Microbial nar-GFP cell sensors reveal oxygen limitations in highly agitated and aerated laboratory-scale fermentors

Background: Small-scale microbial fermentations are often assumed to be homogeneous, and oxygen limitation due to inadequate micromixing is often overlooked as a potential problem. To assess the relative degree of micromixing, and hence propensity for oxygen limitation, a new cellular oxygen sensor has been developed. The oxygen responsive E. coli nitrate reductase (nar) promoter was used to construct an oxygen reporter plasmid (pNar-GFPuv) which allows cell-based reporting of oxygen limitation. Because there are greater than 109 cells in a fermentor, one can outfit a vessel with more than 109 sensors. Our concept was tested in high density, lab-scale ( 5 L), fed-batch, E. coli fermentations operated with varied mixing efficiency - one verses four impellers. Results: In both cases, bioreactors were maintained identically at greater than 80% dissolved oxygen ( DO) during batch phase and at approximately 20% DO during fed-batch phase. Trends for glucose consumption, biomass and DO showed nearly identical behavior. However, fermentations with only one impeller showed significantly higher GFPuv expression than those with four, indicating a higher degree of fluid segregation sufficient for cellular oxygen deprivation. As the characteristic time for GFPuv expression (approx 90 min.) is much larger than that for mixing (approx 10 s), increased specific fluorescence represents an averaged effect of oxygen limitation over time and by natural extension, over space. Conclusion: Thus, the pNar-GFPuv plasmid enabled bioreactor-wide oxygen sensing in that bacterial cells served as individual recirculating sensors integrating their responses over space and time. We envision cell-based oxygen sensors may find utility in a wide variety of bioprocessing applications.open1123sciescopu

Proteomic analysis of Staphylococcus aureus biofilm cells grown under physiologically relevant fluid shear stress conditions

The biofilm forming bacterium Staphylococcus aureus is responsible for maladies ranging from severe skin infection to major diseases such as bacteremia, endocarditis and osteomyelitis. A flow displacement system was used to grow S. aureus biofilms in four physiologically relevant fluid shear rates (50, 100, 500 and 1000 s-1) to identify proteins that are associated with biofilm. Global protein expressions from the membrane and cytosolic fractions of S. aureus biofilm cells grown under the above shear rate conditions are reported. Sixteen proteins in the membrane-enriched fraction and eight proteins in the cytosolic fraction showed significantly altered expression (p < 0.05) under increasing fluid shear. These 24 proteins were identified using nano-LC-ESI-MS/MS. They were found to be associated with various metabolic functions such as glycolysis / TCA pathways, protein synthesis and stress tolerance. Increased fluid shear stress did not influence the expression of two important surface binding proteins: fibronectin-binding and collagen-binding proteins. The reported data suggest that while the general metabolic function of the sessile bacteria is minimal under high fluid shear stress conditions, they seem to retain the binding capacity to initiate new infections.https://doi.org/10.1186/1477-5956-12-2

Aspergillus nidulans Septa Are Indispensable for Surviving Cell Wall Stress

Septation in filamentous fungi is a normal part of development, which involves the formation of cross-hyphal bulkheads, typically containing pores, allowing cytoplasmic streaming between compartments. Based on previous findings regarding septa and cell wall stress, we hypothesized that septa are critical for survival during cell wall stress. To test this hypothesis, we used known Aspergillus nidulans septation-deficient mutants (ΔsepH, Δbud3, Δbud4, and Δrho4) and six antifungal compounds. Three of these compounds (micafungin, Congo red, and calcofluor white) are known cell wall stressors which activate the cell wall integrity signaling pathway (CWIS), while the three others (cycloheximide, miconazole, and 2,3-butanedione monoxime) perturb specific cellular processes not explicitly related to the cell wall. Our results show that deficiencies in septation lead to fungi which are more susceptible to cell wall-perturbing compounds but are no more susceptible to other antifungal compounds than a control. This implies that septa play a critical role in surviving cell wall stress

Isometries of infinite dimensional Hilbert geometries

In this paper we extend two classical results concerning the isometries of strictly convex Hilbert geometries, and the characterisation of the isometry groups of Hilbert geometries on finite dimensional simplices, to infinite dimensions. The proofs rely on a mix of geometric and functional analytic methods

Dynamic Transcriptomic and Phosphoproteomic Analysis During Cell Wall Stress in Aspergillus nidulans

The fungal cell-wall integrity signaling (CWIS) pathway regulates cellular response to environmental stress to enable wall repair and resumption of normal growth. This complex, interconnected, pathway has been only partially characterized in filamentous fungi. To better understand the dynamic cellular response to wall perturbation, a-glucan synthase inhibitor (micafungin) was added to a growing A. nidulans shake-flask culture. From this flask, transcriptomic and phosphoproteomic data were acquired over 10 and 120 min, respectively. To differentiate statistically-significant dynamic behavior from noise, a multivariate adaptive regression splines (MARS) model was applied to both data sets. Over 1800 genes were dynamically expressed and over 700 phosphorylation sites had changing phosphorylation levels upon micafungin exposure. Twelve kinases had altered phosphorylation and phenotypic profiling of all non-essential kinase deletion mutants revealed putative connections between PrkA, Hk-8 –4, and Stk19 and the CWIS pathway. Our collective data implicate actin regulation, endocytosis, and septum formation as critical cellular processes responding to activation of the CWIS pathway, and connections between CWIS and calcium, HOG, and SIN signaling pathways

Best practices for virtual participation in meetings : experiences from synthesis centers

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Corrigendum: Normal Values of Corrected Heart-Rate Variability in 10-Second Electrocardiograms for All Ages

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Resource Selection of Free-ranging Horses Influenced by Fire in Northern Canada

Free-ranging or feral horses (Equus ferus caballus) were important to the livelihood of First Nations and indigenous communities in Canada. The early inhabitants of the boreal region of British Columbia (BC) capitalized on naturally occurring wildfires and anthropogenic burning to provide forage for free-ranging horses and manage habitat for wildlife. This form of pyric herbivory, or grazing driven by fi re via the attraction to the palatable vegetation in recently burned areas, is an evolutionary disturbance process that occurs globally. However, its application to manage forage availability for free-ranging horses has not been studied in northern Canada. Across Canada, there are varying levels of governance for feral and free-ranging horses depending on the provincial jurisdiction and associated legislation. The BC Range Act (Act) allows range tenure holders to free-range horses that they own for commercial operations on Crown land. Big-game guide outfitters as range tenure holders are provided grazing licences or grazing permits under the Act with an approved range use plan. Guide outfitters and other range tenure holders have incorporated fi re ecology as part of their rangeland management in mountainous portions of the boreal forest of northeastern BC to promote mosaics of vegetation height and species composition across the landscape to meet nutritional requirements of their free-ranging horses. Using resource selection function models, we evaluated the influence of pyric herbivory on boreal vegetation and use by horse herds occupying 4 distinct landscapes. We found that horses preferentially selected recently burned areas and areas that burned more frequently when they were available. We also found that horses avoided steep slopes and forest cover types. Fire and the ecological processes associated with it, including pyric herbivory, are important considerations when managing boreal rangelands in northeastern BC. Because historical fi re regimes of the boreal region of Canada differ from the arid regions of the United States inhabited by feral horses, the role of pyric herbivory in altering horse distributions in the United States is limited

A vision for global monitoring of biological invasions

Managing biological invasions relies on good global coverage of species distributions. Accurate information on alien species distributions, obtained from international policy and cross-border co-operation, is required to evaluate trans-boundary and trading partnership risks. However, a standardized approach for systematically monitoring alien species and tracking biological invasions is still lacking. This Perspective presents a vision for global observation and monitoring of biological invasions. We show how the architecture for tracking biological invasions is provided by a minimum information set of Essential Variables, global collaboration on data sharing and infrastructure, and strategic contributions by countries. We show how this novel, synthetic approach to an observation system for alien species provides a tangible and attainable solution to delivering the information needed to slow the rate of new incursions and reduce the impacts of invaders. We identify three Essential Variables for Invasion Monitoring; alien species occurrence, species alien status and alien species impact. We outline how delivery of this minimum information set by joint, complementary contributions from countries and global community initiatives is possible. Country contributions are made feasible using a modular approach where all countries are able to participate and strategically build their contributions to a global information set over time. The vision we outline will deliver wide-ranging benefits to countries and international efforts to slow the rate of biological invasions and minimize their environmental impacts. These benefits will accrue over time as global coverage and information on alien species increases