Investigation of alkaline effects on Protein A affinity ligands and resins using high resolution mass spectrometry.

In this study, the enhanced alkaline stability of Protein A ligands and resins designed by protein engineering approaches is demonstrated. High throughput PreDictor™ plates were used to evaluate and compare the human Immunoglobulin G (IgG) static binding capacities (SBC) of MabSelect SuRe™ and MabSelect™ PrismA affinity chromatography (AC) resins after continuous incubation in 0.1-2.0 M NaOH for 1-72 h. The alkaline effect on the Protein A affinity ligand was studied by high resolution mass spectrometry (MS). The IgG binding capacity of the investigated AC resins show expected declining trends with increasing NaOH concentrations and incubation times. The decrease is larger for MabSelect SuRe than for MabSelect PrismA and occur at lower NaOH concentrations. MabSelect SuRe display high remaining binding capacity even after 72 h continuous incubation in 0.1 M NaOH, while higher concentrations induce an accentuated decline with incubation time. The MabSelect PrismA resin shows almost no effect on the binding capacity even after 72 h incubation in 0.5 M NaOH. Decline in capacity is first observed after 48 h incubation in 1.0 M NaOH, thus displaying the extreme alkaline stability of the PrismA affinity ligand. The MS analysis of the ligands, including a Protein A single B-domain, SuRe-domain and PrismA-domain clearly illustrate the increasing alkaline stability (B-domain < SuRe < PrismA) as the ligand has been refined using a protein engineering approach. Deamidation and ligand degradation could be monitored in relation to NaOH incubation conditions. Enzymatic digestion of MabSelect SuRe and MabSelect PrismA resins after alkaline incubation and LC-MS/MS peptide mapping facilitates identification and quantification of specific deamidation sites on the affinity ligand.

Sarcoptic mange in the wild boar, Sus scrofa, in Sweden.

Sarcoptic mange caused by Sarcoptes scabiei has been present in the Swedish red fox (Vulpes vulpes) population since the 1970s. The disease has been described in other Swedish wildlife species, but not in the wild boar, Sus scrofa, until 2009. Single cases of sarcoptic mange have been diagnosed the last years in the expanding population of wild boar. This study aims to describe the histopathological lesions found on mangy wild boar and compare, by molecular methods, mites from wild boar cases with mites from mangy red foxes, raccoon dogs, and domestic pigs. Mangy wild boar with focal alopecia and clinical signs of pruritis were reported or submitted from various areas in southern Sweden to the National Veterinary Institute, Uppsala. The examined skin samples of wild boar infected with S. scabiei showed limited gross skin lesions, except for cases with severe exudative dermatitis. Histopathology of the affected wild boar skin samples showed an eosinophilic dermatitis with a variable hyperkeratosis and often low number of mites present. To study the relationship of S. scabiei mites isolated from different host species, a population genetics investigation was performed based on microsatellite markers. In total, 225 individual mites from eight individuals of four different host species; red fox (48 mites), wild boar (80 mites), domestic pig (48 mites) and raccoon dog (43 mites), were included in the study. In the phylogenetic analysis, all mites isolated from wild boar clustered together even though they originate from different geographical regions in Sweden. Mites from each individual host showed high similarity. The results indicate that wild boar mites differ from mites both from the red fox, raccoon dog, and domestic pig.

Torix group Rickettsia are widespread in Culicoides biting midges (Diptera: Ceratopogonidae), reach high frequency and carry unique genomic features.

There is increasing interest in the heritable bacteria of invertebrate vectors of disease as they present novel targets for control initiatives. Previous studies on biting midges (Culicoides spp.), known to transmit several RNA viruses of veterinary importance, have revealed infections with the endosymbiotic bacteria, Wolbachia and Cardinium. However, rickettsial symbionts in these vectors are underexplored. Here, we present the genome of a previously uncharacterized Rickettsia endosymbiont from Culicoides newsteadi (RiCNE). This genome presents unique features potentially associated with host invasion and adaptation, including genes for the complete non-oxidative phase of the pentose phosphate pathway, and others predicted to mediate lipopolysaccharides and cell wall modification. Screening of 414 Culicoides individuals from 29 Palearctic or Afrotropical species revealed that Rickettsia represent a widespread but previously overlooked association, reaching high frequencies in midge populations and present in 38% of the species tested. Sequence typing clusters the Rickettsia within the Torix group of the genus, a group known to infect several aquatic and hematophagous taxa. FISH analysis indicated the presence of Rickettsia bacteria in ovary tissue, indicating their maternal inheritance. Given the importance of biting midges as vectors, a key area of future research is to establish the impact of this endosymbiont on vector competence.

Implant stability and bone remodeling up to 84 days of implantation with an initial static strain. An in vivo and theoretical investigation.

OBJECTIVES: When implants are inserted, the initial implant stability is dependent on the mechanical stability. To increase the initial stability, it was hypothesized that bone condensation implants will enhance the mechanical stability initially and that the moderately rough surface will further contribute to the secondary stability by enhanced osseointegration. It was further hypothesized that as the healing progresses the difference in removal torque will diminish. In addition, a 3D model was developed to simulate the interfacial shear strength. This was converted to a theoretical removal torque that was compared to the removal torque obtained in vivo. MATERIAL AND METHODS: Condensation implants, inducing bone strains of 0.015, were installed into the left tibia of 24 rabbits. Non-condensation implants were installed into the right tibia. All implants had a moderately rough surface. The implants had an implantation time of 7, 28, or 84 days before the removal torque was measured. The interfacial shear strength at different healing time was estimated by the means of finite element method. RESULTS: At 7 days of healing, the condensation implant had an increased removal torque compared to the non-bone-condensation implant. At 28 and 84 days of healing, there was no difference in removal torque. The simulated interfacial shear strength ratios of bone condensation implants at different implantation time were in line with the in vivo data. CONCLUSIONS: Moderately rough implants that initially induce bone strain during installation have increased stability during the early healing period. In addition, the finite element method may be used to evaluate differences in interlocking capacity.

Simulation of the mechanical interlocking capacity of a rough bone implant surface during healing.

BACKGROUND: When an implant is inserted in the bone the healing process starts to osseointegrate the implant by creating new bone that interlocks with the implant. Biomechanical interlocking capacity is commonly evaluated in in vivo experiments. It would be beneficial to find a numerical method to evaluate the interlocking capacity of different surface structures with bone. In the present study, the theoretical interlocking capacity of three different surfaces after different healing times was evaluated by the means of explicit finite element analysis. METHODS: The surface topographies of the three surfaces were measured with interferometry and were used to construct a 3D bone-implant model. The implant was subjected to a displacement until failure of the bone-to-implant interface and the maximum force represents the interlocking capacity. RESULTS: The simulated ratios (test/control) seem to agree with the in vivo ratios of Halldin et al. for longer healing times. However the absolute removal torque values are underestimated and do not reach the biomechanical performance found in the study by Halldin et al. which might be a result of unknown mechanical properties of the interface. CONCLUSION: Finite element analysis is a promising method that might be used prior to an in vivo study to compare the load bearing capacity of the bone-to-implant interface of two surface topographies at longer healing times.