Inclusion of maintenance energy improves the intracellular flux predictions of CHO

Chinese hamster ovary (CHO) cells are the leading platform for the production of biopharmaceuticals with human-like glycosylation. The standard practice for cell line generation relies on trial and error approaches such as adaptive evolution and high-throughput screening, which typically take several months. Metabolic modeling could aid in designing better producer cell lines and thus shorten development times. The genome-scale metabolic model (GSMM) of CHO can accurately predict growth rates. However, in order to predict rational engineering strategies it also needs to accurately predict intracellular fluxes. In this work we evaluated the agreement between the fluxes predicted by parsimonious flux balance analysis (pFBA) using the CHO GSMM and a wide range of 13C metabolic flux data from literature. While glycolytic fluxes were predicted relatively well, the fluxes of tricarboxylic acid (TCA) cycle were vastly underestimated due to too low energy demand. Inclusion of computationally estimated maintenance energy significantly improved the overall accuracy of intracellular flux predictions. Maintenance energy was therefore determined experimentally by running continuous cultures at different growth rates and evaluating their respective energy consumption. The experimentally and computationally determined maintenance energy were in good agreement. Additionally, we compared alternative objective functions (minimization of uptake rates of seven nonessential metabolites) to the biomass objective. While the predictions of the uptake rates were quite inaccurate for most objectives, the predictions of the intracellular fluxes were comparable to the biomass objective function.COMET center acib: Next Generation Bioproduction, which is funded by BMK, BMDW, SFG, Standortagentur Tirol, Government of Lower Austria and Vienna Business Agency in the framework of COMET - Competence Centers for Excellent Technologies. The COMET-Funding Program is managed by the Austrian Research Promotion Agency FFG; D.S., J.S., M.W., M.H., D. E.R. This work has also been supported by the PhD program BioToP of the Austrian Science Fund (FWF Project W1224)info:eu-repo/semantics/publishedVersio

A Deep Insight into the Sialome of Rhodnius neglectus, a vector of chagas disease

Background Triatomines are hematophagous insects that act as vectors of Chagas disease. Rhodnius neglectus is one of these kissing bugs found, contributing to the transmission of this American trypanosomiasis. The saliva of hematophagous arthropods contains bioactive molecules responsible for counteracting host haemostatic, inflammatory, and immuneresponses. Methods/Principal Findings Next generation sequencing and mass spectrometry-based protein identification were performed to investigate the content of triatomine R. neglectus saliva.We deposited 4,230 coding DNA sequences (CDS) in GenBank. A set of 636 CDS of proteins of putative secretory nature was extracted from the assembled reads, 73 of them confirmed by proteomic analysis. The sialome of R. neglectus was characterized and serine protease transcripts detected. The presence of ubiquitous protein families was revealed, including lipocalins, serine protease inhibitors, and antigen-5. Metalloproteases, disintegrins, and odorant binding protein families were less abundant. Conclusions/Significance The data presented improve our understanding of hematophagous arthropod sialomes, and aid in understanding hematophagy and the complex interplay among vectors and their vertebrate hosts

Physicochemical characteristics of citrus jelly with non cariogenic and functional sweeteners

In this study the effect of sweeteners with low glycemic index and non-cariogenic characteristics (isomaltulose, oligofructose and tagatose) in jelly prepared with citrus juice has been evaluated considering a citrus jelly formulated with sucrose as reference. The soluble solids, moisture content, pH, water activity, antioxidant capacity, optical and mechanical properties of jelly made using different sweeteners was determined during storage. Besides, mesophilic aerobics and moulds and yeasts was also counted to determine their stability over time. Sensory evaluation of the citrus jelly has also been done. The results showed the antioxidant activity decreased during storage in all formulations. Tagatose increased lightness whereas coordinates a*, b* and chrome of all the jellies prepared using new sweeteners were lower than jellies with sucrose. However, citrus jelly with only oligofructose or tagatose or with the mixture of isomaltulose and tagatose were most closely resembled to the control jelly with respect to mechanical properties. Jelly prepared with the combination of isomaltulose and tagatose in equal proportions obtained the best score in the sensorial analysis.The authors would like to thank the Serigo-Andres family for donating the raw materials, and also the GVA projects GV/2013/029, GV/2014/012 as well as the Universitat Politecnica de Valencia (Spain) for the financial support given to this research study (UPV PAID-06-12 SP20120889).Rubio-Arraez, S.; Capella Hernández, JV.; Castelló Gómez, ML.; Ortolá Ortolá, MD. (2016). Physicochemical characteristics of citrus jelly with non cariogenic and functional sweeteners. 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J Food Sci Tech 52(11):6900–6913Edwards WP (2002) The science of goodies. Acribia S.A, SpainFood and Drug Administration (FDA) (2005) GRAS Notification Isomaltulose (PALATINOSE). http://www.fda.gov/ucm/groups/fdagov-public/@fdagovfoodsgen/documents/document/ucm268989.pdf . Accessed 12 July 2015Food and Drug Administration (FDA) (2010) GRAS Notification Tagatose. GRN No.352. http://www.fda.gov/ucm/groups/fdagov-public/@fdagov-foods-gen/documents/document/ucm269560.pdf . Accessed 12 July 2015Food and Drug Administration (FDA) (2011) GRAS Notification Oligofructose. GRN No.392. http://www.fda.gov/ucm/groups/fdagov-public/@fdagov-foodsgen/documents/document/ucm277112.pdf . Accessed 12 July 2015GME (2015) Gelatine manufactured Europe gelatine properties. http://www.gelatine.org/en/about-gelatine/properties.html . Accessed 12 July 2015ISO (2003) Sensory analysis. Guidelines for the use of quantitative response scales [ref. no.ISO 4121:2003]. 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Draft Genome Sequencing of Giardia intestinalis Assemblage B Isolate GS: Is Human Giardiasis Caused by Two Different Species?

Giardia intestinalis is a major cause of diarrheal disease worldwide and two major Giardia genotypes, assemblages A and B, infect humans. The genome of assemblage A parasite WB was recently sequenced, and the structurally compact 11.7 Mbp genome contains simplified basic cellular machineries and metabolism. We here performed 454 sequencing to 16× coverage of the assemblage B isolate GS, the only Giardia isolate successfully used to experimentally infect animals and humans. The two genomes show 77% nucleotide and 78% amino-acid identity in protein coding regions. Comparative analysis identified 28 unique GS and 3 unique WB protein coding genes, and the variable surface protein (VSP) repertoires of the two isolates are completely different. The promoters of several enzymes involved in the synthesis of the cyst-wall lack binding sites for encystation-specific transcription factors in GS. Several synteny-breaks were detected and verified. The tetraploid GS genome shows higher levels of overall allelic sequence polymorphism (0.5 versus <0.01% in WB). The genomic differences between WB and GS may explain some of the observed biological and clinical differences between the two isolates, and it suggests that assemblage A and B Giardia can be two different species

Assessment of Three Mitochondrial Genes (16S, Cytb, CO1) for Identifying Species in the Praomyini Tribe (Rodentia: Muridae)

The Praomyini tribe is one of the most diverse and abundant groups of Old World rodents. Several species are known to be involved in crop damage and in the epidemiology of several human and cattle diseases. Due to the existence of sibling species their identification is often problematic. Thus an easy, fast and accurate species identification tool is needed for non-systematicians to correctly identify Praomyini species. In this study we compare the usefulness of three genes (16S, Cytb, CO1) for identifying species of this tribe. A total of 426 specimens representing 40 species (sampled across their geographical range) were sequenced for the three genes. Nearly all of the species included in our study are monophyletic in the neighbour joining trees. The degree of intra-specific variability tends to be lower than the divergence between species, but no barcoding gap is detected. The success rate of the statistical methods of species identification is excellent (up to 99% or 100% for statistical supervised classification methods as the k-Nearest Neighbour or Random Forest). The 16S gene is 2.5 less variable than the Cytb and CO1 genes. As a result its discriminatory power is smaller. To sum up, our results suggest that using DNA markers for identifying species in the Praomyini tribe is a largely valid approach, and that the CO1 and Cytb genes are better DNA markers than the 16S gene. Our results confirm the usefulness of statistical methods such as the Random Forest and the 1-NN methods to assign a sequence to a species, even when the number of species is relatively large. Based on our NJ trees and the distribution of all intraspecific and interspecific pairwise nucleotide distances, we highlight the presence of several potentially new species within the Praomyini tribe that should be subject to corroboration assessments