Production and Purification of Filovirus Glycoproteins in Insect and Mammalian Cell Lines.

Filoviruses are highly virulent pathogens capable of causing severe disease. The glycoproteins of filoviruses are the only virally expressed proteins on the virion surface and are required for receptor binding. As such, they are the main candidate vaccine antigen. Despite their virulence, most filoviruses are not comprehensively characterized, and relatively few commercially produced reagents are available for their study. Here, we describe two methods for production and purification of filovirus glycoproteins in insect and mammalian cell lines. Considerations of expression vector choice, modifications to sequence, troubleshooting of purification method, and glycosylation differences are all important for successful expression of filovirus glycoproteins in cell lines. Given the scarcity of commercially available filovirus glycoproteins, we hope our experiences with possible difficulties in purification of the proteins will facilitate other researchers to produce and purify filovirus glycoproteins rapidly.

Tau pathology and cognitive reserve in Alzheimer's disease.

Cognitive reserve (CR) is defined as the ability to maintain functionality despite accumulating pathology. Education has been used as a proxy for CR. For example, by using positron emission tomography imaging, higher educated Alzheimer's disease (AD) patients presented increased amyloid ß pathology than lower educated patients despite equal symptomatology. Whether similar associations exist for in vivo tau pathology remains elusive. We utilized [18F]AV-1451 positron emission tomography imaging to examine whether high-educated AD patients (n = 12) present more severe tau pathology compared with low-educated patients (n = 12) despite equal clinical severity in regions of interest corresponding to the pathologic disease stages defined by Braak & Braak. We report tau pathology in advanced Braak stages associated with parietal and frontal regions in high-educated AD patients, whereas in low-educated AD patients tau accumulation is still confined to lower Braak stages associated with temporal and cingulate regions. Highly educated AD patients seem to be able to tolerate more tau tangle pathology than lower educated patients with comparable cognitive impairment supporting the cognitive reserve hypothesis.

Comparison of N- and O-linked glycosylation patterns of ebolavirus glycoproteins.

Ebolaviruses are emerging pathogens that cause severe and often fatal viral hemorrhagic fevers. Four distinct ebolaviruses are known to cause Ebola virus disease in humans. The ebolavirus envelope glycoprotein (GP1,2) is heavily glycosylated, but the precise glycosylation patterns of ebolaviruses are largely unknown. Here we demonstrate that approximately 50 different N-glycan structures are present in GP1,2 derived from the four pathogenic ebolaviruses, including high mannose, hybrid, and bi-, tri-, and tetra-antennary complex glycans with and without fucose and sialic acid. The overall N-glycan composition is similar between the different ebolavirus GP1,2s. In contrast, the amount and type of O-glycan structures varies widely between ebolavirus GP1,2s. Notably, this O-glycan dissimilarity is also present between two variants of Ebola virus, the original Yambuku variant and the Makona variant responsible for the most recent Western African epidemic. The data presented here should serve as the foundation for future ebolaviral entry and immunogenicity studies.

CSF N-glycan profiles to investigate biomarkers in brain developmental disorders: application to leukodystrophies related to eIF2B mutations.

BACKGROUND: Primary or secondary abnormalities of glycosylation have been reported in various brain diseases. Decreased asialotransferrin to sialotransferrin ratio in cerebrospinal fluid (CSF) is a diagnostic marker of leukodystrophies related to mutations of genes encoding translation initiation factor, EIF2B. We investigated the CSF glycome of eIF2B-mutated patients and age-matched normal individuals in order to further characterize the glycosylation defect for possible use as a biomarker. METHODOLOGY/PRINCIPAL FINDINGS: We conducted a differential N-glycan analysis using MALDI-TOF/MS of permethylated N-glycans in CSF and plasma of controls and eIF2B-mutated patients. We found in control CSF that tri-antennary/bisecting and high mannose structures were highly represented in samples obtained between 1 to 5 years of age, whereas fucosylated, sialylated structures were predominant at later age. In CSF, but not in plasma, of eIF2B-mutated patient samples, we found increased relative intensity of bi-antennary structures and decreased tri-antennary/bisecting structures in N-glycan profiles. Four of these structures appeared to be biomarker candidates of glycomic profiles of eIF2B-related disorders. CONCLUSION: Our results suggest a dynamic development of normal CSF N-glycan profiles from high mannose type structures to complex sialylated structures that could be correlated with postnatal brain maturation. CSF N-glycome analysis shows relevant quantitative changes associated with eIF2B related disorders. This approach could be applied to other neurological disorders involving developmental gliogenesis/synaptogenesis abnormalities.

Discharging neonates with congenital heart disease after cardiac surgery: a practical approach.

Discharging neonates to home after cardiac surgery takes time, effective communication, and a commitment to continuity of care. The efforts of all members of a multidisciplinary team are necessary and valuable to ensure success. The discharge process involves many steps beginning at the time of admission and continuing past the actual discharge date. Discharge planning is an evolving process rather than a single event.

Production of two vaccinating recombinant rotavirus proteins in the milk of transgenic rabbits.

Rotaviruses are the main cause of infantile viral gastroenteritis worldwide leading to approximately 500,000 deaths each year mostly in the developing world. For unknown reasons, live attenuated viruses used in classical vaccine strategies were shown to be responsible for intussusception (a bowel obstruction). New strategies allowing production of safe recombinant non-replicating rotavirus candidate vaccine are thus clearly needed. In this study we utilized transgenic rabbit milk as a source of rotavirus antigens. Individual transgenic rabbit lines were able to produce several hundreds of micrograms per ml of secreted recombinant VP2 and VP6 proteins in their milk. Viral proteins expressed in our model were immunogenic and were shown to induce a significant reduction in viral antigen shedding after challenge with virulent rotavirus in the adult mouse model. To our knowledge, this is the first report of transgenic mammal bioreactors allowing the rapid co-production of two recombinant viral proteins in milk to be used as a vaccine.

The Zodiak workshop: an innovative model for teaching financial management through partnership with industry.

The National Commission on Veterinary Economic Issues (NCVEI) is working to enhance the non-technical skills, knowledge, aptitudes, and attitudes (SKAs) of veterinarians. This report describes the development of an innovative model for teaching the principles of financial management as they apply to the veterinary practice. Zodiak: The Game of Business Finance and Strategy is a "business literacy" game in which players work together in small teams (generally four people) to run a fictional multi-million-dollar company called Zodiak Industries for three "years" in order to learn principles of business finance and strategy. After finishing the 4.5-hour game, participants spend the rest of the workshop making the right "Connections"-exercises designed to connect what they have learned to business strategies, financial statements, and operational tactics drawn from veterinary practice. Issues addressed for the veterinary practice, with parallels drawn to Zodiak, included return on owner investment in a veterinary practice (vs. salary drawn by owner veterinarians); pricing (setting prices, price elasticity of demand, and relationships between volume, quality, and price); human resources and operations management as they relate to profitability and efficiency; cash flow and management of accounts receivable; and commonly used financial benchmarks. Workshop venues have included Michigan State University, The Ohio State University, the University of Illinois, and Purdue University. Financial and in-kind support were provided through partnership with Pharmacia Animal Health (now Pfizer Animal Health) and Hill's Pet Nutrition, Inc. Through course evaluations, participants generally rated the workshop high as an educational experience and indicated that the most important things learned were related to financial management (principles, terminology, and methods). The most enjoyable aspects of the workshop tended to be group discussions, teamwork, the dynamic/interactive environment, and the "game" atmosphere. Based on these experiences, the Zodiak workshop provides a useful model for teaching career development and practice management topics to veterinary students. The business simulation in a workshop format was especially useful for teaching these "non-mainstream" topics, as traditional classroom lecture approaches might not have engaged students sufficiently to achieve effective learning. In addition, the partnership developed between academia and industry offered substantial benefits to both parties. Similar educational approaches should be considered for additional aspects of the non-technical SKAs.

Prolyl hydroxylation of collagen type I is required for efficient binding to integrin alpha 1 beta 1 and platelet glycoprotein VI but not to alpha 2 beta 1.

Collagen is a potent adhesive substrate for cells, an event essentially mediated by the integrins alpha 1 beta 1 and alpha 2 beta 1. Collagen fibrils also bind to the integrin alpha 2 beta 1 and the platelet receptor glycoprotein VI to activate and aggregate platelets. The distinct triple helical recognition motifs for these receptors, GXOGER and (GPO)n, respectively, all contain hydroxyproline. Using unhydroxylated collagen I produced in transgenic plants, we investigated the role of hydroxyproline in the receptor-binding properties of collagen. We show that alpha 2 beta 1 but not alpha 1 beta 1 mediates cell adhesion to unhydroxylated collagen. Soluble recombinant alpha 1 beta 1 binding to unhydroxylated collagen is considerably reduced compared with bovine collagens, but binding can be restored by prolyl hydroxylation of recombinant collagen. We also show that platelets use alpha 2 beta 1 to adhere to the unhydroxylated recombinant molecules, but the adhesion is weaker than on fully hydroxylated collagen, and the unhydroxylated collagen fibrils fail to aggregate platelets. Prolyl hydroxylation is thus required for binding of collagen to platelet glycoprotein VI and to cells by alpha 1 beta 1. These observations give new insights into the molecular basis of collagen-receptor interactions and offer new selective applications for the recombinant unhydroxylated collagen I.