Light Element Imaging Technique at Low Dose Condition by Processing Simultaneously Obtained STEM Images Using a Segmented Detector

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.

As interest in fast real-space frame-rate scanning transmission electron microscopy for both structural and functional characterization of materials increases, so does the need for precise and fast electron detection techniques. Electron counting, with monolithic, segmented, or 4D detectors, has been explored for many years. Recent studies have shown that a retrofittable signal digitizer for a monolithic or segmented detector can be a sustainable and accessible way to enhance the performance of existing detectors, especially for imaging at fast scan speeds. Since such signal digitization uses a threshold on the gradient of the detector signal to identify electron events, appropriate threshold choice is key. Previously, this threshold has been set manually by the operator and is therefore inherently susceptible to human bias. In this work, we introduce an auto-thresholding approach for electron counting to determine the optimal threshold by maximizing the difference in identified counts from a stream with real electron events and a stream with only noise. This leads to easier operation, increased throughput and eliminates human bias in signal digitization. When pixel dwell time becomes shorter than scintillator response time, digitization of the detector signal is needed to avoid artefacts in STEM images. Optimizing the threshold for this digitization process automatically is crucial to achieve high-quality quantitative digitized images, free of human bias for what threshold yields the best digitization.

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.

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.

We have been developing a software system, called Leginon, for the control and acquisition of images from a transmission electron microscope. This software allows for the automatic acquisition of images under low dose conditions from a specimen embedded in vitreous ice. Thousands of images are collected during each session at the microscope. These images are collected at various magnifications and under a variety of conditions. For example, images are collected of the entire grid, individual grid squares, individual holes within the square, and several high magnification images are acquired for one to several targets within each hole (figure 1). in addition, for each identified hole an image is acquired at the low dose focus position. Power spectra are also calculated for each of the high magnification images as well as for the focus images. For managing these images we are using a relational database management system. We have also developed a bench of tools to store, access, and display the data as well as tools for extracting information from the database.A database is a structured collection of data in which the information is classified by categories. Each category is then stored in a table and these tables are linked together by defined relations. Data may be combined from several tables on request. We have been using MySQL as our relational database management system. This allows us both to manage the images and to refer each image to its real context. in other words, through the database, we can keep track of information related to the microscope settings (e.g. defocus, electron dose, magnification, goniometer coordinates etc.), the type of image, and overall information about the specimen and the experiment.

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.

Many nonmalignant hematologic disorders could potentially be treated by genetic correction of as few as 5-10% of target lineage cells. However, immune system clearance of cells expressing gene products perceived as foreign could be limiting. There is evidence that tolerance to foreign proteins can result when myeloablative conditioning is used, but this limits the overall applicability of such techniques. Therefore, we sought to evaluate the engraftment of hematopoietic stem cells carrying a foreign transgene after low-dose irradiation by comparing in vivo survival of murine long-term repopulating cells (LTRC) transduced with either a retroviral vector expressing the bacterial neomycin phosphotransferase gene (neo) or a vector containing neo gene sequences but modified to prevent protein expression (nonexpression). First, marrow cells from congenic donors were transduced with either vector and transplanted into recipients treated with standard dose irradiation of 800 rads. High-level engraftment and gene marking resulted, without differences in the marking levels or pattern of persistence of the cells between cells transduced with either vector. Low-dose irradiation at 100 rads was tested using higher cell doses. Marking levels as high as 10% overall were obtained, again with no differences between mice receiving cells transduced with the neo versus the nonexpression vectors. To investigate a potentially more immunogenic protein, marrow cells were transduced with a vector containing the green fluorescent protein (GFP) gene, and their persistence was studied in recipient mice receiving 100 rads. Stable GFP expression in 5-10% of circulating cells was observed long term. We conclude that even with very low dose conditioning, engraftment by genetically modified LTRC cells at clinically significant levels can be achieved without evidence for clearance of cells known to be expressing immunogenic proteins.

JADAS: a customizable automated data acquisition system and its application to ice-embedded single particles.

Three-dimensional reconstruction of icosahedral particles from single micrographs in real time at the microscope.

Heavy alcohol consumption (Testa & Parks, 1996) and childhood sexual abuse (CSA; Messman-Moore & Long, 2003) have been associated with adult sexual victimization. We examined the social behavior of 42 women under two alcohol conditions (high dose and low dose) in a bar laboratory. Women were videotaped interacting with a man they had just met. Women in the higher dose condition engaged in more open body position and talked, stood, and walked more than women in the lower dose condition. These behaviors are consistent with signs of intoxication or romantic interest. The women in the high-dose condition also frowned more than women in the low-dose condition. An increase in frowning could indicate less comfort or may be considered consistent with an increase in animation during the social interaction given the concomitant increase in other behaviors. Thus, the nonverbal behavior of women in the high-dose condition could be interpreted as mixed signals. CSA victims exhibited fewer head movements (e.g., nods), were less animated, and frowned more than non-CSA victims. These behaviors convey reticence or possibly even anxiety or discomfort during the social interaction. Thus, the nonverbal behavior of women with a history of CSA may convey an unease that could be viewed by a potential perpetrator as vulnerability. Our findings suggest that both acute alcohol consumption and history of CSA may influence nonverbal social behavior and may influence risk for sexual assault by sending mixed cues of romantic interest or signs of vulnerability to potential perpetrators.

A multistage cancer model that describes the putative rate-limiting steps in carcinogenesis is developed and used to investigate the potential impact on cumulative lung cancer incidence of the hormesis mechanisms suggested by Feinendegen and Pollycove. In the model, radiation and endogenous processes damage the DNA of target cells in the lung. Some fraction of the misrepaired or unrepaired DNA damage induces genomic instability and, ultimately, leads to the accumulation of malignant cells. The model explicitly accounts for cell birth and death processes, the clonal expansion of initiated cells, malignant conversion, and a lag period for tumor formation. Radioprotective mechanisms are incorporated into the model by postulating dose and dose-rate-dependent radical scavenging. The accuracy of DNA damage repair also depends on dose and dose rate. As currently formulated, the model is most applicable to low-linear-energy-transfer (LET) radiation delivered at low dose rates. Sensitivity studies are conducted to identify critical model inputs and to help define the shapes of the cumulative lung cancer incidence curves that may arise when dose and dose-rate-dependent cellular defense mechanisms are incorporated into a multistage cancer model. For lung cancer, both linear no-threshold (LNT-), and non-LNT-shaped responses can be obtained. If experiments demonstrate that the effects of DNA damage repair and radical scavenging are enhanced at least three-fold under low-dose conditions, our studies would support the existence of U-shaped responses. The overall fidelity of the DNA damage repair process may have a large impact on the cumulative incidence of lung cancer. The reported studies also highlight the need to know whether or not (or to what extent) multiply damaged DNA sites are formed by endogenous processes. Model inputs that give rise to U-shaped responses are consistent with an effective cumulative lung cancer incidence threshold that may be as high as 300 mGy (4 mGy per year for 75 years) for low-LET radiation.

This study investigated the impact of metallic nanoparticles (NPs) containing copper, silver, copper oxide, and zinc oxide, recognized as potential pollutants, on the structural and compositional aspects of soil microbial communities in comparison to their bulk counterparts. The influence of these nanoparticles was examined at two distinct accumulation levels within the soil ecosystem.The potential effects of metallic nanoparticles in comparison to their bulk counterparts were evaluated in a pot experiment under controlled environmental conditions. High-throughput sequencing of PCR-amplified 16S rRNA and ITS2 marker genes was employed to analyze the impact of NPs and counterparts on bacterial and fungal rhizospheric communities using two dosage levels.Bioinformatic analysis of the obtained sequencing results revealed a distinct metal-dependent differentiation in bacterial and fungal soil community structures. Silver-containing treatments exhibited an enhanced ability to induce changes in both bacterial and fungal communities compared to other metals. Furthermore, treatment dose had a profound differentiation effect on the two microbial communities. The low dose notably influenced bacterial communities to a greater extent compared to the high dose, whereas fungal communities exhibited significant alterations under high-dose conditions rather than under low-dose conditions.This study was co-financed by the European Regional Development Fund of the European Union and Greek national funds through the Rural Development Program (RDP/ΠΑΑ) 2014 – 2020, under the call "Cooperation for environmental projects, environmental practices and actions for climate change” (project code: Μ16SΥΝ2-00354) and by the European Union- Next Generation EU, Greece 2.0 National Recovery and Resilience plan (project code: TAEDR-0535675)

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.

CANNABIS USE AND PSYCHOTIC EXPERIENCES IN A HEALTHY POPULATION SAMPLE

A methodology recently developed by the U.S. EPA for estimating the carcinogenic risks from ionizing radiation is described. For most cancer sites, the risk model is one in which age-specific, relative risk coefficients are obtained by taking a geometric mean of the coefficients derived from the atomic bomb survivor data using two different methods for transporting risks from the Japanese to the U.S. population. The risk models are applied to estimate organ-specific risks per unit dose for a stationary population with mortality rates governed by 1980 U.S. vital statistics. With the exception of breast cancer, low-LET radiogenic cancer risk estimates are reduced by a factor of 2 at low doses and dose rates compared to acute high dose exposure conditions. For low dose (or dose rate) conditions, the risk of inducing a premature cancer death from uniform, whole body, low-LET irradiation is calculated to be 5.1 x 10(-2) Gy-1. Neglecting nonfatal skin cancers, the corresponding incidence risk is 7.6 x 10(-2) Gy-1. High-LET (alpha particle) risks are presumed to increase linearly with dose and to be independent of dose rate. High-LET risks are estimated to be 20 times the low-LET risks estimated under low dose rate conditions, except for leukemia and breast cancer where RBEs of 1 and 10 are adopted, respectively.