Application of a SQL Database for Automated Image Acquisition and Analysis for CRYOEM

Study objectives: To investigate the effect of differing doses of the combination of acetazolamide and dronabinol (Aceta-Dro) on the obstructive sleep apnea (OSA) endotypes and their relationship to OSA severity. Methods: We retrospectively analysed data from a double-blind, randomized, placebo-controlled trial of 3 different doses of Aceta-Dro [Low (125mg/2.5 mg), Medium (250mg/5 mg), High (500mg/10 mg)] in OSA patients. OSA endotypes (collapsibility [VPASSIVE], Loop gain [LG], Arousal threshold [ArTH], and muscle compensation [VCOMP]) were extracted from polysomnography signals using validated algorithms. Linear mixed models were employed to assess the impact of treatment condition on the endotypes. Linear regressions assessed the relationship between the changes in endotypes vs. apnea-hypopnea index (AHI) and to predict treatment response from baseline endotypes. Results: Relative to placebo, Aceta-Dro dose-dependently reduced LG by 11% (p = 0.18), 18.5% (p = 0.022) and 20.5% (p = 0.016). The reduction in LG was associated with a decrease in AHI in only the high dose condition (R2 = 0.78, p = 0.004). Aceta-Dro improved VPASSIVE in the low dose condition (+13%, p = 0.005) only, which was strongly associated with AHI reduction (R2 = 0.71, p = 0.005). Aceta-Dro had no impact on VCOMP or ArTH. A higher baseline LG predicted treatment response in the high dose (R2 = 0.56, p = 0.03), with a similar trend observed in the low dose group. More severe collapsibility (lower VPASSIVE) predicted treatment response to both low (R2 = 0.46, p = 0.07) and medium dose (R2 = 0.50, p = 0.03) conditions.

Cryo-electron microscopy is becoming an increasingly powerful tool for solving the structure of protein complexes and has the potential to address problems that cannot be solved using other methods. The field however suffers from several major disadvantages related to the time required to acquire, process and analyze the data and the tedium of using the current prevailing methods. We have for some time been working towards the goal of developing a system that will result in a 3D map of a macromolecular structure automatically and within hours of inserting a specimen into a transmission electron microscope. We propose that these automated methods for data collection and analysis will have a significant impact in transferring the cryo-electron microscopy technology to the general biological community as well as in increasing the volume of data that can be collected during a single session at the microscope. The Leginon system that we have developed is designed to emulate all of the decisions and actions of a highly trained microscopist in collecting data from a vitreous ice specimen. These include identifying suitable areas of vitreous ice at low magnification, determining the presence and location of specimen on the grid, automatically adjusting imaging parameters (focus, astigmatism) under low dose conditions and acquiring images at high magnification to either film or a digital camera.

Theory of the spatial resolution of (scanning) transmission electron microscopy in liquid water or ice layers

Databases are used in many facets of society by an array of people. While the obvious platform for most database applications has been a relational Data Base management Systems (DBMS), the object-oriented programming languages which are widely used in software development are not well matched to their development. In many ways it is obvious that there is an ‘object-relational’ impedance and mismatch. There are currently only a few courses about object-oriented database development in universities. In this paper, an object-oriented database named db4o is introduced as a viable alternative to traditional database development methods. It is now used in our newly developed object-oriented database course. With a visual object management viewer, db4o helps students to understand basic object-oriented database conception and to grasp programming with the object-oriented programming language.

An efficient database is an essential component of organizing diverse information on image metadata and patient information for research in medical imaging. This paper describes the design, development and deployment of a large database system serving as a brain image repository that can be used across different platforms in various medical researches. It forms the infrastructure that links hospitals and institutions together and shares data among them. The database contains patient-, pathology-, image-, research- and management-specific data. The functionalities of the database system include image uploading, storage, indexing, downloading and sharing as well as database querying and management with security and data anonymization concerns well taken care of. The structure of database is multi-tier client-server architecture with Relational Database Management System, Security Layer, Application Layer and User Interface. Image source adapter has been developed to handle most of the popular image formats. The database has a user interface based on web browsers and is easy to handle. We have used Java programming language for its platform independency and vast function libraries. The brain image database can sort data according to clinically relevant information. This can be effectively used in research from the clinicians’ points of view. The database is suitable for validation of algorithms on large population of cases. Medical images for processing could be identified and organized based on information in image metadata. Clinical research in various pathologies can thus be performed with greater efficiency and large image repositories can be managed more effectively. The prototype of the system has been installed in a few hospitals and is working to the satisfaction of the clinicians.

Electron tomography is the most widely applicable method for obtaining three-dimensional information by electron microscopy. It is, in fact, the only method suitable for investigating pleomorphic structures, such as many supramolecular assemblies, organelles and cells. With the recent development of automated low-dose data-acquisition schemes, it is now possible to study molecules and cells embedded in vitreous ice. This opens up new horizons for investigating the functional organization of cellular components with minimal perturbation of the cellular context. In the present study we used automated electron tomography in conjunction with cryomicroscopy to reveal the internal organization and ultrastructure of mitochondria. The whole isolated cell organelles from Neurospora crassa were quick-frozen and examined in vitrified ice. Single-axis tilt series were recorded with a Philips CM 120 Biotwin under low-dose conditions; the estimated total exposure was 10,000 e−nm−2. The tilt increment was 1° and the tilt series ranged from −65° to +65°. In order to enhance the contrast of the rather thick samples zero-loss energy filtering was employed. The images were recorded with a Ik × Ik CCD camera. The experimental setup has been described by Koster et al2 Three-dimensional reconstructions were performed by weighted backprojection. Prior to the three-dimensional visualization of 3-D data sets a denoising technique based on nonlinear anisotropic diffusion was applied (Figs. 2, 3).

Size distributions of cellulose nanocrystals (CNCs), extracted from softwood pulp via strong sulfuric acid hydrolysis, exhibit large variability when analyzed from transmission electron microscopy (TEM) images. In this article, the causes of this variability are studied and discussed. In order to obtain results comparable with those reported, a reference material of CNCs (CNCD-1) was used to evaluate size distribution. CNC TEM specimens were prepared as-stained and dried with a rapid-flushing staining method or hydrated and embedded in vitreous ice with the plunge-freezing method. Several sets of bright-field TEM (BF-TEM), annular dark-field scanning TEM (ADF-STEM) and cryogenic-TEM (cryo-TEM) images were acquired for size distribution analysis to study the contributing factors. The rapid-flushing staining method was found to be the most effective for contrast enhancement of CNCs, not only revealing the helical structure of single CNCs but also resolving the laterally jointed CNCs. During TEM specimen preparation, CNCs were fractionated on TEM grids driven by the coffee-ring effect, as observed from contrast variation of CNCs with a stain-depth gradient. From the edge to the center of the TEM grids, the width of CNCs increases, while the aspect ratio (length to width) decreases. This fractionated dispersion of CNCs suggests that images taken near the center of a droplet would give a larger mean width. In addition to particle fractionation driven by the coffee-ring effect, the arrangement and orientation of CNC particles on the substrate significantly affect the size measurement when CNC aggregation cannot be resolved in images. The coexistence of asymmetric cross-section CNC particles introduces a large variation in size measurement, as TEM images of CNCs are mixed projections of the width and height of particles. As a demonstration of how this contributes to inflated size measurement, twisted CNC particles, rectangular cross-section particles and end-to-end jointed CNCs were revealed in reconstructed three-dimensional (3D) micrographs by electron tomography (ET).

When searching for viruses in an electron microscope the sample grid constitutes an enormous search area. Here, we present methods for automating the image acquisition process for an automatic virus diagnostic application. The methods constitute a multi resolution approach where we first identify the grid squares and rate individual grid squares based on content in a grid overview image and then detect regions of interest in higher resolution images of good grid squares. Our methods are designed to mimic the actions of a virus TEM expert manually navigating the microscope and they are also compared to the expert’s performance. Integrating the proposed methods with the microscope would reduce the search area by more than 99.99 % and it would also remove the need for an expert to perform the virus search by the microscope.

Cannabidiol (CBD) is a non-psychoactive cannabinoid purported to reduce symptoms of discomfort. Individuals are now using CBD to treat symptoms of multiple sclerosis, seizures, and chronic pain. Animal models indicate that CBD may be effective at reducing inflammation post fatiguing exercise. However, little evidence is available to evaluate these findings in humans. Therefore, the purpose of this investigation was to evaluate the impact of two doses of CBD oil on inflammation (IL-6), performance, and pain after an eccentric loading protocol. Participants (n = 4) participated in three conditions (placebo, low dose, and high dose), in this randomized, counterbalanced design. Each condition took 72 hours to complete, with a 1-week washout period between conditions. At the beginning of each week, participants were subjected to a loading protocol of six sets of ten eccentric only repetitions in the single-arm bicep curl. Participants consumed capsules of either a placebo, low dose (2mg/kg) or high dose (10mg/kg) of CBD oil immediately following the session and continued every twelve hours for 48 hours. Venipunctures were taken before exercise and repeated at 24, 48, and 72 hours post exercise. Blood samples were centrifuged for 15 minutes in gel and lithium heparin vacutainers. Plasma was separated from cells and stored at -80° until analysis. Samples were analyzed using an immunometric assay for IL-6 (ELISA). Data were analyzed using a three (condition) by four (time) repeated measure ANOVA. There were no differences in inflammation between conditions (F(2,6) = 0.726, p = 0.522, np 2 = 0.195) or across time (F(3,9) = 0.752, p = 0.548, np 2 = 0.200), handgrip strength between conditions (F(2,6) = 0.542, p = 0.607, np 2 = .153) or across time (F(3,9) = 2.235, p = .153, np 2 = .427), or bicep curl strength between conditions (F(2,6) = 0.675, p = 0.554, np 2 = .184) or across time (F(3,9) = 3.513, p = .150, np 2 = .539). There were no differences in pain between conditions (F(2,6) = 0.495, p = 0.633, np 2 = .142), but there was a difference across time (F(3,9) = 7.028, p = .010, np 2 = .701). There were no significant interactions to note. Although there was no statistical significance between conditions (likely due to the low sample size), there was a visible increase in IL-6 48 (4.88 ± 6.53) and 72 hours (3.12 ± 4.26) post exercise in the placebo condition which was not observed in the low (48: 0.35 ± 2.22; 72: 1.34 ± 5.6) and high dose condition (48: 1.34 ± 1.34; 72: -0.79 ± 5.34). Future investigations should consider implementing eccentric resistance training across a larger portion of the body to improve ecological validity of the exercise. A larger sample would reduce risk of researchers committing a type II statistical error and give strength to detecting differences between conditions.

The long term goal of this program is to determine the fundamental rules needed to predict the type and yield of damage produced in DNA due to direct effects of ionizing radiation. The focus is on damage to the sugar-phosphate backbone, damage that would lead to strand breaks. Model systems have been chosen that permit various aspects of this problem to be investigated. The emphasis will be on single crystals of monosaccharides, nucleosides, and nucleotides but will also include some powder work on polynucleotides. In these model systems, free radical products and reactions are observed by electron spin resonance (ESR) and electron nuclear double resonance (ENDOR) techniques. The information thus gained is used in constructing rules that predict what primary free radicals are formed in single crystals of model compounds and the reactions stemming from the primary radicals. The formulation of a set of rules that work in model systems will represent a major advance toward formulating a set of rules that predict the direct damage in DNA itself. In a broader context this program is part of the effort to understand and predict the effects of exposure to ionizing radiation received at low dose rates over long periods of time. Assessment of low dose effects requires a basic understanding of the action of radiation at the molecular level. By contributing to that basic understanding, this program will help solve the problems of risk assessment under low dose conditions. 5 refs., 3 figs.

We discuss the important database theme of Data Independence and how object oriented database applications are failing to perceive its benefits, and instead put too much reliance on encapsulation. The P/FDM object-oriented database is based on the functional data model and has a modular design based on data independence, which allows alternative kinds of object storage to be used. In this work, a relational database management system has been used to provide object storage, and we describe how the data access routines have been implemented. The principal query language used with P/FDM is Daplex, which is normally translated to Prolog, including calls to the basic data access routines. The query is optimised to minimise the expected number of calls. This gives very general method execution and pattern matching search. However, much better performance can be achieved for simpler data-intensive Daplex queries against a relational storage module by translating these to a single SQL statement, which is possible because of data independence.

CANNABIS USE AND PSYCHOTIC EXPERIENCES IN A HEALTHY POPULATION SAMPLE

The current protocol describes the modifications of the transmission electron microscope (TEM) setup for in situ light-induced observations. A glass optical fiber inserted into the electron column above the objective lens polepiece, and a laser, an adjustable light source, was used to fabricate the device. After the illuminator has been calibrated using an external measuring system, it allows one to adjust the intensity of the lighting to the needs of the observed process. This lighting system was utilized to image antimicrobial photodynamic therapy phenomena, which are currently the subject of intense research. The sample was prepared by spotting a suspension of bacteria on a carbon, graphene, or silicon nitride substrate, blotting the excess solution, spotting the photosensitizer solution, blotting the excess liquid again, and then assembling the liquid cell with a second substrate or graphene film. The process of the imaging experiment itself includes choosing the right place for observation with the use of low magnification and a minimum dose of electrons, and then cyclical activation of the light source to capture subsequent images at specified intervals with the minimum amount of electrons necessary. The electron dose of each exposure and the time and intensity of lighting used need to be carefully recorded due to the complexity of the observed phenomena as, at the same time, the process is both light- and electron-driven. After the actual experiment is performed, additional control observations must be made, in which the same doses of electrons are used but without additional light influence and smaller doses of electrons are used for higher doses of light. This makes it possible to distinguish light-induced microstructural effects from those caused by electrons in both the fields of life and materials science.

Gait analysis is used to quantify changes in motor function in many rodent models of disease. Despite the importance of assessing gait and motor function in many areas of research, the available commercial options have several limitations such as high cost and lack of accessible, open code. To address these issues, we developed PrAnCER, Paw-Print Analysis of Contrast-Enhanced Recordings, for automated quantification of gait. The contrast-enhanced recordings are produced by using a translucent floor that obscures objects not in contact with the surface, effectively isolating the rat's paw prints as it walks. Using these videos, our simple software program reliably measures a variety of spatiotemporal gait parameters. To demonstrate that PrAnCER can accurately detect changes in motor function, we employed a haloperidol model of Parkinson's disease (PD). We tested rats at two doses of haloperidol: high dose (0.30 mg/kg) and low dose (0.15 mg/kg). Haloperidol significantly increased stance duration and hind paw contact area in the low dose condition, as might be expected in a PD model. In the high dose condition, we found a similar increase in contact area but also an unexpected increase in stride length. With further research, we found that this increased stride length is consistent with the bracing-escape phenomenon commonly observed at higher doses of haloperidol. Thus, PrAnCER was able to detect both expected and unexpected changes in rodent gait patterns. Additionally, we confirmed that PrAnCER is consistent and accurate when compared with manual scoring of gait parameters.

Journal Article Light Element Imaging Technique at Low Dose Condition by Processing Simultaneously Obtained STEM Images Using a Segmented Detector Get access Kousuke Ooe, Kousuke Ooe Institute of Engineering Innovation, School of Engineering, the University of Tokyo, Tokyo, Japan Search for other works by this author on: Oxford Academic Google Scholar Takehito Seki, Takehito Seki Institute of Engineering Innovation, School of Engineering, the University of Tokyo, Tokyo, Japan Search for other works by this author on: Oxford Academic Google Scholar Yuichi Ikuhara, Yuichi Ikuhara Institute of Engineering Innovation, School of Engineering, the University of Tokyo, Tokyo, JapanNanostructure Research Laboratory, Japan Fine Ceramics Center, Nagoya, Japan Search for other works by this author on: Oxford Academic Google Scholar Naoya Shibata Naoya Shibata Institute of Engineering Innovation, School of Engineering, the University of Tokyo, Tokyo, JapanNanostructure Research Laboratory, Japan Fine Ceramics Center, Nagoya, Japan Search for other works by this author on: Oxford Academic Google Scholar Microscopy and Microanalysis, Volume 25, Issue S2, 1 August 2019, Pages 484–485, https://doi.org/10.1017/S1431927619003155 Published: 01 August 2019