Field evaluation of a simple fluorescence method for detection of viable Mycobacterium tuberculosis in sputum specimens during treatment follow-up.

Simple tuberculosis (TB) treatment monitoring tools are needed. We assessed the performance of fluorescein-diacetate (FDA) smear microscopy for detection of viable Mycobacterium tuberculosis in sputum specimens (n = 288) of TB cases under treatment compared to culture (17.4% culture positivity). FDA sensitivity was moderate (83.7% [95% confidence interval {CI}, 70.3 to 92.6]), and specificity was low (66.1% [59.5 to 72.2]). The good negative predictive value (94.8% [90.1 to 97.8]) and negative likelihood ratio (0.2) suggest using this method to rule out treatment failure in settings without access to culture

Nanosize Titanium Dioxide Stimulates Reactive Oxygen Species in Brain Microglia and Damages Neurons in Vitro

BackgroundTitanium dioxide is a widely used nanomaterial whose photo-reactivity suggests that it could damage biological targets (e.g., brain) through oxidative stress (OS).ObjectivesBrain cultures of immortalized mouse microglia (BV2), rat dopaminergic (DA) neurons (N27), and primary cultures of embryonic rat striatum, were exposed to Degussa P25, a commercially available TiO2 nanomaterial. Physical properties of P25 were measured under conditions that paralleled biological measures.FindingsP25 rapidly aggregated in physiological buffer (800–1,900 nm; 25°C) and exposure media (~ 330 nm; 37°C), and maintained a negative zeta potential in both buffer (–12.2 ± 1.6 mV) and media (–9.1 ± 1.2 mV). BV2 microglia exposed to P25 (2.5–120 ppm) responded with an immediate and prolonged release of reactive oxygen species (ROS). Hoechst nuclear stain was reduced after 24-hr (≥100 ppm) and 48-hr (≥2.5 ppm) exposure. Microarray analysis on P25-exposed BV2 microglia indicated up-regulation of inflammatory, apoptotic, and cell cycling pathways and down-regulation of energy metabolism. P25 (2.5–120 ppm) stimulated increases of intracellular ATP and caspase 3/7 activity in isolated N27 neurons (24–48 hr) but did not produce cytotoxicity after 72-hr exposure. Primary cultures of rat striatum exposed to P25 (5 ppm) showed a reduction of immunohistochemically stained neurons and microscopic evidence of neuronal apoptosis after 6-hr exposure. These findings indicate that P25 stimulates ROS in BV2 microglia and is nontoxic to isolated N27 neurons. However, P25 rapidly damages neurons at low concentrations in complex brain cultures, plausibly though microglial generated ROS

Pichia pastoris versus Saccharomyces cerevisiae:a case study on the recombinant production of human granulocyte-macrophage colony-stimulating factor

BACKGROUND: Recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) is a glycoprotein that has been approved by the FDA for the treatment of neutropenia and leukemia in combination with chemotherapies. Recombinant hGM-CSF is produced industrially using the baker's yeast, Saccharomyces cerevisiae, by large-scale fermentation. The methylotrophic yeast, Pichia pastoris, has emerged as an alternative host cell system due to its shorter and less immunogenic glycosylation pattern together with higher cell density growth and higher secreted protein yield than S. cerevisiae. In this study, we compared the pipeline from gene to recombinant protein in these two yeasts. RESULTS: Codon optimization in silico for both yeast species showed no difference in frequent codon usage. However, rhGM-CSF expressed from S. cerevisiae BY4742 showed a significant discrepancy in molecular weight from those of P. pastoris X33. Analysis showed purified rhGM-CSF species with molecular weights ranging from 30 to more than 60 kDa. Fed-batch fermentation over 72 h showed that rhGM-CSF was more highly secreted from P. pastoris than S. cerevisiae (285 and 64 mg total secreted protein/L, respectively). Ion exchange chromatography gave higher purity and recovery than hydrophobic interaction chromatography. Purified rhGM-CSF from P. pastoris was 327 times more potent than rhGM-CSF from S. cerevisiae in terms of proliferative stimulating capacity on the hGM-CSF-dependent cell line, TF-1. CONCLUSION: Our data support a view that the methylotrophic yeast P. pastoris is an effective recombinant host for heterologous rhGM-CSF production

Increasing gene dosage greatly enhances recombinant expression of aquaporins in Pichia pastoris

<p>Abstract</p> <p>Background</p> <p>When performing functional and structural studies, large quantities of pure protein are desired. Most membrane proteins are however not abundantly expressed in their native tissues, which in general rules out purification from natural sources. Heterologous expression, especially of eukaryotic membrane proteins, has also proven to be challenging. The development of expression systems in insect cells and yeasts has resulted in an increase in successful overexpression of eukaryotic proteins. High yields of membrane protein from such hosts are however not guaranteed and several, to a large extent unexplored, factors may influence recombinant expression levels. In this report we have used four isoforms of aquaporins to systematically investigate parameters that may affect protein yield when overexpressing membrane proteins in the yeast <it>Pichia pastoris</it>.</p> <p>Results</p> <p>By comparing clones carrying a single gene copy, we show a remarkable variation in recombinant protein expression between isoforms and that the poor expression observed for one of the isoforms could only in part be explained by reduced transcript levels. Furthermore, we show that heterologous expression levels of all four aquaporin isoforms strongly respond to an increase in recombinant gene dosage, independent of the amount of protein expressed from a single gene copy. We also demonstrate that the increased expression does not appear to compromise the protein folding and the membrane localisation.</p> <p>Conclusions</p> <p>We report a convenient and robust method based on qPCR to determine recombinant gene dosage. The method is generic for all constructs based on the pPICZ vectors and offers an inexpensive, quick and reliable means of characterising recombinant <it>P. pastoris </it>clones. By using this method we show that: (1) heterologous expression of all aquaporins investigated respond strongly to an increase in recombinant gene dosage (2) expression from a single recombinant gene copy varies in an isoform dependent manner (3) the poor expression observed for AtSIP1;1 is mainly caused by posttranscriptional limitations. The protein folding and membrane localisation seems to be unaffected by increased expression levels. Thus a screen for elevated gene dosage can routinely be performed for identification of <it>P. pastoris </it>clones with high expression levels of aquaporins and other classes of membrane proteins.</p

Medium-throughput production of recombinant human proteins: protein production in insect cells.

This chapter describes the step-by-step methods employed by the Structural Genomics Consortium (SGC) for screening and producing proteins in the baculovirus expression vector system (BEVS). This eukaryotic expression system was selected and a screening process established in 2007 as a measure to tackle the more challenging kinase, RNA-DNA processing and integral membrane protein families on our target list. Here, we discuss our platform for identifying soluble proteins from 3 ml of insect cell culture and describe the procedures involved in producing protein from liter-scale cultures. Although not discussed in this chapter, the same process can also be applied to integral membrane proteins (IMPs) with slight adaptations to the purification procedure