Targeted studies using serial block face and focused ion beam scan electron microscopy

This protocol allows for the efficient and effective imaging of cell or tissue samples in three dimensions at the resolution level of electron microscopy. For many years electron microscopy (EM) has remained an inherently two-dimensional technique. With the advent of serial scanning electron microscope imaging techniques (volume EM), using either an integrated microtome or focused ion beam to slice then view embedded tissues, the third dimension becomes easily accessible. Serial block face scanning electron microscopy (SBF-SEM) uses an ultramicrotome enclosed in the SEM chamber. It has the capability to handle large specimens (1,000 mu m x 1,000 mu m) and image large fields of view at small X,Y pixel size, but is limited in the Z dimension by the diamond knife. Focused ion beam SEM (FIB-SEM) is not limited in 3D resolution, (isotropic voxels of <= 5 nm are achievable), but the field of view is much more limited. This protocol demonstrates a workflow for combining the two techniques to allow for finding individual regions of interest (ROls) in a large field and then imaging the subsequent targeted volume at high isotropic voxel resolution. Preparing fixed cells or tissues is more demanding for volume EM techniques due to the extra contrasting needed for efficient signal generation in SEM imaging. Such protocols are time consuming and labor intensive. This protocol also incorporates microwave assisted tissue processing facilitating the penetration of reagents, which reduces the time needed for the processing protocol from days to hours

The Special Case of Sea Mines

In this chapter, work carried out at the Royal Military Academy regarding sea mines and mine countermeasures is summarized. Three sensors used for the detection and identification of sea mines are studied here: sonar, gradiometer and infrared camera. These sensors can be applied to detect different types of sea mines. Some signal and image processing techniques developed to extract relevant information for the detection of underwater objects are presented in this chapter. These techniques are validated using data collected in the frame of different European and NATO projects

An evaluation of pixel-based methods for the detection of floating objects on the sea surface

Ship-based automatic detection of small floating objects on an agitated sea surface remains a hard problem. Our main concern is the detection of floating mines, which proved a real threat to shipping in confined waterways during the first Gulf War, but applications include salvaging, search-and-rescue operation, perimeter, or harbour defense. Detection in infrared (IR) is challenging because a rough sea is seen as a dynamic background of moving objects with size order, shape, and temperature similar to those of the floating mine. In this paper we have applied a selection of background subtraction algorithms to the problem, and we show that the recent algorithms such as ViBe and behaviour subtraction, which take into account spatial and temporal correlations within the dynamic scene, significantly outperformthe more conventional parametric techniques, with only little prior assumptions about the physical properties of the scene

Three-dimensional visualization of APEX2-tagged Erg11 in Saccharomyces cerevisiae using Focused Ion Beam Scanning Electron Microscopy

The determination of the exact location of a protein in the cell is essential to the understanding of biological processes. Here, we report for the first time the visualization of a protein of interest in Saccharomyces cerevisiae using focused ion beam scanning electron microscopy (FIB-SEM). As a proof of concept, the integral endoplasmic reticulum (ER) membrane protein Erg11 has been C-terminally tagged with APEX2, which is an engineered peroxidase that catalyzes an electron-dense deposition of 3,3'-diaminobenzidine (DAB), as such marking the location of the fused protein of interest in electron microscopic images. As DAB is unable to cross the yeast cell wall to react with APEX2, cell walls have been partly removed by the formation of spheroplasts. This has resulted in a clear electron-dense ER signal for the Erg11 protein using FIB-SEM. With this study, we have validated the use of the APEX2 tag for visualization of yeast proteins in electron microscopy. Furthermore, we have introduced a methodology that enables precise and three-dimensional (3D) localization studies in yeast, with nanometer resolution and without the need for antibody staining. Because of these properties, the described technique can offer valuable information on the molecular functions of studied proteins. IMPORTANCE With this study, we have validated the use of the APEX2 tag to define the localization of proteins in the model yeast S. cerevisiae. As such, FIB-SEM can identify the exact 3D location of a protein of interest in the cell with nanometerscale resolution. Such detailed imaging could provide essential information on the elucidation of various biological processes. APEX2, which adds electron density to a fused protein of interest upon addition of the substrate DAB, originally was used in mammalian studies. With this study, we expand its use to protein localization studies in one of the most important models in molecular biology

Statistical real-time model for performance prediction of ship detection from microsatellite electro-optical imagers

For locating maritime vessels longer than 45 meters, such vessels are required to set up an Automatic Identification System (AIS) used by vessel traffic services. However, when a boat is shutting down its AIS, there are no means to detect it in open sea. In this paper, we use Electro-Optical (EO) imagers for noncooperative vessel detection when the AIS is not operational. As compared to radar sensors, EO sensors have lower cost, lower payload, and better computational processing load. EO sensors are mounted on LEO microsatellites. We propose a real-time statistical methodology to estimate sensor Receiver Operating Characteristic (ROC) curves. It does not require the computation of the entire image received at the sensor. We then illustrate the use of this methodology to design a simple simulator that can help sensor manufacturers in optimizing the design of EO sensors for maritime applications

AAV-Tau Mediates Pyramidal Neurodegeneration by Cell-Cycle Re-Entry without Neurofibrillary Tangle Formation in Wild-Type Mice

In Alzheimer's disease tauopathy is considered secondary to amyloid, and the duality obscures their relation and the definition of their respective contributions

Factors associated with self-perceived burden to the primary caregiver in older patients with hematologic malignancies: an exploratory study

Objective: Although cancer patients frequently experience self-perceived burden to others, this perception has not been enough studied. The aim of this study was to investigate the prevalence of selfperceived burden to the primary caregiver (SPB-PC) and associated factors in an older patient population with hematologic malignancies at the time of chemotherapy initiation. Methods: In total, 166 consecutive patients with hematologic malignancies aged ≥65 years were recruited at the time of chemotherapy initiation. Patients’ SPB-PC was assessed using a 100-mm visual analogue scale (VAS). Characteristics potentially associated with SPB-PC, including sociodemographic and medical characteristics, physical functioning status (Karnofsky performance score, activities of daily living (ADL)/instrumental ADL), symptoms (fatigue, pain, nausea, quality of life), psychological distress (Hospital Anxiety and Depression Scale (HADS)), perceived cognitive function (Functional Assessment of Cancer Therapy Cognitive (FACT-Cog) Scale), and patients’/primary caregivers’ personal relationship characteristics (family tie, support), were assessed. Results: Thirty-five percent of patients reported moderate to severe SPB-PC (VAS ≥ 50 mm). Patients’ SPB-PC was associated with lower Karnofsky performance (β = 0.135, p = 0.058) and ADL (β = 0.148, p = 0.037) scores, and higher HADS (β = 0.283, p<0.001) and FACT-Cog perceived cognitive impairments subscale (β = 0.211, p = 0.004) scores. The proportion of explained variance was 23.5%. Conclusions: Health care professionals should be aware that about one third of older cancer patients experience moderate to severe SPB-PC at the time of chemotherapy initiation. They should adapt their support of patients who report such a feeling

Stellate cells, hepatocytes, and endothelial cells imprint the Kupffer cell identity on monocytes colonizing the liver macrophage niche

Macrophages are strongly adapted to their tissue of residence. Yet, little is known about the cell-cell interactions that imprint the tissue-specific identities of macrophages in their respective niches. Using conditional depletion of liver Kupffer cells, we traced the developmental stages of monocytes differentiating into Kupffer cells and mapped the cellular interactions imprinting the Kupffer cell identity. Kupffer cell loss induced tumor necrosis factor (TNF)- and interleukin-1 (IL-1) receptor-dependent activation of stellate cells and endothelial cells, resulting in the transient production of chemokines and adhesion molecules orchestrating monocyte engraftment. Engrafted circulating monocytes transmigrated into the perisinusoidal space and acquired the liver-associated transcription factors inhibitor of DNA 3 (ID3) and liver X receptor-alpha (LXR-alpha). Coordinated interactions with hepatocytes induced ID3 expression, whereas endothelial cells and stellate cells induced LXR-alpha via a synergistic NOTCH-BMP pathway. This study shows that the Kupffer cell niche is composed of stellate cells, hepatocytes, and endothelial cells that together imprint the liver-specific macrophage identity

Osteopontin expression identifies a subset of recruited macrophages distinct from Kupffer cells in the fatty liver

Metabolic-associated fatty liver disease (MAFLD) represents a spectrum of disease states ranging from simple steatosis to non-alcoholic steatohepatitis (NASH). Hepatic macrophages, specifically Kupffer cells (KCs), are suggested to play important roles in the pathogenesis of MAFLD through their activation, although the exact roles played by these cells remain unclear. Here, we demonstrated that KCs were reduced in MAFLD being replaced by macrophages originating from the bone marrow. Recruited macrophages existed in two subsets with distinct activation states, either closely resembling homeostatic KCs or lipid-associated macrophages (LAMs) from obese adipose tissue. Hepatic LAMs expressed Osteopontin, a biomarker for patients with NASH, linked with the development of fibrosis. Fitting with this, LAMs were found in regions of the liver with reduced numbers of KCs, characterized by increased Desmin expression. Together, our data highlight considerable heterogeneity within the macrophage pool and suggest a need for more specific macrophage targeting strategies in MAFLD

Analysis of transition state mimicry by tight binding aminothiazoline inhibitors provides insight into catalysis by human : O-GlcNAcase

The modification of nucleocytoplasmic proteins with O-linked N-Acetylglucosamine (O-GlcNAc) plays diverse roles in multicellular organisms. Inhibitors of O-GlcNAc hydrolase (OGA), the enzyme that removes O-GlcNAc from proteins, lead to increased O-GlcNAc levels in cells and are seeing widespread adoption in the field as a research tool used in cells and in vivo. Here we synthesize and study a series of tight binding carbohydrate-based inhibitors of human OGA (hOGA). The most potent of these 2′-Aminothiazolines binds with a sub-nanomolar Ki value to hOGA (510 ± 50 pM) and the most selective has greater than 1800000-fold selectivity for hOGA over mechanistically related human lysosomal β-hexosaminidase. Structural data of inhibitors in complex with an hOGA homologue reveals the basis for variation in binding among these compounds. Using linear free energy analyses, we show binding of these 2′-Aminothiazoline inhibitors depends on the pKa of the aminothiazoline ring system, revealing the protonation state of the inhibitor is a key driver of binding. Using series of inhibitors and synthetic substrates, we show that 2′-Aminothiazoline inhibitors are transition state analogues of hOGA that bind to the enzyme up to 1-million fold more tightly than the substrate. These collective data support an oxazoline, rather than a protonated oxazolinium ion, intermediate being formed along the reaction pathway. Inhibitors from this series will prove generally useful tools for the study of O-GlcNAc. The new insights gained here, into the catalytic mechanism of hOGA and the fundamental drivers of potency and selectivity of OGA inhibitors, should enable tuning of hOGA inhibitors with desirable properties