Prediction of lymph node metastasis in early colorectal cancer based on histologic images by artificial intelligence.

Risk evaluation of lymph node metastasis (LNM) for endoscopically resected submucosal invasive (T1) colorectal cancers (CRC) is critical for determining therapeutic strategies, but interobserver variability for histologic evaluation remains a major problem. To address this issue, we developed a machine-learning model for predicting LNM of T1 CRC without histologic assessment. A total of 783 consecutive T1 CRC cases were randomly split into 548 training and 235 validation cases. First, we trained convolutional neural networks (CNN) to extract cancer tile images from whole-slide images, then re-labeled these cancer tiles with LNM status for re-training. Statistical parameters of the tile images based on the probability of primary endpoints were assembled to predict LNM in cases with a random forest algorithm, and defined its predictive value as random forest score. We evaluated the performance of case-based prediction models for both training and validation datasets with area under the receiver operating characteristic curves (AUC). The accuracy for classifying cancer tiles was 0.980. Among cancer tiles, the accuracy for classifying tiles that were LNM-positive or LNM-negative was 0.740. The AUCs of the prediction models in the training and validation sets were 0.971 and 0.760, respectively. CNN judged the LNM probability by considering histologic tumor grade.

Cryo-EM in molecular and cellular biology.

This review summarizes the current state of methods and results achievable by cryo-electron microscopy (cryo-EM) imaging for molecular, cell, and structural biologists who wish to understand what is required and how it might help to address their research questions. It covers some of the main issues in sample preparation, microscopes and data collection, image processing, three-dimensional (3D) reconstruction, and validation and interpretation of the resulting EM density maps and atomic models.

Highlights of infectious agents in tissue.

The evolution of the diagnosis of infectious diseases began with the observation of the morphological characteristics of organisms such as ascaris and whipworms, followed by the use of the microscope and haematoxylin and eosin stains, which allowed recognition of microscopic characteristics undetectable with the naked eye, such as the viral cytopathic changes of herpes and the presence of fungi. Patterns of acute and chronic granulomatous inflammation were also observed; these were not specific to the exact aetiology of the disease, which led to the introduction of special methenamine stains for fungi and Ziehl-Neelsen for fungi and mycobacteria. Later, the use of immunohistochemistry was introduced, which acknowledged the use of antibodies to classify microorganisms and detect cases that were either difficult to interpret or in the midst of severe inflammatory processes. Currently, the use of molecular biology has made it possible to reach diagnoses that would have been very difficult to obtain through traditional methods; these techniques show key specific characteristics and facilitate the diagnosis of various infectious pathologies. These new techniques are based on the detection of antigens and nucleic acids of microorganisms, an important advance in the diagnosis of infectious diseases.

Karyotype diversity in cultivated and wild Indian rice through EMA-based chromosome analysis.

India is known for its diverse cultivated and wild rice germplasm. In today's crop improvement programmes, wild relatives are much-needed genetic repository of valuable traits. Analysis of genetic diversity at the chromosomal level is one cost-effective tool to unlock foundational information related to genetics and plant breeding. Presently, enzymatic maceration and air-drying method (EMA) has been applied for the first time in six cultivated and nine wild Indian rice (diploid and tetraploid). EMA method following Giemsa staining has yielded large numbers of cytoplasm free metaphase plates with distinct chromosome morphology. Detailed analysis has revealed karyotype diversities in terms of total chromatin length (TCL), chromosome morphology and location of sat chromosomes within and between the studied species. Most of the cultivated rice has gained additional amount in TCL during the period of domestication in comparison to their progenitor Oryza nivara. Morphological clarity of the small chromosomes of rice was much required and has helped to identify individual chromosomes in the diverse karyotypes. Diversity in landmark SAT chromosomes is another important observation, not reported previously in Indian rice. Present study has shown that in most of the O. sativa members, the 10th pair contains SAT except one where 6th pair is satellited. On the other hand, diversity of SAT in diploid and tetraplod wild species has been recorded on 5th, 7th and 8th chromosome pairs and on 9th, 12th, 22nd and 23rd chromosome pairs, respectively. Karyomorphometric indices has helped to construct dendrogram to elucidate intraspecies and interspecies relationships. Untapped genetic diversity recorded in Indian rice through chromosomal analysis will be useful to the breeders and genome researchers.

NuMorph: Tools for cortical cellular phenotyping in tissue-cleared whole-brain images.

Tissue-clearing methods allow every cell in the mouse brain to be imaged without physical sectioning. However, the computational tools currently available for cell quantification in cleared tissue images have been limited to counting sparse cell populations in stereotypical mice. Here, we introduce NuMorph, a group of analysis tools to quantify all nuclei and nuclear markers within the mouse cortex after clearing and imaging by light-sheet microscopy. We apply NuMorph to investigate two distinct mouse models: a Topoisomerase 1 (Top1) model with severe neurodegenerative deficits and a Neurofibromin 1 (Nf1) model with a more subtle brain overgrowth phenotype. In each case, we identify differential effects of gene deletion on individual cell-type counts and distribution across cortical regions that manifest as alterations of gross brain morphology. These results underline the value of whole-brain imaging approaches, and the tools are widely applicable for studying brain structure phenotypes at cellular resolution.

Isolation, expansion, differentiation, and histological processing of human nasal epithelial cells.

This protocol is intended as a guide for implementing or refining the usage of the air-liquid interface (ALI) model system to generate airway mucociliary tissue in vitro. We present a streamlined protocol for isolating the stem cells from inferior nasal turbinates of donors, allowing for a simple and low-cost supply of primary cells for research. We also provide our detailed protocols for ALI tissue processing and immunofluorescence to aid in the standardization of these techniques between research groups. For complete details on the use and execution of this protocol, please refer to Hussain et al., (2014)Yang et al., (2016)Im et al., (2019).

Isolating and Imaging Live, Intact Pacemaker Regions of Mouse Renal Pelvis by Vibratome Sectioning.

The renal pelvis (RP) is a funnel-shaped, smooth muscle structure that facilitates normal urine transport from the kidney to the ureter by regular, propulsive contractions. Regular RP contractions rely on pacemaker activity, which originates from the most proximal region of the RP at the pelvis-kidney junction (PKJ). Due to the difficulty in accessing and preserving intact preparations of the PKJ, most investigations on RP pacemaking have focused on single-cell electrophysiology and Ca2+ imaging experiments. Although important revelations on RP pacemaking have emerged from such work, these experiments have several intrinsic limitations, including the inability to accurately determine cellular identity in mixed suspensions and the lack of in situ imaging of RP pacemaker activity. These factors have resulted in a limited understanding of the mechanisms that underlie normal rhythmic RP contractions. In this paper, a protocol is described to prepare intact segments of mouse PKJ using a vibratome sectioning technique. By combining this approach with mice expressing cell-specific reporters and genetically encoded Ca2+ indicators, investigators may be able to more accurately study the specific cell types and mechanisms responsible for peristaltic RP contractions that are vital for normal urine transport.

Tutorial: practical considerations for tissue clearing and imaging.

Tissue clearing has become a powerful technique for studying anatomy and morphology at scales ranging from entire organisms to subcellular features. With the recent proliferation of tissue-clearing methods and imaging options, it can be challenging to determine the best clearing protocol for a particular tissue and experimental question. The fact that so many clearing protocols exist suggests there is no one-size-fits-all approach to tissue clearing and imaging. Even in cases where a basic level of clearing has been achieved, there are many factors to consider, including signal retention, staining (labeling), uniformity of transparency, image acquisition and analysis. Despite reviews citing features of clearing protocols, it is often unknown a priori whether a protocol will work for a given experiment, and thus some optimization is required by the end user. In addition, the capabilities of available imaging setups often dictate how the sample needs to be prepared. After imaging, careful evaluation of volumetric image data is required for each combination of clearing protocol, tissue type, biological marker, imaging modality and biological question. Rather than providing a direct comparison of the many clearing methods and applications available, in this tutorial we address common pitfalls and provide guidelines for designing, optimizing and imaging in a successful tissue-clearing experiment with a focus on light-sheet fluorescence microscopy (LSFM).

An optimized method for adult zebrafish brain-tissue dissociation that allows access mitochondrial function under healthy and epileptic conditions.

Mitochondria play key roles in brain metabolism. Not surprisingly, mitochondria dysfunction is a ubiquitous cause of neurodegenerative diseases. In turn, acquired forms of epilepsy etiology is specifically intriguing since mitochondria function and dysfunction remain not completely enlightened. Investigation in the field includes models of epileptic disorder using mainly rodents followed by mitochondrial function evaluation, which in general evidenced controversial data. So, we considered the efforts and limitations in this research field and we took into account that sample preparation and quality are critical for bioenergetics investigation. For these reasons the aim of the present study was to develop a thorough protocol for adult zebrafish brain-tissue dissociation to evaluate oxygen consumption flux and reach the bioenergetics profile in health and models of epileptic disorder in both, in vitro using pentylenetetrazole (PTZ) and N-methyl-D-Aspartic acid (NMDA), and in vivo after kainic acid (KA)-induced status epilepticus. In conclusion, we verify that fire-polished glass Pasteur pipette is eligible to brain-tissue dissociation and to study mitochondrial function and dysfunction in adult zebrafish. The results give evidence for large effect size in increase of coupling efficiency respiration (p/O2) correlated to treatment with PTZ and spare respiratory capacity (SRC) in KA-induced model indicating oxidative phosphorylation (OXPHOS) variable alterations. Further investigation is needed in order to clarify the bioenergetics role as well as other mitochondrial functions in epilepsy.

Histologic Study of the Esophagogastric Junction of Organ Donors Reveals Novel Glandular Structures in Normal Esophageal and Gastric Mucosae.

INTRODUCTION: Whether cardiac mucosa at the esophagogastric junction is normal or metaplastic is controversial. Studies attempting to resolve this issue have been limited by the use of superficial pinch biopsies, abnormal esophagi resected typically because of cancer, or autopsy specimens in which tissue autolysis in the stomach obscures histologic findings. METHODS: We performed histologic and immunohistochemical studies of the freshly fixed esophagus and stomach resected from 7 heart-beating, deceased organ donors with no history of esophageal or gastric disease and with minimal or no histologic evidence of esophagitis and gastritis. RESULTS: All subjects had cardiac mucosa, consisting of a mixture of mucous and oxyntic glands with surface foveolar epithelium, at the esophagogastric junction. All also had unique structures we termed compact mucous glands (CMG), which were histologically and immunohistochemically identical to the mucous glands of cardiac mucosa, under esophageal squamous epithelium and, hitherto undescribed, in uninflamed oxyntic mucosa throughout the gastric fundus. DISCUSSION: These findings support cardiac mucosa as a normal anatomic structure and do not support the hypothesis that cardiac mucosa is always metaplastic. However, they do support our novel hypothesis that in the setting of reflux esophagitis, reflux-induced damage to squamous epithelium exposes underlying CMG (which are likely more resistant to acid-peptic damage than squamous epithelium), and proliferation of these CMG as part of a wound-healing process to repair the acid-peptic damage could result in their expansion to the mucosal surface to be recognized as cardiac mucosa of a columnar-lined esophagus.

Whole-organ analysis of TGF-ß-mediated remodelling of the tumour microenvironment by tissue clearing.

Tissue clearing is one of the most powerful strategies for a comprehensive analysis of disease progression. Here, we established an integrated pipeline that combines tissue clearing, 3D imaging, and machine learning and applied to a mouse tumour model of experimental lung metastasis using human lung adenocarcinoma A549 cells. This pipeline provided the spatial information of the tumour microenvironment. We further explored the role of transforming growth factor-ß (TGF-ß) in cancer metastasis. TGF-ß-stimulated cancer cells enhanced metastatic colonization of unstimulated-cancer cells in vivo when both cells were mixed. RNA-sequencing analysis showed that expression of the genes related to coagulation and inflammation were up-regulated in TGF-ß-stimulated cancer cells. Further, whole-organ analysis revealed accumulation of platelets or macrophages with TGF-ß-stimulated cancer cells, suggesting that TGF-ß might promote remodelling of the tumour microenvironment, enhancing the colonization of cancer cells. Hence, our integrated pipeline for 3D profiling will help the understanding of the tumour microenvironment.

Navigating across multi-dimensional space of tissue clearing parameters.

Optical tissue clearing refers to physico-chemical treatments which make thick biological samples transparent by removal of refractive index gradients and light absorbing substances. Although tissue clearing was first reported in 1914, it was not widely used in light microscopy until 21th century, because instrumentation of that time did not permit to acquire and handle images of thick (mm to cm) samples as whole. Rapid progress in optical instrumentation, computers and software over the last decades made micrograph acquisition of centimeter-thick samples feasible. This boosted tissue clearing use and development. Numerous diverse protocols have been developed. They use organic solvents or water-miscible substances, such as detergents and chaotropic agents; some protocols require application of electric field or perfusion with special devices. There is no 'best-for-all' tissue clearing method. Depending on the case, one or another protocol is more suitable. Most of protocols require days or even weeks to complete, thus choosing an unsuitable protocol may cause an important waste of time. Several inter-dependent parameters should be taken into account to choose a tissue clearing protocol, such as: (1) required image quality (resolution, contrast, signal to noise ratio etc), (2) nature and size of the sample, (3) type of labels, (4) characteristics of the available instrumentation, (5) budget, (6) time budget, and (7) feasibility. Present review focusses on the practical aspects of various tissue clearing techniques. It is aimed to help non-experts to choose tissue clearing techniques which are optimal for their particular cases.

Tissue clearing technique: Recent progress and biomedical applications.

Organisms are inherently three dimensional, thus comprehensive understanding of the complicated biological system requires analysis of organs or even whole bodies in the context of three dimensions. However, this is a tremendous task since the biological specimens are naturally opaque, a major obstacle in whole-body and whole-organ imaging. Tissue clearing technique provides a prospective solution and has become a powerful tool for three-dimensional imaging and quantification of organisms. Tissue clearing technique aims to make tissue transparent by minimizing light scattering and light absorption, thus allowing deep imaging of large volume samples. When combined with diverse molecular labeling methods and high-throughput optical sectioning microscopes, tissue clearing technique enables whole-body and whole-organ imaging at cellular or subcellular resolution, providing detailed and comprehensive information about the intact biological systems. Here, we give an overview of recent progress and biomedical applications of tissue clearing technique. We introduce the mechanisms and basic principles of tissue clearing, and summarize the current tissue clearing methods. Moreover, the available imaging techniques and software packages for data processing are also presented. Finally, we introduce the recent advances in applications of tissue clearing in biomedical fields. Tissue clearing contributes to the investigation of structure-function relationships in intact mammalian organs, and opens new avenues for cellular and molecular mapping of intact human organs. We hope this review contributes to a better understanding of tissue clearing technique and can help researchers to select the best-suited clearing protocol for their experiments.

Practical Approaches for Cryo-FIB Milling and Applications for Cellular Cryo-Electron Tomography.

Cryo-electron tomography (cryo-ET) is a powerful technique to examine cellular structures as they exist in situ. However, direct imaging by TEM for cryo-ET is limited to specimens up to ∼400 nm in thickness, narrowing its applicability to areas such as cellular projections or small bacteria and viruses. Cryo-focused ion beam (cryo-FIB) milling has emerged in recent years as a method to generate thin specimens from cellular samples in preparation for cryo-ET. In this technique, specimens are thinned with a beam of gallium ions to gradually ablate cellular material in order to leave a thin, electron-transparent section (a lamella) through the bulk material. The lamella can be used for high-resolution cryo-ET to visualize cells in 3D in a near-native state. This approach has proved to be robust and relatively simple for new users and exhibits minimal sectioning artifacts. In this chapter, we describe a general approach to cryo-FIB milling for users with prior cryo-EM experience, with extensive notes on operation and troubleshooting.

Optimised tissue clearing minimises distortion and destruction during tissue delipidation.

AIMS: A variety of tissue clearing techniques have been developed to render intact tissue transparent. For thicker samples, additional partial tissue delipidation is required before immersion into the final refractive index (RI)-matching solution, which alone is often inadequate to achieve full tissue transparency. However, it is difficult to determine a sufficient degree of tissue delipidation, excess of which can result in tissue distortion and protein loss. Here, we aim to develop a clearing strategy that allows better monitoring and more precise determination of delipidation progress. METHODS: We combined the detergent sodium dodecyl sulphate (SDS) with OPTIClear, a RI-matching solution, to form a strategy termed Accurate delipidation with Optimal Clearing (Accu-OptiClearing). Accu-OptiClearing allows for a better preview of the final tissue transparency achieved when immersed in OPTIClear alone just before imaging. We assessed for the changes in clearing rate, protein loss, degree of tissue distortion, and preservation of antigens. RESULTS: Partial delipidation using Accu-OptiClearing accelerated tissue clearing and better preserved tissue structure and antigens than delipidation with SDS alone. Despite achieving similar transparency in the final OPTIClear solution, more lipids were retained in samples cleared with Accu-OptiClearing compared to SDS. CONCLUSIONS: Combining the RI-matching solution OPTIClear with detergents, Accu-OptiClearing, can avoid excessive delipidation, leading to accelerated tissue clearing, less tissue damage and better preserved antigens.

A Simplified Brain Blocking Protocol Optimized for the Diagnosis of Neurodegenerative Disease Saves Time and Money While Preserving Anatomic Relationships.

CONTEXT.­: Postmortem evaluation for neurodegenerative disease is expensive in time and materials. These challenges can be met by implementing simpler sampling protocols while preserving anatomic relations. OBJECTIVE.­: To determine the diagnostic effectiveness and cost-effectiveness of a simplified brain blocking protocol compared with the standard blocking protocol used in our Alzheimer's Disease Research Center (ADRC). DESIGN.­: We prospectively compared the neuropathologic diagnoses established from our standard 19-cassette/19 brain sites ADRC protocol to a simplified 6-cassette/12 brain sites protocol in 52 consecutive cases. The simplified protocol generated 14 slides for comparison to 52 slides from our standard protocol. RESULTS.­: Compared with the ADRC protocol the simplified protocol produced Alzheimer Disease Neuropathologic Changes probability scores that were the same in 50 of 52 cases (r = 0.99). Staging for Lewy pathology was equivalent in 45 of 52 (r = 0.98), scoring for cerebral amyloid angiopathy was equivalent in 48 of 52 (r = 0.97), and grading for arteriolosclerosis was the same in 45 of 52 cases (r = 0.92). Progressive supranuclear palsy (n = 4), multiple system atrophy (n = 2), and corticobasal degeneration (n = 1) could be diagnosed by either protocol independently. The estimated savings per case was 72% or $1744.89 ($2436.37 [ADRC] versus $691.48 [simplified]). CONCLUSIONS.­: The diagnosis of neurodegenerative disease at autopsy can be done accurately with a less expensive, simplified protocol. Our protocol is similar to those of previously published approaches, but it has a simpler organization scheme. This method should be valuable to institutions where autopsy cost considerations may be important.

¿Hay suficiente número de plazas hospitalarias para las prácticas de nuestros alumnos de Formación Profesional / Do we have sufficient hospital placements for our pathology technician trainees?

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Ex vivo MR microscopy of a human brain with multiple sclerosis: Visualizing individual cells in tissue using intrinsic iron.

PURPOSE: To perform magnetic resonance microscopy (MRM) on human cortex and a cortical lesion as well as the adjacent normal appearing white matter. To shed light on the origins of MRI contrast by comparison with histochemical and immunostaining. METHODS: 3D MRM at a nominal isotropic resolution of 15 and 18 µm was performed on 2 blocks of tissue from the brain of a 77-year-old man who had MS for 47 years. One block contained normal appearing cortical gray matter (CN block) and adjacent normal appearing white matter (NAWM), and the other also included a cortical lesion (CL block). Postmortem ex-vivo MRI was performed at 11.7T using a custom solenoid coil and T2*-weighted 3D GRE sequence. Histochemical and immunostaining were done after paraffin embedding for iron, myelin, oligodendrocytes, neurons, blood vessels, macrophages and microglia, and astrocytes. RESULTS: MRM could identify individual iron-laden oligodendrocytes with high sensitivity (70% decrease in signal compared to surrounding) in CN and CL blocks, as well as some iron-laden activated macrophages and microglia. Iron-deficient oligodendrocytes seemed to cause relative increase in MRI signal within the cortical lesion. High concentration of myelin in the white matter was primarily responsible for its hypointense appearance relative to the cortex, however, signal variations within NAWM could be attributed to changes in density of iron-laden oligodendrocytes. CONCLUSION: Changes in iron accumulation within cells gave rise to imaging contrast seen between cortical lesions and normal cortex, as well as the patchy signal in NAWM. Densely packed myelin and collagen deposition also contributed to MRM signal changes. Even though we studied only one block each from normal appearing and cortical lesions, such studies can help better understand the origins of histopathological and microstructural correlates of MRI signal changes in multiple sclerosis and contextualize the interpretation of lower-resolution in vivo MRI scans.