CHO-K1 host cells adapted to growth in glutamine-free medium by FACS-assisted evolution

During the process of recombinant cell line optimisation for production of biopharmaceuticals, multiple cellular properties like robustness against stress, the attainment of high cell concentrations and maintenance of high viability must be considered to maximize protein yield. To improve growth and viability, glutamine is supplemented as an alternative energy source for rapidly dividing cells that oxidize glucose inefficiently. However, the resulting by-product ammonia is toxic at high concentrations and has a negative impact on protein glycosylation, a major quality-determining parameter of biopharmaceuticals. In this work, the CHO-K1 cell line was adapted to a chemically defined medium and suspension growth within 3 weeks. Subsequently, the glutamine concentration was stepwise reduced from 8 to 4 and 2 mM. After each reduction, both the final cell concentration in the batch and the viability decreased. To force a rapid evolution of cells to achieve high final cell concentrations, cells were seeded at high densities (10(7) cells/mL) and surviving cells were sorted by FACS or MACS when viability declined to 10% (typically after 24 h). Sorted cells were grown in batch until viability declined to 10% and viable cells recovered again. The final sorted population was able to reach comparable or even better viable cell concentrations and showed a significantly improved viability compared to their ancestors. The 2 mM glutamine-adapted cell line was directly transferred into glutamine-free medium and was able to grow at comparable rates without requiring further adaptation. Cells compensated the lack of glutamine by increasing their consumption of glutamate and aspartate.     

Economic Appraisal of Ontario's Universal Influenza Immunization Program: A Cost-Utility Analysis

Background

In July 2000, the province of Ontario, Canada, initiated a universal influenza immunization program (UIIP) to provide free seasonal influenza vaccines for the entire population. This is the first large-scale program of its kind worldwide. The objective of this study was to conduct an economic appraisal of Ontario''s UIIP compared to a targeted influenza immunization program (TIIP).

Methods and Findings

A cost-utility analysis using Ontario health administrative data was performed. The study was informed by a companion ecological study comparing physician visits, emergency department visits, hospitalizations, and deaths between 1997 and 2004 in Ontario and nine other Canadian provinces offering targeted immunization programs. The relative change estimates from pre-2000 to post-2000 as observed in other provinces were applied to pre-UIIP Ontario event rates to calculate the expected number of events had Ontario continued to offer targeted immunization. Main outcome measures were quality-adjusted life years (QALYs), costs in 2006 Canadian dollars, and incremental cost-utility ratios (incremental cost per QALY gained). Program and other costs were drawn from Ontario sources. Utility weights were obtained from the literature. The incremental cost of the program per QALY gained was calculated from the health care payer perspective. Ontario''s UIIP costs approximately twice as much as a targeted program but reduces influenza cases by 61% and mortality by 28%, saving an estimated 1,134 QALYs per season overall. Reducing influenza cases decreases health care services cost by 52%. Most cost savings can be attributed to hospitalizations avoided. The incremental cost-effectiveness ratio is Can$10,797/QALY gained. Results are most sensitive to immunization cost and number of deaths averted.

Conclusions

Universal immunization against seasonal influenza was estimated to be an economically attractive intervention. Please see later in the article for the Editors'' Summary     

Novel whole-cell antibiotic biosensors for compound discovery

Cells containing reporters which are specifically induced via selected promoters are used in pharmaceutical drug discovery and in environmental biology. They are used in screening for novel drug candidates and in the detection of bioactive compounds in environmental samples. In this study, we generated and validated a set of five Bacillus subtilis promoters fused to the firefly luciferase reporter gene suitable for cell-based screening, enabling the as yet most-comprehensive high-throughput diagnosis of antibiotic interference in the major biosynthetic pathways of bacteria: the biosynthesis of DNA by the yorB promoter, of RNA by the yvgS promoter, of proteins by the yheI promoter, of the cell wall by the ypuA promoter, and of fatty acids by the fabHB promoter. The reporter cells mainly represent novel antibiotic biosensors compatible with high-throughput screening. We validated the strains by developing screens with a set of 14,000 pure natural products, representing a source of highly diverse chemical entities, many of them with antibiotic activity (6% with anti-Bacillus subtilis activity of 相似文献   

MALDI-TOF MS of phosphatidylethanolamines: different adducts cause different post source decay (PSD) fragment ion spectra

Matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS) is increasingly applied to lipids. However, positional acyl chain analysis of lipids by MALDI was so far scarcely described. In this paper, the fragmentation behavior of phosphatidylethanolamine (PE) is investigated by using post-source decay (PSD) MS. In dependence on the investigated adduct, significant differences could be obtained. It will be shown that in particular the negative ion spectra enable the determination of the individual acyl chains as well as their positions (sn-1 or sn-2). Therefore, MALDI-TOF PSD spectra are a real alternative to more sophisticated MS/MS methods.     

Methane and sulfate profiles within the subsurface of a tidal flat are reflected by the distribution of sulfate-reducing bacteria and methanogenic archaea

The anoxic layers of marine sediments are dominated by sulfate reduction and methanogenesis as the main terminal oxidation processes. The aim of this study was to analyze the vertical succession of microbial populations involved in these processes along the first 4.5 m of a tidal-flat sediment. Therefore, a quantitative PCR approach was applied using primers targeting the domains of Bacteria and Archaea, and key functional genes for sulfate reduction (dsrA) and methanogenesis (mcrA). The sampling site was characterized by an unusual sulfate peak at 250 cm depth resulting in separate sulfate-methane transition zones. Methane and sulfate profiles were diametrically opposed, with a methane maximum in the sulfate-depleted zone showing high numbers of archaea and methanogens. The methane-sulfate interfaces harbored elevated numbers of sulfate reducers, and revealed a slight increase in mcrA and archaeal 16S rRNA genes, suggesting sulfate-dependent anaerobic oxidation of methane. A diversity analysis of both functional genes by PCR-denaturing gradient gel electrophoresis revealed a vertical succession of subpopulations that were governed by geochemical and sedimentologic conditions. Along the upper 200 cm, sulfate-reducing populations appeared quite uniform and were dominated by the Deltaproteobacteria. In the layers beneath, an apparent increase in diversity and a shift to the Firmicutes as the predominant group was observed.     

The role of an intracellular cysteine stretch in the sorting of the type II Na/phosphate cotransporter

The type II Na/phosphate cotransporters (NaPi-II) are critical for the control of plasma phosphate levels in vertebrates. NaPi-IIb mediates phosphate uptake from the small intestine followed by glomerular filtration and selective reabsorption from the renal proximal tubule by NaPi-IIa and NaPi-IIc. A C-terminal stretch of cysteine residues represents the hallmark of the NaPi-IIb isoforms. This motif is well conserved among NaPi-IIb type transporters but not found in other membrane proteins. To investigate the role of this motif we analyzed NaPi-II constructs in transiently and stably transfected MDCK cells. This cell line targets the NaPi-IIb isoforms from flounder and mouse to the apical membrane whereas the mouse IIa isoform shows no plasma membrane preference. Different parts of mouse NaPi-IIa and NaPi-IIb C-termini were fused to GFP-tagged flounder NaPi-II. The constructs showed strong staining of the plasma membrane with NaPi-IIb related constructs sorted predominantly apically, the IIa constructs localized apically and basolaterally with slight intracellular retention. When the cysteine stretch was inserted into the NaPi-IIa C-terminus, the construct was retained in a cytoplasmic compartment. 2-bromopalmitate, a specific palmitoylation inhibitor, released the transporter to apical and basolateral membranes. The drug also leads to a redistribution of the NaPi-IIb construct to both plasma membrane compartments. Immunoprecipitation of tagged NaPi-II constructs from [³H]-palmitate labeled MDCK cells indicated that the cysteine stretch is palmitoylated. Our results suggest that the modified cysteine motif prevents the constructs from basolateral sorting. Additional sorting determinants located downstream of the cysteine stretch may release the cargo to the apical compartment.     

Tropheryma whipplei in the Environment: Survey of Sewage Plant Influxes and Sewage Plant Workers

We studied the prevalence of Tropheryma whipplei in influxes to 46 sewage treatment plants and in stool, mouthwash fluids, and dental plaques of 64 healthy workers in those facilities and 146 disease control patients. T. whipplei was found in sewage water, in stool of healthy individuals, and significantly more often in stool of workers exposed to sewage water.