Designing a surgical device for harvesting autologous fascia lata grafts as a minimal invasive procedure]

AIMS: Fascia lata is used in different shapes and sizes as a graft material in surgical procedures. The conventional method of harvesting a fascia lata graft is through a long skin incision on the lateral aspect of the thigh. Minimal invasive procedures have been established to reduce the disadvantages of an extensive surgical approach for obtaining the autotransplant. However, they do not facilitate to suture the remaining fascia after harvesting the transplant and therefore bear the risk of a symptomatic herniation of the muscle belly. The aim of this study was to design a surgical device to harvest a fascia lata graft and close the resulting fascia defect as a minimal invasive procedure. MATERIALS AND METHODS: The prototype was tested in 11 human cadaver specimens. It was introduced subcutanously via two small skin incisions. The device contained a special fixation- and working mechanism which enabled the fascial closure using a continuous suture. After the harvest procedure, both the transplant and the sutured fascia lata were examined. RESULTS: The experiments demonstrated the suitability of this method for minimal invasive harvesting of fascia lata. The removed transplants complied in all experiments with the expected dimensions. The continuous suture of the femoral fascia ran with accurate gaps between the sutures and constant tension without dehiscence. Neither the transplant nor the tissue in the region of harvest have shown unduly macroscopic damage due to the use of the device. CONCLUSION: The designed prototype can be used for harvesting a fascia lata graft and repairing the resulting defect minimal invasively. Clinical implementation seems possible. However, improvements could be made mainly concerning the handling of the device.     

Development of a procedure for coupling the homing device glu-plasminogen to liposomes.

The aim of this study was to find a suitable way of coupling the homing-device glu-plasminogen to the outside of liposomes. The described procedure is based on the reaction of thiol-groups introduced in the protein with thiol-reactive groups of the liposome. Details on the thiolation of proteins with the reagent succinimidyl-S-acetylthioacetate (SATA) were studied for a model-protein, amylase. Increasing the incubation-ratio SATA: amylase resulted in a gradually growing number of introduced thiol-groups, until a maximum of about 5 mol SH per mol amylase was reached. The enzymatic activity of the derivatized protein was even higher than that of native amylase. The thiol-introduction was then applied to glu-plasminogen itself. After activation with SATA, the protein was incubated with liposomes containing the thiol-reactive anchor maleimido-4-(p-phenylbutyrate)-phosphatidylethanolamine (MPB-PE). Under the chosen conditions, incubation of 0.5-2.5 mg/ml protein with 6.0-7.5 mumol/ml phospholipid for 30-120 min resulted in coupling-ratios of 20 to 94 micrograms glu-plasminogen per mumol phospholipid. This corresponds with about 140 to 660 protein molecules per liposome. SATA-derivatization of glu-plasminogen brought about a loss of its enzymatic activity induced by streptokinase. This activity of liposomally coupled plasminogen was about 52 to 74% of the activity of native glu-plasminogen (depending on the coupling-ratio). Although this may seem a significant loss of activity, it was shown that the capacity of liposomal glu-plasminogen to bind to its target, fibrin, was not reduced but several fold higher under the used conditions than that of the free protein. Therefore, the described method for thiol-introduction is an effective way to thiolate amylase without loss of activity, and to bind the homing-device glu-plasminogen to liposomes without substantially interfering with its fibrin-binding/homing capacity.     

Acitivity measurements as a tool to characterize the microbial composition of methanogenic environments

A rapid and reliable method to assess the potential specific activity of methagenic sludge is presented. The method is based on the gas chromatographic analysis for methane in the headspace of closed vials. Gas is sampled with a pressure lock syringe, which allows quantification independent of the pressure prevailing in the vials.The influence of various parameters as substrates, pH, NaCl and NH₄ Cl concentrations on the activity of methaogenic sludge was investigated with this method. Data on the methanogenic activities on different substrate are discussed in terms of different physiological groups present in the sludge.     

Extended push-out test to characterize the failure of bone-implant interface]

To study the mechanical behaviour of the implant-bone interface the push- or pull-out test was overtaken from material science. Most authors equate the maximum load (break point) with the failure of the implant integration. Extending the test procedure by acoustic emission analysis reveals the possibility to detect the failure of the interface more in detail and from its earliest beginning. The development of disconnection between host and implant was found to start long before the ultimate load is reached and can be monitored and quantified during this period. The active interface mechanisms are characterized by the distribution function of acoustic emissions and the number of hits per time defines the kinetics of the failure. From clinical studies a gradual subsidence of loaded implants is known starting long time before the definite implant failure. The presented extension of the push-out test with acoustic emission analysis allows the detection of a critical shear stress tc which demarks the onset of the gradual interface failure. We believe this value to represent the real critical load which should not be exceeded in the clinical application of intraosseous implants.     

Capillary electrophoresis-mass spectrometry as a characterization tool for therapeutic proteins

With the increasing use of capillary electrophoresis (CE) in the biotechnology industry, there is a demand for analytical tools and methodology that can be used to characterize CE profiles. This article describes the implementation and optimization of a robust online CE-mass spectrometry (CE-MS) system used for the characterization of several CE assays developed at Genentech Inc. These assays include CE as a complement to reverse-phase peptide mapping for the identification of small peptides eluting in the void volume, profiling N-linked glycopeptide heterogeneity, and determining O-linked site occupancy. In addition, CE-MS was used to confirm major 8-aminopyrene-1,3,6-trisulfonate (APTS)-labeled glycans released from recombinant antibodies that are routinely profiled by CE-laser-induced fluorescence (CE-LIF). For each study, CE-MS was able to successfully identify components seen in UV or LIF electropherograms, thereby expanding the capability of CE and CE-MS for profiling biomolecules.     

Sensitivity to polyvalent therapeutic staphylococcal bacteriophage as a supplementary criterion of staphylococcal pathogenicity]

It is recommended to use the capacity of pathogenic staphylococci to be lysed by polyvalent therapeutic staphylococcal bacteriophage in the capacity of an additional simple and accessible criterion of staphylococcus pathogenicity. Of 147 strains of the pathogenic plasmacoagulating staphylococci 101 were lysed by the phage and of 166 nonplasmocoagulating nonpathogenic strains--only 6. This test correlated with the other signs of staphylococcus (lecithinase and hemolytic activity). The simplicity and sufficient specificity of this test permits to use it in any practical laboratory. Polyvalent diagnostic phage can be used on the basis of therapeutic bacteriophage by its additional adaptation to the pathogenic strains of staphylococcus.     

Physical and photophysical characterization of a BODIPY phosphatidylcholine as a membrane probe

Lipids containing the dimethyl BODIPY fluorophore are used in cell biology because their fluorescence properties change with fluorophore concentration (C.-S. Chen, O. C. Martin, and R. E. Pagano. 1997. Biophys J. 72:37-50). The miscibility and steady-state fluorescence behavior of one such lipid, 1-palmitoyl-2-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-sn-glycero-3-phosphocholine (PBPC), have been characterized in mixtures with 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC). PBPC packs similarly to phosphatidylcholines having a cis-unsaturated acyl chain and mixes nearly ideally with SOPC, apparently without fluorophore-fluorophore aggregation. Increasing PBPC mole fraction from 0.0 to 1.0 in SOPC membranes changes the emission characteristics of the probe in a continuous manner. Analysis of these changes shows that emission from the excited dimethyl BODIPY monomer self quenches with a critical radius of 25.9 A. Fluorophores sufficiently close (< or =13.7 A) at the time of excitation can form an excited dimer, emission from which depends strongly on total lipid packing density. Overall, the data show that PBPC is a reasonable physical substitute for other phosphatidylcholines in fluid membranes. Knowledge of PBPC fluorescence in lipid monolayers has been exploited to determine the two-dimensional concentration of SOPC in unilamellar, bilayer membranes.     

A measuring device for calculating electrical impedance of the heart in clinical conditions]

Changes in the electrical impedance of tissue can indicate structural changes. This suggests a technique for the noninvasive detection of allograft rejection after heart transplantation. The direct electrical connection to the heart and the application of a measuring current to the myocardium requires a high standard of safety. A device was developed for measuring cardiac impedance using a sinusoidal current of 20 microA at a frequency of 15 kHz. The control logic ensures a slow current onset and also an immediate cessation in case of conductor fracture or excessive voltage. Initial results in patients with normal recovery after heart transplantation revealed a rapid drop in impedance to about 70% of the initial value in the 1st 48 hours and then a stable course. In the sole rejection episode observed so far, the impedance increased again to 85% of the initial value. This paper discusses the technical safety requirements and the design of the device, and presents initial results of clinical examinations.     

Aspergillus nidulans as a model system to characterize the DNA damage response in eukaryotes

Interest in DNA repair in Aspergillus nidulans had mainly grown out of studies of three different biological processes, namely mitotic recombination, inducible responses to detrimental environmental changes, and genetic control of the cell cycle. Ron Morris started the investigation of the genetic control of the cell cycle by screening hundreds of cell cycle temperature sensitive Aspergillus mutants. The sequencing and innovative analysis of these genes revealed not only several components of the cell cycle machinery that are directly involved in checkpoint response, but also components required for DNA replication and DNA damage response machinery. Here, we will provide an overview about currently known aspects of the DNA damage response in A. nidulans. Emphasis is put on analyzed mutants that are available and review epistatic relationships and other interactions among them. Furthermore, a comprehensive list of A. nidulans genes involved in different processes of the DNA damage response, as identified by homology of genome sequences with well-characterized human and yeast DNA repair genes, is shown.     

Microdialysis as a procedure for evaluating intestinal hypoxia--an animal experiment approach]

Usual ICU monitoring of patients with abdominal pathology provides no detailed information about hepatosplanchnic haemodynamics or intestinal metabolism. In our animal experiment, the effects of systemic hypoxia on microdialysis measurements of the peritoneum in comparison with the ischiocrural muscle as reference were investigated in 7 rats. The parameter of interest was the course of glucose metabolism reflecting sympathoadrenergic activity during the experiment. Measurements were obtained at timed intervals at baseline, under hypoxia, and during reoxygenation. After induction of systemic hypoxia, the peritoneal microdialysis showed significantly higher levels of glucose in comparison with the ischiocrural muscle. The results indicate hypermetabolic activity or a hypersympathetic response of the bowel in response to hypoxic stress. In the clinical setting, the bowel has an important role in the development of multiorgan failure. Microdialysis may therefore be an interesting tool for the early detection of hypoxic metabolism during and after abdominal procedures.     

Using Drosophila eye as a model system to characterize the function of mars gene in cell-cycle regulation

Human hepatoma up-regulated protein (HURP), a cell-cycle regulator, is found consistently overexpressed in human hepatocellular carcinoma. At present, the function of HURP in cell-cycle regulation and carcinogenesis remains unclear. In database mining, we have identified a mars gene in Drosophila, which encodes a protein with a high similarity to HURP in its guanylate kinase-associated protein (GKAP) motif. Overexpression but not down-regulation of mars in eye discs resulted in a higher mitotic index along with a high frequency of mitotic defects, including misalignment of chromosomes and mispositioned centrosomes, at the second mitotic wave (SMW). The consequence of mitotic defects impairs cell-cycle progression, and causes cell death posterior to the furrow. Immunocytochemical studies also have indicated that the expression of Mars is cell cycle regulated, and that its subcellular localization is dynamically changed during cell-cycle progression. Furthermore, we also demonstrated that the first 198 amino acids at the N-terminus of Mars are responsible for the degradation of Mars in non-mitotic cells. Together, we report the use Drosophila eye as a model system to characterize the function of the mars gene in cell-cycle regulation.     

Feasibility of using a magnetic tracking device for measuring carpal kinematics

While several different methods have been used to measure carpal kinematics, biplanar radiography is generally considered to be the most accurate and popular one. However, biplanar radiography is tedious and so only pseudo-dynamic kinematics can be measured. Recently, magnetic tracking system has been developed for the measurement of joint kinematics which is versatile and easy to use and so the possibility of measuring motions dynamically. In this study, the capability of a magnetic tracking device to accurately measure carpal kinematics was investigated by comparing it with biplanar radiography. The kinematics of the third metacarpal, scaphoid, and lunate in five fresh cadaveric specimens were measured using both methods as the wrists were placed in eight positions. The finite screw rotation of each bone with respect to the distal radius during selecting the seven wrist motions was calculated for both measuring techniques and compared. In general, the kinematics for all three bones measured by using either magnetic tracking device or biplanar radiography was identical and showed no statistical difference. The averaged differences ranged from 0.0 to 2.0°. These differences were due to the potential effect of the weight of the sensors and the interference of the attaching rod to the surrounding tissue. It is concluded that the application of the magnetic tracking device to carpal kinematics is warranted, if proper technical procedures as suggested are followed.     

Physical characterization of a phytodewatered anaerobic sludge

Some physical parameters were determined on an organic product derived from a new phytodewatering process of anaerobic sludge, in order to verify its possible use as a substrate. Wetting-drying cycles were carried out with a sandy soil, and with mixtures of this soil with the organic product taken before and after the phytodewatering process. The phytodewatering process decreased bulk density while raising aggregate stability and friability.     

The identification of mouse sperm-surface-associated proteins and characterization of their ability to act as decapacitation factors

Mammalian spermatozoa must undergo capacitation before acquiring the ability to fertilize the oocyte. This process is believed to be initiated following the release of surface-associated decapacitation factors that are elaborated by both the epididymis and the male accessory organs. Herein, we report the identification of a number of proteins that are actively released from the surface of mouse spermatozoa during capacitation in vitro. As anticipated, the addition of these factors back to suspensions of mouse spermatozoa was shown to suppress several correlates of the capacitation process. Specifically, they induced a significant, dose-dependent inhibition of the ability of spermatozoa to undergo a progesterone-induced acrosome reaction and to bind to the zona pellucida in vitro. Inhibition of these functions was associated with the suppression of tyrosine phosphorylation in the sperm plasma membrane but had no effect on the phosphorylation of internal proteins in either the sperm head or tail. This inhibitory activity was attributed to a subset of the isolated proteins compromising at least four putative decapacitation factors. These proteins were identified via tandem-mass spectrometry amino acid sequence analysis as plasma membrane fatty acid binding protein, cysteine-rich secretory protein 1 (CRISP1), phosphatidylethanolamine binding protein 1 (PBP), and an unnamed protein product that we have termed decapacitation factor 10 (DF10). Of these proteins, PBP was identified as a primary candidate for a decapacitation factor.     

Mass spectrometry to characterize the binding of a peptide to a lipid surface.

The binding of an amphipathic alpha-helical peptide to small unilamellar lipid vesicles has been examined using chemical derivitization and mass spectrometry. The peptide is derived from the sequence of human apolipoprotein C-II (apoC-II), the protein activator of lipoprotein lipase (LpL). ApoC-II(19-39) forms approximately 60% alpha-helix upon binding to model egg yolk phosphatidylcholine small unilamellar vesicles. Measurement of the affinity of the peptide for lipid by spectrophotometric methods is complicated by the contribution of scattered light to optical signals. Instead, we characterize the binding event using the differential labeling of lysine residues by the lipid- and aqueous-phase cross-linkers, disuccinimidyl suberate (DSS) and bis(sulfosuccinimidyl) suberate (BS(3)), respectively. In aqueous solution, the three lysine residues of the peptide are accessible to both cross-linkers. In the presence of lipid, the C-terminal lysine residue becomes inaccessible to the lipid-phase cross-linker DSS, but remains accessible to the aqueous-phase cross-linker, BS(3). We use mass spectrometry to characterize this binding event and to derive a dissociation constant for the interaction (K(d) = 5 microM). We also provide evidence for the formation of dimeric cross-linked peptide when high densities of peptide are bound to the lipid surface.     

A new optoelectronic measuring device for assessment of leg circumference in comparison with manual measurement]

For compression treatment to be effective in patients with chronic venous insufficiency, it is vital that leg circumference be measured accurately. If compression stockings are custom fit and appropriate for the medical indications, patient compliance will be high. Exact measurements of circumference and length are prerequisites for a good fit. The aim of the present study was to compare an opto-electronic device for the contact-free measurement of calf circumference with the conventional manual method using a tape measure. We investigated the differences between the results obtained with the two methods, and also their reproducibility. Circumferences were measured at defined heights on an anatomically shaped non-yielding leg model and on the leg of a healthy volunteer by 10 different experimenters both with the tape measure and with the opto-electronic device. The calf circumferences measured manually with the tape measure varied significantly more than those measured opto-electronically, both in the leg model and in the leg of the volunteer. A systematic error in the opto-electronic method appears unlikely, since the manual measurements on the leg model were both larger and smaller than those obtained with the opto-electronic device. Reproducibility was exceptionally high with the opto-electronic device (standard deviation 0.11-0.42 cm). The opto-electronic method yields rapid accurate measurements of circumference with excellent intra- and inter-operator reproducibility.     

Isolation and characterization of a novel Enterococcus faecalis bacteriophage phiEF24C as a therapeutic candidate.

Vancomycin-resistant Enterococcus faecalis (VRE) has become a significant threat in nosocomial settings. Bacteriophage (phage) therapy is frequently proposed as a potential alternative therapy for infections caused by this bacterium. To search for candidate therapeutic phages against Enterococcus faecalis infections, 30 Enterococcus faecalis phages were isolated from the environment. One of these, virulent phage phiEF24C, which has a broad host range, was selected for analysis. The plaque-forming ability of phiEF24C was virtually unaffected by differences in the clinical host strains. Furthermore, the phage had a shorter latent period and a larger burst size than ordinary tailed phages, indicating that phiEF24C has effective lytic activity against many Enterococcus faecalis strains, including VRE. Morphological and genomic analyses revealed that phiEF24C is a large myovirus (classified as family Myoviridae morphotype A1) with a linear double-stranded DNA genome of c. 143 kbp. Analyses of the N-terminal amino acid sequences of the virion proteins, together with the morphology and the genome size, speculated that phiEF24C is closely related to other myoviruses of Gram-positive bacteria that have been used experimentally or practically for therapy or prophylaxis. Considering these results, phiEF24C may be a potential candidate therapeutic phage against Enterococcus faecalis infections.     

Ultrastructural biomarkers as tools to characterize the health status of fish in contaminated streams

Ultrastructural biomarkers in gill, liver, andkidney of brown trout (Salmo truttaf. fario) and stone loach (Barbatulabarbatula) were investigated over afive year period. The cellular damage of theorgans was assessed semi-quantitatively basedon a three-step classification ofultrastructural responses. Data obtained forfish exposed under semi-field conditions in twodifferently polluted test streams and in thelaboratory demonstrated that the ultrastructureof the organs can be correlated with differentpollutant exposure conditions. Cellular damagewas generally more severe in fish exposed to acomplexly polluted stream than in those exposedto a moderately polluted stream or to tap waterin the laboratory. Histopathological effects inliver and gill of trout were more pronounced inferal fish than in transplanted fish, whereasresponses in the kidney in both species, and inliver and gill of loach, were similar forintroduced and feral fish. In a laboratoryexperiment where trout were exposed todifferent mixtures of pollutants includingpesticides, PAH, and ammonia, only theultrastructure of kidney and liver showedsignificant differences between the threeexperimental set-ups. In a recovery experiment,where trout were transferred from thesemi-field condition back to the laboratory,ultrastructural investigations showed adifferential capacity of the respective organsto recover from stress under field conditions.Kidney and liver fully recovered after threemonths under control condition whereas gillsdemonstrated only partial recovery.