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Mafalda Ramos,1 John Haughney,2 Nathaniel Henry,3 Leandro Lindner,4 Mark Lamotte1 1Real World Evidence, IMS Health, Zaventem, Belgium; 2Academic Primary Care Division of Applied Health Sciences, University of Aberdeen, Aberdeen, Scotland, 3Health Economics and Outcomes Research, Real World Evidence, IMS Health, London, UK; 4AstraZeneca, Barcelona, Spain Purpose: Aclidinium–formoterol 400/12 µg is a long-acting muscarinic antagonist (LAMA) and a long-acting β2-agonist in a fixed-dose combination used in the management of patients with COPD. This study aimed to assess the cost-effectiveness of aclidinium–formoterol 400/12 µg against the long-acting muscarinic antagonist aclidinium bromide 400 µg.Materials and methods: A five-health-state Markov transition model with monthly cycles was developed using MS Excel to simulate patients with moderate-to-severe COPD and their initial lung-function improvement following treatment with aclidinium–formoterol 400/12 µg or aclidinium 400 µg. Health states were based on severity levels defined by Global Initiative for Chronic Obstructive Lung Disease 2010 criteria. The analysis was a head-to-head comparison without step-up therapy, from the NHS Scotland perspective, over a 5-year time horizon. Clinical data on initial lung-function improvement were provided by a pooled analysis of the ACLIFORM and AUGMENT trials. Management, event costs, and utilities were health state-specific. Costs and effects were discounted at an annual rate of 3.5%. The outcome of the analysis was expressed as cost (UK£) per quality-adjusted life-year (QALY) gained. The analysis included one way and probabilistic sensitivity analyses to investigate the impact of parameter uncertainty on model outputs.Results: Aclidinium–formoterol 400/12 µg provided marginally higher costs (£41) and more QALYs (0.014), resulting in an incremental cost-effectiveness ratio of £2,976/QALY. Sensitivity analyses indicated that results were robust to key parameter variations, and the main drivers were: mean baseline forced expiratory volume in 1 second (FEV1), risk of exacerbation, FEV1 improvement from aclidinium–formoterol 400/12 µg, and lung-function decline. The probability of aclidinium–formoterol 400/12 µg being cost-effective (using a willingness-to-pay threshold of £20,000/QALY) versus aclidinium 400 µg was 79%.Conclusion: In Scotland, aclidinium–formoterol 400/12 µg can be considered a cost-effective treatment option compared to aclidinium 400 µg alone in patients with moderate-to-severe COPD. Keywords: cost-effectiveness, Scotland, Markov model, lung-function improvement, LABA, LAMA

Serial femtosecond crystallography using X-ray free-electron lasers has huge potential for time-resolved structural experiments. Here, the authors present a structure of the light-driven proton pump bacteriorhodopsin using these techniques.

The possibility of using femtosecond pulses from an X-ray free-electron laser to collect diffraction data from protein crystals formed in their native cellular organelle has been explored. X-ray diffraction of submicrometre-sized alcohol oxidase crystals formed in peroxisomes within cells of genetically modified variants of the methylotrophic yeast Hansenula polymorpha is reported and characterized. The observations are supported by synchrotron radiation-based powder diffraction data and electron microscopy. Based on these findings, the concept of in cellulo serial crystallography on protein targets imported into yeast peroxisomes without the need for protein purification as a requirement for subsequent crystallization is outlined.

Cross coupling reactions of organolithiums are challenging due to their high reactivity, though palladium catalysed versions have been reported. Here, the authors report the cross coupling of organolithiums with aryl halides with inexpensive and widely available iron catalysts.

Intensity interferometry is a well known method in astronomy. Recently, a related method called incoherent diffractive imaging (IDI) was proposed to apply intensity correlations of x-ray fluorescence radiation to determine the 3D arrangement of the emitting atoms in a sample. Here we discuss inherent sources of noise affecting IDI and derive a model to estimate the dependence of the signal to noise ratio (SNR) on the photon counts per pixel, the temporal coherence (or number of modes), and the shape of the imaged object. Simulations in two- and three-dimensions have been performed to validate the predictions of the model. We find that contrary to coherent imaging methods, higher intensities and higher detected counts do not always correspond to a larger SNR. Also, larger and more complex objects generally yield a poorer SNR despite the higher measured counts. The framework developed here should be a valuable guide to future experimental design.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

ABSTRACT The role of protists and bacteriophages in bacterial predation in the microbial food web has been well studied. There is mounting evidence that Bdellovibrio and like organisms (BALOs) also contribute to bacterial mortality and, in some cases, more so than bacteriophages. A full understanding of the ecologic function of the microbial food web requires recognition of all major predators and the magnitude of each predator’s contribution. Here we investigated the contribution of Halobacteriovorax, one of the BALOs, and bacteriophages when incubated with their common prey, Vibrio vulnificus, in a seawater microcosm. We observed that Halobacteriovorax was the greatest responder to the prey, increasing 18-fold with a simultaneous 4.4-log-unit reduction of V. vulnificus at 40 h, whereas the bacteriophage population showed no significant increase. In subsequent experiments to formulate a medium that would support the predatory activities and replication of both predators, low-nutrient media favored the predation and replication of the Halobacteriovorax, whereas higher-nutrient media enhanced phage growth. The greatest prey reduction and replication of both Halobacteriovorax and phage were observed in media with moderate nutrient levels. Additional experiments show that the predatory activities of both predators were influenced by environmental conditions, specifically, temperature and salinity. The two predators combined exerted greater control on V. vulnificus, a synergism that may be exploited for practical applications to reduce bacterial populations. These findings suggest that along with bacteriophage and protists, Halobacteriovorax has the potential to have a prominent role in bacterial mortality and cycling of nutrients, two vital ecologic functions. IMPORTANCE Although much has been reported about the marine microbial food web and the role of micropredators, specifically viruses and protists, the contribution of Bdellovibrio-like predators has largely been ignored, posing a major gap in understanding food web processes. A complete scenario of the microbial food web cannot be developed until the roles of all major micropredators and the magnitude of their contributions to bacterial mortality, structuring of microbial communities, and cycling of nutrients are assessed. Here we show compelling evidence that Halobacteriovorax, a predatory bacterium, is a significant contributor to bacterial death and, in some cases, may rival viruses as agents of bacterial mortality. These results advance current understanding of the microbial loop and top-down control on the bacterial community.

The high frequency of treatment-related side effects for men with localized prostate cancer creates uncertainty for treatment outcomes. This study assessed the comparative effectiveness of treatment-related side effects associated with conservative management and cryotherapy in patients with localized prostate cancer. A retrospective longitudinal cohort study was conducted, using the linked data of the Surveillance, Epidemiology, and End Results and Medicare, which included patients diagnosed from 2000 through year 2013, and their Medicare claims information from 2000 through 2014. To compare the differences in baseline characteristics and treatment-related side effects between the study cohorts, χ 2 tests were conducted. Multivariate logistic regression was used to assess the association between treatment selection and side effects. There were 7,998 and 3,051 patients in the conservative management and cryotherapy cohort, respectively. The likelihood of erectile dysfunction, lower urinary tract obstruction, urinary fistula, urinary incontinence, and hydronephrosis was reported to be significantly lower (53%, 35%, 69%, 65%, and 36%, respectively) in the conservative management cohort. Conservative management had a lower likelihood of treatment-related side effects compared to cryotherapy. However, further research is needed to compare other significant long-term outcomes such as costs associated with these treatment choices and quality of life.

Background In statin trials, men and women derived similar relative risk reductions in cardiovascular events per 39 mg/dL low‐density lipoprotein cholesterol (LDL‐C) reduction. We explored whether lower LDL‐C levels and greater LDL‐C percentage reductions than those achieved with statins are associated with reduced major adverse cardiovascular event (MACE) rates in women as well as men. Methods and Results Data pooled from 10 phase 3 ODYSSEY randomized trials (n=4983) comparing alirocumab with control (placebo/ezetimibe) were assessed for association between 39 mg/dL lower on‐treatment LDL‐C and percentage LDL‐C change from baseline, and MACE risk by sex, using multivariable Cox regression. Mean baseline LDL‐C was 135 mg/dL (women) and 121 mg/dL (men). Average on‐treatment LDL‐C levels with alirocumab, ezetimibe, and placebo were 71, 114, and 134 mg/dL, respectively, in women (n=1882) and 52, 93, and 122 mg/dL, respectively, in men (n=3090). Overall, 36.5% and 58.7% of women and men, respectively, achieved on‐treatment LDL‐C

Sarcoidosis is an inflammatory process of unknown etiology, characterized by noncaseating granulomas. Isolated extrapulmonary disease is rare. We present a case of a 60-year-old woman with chronically elevated alkaline phosphatase. Upon obtaining a liver biopsy, granulomatous hepatitis was observed, suggestive of sarcoidosis. No particular treatment was initiated, and 3 years following the onset of elevated alkaline phosphatase, her levels decreased spontaneously.

X-ray free-electron lasers (XFELs) show great promise for macromolecular structure determination from sub-micrometre-sized crystals, using the emerging method of serial femtosecond crystallography. The extreme brightness of the XFEL radiation can multiply ionize most, if not all, atoms in a protein, causing their scattering factors to change during the pulse, with a preferential `bleaching' of heavy atoms. This paper investigates the effects of electronic damage on experimental data collected from a Gd derivative of lysozyme microcrystals at different X-ray intensities, and the degree of ionization of Gd atoms is quantified from phased difference Fourier maps. A pattern sorting scheme is proposed to maximize the ionization contrast and the way in which the local electronic damage can be used for a new experimental phasing method is discussed.

Mix-and-inject serial crystallography (MISC) is a technique designed to image enzyme catalyzed reactions in which small protein crystals are mixed with a substrate just prior to being probed by an X-ray pulse. This approach offers several advantages over flow cell studies. It provides (i) room temperature structures at near atomic resolution, (ii) time resolution ranging from microseconds to seconds, and (iii) convenient reaction initiation. It outruns radiation damage by using femtosecond X-ray pulses allowing damage and chemistry to be separated. Here, we demonstrate that MISC is feasible at an X-ray free electron laser by studying the reaction of M. tuberculosis ß-lactamase microcrystals with ceftriaxone antibiotic solution. Electron density maps of the apo-ß-lactamase and of the ceftriaxone bound form were obtained at 2.8 Å and 2.4 Å resolution, respectively. These results pave the way to study cyclic and non-cyclic reactions and represent a new field of time-resolved structural dynamics for numerous substrate-triggered biological reactions.

Background. Prognostic significance of serum uric acid (SUA) in acute stroke still remains controversial. Objectives. To determine the prevalence of hyperuricemia and its association with outcome of stroke patients in the Douala General Hospital (DGH). Methods. This was a hospital based prospective cohort study which included acute stroke patients with baseline SUA levels and 3-month poststroke follow-up data. Associations between high SUA levels and stroke outcomes were analyzed using multiple logistic regression and survival analysis (Cox regression and Kaplan-Meier). Results. A total of 701 acute stroke patients were included and the prevalence of hyperuricemia was 46.6% with a mean SUA level of 68.625 ± 24 mg/l. Elevated SUA after stroke was associated with death (OR = 2.067; 95% CI: 1.449–2.950; p < 0.001) but did not predict this issue. However, an independent association between increasing SUA concentration and mortality was noted in a Cox proportional hazards regression model (adjusted HR = 1.740; 95% CI: 1.305–2.320; p < 0.001). Furthermore, hyperuricemia was an independent predictor of poor functional outcome within 3 months after stroke (OR = 2.482; 95% CI: 1.399–4.404; p = 0.002). Conclusion. The prevalence of hyperuricemia in black African stroke patients is quite high and still remains a predictor of poor outcome.

CTB-MPR is a fusion protein between the B subunit of cholera toxin (CTB) and the membrane-proximal region of gp41 (MPR), the transmembrane envelope protein of Human immunodeficiency virus 1 (HIV-1), and has previously been shown to induce the production of anti-HIV-1 antibodies with antiviral functions. To further improve the design of this candidate vaccine, X-ray crystallography experiments were performed to obtain structural information about this fusion protein. Several variants of CTB-MPR were designed, constructed and recombinantly expressed in Escherichia coli. The first variant contained a flexible GPGP linker between CTB and MPR, and yielded crystals that diffracted to a resolution of 2.3 Å, but only the CTB region was detected in the electron-density map. A second variant, in which the CTB was directly attached to MPR, was shown to destabilize pentamer formation. A third construct containing a polyalanine linker between CTB and MPR proved to stabilize the pentameric form of the protein during purification. The purification procedure was shown to produce a homogeneously pure and monodisperse sample for crystallization. Initial crystallization experiments led to pseudo-crystals which were ordered in only two dimensions and were disordered in the third dimension. Nanocrystals obtained using the same precipitant showed promising X-ray diffraction to 5 Å resolution in femtosecond nanocrystallography experiments at the Linac Coherent Light Source at the SLAC National Accelerator Laboratory. The results demonstrate the utility of femtosecond X-ray crystallography to enable structural analysis based on nano/microcrystals of a protein for which no macroscopic crystals ordered in three dimensions have been observed before.

A new approach for collecting data from many hundreds of thousands of microcrystals using X-ray pulses from a free-electron laser has recently been developed. Referred to as serial crystallography, diffraction patterns are recorded at a constant rate as a suspension of protein crystals flows across the path of an X-ray beam. Events that by chance contain single-crystal diffraction patterns are retained, then indexed and merged to form a three-dimensional set of reflection intensities for structure determination. This approach relies upon several innovations: an intense X-ray beam; a fast detector system; a means to rapidly flow a suspension of crystals across the X-ray beam; and the computational infrastructure to process the large volume of data. Originally conceived for radiation-damage-free measurements with ultrafast X-ray pulses, the same methods can be employed with synchrotron radiation. As in powder diffraction, the averaging of thousands of observations per Bragg peak may improve the ratio of signal to noise of low-dose exposures. Here, it is shown that this paradigm can be implemented for room-temperature data collection using synchrotron radiation and exposure times of less than 3 ms. Using lysozyme microcrystals as a model system, over 40 000 single-crystal diffraction patterns were obtained and merged to produce a structural model that could be refined to 2.1 Å resolution. The resulting electron density is in excellent agreement with that obtained using standard X-ray data collection techniques. With further improvements the method is well suited for even shorter exposures at future and upgraded synchrotron radiation facilities that may deliver beams with 1000 times higher brightness than they currently produce.

LC/MS quantification of multiple plasma proteins that differ by several orders of magnitude in concentration from a single sample is challenging. We present a strategy that allows the simultaneous determination of the concentration and turnover kinetics of higher and lower abundant proteins from a single digestion mixture. Our attention was directed at a cluster of proteins that interact to affect the absorption and interorgan lipid trafficking. We demonstrate that apos involved in TG metabolism such as apoC2, C3, E, and A4 (micromolar concentration), and apoB48 and apoA5 (single-digit nanomolar concentration) can be quantified from a single digestion mixture. A high degree of correlation between LC/MS and immunobased measurements for apoC2, C3, E, and B48 was observed. Moreover, apoA5 fractional synthesis rate was measured in humans for the first time. Finally, the method can be directly applied to studies involving nonhuman primates because peptide sequences used in the method are conserved between humans and nonhuman primates.

Crystal structure determinations of biological macromolecules are limited by the availability of sufficiently sized crystals and by the fact that crystal quality deteriorates during data collection owing to radiation damage. Exploiting a micrometre-sized X-ray beam, high-precision diffractometry and shutterless data acquisition with a pixel-array detector, a strategy for collecting data from many micrometre-sized crystals presented to an X-ray beam in a vitrified suspension is demonstrated. By combining diffraction data from 80 Trypanosoma brucei procathepsin B crystals with an average volume of 9 µm3, a complete data set to 3.0 Å resolution has been assembled. The data allowed the refinement of a structural model that is consistent with that previously obtained using free-electron laser radiation, providing mutual validation. Further improvements of the serial synchrotron crystallography technique and its combination with serial femtosecond crystallography are discussed that may allow the determination of high-resolution structures of micrometre-sized crystals.

Actin has well established functions in cellular morphogenesis. However, it is not well understood how the various actin assemblies in a cell are kept in a dynamic equilibrium, in particular when cells have to respond to acute signals. Here, we characterize a rapid and transient actin reset in response to increased intracellular calcium levels. Within seconds of calcium influx, the formin INF2 stimulates filament polymerization at the endoplasmic reticulum (ER), while cortical actin is disassembled. The reaction is then reversed within a few minutes. This Calcium-mediated actin reset (CaAR) occurs in a wide range of mammalian cell types and in response to many physiological cues. CaAR leads to transient immobilization of organelles, drives reorganization of actin during cell cortex repair, cell spreading and wound healing, and induces long-lasting changes in gene expression. Our findings suggest that CaAR acts as fundamental facilitator of cellular adaptations in response to acute signals and stress.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Objective & methods: This study tested associations of genotype-predicted activity of CYP3A4, other pharmacogenes, SLC28A7 (rs11648166) and ALPPL2 (rs28845026) with systemic concentrations of the endocrine therapies anastrozole and fulvestrant in SWOG S0226 trial participants. Results: Participants in the anastrozole-only arm with low CYP3A4 activity (i.e. CYP3A4*22 carriers) had higher systemic anastrozole concentrations than patients with high CYP3A4 activity (β-coefficient = 10.03; 95% CI: 1.42, 18.6; p = 0.025). In an exploratory analysis, participants with low CYP2C9 activity had lower anastrozole concentrations and higher fulvestrant concentrations than participants with high CYP2C9 activity. Conclusion: Inherited genetic variation in CYP3A4 and CYP2C9 may affect concentrations of endocrine therapy and may be useful to personalize dosing and improve treatment outcomes.