Doses for experiments with microbeams and microcrystals: Monte Carlo simulations in RADDOSE‐3D

Increasingly, microbeams and microcrystals are being used for macromolecular crystallography (MX) experiments at synchrotrons. However, radiation damage remains a major concern since it is a fundamental limiting factor affecting the success of macromolecular structure determination. The rate of radiation damage at cryotemperatures is known to be proportional to the absorbed dose, so to optimize experimental outcomes, accurate dose calculations are required which take into account the physics of the interactions of the crystal constituents. The program RADDOSE‐3D estimates the dose absorbed by samples during MX data collection at synchrotron sources, allowing direct comparison of radiation damage between experiments carried out with different samples and beam parameters. This has aided the study of MX radiation damage and enabled prediction of approximately when it will manifest in diffraction patterns so it can potentially be avoided. However, the probability of photoelectron escape from the sample and entry from the surrounding material has not previously been included in RADDOSE‐3D, leading to potentially inaccurate does estimates for experiments using microbeams or microcrystals. We present an extension to RADDOSE‐3D which performs Monte Carlo simulations of a rotating crystal during MX data collection, taking into account the redistribution of photoelectrons produced both in the sample and the material surrounding the crystal. As well as providing more accurate dose estimates, the Monte Carlo simulations highlight the importance of the size and composition of the surrounding material on the dose and thus the rate of radiation damage to the sample. Minimizing irradiation of the surrounding material or removing it almost completely will be key to extending the lifetime of microcrystals and enhancing the potential benefits of using higher incident X‐ray energies.     

Synchrotron X-ray micro-computed tomography as a tool for in situ elucidation of insect bacteriomes

Obligate bacterial endosymbionts are common, influential associates of arthropods, and are often found in specific organs termed bacteriomes. Three dimensional images of bacteriomes of the leafhopper Orosius albicinctus (Hemiptera: Cicadellidae) were reconstructed from synchrotron-based X-ray micro-computed tomography (CT). Results show that bilateral bacteriomes are located between the first and second abdominal tergites, are mushroom-shaped and consist two different types of tissue. Fluorescence in situ hybridization reveals that the primary bacterial symbiont Sulcia muelleri is in the ‘cap’ part of the of organ. The technique allows a noninvasive, in situ, means of visualizing bacteriomes and will facilitate understanding their form and function.     

Fusion genes: A promising tool combating against cancer

The driving roles of fusion genes during tumorigenesis have been recognized for decades, with efficacies demonstrated in clinical diagnosis and targeted therapy. With advances in sequencing technologies and computational biology, a surge in the identification of fusion genes has been witnessed during the past decade. The discovery and presence of splicing based fusions in normal tissues have challenged our canonical conceptions on fusion genes and offered us novel medical opportunities. The specificity of fusion genes to neoplastic tissues and their diverse functionalities during carcinogenesis foster them as promising tools in the battle against cancer. It is time to re-visit and comb through our cutting-edge knowledge on fusion genes to accelerate clinical translation of these internal markers. Urged as such, we are encouraged to categorize fusion events according to mechanisms leading to their generation, oncological consequences and clinical implications, offer insights on fusion occurrence across tumors from the system level, highlight feasible practices in fusion-related pharmaceutical development, and identify understudied yet important niches that may lead future research trend in this field.     

Near-infrared spectroscopy: promising diagnostic tool for viral infections

Although several methods, including enzyme-linked immunosorbent assay, polymerase chain reaction, immunofluorescent assay, and Western blotting, have been used for the diagnosis of viral infections, none of them is ideal in terms of cost-effectiveness, speed, and accuracy. Currently, the rate of outbreak of emerging viruses is increasing and therefore the development and establishment of analytical methods for such viral infections are becoming more important. Near-infrared (NIR) spectroscopy is a fast, multicomponent assay that enables non-invasive, non-destructive analysis. Recently, the diagnosis of viral infections using NIR spectroscopy has been attempted. In this review, the potential of the NIR method in the medical and virological fields is discussed.     

BMIT facility at the Canadian Light Source: Advances in X-ray phase-sensitive imaging

The BioMedical Imaging and Therapy (BMIT) facility [1,2] located at the Canadian Light Source, provides synchrotron-specific imaging and radiation therapy capabilities. There are two separate beamlines used for experiments: the bending magnet (05B1-1) and the insertion device (05ID-2) beamline.The bending magnet beamline provides access to monochromatic beam spanning a spectral range of 15–40 keV, and the beam is 240 mm wide in the POE-2 experimental hutch. Users can also perform experiments with polychromatic (pink) beam.The insertion device beamline was officially opened for general user program in 2015. The source for the ID beamline is a multi-pole, superconducting 4.3 T wiggler. The high field gives a critical energy over 20 keV. The optics hutches prepare a beam that is 220 mm wide in the last experimental hutch SOE-1. The monochromatic spectral range spans 25–150+ keV. Several different X-ray detectors are available for both beamlines, with resolutions ranging from 2 μm to 200 μm.BMIT provides a number of imaging techniques including standard absorption X-ray imaging, K-edge subtraction imaging (KES), in-line phase contrast imaging (also known as propagation based imaging, PBI) and Diffraction Enhanced Imaging/Analyzer Based Imaging (DEI/ABI), all in either projection or CT mode. PBI and DEI/ABI are particularly important tools for BMIT users since these techniques enable visualization of soft tissue and allow for low dose imaging.     

Aptamers: A promising tool for cancer imaging,diagnosis, and therapy

Aptamers are a group of molecules, which can specifically bind, track, and inhibit target molecules, comprising DNA aptamers, RNA aptamers, and peptide aptamers. So far, there are much progress about developing novel aptamers and their expansile applications. This prospect systematically introduces the composition and technological evolution of aptamers, and then focuses on the application of aptamers in cancer diagnosis, imaging, and therapy. Following this, we discuss the potential to harness aptamers in discovering the biomarker of stem cells, which is favorable for us to study the normal developmental or abnormal pathological process of tissue and to deliver drugs into target cells or tissues in the future. J. Cell. Biochem. 114: 250–255, 2013. © 2012 Wiley Periodicals, Inc.     

Pseudophosphorylation of cardiac myosin regulatory light chain: a promising new tool for treatment of cardiomyopathy

Many genetic mutations in sarcomeric proteins, including the cardiac myosin regulatory light chain (RLC) encoded by the MYL2 gene, have been implicated in familial cardiomyopathies. Yet, the molecular mechanisms by which these mutant proteins regulate cardiac muscle mechanics in health and disease remain poorly understood. Evidence has been accumulating that RLC phosphorylation has an influential role in striated muscle contraction and, in addition to the conventional modulation via Ca²⁺ binding to troponin C, it can regulate cardiac muscle function. In this review, we focus on RLC mutations that have been reported to cause cardiomyopathy phenotypes via compromised RLC phosphorylation and elaborate on pseudo-phosphorylation rescue mechanisms. This new methodology has been discussed as an emerging exploratory tool to understand the role of phosphorylation as well as a genetic modality to prevent/rescue cardiomyopathy phenotypes. Finally, we summarize structural effects post-phosphorylation, a phenomenon that leads to an ordered shift in the myosin S1 and RLC conformational equilibrium between two distinct states.     

Novel KV7 ion channel openers for the treatment of epilepsy and implications for detrusor tissue contraction

Neuronal voltage-gated potassium channels, K_V7s, are the molecular mediators of the M current and regulate membrane excitability in the central and peripheral neuronal systems. Herein, we report novel small molecule K_V7 openers that demonstrate anti-seizure activities in electroshock and pentylenetetrazol-induced seizure models without influencing Rotarod readouts in mice. The anti-seizure activity was determined to be proportional to the unbound concentration in the brain. K_V7 channels are also expressed in the bladder smooth muscle (detrusor) and activation of these channels may cause localized undesired effects. Therefore, the impact of individual K_V7 isoforms was investigated in human detrusor tissue using a panel of K_V7 openers with distinct activity profiles among K_V7 isoforms. KCNQ4 and KCNQ5 mRNA were highly expressed in detrusor tissue, yet a compound that has significantly reduced activity on homomeric K_V7.4 did not reduce detrusor contraction. This may suggest that the homomeric K_V7.4 channel plays a less significant role in bladder contraction and further investigation is needed.     

X-ray computed tomography: A promising tool to investigate the brachiopod shell interior. Effects on 3D modelling and taxonomy

Barremian and Cenomanian rhynchonelliform brachiopods, one Santonian and one Holocene species (Rhynchonellida and Terebratulida), have been observed using X-ray Computed Tomography in order to investigate the shell interior. Compared to transverse serial sections used formerly, X-ray CT is a promising tool. It permits 3D modelling of the brachidium, which is important for brachiopod classification. Four types of brachidium present during the Cretaceous have been observed (crura for the Rhynchonellida, short loop for Terebratulidina–Terebratulidae, ring-loop for Terebratulidina–Cancellothyrididae and long-loop for Terebratellidina), as well as relationships lophophore/brachidium in a Holocene species. Limitations are discussed, including the nature of the sediments enclosed between the valves, diagenesis, and the necessity to observe several age groups concerning the Terebratellidina. Finally, this non-destructive tool facilitates 3D reconstruction of inaccessible brachidia to improve brachiopod taxonomy and as an aid in curating collections.     

Mass spectrometry: A tool for on-line monitoring of animal cell cultures

The magnetic sector mass spectrometer is able to detect oxygen uptake and carbon dioxide production rates from animal cell cultivations performed in 101 biorectors. Such data have not been available with the use of classic exhaust gas analysis applying paramagnetic analyzers and infra-red sensors due to the insensitivity of the apparatus available. In the course of the present work we were able to demonstrate, that the oxygen uptake rate correlates to the number of viable cells. Additionally oxygen uptake rates supplied on-line information about the actual physiology of the cells: When the rates changed during the cultivation process, this immediately indicated the occurrence of limitations of components in the medium. The information could be useful in timing key events, such as performing splits or harvesting the bioreactor.Abbreviations OUR oxygen uptake rate - CDPR carbon dioxide production rate - RQ respiratory quotient This publication is dedicated to the 65 ^th birthday of Prof. Dr. F. Wagner, University of Braunschweig.     

Embryonic stem cells: a promising tool for cell replacement therapy

Embryonic stem (ES) cells are revolutionizing the field of developmental biology as a potential tool to understand the molecular mechanisms occurring during the process of differentiation from the embryonic stage to the adult phenotype. ES cells harvested from the inner cell mass (ICM) of the early embryo can proliferate indefinitely in vitro while retaining the ability to differentiate into all somatic cells. Emerging results from mice models with ES cells are promising and raising tremendous hope among the scientific community for the ES-cell based cell replacement therapy (CRT) of various severe diseases. ES cells could potentially revolutionize medicine by providing an unlimited renewable source of cells capable of replacing or repairing tissues that have been damaged in almost all degenerative diseases such as diabetes, myocardial infarction and Parkinson's disease. This review updates the progress of ES cell research in CRT, discusses about the problems encountered in the practical utility of ES cells in CRT and evaluates how far this approach is successful experimentally.     

19F NMR: A promising tool for dynamic conformational studies of G protein-coupled receptors

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A validation of SpekPy: A software toolkit for modelling X-ray tube spectra

PurposeTo validate the SpekPy software toolkit that has been developed to estimate the spectra emitted from tungsten anode X-ray tubes. The model underlying the toolkit introduces improvements upon a well-known semi-empirical model of X-ray emission.Materials and methodsUsing the same theoretical framework as the widely-used SpekCalc software, new electron penetration data was simulated using the Monte Carlo (MC) method, alternative bremsstrahlung cross-sections were applied, L-line characteristic emissions were included, and improvements to numerical methods implemented. The SpekPy toolkit was developed with the Python programming language. The toolkit was validated against other popular X-ray spectrum models (50 to 120 kVp), X-ray spectra estimated with MC (30 to 150 kVp) as well as reference half value layers (HVL) associated with numerous radiation qualities from standard laboratories (20 to 300 kVp).ResultsThe toolkit can be used to estimate X-ray spectra that agree with other popular X-ray spectrum models for typical configurations in diagnostic radiology as well as with MC spectra over a wider range of conditions. The improvements over SpekCalc are most evident at lower incident electron energies for lightly and moderately filtered radiation qualities. Using the toolkit, estimations of the HVL over a large range of standard radiation qualities closely match reference values.ConclusionsA toolkit to estimate X-ray spectra has been developed and extensively validated for central-axis spectra. This toolkit can provide those working in Medical Physics and beyond with a powerful and user-friendly way of estimating spectra from X-ray tubes.     

建立犬肺渐次萎陷动物模型的实验研究

目的:术中定位是微小肺癌手术面临的主要难题,而原因则是肺在术中萎陷造成的巨大形变。我们从研究肺的术中萎陷着手,建立肺渐次萎陷动物模型,模拟肺在术中发生的萎陷,用以研究肺萎陷的过程、规律及影响因素,为微小肺癌的术中定位提供理论基础。本文用渐进的人工气胸模拟肺在术中的渐次萎陷过程,探讨简便、有效的肺渐次萎陷动物模型的制作方法。方法:健康成年犬12只机等分入左、右侧手术组麻醉后移至CT扫描床采用切小口置管和胸腔穿刺两种方式制作人工气胸。向胸膜腔内分次、定量地注射气体,使肺逐渐萎陷直至完全萎陷。通过夹闭气管插管协助稳定肺的萎陷状态。将各萎陷状态分别进行CT扫描。结果:所有实验犬均顺利完成实验麻醉良好,无意外情况发生。9只犬给予气管插管,3只未给予气管插管;4只犬采用切小口置管方式制作气胸,8只采用胸腔穿刺方式。气管插管增加了模型制作难度,但有利于稳定肺的萎陷状态;胸腔穿刺方式相对操作更为简便。随着向胸膜腔内注射气体,肺缓慢而均匀地向肺门方向集中,萎陷进程满意。CT扫描记录了肺从膨胀到萎陷的各阶段,经过后期重建再现了肺的萎陷过程。5只犬出现并发症,均通过改变操作得以纠正,未影响实验进程。结论:本研究建立的肺渐次萎陷动物模型,能够很好地模拟肺在术中的萎陷过程,是研究术中肺萎陷的理想动物模型。     

Joint refinement as a tool for thorough comparison between NMR and X-ray data and structures of HU protein

Joint refinement, i.e., the simultaneous refinement of a structure against both nuclear magnetic resonance (NMR) spectroscopic and X-ray crystallographic data, was performed on the HU protein from Bacillus stearothermophilus (HUBst). The procedure was aimed at investigating the compatibility of the two data sets and at identifying conflicting information. Wherever important differences were found, such as peptide flips in the main-chain conformation, the data were further analyzed to find the cause. The NMR data showed some errors arising either from the manual interpretation of the spectra or from the incorrect account for spin diffusion. The most important artefact inherent to the X-ray data is the crystal packing of the molecules: the effects range from the limitation of the freedom of the flexible parts of the HUBst molecule to possibly one of the peptide flips.     

MetaGen: 从KEGG建模代谢网络的新工具

为便于大规模代谢网络的计算,发展了一款方便实用的工具:MetaGen,对Kyoto Encyclopedia of Genesand Genomes(KEGG)中物种特异的各层次代谢系统进行建模,生成的代谢网络以酶图和通路图的方式表示.利用该工具,对人类代谢系统的bow-tie结构进行了初步研究,并以此为例展示了该工具广阔的应用前景.MetaGen利用KEGGweb服务保证建模数据的可靠性,依靠本地关系数据库加速网络建模过程并提供更多的数据管理和利用方式,并结合高级JAVA技术提高代码的可扩展性.MetaGen完全开源,可直接从http://bnct.sourceforge.net/下载.     

Complete microscale profiling of tumor microangiogenesis: A microradiological methodology reveals fundamental aspects of tumor angiogenesis and yields an array of quantitative parameters for its characterization

Complete profiling would substantially facilitate the fundamental understanding of tumor angiogenesis and of possible anti-angiogenesis cancer treatments. We developed an integrated synchrotron-based methodology with excellent performances: detection of very small vessels by high spatial resolution (~ 1 μm) and nanoparticle contrast enhancement, in vivo dynamics investigations with high temporal resolution (~ 1 ms), and three-dimensional quantitative morphology parametrization by computer tracing. The smallest (3–10 μm) microvessels were found to constitute > 80% of the tumor vasculature and exhibit many structural anomalies. Practical applications are presented, including vessel microanalysis in xenografted tumors, monitoring the effects of anti-angiogenetic agents and in vivo detection of tumor vascular rheological properties.