Accuracy and safety of 3D printed surgical guides combined with monitored guidewires for placement of cervicothoracic pedicle screws: Technical note.

Subaxial cervical pedicle screws provide rigid fixation, but their placement poses an important neurovascular injury risk. 3D printed guides have successfully been used to place pedicle screws, but experience in the subaxial cervical spine is limited. We present a case of cervicothoracic dissociation after a pathological fracture due to tumour involvement of the upper thoracic spine, causing paraparesis and intense pain. The cervicothoracic junction is of difficult visualization on fluoroscopy and the patients' severe instability made navigation unreliable. 3D printed individualized guidewire guides were used to help place canulated pedicle screws from C4 to T6. We successfully report the use of impedance guidewire monitoring to prevent pedicle violation and improve procedure safety.

Analysis of in situ manganese(II) oxidation in the Columbia River and offshore plume: linking Aurantimonas and the associated microbial community to an active biogeochemical cycle.

The Columbia River is a major source of dissolved nutrients and trace metals for the west coast of North America. A large proportion of these nutrients are sourced from the Columbia River Estuary, where coastal and terrestrial waters mix and resuspend particulate matter within the water column. As estuarine water is discharged off the coast, it transports the particulate matter, dissolved nutrients and microorganisms forming nutrient-rich and metabolically dynamic plumes. In this study, bacterial manganese oxidation within the plume and estuary was investigated during spring and neap tides. The microbial community proteome was fractionated and assayed for Mn oxidation activity. Proteins from the outer membrane and the loosely bound outer membrane fractions were separated using size exclusion chromatography and Mn(II)-oxidizing eluates were analysed with tandem mass spectrometry to identify potential Mn oxidase protein targets. Multi-copper oxidase (MCO) and haem-peroxidase enzymes were identified in active fractions. T-RFLP profiles and cluster analysis indicates that organisms and bacterial communities capable of oxidizing Mn(II) can be sourced from the Columbia River estuary and nearshore coastal ocean. These organisms are producing up to 10 fM MnO2 cell⁻¹ day⁻¹. Evidence for the presence of Mn(II)-oxidizing bacterial isolates from the genera Aurantimonas, Rhodobacter, Bacillus and Shewanella was found in T-RFLP profiles. Specific Q-PCR probes were designed to target potential homologues of the Aurantimonas manganese oxidizing peroxidase (Mop). By comparing total Mop homologues, Aurantimonas SSU rRNA and total bacterial SSU rRNA gene copies, it appears that Aurantimonas can only account for ~1.7% of the peroxidase genes quantified. Under the broad assumption that at least some of the peroxidase homologues quantified are involved in manganese oxidation, it is possible that other organisms oxidize manganese via peroxidases.

Culturable Rhodobacter and Shewanella species are abundant in estuarine turbidity maxima of the Columbia River.

Measurements of dissolved, ascorbate-reducible and total Mn by ICP-OES revealed significantly higher concentrations during estuarine turbidity maxima (ETM) events, compared with non-events in the Columbia River. Most probable number (MPN) counts of Mn-oxidizing or Mn-reducing heterotrophs were not statistically different from that of other heterotrophs (10³ -104 cells ml⁻¹) when grown in defined media, but counts of Mn oxidizers were significantly lower in nutrient-rich medium (13 cells ml⁻¹). MPN counts of Mn oxidizers were also significantly lower on Mn(III)-pyrophosphate and glycerol (21 cells ml⁻¹). Large numbers of Rhodobacter spp. were cultured from dilutions of 10⁻² to 10⁻5, and many of these were capable of Mn(III) oxidation. Up to c. 30% of the colonies tested LBB positive, and all 77 of the successfully sequenced LBB positive colonies (of varying morphology) yielded sequences related to Rhodobacter spp. qPCR indicated that a cluster of Rhodobacter isolates and closely related strains (95-99% identity) represented approximately 1-3% of the total Bacteria, consistent with clone library results. Copy numbers of SSU rRNA genes for either Rhodobacter spp. or Bacteria were four to eightfold greater during ETM events compared with non-events. Strains of a Shewanella sp. were retrieved from the highest dilutions (10⁻5) of Mn reducers, and were also capable of Mn oxidation. The SSU rRNA gene sequences from these strains shared a high identity score (98%) with sequences obtained in clone libraries. Our results support previous findings that ETMs are zones with high microbial activity. Results indicated that Shewanella and Rhodobacter species were present in environmentally relevant concentrations, and further demonstrated that a large proportion of culturable bacteria, including Shewanella and Rhodobacter spp., were capable of Mn cycling in vitro.

Implicit solvation in the self-consistent mean field theory method: sidechain modelling and prediction of folding free energies of protein mutants.

The Atomic Solvation Parameter (ASP) model is one of the simplest models of solvation, in which the solvation free energy of a molecule is proportional to the solvent accessible surface area (SAS) of its atoms. However, until now this model had not been incorporated into the Self-Consistent Mean Field Theory (SCMFT) method for modelling sidechain conformations in proteins. The reason for this is that SAS is a many-body quantity and, thus, it is not obvious how to define it within the Mean Field (MF) framework, where multiple copies of each sidechain exist simultaneously. Here, we present a method for incorporating an SAS-based potential, such as the ASP model, into SCMFT. The theory on which the method is based is exact within the MF framework, that is, it does not depend on a pairwise or any other approximation of SAS. Therefore, SAS can be calculated to arbitrary accuracy. The method is computationally very efficient: only 7.6% slower on average than the method without solvation. We applied the method to the prediction of sidechain conformation, using as a test set high-quality solution structures of 11 proteins. Solvation was found to substantially improve the prediction accuracy of well-defined surface sidechains. We also investigated whether the methodology can be applied to prediction of folding free energies of protein mutants, using a set of barnase mutants. For apolar mutants, the modest correlation observed between calculated and observed folding free energies without solvation improved substantially when solvation was included, allowing the prediction of trends in the folding free energies of this type of mutants. For polar mutants, correlation was not significant even with solvation. Several other factors also responsible for the correlation were identified and analysed. From this analysis, future directions for applying and improving the present methodology are discussed.

Conformational component in the coupled transfer of multiple electrons and protons in a monomeric tetraheme cytochrome.

Cell metabolism relies on energy transduction usually performed by complex membrane-spanning proteins that couple different chemical processes, e.g. electron and proton transfer in proton-pumps. There is great interest in determining at the molecular level the structural details that control these energy transduction events, particularly those involving multiple electrons and protons, because tight control is required to avoid the production of dangerous reactive intermediates. Tetraheme cytochrome c(3) is a small soluble and monomeric protein that performs a central step in the bioenergetic metabolism of sulfate reducing bacteria, termed "proton-thrusting," linking the oxidation of molecular hydrogen with the reduction of sulfate. The mechano-chemical coupling involved in the transfer of multiple electrons and protons in cytochrome c(3) from Desulfovibrio desulfuricans ATCC 27774 is described using results derived from the microscopic thermodynamic characterization of the redox and acid-base centers involved, crystallographic studies in the oxidized and reduced states of the cytochrome, and theoretical studies of the redox and acid-base transitions. This proton-assisted two-electron step involves very small, localized structural changes that are sufficient to generate the complex network of functional cooperativities leading to energy transduction, while using molecular mechanisms distinct from those established for other Desulfovibrio sp. cytochromes from the same structural family.

[Study of post-myocardial infarction ventricular aneurysms with equilibrium radionuclide angiography. Significance of Fourier analysis]. / Estudo dos aneurismas ventriculares pós-enfarte do miocárdio pela angiografia de radionuclidos de equilíbrio. Importância da análise de Fourier.

The concept of left ventricular aneurysm has been a subject of controversy and it's not yet completely settled. This has some implications on the patients selection for surgery and explains the various non-invasive methods so far developed for the clinical assessment of patients with ventricular aneurysms. Seventy-one patients with ischemic heart disease, 65 with recent myocardial infarction, were studied by equilibrium radionuclide angiography (ERNA) in order to define left ventricular wall motion abnormalities. One hundred ERNA were undertaken, through the usual erytrocites in vivo labelling technic, employing a GE 400 T Gama-Camera GP. Image acquisition was through 400 cardiac cycles, on LAO (30 and 60 degrees) and left lateral. PAGE protocol was employed. Fourier transform was used in the present work to obtain phase and amplitude images, which actually became the main criteria to define aneurysmatic areas. Global ejection fraction, regional wall motion and regional ejection fraction were other parameters investigated. Wall motion abnormalities have been identified in all the 65 patients having suffered a myocardial infarction. Extensive areas of akinesia or localized dyskinesia were present in 40 patients (16.5%), while remainder 25 had just localized hypoakinesia. Phase image enabled the selection of LV areas of contraction delay in 19 of the 40 patients with extensive wall motion abnormalities (Group I). In such Group I we could identify an LV area with contrasting colour, defining the aneurysmatic LV portion. In the order 21 patients with extensive akinesia, no significant changes of colour were present on ventricular phase image (Group II) meaning absence of aneurysm. No phase disturbances were seen in the remainder 25 patients with MI (Group III) and the 6 patients with CAD without MI had normal phase images (Group IV). The percentage of akinetic segments was 39.1 and 35.4 in Group I and II, respectively (p = .53) while it was significantly lower in Group III 17.9%; p < .0001). LV ejection fraction was statistically different in the four groups considered (I = 30.0% +/- 3.7; II = 39.9% +/- 2.9; III = 49.0% +/- 2.5; IV = 62.0% +/- 3.2). The degree of phase delay in aneurysmatic zones was quantified by the phase histogram. Average value of phase for the left ventricle was 129.7 +/- 8.4 degrees, and for the aneurysm it was 238.0 +/- 5.0 with an average phase delay of 104.8 +/- 4.1 degrees. The association of phase and amplitude images and the measurements of phase and amplitude values on the histograms allowed the distinction of akinetic aneurysm (phase delay and low amplitude) from the dyskinetic aneurysms (phase delay and high amplitude). Half of the isotopic diagnosis of aneurysms have been confirmed by classic contrast angiography, and a third of then have undergone surgery. The two dyskinetic aneurysms that have been submitted to aneurysmectomy had pos-operative improvement of LV function. On summary, phase and amplitude analysis by the Fourier method, which is independent of LV geometry, showed that LV regions with high phase values are associated with severe regional wall motion abnormalities and low ejections fractions. The definition of the sequence and amplitude of ventricular contraction allows the distinction of akinetic and dyskinetic aneurysms, anticipating the method as a valid contribution for the screening of patients likely to benefit from aneurysmectomy.

The use of a modular titanium endoprosthesis in skeletal reconstructions after bone tumor resections: method presentation and analysis of 37 cases.

We analyzed 37 patients who underwent segmental wide resection of bone tumors and reconstruction with a modular titanium endoprosthesis at the Orthopaedic Oncology Group, between 1992 and 1998. Twelve patients were male and 25 were female, with a mean age of 30 years (9 - 81). The mean follow-up was 14 months (2 - 48). The diagnoses were: osteosarcoma (14 cases), metastatic carcinoma (10), Ewing's sarcoma (4), giant cell tumor (4), malignant fibrous histiocytoma (3), chondrosarcoma (1), and aneurysmal bone cyst (1). Eleven articulated total knee, 8 partial proximal femur with bipolar acetabulum, 8 partial proximal humerus, 3 total femur, 2 partial proximal tibia, 2 diaphyseal femur, 2 diaphyseal humerus, and 1 total proximal femur with cementless acetabulum endoprosthesis implant procedures were done. The complications related to the procedure included: infection (5 cases), dislocation (3), module loosening (1), and ulnar nerve paresthesia (1). We used the following criteria for the clinical evaluation: presence of pain, range of motion, reconstruction stability, surgical and oncologic complications, and patient acceptance. The results were good in 56.8% of the cases, regular in 32.4% and poor in 10.8%.

Comparative redox and pKa calculations on cytochrome c3 from several Desulfovibrio species using continuum electrostatic methods.

A comparative study of the pH-dependent redox mechanisms of several members of the cytochrome c3 family has been carried out. In a previous work, the molecular determinants of this dependency (the so-called redox-Bohr effect) were investigated for one species using continuum electrostatic methods to find groups with a titrating range and strength of interaction compatible with a mediating role in the redox-Bohr effect. Here we clarify these aspects in the light of new and improved pKa calculations, our findings supporting the hypothesis of propionate D from heme I being the main effector in the pH-dependent modulation of the cytochrome c3 redox potentials in all the c3 molecules studied here. However, the weaker (but significant) role of other titrating groups cannot be excluded, their importance and identity changing with the particular molecule under study. We also calculate the relative redox potentials of the four heme centers among the selected members of the c3 family, using a continuum electrostatic method that takes into account both solvation and interaction effects. Comparison of the calculated values with available data for the microscopic redox potentials was undertaken, the quality of the agreement being dependent upon the choice of the dielectric constant for the protein interior. We find that high dielectric constants give best correlations, while low values result in better magnitudes for the calculated potentials. The possibility that the crystallographic calcium ion in c3 from Desulfovibrio gigas may be present in the solution structure was tested, and found to be likely.

Simulation of electron-proton coupling with a Monte Carlo method: application to cytochrome c3 using continuum electrostatics.

A new method is presented for simulating the simultaneous binding equilibrium of electrons and protons on protein molecules, which makes it possible to study the full equilibrium thermodynamics of redox and protonation processes, including electron-proton coupling. The simulations using this method reflect directly the pH and electrostatic potential of the environment, thus providing a much closer and realistic connection with experimental parameters than do usual methods. By ignoring the full binding equilibrium, calculations usually overlook the twofold effect that binding fluctuations have on the behavior of redox proteins: first, they affect the energy of the system by creating partially occupied sites; second, they affect its entropy by introducing an additional empty/occupied site disorder (here named occupational entropy). The proposed method is applied to cytochrome c3 of Desulfovibrio vulgaris Hildenborough to study its redox properties and electron-proton coupling (redox-Bohr effect), using a continuum electrostatic method based on the linear Poisson-Boltzmann equation. Unlike previous studies using other methods, the full reduction order of the four hemes at physiological pH is successfully predicted. The sites more strongly involved in the redox-Bohr effect are identified by analysis of their titration curves/surfaces and the shifts of their midpoint redox potentials and pKa values. Site-site couplings are analyzed using statistical correlations, a method much more realistic than the usual analysis based on direct interactions. The site found to be more strongly involved in the redox-Bohr effect is propionate D of heme I, in agreement with previous studies; other likely candidates are His67, the N-terminus, and propionate D of heme IV. Even though the present study is limited to equilibrium conditions, the possible role of binding fluctuations in the concerted transfer of protons and electrons under nonequilibrium conditions is also discussed. The occupational entropy contributions to midpoint redox potentials and pKa values are computed and shown to be significant.

Improved modeling of side-chains in proteins with rotamer-based methods: a flexible rotamer model.

Side-chain modeling has a widespread application in many current methods for protein tertiary structure determination, prediction, and design. Of the existing side-chain modeling methods, rotamer-based methods are the fastest and most efficient. Classically, a rotamer is conceived as a single, rigid conformation of an amino acid sidechain. Here, we present a flexible rotamer model in which a rotamer is a continuous ensemble of conformations that cluster around the classic rigid rotamer. We have developed a thermodynamically based method for calculating effective energies for the flexible rotamer. These energies have a one-to-one correspondence with the potential energies of the rigid rotamer. Therefore, the flexible rotamer model is completely general and may be used with any rotamer-based method in substitution of the rigid rotamer model. We have compared the performance of the flexible and rigid rotamer models with one side-chain modeling method in particular (the self-consistent mean field theory method) on a set of 20 high quality crystallographic protein structures. For the flexible rotamer model, we obtained average predictions of 85.8% for chi1, 76.5% for chi1+2 and 1.34 A for root-mean-square deviation (RMSD); the corresponding values for core residues were 93.0%, 87.7% and 0.70 A, respectively. These values represent improvements of 7.3% for chi1, 8.1% for chi1+2 and 0.23 A for RMSD over the predictions obtained with the rigid rotamer model under otherwise identical conditions; the corresponding improvements for core residues were 6.9%, 10.5% and 0.43 A, respectively. We found that the predictions obtained with the flexible rotamer model were also significantly better than those obtained for the same set of proteins with another state-of-the-art side-chain placement method in the literature, especially for core residues. The flexible rotamer model represents a considerable improvement over the classic rigid rotamer model. It can, therefore, be used with considerable advantage in all rotamer-based methods commonly applied to protein tertiary structure determination, prediction, and design and also in predictions of free energies in mutational studies.

The origin of trypsin: evidence for multiple gene duplications in trypsins.

The trypsin family of serine proteases is one of the most studied protein families, with a wealth of amino acid sequence information available in public databases. Since trypsin-like enzymes are widely distributed in living organisms in nature, likely evolutionary scenarios have been proposed. A novel methodology for Fourier transformation of biological sequences (FOTOBIS) is presented. The methodology is well suited for the identification of the size and extent of short repeats in protein sequences. In the present paper the trypsin family of enzymes is analyzed with FOTOBIS and strong evidence for tandem gene duplication is found. A likely evolutionary path for the development of present-day trypsins involved an intrinsic extensive tandem gene duplication of a small DNA fragment of 15-18 nucleotides, corresponding to five or six amino acids. This ancestral trypsin gene was subsequently duplicated, leading to the earliest version of a full-sized trypsin, from which the contemporary trypsins have developed.

Evaluating isovelocity surface area flow convergence method with finite element modeling.

Through numerical experimentation we investigated the isovelocity surface area flow convergence method used in estimating regurgitant valve flow rates. Recent advances in three-dimensional color Doppler flow imaging have created renewed interest in this method. Experimentation was based on the use of depth-averaged finite element models of the left heart. The heart models studied varied from "synthetic" representations to a model of a left heart traced from an actual echocardiographic image of a patient with a prolapsed mitral valve. The isovelocity surface area flow convergence method overestimated regurgitant flow rates throughout the Nyquist limits considered with a critical Nyquist limit in which this overestimation is minimized. The angle dependence of Doppler color flow imaging partially corrects for this overestimation. The isovelocity surface area flow convergence method is a viable alternative to methods currently in use. Through numerical experimentation, we have begun to shed light on the inaccuracies inherent in this flow convergence method.

[Ankle arthrodesis: critical analysis of three techniques]. / Artrodese do tornozelo: análise crítica de três técnicas.

The authors analyze the randomized use of three ankle arthrodesis techniques. Indications for this procedure, which are pain, instability, deformity and articular degeneration, are common to the three groups. In group I the Gatellier-Adams technique was used by transfibular approach. In group II the Kenneth-Johnson technique was adopted, using Calandruccio external fixator. The same surgical procedure was adopted in group III, using crossed canulated screws as fixation device. The specific advantages and disadvantages of each method are discussed. The criteria for evaluation of the results are the final roentgenographic position of the foot and the achievement of union. The critical analysis conclude that the major indications for group I technique are cases of "in situ" arthrodesis, for group II infected cases and for group III cases with severe malalignment.

A study of the contribution of changes in the cerebral blood volume to the haemodynamic response to anoxia in rat brain.

A susceptibility contrast agent which does not pass into the extra-cellular space was used to study the effect of changes in the relative cerebral blood volume (CBV) on the haemodynamic response to anoxia, for both normal and ischaemic brain tissue, in a rat model of acute focal ischaemia. In non-ischaemic tissue a strong CBV component was observed in the haemodynamic response, both during and after anoxia. During anoxia the change in the CBV of the non-ischaemic tissue was estimated to be 40% in the caudate putamen and 70% in the frontal-parietal cortex. For severely ischaemic tissue (ischaemic caudate putamen) there was no change in the CBV during anoxia while in areas of moderate ischaemia (ischaemic frontal parietal cortex) a change of 20% was observed. The effect of the contrast agent on spin-echo images was consistent with a small reduction in the microvascular blood volume of the ischaemic tissue.

Simulation of protein conformational freedom as a function of pH: constant-pH molecular dynamics using implicit titration.

Solution pH is a determinant parameter on protein function and stability, and its inclusion in molecular dynamics simulations is attractive for studies at the molecular level. Current molecular dynamics simulations can consider pH only in a very limited way, through a somewhat arbitrary choice of a set of fixed charges on the titrable sites. Conversely, continuum electrostatic methods that explicitly treat pH effects assume a single protein conformation whose choice is not clearly defined. In this paper we describe a general method that combines both titration and conformational freedom. The method is based on a potential of mean force for implicit titration and combines both usual molecular dynamics and pH-dependent calculations based on continuum methods. A simple implementation of the method, using a mean field approximation, is presented and applied to the bovine pancreatic trypsin inhibitor. We believe that this constant-pH molecular dynamics method, by correctly sampling both charges and conformation, can become a valuable help in the understanding of the dependence of protein function and stability on pH.

Quantification of mitral flow by Doppler color flow mapping.

This study was performed to develop and validate Doppler color flow methods for quantifying forward transmitral flow rates and volumes with isovelocity aliasing contours. We undertook computer modeling of flows and studied an animal model with strictly controlled mitral flows. Finite element analysis was first used to establish the isovelocity surface contours reconstructed from the magnitudes and directions of the velocity vectors proximal to the normal mitral orifice. We modeled finite element-simulated Doppler color flow isovelocity surfaces and computed non-angle-dependent simulated isovelocities to compare them. Then 24 pharmacologically induced hemodynamic states in six sheep in which mitral regurgitation had been previously created surgically were studied. Three methods were used for peak flow (PF) computation: (1) the classic hemispheric methods: PF = 2 pi r2.aliasing velocity; (2) a modified hemispheric method: PF = 2 pi r2.aliasing velocity Vo/Vo-aliasing velocity; and (3) a new segment of sphere method: PF = pi p2.aliasing velocity, where p is the chord from the zenith of the first aliasing contour to the circumference at its base. Mean volume flow was also calculated in combination with phasic flow information from continuous-wave Doppler echocardiography: mean volume flow = PF.VTI/Vmax.heart rate, where VTI and Vmax are the velocity-time integral and maximal velocity of mitral inflow by continuous-wave Doppler echocardiography. Compared with the flow rates obtained by electromagnetic flowmeters, different correlations and agreements were achieved for these methods. Correlation (r = 0.86) and agreement were best for the segment of sphere method for computation of forward mean volume flows in our model. Color flow Doppler quantitation with a segment of sphere or modified hemispheric method appears applicable for quantification of forward transmitral valve flow rates and volumes with reasonable accuracy.

A study of the contribution of changes in the cerebral blood volume to the haemodynamic response to anoxia in rat brain.

A susceptibility contrast agent which does not pass into the extra-cellular space was used to study the effect of changes in the relative cerebral blood volume (CBV) on the haemodynamic response to anoxia, for both normal and ischaemic brain tissue, in a rat model of acute focal ischaemia. In non-ischaemic tissue a strong CBV component was observed in the haemodynamic response, both during and after anoxia. During anoxia the change in the CBV of the non-ischaemic tissue was estimated to be 40% in the caudate putamen and 70% in the frontal-parietal cortex. For severely ischaemic tissue (ischaemic caudate putamen) there was no change in the CBV during anoxia while in areas of moderate ischaemia (ischaemic frontal parietal cortex) a change of 20% was observed. The effect of the contrast agent on spin-echo images was consistent with a small reduction in the microvascular blood volume of the ischaemic tissue.

Cerebrovascular changes in rats during ischemia and reperfusion: a comparison of BOLD and first pass bolus tracking techniques.

Both first pass bolus tracking of a susceptibility contrast agent and blood oxygenation level dependent (BOLD) sequences provide information on the tissue perfusion and the cerebral blood volume, but each sequence has its own particular limitations. In this article, both techniques were used to assess the cerebrovascular changes occurring in a rat model of focal cerebral ischemia with reperfusion after 2 h of ischemia. The blood oxygenation level dependent studies were performed before, during, and after 60 s of anoxia to observe the response of the tissue to a respiratory challenge. Both techniques were able to detect ischemia and reperfusion; however, first pass bolus tracking provided better sensitivity and was easier to interpret. Because the blood oxygenation level dependent sequence did not provide any additional information, bolus tracking would appear to be the method of choice for studies of cerebral ischemia with reperfusion.

[Renal artery angioplasty]. / Angioplastia da artéria renal.

In the 59 hypertensive patients submitted to percutaneous transluminal angioplasty (PTA) of the renal artery, there was an immediate success in the blood pressure in 91.5% and a later one of 79.6%. In these patients we obtained better results: 81.4% in the unilateral lesions, more than in the bilateral ones--72.7%; 82.5% in the renal artery trunk lesions, more than in the ostium ones--71.4%; 88.9% in the lesions of fibromuscular origin, more than in the aterosclerotic ones--75%; 84.4% in up to 55 years old patients, more than in older ones--71.4%. These differences were not significant. The results of renal angioplasty in renovascular hypertension suggest this type of intervention as an alternative treatment.