Rho GTPases show differential sensitivity to nucleotide triphosphate depletion in a model of ischemic cell injury

Rho GTPases are critical for actin cytoskeletal regulation, and alterations in their activity may contribute to altered cytoskeletal organization that characterizes many pathological conditions, including ischemia. G protein activity is a function of the ratio of GTP-bound (active) to GDP-bound (inactive) protein, but the effect of altered energy metabolism on Rho protein activity has not been determined. We used antimycin A and substrate depletion to induce depletion of intracellular ATP and GTP in the kidney proximal tubule cell line LLC-PK10 and measured the activity of RhoA, Rac1, and Cdc42 with GTPase effector binding domains fused to glutathione S-transferase. RhoA activity decreased in parallel with the concentration of ATP and GTP during depletion, so that by 60 min there was no detectable RhoA-GTP, and recovered rapidly when cells were returned to normal culture conditions. Dissociation of the membrane-actin linker ezrin, a target of RhoA signaling, from the cytoskeletal fraction paralleled the decrease in RhoA activity and was augmented by treatment with the Rho kinase inhibitor Y27632. The activity of Cdc42 did not decrease significantly during depletion or recovery. Rac1 activity decreased moderately to a minimum at 30 min of depletion but then increased from 30 to 90 min of depletion, even as ATP and GTP levels continued to fall. Our data are consistent with a principal role for RhoA in cytoskeletal reorganization during ischemia and demonstrate that the activity of Rho GTPases can be maintained even at low GTP concentrations

Rate of Freeze Impacts the Survival and Immune Responses Post Cryoablation of Melanoma.

The emergence of ablative therapies has revolutionized the treatment of inoperable solid tumors. Cryoablation stands out for its uniqueness of operation based on hypothermia, and for its ability to unleash the native tumor antigens, resulting in the generation of anti-tumor immune responses. It is not clearly understood how alterations in the rate of freeze impact the immune response outcomes. In this study, we tested fast freeze and slow freeze rates for their locoregional effectiveness and their ability to elicit immune responses in a B16F10 mouse model of melanoma. Tumor bearing mice treated with fast freeze protocol survived better than the ones treated with slow freeze protocol. Fast freeze resulted in a higher magnitude of CD4 <sup>+</sup> and CD8 <sup>+</sup> T-cell responses, and a significantly extended survival post re-challenge. Thus, fast freeze rate should be applied in any future studies employing cryoablation as an in vivo vaccination tool in conjunction with targeted immunotherapies

Occupational Radiation Exposure Analysis of US ITER DCLL TBM

This report documents an Occupational Radiation Exposure (ORE) analysis that was performed for the US International Thermonuclear Experimental Reactor (ITER) Dual Coolant Lead Lithium (DCLL) Test Blanket Module (TBM). This analysis was performed with the QADMOD dose code for anticipated maintenance activities for this TBM concept and its ancillary systems. The QADMOD code was used to model the PbLi cooling loop of this TBM concept by specifying gamma ray source terms that simulated radioactive material within the piping, valves, heat exchanger, permeator, pump, drain tank, and cold trap of this cooling system. Estimates of the maintenance tasks that will have to be performed and the time required to perform these tasks where developed based on either expert opinion or on industrial maintenance experience for similar technologies. This report details the modeling activity and the calculated doses for the maintenance activities envisioned for the US DCLL TBM

Human brain anatomy reflects separable genetic and environmental components of socioeconomic status

Socioeconomic status (SES) correlates with brain structure, a relation of interest given the long-observed relations of SES to cognitive abilities and health. Yet, major questions remain open, in particular, the pattern of causality that underlies this relation. In an unprecedently large study, here, we assess genetic and environmental contributions to SES differences in neuroanatomy. We first establish robust SES–gray matter relations across a number of brain regions, cortical and subcortical. These regional correlates are parsed into predominantly genetic factors and those potentially due to the environment. We show that genetic effects are stronger in some areas (prefrontal cortex, insula) than others. In areas showing less genetic effect (cerebellum, lateral temporal), environmental factors are likely to be influential. Our results imply a complex interplay of genetic and environmental factors that influence the SES-brain relation and may eventually provide insights relevant to policy

Automatic segmentation of intravital fluorescence microscopy images by K-means clustering of FLIM phasors

Fluorescence lifetime imaging microscopy (FLIM) provides additional contrast for fluorophores with overlapping emission spectra. The phasor approach to FLIM greatly reduces the complexity of FLIM analysis and enables a useful image segmentation technique by selecting adjacent phasor points and labeling their corresponding pixels with different colors. This phasor labeling process, however, is empirical and could lead to biased results. In this Letter, we present a novel and unbiased approach to automate the phasor labeling process using an unsupervised machine learning technique, i.e., K-means clustering. In addition, we provide an open-source, user-friendly program that enables users to easily employ the proposed approach. We demonstrate successful image segmentation on 2D and 3D FLIM images of fixed cells and living animals acquired with two different FLIM systems. Finally, we evaluate how different parameters affect the segmentation result and provide a guideline for users to achieve optimal performance

Functional studies of the kidney of living animals using multicolor 2-photon microscopy

Optical microscopy, when applied to living animals, provides a powerful means of studying cell biology in the most physiologically relevant setting. The ability of two-photon microscopy to collect optical sections deep into biological tissues has opened up the field of intravital microscopy to high-resolution studies of the brain, lens, skin, and tumors. Here we present examples of the way in which two-photon microscopy can be applied to intravital studies of kidney physiology. Because the kidney is easily externalized without compromising its function, microscopy can be used to evaluate various aspects of renal function in vivo. These include cell vitality and apoptosis, fluid transport, receptor-mediated endocytosis, blood flow, and leukocyte trafficking. Efficient two-photon excitation of multiple fluorophores permits comparison of multiple probes and simultaneous characterization of multiple parameters and yields spectral information that is crucial to the interpretation of images containing uncharacterized autofluorescence. The studies described here demonstrate the way in which two-photon microscopy can provide a level of resolution previously unattainable in intravital microscopy, enabling kinetic analyses and physiological studies of the organs of living animals with subcellular resolution

Large-scale 3-dimensional quantitative imaging of tissues: state-of-the-art and translational implications

Recent developments in automated optical sectioning microscope systems have enabled researchers to conduct high resolution, three-dimensional (3D) microscopy at the scale of millimeters in various types of tissues. This powerful technology allows the exploration of tissues at an unprecedented level of detail, while preserving the spatial context. By doing so, such technology will also enable researchers to explore cellular and molecular signatures within tissue and correlate with disease course. This will allow an improved understanding of pathophysiology and facilitate a precision medicine approach to assess the response to treatment. The ability to perform large-scale imaging in 3D cannot be realized without the widespread availability of accessible quantitative analysis. In this review, we will outline recent advances in large-scale 3D imaging and discuss the available methodologies to perform meaningful analysis and potential applications in translational research

Submucosal Tunnel Endoscopic Resection of Gastric Lesion Before Obesity Surgery: a Case Series

Background: Submucosal tumors (SMTs) of the gastrointestinal tract are a rare pathological entity comprising a wide variety of neoplastic and non-neoplastic lesions. Even if most SMTs are benign tumors (e.g., leiomyomas), a smaller portion may have a malignant potential (e.g., gastrointestinal stromal tumor (GIST)). Preoperative diagnosis of SMT in bariatric patients may arise challenging clinical dilemmas. Long-term surveillance may be difficult after bariatric surgery. Moreover, according to SMT location, its presence may interfere with planned surgery. Submucosal tunneling endoscopic resection (STER) has emerged as an effective approach for minimally invasive en bloc excision of SMTs. This is the first case series of STER for SMTs before bariatric surgery. Methods: Seven female patients underwent STER for removal of SMTs before bariatric surgery. All lesions were incidentally diagnosed at preoperative endoscopy. STER procedural steps comprised mucosal incision, submucosal tunneling, lesion enucleation, and closure of mucosal defect. Results: En bloc removal of SMT was achieved in all cases. Mean procedural time was of 45 min (SD 18.6). No adverse event occurred. Mean size of the lesions was 20.6 mm (SD 5.8). Histological diagnoses were 5 leyomiomas, 1 lipoma, and 1 low grade GIST. Bariatric procedure was performed after a mean period of 4.1 months (SD 1.6) from endoscopic resection. Conclusion: STER is a safe and effective treatment for the management of SMT even in bariatric patients awaiting surgery. Preoperative endoscopic resection of SMTs has the advantages of reducing the need for surveillance and removing lesions that could interfere with planned surgery. STER did not altered accomplishment of bariatric procedures