Robotic general surgery: current practice, evidence, and perspective.

BACKGROUND: Robotic technology commenced to be adopted for the field of general surgery in the 1990s. Since then, the da Vinci surgical system (Intuitive Surgical Inc, Sunnyvale, CA, USA) has remained by far the most commonly used system in this domain. The da Vinci surgical system is a master-slave machine that offers three-dimensional vision, articulated instruments with seven degrees of freedom, and additional software features such as motion scaling and tremor filtration. The specific design allows hand-eye alignment with intuitive control of the minimally invasive instruments. As such, robotic surgery appears technologically superior when compared with laparoscopy by overcoming some of the technical limitations that are imposed on the surgeon by the conventional approach. PURPOSE: This article reviews the current literature and the perspective of robotic general surgery. CONCLUSIONS: While robotics has been applied to a wide range of general surgery procedures, its precise role in this field remains a subject of further research. Until now, only limited clinical evidence that could establish the use of robotics as the gold standard for procedures of general surgery has been created. While surgical robotics is still in its infancy with multiple novel systems currently under development and clinical trials in progress, the opportunities for this technology appear endless, and robotics should have a lasting impact to the field of general surgery.

Characterization of endothelial-like cells derived from human mesenchymal stem cells.

BACKGROUND: Blood-derived endothelial progenitor cells (EPC) have been used to treat ischemic disease. However, the number of EPC that can be obtained from adult blood is limited. OBJECTIVE: To characterize endothelial-like cells obtained from human bone marrow and determine their ability to stimulate new blood vessel formation in vivo. METHODS: Mononuclear cells (MNC) were isolated from human bone marrow or umbilical cord blood and cultured in endothelial growth medium (EGM-2). Mesenchymal stem cells (MSC) were isolated from bone marrow and induced to differentiate into endothelial-like cells (MSCE), or adipocytes or osteocytes by growth in EGM-2, adipogenic or osteogenic medium. RESULTS: Cells obtained by culturing bone marrow MNC in EGM-2 formed cord- or tube-like structures when grown on Matrigel(TM) and expressed several endothelial marker proteins. However, cell morphology and the profile of endothelial marker protein expression were different from those of cord blood-derived EPC (cbEPC). Cells with a similar phenotype were obtained by differentiation of MSC into MSCE, which was accompanied by an increase of endothelial marker proteins and a diminished capacity to differentiate into adipocytes. Subcutaneous implantation of MSCE in collagen plugs in non-obese diabetic-severe combined immunodeficient (NOD-SCID) mice resulted in formation of functional blood vessels that had incorporated the MSCE. CONCLUSIONS: Our results show that MSCE and cbEPC are different cell types. The formation of functional blood vessels by MSCE, combined with high yields and a reduced capacity to differentiate into other cell types compared with MSC, makes these cells potentially useful for autologous therapy of ischemic disease.

Defining signal thresholds in DNA microarrays: exemplary application for invasive cancer.

BACKGROUND: Genome-wide or application-targeted microarrays containing a subset of genes of interest have become widely used as a research tool with the prospect of diagnostic application. Intrinsic variability of microarray measurements poses a major problem in defining signal thresholds for absent/present or differentially expressed genes. Most strategies have used fold-change threshold values, but variability at low signal intensities may invalidate this approach and it does not provide information about false-positives and false negatives. RESULTS: We introduce a method to filter false-positives and false-negatives from DNA microarray experiments. This is achieved by evaluating a set of positive and negative controls by receiver operating characteristic (ROC) analysis. As an advantage of this approach, users may define thresholds on the basis of sensitivity and specificity considerations. The area under the ROC curve allows quality control of microarray hybridizations. This method has been applied to custom made microarrays developed for the analysis of invasive melanoma derived tumor cells. It demonstrated that ROC analysis yields a threshold with reduced missclassified genes in microarray experiments. CONCLUSIONS: Provided that a set of appropriate positive and negative controls is included on the microarray, ROC analysis obviates the inherent problem of arbitrarily selecting threshold levels in microarray experiments. The proposed method is applicable to both custom made and commercially available DNA microarrays and will help to improve the reliability of predictions from DNA microarray experiments.

Distribution and dynamics of gap junction channels revealed in living cells.

To study the structural composition and dynamics of gap junctions in living cells, we tagged their subunit proteins, termed connexins, with the autofluorescent tracer green fluorescent protein (GFP) and its cyan (CFP) and yellow (YFP) color variants. Tagged connexins assembled normally and channels were functional. High-resolution fluorescence images of gap junction plaques assembled from CFP and YFP tagged connexins revealed that the mode of channel distribution is strictly dependent on the connexin isoforms. Co-distribution as well as segregation into well-separated domains was observed. Based on accompanying studies we propose that channel distribution is regulated by intrinsic, connexin isoform specific signals. High-resolution time-lapse images revealed that gap junctions, contrary to previous expectations, are dynamic assemblies of channels. Channels within clusters and clusters themselves are mobile and constantly undergo structural rearrangements. Movements are complex and allow channels to move, comparable to other plasma membrane proteins not anchored to cytoskeletal elements. Comprehensive analysis, however, demonstrated that gap junction channel movements are not driven by diffusion described to propel plasma membrane protein movement. Instead, recent studies suggest that movements of gap junction channels are indirect and predominantly propelled by plasma membrane lipid flow that results from metabolic endo- and exocytosis.

Analysis of contrast-enhanced MR images to assess renal function.

The image analysis and kinetic modeling methods used in dynamic contrast-enhanced magnetic resonance imaging of the kidney are reviewed. Image analysis includes various techniques of coregistration and segmentation. Few methods have been completely implemented. Nevertheless, the use of coregistration may become a standard to decrease the effect of motion on abdominal images and improve the quality of the renal signals. Kinetic models are classified into three categories: enhancement-based, external and internal representations. Enhancement-based representations are limited to a basic analysis of the tracer concentration curves in the kidneys. Their relationship to the underlying physiology is complex and undefined. However, they can be used to evaluate the split renal function. External representations assess the kidney input and output. An external representation based on the up-slope of the renal enhancement to calculate the renal perfusion is commonly used because of its simplicity. In contrast, external representation based on deconvolution or identification methods remain underexploited. For glomerular filtration, an internal representation based on a two-compartmental model is mostly used. Internal representations based on multi-compartmental models describe the renal function in a more realistic way. Because of their numerical complexity, these models remain rarely used.

Single channel behavior of matrix porin of Escherichia coli.

Porins are trimeric proteins in the outer membranes of Gram-negative bacteria. Several of them, among them matrix porin of Escherichia coli, form symmetrically voltage-gated ion channels in planar bilayers. Trimers exhibit negative resistance at potentials larger than +/- 90mV. Here we show that, after two pores within a trimer close irreversibly, the remaining third pore shows channel properties distinct from those observed in the trimer. This residual pore exhibits an asymmetric current-voltage dependence with a pronounced polarity-dependent shift toward low potentials and rates of channel-closing and opening that are one to two orders of magnitude faster than those observed for single channels in a reversibly voltage-dependent trimer. Rectification of single channels thus resembles that of a voltage-gated channel type observed in outer membrane patches of E.coli spheroblasts, hinting at the relevance of the phenomenon in vivo.

Artificial transmembrane ion channels from self-assembling peptide nanotubes.

Naturally occurring membrane channels and pores are formed from a large family of diverse proteins, peptides and organic secondary metabolites whose vital biological functions include control of ion flow, signal transduction, molecular transport and production of cellular toxins. But despite the availability of a large amount of biochemical information about these molecules, the design and synthesis of artificial systems that can mimic the biological function of natural compounds remains a formidable task. Here we present a simple strategy for the design of artificial membrane ion channels based on a self-assembled cylindrical beta-sheet peptide architecture. Our systems--essentially stacks of peptide rings--display good channel-mediated ion-transport activity with rates exceeding 10(7) ions s-1, rivalling the performance of many naturally occurring counterparts. Such molecular assemblies should find use in the design of novel cytotoxic agents, membrane transport vehicles and drug-delivery systems.

Plasticity of Escherichia coli porin channels. Dependence of their conductance on strain and lipid environment.

The conductance properties of three members of the porin family which form channels across the outer membrane of Gram-negative bacteria were compared. With their endogenous lipopolysaccharide (LPS) bound, the closely related porins F and C from Escherichia coli reveal significantly different conductance steps and closing potentials, with values of 0.82 nS (nanosiemens) and 89 mV for F-type channels, and 0.49 nS and 158 mV for C-type pores (1 M NaCl), respectively. On the basis of their closing potentials, the two channel types can be distinguished unequivocally. If reconstituted in asolectin and extraneous LPS, porin C forms F-type in addition to C-type channels. Substitution of asolectin by mitochondrial lipids yields the native C-type pores only. Both channel types can be induced to assume the mutually other channel configuration by variation of ionic strength. A multiplicity of channel subtypes is observed by variation of the pH of the medium. The three channels within a trimer are, however, consistently of the same type. Since structural studies have revealed a single channel per monomer, the several conductance steps observed are likely to reflect distinct configurations of the same channel. Best channel recoveries were observed if endogenous LPS remained associated to porin during purification. Significant yields could nevertheless be obtained also if LPS was removed from porin and replaced with various precursors or chemically synthesized analogues. As function requires the presence of glycolipids, yet crystallization is perturbed by heterodisperse endogenous LPS, the smallest monodisperse analogues yielding good channel recovery were determined. The minimal synthetic moiety is a monoglucosaminetetraacyl compound. The characteristics of porin B from E. coli BE are shown to be indistinguishable from those of porin F. The conductance properties of this porin, refolded from random coil configuration, are indistinguishable from those exhibited by native protein. The formation of channels is thus encoded by the sequence of the mature polypeptide alone.

Single channel behavior of recombinant beta 2 gap junction connexons reconstituted into planar lipid bilayers.

The beta 2 gap junction protein (Cx26) was expressed in an insect cell line by infection with a baculovirus vector containing the rat beta 2 cDNA. Isolated beta 2 gap junction connexons were reconstituted into planar lipid bilayers. Single channel activity was observed with a unitary conductance of 35-45 pS in 200 mM KCl. Channels with conductance values of 60 pS and 90-110 pS also coexisted with the lower conducting channel suggesting that there are channels with different conductance properties within a population of connexons. Channel activity was observed at voltages of up to 150 mV. Furthermore, the characterization of these channel properties from the beta 2 connexons that were generated by this heterologous expression system has provided the basis for identifying an endogenous beta 2 connexon channel in material reconstituted from native rat liver gap junctions.

Cell-free synthesis and assembly of connexins into functional gap junction membrane channels.

Several different gap junction channel subunit isotypes, known as connexins, were synthesized in a cell-free translation system supplemented with microsomal membranes to study the mechanisms involved in gap junction channel assembly. Previous results indicated that the connexins were synthesized as membrane proteins with their relevant transmembrane topology. An integrated biochemical and biophysical analysis indicated that the connexins assembled specifically with other connexin subunits. No interactions were detected between connexin subunits and other co-translated transmembrane proteins. The connexins that were integrated into microsomal vesicles assembled into homo- and hetero-oligomeric structures with hydrodynamic properties of a 9S particle, consistent with the properties reported for hexameric gap junction connexons derived from gap junctions in vivo. Further, cell-free assembled homo-oligomeric connexons composed of beta1 or beta2 connexin were reconstituted into synthetic lipid bilayers. Single channel conductances were recorded from these bilayers that were similar to those measured for these connexons produced in vivo. Thus, this is the first direct evidence that the synthesis and assembly of a gap junction connexon can take place in microsomal membranes. Finally, the cell-free system has been used to investigate the properties of alpha1, beta1 and beta2 connexin to assemble into hetero-oligomers. Evidence has been obtained for a selective interaction between individual connexin isotypes and that a signal determining the potential hetero-oligomeric combinations of connexin isotypes may be located in the N-terminal sequence of the connexins.

Decomplementation with cobra venom factor prolongs survival of xenografted islets in a rat to mouse model.

Although the involvement of complement in hyperacute rejection of xenotransplants is well recognized, its role in rejection of devascularized xenografts, such as pancreatic islets, is not completely understood. In this study, we investigated whether complement participates in the immunopathology of xeno-islet transplantation in a concordant rat to mouse model. Rat pancreatic islets were implanted under the kidney capsule of normal and cobra venom factor (CVF)-decomplementized diabetic C57BL/6 mice. Graft survival was monitored by blood glucose levels. Deposition of IgM and C3 on grafted islets in vivo or on isolated islets in vitro (after incubation with normal and decomplementized mouse serum), as well as CD4- and CD8-positive leucocyte infiltration of grafts, was checked by immunohistochemistry. In addition, complement-mediated cytotoxicity on rat islet cells was evaluated by a 3-(4, 5-dimethythiazolyl)-2.5-diphenyl-2H-tetrazolium-bromide (MTT) assay. A significant C3 deposition was found on grafted islets from the first day after transplantation in vivo, as well as on isolated islets after incubation with mouse serum in vitro. By MTT assay, complement-mediated cytotoxicity for islet cells was found. Decomplementation by CVF decreased C3 deposition on either isolated or grafted islets, delayed CD4- and CD8-positive leucocyte infiltration, led to significant inhibition of complement-mediated cytotoxicity for islet cells, and prolonged graft survival (mean survival time 21.3 versus 8.5 days; P<0.01). Our results indicate that decomplementation can prolong the survival time of devascularized xenografts across concordant species. The deposition of complement on transplanted islets may contribute to xenograft rejection by direct cytotoxicity and by promoting leucocyte infiltration.