BHK cells physiological response to spin-filter stress condition

Mammalian cells are the preferable platform for secreted pharmaceutical proteins due to the compatibility of posttranslational modifications performed by these cells with therapeutic applications. However, to obtain a desirable quantity of these proteins, high cell concentrations are also needed. Baby hamster kidney BHK-21/C13 cells, adapted to single-cell suspension culture growth were cultivated in batch mode in a Biostat B (Sartorius AG, Germany) bioreactor with a 1,5 L working volume vessel. The temperature was controlled at 37 ºC, pH at 7.2 with CO­2, agitation at 80 rpm and dissolved oxygen at 50% of air saturation. The culture medium used was IMDM/DMEM supplemented with Fetal Bovine Serum (5%), Pluronic F-68 as a shear protectant (2%) and L-glutamine 4 mM solution (2%). The bioreactor was inoculated with exponential-growing cells previously grown in T-25 and spinner flasks. Relevant data collected from this experiment is shown in Table 1. In order to achieve high cell concentration, the same BHK-21/C13 cells were cultivated in a perfusion set-up, with an internal polyester spin-filter BB-8808571 (Sartorius) with 10 µm diameter pore size attached to the impeller shaft as cell retention device. This set-up is different from the one previously used only by the presence of the internal spin-filter. To start the perfusion, before the feed and withdrawal pumps were turned on, the set-up was operated as a batch process until there were enough cells to start the continuous operation mode. Data collected from this phase was then compared with data obtained from the batch cultivation. Specific growth rates and exponential growth phase duration were similar for both experiments. However, the specific rates of glucose (qGLU) and glutamine (qGLN) consumption were 84% and 32% higher (Table 1), respectively, when compared to the batch cultivation. Similarly, the specific rates of lactate (qLAC) and ammonium (qNH4) formation were 78% and 102% higher, respectively. The specific rates of substrates consumption and of metabolites formation were calculated for the exponential growth phase. It is reported in literature the association of higher specific substrates consumption rates and metabolites production rates with stress factors, and we can associate the presence of the spin-filter with the physiological parameters findings, suggesting that BHK cells were exposed to a more stressful condition. The calculated value, obtained though CFD (computational fluid dynamics), for the wall shear stress of a filter rotating at 100 rpm vary from 1.57 to 1.67 Pa depending on the recirculation rate1. Despite being a small value, the system without the spin-filter already has its intrinsic shear stress value, so the stress caused by the filter must be added to and not considered as the absolute value. It was also reported that shear stress in the range from 0.75 to 1.0 Pa can affect both the viability and morphology of one BHK-21/C13 adherent cell line cultivated up to 24 hours2. Our data indicate that the presence of an internal spin-filter may be associated to cell stress

Cardiovascular toxicity in patients with chronic myeloid leukemia treated with second-generation tyrosine kinase inhibitors in the real-life practice: Identification of risk factors and the role of prophylaxis

No abstract availabl

The Eucalyptus Cuticular Waxes Contribute in Preformed Defense Against Austropuccinia psidii

Austropuccinia psidii, the causal agent of myrtle rust, is a biotrophic pathogen whose growth and development depends on the host tissues. The uredospores of A. psidii infect Eucalyptus by engaging in close contact with the host surface and interacting with the leaf cuticle that provides important chemical and physical signals to trigger the infection process. In this study, the cuticular waxes of Eucalyptus spp. were analyzed to determine their composition or structure and correlation with susceptibility/resistance to A. psidii. Twenty-one Eucalyptus spp. in the field were classified as resistant or susceptible. The resistance/susceptibility level of six Eucalyptus spp. were validated in controlled conditions using qPCR, revealing that the pathogen can germinate on the eucalyptus surface of some species without multiplying in the host. CG-TOF-MS analysis detected 26 compounds in the Eucalyptus spp. cuticle and led to the discovery of the role of hexadecanoic acid in the susceptibility of Eucalyptus grandis and Eucalyptus phaeotricha to A. psidii. We characterized the epicuticular wax morphology of the six previously selected Eucalyptus spp. using scanning electron microscopy and observed different behavior in A. psidii germination during host infection. It was found a correlation of epicuticular morphology on the resistance to A. psidii. However, in this study, we provide the first report of considerable interspecific variation in Eucalyptus spp. on the susceptibility to A. psidii and its correlation with cuticular waxes chemical compounds that seem to play a synergistic role as a preformed defense mechanism

Common and rare variant association analyses in amyotrophic lateral sclerosis identify 15 risk loci with distinct genetic architectures and neuron-specific biology

A cross-ancestry genome-wide association meta-analysis of amyotrophic lateral sclerosis (ALS) including 29,612 patients with ALS and 122,656 controls identifies 15 risk loci with distinct genetic architectures and neuron-specific biology. Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease with a lifetime risk of one in 350 people and an unmet need for disease-modifying therapies. We conducted a cross-ancestry genome-wide association study (GWAS) including 29,612 patients with ALS and 122,656 controls, which identified 15 risk loci. When combined with 8,953 individuals with whole-genome sequencing (6,538 patients, 2,415 controls) and a large cortex-derived expression quantitative trait locus (eQTL) dataset (MetaBrain), analyses revealed locus-specific genetic architectures in which we prioritized genes either through rare variants, short tandem repeats or regulatory effects. ALS-associated risk loci were shared with multiple traits within the neurodegenerative spectrum but with distinct enrichment patterns across brain regions and cell types. Of the environmental and lifestyle risk factors obtained from the literature, Mendelian randomization analyses indicated a causal role for high cholesterol levels. The combination of all ALS-associated signals reveals a role for perturbations in vesicle-mediated transport and autophagy and provides evidence for cell-autonomous disease initiation in glutamatergic neurons

On the mechanisms governing gas penetration into a tokamak plasma during a massive gas injection

A new 1D radial fluid code, IMAGINE, is used to simulate the penetration of gas into a tokamak plasma during a massive gas injection (MGI). The main result is that the gas is in general strongly braked as it reaches the plasma, due to mechanisms related to charge exchange and (to a smaller extent) recombination. As a result, only a fraction of the gas penetrates into the plasma. Also, a shock wave is created in the gas which propagates away from the plasma, braking and compressing the incoming gas. Simulation results are quantitatively consistent, at least in terms of orders of magnitude, with experimental data for a D 2 MGI into a JET Ohmic plasma. Simulations of MGI into the background plasma surrounding a runaway electron beam show that if the background electron density is too high, the gas may not penetrate, suggesting a possible explanation for the recent results of Reux et al in JET (2015 Nucl. Fusion 55 093013)