Construction and analysis of a genetically tuneable lytic phage display system

The Bacteriophage λ capsid protein gpD has been used extensively for fusion polypeptides that can be expressed from plasmids in Escherichia coli and remain soluble. In this study, a genetically controlled dual expression system for the display of enhanced green fluorescent protein (eGFP) was developed and characterized. Wild-type D protein (gpD) expression is encoded by λ Dam15 infecting phage particles, which can only produce a functional gpD protein when translated in amber suppressor strains of E. coli in the absence of complementing gpD from a plasmid. However, the isogenic suppressors vary dramatically in their ability to restore functional packaging to λDam15, imparting the first dimension of decorative control. In combination, the D-fusion protein, gpD::eGFP, was supplied in trans from a multicopy temperature-inducible expression plasmid, influencing D::eGFP expression and hence the availability of gpD::eGFP to complement for the Dam15 mutation and decorate viable phage progeny. Despite being the worst suppressor, maximal incorporation of gpD::eGFP into the λDam15 phage capsid was imparted by the SupD strain, conferring a gpDQ68S substitution, induced for plasmid expression of pD::eGFP. Differences in size, fluorescence and absolute protein decoration between phage preparations could be achieved by varying the temperature of and the suppressor host carrying the pD::eGFP plasmid. The effective preparation with these two variables provides a simple means by which to manage fusion decoration on the surface of phage λ.     

Development and validation of a HPLC method for the quantification of baculovirus particles

A HPLC method using an anion exchange column was developed for the quantification of baculovirus particles. To properly detect the virus eluting from the column, a nucleic acid dye was used to amplify the signal projected by the virus. The viral genome was labeled by incubating the virus with SYBR Green I at 37°C for a minimum of 1h. The virus was specifically eluted from the contaminants in 8.9 min at a NaCl concentration of 480 mM NaCl (in 20 mM Tris-HCl, pH 7.5). The total run time of the method was 25 min. The method resulted in a linear response from 1×10(8) to 5.0×10(10)viral particles (VP/ml). The detection limit was 3.0×10(7) and the quantification limit was 1×10(8)VP/ml. The intra-assay precision was <10% for both purified and crude virus preparations whereas the inter-assay precisions were <5% and <10% for purified and crude virus preparations, respectively. The recovery/accuracy of the method ranged from 78 to 101%. This method is a robust monitoring tool to facilitate research activities with baculovirus vector and accelerate development of baculovirus-based processes for manufacturing of biologics.     

Co-expression vs. co-infection using baculovirus expression vectors in insect cell culture: Benefits and drawbacks

The baculovirus expression vector system (BEVS) is a versatile and powerful platform for protein expression in insect cells. With the ability to approach similar post-translational modifications as in mammalian cells, the BEVS offers a number of advantages including high levels of expression as well as an inherent safety during manufacture and of the final product. Many BEVS products include proteins and protein complexes that require expression from more than one gene. This review examines the expression strategies that have been used to this end and focuses on the distinguishing features between those that make use of single polycistronic baculovirus (co-expression) and those that use multiple monocistronic baculoviruses (co-infection). Three major areas in which researchers have been able to take advantage of co-expression/co-infection are addressed, including compound structure-function studies, insect cell functionality augmentation, and VLP production. The core of the review discusses the parameters of interest for co-infection and co-expression with time of infection (TOI) and multiplicity of infection (MOI) highlighted for the former and the choice of promoter for the latter. In addition, an overview of modeling approaches is presented, with a suggested trajectory for future exploration. The review concludes with an examination of the gaps that still remain in co-expression/co-infection knowledge and practice.     

Impact of Dissolved Oxygen during UV-Irradiation on the Chemical Composition and Function of CHO Cell Culture Media

Ultraviolet (UV) irradiation is advantageous as a sterilization technique in the biopharmaceutical industry since it is capable of targeting non-enveloped viruses that are typically challenging to destroy, as well as smaller viruses that can be difficult to remove via conventional separation techniques. In this work, we investigated the influence of oxygen in the media during UV irradiation and characterized the effect on chemical composition using NMR and LC-MS, as well as the ability of the irradiated media to support cell culture. Chemically defined Chinese hamster ovary cell growth media was irradiated at high fluences in a continuous-flow UV reactor. UV-irradiation caused the depletion of pyridoxamine, pyridoxine, pyruvate, riboflavin, tryptophan, and tyrosine; and accumulation of acetate, formate, kynurenine, lumichrome, and sarcosine. Pyridoxamine was the only compound to undergo complete degradation within the fluences considered; complete depletion of pyridoxamine was observed at 200 mJ/cm². Although in both oxygen- and nitrogen-saturated media, the cell culture performance was affected at fluences above 200 mJ/cm², there was less of an impact on cell culture performance in the nitrogen-saturated media. Based on these results, minimization of oxygen in cell culture media prior to UV treatment is recommended to minimize the negative impact on sensitive media.     

A Dutch guideline for the treatment of scoliosis in neuromuscular disorders

Background

Children with neuromuscular disorders with a progressive muscle weakness such as Duchenne Muscular Dystrophy and Spinal Muscular Atrophy frequently develop a progressive scoliosis. A severe scoliosis compromises respiratory function and makes sitting more difficult. Spinal surgery is considered the primary treatment option for correcting severe scoliosis in neuromuscular disorders. Surgery in this population requires a multidisciplinary approach, careful planning, dedicated surgical procedures, and specialized after care.

Methods

The guideline is based on scientific evidence and expert opinions. A multidisciplinary working group representing experts from all relevant specialties performed the research. A literature search was conducted to collect scientific evidence in answer to specific questions posed by the working group. Literature was classified according to the level of evidence.

Results

For most aspects of the treatment scientific evidence is scarce and only low level cohort studies were found. Nevertheless, a high degree of consensus was reached about the management of patients with scoliosis in neuromuscular disorders. This was translated into a set of recommendations, which are now officially accepted as a general guideline in the Netherlands.

Conclusion

In order to optimize the treatment for scoliosis in neuromuscular disorders a Dutch guideline has been composed. This evidence-based, multidisciplinary guideline addresses conservative treatment, the preoperative, perioperative, and postoperative care of scoliosis in neuromuscular disorders.     

产β—葡萄糖苷酶真菌诱变菌株快速筛选方法

报道了一种快速筛选产β-葡萄糖管酶真菌诱变菌株的方法。该法利用ρNPG经β-葡萄糖苷酶水解,水解产物ρNP在碱性条件下显色的原理。采用自制的特殊初筛平皿.经一年多的实践应用表明此法具有简便、灵敏、筛子消耗量小、筛选效率高等优点。     

N-terminal Domain of Myelin Basic Protein Inhibits Amyloid β-Protein Fibril Assembly

Accumulation of amyloid β-protein (Aβ) into brain parenchymal plaques and the cerebral vasculature is a pathological feature of Alzheimer disease and related disorders. Aβ peptides readily form β-sheet-containing oligomers and fibrils. Previously, we reported a strong interaction between myelin basic protein (MBP) and Aβ peptides that resulted in potent inhibition of fibril assembly (Hoos, M. D., Ahmed, M., Smith, S. O., and Van Nostrand, W. E. (2007) J. Biol. Chem. 282, 9952–9961; Hoos, M. D., Ahmed, M., Smith, S. O., and Van Nostrand, W. E. (2009) Biochemistry 48, 4720–4727). MBP is recognized as a highly post-translationally modified protein. In the present study, we demonstrate that human MBP purified from either brain or a bacterial recombinant expression system comparably bound to Aβ and inhibited Aβ fibril assembly indicating that post-translational modifications are not required for this activity. We also show that purified mouse brain MBP and recombinantly expressed mouse MBP similarly inhibited Aβ fibril formation. Through a combination of biochemical and ultrastructural techniques, we demonstrate that the binding site for Aβ is located in the N-terminal 64 amino acids of MBP and that a stable peptide (MBP1) comprising these residues was sufficient to inhibit Aβ fibrillogenesis. Under conditions comparable with those used for Aβ, the fibrillar assembly of amylin, another amyloidogenic peptide, was not inhibited by MBP1, although MBP1 still bound to it. This observation suggests that the potent inhibitory effect of MBP on fibril formation is not general to amyloidogenic peptides. Finally, MBP1 could prevent the cytotoxic effects of Aβ in primary cortical neurons. Our findings suggest that inhibition of Aβ fibril assembly by MBP, mediated through its N-terminal domain, could play a role in influencing amyloid formation in Alzheimer disease brain and corresponding mouse models.     

The four hexamerin genes in the honey bee: structure,molecular evolution and function deduced from expression patterns in queens,workers and drones

Background  

Hexamerins are hemocyanin-derived proteins that have lost the ability to bind copper ions and transport oxygen; instead, they became storage proteins. The current study aimed to broaden our knowledge on the hexamerin genes found in the honey bee genome by exploring their structural characteristics, expression profiles, evolution, and functions in the life cycle of workers, drones and queens.     

The protective effect of melatonin in lungs of newborn rats exposed to maternal nicotine

We investigated possible healing effects of melatonin (MEL) on biochemical and histological changes in the lungs of rat offspring caused by exposure to nicotine (NT) in utero. Pregnant rats were divided randomly into five groups. The SP group was treated with physiological saline. The EA group was treated with ethyl alcohol. The MEL group was treated with MEL. The NT group was treated with NT. The NT + MEL group was treated with NT and MEL. At the end of the study, the biochemistry and histopathology of lung tissue of the offspring were examined. Reduced alveolar development and increased numbers of alveolar macrophages and mast cells were observed in the NT group compared to the SP, EA and MEL groups. We also found increased malondialdehyde (MDA) levels and decreased total glutathione (GSH) levels in the NT group. Application of MEL ameliorated the histological and biochemical damage caused by NT. The number of alveoli was greater in the NT + MEL group than in the NT group. Also, the increased numbers of alveolar macrophages and mast cells resulting from exposure to NT were decreased following MEL treatment. We found that MEL caused a significant decrease in the level of MDA. Maternal exposure to NT caused significant structural and biochemical changes in the lungs of the offspring and administration of MEL ameliorated the changes.