Serial Sections Research Articles

Abstract B065: Biomarker approach to define tumor subtype in pancreatic ductal adenocarcinoma

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal tumors with a predicted five-year survival rate in 2023 of 12%. Building on prior observations that molecular subtypes correlate with survival and response to treatment, our goal has been to understand the patterns of expression and prognostic correlations of three reciprocally expressed proteins previously identified as surrogate biomarkers of PDAC subtypes. Keratin 17 (K17) and Keratin 5 (K5) have both been identified as biomarkers of the basal subtype whereas GATA6, a transcription factor necessary for normal pancreatic development, is a surrogate marker of the classical subtype associated with longer-term survival. Here, we explore the expression of these three diagnostic biomarkers in PDAC and their specificity to tumor cells. Methods: Immunohistochemical analyses of K17 (KDx Diagnostics), GATA6 (R&D Systems), and KT5 (Leica) were performed on archival primary PDAC specimens from Stony Brook University Hospital (n= 30), by routine immunoperoxidase methods on serial sections. IHC stained slides were scanned using an Olympus VS200 slide scanner and images were imported into QuPath for digital analysis. Regions of interest (ROIs) were defined by the presence of tumor cells and were identified by histological assessment of representative sections. Digital scoring was performed on identified ROIs using the integrated "positive cell detection" function in QuPath and classified into three different stain intensities (1+, 2+, 3+). The threshold for each intensity is as follows: 1+, 0.2; 2+, 0.4; 3+, 1.0. According to the number of detections of each intensity, tumor cells were categorized into negative (0 and 1+) or positive (2+ and 3+). The percentage positive of analyzed biomarker expression within the tumor ROI was calculated by dividing the total number of positive cells by the total number of detected cells. Results: K17 was detected in both well and poorly differentiated PDAC components and in a few benign proliferative ducts of chronic pancreatitis but no staining was detected in stromal cells, benign ductal epithelium, or acinar cells. K5 was detected in a smaller proportion of cells from glandular components of the tumor as well as in most diffusely infiltrative single cells, benign ductal, and acinar cells but not in stromal cells. By contrast, GATA6 was detected in the nuclei of most cancer epithelial cells and was also detected in benign ductal cells, acinar cells, and stromal cells. The proportion of K17 expressing cells exceeded that of K5 positive cells in the great majority of ROIs and across cases. Conclusions: Keratin 17 detects a higher proportion of tumor cells than K5 and shows the lowest level of expression in benign cell types. By contrast, GATA6 IHC shows variable patterns of expression related to K17 or K5 expression and is expressed in the highest proportion of tumor cells, including both well-differentiated, diffusely infiltrative components, and benign tissue components. Citation Format: Carson Ho, Michael Horowitz, Lyanne Oblein, Shayan Sarkar, Soma Kobayashi, Karen Bai, Natalia Marchenko, Luisa Escobar-Hoyos, Kenneth Shroyer. Biomarker approach to define tumor subtype in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr B065.

Abstract B064: Redefining phenotypic heterogeneity of pancreatic ductal adenocarcinoma: A bottom-up approach

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) tumor inter-patient heterogeneity has been well described with two major subtypes (classical and basal-like). An important intra-patient heterogeneity has not yet been extensively studied due to the lack of standardized and easily accessible high throughput methods compatible with clinical routine. Material and Methods: To build an immunohistochemical (IHC) tool capable of differentiating classical and basal-like tumors, relevant antibodies were selected using a multi-step process. The successive stages of an in-silico selection from the bulk transcriptome of 309 PDACs and 30 patient-derived xenografts followed by the validation on tissue microarrays in a cohort of PDAC (n=50) were conducted. We used our final IHC panel on whole slides of serial histology sections in 95 resected PDACs. After image registration, we performed a tile-based-analysis in tumoral and pre-neoplastic areas and a k-means clustering to identify relevant marker combinations. Results: Our final selection of the IHC panel included GATA6, CLN18, TFF1, MUC16, S100A2, KRT17, PanBasal. Among the 44,042 tumor tiles analyzed, 4 different phenotypes were identified: 1 classical (CLN18+, TFF1+, GATA6+), 1 intermediate (KRT17+) and 2 basal-like (MUC16+ vs S100A2+) with specific biological properties. A minor basal contingent drastically reduced overall survival (OS) in the multivariate analysis (HR=2.36, p=0.01). In contrast, the presence of the intermediate and classical clusters did not impact OS. However, among patients with PDAC expressing the classical cluster, a tremendous difference of OS was observed according to the co-occurence of a basal cluster (HR=0.34, p=0.003). These prognostic associations have been validated in an independent cohort of 150 patients with PDAC on tissue microarrays. By analyzing preneoplastic lesions, we showed the evolution process of pancreatic carcinogenesis going from a classical toward a basal through an early intermediate phenotype. Conclusion: Our restricted panel of antibodies could help pathologists to predict the high degree of PDAC heterogeneity, ultimately leading to an improvement of patient management. Citation Format: Marc Hilmi, Flore Delecourt, Jerôme Rafenne, Taib Bourega, Nelson Dusetti, Juan Iovanna, Yuna Blum, Magali Richard, Cindy Neuzillet, Anne Couvelard, Louis De Mestier, Vinciane Rebours, Rémy Nicolle, Jérôme Cros. Redefining phenotypic heterogeneity of pancreatic ductal adenocarcinoma: A bottom-up approach [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr B064.

Pudgy mouse rib deformities emanate from abnormal paravertebral longitudinal cartilage/bone accumulations.

The pudgy (pu/pu) mouse, caused by a recessive mutation in the Notch family Delta like-3 gene (Dll3), has severe rib, vertebral body and intervertebral disc abnormalities. Using whole-mount preparations and serial histologic sections we demonstrate: 1) localized paravertebral longitudinal cartilage/bone accumulations (PVLC/BAs) invariably associated with branched, fused and asymmetrically spaced ribs that emanate from it laterally; 2) abnormal rib formation immediately adjacent to abnormal vertebral body and intervertebral disc formation in asymmetric right/left fashion; and 3) patterns of rib deformation that differ in each mouse. Normal BALB/c embryo and age-matched non-affected pu/+ mice assessments allow for pu/pu comparisons. The Dll3 Notch family gene is involved in normal somitogenesis via the segmentation clock mechanism. Although pathogenesis of rib deformation is initially triggered by the Dll3 gene mutation, these findings of abnormal asymmetric costo-vertebral region structure imply that differing patterns cannot be attributed to this single gene mutation alone. All findings implicate a dual mechanism of malformation: the Dll3 gene mutation leading to subtle timing differences in traveling oscillation waves of the segmentation clock and further subsequent misdirection of tissue formation by altered chemical reaction-diffusion and epigenetic landscape responses. PVLC/BAs appear as primary supramolecular structures underlying severe rib malformation associated both with time-sensitive segmentation clock mutations and subsequent reactions.

JCAD deficiency attenuates activation of hepatic stellate cells and cholestatic fibrosis.

Cholestatic liver diseases including primary biliary cholangitis (PBC) are associated with active hepatic fibrogenesis, which ultimately progresses to cirrhosis. Activated hepatic stellate cells (HSCs) are the main fibrogenic effectors in response to cholangiocyte damage. JCAD regulates cell proliferation and malignant transformation in nonalcoholic steatoheaptitis-associated hepatocellular carcinoma (NASH-HCC). However, its participation in cholestatic fibrosis has not been explored yet. Serial sections of liver tissue of PBC patients were stained with immunofluorescence. Hepatic fibrosis was induced by bile duct ligation (BDL) in wild-type (WT), global JCAD knockout mice (JCAD-KO) and HSC-specific JCAD knockout mice (HSC-JCAD-KO), and evaluated by histopathology and biochemical tests. In situ-activated HSCs isolated from BDL mice were used to determine effects of JCAD on HSC activation. In consistence with staining of liver sections from PBC patients, immunofluorescent staining revealed that JCAD expression was identified in smooth muscle α-actin (α-SMA)-positive fibroblast-like cells and was significantly up-regulated in WT mice with BDL. JCAD deficiency remarkably ameliorated BDL-induced hepatic injury and fibrosis, as documented by liver hydroxyproline content, when compared to WT mice with BDL. Histopathologically, collagen deposition was dramatically reduced in both JCAD-KO and HSC-JCAD-KO mice compared to WT mice, as visualized by Trichrome staining and semi-quantitative scores. Moreover, JCAD deprivation significantly attenuated in situ HSC activation and reduced expression of fibrotic genes after BDL. JCAD deficiency effectively suppressed hepatic fibrosis induced by BDL in mice, and the underlying mechanisms are largely through suppressed Hippo-YAP signaling activity in HSCs.

Image quality evaluation for FIB-SEM images.

Focused ion beam scanning electron microscopy (FIB-SEM) tomography is a serial sectioning technique where an FIB mills off slices from the material sample that is being analysed. After every slicing, an SEM image is taken showing the newly exposed layer of the sample. By combining all slices in a stack, a 3D image of the material is generated. However, specific artefacts caused by the imaging technique distort the images, hampering the morphological analysis of the structure. Typical quality problems in microscopy imaging are noise and lack of contrast or focus. Moreover, specific artefacts are caused by the FIB milling, namely, curtaining and charging artefacts. We propose quality indices for the evaluation of the quality of FIB-SEM data sets. The indices are validated on real and experimental data of different structures andmaterials.

Palps across the tree - the neuronal innervation and development of sensory head appendages in Annelida.

Polychaetes inhabit a wide variety of habitats and show a great morphological diversity. In this context, a key morphological structure for adapting to their individual lifestyles and ecological niches are the prominent head appendages. In the last years more and more studies focused on the mainly sensory annelid head appendages - namely the antennae, palps, buccal lips and cirri - to unravel the evolutionary origin and phylogeny of Annelida. Unfortunately, comparable data for most of the polychaete families are lacking so far, especially when it comes to features of the larval anterior nervous system and the related innervation and potential homology of these head appendages. In this study, we therefore use an integrative morphological approach including immunohistochemistry and confocal laser scanning microscopy in combination with histological serial sections and 3D-visualizations. With special focus on the palp-like appendages, our data provides a closer look into the development of the larval anterior nervous system and the related sensory structures of three polychaete families representing major groups of the annelid tree of life. Hence, we investigate members of the palaeoannelid Magelonidae as well as basally-branching Amphinomidae, and the pleistoannelid Spionidae forming a taxon deeply nested within Sedentaria. Our comparative data of larval and adult neuronal features support the homology of feeding-palps across the annelid tree. Furthermore, our observations show that larval palps gradually transform into the adult ones while keeping a very similar neuronal innervation pattern. Solely for Amphinomidae a loss of larval palps during ontogenesis has to be assumed. Therefore, our investigations uncover important and so far unknown details in terms of structural homology across Annelida and provide important results necessary for our understanding of annelid evolution.

Petascale pipeline for precise alignment of images from serial section electron microscopy

The reconstruction of neural circuits from serial section electron microscopy (ssEM) images is being accelerated by automatic image segmentation methods. Segmentation accuracy is often limited by the preceding step of aligning 2D section images to create a 3D image stack. Precise and robust alignment in the presence of image artifacts is challenging, especially as datasets are attaining the petascale. We present a computational pipeline for aligning ssEM images with several key elements. Self-supervised convolutional nets are trained via metric learning to encode and align image pairs, and they are used to initialize iterative fine-tuning of alignment. A procedure called vector voting increases robustness to image artifacts or missing image data. For speedup the series is divided into blocks that are distributed to computational workers for alignment. The blocks are aligned to each other by composing transformations with decay, which achieves a global alignment without resorting to a time-consuming global optimization. We apply our pipeline to a whole fly brain dataset, and show improved accuracy relative to prior state of the art. We also demonstrate that our pipeline scales to a cubic millimeter of mouse visual cortex. Our pipeline is publicly available through two open source Python packages.

Whole-Cell Electron Tomography Analysis of Vacuole Biogenesis.

Vacuoles in plant cells are the most prominent organelles that harbor distinctive features, including lytic function, storage of proteins and sugars, balance of cell volume, and defense responses. Despite their dominant size and functional versatility, the nature and biogenesis of vacuoles in plants per se remain elusive and several models have been proposed. Recently, we used the whole-cell 3D electron tomography (ET) technique to study vacuole formation and distribution at nanometer resolution and demonstrated that small vacuoles are derived from multivesicular body maturation and fusion. Good sample preparation is a critical step to get high-quality electron tomography images. In this chapter, we provide detailed sample preparation methods for high-resolution ET in Arabidopsis thaliana root cells, including high-pressure freezing, subsequent freeze-substitution fixation, embedding, and serial sectioning.

Determination of the embryotoxic effects of propofol injected into eggs on the cerebellum and spinal cord using histologic methods: an animal study.

This study aims to determine the possible embryotoxic effects of propofol on the cerebellum and spinal cord using fertile chicken eggs. A total of 430 fertile eggs were divided into 5 groups: control, saline, 2.5 mg.kg-1, 12.5 mg.kg-1, and 37.5 mg.kg-1 propofol. Injections were made immediately before incubation via the air chamber. On the 15th, 18th, and 21st day of incubation, 6 embryos from each group were evaluated. Serial paraffin sections taken from the cerebellum and spinal cord were stained with hematoxylin-eosin, Kluver-Barrera, toluidine blue, and periodic acid-Schiff's reaction. The outer granular layer and total cortex thickness were measured, and the linear density of the Purkinje cells was determined. The ratios of the substantia grisea surface area to the total surface area of the spinal cord were calculated. The transverse and longitudinal diameters of the canalis centralis were also assessed. No structural malformation was observed in any embryos examined macroscopically. No significant difference was observed between the groups in terms of development and histologic organization of the cerebellum and spinal cord. However, on the 15th, 18th, and 21st day, the outer granular layer (p < 0.001 for all days) and the total cortex thickness (p < 0.01, p < 0.001, and p < 0.001, respectively) decreased significantly in different propofol dose groups in varying degrees in the cerebellum. Similarly, in the spinal cord, there were significant changes in the ratios of the substantia grisea surface area to the total surface area (p < 0.01 and p < 0.001, respectively). It was concluded that the in-ovo-administered propofol given immediately before incubation has adverse effects on the developing cerebellum and spinal cord. Therefore, it is important for anesthesiologists always to remain vigilant when treating female patients of childbearing age.

ULTRASTRUCTURAL ASPECTS OF TWO MORPHOTYPES OF GRAM-NEGATIVE BACTERIA LOCATED IN THE ENDOMYSIUM OF THE SKELETAL MUSCLE OF Trachurus trecae (CARANGIDAE) IN ANGOLA

Ultrastructural and histopathological ocasional parasite survey of some samples of the teleostean, Trachurus trecae (Carangidae), collected on the Atlantic coast of Angola were found infected by prokaryotic organisms identified as belonging to two morphotypes of Gram-negative bacteria located inside on extracellular space of the connective tissue of the endomysium among the skeletal muscle fibers. By serial ultrathin sections was observed that the majority of bacteria were cylindrical morphology, some appeared in division measuring 2.8 ± 0.7 µm long x 0.9 ± 0.3 µm in diameter, other having spherical form measuring 2.8 ± 0.4 µm in diameter. The ultrastructural organization of the myofibres contacting with of region where the bacteria were located, present the myofibrils ultrastructurally disorganized, without the typical muscular striations. The pathogenic effects of the two bacterial association seemed to give rise to the myofibrils lysis and concomitant disintegration and rupture of the wall of the muscle fibres, forming a recess where the bacteria live in close proximity. Muscle fiber wall in contact with the local where the bacteria were localized appeared lysed, disorganized and without the typical striations where, among them, some bacteria were observed. Some aspects of apparent penetration of bacteria through the degraded wall of the myofibers into the interior of the fibers, were observed and discussed. This is the first description of the pathogenic activity of bacteria in muscle tissue of a teleost from the aquatic fauna of Angola.

Three-dimensional model of normal human dermal tissue using serial tissue sections.

Background: This study aims to construct a three-dimensional model of skin dermis utilizing continuous tissue sections, with the primary objective of obtaining anatomical structure data for normal human dermal tissues. Methods: Normal skin tissue specimens were acquired, paraffin-embedded, and subjected to HE staining. Panoramic images of skin sections were captured using a microscope. Tissue section images were aligned using the SIFT and StackReg image alignment methods, with analysis conducted using the OpenCV module. Mimics17 software facilitated the reconstruction of the skin dermal 3D model, enabling the calculation of dermal porosity and the void diameter. Results: Panoramic skin slices exhibited high-resolution differentiation of dermal fibers and cellular structures. Both SIFT and StackReg image alignment methods yielded similar results, although the SIFT method demonstrated greater robustness. Successful reconstruction of the three-dimensional dermal structure was achieved. Quantitative analysis revealed a dermal porosity of 18.96 ± 4.41% and an average pore diameter of 219.29 ± 34.27 μm. Interestingly, the porosity of the dermis exhibited a gradual increase from the papillary layer to the fourth layer, followed by a transient decrease and then a gradual increase. The distribution of the mean pore diameter mirrored the pattern observed in porosity distribution. Conclusion: Utilizing the continuous skin tissue slice reconstruction technique, this study successfully reconstructed a high-precision three-dimensional tissue structure of the skin. The quantitative analysis of dermal tissue porosity and average pore diameter provides a standardized dataset for the development of biomimetic tissue-engineered skin.

Resolving the In Situ Three-Dimensional Structure of Fly Mechanosensory Organelles Using Serial Section Electron Tomography.

Mechanosensory organelles (MOs) are specialized subcellular entities where force-sensitive channels and supporting structures (e.g., microtubule cytoskeleton) are organized in an orderly manner. The delicate structure of MOs needs to be resolved to understand the mechanisms by which they detect forces and how they are formed. Here, we describe a protocol that allows obtaining detailed information about the nanoscopic ultrastructure of fly MOs by using serial section electron tomography (SS-ET). To preserve fine structural details, the tissues are cryo-immobilized using a high-pressure freezer followed by freeze-substitution at low temperature and embedding in resin at room temperature. Then, sample sections are prepared and used to acquire the dual-axis tilt series images, which are further processed for tomographic reconstruction. Finally, tomograms of consecutive sections are combined into a single larger volume using microtubules as fiducial markers. Using this protocol, we managed to reconstruct the sensory organelles, which provide novel molecular insights as to how fly mechanosensory organelles work and are formed. Based on our experience, we think that, with minimal modifications, this protocol can be adapted to a wide range of applications using different cell and tissue samples. Key features • Resolving the high-resolution 3D ultrastructure of subcellular organelles using serial section electron tomography (SS-ET). • Compared with single-axis tilt series, dual-axis tilt series provides a much wider coverage of Fourier space, improving resolution and features in the reconstructed tomograms. • The use of high-pressure freezing and freeze-substitution maximally preserves the fine structural details.

Life history of a high‐class noblewoman from the late Shu state in the Chengdu Plain during the Eastern Zhou period (770–221 BC): Childhood stresses and stable life

AbstractThis preliminary study aims at reconstructing the life history, including dietary patterns, mobility, and health conditions, of a high‐class noblewoman (M154) from the late Shu state (晚蜀), the period of Kai Ming regime (開明氏), in the Shuangyuan Village Cemetery of the Eastern Zhou period (770–221 BC), Chengdu Plain, China, as a way to provide a glimpse into the human lifeway in the late Shu culture period under the impact of non‐local cultures. We combined skeletal pathological observations with multi‐isotope (carbon, nitrogen, and oxygen) analyses of bone collagen, enamel, and dentin serial sections and analyzed multi‐tooth enamel from the remaining 10 humans in the cemetery as comparative data. The results suggest that the occupant of M154, a female who died at around 30 years of age, consumed mainly rice (C3‐based crop) and high animal protein throughout her life, was weaned at 2–3 years of age, experienced physiological stresses after weaning and at 7–8 years of age but recovered from both rapidly (less than a year), which probably was related to better care, and that the timing of physiological stress embodied in isotopic profiles from incremental dentin sections is consistent with the observed enamel hypoplasia and cribra orbitalia. She more likely lived with the local population from childhood to adolescence and did not experience long‐distance migrations. After 7–8 years of age, she consumed relatively stable food resources and lived a stable life until death. Here, we show the stable and well‐cared‐for life history of a high‐ranking noble of the late Shu state, providing new insights into the study of the ancient Shu people and a reference for further research.

Role of Angiogenesis and Endothelialmesenchymal Transition in Bone Marrow Fibrosis Associated with Haematopoietic Neoplasms: A Cross-sectional Immunohistochemical Analysis

Introduction: The bone marrow examination is an essential investigation in the diagnosis and management of many haematological disorders. The integration of all investigations, including peripheral blood analysis, bone marrow aspirate, and trephine biopsy findings, along with supplementary tests such as immunophenotyping, cytogenetic analysis, and molecular genetic studies, is crucial for arriving at a final diagnosis. Aim: To assess the presence of reticulin fibres in bone marrow biopsy sections in haematological malignancies, to evaluate the grade of BMF associated with haematological malignancies and to assess the role of angiogenesis using IHC markers in various haematological malignancies. Materials and Methods: A cross-sectional study was conducted in the Department of Pathology at the National Institute of Pathology, Safdarjung Hospital, New Delhi, India in 2009 for a duration of 18 months. Thirty-eight patients with a diagnosis of Acute Myeloid Leukaemia (AML), Acute Lymphoblastic Leukaemia (ALL), Chronic Myeloid Leukaemia (CML), and Chronic Lymphoproliferative Disorder (CLPD) were studied. Bone marrow biopsies were taken, fixed in 10% formalin, and decalcified in 10% Ethylene Diamine Tetraacetic Acid (EDTA). Routine paraffin embedding was performed, and serial sections of 4 µm were obtained on poly-L-lysine-coated slides for Immunohistochemistry (IHC) {Vimentin, Vascular Endothelial Growth Factor (VEGF), CD-34, Smooth Muscle Actin (SMA)}. The presence of reticulin fibres in the bone marrow biopsy sections was assessed using two special stains: Gomori’s Silver Impregnation and Masson’s Trichrome. Fibrosis was quantified according to the Baurmeister 0-4 grading system of Bone Marrow Fibrosis (BMF). Results: The results of the present study suggest that Endothelial to Mesenchymal Transition (EndMT) may play a role in the pathogenesis of BMF. Various grades of fibrosis were observed, with 15 cases (39.47%) in Grade 3, followed by 11 cases (28.95%) in Grade 2, 8 cases (21.05%) in Grade 1, and 4 cases (10.53%) in Grade 4. Conclusion: BMF was a significant finding even in the early stages of the majority of the lesions studied and was closely linked with angiogenesis. This study showed that angiogenesis plays an important role in the pathogenesis of haematological neoplasms and that VEGF is a prominent stimulus in the majority of these disorders. Additionally, this study suggests that EndMT has a possible role in the pathogenesis of BMF.Introduction: The bone marrow examination is an essential investigation in the diagnosis and management of many haematological disorders. The integration of all investigations, including peripheral blood analysis, bone marrow aspirate, and trephine biopsy findings, along with supplementary tests such as immunophenotyping, cytogenetic analysis, and molecular genetic studies, is crucial for arriving at a final diagnosis. Aim: To assess the presence of reticulin fibres in bone marrow biopsy sections in haematological malignancies, to evaluate the grade of BMF associated with haematological malignancies and to assess the role of angiogenesis using IHC markers in various haematological malignancies. Materials and Methods: A cross-sectional study was conducted in the Department of Pathology at the National Institute of Pathology, Safdarjung Hospital, New Delhi, India in 2009 for a duration of 18 months. Thirty-eight patients with a diagnosis of Acute Myeloid Leukaemia (AML), Acute Lymphoblastic Leukaemia (ALL), Chronic Myeloid Leukaemia (CML), and Chronic Lymphoproliferative Disorder (CLPD) were studied. Bone marrow biopsies were taken, fixed in 10% formalin, and decalcified in 10% Ethylene Diamine Tetraacetic Acid (EDTA). Routine paraffin embedding was performed, and serial sections of 4 µm were obtained on poly-L-lysine-coated slides for Immunohistochemistry (IHC) {Vimentin, Vascular Endothelial Growth Factor (VEGF), CD-34, Smooth Muscle Actin (SMA)}. The presence of reticulin fibres in the bone marrow biopsy sections was assessed using two special stains: Gomori’s Silver Impregnation and Masson’s Trichrome. Fibrosis was quantified according to the Baurmeister 0-4 grading system of Bone Marrow Fibrosis (BMF). Results: The results of the present study suggest that Endothelial to Mesenchymal Transition (EndMT) may play a role in the pathogenesis of BMF. Various grades of fibrosis were observed, with 15 cases (39.47%) in Grade 3, followed by 11 cases (28.95%) in Grade 2, 8 cases (21.05%) in Grade 1, and 4 cases (10.53%) in Grade 4. Conclusion: BMF was a significant finding even in the early stages of the majority of the lesions studied and was closely linked with angiogenesis. This study showed that angiogenesis plays an important role in the pathogenesis of haematological neoplasms and that VEGF is a prominent stimulus in the majority of these disorders. Additionally, this study suggests that EndMT has a possible role in the pathogenesis of BMF.

Electron Microscopy Techniques for 3D Plant ER Imaging.

The endoplasmic reticulum (ER) forms an extensive network in plant cells. In leaf cells and vacuolated root cells it is mainly restricted to the cortex, whereas in the root meristem the cortical and cytoplasmic ER takes up a large volume throughout the entire cell. Only 3D electron microscopy provides sufficient resolution to understand the spatial organization of the ER in the root. Here we present two protocols for 3D EM imaging of the ER across a range of scales. For large-scale ER structure analysis, we describe selective ER staining with ZIO that allows for automated or semi-automated ER segmentation. For smaller regions of ER, we describe high-pressure freezing, which enables almost instantaneous fixation of plant tissues but without organelle specific staining. These fixation and staining techniques are suitable for a range of imaging modalities, including serial sections, array tomography, serial block face-scanning electron microscopy (SBF-SEM), or focused ion beam (FIB) SEM.

Comparative Analysis of Internal Porosity in AM Ti64 using X-ray Computed Tomography and Mechanical Polishing Serial Sectioning

Comparative Analysis of Internal Porosity in AM Ti64 using X-ray Computed Tomography and Mechanical Polishing Serial Sectioning

Array tomography of in vivo labeled synaptic receptors.

Array tomography (AT) allows one to localize sub-cellular components within the structural context of cells in 3D through the imaging of serial sections. Using this technique, the z-resolution can be improved physically by cutting ultra-thin sections. Nevertheless, conventional immunofluorescence staining of those sections is time consuming and requires relatively large amounts of costly antibody solutions. Moreover, epitopes are only readily accessible at the section's surface, leaving the volume of the serial sections unlabeled. Localization of receptors at neuronal synapses in 3D in their native cellular ultrastructural context is important for understanding signaling processes. Here, we present in vivo labeling of receptors via fluorophore-coupled tags in combination with super-resolution AT. We present two workflows where we label receptors at the plasma membrane: first, in vivo labeling via microinjection with a setup consisting of readily available components and self-manufactured microscope table equipment and second, live receptor labeling by using a cell-permeable tag. To take advantage of a near-to-native preservation of tissues for subsequent scanning electron microscopy (SEM), we also apply high-pressure freezing and freeze substitution. The advantages and disadvantages of our workflows are discussed.

Modeling of human placental capillaries by using a unique 3d reconstruction program

Purpose: Visualization of the three-dimensional structures is performed by examining two-dimensional sections in histology. It is not always possible to comprehend structure-function integrity. Therefore, it was planned to obtain three-dimensional (3D) models of the capillaries in the placenta terminal villus by using the program developed in the Başkent University’s laboratory and used in studies in different disciplines. The program’s suitability for histology studies will also be assessed.&#x0D; Materials and Methods: A term placenta tissue sample was obtained from a 33-year-old healthy woman. Following the plastic embedding procedure, semi-thin serial sections were taken from these blocks. The sections were stained using toluidine blue and examined and photographed by using a light microscope. The 3D modeling procedure was conducted on the light micrographs of serial sections in order to reconstruct villous capillaries. &#x0D; Results: 3D images were obtained from two-dimensional light micrographs by using the same program. It was determined that the vessels running parallel to each other branched harmoniously, forming a capillary network in the terminal villus. Additionally, challenges encountered during the utilization of this program in histological studies are also presented in this study. &#x0D; Conclusion: Fetal blood reaches every part of the terminal villus through the branching capillary network. This feature would increase the surface area of vasculo-syncytial membrane and facilitate feto-maternal exchange. This can be clearly visualized by using 3D models. Three-dimensional modeling helps to understand the structure of all organs and tissues. Given that the current program is freely accessible, it will not incur additional costs for researchers and is open to further improvement by the programmer (erhankiziltan@gmail.com).

Predicting Neoadjuvant Treatment Response in Triple-Negative Breast Cancer Using Machine Learning.

Neoadjuvant chemotherapy (NAC) is the standard treatment for early-stage triple negative breast cancer (TNBC). The primary endpoint of NAC is a pathological complete response (pCR). NAC results in pCR in only 30-40% of TNBC patients. Tumor-infiltrating lymphocytes (TILs), Ki67 and phosphohistone H3 (pH3) are a few known biomarkers to predict NAC response. Currently, systematic evaluation of the combined value of these biomarkers in predicting NAC response is lacking. In this study, the predictive value of markers derived from H&E and IHC stained biopsy tissue was comprehensively evaluated using a supervised machine learning (ML)-based approach. Identifying predictive biomarkers could help guide therapeutic decisions by enabling precise stratification of TNBC patients into responders and partial or non-responders. Serial sections from core needle biopsies (n = 76) were stained with H&E and immunohistochemically for the Ki67 and pH3 markers, followed by whole-slide image (WSI) generation. The serial section stains in H&E stain, Ki67 and pH3 markers formed WSI triplets for each patient. The resulting WSI triplets were co-registered with H&E WSIs serving as the reference. Separate mask region-based CNN (MRCNN) models were trained with annotated H&E, Ki67 and pH3 images for detecting tumor cells, stromal and intratumoral TILs (sTILs and tTILs), Ki67+, and pH3+ cells. Top image patches with a high density of cells of interest were identified as hotspots. Best classifiers for NAC response prediction were identified by training multiple ML models and evaluating their performance by accuracy, area under curve, and confusion matrix analyses. Highest prediction accuracy was achieved when hotspot regions were identified by tTIL counts and each hotspot was represented by measures of tTILs, sTILs, tumor cells, Ki67+, and pH3+ features. Regardless of the hotspot selection metric, a complementary use of multiple histological features (tTILs, sTILs) and molecular biomarkers (Ki67 and pH3) resulted in top ranked performance at the patient level. Overall, our results emphasize that prediction models for NAC response should be based on biomarkers in combination rather than in isolation. Our study provides compelling evidence to support the use of ML-based models to predict NAC response in patients with TNBC.

Investigation of Histological and Histochemical Features of Mouse Placenta in Different Periods of Pregnancy

This study aimed to investigate the histological and histochemical properties of mouse placenta samples from different periods of pregnancy. For this purpose, mouse placenta samples were collected on the fourth, tenth, and seventeenth days of pregnancy and blocked in paraffin. Serial sections of 5μ thickness were taken at 50μ intervals. Histological and histochemical staining methods were applied to the sections. As a result, histological and histochemical characteristics of mouse placenta from different periods of pregnancy were determined.