ABC for GRASPing enzyme kinetics in metabolic models

Large scale kinetic models of metabolism are required to explore and explain the molecular basis for homeostasis, the self-regulating processes evolved to maintain metabolic equilibrium. Studying homeostasis is relevant for the understanding and treatment of complex diseases, particular with the emergence of personalized medicine. It is equally important when we seek to repurpose the cellular machinery for the production of desired chemicals, materials and pharmaceuticals. In the latter process, the cells’ homeostatic control mechanisms must be either disabled or exploited. However, estimation of in vivo parameters is hard due to the large amount of data needed and the fact that homeostatic control typically renders many parameters practically unobservable. We have developed a General Reaction Assembly and Sampling Platform (GRASP) capable of consistently parameterizing and sampling thermodynamically feasible kinetics using minimal reference data (1). The former integrates the concerted MWC model and elementary reaction formalism to describe both simple and complex (e.g., allosteric) kinetics. Application of this approach enabled assessment of the impact of thermodynamics on reaction kinetics, as well as the exploration of complex allosteric behaviours (2). Formulation of the sampling problem within the Bayesian framework provides a natural interface for the addition of experimental data, hereby improving sampling accuracy. Approximate Bayesian Computation (ABC) methods were used to unravel the dynamic properties of metabolic networks (3). A posteriori analysis of the parameter distribution enabled prediction of metabolic states, identification of critical parameters as well as unravelling the control structure of the network. We have illustrated the capabilities of our framework in several cases ranging from simple enzymatic mechanisms to tightly-regulated pathways. Overall, this framework demonstrates that detailed kinetic representation is possible without sacrificing complexity and with low amounts of data. Expanding this framework to large metabolic networks requires further algorithmic advances, including the development of efficient and robust sequential sampling schemes. Recent advances in algorithmics will be Please click Additional Files below to see the full abstract

SUN-projektet: Skolebaseret udvikling af naturfag og kapacitet i gymnasiet

I artiklen beskrives det treårige udviklingsprojekt Skolebaseret Udvikling i Naturfag (SUN) som på samme tid har tilgodeset udvikling af konkret naturfagsundervisning og udvikling af de deltagende gymnasiers kapacitet til fremadrettet at tage hånd om egne naturfaglige udviklingsbehov. Vi beskriver det forskningsbaserede forløbsdesign med målrettede indsatser ift. naturfaglige udviklingsgrupper, fagteams, faglige koordinatorer og ledelse samt videregiver vore erfaringer på godt og ondt fra implementeringen. Endelig diskuterer vi kritiske lærestykker af relevans for fremtidige designere, afviklere eller brugere/rekvirenter af professionelle udviklingsforløb med tilsvarende ambitiøst dobbeltsigte, det være sig i gymnasiet eller grundskolen

Polyclonal production of antibodies using CHO cell line mixtures

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Transcriptional response to recombinant protein production in isogenic multi-copy CHO cells

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Expression of anti-apoptotic genes to enhance rAAV production

Recombinant Adeno-associated viruses (rAAV) are becoming popular as viral vector delivery systems for gene therapy. Currently, two rAAV-based products are available on the market and several clinical trials are on-going with promising results for diseases such as hemophilia or muscular dystrophy1,2. The most common method for manufacturing rAAV products is by triple transfection with a plasmid containing the gene of interested flanked by ITR sequences, a plasmid with adenovirus helper genes and a plasmid with AAV helper genes. The scalability of transfection processes has been disputed for a long time. However, in rAAV production it might be the only feasible option until a proficient stable cell line is developed solving all current challenges including, but not limited to: low cell density at transfection and interruption of cell replication. Another bottleneck in all cell culture-based process is the duration of the culture. Typically, the viable cell density increases until a stationary phase is reached in which, after a specific amount of time depending on the cell line, apoptosis begins and viability rapidly falls. In rAAV production processes, apoptosis is triggered after transfection by AAV helper genes (mostly Rep78 and Rep 52) which are toxic for mammalian cells, reducing the duration of the cell culture decreasing the productivity of the process3,4 . To overcome this challenge, several anti-apoptotic genes from different origins, such as human (bcl-2, bcl-xL and mcl-1), hamster (bcl-2 and bcl-xL) and viral genes (bhrf-1, p25 and vBcl-2), have been expressed in this study as well as a recombinant protein-based product to mimic the production of rAAV. Even though some genes were already reported in the literature, a detailed comparison could not be performed as random integration was used in the development of most of the cell lines. In this novel study, targeted integration was used to develop isogenic cell lines with one copy of both the anti-apoptotic gene and the protein product. As expected, the expression of anti-apoptotic genes increased the duration of the cell culture proving its potential as a tool to enhance the performance of rAAV production processes. Consistent with the literature, some genes were also able to improve the specific productivity of the protein product by a significant amount. In fact, one gene that was not studied previously outperformed the other candidates. Regarding the metabolism, consumption rates of glucose and production rates of lactate were analyzed to have a better understanding of the effect of these genes on the metabolism of the mammalian cell. Considering all the available results, we propose the use of anti-apoptotic genes to enhance the production of rAAV with mammalian cells. References 1. Verdera HC, Kuranda K, Mingozzi F. AAV Vector Immunogenicity in Humans: A Long Journey to Successful Gene Transfer. Mol Ther. 2020;28(3):723-746. doi:10.1016/j.ymthe.2019.12.010 2. Van Vliet KM, Blouin V, Brument N, Agbandje-McKenna M, Snyder RO. The Role of the Adeno-Associated Virus Capsid in Gene Transfer. Vol 437.; 2008. doi:10.1007/978-1-59745-210-6_2 3. Matsushita T, Okada T, Inaba T, Mizukami H, Ozawa K, Colosi P. The adenovirus E1A and E1B19K genes provide a helper function for transfection-based adeno-associated virus vector production. J Gen Virol. 2004;85(Pt 8):2209-2214. doi:10.1099/vir.0.79940-0 4. Schmidt M, Afione S, Kotin RM. Adeno-Associated Virus Type 2 Rep78 Induces Apoptosis through Caspase Activation Independently of p53. J Virol. 2000;74(20):9441-9450. doi:10.1128/jvi.74.20.9441-9450.200

Expression of anti-apoptotic genes to enhance rAAV production

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Cell-free pipeline for discovery of thermotolerant xylanases and endo-1,4-β-glucanases

The rapid expansion in the number of sequenced genomes and metagenomes provides an exceptional resource for mining of the enzymes with biotechnologically relevant properties. However, the majority of protein production and analysis methods are not sufficiently cost-efficient and scalable to experimentally verify the results of computational genomic mining. Here, we present a pipeline based on Leishmania tarentolae cell-free system that was used to characterize 30 putative thermostable endo-1,4-beta-glucanases and xylanases identified in public genomic databases. In order to analyse the recombinant proteins without purification, novel high-throughput assays for glucanase and xylanase activities were developed. The assays rely on solubilisation of labelled particulate substrates performed in multiwell plates. Using this approach both acidophilic and thermophilic enzymes were identified. The developed approach enables rapid discovery of new biotechnologically useful enzymes

Acute and Subchronic Airway Inflammation after Intratracheal Instillation of Quartz and Titanium Dioxide Agglomerates in Mice

This study investigated the acute and subchronic inflammatory effects of micrometer-size (micro-size) and nanometer-size (nano-size) particles after intratracheal (i.t.) installation in mice. The role of the type of compound, polymorphism, and size of the particles was investigated. Studied compounds were the two micro-size reference quartzes, SRM1878a and DQ12, a micro- and nano-size rutile titanium dioxide (TiO2), a nano-size anatase, and an amorphous TiO2. Particles were administered by a single i.t. instillation in mice at a fixed dose of 5, 50, and 500 μg, respectively. Inflammation was evaluated from the bronchoalveolar lavage fluid (BALF) content of inflammatory cells, the cytokines tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6), as well as from lung histology. Evaluations were at 24 h (acute effects) and 3 months (subchronic effects) after instillations. Both types of quartz induced a dose-dependent acute increase of neutrophils, IL-6, and total protein in BALF. Limited subchronic inflammation was observed. All types of TiO2 induced a dose-dependent acute increase of neutrophils in BALF. In the acute phase, micro- and nano-size rutile and nano-size amorphous TiO2 induced elevated levels of IL-6 and total protein in BALF at the highest dose. At the nano-size rutile and amorphous TiO2, subchronic lung inflammation was apparent from a dose-dependent increase in BALF macrophages. Histology showed little inflammation overall. The two types of quartz showed virtually similar inflammatory effects. Nearly similar effects were observed for two sizes of rutile TiO2. Differences were seen between the different polymorphs of nano-size TiO2, with rutile being the most inflammogenic and amorphous being the most potent in regard to acute tissue damage

A randomized placebo-controlled trial of convalescent plasma for adults hospitalized with COVID-19 pneumonia

Passive immunotherapy with convalescent plasma may be the only available agent during the early phases of a pandemic. Here, we report safety and efficacy of high-titer convalescent plasma for COVID-19 pneumonia. Double-blinded randomized multicenter placebo-controlled trial of adult patients hospitalized with COVID-19 pneumonia. The intervention was COVID-19 convalescent plasma and placebo was saline allocated 2:1. The primary outcome was clinical status 14 days after the intervention evaluated on a clinical ordinal scale. The trial was registered at ClinicalTrials.Gov, NCT04345289, 14/04/2020. The CCAP-2 trial was terminated prematurely due to futility. Of 147 patients randomized, we included 144 patients in the modified intention-to-treat population. The ordinal clinical status 14 days post-intervention was comparable between treatment groups (odds ratio (OR) 1.41, 95% confidence interval (CI) 0.72–2.09). Results were consistent when evaluating clinical progression on an individual level 14 days after intervention (OR 1.09; 95% CI 0.46–1.73). No significant differences in length of hospital stay, admission to ICU, frequency of severe adverse events or all-cause mortality during follow-up were found between the intervention and the placebo group. Infusion of convalescent plasma did not influence clinical progression, survival or length of hospitalization in patients with COVID-19 pneumonia