A screening platform to monitor RNA processing and protein-RNA interactions in ribonuclease P uncovers a small molecule inhibitor.

Ribonucleoprotein (RNP) complexes and RNA-processing enzymes are attractive targets for antibiotic development owing to their central roles in microbial physiology. For many of these complexes, comprehensive strategies to identify inhibitors are either lacking or suffer from substantial technical limitations. Here, we describe an activity-binding-structure platform for bacterial ribonuclease P (RNase P), an essential RNP ribozyme involved in 5' tRNA processing. A novel, real-time fluorescence-based assay was used to monitor RNase P activity and rapidly identify inhibitors using a mini-helix and a pre-tRNA-like bipartite substrate. Using the mini-helix substrate, we screened a library comprising 2560 compounds. Initial hits were then validated using pre-tRNA and the pre-tRNA-like substrate, which ultimately verified four compounds as inhibitors. Biolayer interferometry-based binding assays and molecular dynamics simulations were then used to characterize the interactions between each validated inhibitor and the P protein, P RNA and pre-tRNA. X-ray crystallographic studies subsequently elucidated the structure of the P protein bound to the most promising hit, purpurin, and revealed how this inhibitor adversely affects tRNA 5' leader binding. This integrated platform affords improved structure-function studies of RNA processing enzymes and facilitates the discovery of novel regulators or inhibitors.

Genes regulating hormone stimulus and response to protein signaling revealed differential expression pattern during porcine oocyte in vitro maturation, confirmed by lipid concentration.

Genes influencing oocyte maturation may be valuable for predicting their developmental potential, as well as discerning the mechanistic pathways regulating oocyte development. In the presented research microarray gene expression analysis of immature and in vitro matured porcine oocytes was performed. Two groups of oocytes were compared in the study: before (3 × n = 50) and after in vitro maturation (3 × n = 50). The selection of viable oocytes was performed using the brilliant cresyl blue (BCB) test. Furthermore, microarrays and RT-qPCR was used to analyze the transcriptome of the oocytes before and after IVM. The study focused on the genes undergoing differential expression in two gene-ontology groups: "Cellular response to hormone stimulus" and "Cellular response to unfolded protein", which contain genes that may directly or indirectly be involved in signal transduction during oocyte maturation. Examination of all the genes of interest showed a lower level of their expression after IVM. From the total number of genes in these gene ontologies ten of the highest change in expression were identified: FOS, ID2, BTG2, CYR61, ESR1, AR, TACR3, CCND2, EGR2 and TGFBR3. The successful maturation of the oocytes was additionally confirmed with the use of lipid droplet assay. The genes were briefly described and related to the literature sources, to investigate their potential roles in the process of oocyte maturation. The results of the study may serve as a basic molecular reference for further research aimed at improving the methods of oocyte in vitro maturation, which plays an important role in the procedures of assisted reproduction.

Molecular analysis of H&E- and Papanicolau-stained samples-systematic review.

Molecular pathology allows the identification of causative agents in infectious diseases and detection of biomarkers important for prediction of disease susceptibility, diagnosis and personalized therapy. Accordingly, nucleic acid-based methods have gained a special role in clinical laboratories particularly to evaluate solid and hematological tumors. Extraction of nucleic acids is commonly performed in microdissected formalin-fixed paraffin-embedded (FFPE) or cytological samples that had been previously evaluated through the use of hematoxylin and eosin (H&E) or Papanicolau (Pap) stains, respectively. Although the effect of both stains on nucleic acids integrity has been explored by several authors, the results are not consistent and require further examination. Accordingly, the goal of this review was to assess the influence of H&E and Pap stains on DNA and RNA integrity and to address the mechanism by which each staining compromises molecular based-analysis. The analyzed studies demonstrate that H&E- and Pap-staining result in low DNA recovery and some degree of DNA fragmentation. Additionally, it is concluded that hemalum inhibits PCR by interfering with DNA extraction, preventing DNA polymerase attachment and possibly by rescuing divalent cations. Accordingly, proper sample purification and adjustment of PCR conditions are of key importance to achieve satisfactory results by PCR in H&E- and Pap-stained samples. Furthermore, although H&E results in RNA fragmentation, it is possible to perform expression analysis in H&E-stained frozen sections, using RNase-free conditions, low amounts of hematoxylin and a rapid protocol from sample collection to RNA analysis. It The effect of Pap-staining on RNA integrity remains to be determined.

Histological validation of adipogenic differentiation potential of ASC on collagen-based 2D scaffolds.

Determination of the adipogenic potential and behavior of adipose-derived mesenchymal stem/stromal cells (ASCs) is particularly relevant for their potential clinical application in regenerative medicine, especially when regeneration is supported by biomaterials or scaffolds. Scaffolds need to be able to induce tissue repair and limit undesired adipogenic differentiation. Depending on the scaffold employed, determination of cell behavior may be hindered by material interference with staining, which will limit either cells identification or dye quantification. Collagen is a promising biomaterial in regenerative medicine, however, histological analysis of cells cultured on collagen-based scaffolds is challenging. Here we describe a new histological method based on iron hematoxylin combined with Oil red O (ORO) staining, for the determination of the adipogenic differentiation of ASCs cultivated on a collagen-based 2D scaffold. ASCs were seeded on collagen films or plastic, differentiated into adipocytes for 14 days, and then stained with either ORO or iron hematoxylin and ORO combined. The collagen films avidly absorbed the ORO dye; conventional staining and quantification by dye extraction failed to discriminate between differentiated and undifferentiated cells on the films. On the contrary, the iron hematoxylin-ORO combination provided a quantitative and more reliable determination of adipocytes based on single cell count. This method is particularly recommended for determining the adipogenic differentiation potential of ASCs and other cell types grown on highly absorptive materials that need to be validated for their potential use in bioengineering and regenerative medicine.

Support Vector Machine Classification of Nonmelanoma Skin Lesions Based on Fluorescence Lifetime Imaging Microscopy.

Early diagnosis of malignant skin lesions is critical for prompt treatment and a clinical prognosis of skin cancers. However, it is difficult to precisely evaluate the development stage of nonmelanoma skin cancers because they are derived from the same tissues as a result of the uncontrolled growth of abnormal squamous keratinocytes in the epidermis layer of the skin. In the present study, we developed a linear-kernel support vector machine (LSVM) model to distinguish basal cell carcinoma (BCC) from actinic keratosis (AK) and Bowen's disease (BD). The input parameters of the LSVM model consist of appropriate lifetime components and entropy values, which were extracted from two-photon fluorescence lifetime imaging of hematoxylin and eosin (H&E)-stained biopsy sections. Different features used as inputs for SVM training were compared and evaluated. In constructing the SVM models, features obtained from the lifetime (τ2) of the second component were found to be significantly more predictive than the average fluorescence lifetime (τm) in terms of diagnostic accuracy, sensitivity, and specificity. The above findings were confirmed on the basis of the receiver operating characteristic (ROC) curves of diagnostic models. Shannon entropy was added to the SVM models as an independent feature to further improve the diagnostic accuracy. Therefore, fluorescence lifetime analysis and entropy calculations can provide highly informative features for the accurate detection of skin neoplasm disorders. In summary, fluorescence lifetime imaging microscopy (FLIM) combined with the SVM classification exhibited great potential for developing an effective computer-aided diagnostic criterion and accurate cancer detection in dermatology.

Quantitative pixel intensity- and color-based image analysis on minimally compressed files: implications for whole-slide imaging.

Current best practice in the quantitative analysis of microscopy images dictates that image files should be saved in a lossless format such as TIFF. Use of lossy files, including those processed with the JPEG algorithm, is highly discouraged due to effects of compression on pixel characteristics. However, with the growing popularity of whole-slide imaging (WSI) and its attendant large file sizes, compressed image files are becoming more prevelent. This prompted us to perform a color-based quantitative pixel analysis of minimally compressed WSI images. Sections from three tissues stained with one of three reagents representing the colors blue (hematoxylin), red (Oil-Red-O), and brown (immunoperoxidase) were scanned with a whole slide imager in triplicate at 20x, 40x, and 63x magnifications. The resulting files were in the form of a BigTIFF with a JPEG compression automatically applied during acquisition. Images were imported into analysis software, six regions of interest were applied to various morphological locations, and the areas assessed for the color of interest. Whereas the number of designated weakly or strongly positive pixels was variable across the triplicate scans for the individual regions of interest, the total number of positive pixels was consistent. These results suggest that total positivity for a specific color representing a histochemical or immunohistochemical stain can be adequately quantitated on compressed images, but degrees of positivity (e.g., weak vs. strong) may not be as reliable. However, it is important to assess individual whole-slide imagers, file compression level and algorithm, and analysis software for reproducibility.

Robust Cell Detection and Segmentation for Image Cytometry Reveal Th17 Cell Heterogeneity.

Image cytometry enables quantitative cell characterization with preserved tissue architecture; thus, it has been highlighted in the advancement of multiplex immunohistochemistry (IHC) and digital image analysis in the context of immune-based biomarker monitoring associated with cancer immunotherapy. However, one of the challenges in the current image cytometry methodology is a technical limitation in the segmentation of nuclei and cellular components particularly in heterogeneously stained cancer tissue images. To improve the detection and specificity of single-cell segmentation in hematoxylin-stained images (which can be utilized for recently reported 12-biomarker chromogenic sequential multiplex IHC), we adapted a segmentation algorithm previously developed for hematoxlin and eosin-stained images, where morphological features are extracted based on Gabor-filtering, followed by stacking of image pixels into n-dimensional feature space and unsupervised clustering of individual pixels. Our proposed method showed improved sensitivity and specificity in comparison with standard segmentation methods. Replacing previously proposed methods with our method in multiplex IHC/image cytometry analysis, we observed higher detection of cell lineages including relatively rare TH 17 cells, further enabling sub-population analysis into TH 1-like and TH 2-like phenotypes based on T-bet and GATA3 expression. Interestingly, predominance of TH 2-like TH 17 cells was associated with human papilloma virus (HPV)-negative status of oropharyngeal squamous cell carcinoma of head and neck, known as a poor-prognostic subtype in comparison with HPV-positive status. Furthermore, TH 2-like TH 17 cells in HPV-negative head and neck cancer tissues were spatiotemporally correlated with CD66b+ granulocytes, presumably associated with an immunosuppressive microenvironment. Our cell segmentation method for multiplex IHC/image cytometry potentially contributes to in-depth immune profiling and spatial association, leading to further tissue-based biomarker exploration. © 2019 International Society for Advancement of Cytometry.

Solochrome cyanine: A histological stain for cobalt-chromium wear particles in metal-on-metal periprosthetic tissues.

Metal-on-metal (MoM) hip arthroplasties produce abundant implant-derived wear debris composed mainly of cobalt (Co) and chromium (Cr). Cobalt-chromium (Co-Cr) wear particles are difficult to identify histologically and need to be distinguished from other wear particle types and endogenous components (e.g., haemosiderin, fibrin) which may be present in MoM periprosthetic tissues. In this study we sought to determine whether histological stains that have an affinity for metals are useful in identifying Co-Cr wear debris in MoM periprosthetic tissues. Histological sections of periprosthetic tissue from 30 failed MoM hip arthroplasties were stained with haematoxylin-eosin (HE), Solochrome Cyanine (SC), Solochrome Azurine (SA) and Perls' Prussian Blue (PB). Sections of periprosthetic tissue from 10 cases of non-MoM arthroplasties using other implant biomaterials, including titanium, ceramic, polymethylmethacrylate (PMMA) and ultra-high molecular weight polyethylene (UHMWP) were similarly analysed. Sections of 10 cases of haemosiderin-containing knee tenosynovial giant cell tumour (TSGCT) were also stained with HE, SC, SA and PB. In MoM periprosthetic tissues, SC stained metal debris in phagocytic macrophages and in the superficial necrotic zone which exhibited little or no trichrome staining for fibrin. In non-MoM periprosthetic tissues, UHMWP, PMMA, ceramic and titanium particles were not stained by SC. Prussian Blue, but not SC or SA, stained haemosiderin deposits in MoM periprosthetic tissues and TSGT. Our findings show that SC staining (most likely Cr-associated) is useful in distinguishing Co-Cr wear particles from other metal/non-metal wear particles types in histological preparations of periprosthetic tissue and that SC reliably distinguishes haemosiderin from Co-Cr wear debris.

Enhanced Visualization of Hematoxylin and Eosin Stained Pathological Characteristics by Phasor Approach.

The phasor approach to fluorescence lifetime imaging microscopy (FLIM) is used to identify different types of tissues from hematoxylin and eosin (H&E) stained basal cell carcinoma (BCC) sections. The results suggest that working directly on the phasor space with the clustering assignment achieves immunofluorescence like simultaneous five or six-color imaging by using multiplexed fluorescence lifetimes of H&E. The phase approach is of particular effectiveness for enhanced visualization of the abnormal morphology of a suspected nidus. Moreover, the phasor approach to H&E FLIM data can determine the actual paths or the infiltrating trajectories of basophils and immune cells associated with the preneoplastic or neoplastic skin lesions. The integration of the phasor approach with routine histology proved its available value for skin cancer prevention and early detection. We therefore believe that the phasor analysis of H&E tissue sections is an enhanced visualization tool with the potential to simplify the preparation process of special staining and serve as color contrast aided imaging in clinical pathological examination.

A model of the spatial tumour heterogeneity in colorectal adenocarcinoma tissue.

BACKGROUND: There have been great advancements in the field of digital pathology. The surge in development of analytical methods for such data makes it crucial to develop benchmark synthetic datasets for objectively validating and comparing these methods. In addition, developing a spatial model of the tumour microenvironment can aid our understanding of the underpinning laws of tumour heterogeneity. RESULTS: We propose a model of the healthy and cancerous colonic crypt microenvironment. Our model is designed to generate synthetic histology image data with parameters that allow control over cancer grade, cellularity, cell overlap ratio, image resolution, and objective level. CONCLUSIONS: To the best of our knowledge, ours is the first model to simulate histology image data at sub-cellular level for healthy and cancerous colon tissue, where the cells have different compartments and are organised to mimic the microenvironment of tissue in situ rather than dispersed cells in a cultured environment. Qualitative and quantitative validation has been performed on the model results demonstrating good similarity to the real data. The simulated data could be used to validate techniques such as image restoration, cell and crypt segmentation, and cancer grading.

The Safe Labiaplasty: A Study of Nerve Density in Labia Minora and Its Implications.

BACKGROUND: Surgical techniques to alleviate labia minora hypertrophy are gaining popularity. Due to the rapidly growing number of labiaplasties performed around the world, there is concern for the safety of these procedures with respect to maintaining sensitivity to the genitalia and/or implications for sexual arousal. OBJECTIVES: An anatomic study aimed at identifying the nerve density distribution of the labia minora was performed to provide unique insight into performing labiaplasty while preserving sensation. METHODS: Four fresh tissue cadaver labia minora were analyzed. Each labia minora was divided into 6 anatomic areas. The samples from each of the 6 anatomic locations were analyzed for presence of nerve bundles using both a routine hematoxylin and eosin (H&E) stain and a confirmatory immunohistochemical staining for S100 protein. Nerve density was analyzed under light microscopy, counted, and then expressed as percentage nerve density as well as number of bundles per square millimeter. RESULTS: Upon gross analysis, the raw data reveal that labia minora have a heterogeneous population of sensory nerves. When looking at percent nerve density, the data do not reveal any statistical differences between the anatomic locations. CONCLUSIONS: Most labiaplasty techniques can be performed safely and are unlikely to cause loss of sensation as the nerve density distribution in labia minora is heterogeneous.

A novel domino cyclization for the stereoselective synthesis of indeno[2,1-c]pyran and cyclopenta[c]pyran derivatives.

A novel bicyclization of 2-(2-(hydroxymethyl)-1-methylene-2,3-dihydro-1H-inden-2-yl)ethanol with aldehydes in the presence of 10 mol% BF3·OEt2 in dichloromethane at 0-25 °C affords the biologically relevant indeno[2,1-c]pyran scaffolds in good yields with high selectivity. Similarly the bicyclization of 2-(1-(hydroxymethyl)-2-methylenecyclopentyl)ethanol with aldehydes generates the corresponding cyclopenta[c]pyran derivatives under similar conditions. This method is very useful to produce hematoxylin and brazilin like scaffolds.

Automated image analysis method for detecting and quantifying macrovesicular steatosis in hematoxylin and eosin-stained histology images of human livers.

Large-droplet macrovesicular steatosis (ld-MaS) in more than 30% of liver graft hepatocytes is a major risk factor for liver transplantation. An accurate assessment of the ld-MaS percentage is crucial for determining liver graft transplantability, which is currently based on pathologists' evaluations of hematoxylin and eosin (H&E)-stained liver histology specimens, with the predominant criteria being the relative size of the lipid droplets (LDs) and their propensity to displace a hepatocyte's nucleus to the cell periphery. Automated image analysis systems aimed at objectively and reproducibly quantifying ld-MaS do not accurately differentiate large LDs from small-droplet macrovesicular steatosis and do not take into account LD-mediated nuclear displacement; this leads to a poor correlation with pathologists' assessments. Here we present an improved image analysis method that incorporates nuclear displacement as a key image feature for segmenting and classifying ld-MaS from H&E-stained liver histology slides. 52,000 LDs in 54 digital images from 9 patients were analyzed, and the performance of the proposed method was compared against the performance of current image analysis methods and the ld-MaS percentage evaluations of 2 trained pathologists from different centers. We show that combining nuclear displacement and LD size information significantly improves the separation between large and small macrovesicular LDs (specificity = 93.7%, sensitivity = 99.3%) and the correlation with pathologists' ld-MaS percentage assessments (linear regression coefficient of determination = 0.97). This performance vastly exceeds that of other automated image analyzers, which typically underestimate or overestimate pathologists' ld-MaS scores. This work demonstrates the potential of automated ld-MaS analysis in monitoring the steatotic state of livers. The image analysis principles demonstrated here may help to standardize ld-MaS scores among centers and ultimately help in the process of determining liver graft transplantability.

Application of the CellDetect® staining technique in diagnosis of human cervical cancer.

BACKGROUND: CellDetect® staining technique is a newly invented technique for cancer diagnosis. It easily distinguishes between normal and neoplastic cells including pre-cancer and squamous cell carcinoma (SCC) cells, based on staining color and morphology. In this study, application of CellDetect® staining technique was assessed in diagnosis of human cervical cancer as compared with hematoxylin and eosin (H&E) staining in conventional slides and Thinprep cytologic test (TCT) smears. METHODS: The conventional slides and TCT smears of 600 patients were stained and observed while comparing with H&E staining to assess sensitivity and specificity of CellDetect® staining technique in diagnosis of cervical cancer. Conventional smear slides (440 cases) were fixed in 95% ethanol or with CYTOFIX® Spray. TCT smears (160 cases) were processed based on manual. The paraffin sections from cervical intraepithelium neoplasia (CIN) 2-3 and SCC cases were prepared by biopsy. RESULTS: CellDetect® staining exhibited well cell morphology, simultaneously, showed dual color discrimination, the stain targeted cytoplasm in normal cells in green and dysplastic cells or neoplastic cells in purple/red. Both cervical cell smears or both fixation methods in conventional slides did not affect CellDetect® staining diagnosis, especially in tissue biopsies CellDetect® staining exhibited well epithelium layers to benefit the diagnosis of CIN grade. The sensitivity and specificity of CellDetect® staining technology in diagnosing CIN and SCC were 94.34% and 88.73%, respectively. CONCLUSIONS: CellDetect® staining technique provided a dual color discrimination and morphological analysis. It has the potential to become one of the most effective methods for cervical screening and early diagnosis.

Female rat hippocampal cell density after conditioned place preference.

The hippocampus is important for learning tasks, such as conditioned place preference (CPP), which is widely used as a model for studying the reinforcing effects of drugs with dependence liability. Long-term opiate use may produce maladaptive plasticity in the brain structures involved in learning and memory, such as the hippocampus. We investigated the phenomenon of conditioning with morphine on the cell density of female rat hippocampus. Forty-eight female Wistar rats weighing on average 200-250 g were used. Rats were distributed into eight groups. Experimental groups received morphine daily (three days) at different doses (2.5, 5, 7.5 mg/kg) and the control-saline group received normal saline (1 ml/kg), and then the CPP test was performed. Three sham groups received only different doses (2.5, 5, 7.5 mg/kg) of morphine without CPP test. Forty-eight hours after behavioural testing animals were decapitated under chloroform anaesthesia and their brains were fixed, and after tissue processing, slices were stained with cresyl violet for neurons and phosphotungstic acid haematoxylin for astrocytes. The maximum response was obtained with 5 mg/kg of morphine. The density of neurons in CA1 and CA3 areas of hippocampus after injection of morphine and CPP was decreased. The number of astrocytes in different areas of hippocampus was increased after injection of morphine and CPP. It seems that the effective dose was 5 mg/kg, as it led to the CPP. We concluded that both injection of mor phine and CPP can decrease the density of neurons and also increase the number of astrocytes in the rat hippocampus.

[Pathological assessment of bone sarcomas]. / Pathologische Begutachtung von Knochensarkomen.

Bone tumors are very rare. Diagnosis and treatment is an interdisciplinary task for experienced radiologists, pathologist, and surgeons that is ideally performed in specialized centers. For optimal processing of bone specimens, basic laboratory equipment and special techniques are required. The cornerstone of the histological diagnosis remains H&E staining, supplemented by special stains, immunohistochemistry, and molecular techniques. For an appropriate diagnosis, data on clinical history, age, location, topography within bone, and imaging are required. Major differences between histological and radiological diagnosis have to be clarified before starting treatment (e.g., by involving a reference registry).

Deep learning for rapid virtual H&E staining of label-free glioma tissue from hyperspectral images.

Hematoxylin and eosin (H&E) staining is a crucial technique for diagnosing glioma, allowing direct observation of tissue structures. However, the H&E staining workflow necessitates intricate processing, specialized laboratory infrastructures, and specialist pathologists, rendering it expensive, labor-intensive, and time-consuming. In view of these considerations, we combine the deep learning method and hyperspectral imaging technique, aiming at accurately and rapidly converting the hyperspectral images into virtual H&E staining images. The method overcomes the limitations of H&E staining by capturing tissue information at different wavelengths, providing comprehensive and detailed tissue composition information as the realistic H&E staining. In comparison with various generator structures, the Unet exhibits substantial overall advantages, as evidenced by a mean structure similarity index measure (SSIM) of 0.7731 and a peak signal-to-noise ratio (PSNR) of 23.3120, as well as the shortest training and inference time. A comprehensive software system for virtual H&E staining, which integrates CCD control, microscope control, and virtual H&E staining technology, is developed to facilitate fast intraoperative imaging, promote disease diagnosis, and accelerate the development of medical automation. The platform reconstructs large-scale virtual H&E staining images of gliomas at a high speed of 3.81 mm2/s. This innovative approach will pave the way for a novel, expedited route in histological staining.

Hematoxylin staining reveals a decrease in nucleolar diameter of pig oocytes before germinal vesicle breakdown.

During oocyte growth, the morphology of the nucleolus changes into a compact and homogenous structure. The compact nucleoli in full-grown oocytes are not stained by aceto-orcein staining or immunofluorescence staining. In this study, we developed a hematoxylin staining method for pig oocytes in whole-mount preparations to visualize the nucleoli. Nucleoli of growing and full-grown oocytes were stained blue with hematoxylin. Using this staining method, the changes in the oocyte nucleolus during maturation were examined. The nucleolar diameter gradually decreased in maturing oocytes (10.7 ± 0.1 µm to 9.0 ± 0.7 µm, P<0.05) before germinal vesicle breakdown (GVBD). The results suggest that the nucleolar volume of oocytes decreases before GVBD.