Metabolomics analysis of soy hydrolysates for the identification of productivity markers of mammalian cells for manufacturing therapeutic proteins

Soy hydrolysates are widely used as a nutrient supplement in mammalian cell culture for the production of recombinant proteins. The batch‐to‐batch variability of a soy hydrolysate often leads to productivity differences. This report describes our metabolomics platform, which includes a battery of LC‐MS/MS modes of operation, and advanced data analysis software for automated data processing. The platform was successfully used for screening productivity markers in soy hydrolysates during the production of two therapeutic antibodies in two Chinese hamster ovary cell lines. A total of 123 soy hydrolysate batches were analyzed, from which 62 batches were used in the production runs of cell line #1 and 12 batches were used in the production runs of cell line #2. For cell line #1, out of 19 amino acids, 106 other metabolites and 4,131 peptides identified in the soy hydrolysate batches being used, several nucleosides and short hydrophobic peptides showed negative correlation with antibody titer, while ornithine, citrulline and several amino acids and organic acids correlated positively with titer. For cell line #2, only ornithine and citrulline showed strong positive correlation. When ornithine was spiked into the culture media, both cell lines demonstrated accelerated cell growth, indicating ornithine as a root cause of the performance difference. It is proposed that better soy hydrolysate performance resulted from better bacterial fermentation during the hydrolysate production. A few selected markers were used to predict the performance of other soy hydrolysate batches for cell line #1. The predicted titers agreed with the experimental values with good accuracy. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:522–531, 2015     

Transient Influx of Nickel in Root Mitochondria Modulates Organic Acid and Reactive Oxygen Species Production in Nickel Hyperaccumulator Alyssum murale

Mitochondria are important targets of metal toxicity and are also vital for maintaining metal homeostasis. Here, we examined the potential role of mitochondria in homeostasis of nickel in the roots of nickel hyperaccumulator plant Alyssum murale. We evaluated the biochemical basis of nickel tolerance by comparing the role of mitochondria in closely related nickel hyperaccumulator A. murale and non-accumulator Alyssum montanum. Evidence is presented for the rapid and transient influx of nickel in root mitochondria of nickel hyperaccumulator A. murale. In an early response to nickel treatment, substantial nickel influx was observed in mitochondria prior to sequestration in vacuoles in the roots of hyperaccumulator A. murale compared with non-accumulator A. montanum. In addition, the mitochondrial Krebs cycle was modulated to increase synthesis of malic acid and citric acid involvement in nickel hyperaccumulation. Furthermore, malic acid, which is reported to form a complex with nickel in hyperaccumulators, was also found to reduce the reactive oxygen species generation induced by nickel. We propose that the interaction of nickel with mitochondria is imperative in the early steps of nickel uptake in nickel hyperaccumulator plants. Initial uptake of nickel in roots results in biochemical responses in the root mitochondria indicating its vital role in homeostasis of nickel ions in hyperaccumulation.     

Comparative life cycle assessment of autotrophic cultivation of Scenedesmus dimorphus in raceway pond coupled to biodiesel and biogas production

Life cycle assessment (LCA) of indigenous freshwater microalgae, Scenedesmus dimorphus, cultivation in open raceway pond and its conversion to biodiesel and biogas were carried out. The LCA inventory inputs for the biogas scenario was entirely based on primary data obtained from algal cultivation (in pilot scale raceway pond), harvesting, and biogas production; while only the downstream processing involved in biodiesel production namely drying, reaction and purification were based on secondary data. Overall, eight scenarios were modeled for the integrated process involving: algae-based CO₂ capture and downstream processing scenarios for biodiesel and biogas along with impact assessment of nutrient addition and extent of recycling in a life cycle perspective. The LCA results indicated a huge energy deficit and net CO₂ negative in terms of CO₂ capture for both the biodiesel and biogas scenarios, majorly due to lower algal biomass productivity and higher energy requirements for culture mixing. The sensitivity analysis indicated that variability in the biomass productivity has predominant effect on the primary energy demand and global warming potential (GWP, kg CO₂ eq.) followed by specific energy consumption for mixing algal culture. Furthermore, the LCA results indicated that biogas conversion route from microalgae was more energy efficient and sustainable than the biodiesel route. The overall findings of the study suggested that microalgae-mediated CO₂ capture and conversion to biodiesel and biogas production can be energy efficient at higher biomass productivity (> 10 g m⁻² day⁻¹) and via employing energy-efficient systems for culture mixing (< 2 W m⁻³).

     

IL-8 directly enhanced endothelial cell survival,proliferation, and matrix metalloproteinases production and regulated angiogenesis

IL-8, a member of the chemokine family, has been shown to play an important role in tumor growth, angiogenesis, and metastasis. The objective of this study was to determine the mechanism of IL-8-mediated angiogenesis. We examined the direct role of IL-8 in angiogenesis by examining IL-8 receptor expression on endothelial cells and their proliferation, survival, and matrix metalloproteinases (MMPs) production. We demonstrate that HUVEC and human dermal microvascular endothelial cells constitutively express CXCR1 and CXCR2 mRNA and protein. Recombinant human IL-8 induced endothelial cell proliferation and capillary tube organization while neutralization of IL-8 by anti-IL-8 Ab blocks IL-8-mediated capillary tube organization. Incubation of endothelial cells with IL-8 inhibited endothelial cell apoptosis and enhanced antiapoptotic gene expression. Endothelial cells incubated with IL-8 had higher levels of Bcl-x(L):Bcl-x(S) and Bcl-2:Bax ratios. Furthermore, incubation of endothelial cells with IL-8 up-regulated MMP-2 and MMP-9 production and mRNA expression. Our data suggest that IL-8 directly enhanced endothelial cell proliferation, survival, and MMP expression in CXCR1- and CXCR2-expressing endothelial cells and regulated angiogenesis.     

A high throughput mechanical screening device for cartilage tissue engineering

Articular cartilage enables efficient and near-frictionless load transmission, but suffers from poor inherent healing capacity. As such, cartilage tissue engineering strategies have focused on mimicking both compositional and mechanical properties of native tissue in order to provide effective repair materials for the treatment of damaged or degenerated joint surfaces. However, given the large number design parameters available (e.g. cell sources, scaffold designs, and growth factors), it is difficult to conduct combinatorial experiments of engineered cartilage. This is particularly exacerbated when mechanical properties are a primary outcome, given the long time required for testing of individual samples. High throughput screening is utilized widely in the pharmaceutical industry to rapidly and cost-effectively assess the effects of thousands of compounds for therapeutic discovery. Here we adapted this approach to develop a high throughput mechanical screening (HTMS) system capable of measuring the mechanical properties of up to 48 materials simultaneously. The HTMS device was validated by testing various biomaterials and engineered cartilage constructs and by comparing the HTMS results to those derived from conventional single sample compression tests. Further evaluation showed that the HTMS system was capable of distinguishing and identifying ‘hits’, or factors that influence the degree of tissue maturation. Future iterations of this device will focus on reducing data variability, increasing force sensitivity and range, as well as scaling-up to even larger (96-well) formats. This HTMS device provides a novel tool for cartilage tissue engineering, freeing experimental design from the limitations of mechanical testing throughput.     

Extracellular red Monascus pigment-mediated rapid one-step synthesis of silver nanoparticles and its application in biomedical and environment

Bioprocess and Biosystems Engineering - The development of a safe and eco-friendly method for metal nanoparticle synthesis has an increasing demand, due to emerging environmental and biological...     

UBQLN4 Represses Homologous Recombination and Is Overexpressed in Aggressive Tumors

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Evaluation of a New Cryptococcal Antigen Lateral Flow Immunoassay in Serum,Cerebrospinal Fluid and Urine for the Diagnosis of Cryptococcosis: A Meta-Analysis and Systematic Review

BackgroundA new lateral flow immunoassay (LFA) for the detection of cryptococcal antigen was developed.ObjectiveWe aimed to systematically review all relevant studies to evaluate the diagnostic accuracy of the cryptococcal antigen LFA on serum, CSF and urine specimens.MethodsWe searched public databases including PubMed, Web of Science, Elsevier Science Direct and Cochrane Library for the English-language literature published up to September 2014. We conducted meta-analyses of sensitivity, specificity, positive likelihood ratio (PLR), negative likelihood ratio (NLR) and diagnostic odds ratios (DOR) and SROC of LFA in serum and CSF, respectively. The sensitivity of LFA in urine was also analyzed. Subgroup analyses were carried out to analyze the potential heterogeneity.Results12 studies were included in this study. The pooled sensitivity and specificity values of LFA in serum were 97.6% (95% CI, 95.6% to 98.9%) and 98.1% (95% CI, 97.4% to 98.6%), respectively. The average PLR of LFA in serum was 43.787 (95% CI, 22.60–84.81) and the NLR was 0.03 (95% CI, 0.01–0.09). The pooled DOR was 2180.30 (95% CI, 868.92–5471.00) and the AUC was 0.9968. The pooled sensitivity and specificity values of LFA in CSF were 98.9% (95% CI, 97.9% to 99.5%) and 98.9% (95% CI, 98.0% to 99.5%), respectively. The average PLR of LFA in serum was 48.83 (95% CI, 21.59–110.40) and the NLR was 0.02 (95% CI, 0.01–0.04). The pooled DOR was 2931.10 (95% CI, 1149.20–7475.90) and the AUC was 0.9974. The pooled sensitivity value of LFA in urine was 85.0% (95% CI, 78.7% to 90.1%)ConclusionsThe study demonstrates a very high accuracy of LFA in serum and CSF for the diagnosis of cryptococcosis in patients at risk. LFA in urine can be a promising sample screening tool for early diagnosis of cryptococcosis.