Controlling pH in shake flasks using polymer-based controlled-release discs with pre-determined release kinetics

Background  

There are significant differences in the culture conditions between small-scale screenings and large-scale fermentation processes. Production processes are usually conducted in fed-batch cultivation mode with active pH-monitoring and control. In contrast, screening experiments in shake flasks are usually conducted in batch mode without active pH-control, but with high buffer concentrations to prevent excessive pH-drifts. These differences make it difficult to compare results from screening experiments and laboratory and technical scale cultivations and, thus, complicate rational process development. In particular, the pH-value plays an important role in fermentation processes due to the narrow physiological or optimal pH-range of microorganisms. To reduce the differences between the scales and to establish a pH-control in shake flasks, a newly developed easy to use polymer-based controlled-release system is presented in this paper. This system consists of bio-compatible silicone discs embedding the alkaline reagent Na₂CO₃. Since the sodium carbonate is gradually released from the discs in pre-determined kinetics, it will ultimately compensate the decrease in pH caused by the biological activity of microorganisms.     

Engineering of the E. coli Outer Membrane Protein FhuA to overcome the Hydrophobic Mismatch in Thick Polymeric Membranes

Background

There is an urgent need to develop safe and effective adjuvants for the new generation of subunit vaccines. We developed the tubular immunostimulating complex (TI-complex) as a new nanoparticulate antigen delivery system. The morphology and composition of TI-complexes principally differ from the known vesicular immunostimulating complexes (ISCOMs). However, methodology for the preparation of TI-complexes has suffered a number of shortcomings. The aim of the present work was to obtain an antigen carrier consisting of triterpene glycosides from Cucumaria japonica, cholesterol, and monogalactosyldiacylglycerol from marine macrophytes with reproducible properties and high adjuvant activity.

Results

The cucumarioside A₂-2 - cholesterol - MGalDG ratio of 6:2:4 (by weight) was found to provide the most effective formation of TI-complexes and the minimum hemolytic activity in vitro. Tubules of TI-complexes have an outer diameter of about 16 nm, an inner diameter of 6 nm, and a length of 500 nm. A significant dilution by the buffer gradually destroyed the tubular nanoparticles. The TI-complex was able to increase the immunogenicity of the protein antigens from Yersinia pseudotuberculosis by three to four times.

Conclusions

We propose an optimized methodology for the preparation of homogeneous TI-complexes containing only tubular particles, which would achieve reproducible immunization results. We suggest that the elaborated TI-complexes apply as a universal delivery system for different subunit antigens within anti-infectious vaccines and enhance their economic efficacy and safety.     

Human α-mannosidase produced in transgenic tobacco plants is processed in human α-mannosidosis cell lines

Deficiency in human lysosomal α-mannosidase (MAN2B1) results in α-mannosidosis, a lysosomal storage disorder; patients present a wide range of neurological, immunological, and skeletal symptoms caused by a multisystemic accumulation of mannose-containing oligosaccharides. Here, we describe the expression of recombinant MAN2B1 both transiently in Nicotiana benthamiana leaves and in the leaves and seeds of stably transformed N. tabacum plants. After purification from tobacco leaves, the recombinant enzyme was found to be N-glycosylated and localized in vacuolar compartments. In the fresh leaves of tobacco transformants, MAN2B1 was measured at 10,200 units/kg, and the purified enzyme from these leaves had a specific activity of 32-45 U/mg. Furthermore, tobacco-produced MAN2B1 was biochemically similar to the enzyme purified from human tissues, and it was internalized and processed by α-mannosidosis fibroblast cells. These results strongly indicate that plants can be considered a promising expression system for the production of recombinant MAN2B1 for use in enzyme replacement therapy.     

Potential role of nonstatin cholesterol lowering agents

Although statins, 3β-hydroxy-3β-methylglutaryl coenzyme A reductase (HMGR) inhibitors, have revolutionized the management of cardiovascular diseases by lowering serum low density lipoproteins, many patients suffer from their side effects. Whether the statin side effects are related to their intrinsic toxicity or to the decrease of HMGR main isoprenoid end products, which are essential compounds for cell viability, is still debated. In addition to HMGR, the key and rate limiting step of cholesterol synthesis, many enzymes are involved in this multi-step pathway whose inhibition could be taken into account for a "nonstatin approach" in the management of hypercholesterolemia. In particular, due to their unique position downstream from HMGR, the inhibition of squalene synthase, farnesyl diphosphate farnesyltransferase (FDFT1), squalene epoxidase (SQLE), and oxidosqualene cyclase:lanosterol synthase (OSC) should decrease plasma levels of cholesterol without affecting ubiquinone, dolichol, and isoprenoid metabolism. Thus, although FDFT1, SQLE and OSC are little studied, they should be considered as perspective targets for the development of novel drugs against hypercholesterolemia. Here, structure-function relationships of FDFT1, SQLE, and OSC are reviewed highlighting the advantages that the downstream inhibition of HMGR could provide when compared to the statin-based therapy.     

Relative burden of large CNVs on a range of neurodevelopmental phenotypes

While numerous studies have implicated copy number variants (CNVs) in a range of neurological phenotypes, the impact relative to disease severity has been difficult to ascertain due to small sample sizes, lack of phenotypic details, and heterogeneity in platforms used for discovery. Using a customized microarray enriched for genomic hotspots, we assayed for large CNVs among 1,227 individuals with various neurological deficits including dyslexia (376), sporadic autism (350), and intellectual disability (ID) (501), as well as 337 controls. We show that the frequency of large CNVs (>1 Mbp) is significantly greater for ID-associated phenotypes compared to autism (p = 9.58 × 10(-11), odds ratio = 4.59), dyslexia (p = 3.81 × 10(-18), odds ratio = 14.45), or controls (p = 2.75 × 10(-17), odds ratio = 13.71). There is a striking difference in the frequency of rare CNVs (>50 kbp) in autism (10%, p = 2.4 × 10(-6), odds ratio = 6) or ID (16%, p = 3.55 × 10(-12), odds ratio = 10) compared to dyslexia (2%) with essentially no difference in large CNV burden among dyslexia patients compared to controls. Rare CNVs were more likely to arise de novo (64%) in ID when compared to autism (40%) or dyslexia (0%). We observed a significantly increased large CNV burden in individuals with ID and multiple congenital anomalies (MCA) compared to ID alone (p = 0.001, odds ratio = 2.54). Our data suggest that large CNV burden positively correlates with the severity of childhood disability: ID with MCA being most severely affected and dyslexics being indistinguishable from controls. When autism without ID was considered separately, the increase in CNV burden was modest compared to controls (p = 0.07, odds ratio = 2.33).     

Mind the gap: keeping UV lesions in check

Cells respond to genotoxic insults by triggering a DNA damage checkpoint surveillance mechanism and by activating repair pathways. Recent findings indicate that the two processes are more related than originally thought. Here we discuss the mechanisms involved in responding to UV-induced lesions in different phases of the cell cycle and summarize the most recent data in a model where Nucleotide Excision Repair (NER) and exonucleolytic activities act in sequence leading to checkpoint activation in non replicating cells. The critical trigger is likely represented by problematic intermediates that cannot be completely or efficiently repaired by NER. In S phase cells, on the other hand, the replicative polymerases, blocked by bulky UV lesions, re-initiate DNA synthesis downstream of the lesions, leaving behind a ssDNA tract. If these gaps are not rapidly refilled, checkpoint kinases will be activated.     

Workshops Without Walls: broadening access to science around the world

The National Aeronautics and Space Administration (NASA) Astrobiology Institute (NAI) conducted two "Workshops Without Walls" during 2010 that enabled global scientific exchange--with no travel required. The second of these was on the topic "Molecular Paleontology and Resurrection: Rewinding the Tape of Life." Scientists from diverse disciplines and locations around the world were joined through an integrated suite of collaborative technologies to exchange information on the latest developments in this area of origin of life research. Through social media outlets and popular science blogs, participation in the workshop was broadened to include educators, science writers, and members of the general public. In total, over 560 people from 31 US states and 30 other nations were registered. Among the scientific disciplines represented were geochemistry, biochemistry, molecular biology and evolution, and microbial ecology. We present this workshop as a case study in how interdisciplinary collaborative research may be fostered, with substantial public engagement, without sustaining the deleterious environmental and economic impacts of travel.