Effect of different UCOE-promoter combinations in creation of engineered cell lines for the production of Factor VIII

<p>Abstract</p> <p>Background</p> <p>The most common approach used in generating cell lines for the production of therapetic proteins relies on gene amplification induced by a drug resistance gene e. g., DHFR and glutamine synthetase. Practically, this results in screening large number of clones for the one that expresses high levels of the biologic in a stable manner. The inefficiency of mammalian vector systems to express proteins in a stable manner typically involves silencing of the exogenous gene resulting from modifications such as methylation of CpG DNA sequences, histone deacetylation and chromatin condensation. The use of un-methylated CpG island fragments from housekeeping genes referred to as UCOE (ubiquitous chromatin opening elements) in plasmid vectors is now well established for increased stability of transgene expression. However, few UCOE-promoter combinations have been studied to date and in this report we have tested 14 different combinations.</p> <p>Findings</p> <p>In this report we describe studies with two different UCOEs (the 1.5 Kb human RNP fragment and the 3.2 Kb mouse RPS3 fragment) in combination with various promoters to express a large protein (B domain deleted factor VIII; BDD-FVIII) in a production cell line, BHK21. We show here that there are differences in expression of BDD-FVIII by the different UCOE-promoter combinations in both attached and serum free suspension adapted cells. In all cases, the 1.5 Kb human RNP UCOE performed better in expressing BDD-FVIII than their corresponding 3.2 Kb mouse RPS3 UCOE. Surprisingly, in certain scenarios described here, expression from a number of promoters was equivalent or higher than the commonly used and industry standard human CMV promoter.</p> <p>Conclusion</p> <p>This study indicates that certain UCOE-promoter combinations are better than others in expressing the BDD-FVIII protein in a stable manner in BHK21 cells. An empirical study such as this is required to determine the best combination of UCOE-promoter in a vector for a particular production cell line.</p

Changes in structural attributes of plant communities along disturbance gradients in a dry deciduous forest of Western Ghats, India

Changes in tree and understory plant diversity and community composition in two sites at different disturbance levels were studied on the Anaikatty hills, Western Ghats. Systematic sampling using small scale permanent quadrates (50 x 20 m for trees, 5 x 5 m for shrubs/saplings, 1 x 1 m for herbs/seedlings) enumerated 3,376 individuals of trees (106 species), 8,599 of individuals shrubs (122 species) and 16,659 individuals of herbs (145 species). Among the two sites, species richness and diversity were highest for low disturbed stand (98 and 3.9, respectively) compared to high disturbed site (45 and 2.71, respectively). Result of cluster analysis showed that two distinct clusters were formed on the basis of disturbance of the area in concordance with our field observation. A total of 37 species were common to both sites, sixty one species exclusively found in low disturbed site and eight species were pertained to highly disturbed site. Mann-Whitney test based on Monte Carlo approximation at 95% confidence levels indicated that both populations were not entirely different. The clear difference was only observed for average basal area of trees, density of seedlings, number of species, density and diversity for shrubs and number of species and diversity for herb. The species composition were different in two stand i.e., Nothopegia racemosa-Albizia amara-Maba neilghrrensis in low disturbed stand and Albizia amara-Pleiospermium alatum-Bauhinia racemosa in high disturbed stand. The major disturbance factors identification using spearman rank correlation indicated that the disturbance in low disturbed habitats were mostly from past logging followed by cutting and illicit felling and grazing, while in high disturbed habitats, it was human presence, past logging and lopping and fuel wood collection

Diagnosis and management of pulmonary nodules

There are an increased number of pulmonary nodules discovered on CT scan images in part due to those performed for lung cancer screening. Risk stratification and patient involvement is critical in determining management ranging from interval imaging to invasive biopsy or surgery. A definitive diagnosis requires tissue biopsy. The choice of a particular biopsy technique depends on the risks/benefits of the procedure, the diagnostic yield and local expertise. This review will focus on the evaluation and management of pulmonary nodules based on the Fleischner Society and American College of Chest Physician guidelines. There have been recent changes to both societies' recommendations for incidental detection of solid and subsolid nodules, risk stratification, imaging, minimally invasive diagnostic techniques and definitive surgical options

Investigating the role of mitochondria in type 2 diabetes lessons from lipidomics and proteomics studies of skeletal muscle and liver.

Mitochondrial dysfunction is discussed as a key player in the pathogenesis of type 2 diabetes mellitus (T2Dm), a highly prevalent disease rapidly developing as one of the greatest global health challenges of this century. Data however about the involvement of mitochondria, central hubs in bioenergetic processes, in the disease development are still controversial. Lipid and protein homeostasis are under intense discussion to be crucial for proper mitochondrial function. Consequently proteomics and lipidomics analyses might help to understand how molecular changes in mitochondria translate to alterations in energy transduction as observed in the healthy and metabolic diseases such as T2Dm and other related disorders. Mitochondrial lipids integrated in a tool covering proteomic and functional analyses were up to now rarely investigated, although mitochondria]. lipids might provide a possible lynchpin in the understanding of type 2 diabetes development and thereby prevention. In this chapter state-of-the-art analytical strategies, pre -analytical aspects, potential pitfalls as well as current proteomics and lipidomics-based knowledge about the pathophysiological role of mitochondria in the pathogenesis of type 2 diabetes will be discussed