Skeletonization of Volumetric Angiograms for Display

The display of three-dimensional angiograms can benefit from the knowledge of quantitative shape features such as tangent and curvature of the centerline of vessels. These can be obtained from a curve-like skeleton representation. If connectivity and topology are preserved, and if geometrical constraints such as smoothness and centeredness are satisfied, it is possible to estimate length, orientation, curvature, and torsion. It is also required that no part of the original object be left unrepresented. An efficient method for the identification of such shape components is developed. First, a suitable representation is obtained using a voxel coding approach to yield connected and labeled unit-thick paths. The desired features are estimated from a smoothed version of the skeleton produced by a moving average filter. The computational cost is linear, of the order of N_object, the total number of object voxels contained in the binary volumetric data. The method is also shown to be robust to boundary noise. Examples are discussed.     

Ribosome display: next-generation display technologies for production of antibodies in vitro

Antibodies represent an important and growing class of biologic research reagents and biopharmaceutical products. They can be used as therapeutics in a variety of diseases. With the rapid expansion of proteomic studies and biomarker discovery, there is a need for the generation of highly specific binding reagents to study the vast number of proteins encoded by the genome. Display technologies provide powerful tools for obtaining antibodies. Aside from the preservation of natural antibody repertoires, they are capable of exploiting diversity by DNA recombination to create very large libraries for selection of novel molecules. In contrast to in vivo immunization processes, display technologies allow selection of antibodies under in vitro-defined selection condition(s), resulting in enrichment of antibodies with desired properties from large populations. In addition, in vitro selection enables the isolation of antibodies against difficult antigens including self-antigens, and this can be applied to the generation of human antibodies against human targets. Display technologies can also be combined with DNA mutagenesis for antibody evolution in vitro. Some methods are amenable to automation, permitting high-throughput generation of antibodies. Ribosome display is considered as representative of the next generation of display technologies since it overcomes the limitations of cell-based display methods by using a cell-free system, offering advantages of screening larger libraries and continuously expanding new diversity during selection. Production of display-derived antibodies can be achieved by choosing one of a variety of prokaryotic and eukaryotic cell-based expression systems. In the near future, cell-free protein synthesis may be developed as an alternative for large-scale generation of antibodies.     

Phage display antibodies for diagnostic applications

With major developments in molecular biology, numerous display technologies have been successfully introduced for recombinant antibody production. Even so, phage display still remains the gold standard for recombinant antibody production. Its success is mainly attributed to the robust nature of phage particles allowing for automation and adaptation to modifications. The generation of monospecific binders provides a vital tool for diagnostics at a lower cost and higher efficiency. The flexibility to modify recombinant antibodies allows great applicability to various platforms for use. This review presents phage display technology, application and modifications of recombinant antibodies for diagnostics.     

Engineering M13 for phage display

Phage display is achieved by fusing polypeptide libraries to phage coat proteins. The resulting phage particles display the polypeptides on their surfaces and they also contain the encoding DNA. Library members with particular functions can be isolated with simple selections and polypeptide sequences can be decoded from the encapsulated DNA. The technology's success depends on the efficiency with which polypeptides can be displayed on the phage surface, and significant progress has been made in engineering M13 bacteriophage coat proteins as improved phage display platforms. Functional display has been achieved with all five M13 coat proteins, with both N- and C-terminal fusions. Also, coat protein mutants have been designed and selected to improve the efficiency of heterologous protein display, and in the extreme case, completely artificial coat proteins have been evolved specifically as display platforms. These studies demonstrate that the M13 phage coat is extremely malleable, and this property can be used to engineer the phage particle specifically for phage display. These improvements expand the utility of phage display as a powerful tool in modern biotechnology.     

Homogenized constrained mixture models for anisotropic volumetric growth and remodeling

Constrained mixture models for soft tissue growth and remodeling have attracted increasing attention over the last decade. They can capture the effects of the simultaneous presence of multiple constituents that are continuously deposited and degraded at in general different rates, which is important to understand essential features of living soft tissues that cannot be captured by simple kinematic growth models. Recently the novel concept of homogenized constrained mixture models was introduced. It was shown that these models produce results which are very similar (and in certain limit cases even identical) to the ones of constrained mixture models based on multi-network theory. At the same time, the computational cost and complexity of homogenized constrained mixture models are much lower. This paper discusses the theory and implementation of homogenized constrained mixture models for anisotropic volumetric growth and remodeling in three dimensions. Previous constrained mixture models of volumetric growth in three dimensions were limited to the special case of isotropic growth. By numerical examples, comparison with experimental data and a theoretical discussion, we demonstrate that there is some evidence raising doubts whether isotropic growth models are appropriate to represent growth and remodeling of soft tissue in the vasculature. Anisotropic constrained mixture models, as introduced in this paper for the first time, may be required to avoid unphysiological results in simulations of vascular growth and remodeling.     

Phage display for detection of biological threat agents

The essential element of any immuno-based detector device is the probe that binds analyte and, as a part of the analytical platform, generates a measurable signal. The present review summarizes the state of the art in development of the probes for detection of the biological threat agents: toxins, bacteria, spores and viruses. Traditionally, the probes are antibodies, which are isolated from sera of immunized animals or culture media of hybridomas. However, the "natural" antibodies may have limited application in the new generation of real-time field detectors and monitoring systems, where stress-resistant and inexpensive long-livers are required. Phage display is a newcomer in the detection area, whose expertise is development of molecular probes for targeting of various biological structures. The probes can be selection from about billion clone libraries of recombinant phages expressing on their surface a vast variety of peptides and proteins, including antigen-binding fragments of antibodies. The selection procedure, like kind of affinity chromatography, allows separating of phage binders, which are propagated in Escherichia coli bacterial cells and purified using inexpensive technology. Although phage display traditionally is focused more on development of medical preparations and studying molecular recognition in biological systems, there are some examples of its successful use for detection, which are presented in the review. To be used as probes for detection, peptides and antibodies identified by phage display are usually chemically synthesized or produced in bacteria. Another interesting aspect is using of the selected phage itself as a probe in detector devices, like sort of substitute antibodies. This idea is illustrated in the review by "detection" of beta-galactosidase from E. coli with "landscape" phage displaying a dense array of peptide binders on the surface.     

The scope of phage display for membrane proteins

Numerous examples of phage display applied to soluble proteins demonstrate the power of the technique for protein engineering, affinity reagent discovery and structure-function studies. Recent reports have expanded phage display to include membrane proteins (MPs). The scope and limitations of MP display remain undefined. Therefore, we report data from the phage display of representative types of membrane-associated proteins including plasma, nuclear, peripheral, single and multipass. The peripheral MP neuromodulin displays robustly with packaging by conventional M13-KO7 helper phage. The monotopic MP Nogo-66 can also display on the phage surface, if packaged by the modified M13-KO7⁺ helper phage. The modified phage coat of KO7⁺ can better mimic the zwitterionic character of the plasma membrane. Four examples of putatively α-helical, integral MPs failed to express as fusions to an anchoring phage coat protein and therefore did not display on the phage surface. However, the β-barrel MPs ShuA (Shigella heme uptake A) and MOMP (major outer membrane protein), which pass through the membrane 22 and 16 times, respectively, can display surprisingly well on the surfaces of both conventional and KO7⁺ phages. The results provide a guide for protein engineering and large-scale mutagenesis enabled by the phage display of MPs.     

A rapid volumetric method for the analysis of the food of fishes

A volumetric method in which the stomach contents are squashed to a uniform depth and the volume is derived from the area of the squash has been evaluated. The apparatus is simple in constiuction and the method applicable to a wide range of fish sizes. The overall error of the method may be as low as 3.5 % for well macerated material, though for hard bodied food animals the coefficient of variation may be up to 8 %. These volumetric determinations are not directly proportional to gravimetric measurements but logarithmic conversion curves are readily derived.     

Ribosome display: cell-free protein display technology.

Ribosome display is a cell-free system for the in vitro selection of proteins and peptides from large libraries. It uses the principle of coupling individual nascent proteins (phenotypes) to their corresponding mRNA (genotypes), through the formation of stable protein-ribosome-mRNA (PRM) complexes. This permits the simultaneous isolation of a functional nascent protein, through affinity for a ligand, together with the encoding mRNA, which is then converted and amplified as DNA for further manipulation, including repeated cycles or protein expression. Ribosome display has a number of advantages over cell-based systems such as phage display; in particular, it can display very large libraries without the restriction of bacterial transformation. It is also suitable for generating toxic, proteolytically sensitive and unstable proteins, and allows the incorporation of modified amino acids at defined positions. In combination with polymerase chain reaction (PCR)-based methods, mutations can be introduced efficiently into the selected DNA pool in subsequent cycles, leading to continuous DNA diversification and protein selection (in vitro protein evolution). Both prokaryotic and eukaryotic ribosome display systems have been developed and each has its own distinctive features. In this paper, ribosome display systems and their application in selection and evolution of proteins are reviewed.     

Differential display

Characterization of regulated gene expression in eukaryotic cells is essential for studying cell growth and differentiation as well as for understanding the molecular mechanisms of diseases. Differential display was developed for such comparative studies by allowing a systematic and nonbiased screening for molecular differences at the level of mRNA expression between or among different cells or tissues. The essence of the method is to amplify messenger RNA 3′ termini using a pair of anchored oligo-dT primer and a short primer with an arbitrary sequence. The amplified cDNAs labeled with radioisotope are then distributed on a denaturing polyacrylamide gel and visualized by autoradiography. Side-by-side comparison of mRNA species from two or more related samples allows identification of both up- and downregulation genes of interest. Some of the most recent improvements have been incorporated into this general protocol for differential display.     

Development and validation of a graphic method for spatial interpretation and evaluation of biplanar coronary angiograms]

Visual evaluation and measurement of the lesion geometry is impaired by the foreshortening of the radiographically displayed target coronary segment. For this reason, we developed and validated a simple graphic procedure that facilitates the spatial interpretation and allows an objectifiable assessment of the view of the target coronary segment. The method computes a spatial axis of the coronary segment imaged in two planes and determines its inclination in the radiation path of each projection view. A triangle made up of the axis of the imaged segment, the unforeshortened axis of the imaged segment and the foreshortening height is displayed in each projection plane. The shape of the triangle indicates the degree of foreshortening while its position in the angiogram indicates the orientation of the spatial axis relative to the observer. The method was validated by comparing calculated and true foreshortening in the radiographic views of a centimetre grid obtained in the usual angiographic projections. The method has an accuracy and precision of 0.05 +/- 0.62%. It is applied clinically to evaluate biplane segmental visualization during coronary interventions and to select valid segmental views for measurements during quantitative biplane coronary angiography. This application may considerably facilitate the interpretation and assessment of biplane angiograms.     

A novel method for volumetric MRI response assessment of enhancing brain tumors

Current radiographic response criteria for brain tumors have difficulty describing changes surrounding postoperative resection cavities. Volumetric techniques may offer improved assessment, however usually are time-consuming, subjective and require expert opinion and specialized magnetic resonance imaging (MRI) sequences. We describe the application of a novel volumetric software algorithm that is nearly fully automated and uses standard T1 pre- and post-contrast MRI sequences. T1-weighted pre- and post-contrast images are automatically fused and normalized. The tumor region of interest is grossly outlined by the user. An atlas of the nasal mucosa is automatically detected and used to normalize levels of enhancement. The volume of enhancing tumor is then automatically calculated. We tested the ability of our method to calculate enhancing tumor volume with resection cavity collapse and when the enhancing tumor is obscured by subacute blood in a resection cavity. To determine variability in results, we compared narrowly-defined tumor regions with tumor regions that include adjacent meningeal enhancement and also compared different contrast enhancement threshold levels used for the automatic calculation of enhancing tumor volume. Our method quantified enhancing tumor volume despite resection cavity collapse. It detected tumor volume increase in the midst of blood products that incorrectly caused decreased measurements by other techniques. Similar trends in volume changes across scans were seen with inclusion or exclusion of meningeal enhancement and despite different automated thresholds for tissue enhancement. Our approach appears to overcome many of the challenges with response assessment of enhancing brain tumors and warrants further examination and validation.