Synthetic Modular Antibody Construction by Using the SpyTag/SpyCatcher Protein-Ligase System

Efforts to engineer recombinant antibodies for specific diagnostic and therapy applications are time consuming and expensive, as each new recombinant antibody needs to be optimized for expression, stability, bio-distribution, and pharmacokinetics. We have developed a new way to construct recombinant antibody-like “devices” by using a bottom-up approach to build them from well-behaved discrete recombinant antibody domains or “parts”. Studies on antibody structure and function have identified antibody constant and variable domains with specific functions that can be expressed in isolation. We used the SpyTag/SpyCatcher protein ligase to join these parts together, thereby creating devices with desired properties based on summed properties of parts and in configurations that cannot be obtained by using genetic engineering. This strategy will create optimized recombinant antibody devices at reduced costs and with shortened development times.     

Mesh refinement and iterative solution methods for finite element computations

Global and element residuals are introduced to determine a posteriori, computable, error bounds for finite element computations on a given mesh. The element residuals provide a criterion for determining where a finite element mesh requires refinement. This indicator is implemented in an algorithm in a finite element research program. There it is utilized to automatically refine the mesh for sample two-point problems exhibiting boundary layer and interior layer solutions. Results for both linear and nonlinear problems are presented. An important aspect of this investigation concerns the use of adaptive refinement in conjunction with iterative methods for system solution. As the mesh is being enriched through the refinement process, the solution on a given mesh provides an accurate starting iterate for the next mesh, and so on. A wide range of iterative methods are examined in a feasibility study and strategies for interweaving refinement and iteration are compared.     

Influence of glycerol on the mechanical reversibility and thermal damage susceptibility of collagenous tissues

Clinical procedures wherein supraphysiologic temperatures must be achieved in deep layers of tissue via light are often compromised by optical scattering and absorption. Optical clearing of tissue superficial to the target improves the efficacy of such procedures. Glycerol is an attractive chemical agent for achieving dramatic reductions in tissue turbidity, but its net effects on healthy tissue are not fully understood. In this paper, we investigate possible alterations of biaxial mechanical properties in a model collagenous tissue, bovine epicardium, induced by glycerol. Furthermore, we examine the effects of glycerol on the biaxial thermomechanical properties of epicardium constrained at near-physiologic length. It is seen that mechanical changes induced by glycerol are fully reversed upon rehydration in normal saline. Moreover, glycerol protects cleared tissue by increasing its thermal stability and minimizing thermal alterations of mechanical properties.     

Short communication: Lymphocyte proliferative responses in cattle naturally infected with digital dermatitis consist of CD8+ and γδ-T cells but lack CD4+ T cells

Digital dermatitis is an infectious disease of cattle and the leading cause of lameness. This disease is complicated by the reoccurrence of the lesions and the observation of lesions on more than one limb at different time points, indicating infection may not result in a protective immune response. The objective of this study was to characterize the peripheral blood cellular response in naturally infected and naïve cattle to bacterial antigens derived from pathogens associated with digital dermatitis lesions. Peripheral blood mononuclear cells were isolated from dairy cattle identified as having active or chronic lesions during routine hoof-trimming. Following bacterial antigen stimulation, cells were analyzed for proliferation and phenotype by flow cytometry, and culture supernatants were analyzed for IFN-γ secretion. Digital-dermatitis-infected animals had greater serum antibody titers to treponemal antigens, higher percentages of proliferating CD8+, γδ-T cells, and B cells, and increased IFN-γ secretion in vitro when compared with responses of naïve animals. No increase in proliferation of CD4+ T cells was detected in infected or naïve cattle. Although CD8+ and γδ-T cell responses may be antigen specific, the memory nature or long-lived response is yet unknown. The lack of responsiveness of CD4+ memory cells to treponemal antigens could explain the high rate of reoccurrence of digital dermatitis in infected animals.     

The survival of foodborne pathogens during domestic washing-up and subsequent transfer onto washing-up sponges,kitchen surfaces and food

In this study, the survival of Salmonella, Campylobacter and Escherichia coli O157: H7, when exposed to a range of constant temperatures (47-60 degrees C), in hard or soft water, in the presence/absence of detergent (0-0.3%) and organic matter, and during drying, was investigated. Further experiments used a washing-up process simulation, where soiled dishes contaminated with bacteria were washed in a bowl of warm water containing detergent. In addition, this study considered the risk of bacterial transfer onto (1) sterile dishes and sponges via contaminated water, (2) kitchen surfaces wiped with a contaminated sponge, (3) items placed in direct contact with a contaminated kitchen surface, (4) food placed on a contaminated dish or (5) dishes from contaminated food. A proportion of dishes remained contaminated with all pathogen types after a typical washing-up. Water hardness did not appear to affect survival. E. coli, and to a lesser extent Salmonella, survived towel- or air-drying on dishes and after towel-drying the cloth became contaminated on every occasion, regardless of the test organism. A proportion of sterile dishes washed after contaminated dishes became contaminated with pathogens but transfer from dishes onto food was rare. Washing-up sponges frequently became contaminated with pathogens. The results of this study highlight the potential for survival and cross contamination of food borne pathogens in the kitchen environment.     

Gold complexes for nonlinear optics

The nonlinear optical (NLO) properties of gold complexes are reviewed. Experimental procedures which have been used to determine NLO behaviour of gold complexes are summarized, structure / NLO property correlations are presented, and the potential of gold complexes for nonlinear optics is discussed.     

Optimized microvessel density analysis improves prediction of cancer stage from prostate needle biopsies

During angiogenesis in the chorioallantoic membrane (CAM) of the chick, capillary proliferation occurs primarily by intussusceptive growth. Previously, we reported that such growth in the CAM proceeded without substantial macromolecular extravasation. Neovascularization involving capillary sprout formation, on the other hand, has been associated with a concomitant loss of endothelial selectivity. Thus, the present study tested the hypothesis that endothelial selectivity during angiogenesis is dependent on the mode of microvascular growth. Capillary sprout formation occurs in peripheral regions of the CAM, in addition to the more centrally located areas of intussuceptive growth. In this study, angiogenic endothelial permselectivities were evaluated in these respective areas of CAM microvascular growth by intravital fluorescent microscopy of a graded series of FITC-dextrans. In both cases, the angiogenic endothelia restricted extravasation of macromolecules > or = 20 kDa. Furthermore, capillary sprout endothelia, like the intussusceptive CAM endothelia, remained tightly sealed at the junctional clefts. Thus, angiogenic endothelial permselectivity in the CAM is not dependent on the mode of microvascular growth. Whether distinct cellular mechanisms are operable in capillary endothelial sprouts of the CAM, relative to those of other proliferating sprout endothelia, remains to be tested.     

The effect of prostate volume, age, total prostate specific antigen level and acute inflammation on the percentage of free serum prostate specific antigen levels in men without clinically detectable prostate cancer

PURPOSE: We determine the influence of age, prostate volume, total serum prostate specific antigen (PSA) level and histological evidence of acute inflammation in biopsy specimens on the percent free serum PSA level in men without clinically detectable prostate cancer. MATERIALS AND METHODS: We studied 70 men with total PSA levels of 2.6 to 9.9 ng./ml. who had undergone at least 3 sets of prostate biopsies that were negative for cancer as part of our PSA based prostate cancer screening program. Total and free PSA levels were measured using Hybritech immunoassays. Prostate volume and the presence of acute inflammation were determined from the most recent transrectal ultrasonography and prostate needle biopsy. RESULTS: Percent free PSA levels correlated significantly with age (r = 0.48, p = 0.0001) and prostate volume (r = 0.44, p = 0.0002) but not with total PSA (r = 0.04, p = 0.7). The mean percent free PSA did not differ for those with or without acute inflammation. Multivariate regression models demonstrated that age and prostate volume were significant predictors of percent free PSA. CONCLUSIONS: Among men without detectable prostate cancer and a total PSA level between 2.6 and 9.9 ng./ml. percent free serum PSA was higher in older men and in men with a larger prostate gland but was not influenced by total PSA level or the presence of acute inflammation in the prostatic biopsy specimen.