The Distinctive Regulation of Cyanobacterial Glutamine Synthetase

Glutamine synthetase (GS) features prominently in bacterial nitrogen assimilation as it catalyzes the entry of bioavailable nitrogen in form of ammonium into cellular metabolism. The classic example, the comprehensively characterized GS of enterobacteria, is subject to exquisite regulation at multiple levels, among them gene expression regulation to control GS abundance, as well as feedback inhibition and covalent modifications to control enzyme activity. Intriguingly, the GS of the ecologically important clade of cyanobacteria features fundamentally different regulatory systems to those of most prokaryotes. These include the interaction with small proteins, the so-called inactivating factors (IFs) that inhibit GS linearly with their abundance. In addition to this protein interaction-based regulation of GS activity, cyanobacteria use alternative elements to control the synthesis of GS and IFs at the transcriptional level. Moreover, cyanobacteria evolved unique RNA-based regulatory mechanisms such as glutamine riboswitches to tightly tune IF abundance. In this review, we aim to outline the current knowledge on the distinctive features of the cyanobacterial GS encompassing the overall control of its activity, sensing the nitrogen status, transcriptional and post-transcriptional regulation, as well as strain-specific differences.Deutsche Forschungsgemeinschaft KL 3114/2-1Ministerio de Economía y Competitividad BIO2016-75634-PFEDER BIO2016-75634-

Prospective monitoring of cefepime in intensive care unit adult patients

INTRODUCTION: Cefepime has been associated with a greater risk of mortality than other beta-lactams in patients treated for severe sepsis. Hypotheses for this failure include possible hidden side-effects (for example, neurological) or inappropriate pharmacokinetic/pharmacodynamic (PK/PD) parameters for bacteria with cefepime minimal inhibitory concentrations (MIC) at the highest limits of susceptibility (8 mg/l) or intermediate-resistance (16 mg/l) for pathogens such as Enterobacteriaceae, Pseudomonas aeruginosa and Staphylococcus aureus. We examined these issues in a prospective non-interventional study of 21 consecutive intensive care unit (ICU) adult patients treated with cefepime for nosocomial pneumonia. METHODS: Patients (median age 55.1 years, range 21.8 to 81.2) received intravenous cefepime at 2 g every 12 hours for creatinine clearance (CLCr) >or= 50 ml/min, and 2 g every 24 hours or 36 hours for CLCr < 50 ml/minute. Cefepime plasma concentrations were determined at several time-points before and after drug administration by high-pressure liquid chromatography. PK/PD parameters were computed by standard non-compartmental analysis. RESULTS: Seventeen first-doses and 11 steady states (that is, four to six days after the first dose) were measured. Plasma levels varied greatly between individuals, from two- to three-fold at peak-concentrations to up to 40-fold at trough-concentrations. Nineteen out of 21 (90%) patients had PK/PD parameters comparable to literature values. Twenty-one of 21 (100%) patients had appropriate duration of cefepime concentrations above the MIC (T>MIC >or= 50%) for the pathogens recovered in this study (MIC <or= 4 mg/l), but only 45 to 65% of them had appropriate coverage for potential pathogens with cefepime MIC >or= 8 mg/l. Moreover, 2/21 (10%) patients with renal impairment (CLCr < 30 ml/minute) demonstrated accumulation of cefepime in the plasma (trough concentrations of 20 to 30 mg/l) in spite of dosage adjustment. Both had symptoms compatible with non-convulsive epilepsy (confusion and muscle jerks) that were not attributed to cefepime-toxicity until plasma levels were disclosed to the caretakers and symptoms resolved promptly after drug arrest. CONCLUSIONS: These empirical results confirm the suspected risks of hidden side-effects and inappropriate PK/PD parameters (for pathogens with upper-limit MICs) in a population of ICU adult patients. Moreover, it identifies a safety and efficacy window for cefepime doses of 2 g every 12 hours in patients with a CLCr >or= 50 ml/minute infected by pathogens with cefepime MICs <or= 4 mg/l. On the other hand, prompt monitoring of cefepime plasma levels should be considered in case of lower CLCr or greater MICs

Virus genomes reveal factors that spread and sustained the Ebola epidemic.

The 2013-2016 West African epidemic caused by the Ebola virus was of unprecedented magnitude, duration and impact. Here we reconstruct the dispersal, proliferation and decline of Ebola virus throughout the region by analysing 1,610 Ebola virus genomes, which represent over 5% of the known cases. We test the association of geography, climate and demography with viral movement among administrative regions, inferring a classic 'gravity' model, with intense dispersal between larger and closer populations. Despite attenuation of international dispersal after border closures, cross-border transmission had already sown the seeds for an international epidemic, rendering these measures ineffective at curbing the epidemic. We address why the epidemic did not spread into neighbouring countries, showing that these countries were susceptible to substantial outbreaks but at lower risk of introductions. Finally, we reveal that this large epidemic was a heterogeneous and spatially dissociated collection of transmission clusters of varying size, duration and connectivity. These insights will help to inform interventions in future epidemics

The Novel PII-Interacting Protein PirA Controls Flux into the Cyanobacterial Ornithine-Ammonia Cycle

Among prokaryotes, cyanobacteria have an exclusive position as they perform oxygenic photosynthesis. Cyanobacteria substantially differ from other bacteria in further aspects, e.g., they evolved a plethora of unique regulatory mechanisms to control primary metabolism. This is exemplified by the regulation of glutamine synthetase (GS) via small proteins termed inactivating factors (IFs). Here, we reveal another small protein, encoded by the ssr0692 gene in the model strain Synechocystis sp. PCC 6803, that regulates flux into the ornithine-ammonia cycle (OAC), the key hub of cyanobacterial nitrogen stockpiling and remobilization. This regulation is achieved by the interaction with the central carbon/nitrogen control protein PII, which commonly controls entry into the OAC by activating the key enzyme of arginine synthesis, N-acetyl-L-glutamate kinase (NAGK). In particular, the Ssr0692 protein competes with NAGK for PII binding and thereby prevents NAGK activation, which in turn lowers arginine synthesis. Accordingly, we termed it PII-interacting regulator of arginine synthesis (PirA). Similar to the GS IFs, PirA accumulates in response to ammonium upshift due to relief from repression by the global nitrogen control transcription factor NtcA. Consistent with this, the deletion of pirA affects the balance of metabolite pools of the OAC in response to ammonium shocks. Moreover, the PirA-PII interaction requires ADP and is prevented by PII mutations affecting the T-loop conformation, the major protein interaction surface of this signal processing protein. Thus, we propose that PirA is an integrator determining flux into N storage compounds not only depending on the N availability but also the energy state of the cell.German Research Foundation (DFG) KL 3114/2-1, Fo195/17-1, HA2002/22-2 and HA 2002/23-1Agencia Estatal de Investigación (AEI) BIO2016-75634-P and PID2019-104513GB-100/AEI/10.13039/501100011033Junta de Andalucía BIO-028

Variability of Voriconazole Plasma Levels Measured by New High-Performance Liquid Chromatography and Bioassay Methods

Voriconazole (VRC) is a broad-spectrum antifungal triazole with nonlinear pharmacokinetics. The utility of measurement of voriconazole blood levels for optimizing therapy is a matter of debate. Available high-performance liquid chromatography (HPLC) and bioassay methods are technically complex, time-consuming, or have a narrow analytical range. Objectives of the present study were to develop new, simple analytical methods and to assess variability of voriconazole blood levels in patients with invasive mycoses. Acetonitrile precipitation, reverse-phase separation, and UV detection were used for HPLC. A voriconazole-hypersusceptible Candida albicans mutant lacking multidrug efflux transporters (cdr1Δ/cdr1Δ, cdr2Δ/cdr2Δ, flu1Δ/flu1Δ, and mdr1Δ/mdr1Δ) and calcineurin subunit A (cnaΔ/cnaΔ) was used for bioassay. Mean intra-/interrun accuracies over the VRC concentration range from 0.25 to 16 mg/liter were 93.7% ± 5.0%/96.5% ± 2.4% (HPLC) and 94.9% ± 6.1%/94.7% ± 3.3% (bioassay). Mean intra-/interrun coefficients of variation were 5.2% ± 1.5%/5.4% ± 0.9% and 6.5% ± 2.5%/4.0% ± 1.6% for HPLC and bioassay, respectively. The coefficient of concordance between HPLC and bioassay was 0.96. Sequential measurements in 10 patients with invasive mycoses showed important inter- and intraindividual variations of estimated voriconazole area under the concentration-time curve (AUC): median, 43.9 mg · h/liter (range, 12.9 to 71.1) on the first and 27.4 mg · h/liter (range, 2.9 to 93.1) on the last day of therapy. During therapy, AUC decreased in five patients, increased in three, and remained unchanged in two. A toxic encephalopathy probably related to the increase of the VRC AUC (from 71.1 to 93.1 mg · h/liter) was observed. The VRC AUC decreased (from 12.9 to 2.9 mg · h/liter) in a patient with persistent signs of invasive aspergillosis. These preliminary observations suggest that voriconazole over- or underexposure resulting from variability of blood levels might have clinical implications. Simple HPLC and bioassay methods offer new tools for monitoring voriconazole therapy

La fabrication du regard dans l’apprentissage du métier

Ce dossier thématique contribue à l’anthropologie du regard et de l’expertise professionnelle. Il documente les formes de la transmission du savoir-voir dans des métiers où les façons de voir sont éprouvées, questionnées et reconfigurées. Il traite de la construction du savoir-voir, sa dimension multisensorielle et multimodale, les méthodologies permettant de documenter son apprentissage et son développement, et ses étayages techniques. La diversité des terrains, des méthodologies et des approches documente le rôle des habiletés visuelles dans la production, la circulation et la transformation des façons de voir de différentes communautés de pratique. This Special Issue focuses on the anthropology of gaze and of professional expertise. It documents the forms of teaching of the ability to see in professions whose “ways of seeing” are tested, questioned, and reshaped. It discusses the construction of the ability to see, its multisensorial and multimodal dimension, methodologies for exploring the learning and development of professional vision, and the technical artifacts supporting it. The diversity of fields and approaches which explore the role of visual skills in the production, diffusion, and transformation of the “ways of seeing” characterizing different communities of practice. Este dossier temático contribuye a la antropología de la mirada y a la pericia profesional. Documenta la transmisión del saber-ver en las profesiones en las que se experimentan, cuestionan y reconfiguran las formas de ver. Se trata de la construcción del saber-ver, su dimensión multisensorial y multimodal, las metodologías utilizadas para documentar su aprendizaje y desarrollo, y sus fundamentos técnicos. La diversidad de terrenos, metodologías y enfoques documenta el papel de las habilidades visuales en la producción, circulación y transformación de las formas de ver en las diferentes comunidades de práctica