Biosynthesis of the proteasome inhibitor syringolin A: the ureido group joining two amino acids originates from bicarbonate

<p>Abstract</p> <p>Background</p> <p>Syringolin A, an important virulence factor in the interaction of the phytopathogenic bacterium <it>Pseudomonas syringae </it>pv. <it>syringae </it>B728a with its host plant <it>Phaseolus vulgaris </it>(bean), was recently shown to irreversibly inhibit eukaryotic proteasomes by a novel mechanism. Syringolin A is synthesized by a mixed non-ribosomal peptide synthetase/polyketide synthetase and consists of a tripeptide part including a twelve-membered ring with an N-terminal valine that is joined to a second valine via a very unusual ureido group. Analysis of sequence and architecture of the syringolin A synthetase gene cluster with the five open reading frames <it>sylA-sylE </it>allowed to formulate a biosynthesis model that explained all structural features of the tripeptide part of syringolin A but left the biosynthesis of the unusual ureido group unaccounted for.</p> <p>Results</p> <p>We have cloned a 22 kb genomic fragment containing the <it>sylA-sylE </it>gene cluster but no other complete gene into the broad host range cosmid pLAFR3. Transfer of the recombinant cosmid into <it>Pseudomonas putida </it>and <it>P. syringae </it>pv. <it>syringae </it>SM was sufficient to direct the biosynthesis of <it>bona fide </it>syringolin A in these heterologous organisms whose genomes do not contain homologous genes. NMR analysis of syringolin A isolated from cultures grown in the presence of NaH¹³CO₃revealed preferential ¹³C-labeling at the ureido carbonyl position.</p> <p>Conclusion</p> <p>The results show that no additional syringolin A-specific genes were needed for the biosynthesis of the enigmatic ureido group joining two amino acids. They reveal the source of the ureido carbonyl group to be bicarbonate/carbon dioxide, which we hypothesize is incorporated by carbamylation of valine mediated by the <it>sylC </it>gene product(s). A similar mechanism may also play a role in the biosynthesis of other ureido-group-containing NRPS products known largely from cyanobacteria.</p

AMANDA : an autonomous self-powered miniaturized smart sensing embedded system

​© 2019 IEEE. Personal use of this material is permitted. Permission from IEEE must be obtained for all other uses, in any current or future media, including reprinting/republishing this material for advertising or promotional purposes, creating new collective works, for resale or redistribution to servers or lists, or reuse of any copyrighted component of this work in other works.This paper introduces an Autonomous Smart Sensing Card (ASSC), an embedded system that will be powered indoors and outdoors by harvested energy, have miniaturized dimensions and serve multi-sensorial IoT applications for smart living and working environments. It will consist of a combination of newly developed and optimized off-the-shelf or close-tocommercialization technologies such as PV harvesters, energy storage and power management units, MCUs and sensors, all packed with a form factor under 3mm in thickness. The system will introduce technical breakthroughs that will boost further miniaturization, a small footprint, ultra-low power consumption as well as short- and long-range communications

Replicative senescence of mesenchymal stem cells causes DNA-methylation changes which correlate with repressive histone marks

Cells in culture undergo replicative senescence. In this study, we analyzed functional, genetic and epigenetic sequels of long-term culture in human mesenchymal stem cells (MSC). Already within early passages the fibroblastoid colonyforming unit (CFU-f) frequency and the differentiation potential of MSC declined significantly. Relevant chromosomal aberrations were not detected by karyotyping and SNP-microarrays. Subsequently, we have compared DNA-methylation profiles with the Infinium HumanMethylation27 Bead Array and the profiles differed markedly in MSC derived from adipose tissue and bone marrow. Notably, all MSC revealed highly consistent senescence-associated modifications at specific CpG sites. These DNA-methylation changes correlated with histone marks of previously published data sets, such as trimethylation of H3K9, H3K27 and EZH2 targets. Taken together, culture expansion of MSC has profound functional implications - these are hardly reflected by genomic instability but they are associated with highly reproducible DNA-methylation changes which correlate with repressive histone marks. Therefore replicative senescence seems to be epigenetically controlled

Mobilisation of critically ill patients receiving norepinephrine: a retrospective cohort study

Background: Mobilisation and exercise intervention in general are safe and feasible in critically ill patients. For patients requiring catecholamines, however, doses of norepinephrine safe for mobilisation in the intensive care unit (ICU) are not defined. This study aimed to describe mobilisation practice in our hospital and identify doses of norepinephrine that allowed a safe mobilisation. Methods: We conducted a retrospective single-centre cohort study of 16 ICUs at a university hospital in Germany with patients admitted between March 2018 and November 2021. Data were collected from our patient data management system. We analysed the effect of norepinephrine on level (ICU Mobility Scale) and frequency (units per day) of mobilisation, early mobilisation (within 72 h of ICU admission), mortality, and rate of adverse events. Data were extracted from free-text mobilisation entries using supervised machine learning (support vector machine). Statistical analyses were done using (generalised) linear (mixed-effect) models, as well as chi-square tests and ANOVAs. Results: A total of 12,462 patients were analysed in this study. They received a total of 59,415 mobilisation units. Of these patients, 842 (6.8%) received mobilisation under continuous norepinephrine administration. Norepinephrine administration was negatively associated with the frequency of mobilisation (adjusted difference -0.07 mobilisations per day; 95% CI - 0.09, - 0.05; p 0.1). Higher compared to lower doses of norepinephrine did not lead to a significant increase in adverse events in our practice (p > 0.1). We identified that mobilisation was safe with up to 0.20 mu g/kg/min norepinephrine for out-of-bed (IMS >= 2) and 0.33 mu g/kg/min for in-bed (IMS 0-1) mobilisation. Conclusions: Mobilisation with norepinephrine can be done safely when considering the status of the patient and safety guidelines. We demonstrated that safe mobilisation was possible with norepinephrine doses up to 0.20 mu g/kg/min for out-of-bed (IMS >= 2) and 0.33 mu g/kg/min for in-bed (IMS 0-1) mobilisation

Molecular basis of FIR-mediated c-myc transcriptional control

The far upstream element (FUSE) regulatory system promotes a peak in the concentration of c-Myc during cell cycle. First, the FBP transcriptional activator binds to the FUSE DNA element upstream of the c-myc promoter. Then, FBP recruits its specific repressor (FIR), which acts as an on/off transcriptional switch. Here we describe the molecular basis of FIR recruitment, showing that the tandem RNA recognition motifs of FIR provide a platform for independent FUSE DNA and FBP protein binding and explaining the structural basis of the reversibility of the FBP-FIR interaction. We also show that the physical coupling between FBP and FIR is modulated by a flexible linker positioned sequentially to the recruiting element. Our data explain how the FUSE system precisely regulates c-myc transcription and suggest that a small change in FBP-FIR affinity leads to a substantial effect on c-Myc concentration.MRC Grant-in-aid U11757455

RNA reference materials with defined viral RNA loads of SARS-CoV-2—A useful tool towards a better PCR assay harmonization

SARS-CoV-2, the cause of COVID-19, requires reliable diagnostic methods to track the circulation of this virus. Following the development of RT-qPCR methods to meet this diagnostic need in January 2020, it became clear from interlaboratory studies that the reported Ct values obtained for the different laboratories showed high variability. Despite this the Ct values were explored as a quantitative cut off to aid clinical decisions based on viral load. Consequently, there was a need to introduce standards to support estimation of SARS-CoV-2 viral load in diagnostic specimens. In a collaborative study, INSTAND established two reference materials (RMs) containing heat-inactivated SARS-CoV-2 with SARS-CoV-2 RNA loads of ~107 copies/mL (RM 1) and ~106 copies/mL (RM 2), respectively. Quantification was performed by RT-qPCR using synthetic SARS-CoV-2 RNA standards and digital PCR. Between November 2020 and February 2021, German laboratories were invited to use the two RMs to anchor their Ct values measured in routine diagnostic specimens, with the Ct values of the two RMs. A total of 305 laboratories in Germany were supplied with RM 1 and RM 2. The laboratories were requested to report their measured Ct values together with details on the PCR method they used to INSTAND. This resultant 1,109 data sets were differentiated by test system and targeted gene region. Our findings demonstrate that an indispensable prerequisite for linking Ct values to SARS-CoV-2 viral loads is that they are treated as being unique to an individual laboratory. For this reason, clinical guidance based on viral loads should not cite Ct values. The RMs described were a suitable tool to determine the specific laboratory Ct for a given viral load. Furthermore, as Ct values can also vary between runs when using the same instrument, such RMs could be used as run controls to ensure reproducibility of the quantitative measurements.Peer Reviewe

Energy autonomous wireless sensing node working at 5 Lux from a 4 cm2 solar cell

Harvesting energy for IoT nodes in places that are permanently poorly lit is important, as many such places exist in buildings and other locations. The need for energy-autonomous devices working in such environments has so far received little attention. This work reports the design and test results of an energy-autonomous sensor node powered solely by solar cells. The system can cold-start and run in low light conditions (in this case 20 lux and below, using white LEDs as light sources). Four solar cells of 1 cm2 each are used, yielding a total active surface of 4 cm2. The system includes a capacitive sensor that acts as a touch detector, a crystal-accurate real-time clock (RTC), and a Cortex-M3-compatible microcontroller integrating a Bluetooth Low Energy radio (BLE) and the necessary stack for communication. A capacitor of 100 μF is used as energy storage. A low-power comparator monitors the level of the energy storage and powers up the system. The combination of the RTC and touch sensor enables the MCU load to be powered up periodically or using an asynchronous user touch activity. First tests have shown that the system can perform the basic work of cold-starting, sensing, and transmitting frames at +0 dBm, at illuminances as low as 5 lux. Harvesting starts earlier, meaning that the potential for full function below 5 lux is present. The system has also been tested with other light sources. The comparator is a test chip developed for energy harvesting. Other elements are off-the-shelf components. The use of commercially available devices, the reduced number of parts, and the absence of complex storage elements enable a small node to be built in the future, for use in constantly or intermittently poorly lit places

Phenomenology without Representation

I criticise a recent variety of argument for the representational theory of experience, which holds that the very idea of perceptual experience entails the representational view. I argue that the representational view is not simply obvious, nor is it contained in the mere idea of the world looking some way. I also clarify and re-present an argument against the representational view due to Charles Travis