Long-range electron transfer in structurally engineered pentaammineruthenium (histidine-62) cytochrome c

In many biological processes, long-range electron transfer (ET) plays a key role. When the three-dimensional structures of proteins are accurately known, use of modified proteins and protein-protein complexes provides an experimental approach to study ET rates between two metal centers. For Ru(His)- modified proteins, the introduction of histidine residues at any desired surface location by site-directed mutagenesis opens the way for systematic investigations of ET pathways

Diabatic processes and the evolution of two contrasting summer extratropical cyclones

Extratropical cyclones are typically weaker and less frequent in summer as a result of differences in the background state flow and diabatic processes with respect to other seasons. Two extratropical cyclones were observed in summer 2012 with a research aircraft during the DIAMET (DIAbatic influences on Mesoscale structure in ExTratropical storms) field campaign. The first cyclone deepened only down to 995 hPa; the second cyclone deepened down to 978 hPa and formed a potential vorticity (PV) tower, a frequent signature of intense cyclones. The objectives of this article are to quantify the effects of diabatic processes and their parametrizations on cyclone dynamics. The cyclones were analyzed through numerical simulations incorporating tracers for the effects of diabatic processes on potential temperature and PV. The simulations were compared with radar rainfall observations and dropsonde measurements. It was found that the observed maximum vapor flux in the stronger cyclone was twice as strong as in the weaker cyclone; the water vapor mass flow along the warm conveyor belt of the stronger cyclone was over half that typical in winter. The model overestimated water vapor mass flow by approximately a factor of two due to deeper structure in the rearwards flow and humidity in the weaker case. An integral tracer interpretation is introduced, relating the tracers with cross-isentropic mass transport and circulation. It is shown that the circulation around the cyclone increases much more slowly than the amplitude of the diabatically-generated PV tower. This effect is explained using the PV impermeability theorem

Mass spectrometry hybridized with gas-phase InfraRed spectroscopy for glycan sequencing

International audiencePrecise structural differentiation of often isomeric glycans is important given their roles in numerous biological processes. Mass spectrometry (MS) (and tandem MS) is one of the analytical techniques at the forefront of glycan analysis given its speed, sensitivity in producing structural information as well as the fact it can be coupled to other orthogonal analytical techniques such as liquid chromatography (LC) and ion mobility spectrometry (IMS). This review describes another family of techniques that are more commonly being hybridized to MS(/MS) namely gas-phase infrared (IR) spectroscopy, whose rise is in part due to the development and improved accessibility of tunable IR lasers. Gas-phase IR can often differentiate fine isomeric differences ubiquitous within carbohydrates that MS may be 'blind' to. There are also examples of cryogenic gas-phase IR spectroscopy with much greater spectral resolution as well as hybridizing with separative methods (LC, IMS). Furthermore, collision-induced dissociation (CID) product ions can also be probed by IR, which may be beneficial to deconvolute spectra, aid analysis and build spectral libraries, thus generating novel opportunities for fragment-based approaches to analyze glycans

Synthesis and evaluation of halogenated nitrophenoxazinones as nitroreductase substrates for the detection of pathogenic bacteria

The synthesis and microbiological evaluation of 7-, 8- and 9-nitro-1,2,4-trihalogenophenoxazin-3-one substrates with potential in the detection of nitroreductase-expressing pathogenic microorganisms are described. The 7- and 9-nitrotrihalogenophenoxazinone substrates were reduced by most Gram negative microorganisms and were inhibitory to the growth of certain Gram positive bacteria; however, the majority of Gram positive strains that were not inhibited by these agents, along with the two yeast strains evaluated, did not reduce the substrates. These observations suggest there are differences in the active site structures and substrate requirements of the nitroreductase enzymes from different strains; such differences may be exploited in the future for differentiation between pathogenic microorganisms. The absence of reduction of the 8-nitrotrihalogenophenoxazinone substrates is rationalized according to their electronic properties and correlates well with previous findings

Plasma and serum oxylipin, endocannabinoid, bile acid, steroid, fatty acid and nonsteroidal anti-inflammatory drug quantification in a 96-well plate format

The goal of this research was to develop a high-throughput, cost-effective method for metabolic profiling of lipid mediators and hormones involved in the regulation of inflammation and energy metabolism, along with polyunsaturated fatty acids and common over-the-counter non-steroidal anti-inflammatory drugs (NSAIDs). We describe a 96-well plate protein precipitation and filtration procedure for 50 μL of plasma or serum in the presence of 37 deuterated analogs and 2 instrument internal standards. Data is acquired in two back-to-back UPLC-MS/MS analyses using electrospray ionization with positive/negative switching and scheduled multiple reaction monitoring for the determination of 145 compounds, including oxylipins, endocannabinoids and like compounds, bile acids, glucocorticoids, sex steroids, polyunsaturated fatty acids, and 3 NSAIDs. Intra- and inter-batch variability was <25% for >70% of metabolites above the LOQ in both matrices, but higher inter-batch variability was observed for serum oxylipins and some bile acids. Results for NIST Standard Reference Material 1950, compared favorably with the 20 certified metabolite values covered by this assay, and we provide new data for oxylipins, N-acylethanolamides, glucocorticoids, and 17-hydroxy-progesterone in this material. Application to two independent cohorts of elderly men and women showed the routine detection of 86 metabolites, identified fasting state influences on essential fatty acid-derived oxylipins, N-acylethanolamides and conjugated bile acids, identified rare presence of high and low testosterone levels and the presence of NSAIDs in ∼10% of these populations. The described method appears valuable for investigations in large cohort studies to provide insight into metabolic cross-talk between the array of mediators assessed here

Learning to laugh : children and being human in early modern thought

This essay explores the construction of the human in early modern English thought, and uses discussions of the nature and use of laughter as a distinguishing feature of humanity from classical arguments as well as early modern ones. Using these classical, reformed English discussions of education and of the nature of children reveals an anxiety about the status of the child. Laughing appropriately - using tile mind and not merely the body - is a key feature of being human, and as such, the child's lack of "true' laughter reveals that child's status to be never always-already human. "Human' is a created rather than merely a natural status

Editorial: Ethics, Values, and Designer Responsibility

As we rely upon increasingly complex sociotechnical systems to support ourselves and, by extension, the structures of society, it becomes yet more important to consider how ethics and values intertwine in design activity. Numerous methods that address issues related to ethics and value-centeredness in design activity exist, but it is unclear what role the design research and practice communities should play in shaping the future of these design approaches. Importantly, how might researchers and practitioners become more aware of the normative assumptions that underlie both their design activity and the design artifacts that result

The OECD Program to Validate the Rat Hershberger Bioassay to Screen Compounds for in Vivo Androgen and Antiandrogen Responses: Phase 2 Dose–Response Studies

OBJECTIVE: The Organisation for Economic Co-operation and Development (OECD) has completed phase 2 of an international program to validate the rodent Hershberger bioassay. DESIGN: The Hershberger bioassay is designed to identify suspected androgens and antiandrogens based on changes in the weights of five androgen-responsive tissues (ventral prostate, paired seminal vesicles and coagulating glands, the levator ani and bulbocavernosus muscles, the glans penis, and paired Cowper’s or bulbourethral glands). Protocol sensitivity and reproducibility were tested using two androgen agonists (17α-methyl testosterone and 17β-trenbolone), four antagonists [procymi-done, vinclozolin, linuron, and 1,1-dichoro-2,2-bis-(p-chlorophenyl)ethylene (p,p’-DDE)], and a 5α-reductase inhibitor (finasteride). Sixteen laboratories from seven countries participated in phase 2. RESULTS: In 40 of 41 studies, the laboratories successfully detected substance-related weight changes in one or more tissues. The one exception was with the weakest antiandrogen, linuron, in a laboratory with reduced sensitivity because of high coefficients of variation in all tissue weights. The protocols performed well under different experimental conditions (e.g., strain, diet, housing protocol, bedding, vehicle). There was good agreement and reproducibility among laboratories with regard to the lowest dose inducing significant effects on tissue weights. CONCLUSIONS: The results show that the OECD Hershberger bioassay protocol is reproducible and transferable across laboratories with androgen agonists, weak androgen antagonists, and a 5α-reductase inhibitor. The next validation phase will employ coded test substances, including positive substances and negative substances having no androgenic or antiandrogenic activity

Phosphonopeptides Revisited, in an Era of Increasing Antimicrobial Resistance

Given the increase in resistance to antibacterial agents, there is an urgent need for the development of new agents with novel modes of action. As an interim solution, it is also prudent to reinvestigate old or abandoned antibacterial compounds to assess their efficacy in the context of widespread resistance to conventional agents. In the 1970s, much work was performed on the development of peptide mimetics, exemplified by the phosphonopeptide, alafosfalin. We investigated the activity of alafosfalin, di-alanyl fosfalin and β-chloro-L-alanyl-β-chloro-L-alanine against 297 bacterial isolates, including carbapenemase-producing Enterobacterales (CPE) (n = 128), methicillin-resistant Staphylococcus aureus (MRSA) (n = 37) and glycopeptide-resistant enterococci (GRE) (n = 43). The interaction of alafosfalin with meropenem was also examined against 20 isolates of CPE. The MIC50 and MIC90 of alafosfalin for CPE were 1 mg/L and 4 mg/L, respectively and alafosfalin acted synergistically when combined with meropenem against 16 of 20 isolates of CPE. Di-alanyl fosfalin showed potent activity against glycopeptide-resistant isolates of Enterococcus faecalis (MIC90; 0.5 mg/L) and Enterococcus faecium (MIC90; 2 mg/L). Alafosfalin was only moderately active against MRSA (MIC90; 8 mg/L), whereas β-chloro-L-alanyl-β-chloro-L-alanine was slightly more active (MIC90; 4 mg/L). This study shows that phosphonopeptides, including alafosfalin, may have a therapeutic role to play in an era of increasing antibacterial resistance