Three new satellite sequences and a mobile element found inside HSP70 introns of the Mediterranean mussel (Mytilus galloprovincialis).

We report the characterization of 3 new repetitive sequences from the bivalve mollusc Mytilus galloprovincialis, designated Mg1, Mg2, and Mg3, with monomer lengths of 169, 260, and 70 bp, respectively. The 3 repeats together constitute approximately 7.8% of the M. galloprovincialis genome and were found, together with ApaI-type 2 repeats, inside the introns of 2 genes of the HSP70 family, hsc70 and hsc71. Both the monomer length and the genomic content of the repeats indicate satellite sequences. The Mg1 repetitive region and its flanking sequences exhibit significant homology to CvE, a member of the Pearl family of mobile elements found in the eastern oyster (Crassostrea virginica). Thus, the whole homologous region is designated MgE, the first putative transposable element characterized in M. galloprovincialis. The ApaI, Mg2, and Mg3 repeats are continuously arranged inside the introns of both the hsc70 and hsc71 genes. The presence of perfect inverted repeats flanking the ApaI-Mg2-Mg3 repetitive region, as well as a sequence analysis of the repeats, indicates a transposition-like insertion of this region. The genes of the HSP70 family are highly conserved, and the presence of repetitive DNA or of mobile elements inside their introns is reported here for the first time.     

Microdamage evaluation in human trabecular bone based on nonlinear ultrasound vibro-modulation (NUVM)

The primary aim of this work is to investigate the potential of nonlinear ultrasound for microdamage detection in human bone. Microdamage evaluation in human bone is of great importance, because it is considered a significant parameter for characterizing fracture risk. Experiments employing nonlinear acoustic vibro-modulation were carried out in human femoral trabecular specimens removed during surgery. A frequency mixing (inter-modulation) was observed between an ultrasound wave, propagating in the bone, and a low-frequency vibration applied directly to the bone specimens. The appearance of side frequencies, which are related to the vibrational excitation, around the fundamental ultrasound frequency manifests the modulation nonlinear phenomenon. Instead of inducing microdamage by mechanical fatigue loading, specimens with different degree of osteoporosis were used. The experiments demonstrated that osteoporotic bone exhibits stronger nonlinearity compared to healthy bone presenting significant increase of the modulation amplitude with increasing degree of osteoporosis. The obtained results indicate that, in contrast to conventional hysteretic nonlinearity, dissipative acoustic nonlinearity can be of significance in the generation of nonlinear modulation effects. In the proposed technique the size and the shape of samples are not crucial compared to nonlinear resonant ultrasound spectroscopy (NRUS). Furthermore, the method is sensitive to the presence of microdamage, non-invasive, easy to implement and most important, it can be proved valuable tool for in vivo bone damage characterization.     

Static and fatigue strength of a fixation device transducer for measuring anterior cruciate ligament graft tension

To determine which exercises do not overload the graft-fixation complex during intensive rehabilitation from reconstructive surgery of the anterior cruciate ligament (ACL), it would be useful to measure ACL graft loads during rehabilitative activities in vivo in humans. A previous paper by Ventura et al. (1998) reported on the design of an implantable transducer integrated into a femoral fixation device and demonstrated that the transducer could be calibrated to measure graft loads to better than 10 percent full-scale error in cadaveric knees. By measuring both the static and fatigue strengths of the transducer, the purpose of the present study was to determine whether the transducer could be safely implanted in humans without risk of structural failure. Eight devices were loaded to failure statically. Additionally, seven devices were tested using the up-and-down method to estimate the median fatigue strength at a life of 225,000 cycles. The average ultimate strength was 1856 +/- 74 N and the median fatigue strength was 441 N at a life of 225,000 cycles. The maximum graft load during normal daily activities is estimated to be 500 N and the 225,000 cycle life corresponds to that of the average healthy individual during a 12-week period. Considering that patients who have had an ACL reconstruction are less ambulatory than normal immediately following surgery and that biologic incorporation of the graft should be well developed by 12 weeks thus decreasing the load transmitted to the fixation device, the FDT can be safely implanted in humans without undue risk of structural failure.     

Cathepsin B promotes both motility and invasiveness of oral carcinoma cells

We previously demonstrated that overexpression of cathepsin B (CB) protease in oral squamous cell carcinomas correlated positively with advanced tumor stage and poor histologic malignancy grade. Here we examined whether CB contributes to the invasiveness of oral carcinoma cells. For RNA-mediated inhibition, two ribozymes that target CB mRNA were designed and stably expressed in the oral squamous cell carcinoma cell line 1386Tu. Both ribozymes diminished expression of CB mRNA, protein, and activity, without affecting cathepsin D or beta-actin, as determined by quantitative real-time PCR, Western blots, and protease activity assays. Matrigel invasion assays showed that the invasiveness of the cells was significantly reduced by the expressed ribozymes and, surprisingly, the motilities of the ribozyme-transfected cells were also diminished. Our results document a direct role for CB in promoting oral cancer spread and invasion, and open the possibility of controlling oral carcinoma malignancy and metastasis by targeting CB with RNA inhibitor strategies.     

ATTRACT: protein-protein docking in CAPRI using a reduced protein model

Protein-protein complex structures have been predicted for CAPRI Rounds 3 and 5 using a reduced protein model. Proteins are represented by up to 3 pseudoatoms per amino acid. The docking approach termed ATTRACT is based on energy minimization in translational and rotational degrees of freedom of one protein with respect to another protein. The reduced protein model allows one to perform systematic docking minimization of many thousand start structures in reasonable computer time. Flexibility of critical surface side-chains can be accounted for by a multiple conformational copy approach. The multicopy approach allows simultaneous adjustment of side-chain conformations and optimization of translational and rotational degrees of freedom of one protein with respect to the partner during docking. For 3 (Targets 8, 14, and 19) out of 5 CAPRI targets, the approach resulted in predictions in close agreement with experiment [root-mean-square deviation (RMSD) of backbone atoms within 10 A of the protein-protein interface < 1.8 A]. The comparison of predicted and experimental structures of the CAPRI targets indicates that besides local conformational changes (e.g., changes in side-chain conformations), global conformational changes of the protein backbone can be critical for complex formation. These conformational changes not accounted for during docking are a likely reason for the unrealistic predictions in 2 cases (Targets 9 and 18).     

Preservation Methods Alter Carbon and Nitrogen Stable Isotope Values in Crickets (Orthoptera: Grylloidea)

Stable isotope analysis (SIA) is an important tool for investigation of animal dietary habits for determination of feeding niche. Ideally, fresh samples should be used for isotopic analysis, but logistics frequently demands preservation of organisms for analysis at a later time. The goal of this study was to establish the best methodology for preserving forest litter-dwelling crickets for later SIA analysis without altering results. We collected two cricket species, Phoremia sp. and Mellopsis doucasae, from which we prepared 70 samples per species, divided among seven treatments: (i) freshly processed (control); preserved in fuel ethanol for (ii) 15 and (iii) 60 days; preserved in commercial ethanol for (iv) 15 and (v) 60 days; fresh material frozen for (vi) 15 and (vii) 60 days. After oven drying, samples were analyzed for δ ¹⁵N, δ ¹³C values, N(%), C(%) and C/N atomic values using continuous flow isotope ratio mass spectrometry. All preservation methods tested, significantly impacted δ ¹³C and δ ¹⁵N and C/N atomic values. Chemical preservatives caused δ ¹³C enrichment as great as 1.5‰, and δ ¹⁵N enrichment as great as 0.9‰; the one exception was M. doucasae stored in ethanol for 15 days, which had δ ¹⁵N depletion up to 1.8‰. Freezing depleted δ ¹³C and δ ¹⁵N by up to 0.7 and 2.2‰, respectively. C/N atomic values decreased when stored in ethanol, and increased when frozen for 60 days for both cricket species. Our results indicate that all preservation methods tested in this study altered at least one of the tested isotope values when compared to fresh material (controls). We conclude that only freshly processed material provides adequate SIA results for litter-dwelling crickets.