Combination of high-throughput microfluidics and FACS technologies to leverage the numbers game in natural product discovery

High-throughput platforms facilitating screening campaigns of environmental samples are needed to discover new products of natural origin counteracting the spreading of antimicrobial resistances constantly threatening human and agricultural health. We applied a combination of droplet microfluidics and fluorescence-activated cell sorting (FACS)-based technologies to access and assess a microbial environmental sample. The cultivation performance of our microfluidics workflow was evaluated in respect to the utilized cultivation media by Illumina amplicon sequencing of a pool of millions of droplets, respectively. This enabled the rational selection of a growth medium supporting the isolation of microbial diversity from soil (five phyla affiliated to 57 genera) including a member of the acidobacterial subgroup 1 (genus Edaphobacter). In a second phase, the entire diversity covered by 1071 cultures was used for an arrayed bioprospecting campaign, resulting in > 6000 extracts tested against human pathogens and agricultural pests. After redundancy curation by using a combinatorial chemical and genomic fingerprinting approach, we assigned the causative agents present in the extracts. Utilizing UHPLC-QTOF-MS/MS-guided fractionation and microplate-based screening assays in combination with molecular networking the production of bioactive ionophorous macrotetrolides, phospholipids, the cyclic lipopetides massetolides E, F, H and serratamolide A and many derivatives thereof was shown.     

“Silenced” polydendrocytes: a new cell type within the oligodendrocyte progenitor cell population?

Oligodendrocyte progenitor cells (OPCs) were first described more than two decades ago. Novel labeling techniques have shown them to be cells with more than just progenitor functions, with their classification as a fourth glial cell type in addition to astrocytes, oligodendrocytes, and microglial cells. Another term used for this cell type is polydendrocytes, owing to both their morphology and to the evolving knowledge about their diverse functions. Recently, an exclusive hallmark of neurons—the generation of action potentials—became debatable, because a subset of polydendrocytes was reported to generate action potentials in response to adequate stimuli. The new technique of inducible reporter gene expression has brought new insights into the fate and function of polydendrocytes. In recent studies, so-called “silenced” OPCs were detected in cortical tissue, and which underwent proliferation with subsequent cell cycle exit, but without any signs of differentiation. Within this review, we focus on the identification of this new subset of polydendrocytes and their possible functions within cortical networks.     

Changes in heart rate variability of athletes during a training camp.

Heart rate variability (HRV) reflects regulatory processes of the cardiovascular system and reveals fractal characteristics. In this paper we investigated standard HRV parameters and scaling characteristics in ten athletes before, during, and after a 2-week training camp to assess the effects of short-term overtraining on cardiovascular control. High-resolution ECGs were recorded over 30 min under resting conditions 1 week before the training camp, after 1 week of training in the camp, and after 3-4 days of recovery. Standard HRV analysis was performed according to Task Force recommendations. Scaling characteristics were assessed, applying detrended fluctuation analysis (DFA). Standard HRV analysis showed significant changes in meanNN and rmssd during the training camp. DFA revealed three distinct regions of scale-invariance and significant alterations during the training camp. In conclusion, HRV might be used to monitor the training state in athletes.     

Large-conductance calcium-activated potassium channel activity is absent in human and mouse neutrophils and is not required for innate immunity

Large-conductance Ca²⁺-activated K⁺ (BK) channels are reported to be essential for NADPH oxidase-dependent microbial killing and innate immunity in leukocytes. Using human peripheral blood and mouse bone marrow neutrophils, pharmacological targeting, and BK channel gene-deficient (BK^–/–) mice, we stimulated NADPH oxidase activity with 12-O-tetradecanoylphorbol-13-acetate (PMA) and performed patch-clamp recordings on isolated neutrophils. Although PMA stimulated NADPH oxidase activity as assessed by O₂^– and H₂O₂ production, our patch-clamp experiments failed to show PMA-activated BK channel currents in neutrophils. In our studies, PMA induced slowly activating currents, which were insensitive to the BK channel inhibitor iberiotoxin. Instead, the currents were blocked by Zn²⁺, which indicates activation of proton channel currents. BK channels are gated by elevated intracellular Ca²⁺ and membrane depolarization. We did not observe BK channel currents, even during extreme depolarization to +140 mV and after elevation of intracellular Ca²⁺ by N-formyl-L-methionyl-L-leucyl-phenylalanine. As a control, we examined BK channel currents in cerebral and tibial artery smooth muscle cells, which showed characteristic BK channel current pharmacology. Iberiotoxin did not block killing of Staphylococcus aureus or Candida albicans. Moreover, we addressed the role of BK channels in a systemic S. aureus and Yersinia enterocolitica mouse infection model. After 3 and 5 days of infection, we found no differences in the number of bacteria in spleen and kidney between BK^–/– and BK^+/+ mice. In conclusion, our experiments failed to identify functional BK channels in neutrophils. We therefore conclude that BK channels are not essential for innate immunity. killing assay; reactive oxygen species; BK-deficient mice; mice infection     

Redox regulation of chloroplast enzymes in Galdieria sulphuraria in view of eukaryotic evolution

Redox modulation is a general mechanism for enzyme regulation, particularly for the post-translational regulation of the Calvin cycle in chloroplasts of green plants. Although red algae and photosynthetic protists that harbor plastids of red algal origin contribute greatly to global carbon fixation, relatively little is known about post-translational regulation of chloroplast enzymes in this important group of photosynthetic eukaryotes. To address this question, we used biochemistry, phylogenetics and analysis of recently completed genome sequences. We studied the functionality of the chloroplast enzymes phosphoribulokinase (PRK, EC 2.7.1.19), NADP-dependent glyceraldehyde 3-phosphate dehydrogenase (NADP-GAPDH, GapA, EC 1.2.1.13), fructose 1,6-bisphosphatase (FBPase, EC 3.1.3.11) and glucose 6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49), as well as NADP-malate dehydrogenase (NADP-MDH, EC 1.1.1.37) in the unicellular red alga Galdieria sulphuraria (Galdieri) Merola. Despite high sequence similarity of G. sulphuraria proteins to those of other photosynthetic organisms, we found a number of distinct differences. Both PRK and GAPDH co-eluted with CP12 in a high molecular weight complex in the presence of oxidized glutathione, although Galdieria CP12 lacks the two cysteines essential for the formation of the N-terminal peptide loop present in higher plants. However, PRK inactivation upon complex formation turned out to be incomplete. G6PDH was redox modulated, but remained in its tetrameric form; FBPase was poorly redox regulated, despite conservation of the two redox-active cysteines. No indication for the presence of plastidic NADP-MDH (and other components of the malate valve) was found.     

A novel approach to sequence validating protein expression clones with automated decision making

Background  

Whereas the molecular assembly of protein expression clones is readily automated and routinely accomplished in high throughput, sequence verification of these clones is still largely performed manually, an arduous and time consuming process. The ultimate goal of validation is to determine if a given plasmid clone matches its reference sequence sufficiently to be "acceptable" for use in protein expression experiments. Given the accelerating increase in availability of tens of thousands of unverified clones, there is a strong demand for rapid, efficient and accurate software that automates clone validation.     

Mitophagy, mitochondrial dynamics and the general stress response in yeast

Autophagy is a fundamental cellular process promoting survival under various environmental stress conditions. Selective types of autophagy have gained much interest recently as they are involved in specific quality control mechanisms removing, for example, aggregated proteins or dysfunctional mitochondria. This is considered to counteract the development of a number of neurodegenerative disorders and aging. Here we review the role of mitophagy and mitochondrial dynamics in ensuring quality control of mitochondria. In particular, we provide possible explanations why mitophagy in yeast, in contrast with the situation in mammals, was found to be independent of mitochondrial fission. We further discuss recent findings linking these processes to nutrient sensing pathways and the general stress response in yeast. In particular, we propose a model for how the stress response protein Whi2 and the Ras/PKA (protein kinase A) signalling pathway are possibly linked and thereby regulate mitophagy.     

Microbial diversity and stratification of South Pacific abyssal marine sediments

Abyssal marine sediments cover a large proportion of the ocean floor, but linkages between their microbial community structure and redox stratification have remained poorly constrained. This study compares the downcore gradients in microbial community composition to porewater oxygen and nitrate concentration profiles in an abyssal marine sediment column in the South Pacific Ocean. Archaeal 16S rRNA clone libraries showed a stratified archaeal community that changed from Marine Group I Archaea in the aerobic and nitrate-reducing upper sediment column towards deeply branching, uncultured crenarchaeotal and euryarchaeotal lineages in nitrate-depleted, anaerobic sediment horizons. Bacterial 16S rRNA clone libraries revealed a similar shift on the phylum and subphylum level within the bacteria, from a complex community of Alpha-, Gamma- and Deltaproteobacteria, Actinobacteria and Gemmatimonadetes in oxic surface sediments towards uncultured Chloroflexi and Planctomycetes in the anaerobic sediment column. The distinct stratification of largely uncultured bacterial and archaeal groups within the oxic and nitrate-reducing marine sediment column provides initial constraints for their microbial habitat preferences.