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We report a systematic diffraction study of two “water-in-salt” electrolytes and a “water-in-bisalt” electrolyte combining high-energy X-ray diffraction (HEXRD) with polarized and unpolarized neutron diffraction (ND) on both H(2)O and D(2)O solutions. The measurements provide three independent combinations of correlations between the different pairs of atom types that reveal the short- and intermediate-range order in considerable detail. The ND interference functions show pronounced peaks around a scattering vector Q ∼ 0.5 Å(−1) that change dramatically with composition, indicating significant rearrangements of the water network on a length scale around 12 Å. The experimental results are compared with two sets of Molecular Dynamics (MD) simulations, one including polarization effects and the other based on a non-polarizable force field. The two simulations reproduce the general shapes of the experimental structure factors and their changes with concentration, but differ in many detailed respects, suggesting ways in which their force fields might be modified to better represent the actual systems.

Interactions of the RNA polymerase II (Pol II) preinitiation complex (PIC) and paused early elongation complexes with the first downstream (+1) nucleosome are thought to be functionally important. However, current methods are limited for investigating these relationships, both for cellular chromatin and the human cytomegalovirus (HCMV) genome. Digestion with human DNA fragmentation factor (DFF) before immunoprecipitation (DFF-ChIP) precisely revealed both similarities and major differences in PICs driven by TBP on the host genome in comparison with PICs driven by TBP or the viral-specific, late initiation factor UL87 on the viral genome. Host PICs and paused Pol II complexes are frequently found in contact with the +1 nucleosome and paused Pol II can also be found in a complex involved in the initial invasion of the +1 nucleosome. In contrast, viral transcription complexes have very limited nucleosomal interactions, reflecting a relative lack of chromatinization of transcriptionally active regions of HCMV genomes.

Tafenoquine has been licensed for the single-dose radical cure of Plasmodium vivax in adults; however, it is only recommended in patients with > 70% of normal glucose-6-phosphate dehydrogenase (G6PD) activity. Because this may hinder widespread use, we investigated sex-based treatment strategies in which all adult patients are tested with a qualitative G6PD rapid diagnostic test (RDT). Glucose-6-phosphate dehydrogenase normal males are prescribed tafenoquine in all three strategies, whereas G6PD normal females are prescribed either a low-dose 14-day primaquine regimen (PQ14, total dose 3.5 mg/kg) or a high-dose 7-day primaquine regimen (PQ7, total dose 7 mg/kg), or referred to a healthcare facility for quantitative G6PD testing before prescribing tafenoquine. Patients testing G6PD deficient are prescribed a weekly course of primaquine for 8 weeks. We compared the cost-effectiveness of these three strategies to usual care in four countries using a decision tree model. Usual care in Ethiopia does not include radical cure, whereas Afghanistan, Indonesia, and Vietnam prescribe PQ14 without G6PD screening. The cost per disability-adjusted life-year (DALY) averted was expressed through incremental cost-effectiveness ratios (ICERs). Compared with usual care, the ICERs for a sex-based treatment strategy with PQ7 for females from a healthcare provider perspective were $127 per DALY averted in Vietnam, $466 in Ethiopia, $1,089 in Afghanistan, and $4,443 in Indonesia. The PQ14 and referral options cost more while averting fewer DALYs than PQ7. This study provides an alternative cost-effective mode of rolling out tafenoquine in areas where initial testing with only a G6PD RDT is feasible.

Guanine-rich nucleic acid sequences have a tendency to form four-stranded non-canonical motifs known as G-quadruplexes. These motifs may adopt a wide range of structures characterized by size, strand orientation, guanine base conformation, and fold topology. Using three K+-bound model systems, we show that vibrational coupling between guanine C6=O and ring modes varies between parallel-stranded and antiparallel-stranded G-quadruplexes, and that such structures can be distinguished by comparison of polarization dependent cross-peaks in their two-dimensional infrared (2D IR) spectra. Combined with previously defined vibrational frequency trends, this analysis reveals key features of a 30-nucleotide unimolecular variant of the Bcl-2 proximal promoter that are consistent with its reported structure. This study shows that 2D IR spectroscopy is a convenient method for analyzing G-quadruplex structures that can be applied to complex sequences where traditional high-resolution methods are limited by solubility and disorder.

Significance Sufficient immune coverage of the peptide universe within a finite host requires highly degenerate T cell receptors (TCRs). However, this inherent need for antigen cross-recognition is associated with a high risk of autoimmunity, which can only be mitigated by a process of adaptable specificity. We describe a mechanism that resolves this conundrum by allowing individual clonotypes to focus on specific peptide ligands without alterations to the structure of the TCR., CD8+ T cells are inherently cross-reactive and recognize numerous peptide antigens in the context of a given major histocompatibility complex class I (MHCI) molecule via the clonotypically expressed T cell receptor (TCR). The lineally expressed coreceptor CD8 interacts coordinately with MHCI at a distinct and largely invariant site to slow the TCR/peptide-MHCI (pMHCI) dissociation rate and enhance antigen sensitivity. However, this biological effect is not necessarily uniform, and theoretical models suggest that antigen sensitivity can be modulated in a differential manner by CD8. We used two intrinsically controlled systems to determine how the relationship between the TCR/pMHCI interaction and the pMHCI/CD8 interaction affects the functional sensitivity of antigen recognition. Our data show that modulation of the pMHCI/CD8 interaction can reorder the agonist hierarchy of peptide ligands across a spectrum of affinities for the TCR.

Background and aims We examined HbA1c and cardiovascular risk factors with a median follow-up of 44 months therapy with dapagliflozin. Methods We undertook a clinical practice evaluation of 101 patients attending our clinic. Results Dapagliflozin resulted in a significant reduction in HbA1c 82.6 ± 15.7 v 68.7 ± 17.8 mmol/mol. Conclusion Dapagliflozin maintains glycaemic control along with sustained improvements in weight and no decline in renal function.

Transcription by RNA polymerase II (Pol II) is controlled during initiation, elongation, and termination by a large variety of transcription factors, the state of chromatin modifications, and environmental conditions. Herein we describe experimental approaches for the examination of Pol II transcription at semi-global and genome-wide scales through analysis of nascent Pol II transcripts. We begin with a description of the nuclear walk-on (NWO) assay, which involves rapid isolation of nuclei in the presence of EDTA, followed by extension of about a quarter of the nascent transcripts with (32)P-CTP. Labeled nascent transcripts are then analyzed by denaturing PAGE and phosphorimaging followed by densitometry analysis to quantify the signal on the gel. A parallel reaction containing α-amanitin to inhibit Pol II reveals transcription due to Pol I and Pol III, which can be subtracted to yield a profile of Pol II transcription. We then describe how to use the NWO as a front end for PRO-Seq and PRO-Cap methods, which permit the genome-wide characterization of Pol II transcription at nucleotide resolution and provide precise information about sites of transcription initiation and pausing. We discuss strategies for optimizing sequencing methods that capture nascent Pol II transcripts, methods of bias reduction, and approaches for normalizing these and other sequencing datasets using spike-in controls.

Mitochondrial outer membrane permeabilization, which is a critical step in apoptosis, is initiated upon transmembrane insertion of the C-terminal α-helix (α9) of the proapoptotic Bcl-2 family protein BAX. The isolated α9 fragment (residues 173-192) is also competent to disrupt model membranes, and the structures of its membrane-associated oligomers are of interest in understanding the potential roles of this sequence in apoptosis. Here, we used ultrafast two-dimensional infrared (2D IR) spectroscopy, thioflavin T binding, and transmission electron microscopy to show that the synthetic BAX α9 peptide (α9p) forms amyloid aggregates in aqueous environments and on the surfaces of anionic small unilamellar vesicles. Its inherent amyloidogenicity was predicted by sequence analysis, and 2D IR spectra reveal that vesicles modulate the β-sheet structures of insoluble aggregates, motivating further examination of the formation or suppression of BAX amyloids in apoptosis.

Mitochondrial outer membrane permeabilization, which is a critical step in apoptosis, is initiated upon transmembrane insertion of the C-terminal α-helix (α9) of the pro-apoptotic Bcl-2 family protein BAX. The isolated α9 fragment (residues 173-192) is also competent to disrupt model membranes, and the structures of its membrane-associated oligomers are of interest in understanding the potential roles of this sequence in apoptosis. Here, we used ultrafast two-dimensional infrared (2D IR) spectroscopy, thioflavin T binding, and transmission electron microscopy to show that the synthetic BAX α9 peptide (α9p) forms amyloid aggregates in solution and on the surfaces of anionic small unilamellar vesicles (SUVs). Its inherent amyloidogenicity was predicted by sequence analysis, and 2D IR spectra reveal that SUVs modulate the β-sheet structures of the resulting amyloid species. These results contradict prior models of transmembrane α9p pores and motivate further examination of the formation or suppression of BAX amyloids in apoptosis.

Here we report a novel microelectrode array recording approach to measure tonic (resting) and phasic release of dopamine (DA) in DA-rich areas such as the rat striatum and nucleus accumbens. The resulting method is tested in intact central nervous system (CNS) and in animals with extensive loss of the DA pathway using the neurotoxin, 6-hydroxyDA (6-OHDA). The self-referencing amperometric recording method employs Nafion-coated with and without m-phenylenediamine recording sites that through real-time subtraction allow for simultaneous measures of tonic DA levels and transient changes due to depolarization and amphetamine-induced release. The recording method achieves low-level measures of both tonic and phasic DA with decreased recording drift allowing for enhanced sensitivity normally not achieved with electrochemical sensors in vivo.

"Water-in-salt" (WIS) and "water-in-bisalt" (WIBS) electrolytes have recently been developed for Li-ion batteries, combining the safety and environmental friendliness of aqueous electrolytes with a larger operating window made possible by a solid-electrolyte interphase. We report quasielastic neutron scattering (QENS) measurements on solutions of a WIS electrolyte at two concentrations, 13.9 and 21 m (molal) lithium bis(trifluoromethane)sulfonimide LiTFSI in H2O/D2O and a WIBS electrolyte at (21 m LiTFSI + 7 m lithium triflate (LiOTf)) in H2O/D2O. The data were Fourier transformed to obtain experimental intermediate scattering functions (ISFs) and compared with corresponding quantities obtained from molecular dynamics (MD) simulations. Both QENS and MD ISFs could be fitted well by a single stretched exponential function to obtain apparent translational diffusion coefficients for the water molecules. The QENS values agree well with the MD simulations for the 13.9 and 21 m solutions, but MD simulations predict a slower relaxation of water compared to QENS for the WIBS electrolyte. Comparison of the incoherent and coherent scattering reveals much faster water dynamics compared with structural relaxation of the ionic framework, consistent with the nanodomain picture where the lithium diffusion occurs through the tortuous water domain around the slower relaxing ionic matrix, leading to highly non-Gaussian water motion.

Background The radical cure of Plasmodium vivax and P. ovale requires treatment with primaquine or tafenoquine to clear dormant liver stages. Either drug can induce haemolysis in individuals with glucose-6-phosphate dehydrogenase (G6PD) deficiency, necessitating screening. The reference diagnostic method for G6PD activity is ultraviolet (UV) spectrophotometry; however, a universal G6PD activity threshold above which these drugs can be safely administered is not yet defined. Our study aimed to quantify assay-based variation in G6PD spectrophotometry and to explore the diagnostic implications of applying a universal threshold. Methods and findings Individual-level data were pooled from studies that used G6PD spectrophotometry. Studies were identified via PubMed search (25 April 2018) and unpublished contributions from contacted authors (PROSPERO: CRD42019121414). Studies were excluded if they assessed only individuals with known haematological conditions, were family studies, or had insufficient details. Studies of malaria patients were included but analysed separately. Included studies were assessed for risk of bias using an adapted form of the Quality Assessment of Diagnostic Accuracy Studies-2 (QUADAS-2) tool. Repeatability and intra- and interlaboratory variability in G6PD activity measurements were compared between studies and pooled across the dataset. A universal threshold for G6PD deficiency was derived, and its diagnostic performance was compared to site-specific thresholds. Study participants (n = 15,811) were aged between 0 and 86 years, and 44.4% (7,083) were women. Median (range) activity of G6PD normal (G6PDn) control samples was 10.0 U/g Hb (6.3–14.0) for the Trinity assay and 8.3 U/g Hb (6.8–15.6) for the Randox assay. G6PD activity distributions varied significantly between studies. For the 13 studies that used the Trinity assay, the adjusted male median (AMM; a standardised metric of 100% G6PD activity) varied from 5.7 to 12.6 U/g Hb (p < 0.001). Assay precision varied between laboratories, as assessed by variance in control measurements (from 0.1 to 1.5 U/g Hb; p < 0.001) and study-wise mean coefficient of variation (CV) of replicate measures (from 1.6% to 14.9%; p < 0.001). A universal threshold of 100% G6PD activity was defined as 9.4 U/g Hb, yielding diagnostic thresholds of 6.6 U/g Hb (70% activity) and 2.8 U/g Hb (30% activity). These thresholds diagnosed individuals with less than 30% G6PD activity with study-wise sensitivity from 89% (95% CI: 81%–94%) to 100% (95% CI: 96%–100%) and specificity from 96% (95% CI: 89%–99%) to 100% (100%–100%). However, when considering intermediate deficiency (, Daniel Pfeffer and coauthors report on the assessment of glucose-6-phosphate dehydrogenase activity, which is required for safe use of some malaria treatments., Author summary Why was this study done? Complete cure of vivax malaria, the most geographically widespread malaria species, requires the use of 8-aminoquinoline drugs to clear dormant liver stages of the parasite (‘radical cure’); however, these drugs can cause severe haemolysis in individuals with glucose-6-phosphate dehydrogenase (G6PD) deficiency. Ultraviolet (UV) spectrophotometry is used as the reference test to measure G6PD activity, for validating new point-of-care diagnostics, and to determine population-specific definitions of G6PD deficiency. Currently, there is no universal threshold to define G6PD deficiency, and each laboratory must invest time and resources to derive site- and laboratory-specific definitions of G6PD deficiency. What did the researchers do and find? We pooled measurements of G6PD activity from studies conducted across different countries and laboratories worldwide. We assessed the comparability of spectrophotometry results between these laboratories to see whether a universal definition and diagnostic cutoff for G6PD deficiency could be determined. There was substantial variation in the performance and absolute measurements of spectrophotometry conducted in different laboratories, hindering the definition of a universal cutoff for G6PD deficiency. What do these findings mean? These findings highlight the importance of quality-control measures to minimise the influence of laboratory procedures on observed measurements. The data suggest that while a robust universal, assay-specific G6PD activity cutoff value can be established for diagnosis of severe G6PD deficiency (

The neglected tropical disease Buruli ulcer (BU) is an infection of subcutaneous tissue with Mycobacterium ulcerans. There is no effective BU vaccine. Here, we assessed an experimental prime-boost vaccine in a low-dose murine tail infection model. We used the enoyl-reductase (ER) domain of the M. ulcerans mycolactone polyketide synthases electrostatically coupled with a previously described TLR-2 agonist-based lipopeptide adjuvant, R4Pam2Cys. Mice were vaccinated and then challenged via tail inoculation with 14-20 colony forming units (CFU) of an engineered bioluminescent strain of M. ulcerans. Mice receiving either the experimental ER vaccine or Mycobacterium bovis Bacille Calmette-Guérin (BCG) were equally well protected, with both groups faring significantly better than non-vaccinated animals (p-γ produced in the spleen best predicted control of infection across all vaccine groups. Univariate logistic regression then revealed vaccine-specific profiles of protection. High titres of ER-specific IgG serum antibodies together with IL-2 and IL-4 in the draining lymph node (DLN) were associated with protection induced by the experimental ER vaccine. In contrast, high titres of IL-6, TNF-α, IFN-γ and IL-10 in the DLN and low IFNγ titres in the spleen were associated with protection following BCG vaccination. This study suggests an effective BU vaccine must induce localized, tissue-specific immune profiles with controlled inflammatory responses at the site of infection.

Financial support from the EPSRC Catalysis Hub (Suwardiyanto) and an Industrial CASE Award (EPSRC/BP Chemicals) (Price) are also acknowledged. Synchrotron infrared micro-spectroscopy has been applied to measure in situ the reaction of dimethylether in single crystals of the silicoaluminophosphate STA-7. The crystals are found to contain a uniform and homogeneous distribution of acidic hydroxyl groups. Dimethylether is hydrogen bonded to the hydroxyl groups at low temperatures, but evidence is found for dissociation to form surface methoxy groups above 473 K, and aromatic hydrocarbon pool species above 573 K. From time resolved infrared measurements coupled with MS analysis of evolved products it is concluded that alkene formation occurs via a direct mechanism from reaction of dimethylether with surface methoxy groups. Publisher PDF

Significance Transcription by RNA Polymerase II (Pol II) is a highly dynamic process that is tightly regulated at each step of the transcription cycle. We generated GFP-RPB1 knockin cells and developed photobleaching of endogenous Pol II combined with computational modeling to study the in vivo dynamics of Pol II in real time. This approach allowed us to dissect promoter-paused Pol II from initiating and elongating Pol II and showed that initiation and promoter proximal pausing are surprisingly dynamic events, due to premature termination of Pol II. Our study provides new insights into Pol II dynamics and suggests that the iterative release and reinitiation of promoter-bound Pol II is an important component of transcriptional regulation., Initiation and promoter-proximal pausing are key regulatory steps of RNA Polymerase II (Pol II) transcription. To study the in vivo dynamics of endogenous Pol II during these steps, we generated fully functional GFP-RPB1 knockin cells. GFP-RPB1 photobleaching combined with computational modeling revealed four kinetically distinct Pol II fractions and showed that on average 7% of Pol II are freely diffusing, while 10% are chromatin-bound for 2.4 seconds during initiation, and 23% are promoter-paused for only 42 seconds. This unexpectedly high turnover of Pol II at promoters is most likely caused by premature termination of initiating and promoter-paused Pol II and is in sharp contrast to the 23 minutes that elongating Pol II resides on chromatin. Our live-cell–imaging approach provides insights into Pol II dynamics and suggests that the continuous release and reinitiation of promoter-bound Pol II is an important component of transcriptional regulation.

Naturally occurring paramagnetic species (PS), such as free radicals and paramagnetic metalloproteins, play an essential role in a multitude of critical physiological processes including metabolism, cell signaling and immune response. These highly dynamic species can also act as intrinsic biomarkers for a variety of disease states whilst synthetic para-magnetic probes targeted to specific sites on biomolecules enable the study of functional information such as tissue oxygenation and redox status in living systems. The work presented herein describes a new sensing method that exploits the spin dependent emission of photoluminescence (PL) from an ensemble of nitrogen vacancy centers in diamond for rapid, non-destructive detection of PS in living systems. Uniquely this approach involves simple measurement protocols that assess PL contrast with and without the application of microwaves. The method is demonstrated to detect concentrations of paramagnetic salts in solution and the widely used magnetic resonance imaging contrast agent Gadobutrol with a limit of detection of less than 10 attomol over a 100 micron x 100 micron field of view. Real time monitoring of changes in the concentration of paramagnetic salts is demonstrated with image exposure times of 20 ms. Further, dynamic tracking of chemical reactions is demonstrated via the conversion of low spin cyanide coordinated Fe3+ to hexaaqua Fe3+ under acidic conditions. Finally, the capability to map paramagnetic species in model cells with sub-cellular resolution is demonstrated using lipid membranes containing gadolinium labelled phospholipids under ambient conditions in the order of minutes. Overall, this work introduces a new sensing approach for the realization of fast, sensitive imaging of PS in a widefield format that is readily deployable in biomedical settings., 6 Figures

Background Blood sample hemolysis affects pre-analytical quality and may cause pseudohyperkalemia. We established a statistical model to estimate the corrected potassium (K+) in serum. Methods Serum K+ and H index were analyzed, and blood cell index was obtained from the examined Full Blood Examination (FBE) results. A linear-regression model was developed using hemolysis (H) index, K+ and covariates of blood cell index from 139 cell lysates of blood samples. The model was then validated against 26 in vitro physically hemolyzed serum samples. Results The final model selected H index, hemoglobin concentration (HGB), and hematocrit (HCT) as important predictors in estimating the K+ content. The model was validated against artificially hemolyzed serum samples, which returned a correlation of 0.942 between observed and predicted net K+ increase by hemolysis. The predictors H index, HCT, and HB contributed 93.7%, 3.5% and 2.8% to the model R2, respectively. Conclusion In vitro hemolysis induced pseudohyperkalemia could be accurately predicted and restored by our model for clinical application.

Summary Background Since the 1918 influenza pandemic, non-randomised studies and small clinical trials have suggested that convalescent plasma or anti-influenza hyperimmune intravenous immunoglobulin (hIVIG) might have clinical benefit for patients with influenza infection, but definitive data do not exist. We aimed to evaluate the safety and efficacy of hIVIG in a randomised controlled trial. Methods This randomised, double-blind, placebo-controlled trial was planned for 45 hospitals in Argentina, Australia, Denmark, Greece, Mexico, Spain, Thailand, UK, and the USA over five influenza seasons from 2013–14 to 2017–18. Adults (≥18 years of age) were admitted for hospital treatment with laboratory-confirmed influenza A or B infection and were randomly assigned (1:1) to receive standard care plus either a single 500-mL infusion of high-titre hIVIG (0·25 g/kg bodyweight, 24·75 g maximum; hIVIG group) or saline placebo (placebo group). Eligible patients had a National Early Warning score of 2 points or greater at the time of screening and their symptoms began no more than 7 days before randomisation. Pregnant and breastfeeding women were excluded, as well as any patients for whom the treatment would present a health risk. Separate randomisation schedules were generated for each participating clinical site using permuted block randomisation. Treatment assignments were obtained using a web-based application by the site pharmacist who then masked the solution for infusion. Patients and investigators were masked to study treatment. The primary endpoint was a six-category ordinal outcome of clinical status at day 7, ranging in severity from death to resumption of normal activities after discharge. The choice of day 7 was based on haemagglutination inhibition titres from a pilot study. It was analysed with a proportional odds model, using all six categories to estimate a common odds ratio (OR). An OR greater than 1 indicated that, for a given category, patients in the hIVIG group were more likely to be in a better category than those in the placebo group. Prespecified primary analyses for safety and efficacy were based on patients who received an infusion and for whom eligibility could be confirmed. This trial is registered with ClinicalTrials.gov , NCT02287467 . Findings 313 patients were enrolled in 34 sites between Dec 11, 2014, and May 28, 2018. We also used data from 16 patients enrolled at seven of the 34 sites during the pilot study between Jan 15, 2014, and April 10, 2014. 168 patients were randomly assigned to the hIVIG group and 161 to the placebo group. 21 patients were excluded (12 from the hIVIG group and 9 from the placebo group) because they did not receive an infusion or their eligibility could not be confirmed. Thus, 308 were included in the primary analysis. hIVIG treatment produced a robust rise in haemagglutination inhibition titres against influenza A and smaller rises in influenza B titres. Based on the proportional odds model, the OR on day 7 was 1·25 (95% CI 0·79–1·97; p=0·33). In subgroup analyses for the primary outcome, the OR in patients with influenza A was 0·94 (0·55–1·59) and was 3·19 (1·21–8·42) for those with influenza B (interaction p=0·023). Through 28 days of follow-up, 47 (30%) of 156 patients in the hIVIG group and in 45 (30%) of 152 patients in the placebo group had the composite safety outcome of death, a serious adverse event, or a grade 3 or 4 adverse event (hazard ratio [HR] 1·06, 95% CI 0·70–1·60; p=0·79). Six (4%) patients in the hIVIG group and five (3%) in the placebo group died, but these deaths were not necessarily related to treatment. Interpretation When administered alongside standard care (most commonly oseltamivir), hIVIG was not superior to placebo for adults hospitalised with influenza infection. By contrast with our prespecified subgroup hypothesis that hIVIG would result in more favourable responses in patients with influenza A than B, we found the opposite effect. The clinical benefit of hIVIG for patients with influenza B is supported by antibody affinity analyses, but confirmation is warranted. Funding NIAID and NIH. Partial support was provided by the Medical Research Council (MRC_UU_12023/23) and the Danish National Research Foundation.

BackgroundAcute respiratory distress syndrome (ARDS) is heterogeneous. As an indication of the heterogeneity of ARDS, there are patients whose syndrome improves rapidly (i.e., within 24 hours), others whose hypoxemia improves gradually and still others whose severe hypoxemia persists for several days. The latter group of patients with persistent severe ARDS poses challenges to clinicians. We attempted to assess the baseline characteristics and outcomes of persistent severe ARDS and to identify which variables are useful to predict it.MethodsA secondary analysis of patient-level data from the ALTA, EDEN and SAILS ARDSNet clinical trials was conducted. We defined persistent severe ARDS as a partial pressure of arterial oxygen to fraction of inspired oxygen ratio (PaO2:FiO2) of equal to or less than 100 mmHg on the second study day following enrollment. Regularized logistic regression with an L1 penalty [Least Absolute Shrinkage and Selection Operator (LASSO)] techniques were used to identify predictive variables of persistent severe ARDS.ResultsOf the 1531 individuals with ARDS alive on the second study day after enrollment, 232 (15%) had persistent severe ARDS. Of the latter, 100 (43%) individuals had mild or moderate hypoxemia at baseline. Usage of vasopressors was greater [144/232 (62%) versus 623/1299 (48%); pConclusionsPatients with persistent severe ARDS have distinct baseline characteristics and poor prognosis. Identifying such patients at enrollment may be useful for the prognostic enrichment of trials.

Background Although GABA is the major inhibitory neurotransmitter in the CNS, quantifying in vivo GABA levels has been challenging. The ability to co-monitor both GABA and the major excitatory neurotransmitter, glutamate, would be a powerful tool in both research and clinical settings. New method Ceramic-based microelectrode arrays (MEAs) were used to quantify gamma-aminobutyric acid (GABA) by employing a dual-enzyme reaction scheme including GABase and glutamate oxidase (GluOx). Glutamate was simultaneously quantified on adjacent recording sites coated with GluOx alone. Endogenous glutamate was subtracted from the combined GABA and glutamate signal to yield a pure GABA concentration. Results Electrode sensitivity to GABA in conventional, stirred in vitro calibrations at pH 7.4 did not match the in vivo sensitivity due to diffusional losses. Non-stirred calibrations in agarose or stirred calibrations at pH 8.6 were used to match the in vivo GABA sensitivity. In vivo data collected in the rat brain demonstrated feasibility of the GABA/glutamate MEA including uptake of locally applied GABA, KCl-evoked GABA release and modulation of endogenous GABA with vigabatrin. Comparison with existing methods Implantable enzyme-coated microelectrode arrays have better temporal and spatial resolution than existing off-line methods. However, interpretation of results can be complicated due to the multiple recording site and dual enzyme approach. Conclusions The initial in vitro and in vivo studies supported that the new MEA configuration may be a viable platform for combined GABA and glutamate measures in the CNS extending the previous reports to in vivo GABA detection. The challenges of this approach are emphasized.

Summary Primary adrenal insufficiency secondary to syphilis is extremely rare, with only five cases being reported in the literature. We report a case of adrenal insufficiency as a manifestation of Treponema pallidum infection (tertiary syphilis). A 69-year-old, previously fit and well Caucasian male was found to have adrenal insufficiency after being admitted with weight loss, anorexia and postural dizziness resulting in a fall. Biochemical testing showed hyponatraemia, hyperkalaemia, and an inadequate response to Synacthen testing, with a peak cortisol level of 302 nmol/L after administration of 250 µg Synacthen. Abdominal imaging revealed bilateral adrenal hyperplasia with inguinal and retroperitoneal lymphadenopathy. He was started on hydrocortisone replacement; however, it was not until he re-attended ophthalmology with a red eye and visual loss 1 month later, that further work-up revealed the diagnosis of tertiary syphilis. Following a course of penicillin, repeat imaging 5 months later showed resolution of the abnormal radiological appearances. However, adrenal function has not recovered and 3 years following initial presentation, the patient remains on both glucocorticoid and mineralocorticoid replacement. In conclusion, this case highlights the importance of considering syphilis as a potential differential diagnosis in patients presenting with adrenal insufficiency and bilateral adrenal masses, given the recent re-emergence of this condition. The relative ease of treating infectious causes of adrenal lesions makes accurate and timely diagnosis crucial. Learning points: Infectious causes, including syphilis, should be excluded before considering adrenalectomy or biopsy for any patient presenting with an adrenal mass. It is important to perform a full infection screen including tests for human immunodeficiency virus, other blood-borne viruses and concurrent sexually transmitted diseases in patients presenting with bilateral adrenal hyperplasia with primary adrenal insufficiency. Awareness of syphilis as a potential differential diagnosis is important, as it not only has a wide range of clinical presentations, but its prevalence has been increasing in recent times.

Two peptides were purified from intestinal extracts of a sea cucumber, Holothuria glaberrima, by high pressure liquid chromatography (HPLC). The peptides were detected by a radioimmunoassay (RIA) based on an antiserum raised to the molluscan peptide, pGlu-Asp-Pro-Phe-Leu-Arg-Phe-NH2 (pQDPFLRFamide). Automated sequencing and mass spectrometry indicate that the isolated peptides are: Gly-Phe-Ser-Lys-Leu-Tyr-Phe-NH2 (GFSKLYFamide) and Ser-Gly-Tyr-Ser-Val-Leu-Tyr-Phe-NH2 (SGYSVLYFamide). These are the first peptides to have been isolated from a member of the echinoderm class Holothuroidea. The antiserum used in the RIA of the peptides was also employed in localizing immunoreactive nerve cells and fibers in the intestine of H. glaberrima. The immunohistochemical results suggest that these peptides might be responsible for the FMRFamide-like immunoreactivity reported earlier. Sequence similarities between GFSKLYFamide, SGYSVLYFamide, and a pair of peptides previously isolated from starfish lead us to propose that all four molecules are members of a family of peptides acting as neurotransmitters in echinoderms.

Five neuropeptides were isolated from CNS extracts of the horseshoe crab Limulus polyphemus by high pressure liquid chromatography (HPLC). The peptides were identified by radioimmunoassays (RIAs) based on two antisera raised to FMRFamide-related peptides (FaRPs). The purified peptides were analyzed by automated sequencing and mass spectrometry, and the following sequences were obtained: DEGHKMLYFamide, GHSLLHFamide, PDHHMMYFamide, DHGNMLYFamide, and GGRSPSLRLRFamide. The first four peptides are members of a novel family with virtually no relationship to FMRFamide. GGRSPSLRLRFamide, on the basis of structural similarity, becomes the second member of a class of FaRPs known previously only from a peptide isolated from mosquito heads. At least one member of the novel family (GHSLLHFamide) inhibits the isolated heart of Limulus.

The SCPs3 are a small peptide family, characterized in gastropods, and implicated in the control of the cardiovascular system and the muscles involved in feeding and gut motility. We aimed to determine the manifestation of this peptide family in the class Bivalvia. Acetone extracts of whole bivalves were fractionated by high pressure liquid chromatography (HPLC), and reactive peaks were identified by radioimmunoassay (RIA). After purification, sequencing, and analysis by mass spectroscopy, three peptides were identified in the clam Mercenaria mercenaria: IAMSFYFPRMamide, AMSFYFPRMamide, and YFAFPRQamide4. SCP-related peptides from two other species were also sequenced: APKYFYFPRMamide and SAFYFPRMamide from an oyster, Crassostrea virginica; and AMSFYFPRMamide (identical to one of the clam peptides) from a cockle, Dinocardium robustum. The tissue distribution and pharmacological actions of the clam SCPs were determined in M. mercenaria, as follows. The levels of peptide in extracts of 12 tissues were estimated by RIA. The largest concentrations of SCP occur in the palps and the visceral ganglia; the levels in the cerebral and pedal ganglia, the rectum, intestinal typhlosole, and gills were substantially lower; and the smallest amounts were found in the heart and the style sac typhlosoles. Immunohistochemistry revealed many cell bodies in the periphery of the ganglia and fibers in the neuropil. Immunoreactive, varicose fibers also occur in the typhlosoles of the intestine and style sac, and in the rectum, gill, and palps. The atrioventricular valves, but not the atria or ventricle proper, contain immunoreactive fibers. Synthetic clam SCPs were assayed on the rectum, the typhlosoles of the intestine and style sac, and the ventricle, all isolated in an organ bath. At low to moderate doses, the SCPs relaxed the muscles of the rectum; higher doses had biphasic actions. The muscles of the intestinal and style sac typhlosoles were relaxed, and spontaneous rhythmicity was slowed by the SCPs. Most ventricles were unresponsive. We conclude that the SCPs isolated in bivalves--though distinctive--are true homologs of those in gastropods. Moreover, the bivalve peptides also serve similar roles, controlling feeding and digestion, and perhaps even cardioactivity.

We have reanalyzed quasielastic neutron scattering data measured for three prototypical ionic liquids characterized by increasingly longer alkyl chains using two standard models of different complexity. The first one contains only two main components representing a long-range translational diffusion process plus a faster localized-like motion. The second model includes an additional component in order to account for the possibility of having more than one type of localized motion with different characteristic time scales. Our results show the complexity of the relaxation landscape of ionic liquids, indicating that even the more complex model provides only a qualitative average picture of such complex dynamics. The comparison of both models allows to asses also the robustness of the parameters obtained from the fits. Finally the results for the three different ionic liquids are compared in order to determine the effect of the length of the alkyl chain in the translational dynamics of ionic liquids, showing a clear decrease in diffusivity when passing from the ethyl (n=2) to the butyl (n=4) alkyl chain, but a much smaller effect when adding two extra C atoms to the chain.We have reanalyzed quasielastic neutron scattering data measured for three prototypical ionic liquids characterized by increasingly longer alkyl chains using two standard models of different complexity. The first one contains only two main components representing a long-range translational diffusion process plus a faster localized-like motion. The second model includes an additional component in order to account for the possibility of having more than one type of localized motion with different characteristic time scales. Our results show the complexity of the relaxation landscape of ionic liquids, indicating that even the more complex model provides only a qualitative average picture of such complex dynamics. The comparison of both models allows to asses also the robustness of the parameters obtained from the fits. Finally the results for the three different ionic liquids are compared in order to determine the effect of the length of the alkyl chain in the translational dynamics of ionic liquids, showing...

This research was supported by Silver Lake Resources through an Honours project by S. E. Hull and through ongoing research as part of ARC Linkage Project LP110200747.

BackgroundRegular use of inhaled corticosteroids (ICS) in patients with obstructive lung diseases has been associated with a higher risk of pneumonia, particularly in COPD. The risk of pneumonia has not been previously evaluated in relation to ICS particle size and dose used.MethodsHistorical cohort, UK database study of 23,013 patients with obstructive lung disease aged 12-80 years prescribed extra-fine or fine-particle ICS. The endpoints assessed during the outcome year were diagnosis of pneumonia, acute exacerbations and acute respiratory events in relation to ICS dose. To determine the association between ICS particle size, dose and risk of pneumonia in unmatched and matched treatment groups, logistic and conditional logistic regression models were used.Results14788 patients were stepped-up to fine-particle ICS and 8225 to extra-fine ICS. On unmatched analysis, patients stepping-up to extra-fine ICS were significantly less likely to be coded for pneumonia (adjusted odds ratio [aOR] 0.60; 95% CI 0.37, 0.97]); experience acute exacerbations (adjusted risk ratio [aRR] 0.91; 95% CI 0.85, 0.97); and acute respiratory events (aRR 0.90; 95% CI 0.86, 0.94) compared with patients stepping-up to fine-particle ICS. Patients prescribed daily ICS doses in excess of 700 mcg (fluticasone propionate equivalent) had a significantly higher risk of pneumonia (OR [95%CI] 2.38 [1.17, 4.83]) compared with patients prescribed lower doses, irrespective of particle size.ConclusionsThese findings suggest that patients with obstructive lung disease on extra-fine particle ICS have a lower risk of pneumonia than those on fine-particle ICS, with those receiving higher ICS doses being at a greater risk.

This work was supported by the European Community Seventh Framework Program (FP7/2007-2013) [grant agreement number 608490] (project M4CO2). A.O. would like to acknowledge the SCI for awarding a scholarship for her PhD research. G.M. thanks Institut Universitaire de France for its support. S.S. would like to thank the EPSRC for support EP/M506631/1. S.E.A would like to thank the Royal Society and Wolfson Foundation for a merit award. Polymorphs of Zn(2-nIm)2 (compound 1 ) and Co(2-nIm)2 (compounds 2 and 3 ) (2-nIm = 2-nitroimidazole) have been prepared by two routes: solvothermal synthesis and recrystallisation of ZIF-65(Zn/Co). Compounds 1 and 2 are isostructural, with a tetrahedrally-connected framework topology related to, but different from, that of tridymite (lonsdaleite). Single crystal X-ray diffraction analysis showed that in compound 1 (Pccn, Z = 8; a = 8.462(8) Å, b = 14.549(15) Å, c = 18.799(18) Å, V = 2314(4) Å3) there is rotational disorder for two of the three crystallographically-distinct linker types, which has been investigated computationally and by solid-state NMR spectroscopy. Detailed adsorption studies on a sample of 1 prepared by recrystallisation show 1.1 mmol g-1 uptake of CO2 at 0.1 bar (25 °C) with high affinity for CO2 over CH4 and N2 (adsorption enthalpies of 39.5, 26.0 and 18.5 kJ mol-1, respectively). A cobalt analogue (compound 2 ) with improved water stability (but lower porosity) has also been prepared. Changing the conditions of synthesis and recrystallisation gives rise to a cobalt 2-nitroimidazolate (Co(2-nIm)2, compound 3 ), which has a layered structure (I41/amd, a = 6.025(18) Å, c = 26.95(8) Å, V = 978.3(5) Å3) containing sheets of tetrahedrally-connected Co2+ cations composed of four membered rings, without porosity. Postprint Postprint

We used the aerodynamic levitation technique combined with CO2 laser heating to study the structure of liquid yttrium aluminates above their melting point with neutron diffraction. For various yttria contents, we determined the structure factors and corresponding pair correlation functions describing the short-range order in the liquids. In particular, we derived Al–O and Y–O bond distances and coordination numbers. Experimental data are compared with ab initio molecular dynamics, carried out using the VASP code where the interatomic forces are obtained from density functional theory. In particular, partial pair correlation functions have been calculated and are in relatively good agreement with the experimental observations.

The influence of water on the relaxation of a prototype ionic liquid (IL) C8mimBF4 is examined in the IL-rich regime combining quasi-elastic neutron scattering (QENS) and molecular dynamics (MD) simulations. The QENS and MD simulations results for relaxation of IL and the equimolar mixture with water probed by the dynamics of the C8mim hydrogen atoms in the time range of 2 ps to 1 ns are in excellent agreement. The QENS data show that translational relaxation increases by a factor of 7 on the addition of water, while rotational relaxation involving multiple processes fitted by a KWW function with low β values is speeded up by a factor of 3 on the time scale of QENS measurements. The MD simulations show that the cation diffusion coefficient, inverse viscosity, and ionic conductivity increase on the addition of water, consistent with the very small change in ionicity. The difficulties in obtaining rotational and translational diffusion coefficients from fits to QENS experiments of pure ILs and IL–water mixt...

Process design studies are described for a new type of VOC recovery system which uses microwave heating to regenerate adsorbents. Microwave regeneration systems create a highly concentrated effluent from which the VOCs can be recovered by condensation at near-ambient temperatures. Important design considerations, predicated on experimental work and model development, are identified and discussed. Parametric studies are then described that identify the optimal adsorbent selection, operating cycle, recovery configuration, regeneration pressure, regeneration final coverage, and column configuration. In general, it was found that microwave regenerated adsorption systems favor the use of low dielectric loss-factor polymeric adsorbents and operation under low pressure conditions (about 5 torr absolute pressure).

Microwave regeneration of adsorbents facilitates the recovery of volatile organic compounds (VOCs) by decoupling the bed heating from the stripping gas. This makes possible the creation of a highly-concentrated regeneration effluent from which the VOCs can be recovered by condensation at near-ambient temperatures. The economic feasibility of two novel microwave-regenerated adsorption systems was evaluated by systematically comparing the capital and operating costs of the proposed systems with 10 conventional VOC control technologies. The microwave systems were found to have similar capital and operating costs to conventional steam regeneration systems and, therefore, may present an attractive alternative for recovering water-miscible solvents. In general, the cost of the microwave subsystem is a relatively small component of the overall system costs, and the microwave power requirements are within the range of commercially available generators, even for large emission streams.

Capping of nascent RNA polymerase II (Pol II) transcripts is required for gene expression and the first two steps are catalyzed by separate 5' triphosphatase and guanylyltransferase activities of the human capping enzyme (HCE). The cap is added co-transcriptionally, but how the two activities are coordinated is unclear. Our previous in vitro work has suggested that an unidentified factor modulates the minimum length at which nascent transcripts can be capped. Using the same well-established in vitro system with hydrogen peroxide as a capping inhibitor, we show that this unidentified factor targets the guanylyltransferase activity of HCE. We also uncover the mechanism of HCE inhibition by hydrogen peroxide, and by using mass spectrometry demonstrate that the active site cysteine residue of the HCE triphosphatase domain becomes oxidized. Using recombinant proteins for the two separated HCE domains, we provide evidence that the triphosphatase normally acts on transcripts shorter than can be acted upon by the guanylyltransferase. Our further characterization of the capping reaction dependence on transcript length and its interaction with the unidentified modulator of capping raises the interesting possibility that the capping reaction could be regulated.

The structure of the nanoporous metal–organic framework Cu3(BTC)2 (BTC = 1,3,5-benzenetricarboxylate) with a variety of molecular guests was studied in situ using single crystal X-ray diffraction. By collecting crystal structure data for a series of guests within the same host crystal, insights into the molecular interactions underpinning guest adsorption processes have been gained. Adsorption behaviors are influenced strongly by both enthalpic and entropic thermodynamic, as well as interpore steric (size-exclusion) effects, and we note correlations between guest attributes and these effects. Vapor adsorption measurements revealed a guest uptake capacity inversely proportional to guest size. Correspondingly, structural results show that guests reside in the smallest pores accessible to them. Interpore steric effects for larger guests cause these to be excluded from the smallest pores, and this corresponds to lower total uptake. Both hydrophilic and lipophilic small guests adsorb favorably into the 5 A dia...

Phosphorylation of the C-terminal domain (CTD) of the large subunit of human RNA polymerase II (Pol II) is regulated during the transcription cycle by the combined action of specific kinases and phosphatases. Pol II enters into the preinitiation complex (PIC) unphosphorylated, but is quickly phosphorylated by Cdk7 during initiation. How phosphatases alter the pattern and extent of CTD phosphorylation at this early stage of transcription is not clear. We previously demonstrated the functional association of an early-acting, magnesium-independent phosphatase with early elongation complexes. Here we show that Ssu72 is responsible for that activity. We found that the phosphatase enters the transcription cycle during the formation of PICs and that Ssu72 is physically associated with very early elongation complexes. The association of Ssu72 with elongation complexes was stable to extensive washing with up to 200 mM KCl. Interestingly, Ssu72 ceased to function on complexes that contained RNA longer than 28 nt. However, when PICs were washed before initiation, the strict cutoff at 28 nt was lost. This suggests that factor(s) are important for the specific regulation of Ssu72 function during the transition between initiation and pausing. Overall, our results demonstrate when Ssu72 can act on early transcription complexes and suggest that Ssu72 may also function in the PIC prior to initiation.

It is pointed out that hydrogen/deuterium substitution, frequently used in neutron scattering studies of the structure and dynamics of hydrogenous samples, can have significant effects on structure, phase behavior and protein stability. The effects must be kept in mind in the interpretation of such experiments. In suitable cases, these effects can be mitigated by an appropriate choice of experimental conditions.

The influence of water on the structure of a prototype ionic liquid (IL) 1-octyl-3-methylimidazolium tetrafluoroborate (C8mimBF4) is examined in the IL-rich regime using high-energy X-ray diffraction (HEXRD) and molecular dynamics (MD) simulations. A many-body polarizable force field APPLE&P was developed for C8mimBF4-water mixture. It predicts structure factors of pure IL and IL-water mixture in excellent agreement with the HEXRD experiments. The MD results provide detailed insights into the structural changes from the partial structure factors, 2-D projections of the simulation box and 3-D distribution functions. Water partitioning with IL and its competition with BF4(-) for complexing the imidazolium rings was examined. The added water molecules occupy a diffuse coordination shell around the imidazolium ring but are not present around the alkyl tail. The strong coordination of the fluorine atoms of the BF4(-) anions to the imidazolium ring is not significantly changed by the addition of water. A complementary packing of water and imidazolium around BF4(-) was found. These results are consistent with the very small differences in the average structure between the pure IL and the mixture.

An important function of the fat body in adult female mosquitoes is the conversion of blood meal derived amino acids (AA) into massive amounts of yolk protein precursors. A highly efficient transport mechanism for AAs across the plasma membrane of the fat body trophocytes is essential in order to deliver building blocks for the rapid synthesis of large amounts of these proteins. This mechanism consists in part of AA transporter proteins from the solute carrier family. These transporters have dual function; they function as transporters and participate in the nutrient signal transduction pathway that is activated in the fat body after a blood meal. In this study we focused on the solute carrier 7 family (SLC7), a family of AA transporters present in all metazoans that includes members with strong substrate specificity for cationic AAs.We identified 11 putative SLC7 transporters in the genome sequence of Aedes aegypti. Phylogenetic analysis puts five of these in the cationic AA transporter subfamily (CAT) and six in the heterodimeric AA transporter (HAT) subfamily. All 11 A. aegypti SLC7 genes are expressed in adult females. Expression profiles are dynamic after a blood meal. We knocked down six fat body-expressed SLC7 transporters using RNAi and found that these 'knockdowns' reduced AA-induced TOR signaling. We also determined the effect these knockdowns had on the number of eggs deposited following a blood meal.Our analysis stresses the importance of SLC7 transporters in TOR signaling pathway and mosquito reproduction.

Our previous work has correlated permanent alterations in the rat neurosecretory machinery with epileptogenesis. Such findings highlighted the need for a greater understanding of the molecular mechanisms underlying epilepsy so that novel therapeutic regimens can be designed. To this end, we examined kindling in transgenic mice with a defined reduction of a key element of the neurosecretory machinery: the v-SNARE (vesicle-bound SNAP [soluble NSF attachment protein] receptor), synaptobrevin/vesicle-associated membrane protein 2 (VAMP2). Initial analysis of biochemical markers, which previously displayed kindling-dependent alterations in rat hippocampal synaptosomes, showed similar trends in both wild-type and VAMP2(+/-) mice, demonstrating that kindled rat and mouse models are comparable. This report focuses on the effects that a ~50% reduction of synaptosomal VAMP2 has on the progression of electrical kindling and on glutamate release in hippocampal subregions. Our studies show that epileptogenesis is dramatically attenuated in VAMP2(+/-) mice, requiring both higher current and more stimulations to reach a fully kindled state (two successive Racine stage 5 seizures). Progression through the five identifiable Racine stages was slower and more variable in the VAMP2(+/-) animals compared with the almost linear progression seen in wild-type littermates. Consistent with the expected effects of reducing a major neuronal v-SNARE, glutamate-selective, microelectrode array (MEA) measurements in specific hippocampal subregions of VAMP2(+/-) mice showed significant reductions in potassium-evoked glutamate release. Taken together these studies demonstrate that manipulating the levels of the neurosecretory machinery not only affects neurotransmitter release but also mitigates kindling-induced epileptogenesis.

Anautogenous mosquitoes depend on vertebrate blood as nutrient source for their eggs. A highly efficient set of membrane transporters mediates the massive movement of nutrient amino acids between mosquito tissues after a blood meal. Here we report the characterization of the amino-acid transporter Slimfast (Slif) from the yellow-fever mosquito Aedes aegypti using codon-optimized heterologous expression. Slif is a well-known component of the target-of-rapamycin signalling pathway and fat body nutrient sensor, but its substrate specificity and transport mechanism were unknown. We found that Slif transports essential cationic and neutral amino acids with preference for arginine. It has an unusual dual-affinity mechanism with only the high affinity being Na+ dependent. Tissue-specific expression and blood meal-dependent regulation of Slif are consistent with conveyance of essential amino acids from gut to fat body. Slif represents a novel transport system and type of transceptor for sensing and transporting essential amino acids during mosquito reproduction., Anautogenous mosquitoes need to obtain essential amino acids from a blood meal for reproduction. Here, the authors examine the amino acid transporter Slimfast from the yellow-fever mosquito and describe both its specificity and mechanism of action.

Because of a preconceived notion that eliminating reactive metabolite (RM) formation with new drug candidates could mitigate the risk of idiosyncratic drug toxicity, the potential for RM formation is routinely examined as part of lead optimization efforts in drug discovery. Likewise, avoidance of "structural alerts" is almost a norm in drug design. However, there is a growing concern that the perceived safety hazards associated with structural alerts and/or RM screening tools as standalone predictors of toxicity risks may be over exaggerated. In addition, the multifactorial nature of idiosyncratic toxicity is now well recognized based upon observations that mechanisms other than RM formation (e.g., mitochondrial toxicity and inhibition of bile salt export pump (BSEP)) also can account for certain target organ toxicities. Hence, fundamental questions arise such as: When is a molecule that contains a structural alert (RM positive or negative) a cause for concern? Could the molecule in its parent form exert toxicity? Can a low dose drug candidate truly mitigate metabolism-dependent and -independent idiosyncratic toxicity risks? In an effort to address these questions, we have retrospectively examined 68 drugs (recalled or associated with a black box warning due to idiosyncratic toxicity) and the top 200 drugs (prescription and sales) in the United States in 2009 for trends in physiochemical characteristics, daily doses, presence of structural alerts, evidence for RM formation as well as toxicity mechanism(s) potentially mediated by parent drugs. Collectively, our analysis revealed that a significant proportion (∼78-86%) of drugs associated with toxicity contained structural alerts and evidence indicating that RM formation as a causative factor for toxicity has been presented in 62-69% of these molecules. In several cases, mitochondrial toxicity and BSEP inhibition mediated by parent drugs were also noted as potential causative factors. Most drugs were administered at daily doses exceeding several hundred milligrams. There was no obvious link between idiosyncratic toxicity and physicochemical properties such as molecular weight, lipophilicity, etc. Approximately half of the top 200 drugs for 2009 (prescription and sales) also contained one or more alerts in their chemical architecture, and many were found to be RM-positive. Several instances of BSEP and mitochondrial liabilities were also noted with agents in the top 200 category. However, with relatively few exceptions, the vast majority of these drugs are rarely associated with idiosyncratic toxicity, despite years of patient use. The major differentiating factor appeared to be the daily dose; most of the drugs in the top 200 list are administered at low daily doses. In addition, competing detoxication pathways and/or alternate nonmetabolic clearance routes provided suitable justifications for the safety records of RM-positive drugs in the top 200 category. Thus, while RM elimination may be a useful and pragmatic starting point in mitigating idiosyncratic toxicity risks, our analysis suggests a need for a more integrated screening paradigm for chemical hazard identification in drug discovery. Thus, in addition to a detailed assessment of RM formation potential (in relationship to the overall elimination mechanisms of the compound(s)) for lead compounds, effects on cellular health (e.g., cytotoxicity assays), BSEP inhibition, and mitochondrial toxicity are the recommended suite of assays to characterize compound liabilities. However, the prospective use of such data in compound selection will require further validation of the cellular assays using marketed agents. Until we gain a better understanding of the pathophysiological mechanisms associated with idiosyncratic toxicities, improving pharmacokinetics and intrinsic potency as means of decreasing the dose size and the associated "body burden" of the parent drug and its metabolites will remain an overarching goal in drug discovery.

The metal-organic host material [{Co(III)(2)(bpbp)(O(2))}(2)bdc](PF(6))(4) (1·2O(2); bpbp(-) = 2,6-bis(N,N-bis(2-pyridylmethyl)aminomethyl)-4-tert-butylphenolato; bdc(2-) = 1,4-benzenedicarboxylato) displays reversible chemisorptive desorption and resorption of dioxygen through conversion to the deoxygenated Co(II) form [{Co(II)(2)(bpbp)}(2)bdc](PF(6))(4) (1). Single crystal X-ray diffraction analysis indicates that the host lattice 1·2O(2), achieved through desorption of included water guests from the as-synthesized phase 1·2O(2)·3H(2)O, consists of an ionic lattice containing discrete tetranuclear complexes, between which lie void regions that allow the migration of dioxygen and other guests. Powder X-ray diffraction analyses indicate that the host material retains crystallinity through the dioxygen desorption/chemisorption processes. Dioxygen chemisorption measurements on 1 show near-stoichiometric uptake of dioxygen at 5 mbar and 25 °C, and this capacity is largely retained at temperatures above 100 °C. Gas adsorption isotherms of major atmospheric gases on both 1 and 1·2O(2) indicate the potential suitability of this material for air separation, with a O(2)/N(2) selectivity factor of 38 at 1 atm. Comparison of oxygen binding in solution and in the solid state indicates a dramatic increase in binding affinity to the complex when it is incorporated in a porous solid.

We present results of parallel quasielastic neutron scattering (QENS) experiments and molecular dynamics numerical simulations for the dynamics of a prototype ionic liquid, 1-ethyl-3-methyl-imidazolium bromide. Differences and similarities with those from the crystal phase are also discussed. Both experiment and simulation demonstrate that, in the length and time scales being probed here (fractions of a nm and a few ps), the dynamics are dominated by activated translational diffusion in the liquid phase and reorientations of the ethyl groups in both solid and liquid.

The reactions between Ln(NO 3 ) 3 ·nH 2 O and (Bu 3 NH) 3 [W-(CN) 8 ]·H 2 O have led to two series of octacyanometallate-based complexes: Ln(H 2 C) 5 [W(CN) 8 ] [Ln = La(1), Pr(2), Nd(3), Eu(4), Gd(5)] and Ln(H 2 O) 4 [W(CN) 8 ] [Ln = Ho(6), Er(7), Tm(8), Lu(9)]. The crystal structures of 1-9 have two-dimensional corrugated layers in which the Ln III and W V centres are linked in an alternating fashion. Thermogravimetric (TG) and powder XRD results reveal the presence of a phase transformation in the Ln III -W V system with increasing atomic number of the Ln III atoms.

Although Lepomis species are abundant in a wide variety of habitats throughout North America and could serve as potentially valuable biomonitoring tools, few studies have examined the induction of pollutant biomarkers in this genus. We hypothesized that the induction of cytochrome P-450 1A (CYP1A), a sensitive and widely used indicator of response to aquatic contaminants, would serve as an effective biomarker of organic pollutant exposure in Lepomis species. We examined the response of CYP1A and two of the major pollutant-responsive phase II enzymes, glutathione S-transferase (GST), and uridine diphosphate glucuronyltransferase (UDPGT), in Lepomis exposed to organic pollutants under laboratory and field conditions. Two Lepomis species (longear sunfish, Lepomis megalottis and bluegill, Lepomis macrochirus) were exposed in the laboratory via intraperitoneal injection to corn oil (vehicle), benzo(a)pyrene (BaP) (10 and 50 mg/kg), a polynuclear aromatic hydrocarbon (PAH) or 3,4,3′,4′-tetrachlorobiphenyl (PCB 77) (0.1 and 1.0 mg/kg), a dioxin-like planar halogenated aromatic hydrocarbon (HAH), and sacrificed 2 (BaP) or 7 (corn oil, PCB77) days later. Lepomis hepatic CYP1A exhibited differential sensitivity to these two classes of environmental contaminants. CYP1A activity was weakly induced in bluegill exposed to 1.0 mg/kg PCB 77 (3 fold induction over controls) but strongly induced in both bluegill and longear sunfish exposed to 50 mg/kg BaP (37 and 15 fold induction over controls, respectively). In contrast, hepatic GST activity in both species remained unchanged following the treatment with either compound and hepatic UDPGT activity, which was assessed only in BaP-treated longear sunfish, was unaffected by that chemical, indicating these phase II enzymes may not be sensitive pollutant biomarkers in this genus. Further, longear sunfish collected from a PCB contaminated site displayed relatively low levels of CYP1A activity despite PCB body burdens associated with strong induction of CYP1A activity in other fish species. The strong induction of CYP1A by BaP with much weaker CYP1A response to PCB indicates that CYP1A in Lepomis sp. could be an excellent biomarker for PAH pollution, but may not be a reliable indicator of site contamination by halogenated hydrocarbons. We conclude that Lepomis species provide a useful model for examining the regulation and potential consequences of differential pollutant sensitivity, but that CYP1A in these species should be used with caution as an indicator of halogenated contaminants.

Neutron diffraction with isotopic substitution (NDIS) experiments have been used to examine the structuring of aqueous solutions of pyridine. A new method is described for extracting the structure factors relating to intermolecular correlations from neutron scattering experiments on liquid solutions of complex molecular species. This approach performs experiments at different concentrations and exploits the intramolecular coordination number concentration invariance (ICNCI) to separate the internal and intermolecular contributions to the total intensities. The ability of this method to deconvolute molecular and intermolecular correlations is tested and demonstrated using simulated neutron scattering results predicted from molecular dynamics simulations of aqueous solutions of the polyatomic solute pyridine in which the inter- and intramolecular terms are known. The method is then implemented using neutron scattering measurements on solutions of pyridine. The results confirm that pyridine shows a significant propensity to aggregate in solution and demonstrate the prospects for the future application of the ICNCI approach to the study of large polyatomic solutes using next-generation neutron sources and detectors.

The statistical copolymerization of methyl methacrylate (MMA) with varying amounts of a disulfide-based dimethacrylate (DSDMA) branching comonomer in toluene at 90 °C can lead to highly branched soluble methacrylic copolymers under appropriate conditions. This model system is utilized in order to examine the following points: (i) the relative merits of using reversible addition−fragmentation chain transfer (RAFT) polymerization and atom transfer radical polymerization (ATRP) in such syntheses; (ii) the effect of varying the number of DSDMA units per primary chain; (iii) the effect of varying the initial monomer concentration. Kinetic studies of the linear RAFT and ATRP homopolymerizations conducted in the absence of any DSDMA confirmed their living character at 10, 30, and 50 wt % [MMA]0, where the former monomer concentration approximately corresponds to the critical overlap concentration, c*, for linear poly(methyl methacrylate) (PMMA) chains with a mean degree of polymerization of 50. HPLC analysis of ...