Publications by authors named "Sean McSweeney"

119 Publications

Sounding out falsified medicines from genuine medicines using Broadband Acoustic Resonance Dissolution Spectroscopy (BARDS).

Sci Rep 2021 Jun 16;11(1):12643. Epub 2021 Jun 16.

School of Chemistry, Analytical and Biological Chemistry Research Facility (ABCRF), University College Cork, Cork, Ireland.

The trade in falsified medicine has increased significantly and it is estimated that global falsified sales have reached $100 billion in 2020. The EU Falsified Medicines Directive states that falsified medicines do not only reach patients through illegal routes but also via the legal supply chain. Falsified medicines can contain harmful ingredients. They can also contain too little or too much active ingredient or no active ingredient at all. BARDS (Broadband Acoustic Resonance Dissolution Spectroscopy) harnesses an acoustic phenomenon associated with the dissolution of a sample (tablet or powder). The resulting acoustic spectrum is unique and intrinsic to the sample and can be used as an identifier or signature profile. BARDS was evaluated in this study to determine whether a product is falsified or genuine in a rapid manner and at lower cost than many existing technologies. A range of genuine and falsified medicines, including falsified antimalarial tablets from south-east Asia, were tested, and compared to their counterpart genuine products. Significant differences between genuine and falsified doses were found in their acoustic signatures as they disintegrate and dissolve. Principal component analysis was employed to differentiate between the genuine and falsified medicines. This demonstrates that the tablets and capsules included here have intrinsic acoustic signatures which could be used to screen the quality of medicines.
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http://dx.doi.org/10.1038/s41598-021-90323-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8209214PMC
June 2021

Structural basis for SARS-CoV-2 envelope protein recognition of human cell junction protein PALS1.

Nat Commun 2021 06 8;12(1):3433. Epub 2021 Jun 8.

Biology Department, Brookhaven National Laboratory, Upton, NY, USA.

The COVID-19 pandemic, caused by the SARS-CoV-2 virus, has created global health and economic emergencies. SARS-CoV-2 viruses promote their own spread and virulence by hijacking human proteins, which occurs through viral protein recognition of human targets. To understand the structural basis for SARS-CoV-2 viral-host protein recognition, here we use cryo-electron microscopy (cryo-EM) to determine a complex structure of the human cell junction protein PALS1 and SARS-CoV-2 viral envelope (E) protein. Our reported structure shows that the E protein C-terminal DLLV motif recognizes a pocket formed exclusively by hydrophobic residues from the PDZ and SH3 domains of PALS1. Our structural analysis provides an explanation for the observation that the viral E protein recruits PALS1 from lung epithelial cell junctions. In addition, our structure provides novel targets for peptide- and small-molecule inhibitors that could block the PALS1-E interactions to reduce E-mediated virulence.
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http://dx.doi.org/10.1038/s41467-021-23533-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8187709PMC
June 2021

The temperature-dependent conformational ensemble of SARS-CoV-2 main protease (M ).

bioRxiv 2021 May 3. Epub 2021 May 3.

The COVID-19 pandemic, instigated by the SARS-CoV-2 coronavirus, continues to plague the globe. The SARS-CoV-2 main protease, or M , is a promising target for development of novel antiviral therapeutics. Previous X-ray crystal structures of M were obtained at cryogenic temperature or room temperature only. Here we report a series of high-resolution crystal structures of unliganded M across multiple temperatures from cryogenic to physiological, and another at high humidity. We interrogate these datasets with parsimonious multiconformer models, multi-copy ensemble models, and isomorphous difference density maps. Our analysis reveals a temperature-dependent conformational landscape for M , including a mobile water interleaved between the catalytic dyad, mercurial conformational heterogeneity in a key substrate-binding loop, and a far-reaching intramolecular network bridging the active site and dimer interface. Our results may inspire new strategies for antiviral drug development to counter-punch COVID-19 and combat future coronavirus pandemics.
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http://dx.doi.org/10.1101/2021.05.03.437411DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8109201PMC
May 2021

Prostate Cancer Mortality Associated with Aggregate Polymorphisms in Androgen-Regulating Genes: The Atherosclerosis Risk in the Communities (ARIC) Study.

Cancers (Basel) 2021 Apr 19;13(8). Epub 2021 Apr 19.

Division of Hematology, Oncology and Transplantation, University of Minnesota Medical School, Minneapolis, MN 55455, USA.

Genetic variations in androgen metabolism may influence prostate cancer (PC) prognosis. Clinical studies consistently linked PC prognosis with four single nucleotide polymorphisms (SNPs) in the critical androgen-regulating genes: 3-beta-hydroxysteroid dehydrogenase () rs1047303, 5-alpha-reductase 2 () rs523349, and solute carrier organic ion () rs1789693 and rs12422149. We tested the association of four androgen-regulating SNPs, individually and combined, with PC-specific mortality in the ARIC population-based prospective cohort. Men diagnosed with PC (N = 622; 79% White, 21% Black) were followed for death (N = 350) including PC death (N = 74). Cox proportional hazards regression was used to estimate hazard ratios (HR) and 95%CI adjusting for center, age, stage, and grade at diagnosis using separate hazards for races. A priori genetic risk score (GRS) was created as the unweighted sum of risk alleles in the four pre-selected SNPs. The gain-of-function rs1047303C allele was associated PC-specific mortality among men with metastatic PC at diagnosis (HR = 4.89 per risk allele, = 0.01). Higher GRS was associated with PC-specific mortality (per risk allele: HR = 1.26, = 0.03). We confirmed that the gain-of-function allele in rs1047303 is associated with greater PC mortality in men with metastatic disease. Additionally, our findings suggest a cumulative effect of androgen-regulating genes on PC-specific mortality; however, further validation is required.
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http://dx.doi.org/10.3390/cancers13081958DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8072683PMC
April 2021

Sounding out stability of enteric coated dosage forms using Broadband Acoustic Resonance Dissolution Spectroscopy (BARDS).

Int J Pharm 2021 Jun 20;602:120614. Epub 2021 Apr 20.

School of Chemistry, University College Cork, Cork, Ireland; Analytical and Biological Chemistry Research Facility (ABCRF), University College Cork, Cork, Ireland; BARDS Acoustic Science Labs, Bio-Innovation Centre, UCC, Cork, Ireland. Electronic address:

Stability testing is essential in the pharmaceutical industry to determine product shelf- life and the conditions under which drug products should be stored. Stability testing involves a complex set of procedures, considerable cost, time, and scientific expertise to build quality, efficacy and safety in a drug formulation. This paper highlights a new complementary approach to stability testing called Broadband Acoustic Resonance Dissolution Spectroscopy (BARDS). BARDS measurements are based on reproducible changes in the compressibility of a solvent during dissolution. It is monitored acoustically via associated changes in the frequency of induced acoustic resonances. This study presents a novel approach to track the change of various drug formulations to determine the formulation's stability. Pellets, tablet and multiple-unit pellet system (MUPS) formulations were investigated to examine the effect of polymer coating and formulation core degradation over time. In combination with minimal usage of Ultra Violet - Visible Spectroscopy, BARDS can effectively track these changes. The technique offers a rapid approach to characterizing pharmaceutical formulations. BARDS can enable rapid development of solid drug formulation dissolution and disintegration testing as an In-Process Control test and drug stability analysis. The data show that a solid oral dose formulation has an intrinsic acoustic signature specific to the method of manufacture, excipient composition and elapsed time since the production of a product. BARDS data are also indicative of which aspect of a formulation may be unstable, whether a coating, sub-coating or core. It is potentially a time-efficient, cost-effective and greener approach to testing coating stability, disintegration and overall formulation stability.
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http://dx.doi.org/10.1016/j.ijpharm.2021.120614DOI Listing
June 2021

FMX - the Frontier Microfocusing Macromolecular Crystallography Beamline at the National Synchrotron Light Source II.

J Synchrotron Radiat 2021 Mar 25;28(Pt 2):650-665. Epub 2021 Feb 25.

Photon Sciences, Brookhaven National Laboratory, Upton, NY 11973, USA.

Two new macromolecular crystallography (MX) beamlines at the National Synchrotron Light Source II, FMX and AMX, opened for general user operation in February 2017 [Schneider et al. (2013). J. Phys. Conf. Ser. 425, 012003; Fuchs et al. (2014). J. Phys. Conf. Ser. 493, 012021; Fuchs et al. (2016). AIP Conf. Proc. SRI2015, 1741, 030006]. FMX, the micro-focusing Frontier MX beamline in sector 17-ID-2 at NSLS-II, covers a 5-30 keV photon energy range and delivers a flux of 4.0 × 10 photons s at 1 Å into a 1 µm × 1.5 µm to 10 µm × 10 µm (V × H) variable focus, expected to reach 5 × 10 photons s at final storage-ring current. This flux density surpasses most MX beamlines by nearly two orders of magnitude. The high brightness and microbeam capability of FMX are focused on solving difficult crystallographic challenges. The beamline's flexible design supports a wide range of structure determination methods - serial crystallography on micrometre-sized crystals, raster optimization of diffraction from inhomogeneous crystals, high-resolution data collection from large-unit-cell crystals, room-temperature data collection for crystals that are difficult to freeze and for studying conformational dynamics, and fully automated data collection for sample-screening and ligand-binding studies. FMX's high dose rate reduces data collection times for applications like serial crystallography to minutes rather than hours. With associated sample lifetimes as short as a few milliseconds, new rapid sample-delivery methods have been implemented, such as an ultra-high-speed high-precision piezo scanner goniometer [Gao et al. (2018). J. Synchrotron Rad. 25, 1362-1370], new microcrystal-optimized micromesh well sample holders [Guo et al. (2018). IUCrJ, 5, 238-246] and highly viscous media injectors [Weierstall et al. (2014). Nat. Commun. 5, 3309]. The new beamline pushes the frontier of synchrotron crystallography and enables users to determine structures from difficult-to-crystallize targets like membrane proteins, using previously intractable crystals of a few micrometres in size, and to obtain quality structures from irregular larger crystals.
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http://dx.doi.org/10.1107/S1600577520016173DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7941291PMC
March 2021

RNA-binding protein DDX3 mediates posttranscriptional regulation of androgen receptor: A mechanism of castration resistance.

Proc Natl Acad Sci U S A 2020 11 26;117(45):28092-28101. Epub 2020 Oct 26.

Department of Urology, University of Wisconsin School of Medicine and Public Health, Madison, WI 53705;

Prostate cancer (CaP) driven by androgen receptor (AR) is treated with androgen deprivation; however, therapy failure results in lethal castration-resistant prostate cancer (CRPC). AR-low/negative (ARL/-) CRPC subtypes have recently been characterized and cannot be targeted by hormonal therapies, resulting in poor prognosis. RNA-binding protein (RBP)/helicase DDX3 (DEAD-box helicase 3 X-linked) is a key component of stress granules (SG) and is postulated to affect protein translation. Here, we investigated DDX3-mediated posttranscriptional regulation of AR mRNA (messenger RNA) in CRPC. Using patient samples and preclinical models, we objectively quantified DDX3 and AR expression in ARL/- CRPC. We utilized CRPC models to identify DDX3:AR mRNA complexes by RNA immunoprecipitation, assess the effects of DDX3 gain/loss-of-function on AR expression and signaling, and address clinical implications of targeting DDX3 by assessing sensitivity to AR-signaling inhibitors (ARSI) in CRPC xenografts in vivo. ARL/- CRPC expressed abundant AR mRNA despite diminished levels of AR protein. DDX3 protein was highly expressed in ARL/- CRPC, where it bound to AR mRNA. Consistent with a repressive regulatory role, DDX3 localized to cytoplasmic puncta with SG marker PABP1 in CRPC. While induction of DDX3-nucleated SGs resulted in decreased AR protein expression, inhibiting DDX3 was sufficient to restore 1) AR protein expression, 2) AR signaling, and 3) sensitivity to ARSI in vitro and in vivo. Our findings implicate the RBP protein DDX3 as a mechanism of posttranscriptional regulation for AR in CRPC. Clinically, DDX3 may be targetable for sensitizing ARL/- CRPC to AR-directed therapies.
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http://dx.doi.org/10.1073/pnas.2008479117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7668093PMC
November 2020

The state of the art in kidney and kidney tumor segmentation in contrast-enhanced CT imaging: Results of the KiTS19 challenge.

Med Image Anal 2021 01 2;67:101821. Epub 2020 Oct 2.

Carleton College, Northfield, United States.

There is a large body of literature linking anatomic and geometric characteristics of kidney tumors to perioperative and oncologic outcomes. Semantic segmentation of these tumors and their host kidneys is a promising tool for quantitatively characterizing these lesions, but its adoption is limited due to the manual effort required to produce high-quality 3D segmentations of these structures. Recently, methods based on deep learning have shown excellent results in automatic 3D segmentation, but they require large datasets for training, and there remains little consensus on which methods perform best. The 2019 Kidney and Kidney Tumor Segmentation challenge (KiTS19) was a competition held in conjunction with the 2019 International Conference on Medical Image Computing and Computer Assisted Intervention (MICCAI) which sought to address these issues and stimulate progress on this automatic segmentation problem. A training set of 210 cross sectional CT images with kidney tumors was publicly released with corresponding semantic segmentation masks. 106 teams from five continents used this data to develop automated systems to predict the true segmentation masks on a test set of 90 CT images for which the corresponding ground truth segmentations were kept private. These predictions were scored and ranked according to their average Sørensen-Dice coefficient between the kidney and tumor across all 90 cases. The winning team achieved a Dice of 0.974 for kidney and 0.851 for tumor, approaching the inter-annotator performance on kidney (0.983) but falling short on tumor (0.923). This challenge has now entered an "open leaderboard" phase where it serves as a challenging benchmark in 3D semantic segmentation.
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http://dx.doi.org/10.1016/j.media.2020.101821DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7734203PMC
January 2021

A self-supervised workflow for particle picking in cryo-EM.

IUCrJ 2020 Jul 23;7(Pt 4):719-727. Epub 2020 Jun 23.

Biology Department, Brookhaven National Laboratory, Upton, NY 11973, USA.

High-resolution single-particle cryo-EM data analysis relies on accurate particle picking. To facilitate the particle picking process, a self-supervised workflow has been developed. This includes an iterative strategy, which uses a 2D class average to improve training particles, and a progressively improved convolutional neural network for particle picking. To automate the selection of particles, a threshold is defined (%/Res) using the ratio of percentage class distribution and resolution as a cutoff. This workflow has been tested using six publicly available data sets with different particle sizes and shapes, and can automatically pick particles with minimal user input. The picked particles support high-resolution reconstructions at 3.0 Å or better. This workflow is a step towards automated single-particle cryo-EM data analysis at the stage of particle picking. It may be used in conjunction with commonly used single-particle analysis packages such as , , , and .
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http://dx.doi.org/10.1107/S2052252520007241DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7340252PMC
July 2020

Public Perceptions of Artificial Intelligence and Robotics in Medicine.

J Endourol 2020 10 29;34(10):1041-1048. Epub 2020 Sep 29.

Department of Urology, University of Minnesota, Minneapolis, Minnesota, USA.

To understand better the public perception and comprehension of medical technology such as artificial intelligence (AI) and robotic surgery. In addition to this, to identify sensitivity to their use to ensure acceptability and quality of counseling. A survey was conducted on a convenience sample of visitors to the MN Minnesota State Fair ( = 264). Participants were randomized to receive one of two similar surveys. In the first, a diagnosis was made by a physician and in the second by an AI application to compare confidence in human and computer-based diagnosis. The median age of participants was 45 (interquartile range 28-59), 58% were female ( = 154) 42% male ( = 110), 69% had completed at least a bachelor's degree, 88% were Caucasian ( = 233) 12% ethnic minorities ( = 31) and were from 12 states, mostly from the Upper Midwest. Participants had nearly equal trust in AI physician diagnoses. However, they were significantly more likely to trust an AI diagnosis of cancer over a doctor's diagnosis when responding to the version of the survey that suggested that an AI could make medical diagnoses ( = 9.32e-06). Though 55% of respondents ( = 145) reported that they were uncomfortable with automated robotic surgery, the majority of the individuals surveyed (88%) mistakenly believed that partially autonomous surgery was already happening. Almost all (94%,  = 249) stated that they would be willing to pay for a review of medical imaging by an AI if available. Most participants express confidence in AI providing medical diagnoses, sometimes even over human physicians. Participants generally express concern with surgical AI, but they mistakenly believe that it is already being performed. As AI applications increase in medical practice, health care providers should be cognizant of the potential amount of misinformation and sensitivity that patients have to how such technology is represented.
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http://dx.doi.org/10.1089/end.2020.0137DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7578175PMC
October 2020

Structural basis for Ca-dependent activation of a plant metacaspase.

Nat Commun 2020 05 7;11(1):2249. Epub 2020 May 7.

Biology Department, Brookhaven National Laboratory, Upton, NY, USA.

Plant metacaspases mediate programmed cell death in development, biotic and abiotic stresses, damage-induced immune response, and resistance to pathogen attack. Most metacaspases require Ca for their activation and substrate processing. However, the Ca-dependent activation mechanism remains elusive. Here we report the crystal structures of Metacaspase 4 from Arabidopsis thaliana (AtMC4) that modulates Ca-dependent, damage-induced plant immune defense. The AtMC4 structure exhibits an inhibitory conformation in which a large linker domain blocks activation and substrate access. In addition, the side chain of Lys225 in the linker domain blocks the active site by sitting directly between two catalytic residues. We show that the activation of AtMC4 and cleavage of its physiological substrate involve multiple cleavages in the linker domain upon activation by Ca. Our analysis provides insight into the Ca-dependent activation of AtMC4 and lays the basis for tuning its activity in response to stresses for engineering of more sustainable crops for food and biofuels.
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http://dx.doi.org/10.1038/s41467-020-15830-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7206013PMC
May 2020

ID30A-3 (MASSIF-3) - a beamline for macromolecular crystallography at the ESRF with a small intense beam.

J Synchrotron Radiat 2020 May 29;27(Pt 3):844-851. Epub 2020 Apr 29.

European Synchrotron Radiation Facility, 71 Avenue des Martyrs, 38000 Grenoble, France.

ID30A-3 (or MASSIF-3) is a mini-focus (beam size 18 µm × 14 µm) highly intense (2.0 × 10 photons s), fixed-energy (12.81 keV) beamline for macromolecular crystallography (MX) experiments at the European Synchrotron Radiation Facility (ESRF). MASSIF-3 is one of two fixed-energy beamlines sited on the first branch of the canted undulator setup on the ESRF ID30 port and is equipped with a MD2 micro-diffractometer, a Flex HCD sample changer, and an Eiger X 4M fast hybrid photon-counting detector. MASSIF-3 is recommended for collecting diffraction data from single small crystals (≤15 µm in one dimension) or for experiments using serial methods. The end-station has been in full user operation since December 2014, and here its current characteristics and capabilities are described.
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http://dx.doi.org/10.1107/S1600577520004002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7206554PMC
May 2020

The sound of tablets during coating erosion, disintegration, deaggregation and dissolution.

Int J Pharm 2020 Apr 9;580:119216. Epub 2020 Mar 9.

Department of Chemistry, Analytical and Biological Chemistry Research Facility (ABCRF), University College Cork, Ireland; BARDS Acoustic Science Labs, Bio-Innovation Centre, UCC, Ireland. Electronic address:

This research aims to address a gap in our understanding of the mechanisms by which pharmaceutical tablets achieve highly reproducible and predictable drug release. The present industrial and regulatory practice is centred around tablet dissolution, i.e. what follows disintegration, yet the vast majority of problems that are found in formulation dissolution testing can be traced back to the erratic disintegration behaviour of the medicinal product. It is only due to the distinct lack of quantitative measurement techniques for disintegration analysis that this situation arises. Current methods involve costly, and time-consuming test equipment, resulting in a need for more simple, green and efficient methods which have the potential to enable rapid development and to accelerate routine solid drug formulation dissolution and disintegration testing. In this study, we present a novel approach to track several sequential tablet dissolution processes, including coating erosion, disintegration, deaggregation and dissolution using Broadband Acoustic Resonance Dissolution Spectroscopy (BARDS). BARDS, in combination with minimal usage of UV spectroscopy, can effectively track these processes. The data also show that a solid oral dose formulation has an intrinsic acoustic signature which is specific to the method of manufacture and excipient composition.
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http://dx.doi.org/10.1016/j.ijpharm.2020.119216DOI Listing
April 2020

: microcrystal data assembly using Python.

J Appl Crystallogr 2020 Feb 1;53(Pt 1):277-281. Epub 2020 Feb 1.

Biology Department, Brookhaven National Laboratory, Upton, NY 11973, USA.

The recent developments at microdiffraction X-ray beamlines are making microcrystals of macromolecules appealing subjects for routine structural analysis. Microcrystal diffraction data collected at synchrotron microdiffraction beamlines may be radiation damaged with incomplete data per microcrystal and with unit-cell variations. A multi-stage data assembly method has previously been designed for microcrystal synchrotron crystallography. Here the strategy has been implemented as a Python program for microcrystal data assembly (). optimizes microcrystal data quality including weak anomalous signals through iterative crystal and frame rejections. Beyond microcrystals, may be applicable for assembling data sets from larger crystals for improved data quality.
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http://dx.doi.org/10.1107/S160057671901673XDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6998775PMC
February 2020

Tracking Yeast Metabolism and the Crabtree Effect in Real Time via CO Production using Broadband Acoustic Resonance Dissolution Spectroscopy (BARDS).

J Biotechnol 2020 Jan 30;308:63-73. Epub 2019 Nov 30.

School of Chemistry, Analytical and Biological Chemistry Research Facility (ABCRF), University College Cork, Ireland. Electronic address:

In this study, a new approach to measure metabolic activity of yeast via the Crabtree effect is described. BARDS is an analytical technique developed to aid powder and tablet characterisation by monitoring changes in the compressibility of a solvent during solute dissolution. It is a rapid and simple method which utilises a magnetic stir bar to mix added solute and induce the acoustic resonance of a vessel containing a fixed volume of solvent. In this study it is shown that initiation of fermentation in a yeast suspension, in aqueous buffer, is accompanied by reproducible changes in the frequency of induced acoustic resonance. These changes signify increased compressibility of the suspension due to CO release by the yeast. A simple standardised BARDS protocol reveals yeast carbon source preferences and can generate quantitative kinetic data on carbon source metabolism which are characteristic of each yeast strain. The Crawford-Woods equation can be used to quantify total gaseous CO produced by a given number of viable yeast when supplied with a fixed amount of carbon source. This allows for a value to be calculated for the amount of gaseous CO produced by each yeast cell. The approach has the potential to transform the way in which yeast metabolism is tracked and potentially provide an orthogonal or surrogate method to determining viability, vitality and attenuation measurements in the future.
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http://dx.doi.org/10.1016/j.jbiotec.2019.11.016DOI Listing
January 2020

Structure of the dihydrolipoamide succinyltransferase catalytic domain from Escherichia coli in a novel crystal form: a tale of a common protein crystallization contaminant.

Acta Crystallogr F Struct Biol Commun 2019 Sep 29;75(Pt 9):616-624. Epub 2019 Aug 29.

National Synchrotron Light Source II, Brookhaven National Laboratory, Upton, NY 11973-5000, USA.

The crystallization of amidase, the ultimate enzyme in the Trp-dependent auxin-biosynthesis pathway, from Arabidopsis thaliana was attempted using protein samples with at least 95% purity. Cube-shaped crystals that were assumed to be amidase crystals that belonged to space group I4 (unit-cell parameters a = b = 128.6, c = 249.7 Å) were obtained and diffracted to 3.0 Å resolution. Molecular replacement using structures from the PDB containing the amidase signature fold as search models was unsuccessful in yielding a convincing solution. Using the Sequence-Independent Molecular replacement Based on Available Databases (SIMBAD) program, it was discovered that the structure corresponded to dihydrolipoamide succinyltransferase from Escherichia coli (PDB entry 1c4t), which is considered to be a common crystallization contaminant protein. The structure was refined to an R of 23.0% and an R of 27.2% at 3.0 Å resolution. The structure was compared with others of the same protein deposited in the PDB. This is the first report of the structure of dihydrolipoamide succinyltransferase isolated without an expression tag and in this novel crystal form.
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http://dx.doi.org/10.1107/S2053230X19011488DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6718150PMC
September 2019

Post-translational Protein Acetylation: An Elegant Mechanism for Bacteria to Dynamically Regulate Metabolic Functions.

Front Microbiol 2019 12;10:1604. Epub 2019 Jul 12.

Health Sciences Division, Department of Microbiology and Immunology, Stritch School of Medicine, Loyola University Chicago, Maywood, IL, United States.

Post-translational modifications (PTM) decorate proteins to provide functional heterogeneity to an existing proteome. The large number of known PTMs highlights the many ways that cells can modify their proteins to respond to diverse stimuli. Recently, PTMs have begun to receive increased interest because new sensitive proteomics workflows and structural methodologies now allow researchers to obtain large-scale, in-depth and unbiased information concerning PTM type and site localization. However, few PTMs have been extensively assessed for functional consequences, leaving a large knowledge gap concerning the inner workings of the cell. Here, we review understanding of -𝜀-lysine acetylation in bacteria, a PTM that was largely ignored in bacteria until a decade ago. Acetylation is a modification that can dramatically change the function of a protein through alteration of its properties, including hydrophobicity, solubility, and surface properties, all of which may influence protein conformation and interactions with substrates, cofactors and other macromolecules. Most bacteria carry genes predicted to encode the lysine acetyltransferases and lysine deacetylases that add and remove acetylations, respectively. Many bacteria also exhibit acetylation activities that do not depend on an enzyme, but instead on direct transfer of acetyl groups from the central metabolites acetyl coenzyme A or acetyl phosphate. Regardless of mechanism, most central metabolic enzymes possess lysines that are acetylated in a regulated fashion and many of these regulated sites are conserved across the spectrum of bacterial phylogeny. The interconnectedness of acetylation and central metabolism suggests that acetylation may be a response to nutrient availability or the energy status of the cell. However, this and other hypotheses related to acetylation remain untested.
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http://dx.doi.org/10.3389/fmicb.2019.01604DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6640162PMC
July 2019

A rapid in-process control (IPC) test to monitor the functionality of taste masking polymer coatings using Broadband Acoustic Resonance Dissolution Spectroscopy (BARDS).

Int J Pharm 2019 Sep 24;568:118559. Epub 2019 Jul 24.

Department of Chemistry, Analytical and Biological Chemistry Research Facility (ABCRF), University College Cork, Ireland; BARDS Acoustic Science Labs, Bio-Innovation Centre, UCC, Ireland. Electronic address:

Monitoring of the coating end-point of functional coatings during the coating application process is desirable. Since currently available PAT methods require expensive test equipment, there is a need for a rapid test that can easily be applied without major investment. BARDS is a novel technique that has the potential to economise the production process of these kinds of pellet and tablet formulations. The thickness of a controlled release coating is a key factor that determines the release rate of the drug in the gastro-intestinal tract or other targeted functionalities such as taste masking or moisture protection. Correspondingly, the amount of drug per unit mass of pellets decreases with increasing thickness of the functional coating. In this study, the functional polymer loading of the coating process is investigated by testing pellets via BARDS technology (Broadband Acoustic Resonance Dissolution Spectroscopy). The technique offers a rapid approach (<200 s) to characterising functional coatings at-line during their manufacture. Measurements are based on reproducible changes in the compressibility of a solvent during dissolution which is monitored acoustically via associated changes in the frequency of induced acoustic resonances. In case of enteric coatings a steady state acoustic lag time is associated with the erosion of the enteric coatings in acidic dissolution test media. This lag time is indicative of the coating layer thickness, assuming that the quality of the film coating is high. BARDS represents a possible future surrogate test for IPC testing, as a PAT method and possibly also for conventional USP dissolution testing. BARDS data correlate directly with the thickness of the functional coating, its integrity and also with the drug loading as validated by UV-Vis spectroscopy.
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http://dx.doi.org/10.1016/j.ijpharm.2019.118559DOI Listing
September 2019

Synchrotron microcrystal native-SAD phasing at a low energy.

IUCrJ 2019 Jul 3;6(Pt 4):532-542. Epub 2019 May 3.

Biology Department, Brookhaven National Laboratory, Upton, NY 11973, USA.

structural evaluation of native biomolecules from single-wavelength anomalous diffraction (SAD) is a challenge because of the weakness of the anomalous scattering. The anomalous scattering from relevant native elements - primarily sulfur in proteins and phospho-rus in nucleic acids - increases as the X-ray energy decreases toward their -edge transitions. Thus, measurements at a lowered X-ray energy are promising for making native SAD routine and robust. For microcrystals with sizes less than 10 µm, native-SAD phasing at synchrotron microdiffraction beamlines is even more challenging because of difficulties in sample manipulation, diffraction data collection and data analysis. Native-SAD analysis from microcrystals by using X-ray free-electron lasers has been demonstrated but has required use of thousands of thousands of microcrystals to achieve the necessary accuracy. Here it is shown that by exploitation of anomalous microdiffraction signals obtained at 5 keV, by the use of polyimide wellmounts, and by an iterative crystal and frame-rejection method, microcrystal native-SAD phasing is possible from as few as about 1 200 crystals. Our results show the utility of low-energy native-SAD phasing with microcrystals at synchrotron microdiffraction beamlines.
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http://dx.doi.org/10.1107/S2052252519004536DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6608635PMC
July 2019

MXCuBE2: the dawn of MXCuBE Collaboration.

J Synchrotron Radiat 2019 Mar 22;26(Pt 2):393-405. Epub 2019 Feb 22.

MAX IV Laboratory, Lund University, SE-221 00 Lund, Sweden.

MXCuBE2 is the second-generation evolution of the MXCuBE beamline control software, initially developed and used at ESRF - the European Synchrotron. MXCuBE2 extends, in an intuitive graphical user interface (GUI), the functionalities and data collection methods available to users while keeping all previously available features and allowing for the straightforward incorporation of ongoing and future developments. MXCuBE2 introduces an extended abstraction layer that allows easy interfacing of any kind of macromolecular crystallography (MX) hardware component, whether this is a diffractometer, sample changer, detector or optical element. MXCuBE2 also works in strong synergy with the ISPyB Laboratory Information Management System, accessing the list of samples available for a particular experimental session and associating, either from instructions contained in ISPyB or from user input via the MXCuBE2 GUI, different data collection types to them. The development of MXCuBE2 forms the core of a fruitful collaboration which brings together several European synchrotrons and a software development factory and, as such, defines a new paradigm for the development of beamline control platforms for the European MX user community.
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http://dx.doi.org/10.1107/S1600577519001267DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6412183PMC
March 2019

Low-dose CT imaging of the acute abdomen using model-based iterative reconstruction: a prospective study.

Emerg Radiol 2019 Apr 17;26(2):169-177. Epub 2018 Nov 17.

Department of Radiology, Cork University Hospital, University College Cork, Cork, Ireland.

Objectives: Performance of a modified abdominopelvic CT protocol reconstructed using full iterative reconstruction (IR) was assessed for imaging patients presenting with acute abdominal symptoms.

Materials And Methods: Fifty-seven patients (17 male, 40 female; mean age of 56.5 ± 8 years) were prospectively studied. Low-dose (LD) and conventional-dose (CD) CTs were contemporaneously acquired between November 2015 and March 2016. The LD and CD protocols imparted radiation exposures approximating 10-20% and 80-90% those of routine abdominopelvic CT, respectively. The LD images were reconstructed with model-based iterative reconstruction (MBIR), and CD images with hybrid IR (40% adaptive statistical iterative reconstruction (ASIR)). Image quality was assessed quantitatively and qualitatively. Independent clinical interpretations were performed with a 6-week delay between reviews.

Results: A 74.7% mean radiation dose reduction was achieved: LD effective dose (ED) 2.38 ± 1.78 mSv (size-specific dose estimate (SSDE) 3.77 ± 1.97 mGy); CD ED 7.04 ± 4.89 mSv (SSDE 10.74 ± 5.5 mGy). LD-MBIR images had significantly lower objective and subjective image noise compared with CD-ASIR (p < 0.0001). Noise reduction for LD-MBIR studies was greater for patients with BMI < 25 kg/m than those with BMI ≥ 25 kg/m (5.36 ± 3.2 Hounsfield units (HU) vs. 4.05 ± 3.1 HU, p < 0.0001). CD-ASIR studies had significantly better contrast resolution, and diagnostic acceptability (p < 0.0001 for all). LD-MBIR studies had significantly lower streak artifact (p < 0.0001). There was no difference in sensitivity for primary findings between the low-dose and conventional protocols with the exception of one case of enteritis.

Conclusions: Low-dose abdominopelvic CT performed with MBIR is a feasible radiation dose reduction strategy for imaging patients presenting with acute abdominal pain.
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http://dx.doi.org/10.1007/s10140-018-1658-zDOI Listing
April 2019

Using sound pulses to solve the crystal-harvesting bottleneck.

Acta Crystallogr D Struct Biol 2018 Oct 2;74(Pt 10):986-999. Epub 2018 Oct 2.

Universidade Federal de Minas Gerais, 31270-901 Belo Horizonte-MG, Brazil.

Crystal harvesting has proven to be difficult to automate and remains the rate-limiting step for many structure-determination and high-throughput screening projects. This has resulted in crystals being prepared more rapidly than they can be harvested for X-ray data collection. Fourth-generation synchrotrons will support extraordinarily rapid rates of data acquisition, putting further pressure on the crystal-harvesting bottleneck. Here, a simple solution is reported in which crystals can be acoustically harvested from slightly modified MiTeGen In Situ-1 crystallization plates. This technique uses an acoustic pulse to eject each crystal out of its crystallization well, through a short air column and onto a micro-mesh (improving on previous work, which required separately grown crystals to be transferred before harvesting). Crystals can be individually harvested or can be serially combined with a chemical library such as a fragment library.
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http://dx.doi.org/10.1107/S2059798318011506DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6173054PMC
October 2018

High-speed raster-scanning synchrotron serial microcrystallography with a high-precision piezo-scanner.

J Synchrotron Radiat 2018 Sep 23;25(Pt 5):1362-1370. Epub 2018 Aug 23.

Photon Sciences, Brookhaven National Laboratory, Upton, NY 11973, USA.

The Frontier Microfocus Macromolecular Crystallography (FMX) beamline at the National Synchrotron Light Source II with its 1 µm beam size and photon flux of 3 × 10 photons s at a photon energy of 12.66 keV has reached unprecedented dose rates for a structural biology beamline. The high dose rate presents a great advantage for serial microcrystallography in cutting measurement time from hours to minutes. To provide the instrumentation basis for such measurements at the full flux of the FMX beamline, a high-speed, high-precision goniometer based on a unique XYZ piezo positioner has been designed and constructed. The piezo-based goniometer is able to achieve sub-100 nm raster-scanning precision at over 10 grid-linepairs s frequency for fly scans of a 200 µm-wide raster. The performance of the scanner in both laboratory and serial crystallography measurements up to the maximum frame rate of 750 Hz of the Eiger 16M's 4M region-of-interest mode has been verified in this work. This unprecedented experimental speed significantly reduces serial-crystallography data collection time at synchrotrons, allowing utilization of the full brightness of the emerging synchrotron radiation facilities.
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http://dx.doi.org/10.1107/S1600577518010354DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6140394PMC
September 2018

ID30B - a versatile beamline for macromolecular crystallography experiments at the ESRF.

J Synchrotron Radiat 2018 Jul 27;25(Pt 4):1249-1260. Epub 2018 Jun 27.

European Synchrotron Radiation Facility, 71 avenue des Martyrs, Grenoble 38043, France.

ID30B is an undulator-based high-intensity, energy-tuneable (6.0-20 keV) and variable-focus (20-200 µm in diameter) macromolecular crystallography (MX) beamline at the ESRF. It was the last of the ESRF Structural Biology Group's beamlines to be constructed and commissioned as part of the ESRF's Phase I Upgrade Program and has been in user operation since June 2015. Both a modified microdiffractometer (MD2S) incorporating an in situ plate screening capability and a new flexible sample changer (the FlexHCD) were specifically developed for ID30B. Here, the authors provide the current beamline characteristics and detail how different types of MX experiments can be performed on ID30B (http://www.esrf.eu/id30b).
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http://dx.doi.org/10.1107/S1600577518007166DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6038607PMC
July 2018

A new MR-SAD algorithm for the automatic building of protein models from low-resolution X-ray data and a poor starting model.

IUCrJ 2018 Mar 25;5(Pt 2):166-171. Epub 2018 Jan 25.

Department of Biophysical Structural Chemistry, Leiden University, Einsteinweg 55, 2333 CC Leiden, The Netherlands.

Determining macromolecular structures from X-ray data with resolution worse than 3 Å remains a challenge. Even if a related starting model is available, its incompleteness or its bias together with a low observation-to-parameter ratio can render the process unsuccessful or very time-consuming. Yet, many biologically important macromolecules, especially large macromolecular assemblies, membrane proteins and receptors, tend to provide crystals that diffract to low resolution. A new algorithm to tackle this problem is presented that uses a multivariate function to simultaneously exploit information from both an initial partial model and low-resolution single-wavelength anomalous diffraction data. The new approach has been used for six challenging structure determinations, including the crystal structures of membrane proteins and macromolecular complexes that have evaded experts using other methods, and large structures from a 3.0 Å resolution F-ATPase data set and a 4.5 Å resolution SecYEG-SecA complex data set. All of the models were automatically built by the method to values of between 28.9 and 39.9% and were free from the initial model bias.
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http://dx.doi.org/10.1107/S2052252517017961DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5947721PMC
March 2018

Sample manipulation and data assembly for robust microcrystal synchrotron crystallography.

IUCrJ 2018 May 19;5(Pt 3):238-246. Epub 2018 Apr 19.

Photon Science Directorate, NSLS-II, Brookhaven National Laboratory, Upton, NY 11973, USA.

With the recent developments in microcrystal handling, synchrotron microdiffraction beamline instrumentation and data analysis, microcrystal crystallo-graphy with crystal sizes of less than 10 µm is appealing at synchrotrons. However, challenges remain in sample manipulation and data assembly for robust microcrystal synchrotron crystallography. Here, the development of micro-sized polyimide well-mounts for the manipulation of microcrystals of a few micrometres in size and the implementation of a robust data-analysis method for the assembly of rotational microdiffraction data sets from many microcrystals are described. The method demonstrates that microcrystals may be routinely utilized for the acquisition and assembly of complete data sets from synchrotron microdiffraction beamlines.
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http://dx.doi.org/10.1107/S2052252518005389DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5929371PMC
May 2018

Broadband Acoustic Resonance Dissolution Spectroscopy (BARDS): A rapid test for enteric coating thickness and integrity of controlled release pellet formulations.

Int J Pharm 2018 Jun 12;544(1):31-38. Epub 2018 Apr 12.

Department of Chemistry, Analytical and Biological Chemistry Research Facility (ABCRF), University College Cork, Ireland. Electronic address:

There are no rapid dissolution based tests for determining coating thickness, integrity and drug concentration in controlled release pellets either during production or post-production. The manufacture of pellets requires several coating steps depending on the formulation. The sub-coating and enteric coating steps typically take up to six hours each followed by additional drying steps. Post production regulatory dissolution testing also takes up to six hours to determine if the batch can be released for commercial sale. The thickness of the enteric coating is a key factor that determines the release rate of the drug in the gastro-intestinal tract. Also, the amount of drug per unit mass decreases with increasing thickness of the enteric coating. In this study, the coating process is tracked from start to finish on an hourly basis by taking samples of pellets during production and testing those using BARDS (Broadband Acoustic Resonance Dissolution Spectroscopy). BARDS offers a rapid approach to characterising enteric coatings with measurements based on reproducible changes in the compressibility of a solvent due to the evolution of air during dissolution. This is monitored acoustically via associated changes in the frequency of induced acoustic resonances. A steady state acoustic lag time is associated with the disintegration of the enteric coatings in basic solution. This lag time is pH dependent and is indicative of the rate at which the coating layer dissolves. BARDS represents a possible future surrogate test for conventional USP dissolution testing as its data correlates directly with the thickness of the enteric coating, its integrity and also with the drug loading as validated by HPLC.
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http://dx.doi.org/10.1016/j.ijpharm.2018.04.018DOI Listing
June 2018

Contactless, probeless and non-titrimetric determination of acid-base reactions using broadband acoustic resonance dissolution spectroscopy (BARDS).

Analyst 2018 Feb;143(4):956-962

School of Chemistry, and Analytical and Biological Chemistry Research Facility (ABCRF), University College Cork (UCC), Cork, Ireland.

pH determination is a routine measurement in scientific laboratories worldwide. Most major advances in pH measurement were made in the 19th and early 20th century. pH measurements are critical for the determination of acid base reactions. This study demonstrates how an acid-base reaction can be monitored without the use of a pH probe, indicator and titres of reagent. The stoichiometric reaction between carbonate and HCl acid yields specific quantities of CO, which causes reproducible changes to the compressibility of the solvent. This in turn slows down the speed of sound in solution which is induced by a magnetic follower gently tapping the inner wall of the vessel. As a consequence the frequencies of the acoustic resonances in the vessel are reduced. This approach is called Broadband Acoustic Resonance Dissolution Spectroscopy (BARDS) which harnesses this phenomenon for many applications. The acid-carbonate experiments have also been validated using HSO acid and using both potassium and sodium counterions for the carbonate. This method can be used to interrogate strong acid-base reactions in a rapid and non-invasive manner using carbonate as the base. The data demonstrate the first example of a reactant also acting as an indicator. The applicability of the method to weak acids has yet to be determined. A novel conclusion from the study is that a person with a well-trained ear is capable of determining the concentration and pH of a strong acid just by listening. This brings pH measurement into the realm of human perception.
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http://dx.doi.org/10.1039/c7an01447cDOI Listing
February 2018

Broadband Acoustic Resonance Dissolution Spectroscopy (BARDS): A Novel Approach To Investigate the Wettability of Pharmaceutical Powder Blends.

Mol Pharm 2018 01 29;15(1):31-39. Epub 2017 Nov 29.

Department of Chemistry, Analytical and Biological Chemistry Research Facility (ABCRF), University College Cork , Cork, T12 YN60, Ireland.

The ability of broadband acoustic resonance dissolution spectroscopy (BARDS) to assess the wettability of powder blends is investigated. BARDS is a novel analytical technology developed on the basis of the change in acoustic phenomena observed when material is added into a solvent under resonance. Addition of solid material to the solvent results in the introduction of gas (air) into the solvent, changing the compressibility of the solvent system, and reducing the velocity of sound in the solvent. As a material is wetted and dissolved, the gas is released from the solvent and resonance frequency is altered. The main purpose of this work is to demonstrate the ability of BARDS to assess differences in the wetting behavior of tablet excipients (microcrystalline cellulose (MCC) and magnesium stearate (MgSt)) and a model drug (metoclopramide hydrochloride) as single component powders and multicomponent powder blends. BARDS acoustic responses showed a prolonged release of gas for the powdered blends with lubricant compared to unlubricated blends. As the elimination of gas from the solvent was assumed to follow first order elimination kinetics, a compressible gas elimination rate constant was calculated from the log plots of the gas volume profiles. The gas elimination rate constant was used as a parameter to compare the release of gas from the powder introduced to the solvent and hence the powder wetting behavior. A lower gas elimination rate constant was measured for lubricated blends compared to nonlubricated blends, suggesting the prolonged hydration of lubricated blends. Standard wetting techniques such as contact angle measurements and wetting time analysis were also used to analyze the blends and confirmed differences in wetting behavior determined by BARDS. The study results demonstrate the capability of BARDS as a rapid, analytical tool to determine the wetting behavior of the pharmaceutical powder blends and the potential of BARDS as a process analytical technology (PAT) tool.
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http://dx.doi.org/10.1021/acs.molpharmaceut.7b00658DOI Listing
January 2018

RoboDiff: combining a sample changer and goniometer for highly automated macromolecular crystallography experiments.

Acta Crystallogr D Struct Biol 2016 08 27;72(Pt 8):966-75. Epub 2016 Jul 27.

European Synchrotron Radiation Facility, 71 Avenue des Martyrs, CS 40220, F-38043 Grenoble, France.

Automation of the mounting of cryocooled samples is now a feature of the majority of beamlines dedicated to macromolecular crystallography (MX). Robotic sample changers have been developed over many years, with the latest designs increasing capacity, reliability and speed. Here, the development of a new sample changer deployed at the ESRF beamline MASSIF-1 (ID30A-1), based on an industrial six-axis robot, is described. The device, named RoboDiff, includes a high-capacity dewar, acts as both a sample changer and a high-accuracy goniometer, and has been designed for completely unattended sample mounting and diffraction data collection. This aim has been achieved using a high level of diagnostics at all steps of the process from mounting and characterization to data collection. The RoboDiff has been in service on the fully automated endstation MASSIF-1 at the ESRF since September 2014 and, at the time of writing, has processed more than 20 000 samples completely automatically.
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http://dx.doi.org/10.1107/S205979831601158XDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4973212PMC
August 2016
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