Lipidomics for robust high performance process development

As the biopharmaceutical industry reduces the risk of potential contaminations by adventitious agents and increases process yields, high performance cell culture processes have been developed that rely on animal-free peptide-free, protein-free chemically defined and lipid-free media. These processes rely on cell lines that have been adapted to these lipid-free production conditions and have developed very effective lipid production capacities. These lipid-production capacities result in new challenges in the harvest and purification steps such as filterability, ability to clean resins and resin reuse. This oral presentation will show case how lipidomics can provide insights and opportunities to control the interactions between high performance bioreactor production processes, harvest conditions and purification. Results obtained across large scale production processes of three different monoclonal antibodies will be displayed. The importance of controlling lipid biosynthesis and the presence of lipids in the cell culture fluid prior to affinity capture chromatography will be discussed. Three different control strategies will be showcased and their pros and cons in terms of process yields, robustness of the harvest and impact on the purification process post-harvest explaine

Genome-wide transcriptional analysis suggests hydrogenase- and nitrogenase-mediated hydrogen production in Clostridium butyricum CWBI 1009

[en] Background: Molecular hydrogen, given its pollution-free combustion, has great potential to replace fossil fuels infuture transportation and energy production. However, current industrial hydrogen production processes, such assteam reforming of methane, contribute significantly to the greenhouse effect. Therefore alternative methods, inparticular the use of fermentative microorganisms, have attracted scientific interest in recent years. However thelow overall yield obtained is a major challenge in biological H2 production. Thus, a thorough and detailedunderstanding of the relationships between genome content, gene expression patterns, pathway utilisation andmetabolite synthesis is required to optimise the yield of biohydrogen production pathways.Results: In this study transcriptomic and proteomic analyses of the hydrogen-producing bacterium Clostridiumbutyricum CWBI 1009 were carried out to provide a biomolecular overview of the changes that occur when themetabolism shifts to H2 production. The growth, H2-production, and glucose-fermentation profiles were monitoredin 20 L batch bioreactors under unregulated-pH and fixed-pH conditions (pH 7.3 and 5.2). Conspicuous differenceswere observed in the bioreactor performances and cellular metabolisms for all the tested metabolites, and theywere pH dependent. During unregulated-pH glucose fermentation increased H2 production was associated withconcurrent strong up-regulation of the nitrogenase coding genes. However, no such concurrent up-regulation ofthe [FeFe] hydrogenase genes was observed. During the fixed pH 5.2 fermentation, by contrast, the expressionlevels for the [FeFe] hydrogenase coding genes were higher than during the unregulated-pH fermentation, whilethe nitrogenase transcripts were less abundant. The overall results suggest, for the first time, that environmentalfactors may determine whether H2 production in C. butyricum CWBI 1009 is mediated by the hydrogenases and/orthe nitrogenase.Conclusions: This work, contributing to the field of dark fermentative hydrogen production, provides amultidisciplinary approach for the investigation of the processes involved in the molecular H2 metabolism ofclostridia. In addition, it lays the groundwork for further optimisation of biohydrogen production pathways basedon genetic engineering techniques.info:eu-repo/semantics/publishe

Developmentally regulated cleavage of tRNAs in the bacterium Streptomyces coelicolor

The ability to sense and respond to environmental and physiological signals is critical for the survival of the soil-dwelling Gram-positive bacterium Streptomyces coelicolor. Nutrient deprivation triggers the onset of a complex morphological differentiation process that involves the raising of aerial hyphae and formation of spore chains, and coincides with the production of a diverse array of clinically relevant antibiotics and other secondary metabolites. These processes are tightly regulated; however, the genes and signals involved have not been fully elucidated. Here, we report a novel tRNA cleavage event that follows the same temporal regulation as morphological and physiological differentiation, and is growth medium dependent. All tRNAs appear to be susceptible to cleavage; however, there appears to be a bias towards increased cleavage of those tRNAs that specify highly utilized codons. In contrast to what has been observed in eukaryotes, accumulation of tRNA halves in S. coelicolor is not significantly affected by amino acid starvation, and is also not affected by induction of the stringent response or inhibition of ribosome function. Mutants defective in aerial development and antibiotic production exhibit altered tRNA cleavage profiles relative to wild-type strains

Arabidopsis thaliana XRN2 is required for primary cleavage in the pre-ribosomal RNA

Three Rat1/Xrn2 homologues exist in Arabidopsis thaliana: nuclear AtXRN2 and AtXRN3, and cytoplasmic AtXRN4. The latter has a role in degrading 3′ products of miRNA-mediated mRNA cleavage, whereas all three proteins act as endogenous post-transcriptional gene silencing suppressors. Here we show that, similar to yeast nuclear Rat1, AtXRN2 has a role in ribosomal RNA processing. The lack of AtXRN2, however, does not result in defective formation of rRNA 5′-ends but inhibits endonucleolytic cleavage at the primary site P in the pre-rRNA resulting in the accumulation of the 35S* precursor. This does not lead to a decrease in mature rRNAs, as additional cleavages occur downstream of site P. Supplementing a P-site cleavage-deficient xrn2 plant extract with the recombinant protein restores processing activity, indicating direct participation of AtXRN2 in this process. Our data suggest that the 5′ external transcribed spacer is shortened by AtXRN2 prior to cleavage at site P and that this initial exonucleolytic trimming is required to expose site P for subsequent endonucleolytic processing by the U3 snoRNP complex. We also show that some rRNA precursors and excised spacer fragments that accumulate in the absence of AtXRN2 and AtXRN3 are polyadenylated, indicating that these nucleases contribute to polyadenylation-dependent nuclear RNA surveillance

Suicide and Suicide Risk in Lesbian, Gay, Bisexual, and Transgender Populations: Review and Recommendations

Despite strong indications of elevated risk of suicidal behavior in lesbian, gay, bisexual, and transgender people, limited attention has been given to research, interventions or suicide prevention programs targeting these populations. This article is a culmination of a three-year effort by an expert panel to address the need for better understanding of suicidal behavior and suicide risk in sexual minority populations, and stimulate the development of needed prevention strategies, interventions and policy changes. This article summarizes existing research findings, and makes recommendations for addressing knowledge gaps and applying current knowledge to relevant areas of suicide prevention practice

Modeling the dependence of respiration and photosynthesis upon light, acetate, carbon dioxide, nitrate and ammonium in Chlamydomonas reinhardtii using design of experiments and multiple regression

Background: In photosynthetic organisms, the influence of light, carbon and inorganic nitrogen sources on the cellular bioenergetics has extensively been studied independently, but little information is available on the cumulative effects of these factors. Here, sequential statistical analyses based on design of experiments (DOE) coupled to standard least squares multiple regression have been undertaken to model the dependence of respiratory and photosynthetic responses (assessed by oxymetric and chlorophyll fluorescence measurements) upon the concomitant modulation of light intensity as well as acetate, CO2, nitrate and ammonium concentrations in the culture medium of Chlamydomonas reinhardtii. The main goals of these analyses were to explain response variability (i.e. bioenergetic plasticity) and to characterize quantitatively the influence of the major explanatory factor(s). Results: For each response, 2 successive rounds of multiple regression coupled to one-way ANOVA F-tests have been undertaken to select the major explanatory factor(s) (1st-round) and mathematically simulate their influence (2nd-round). These analyses reveal that a maximal number of 3 environmental factors over 5 is sufficient to explain most of the response variability, and interestingly highlight quadratic effects and second-order interactions in some cases. In parallel, the predictive ability of the 2nd-round models has also been investigated by k-fold cross-validation and experimental validation tests on new random combinations of factors. These validation procedures tend to indicate that the 2nd-round models can also be used to predict the responses with an inherent deviation quantified by the analytical error of the models. Conclusions: Altogether, the results of the 2 rounds of modeling provide an overview of the bioenergetic adaptations of C. reinhardtii to changing environmental conditions and point out promising tracks for future in-depth investigations of the molecular mechanisms underlying the present observations