees C, T-s of 44.8 degrees C, and RH of 36.4%. The films produced under these conditions displayed high mechanical strength (7.0 MPa), low solubility (38.3%). and satisfactory elongation values (14.6%). This study showed that achira flour is a promising source for the development of biodegradable films with good mechanical properties, low water vapor permeability, and solubility compared to films based on other tubers. (c) 2011 Elsevier Ltd. All rights reserved.

Cassava starch biodegradable films: Influence of glycerol and clay nanoparticles content on tensile and barrier properties and glass transition temperature

In this this study, glycerol content and its incorporation method on tensile and barrier properties of biodegradable films (BF) based on cassava starch were analyzed. ANOVA showed that the glycerol incorporation method did not influence the results (P > 0.05), however the glycerol content influenced significantly the tensile and barrier properties of the films (P < 0.05). Films prepared with lower glycerol content presented better tensile and barrier properties than films with higher content. Films were then prepared with addition of clay nanoparticles and their tensile and barrier properties and glass transition temperature were measured. ANOVA indicated that both glycerol and clay nanoparticles influenced significantly the tensile and barrier properties (P < 0.05), diminishing film permeability when clay nanoparticles were present, while the glass transition temperature was not influenced (P > 0.05). (C) 2011 Elsevier Ltd. All rights reserved.

Late clinical outcomes after implantation of drug-eluting stents coated with biodegradable polymers: 3-year follow-up of the PAINT randomised trial

Aims: The long-term clinical performance of drug-eluting stents (DES) coated with biodegradable polymers is poorly known. Methods and results: A total of 274 coronary patients were randomly allocated to paclitaxel-eluting stents, sirolimus-eluting stents, or bare metal stems (2:2:1 ratio). The two DES used the same biodegradable polymers and were identical except for the drug. At three years, the pooled DES population had similar rates of cardiac death or myocardial infarction (9.0% vs. 7.1; p=0.6), but lower risk of repeat interventions (10.0% vs. 29.9%; p<0.01) than controls with bare stents. The cumulative 3-year incidence of definite or probable stent thrombosis in the pooled DES group was 2.3% (first year: 1.8%; second year: 0.4%; third year: zero). There were no significant differences in outcomes between paclitaxel- and sirolimus-eluting stents. Conclusions: The biodegradable-polymer coated DES releasing either paclitaxel or sirolimus were effective in reducing the 3-year rate of re-interventions.

Differences in walking attainment ages between low-risk preterm and healthy full-term infants

Objective: To compare gross motor development of preterm infants (PT) without cerebral palsy with healthy full-term (FT) infants, according to Alberta Infant Motor Scale (AIMS); to compare the age of walking between PT and FT; and whether the age of walking in PT is affected by neonatal variables. Methods: Prospective study compared monthly 101 PT and 52 FT, from the first visit, until all AIMS items had been observed. Results: Mean scores were similarity in their progression, except from the eighth to tenth months. FT infants were faster in walking attainment than PT. Birth weight and length and duration of neonatal nursery stay were related to walking delay. Conclusion: Gross motor development between PT and FT were similar, except from the eighth to tenth months of age. PT walked later than FT infants and predictive variables were birth weight and length, and duration of neonatal intensive unit stay.

Electrophysiological study of hearing in full-term small-for-gestational-age newborns

PURPOSE: To describe the Brainstem Auditory Evoked Potential (BAEP) results of full-term small-for-gestational-age newborns, comparing them to the results of full-term appropriate-for-gestational-age newborns, in order to verify whether the small-for-gestational-age condition is a risk indicator for retrocochlear hearing impairment. METHODS: This multicentric prospective cross-sectional study assessed 86 full-term newborns - 47 small- (Study Group) and 39 appropriate-for-gestational-age (Control Group - of both genders, with ages between 2 and 12 days. Newborns with presence of transient evoked otoacoustic emissions and type A tympanometry were included in the study. Quantitative analysis was based on the mean and standard deviation of the absolute latencies of waves I, III and V and interpeak intervals I-III, III-V and I-V, for each group. For qualitative analysis, the BAEP results were classified as normal or altered by analyzing these data considering the age range of the newborn at the time of testing. RESULTS: In the Study Group, nine of the 18 (38%) subjects with altered BAEP results had the condition of small-for-gestational-age as the only risk factor for hearing impairments. In the Control Group, seven (18%) had altered results. Female subjects from the Study Group tended to present more central alterations. In the Control Group, the male group tended to have more alterations. CONCLUSION: Full-term children born small or appropriate for gestational age might present transitory or permanent central hearing impairments, regardless of the presence of risk indicators.