Electron Microscopic Level Research Articles

Surfactant dysfunction and alveolar collapse are linked with fibrotic septal wall remodeling in the TGF-β1-induced mouse model of pulmonary fibrosis

In human idiopathic pulmonary fibrosis (IPF), collapse of distal airspaces occurs in areas of the lung not (yet) remodeled. Mice lungs overexpressing transforming growth factor-β1 (TGF-β1) recapitulate this abnormality: surfactant dysfunction results in alveolar collapse preceding fibrosis and loss of alveolar epithelial type II (AE2) cells' apical membrane surface area. Here we examined whether surfactant dysfunction-related alveolar collapse due to TGF-β1 overexpression is linked to septal wall remodeling and AE2 cell abnormalities. Three and 6 days after gene transfer of TGF-β1, mice received either intratracheal surfactant (Surf-groups: Curosurf®, 100 mg/kg bodyweight) or 0.9% NaCl (Saline-groups). On days 7 (D7) and 14 (D14), lung mechanics were assessed followed by design-based stereology at light and electron microscopic level to quantify structures. Compared with Saline, Surf showed significantly improved tissue elastance, increased numbers of open alveoli, as well as reduced alveolar size heterogeneity on D7. Deterioration in lung mechanics was highly correlated to the loss of open alveoli. On D14, lung mechanics, number of open alveoli, and alveolar size heterogeneity remained significantly improved in the Surf-group. Volumes of extracellular matrix and collagen fibrils in septal walls were significantly reduced, whereas the apical membrane surface area of AE2 cells was increased in Surf compared with Saline. In remodeled tissue with collapsed alveoli, three-dimensional reconstruction of AE2 cells based on scanning electron microscopy array tomography revealed that AE2 cells were trapped without contact to airspaces in the TGF-β1 mouse model. Similar observations were made in human IPF. Based on correlation analyses, the number of open alveoli and of alveolar size heterogeneity were highly linked with the loss of apical membrane surface area of AE2 cells and deposition of collagen fibrils in septal walls on D14. In conclusion, surfactant replacement therapy stabilizes alveoli and prevents extracellular matrix deposition in septal walls in the TGF-β1 model.

Osteogenic Cell Behavior on Titanium Surfaces in Hard Tissue.

It is challenging to remove dental implants once they have been inserted into the bone because it is hard to visualize the actual process of bone formation after implant installation, not to mention the cellular events that occur therein. During bone formation, contact osteogenesis occurs on roughened implant surfaces, while distance osteogenesis occurs on smooth implant surfaces. In the literature, there have been many in vitro model studies of bone formation on simulated dental implants using flattened titanium (Ti) discs; however, the purpose of this study was to identify the in vivo cell responses to the implant surfaces on actual, three-dimensional (3D) dental Ti implants and the surrounding bone in contact with such implants at the electron microscopic level using two different types of implant surfaces. In particular, the different parts of the implant structures were scrutinized. In this study, dental implants were installed in rabbit tibiae. The implants and bone were removed on day 10 and, subsequently, assessed using scanning electron microscopy (SEM), immunofluorescence microscopy (IF), transmission electron microscopy (TEM), focused ion-beam (FIB) system with Cs-corrected TEM (Cs-STEM), and confocal laser scanning microscopy (CLSM)—which were used to determine the implant surface characteristics and to identify the cells according to the different structural parts of the turned and roughened implants. The cell attachment pattern was revealed according to the different structural components of each implant surface and bone. Different cell responses to the implant surfaces and the surrounding bone were attained at an electron microscopic level in an in vivo model. These results shed light on cell behavioral patterns that occur during bone regeneration and could be a guide in the use of electron microscopy for 3D dental implants in an in vivo model.

Autopsy and Forensic Study on a Rare Human Corpse Preserved Over Two Thousand Years: The Mawangdui Ancient Cadaver.

In 1972, an enormous tomb site was found in the eastern suburb of Changsha, the capital city of Hunan Province, which led to the discovery of Mawangdui tomb No. 1, and soon thereafter tombs Nos. 2 and 3. These tombs were dated back to the Western Han Dynasty (206 BC-24 AD) in Chinese history. Along with numerous precious historic relics unearthed as grave goods, a well-preserved female cadaver was the most unprecedented, which was considered as one of the world's greatest archeological discoveries in the 20th century. The cadaver was initially examined through autopsy and X-ray imaging, with biopsies from multiple body parts analyzed histologically at the light and electron microscopic levels. In this review, we summarize the major imaging and autopsy findings from the cadaver indicative of remarkable preservation of some histological, cellular, and molecular constituents of the body. A forensic assessment of antemortem illnesses and potential cause of death of the subject are also noted.

Complement component 1q subcomponent binding protein in the brain of the rat

Complement component 1q subcomponent binding protein (C1qbp) is a multifunctional protein involved in immune response, energy homeostasis of cells as a plasma membrane receptor, and a nuclear, cytoplasmic or mitochondrial protein. Recent reports suggested its neuronal function, too, possibly in axon maintenance, synaptic function, and neuroplasticity. Therefore, we addressed to identify C1qbp in the rat brain using in situ hybridization histochemistry and immunolabelling at light and electron microscopic level. C1qbp has a topographical distribution in the brain established by the same pattern of C1qbp mRNA-expressing and protein-containing neurons with the highest abundance in the cerebral cortex, anterodorsal thalamic nucleus, hypothalamic paraventricular (PVN) and arcuate nuclei, spinal trigeminal nucleus. Double labelling of C1qbp with the neuronal marker NeuN, with the astrocyte marker S100, and the microglia marker Iba1 demonstrated the presence of C1qbp in neurons but not in glial cells in the normal brain, while C1qbp appeared in microglia following their activation induced by focal ischemic lesion. Only restricted neurons expressed C1qbp, for example, in the PVN, magnocellular neurons selectively contained C1qbp. Further double labelling by using the mitochondria marker Idh3a antibody suggested the mitochondrial localization of C1qbp in the brain, confirmed by correlated light and electron microscopy at 3 different brain regions. Post-embedding immunoelectron microscopy also suggested uneven C1qbp content of mitochondria in different brain areas but also heterogeneity within single neurons. These data suggest a specific function of C1qbp in the brain related to mitochondria, such as the regulation of local energy supply in neuronal cells.

Spermatogenesis and lobular cyst type of testes organization in marine gastropod Littorina saxatilis (Olivi 1792).

Although individual stages of spermatogenesis in Littorina saxatilis are well studied at the electron microscopic level, the gonad structure and the spatial localization of gametes at different stages of maturation remain unclear. Using differential-interference contrast (DIC) for observations of fresh tissue we show that the mature testis consists of numerous ovoid lobules forming larger lobes. The lobules of intact mature testes of L. saxatilis are filled with randomly arranged multicellular cysts containing gametes at different stages of maturation. Gametes within a cyst are highly synchronized in respect of the differentiation degree. At the same time, no spatial gradient in the arrangement of cysts according to the maturation degree of gametes in them was observed in any of the studied lobules. The male gonads contain cysts with early spermatids, mid, late spermatids, and spermatozoa. Using silver-staining, DAPI, and chromomycin A3 (CMA3) staining, we identify 20 main types of nucleus organization in differentiating sperm. Premature and mature male gonads contain cysts with a mosaic arrangement as well as rare solitary cyst cells, goniablast cysts, or separate spermatogonia in between them. Our data indicate that the testis structure in L. saxatilis cannot be attributed to the tubular type, as previously thought. It corresponds to the lobular cyst type but individual lobules contain cysts with gametes at the same stage of development. It is similar to the testis structure of several fishes, amphibians, and Drosophila melanogaster. This type of the gonad organization has never been described in gastropods before.

Evaluation of medial division of the medial geniculate (MGM) and posterior intralaminar nucleus (PIN) inputs to the rat auditory cortex, amygdala, and striatum.

The medial division of the medial geniculate (MGM) and the posterior intralaminar nucleus (PIN) are association nuclei of the auditory thalamus. We made tracer injections in these nuclei to evaluate/compare their presynaptic terminal and postsynaptic target features in auditory cortex, amygdala and striatum, at the light and electron microscopic levels. Cortical labeling was concentrated in Layer 1 but in other layers distribution was location-dependent. In cortical areas designated dorsal, primary and ventral (AuD, Au1, AuV) terminals deep to Layer 1 were concentrated in infragranular layers and sparser in the supragranular and middle layers. In ectorhinal cortex (Ect), distributions below Layer 1 changed with concentrations in supragranular and middle layers. In temporal association cortex (TeA) terminal distributions below Layer 1 was intermediate between AuV/1/D and Ect. In amygdala and striatum, terminal concentrations were higher in striatum but not as dense as in cortical Layer 1. Ultrastructurally, presynaptic terminal size was similar in amygdala, striatum or cortex and in all cortical layers. Postsynaptically MGM/PIN terminals everywhere synapsed on spines or small distal dendrites but as a population the postsynaptic structures in cortex were larger than those in the striatum. In addition, primary cortical targets of terminals were larger in primary cortex than in area Ect. Thus, although postsynaptic size may play some role in changes in synaptic influence between areas it appears that terminal size is not a variable used for that purpose. In auditory cortex, cortical subdivision-dependent changes in the terminal distribution between cortical layers may also play a role.

Identification of PSD-95 in the Postsynaptic Density Using MiniSOG and EM Tomography.

Combining tomography with electron microscopy (EM) produces images at definition sufficient to visualize individual protein molecules or molecular complexes in intact neurons. When freeze-substituted hippocampal cultures in plastic sections are imaged by EM tomography, detailed structures emerging from 3D reconstructions reveal putative glutamate receptors and membrane-associated filaments containing scaffolding proteins such as postsynaptic density (PSD)-95 family proteins based on their size, shape, and known distributions. In limited instances, structures can be identified with enhanced immuno-Nanogold labeling after light fixation and subsequent freeze-substitution. Molecular identification of structure can be corroborated in their absence after acute protein knockdown or gene knockout. However, additional labeling methods linking EM level structure to molecules in tomograms are needed. A recent development for labeling structures for TEM employs expression of endogenous proteins carrying a green fluorescent tag, miniSOG, to photoconvert diaminobenzidine (DAB) into osmiophilic polymers. This approach requires initial mild chemical fixation but many of structural features in neurons can still be discerned in EM tomograms. The photoreaction product, which appears as electron-dense, fine precipitates decorating protein structures in neurons, may diffuse to fill cytoplasm of spines, thus obscuring specific localization of proteins tagged with miniSOG. Here we develop an approach to minimize molecular diffusion of the DAB photoreaction product in neurons, which allows miniSOG tagged molecule/complexes to be identified in tomograms. The examples reveal electron-dense clusters of reaction product labeling membrane-associated vertical filaments, corresponding to the site of miniSOG fused at the C-terminal end of PSD-95-miniSOG, allowing identification of PSD-95 vertical filaments at the PSD. This approach, which results in considerable improvement in the precision of labeling PSD-95 in tomograms without complications due to the presence of antibody complexes in immunogold labeling, may be applicable for identifying other synaptic proteins in intact neurons.

Presynaptic increase in IP3 receptor type 1 concentration in the early phase of hippocampal synaptic plasticity

The inositol 1,4,5-trisphosphate receptor (IP3R) subtype IP3R1 is highly enriched in the brain, including hippocampal neurons. It plays an important function in regulating intracellular calcium concentrations. Residing on the smooth endoplasmic reticulum (sER), the IP3R1 mobilizes calcium into the cytosol upon binding the intracellular signaling molecule IP3, whose concentration is increased by stimulating certain metabotropic glutamate receptors. Increased calcium may mediate synaptic changes occurring during long-term plasticity, which includes molecular mechanisms underlying memory encoding. The exact synaptic localization of IP3R1 in the central nervous system (CNS) remains unclear. We hypothesized that IP3R1, in addition to its known expression in soma and dendritic shafts of hippocampal CA1 pyramidal neurons, also may be present in postsynaptic spines. Moreover, we hypothesized that IP3R1 may be present in presynaptic terminals as well, given the importance of calcium in regulating presynaptic neurotransmitter exocytosis. To test these two hypotheses, we used IP3R1 immunocytochemistry at the light and electron microscopical levels in the CA1 area of the hippocampus. Furthermore, we hypothesized that induction of long-term potentiation (LTP) would be accompanied by an increase in synaptic IP3R1 concentrations, thereby facilitating synaptic mechanisms of long term plasticity. To investigate this, we used quantitative immunogold electron microscopy to determine possible changes in IP3R1 concentration in sub-synaptic compartments before and five minutes after high frequency tetanizations. Firstly, our data confirm localization of IP3R1 in both presynaptic terminals and postsynaptic spines. Secondly, the concentration of IP3R1 after tetanization was significantly increased in the presynaptic compartment, suggesting a presynaptic role of IP3R1 in early phases of synaptic plasticity. It is therefore possible that IP3R1 is involved in modulating neurotransmitter release by regulating calcium homeostasis presynaptically.

Neurocan Inhibits Semaphorin 3F Induced Dendritic Spine Remodeling Through NrCAM in Cortical Neurons.

Neurocan is a chondroitin sulfate proteoglycan present in perineuronal nets, which are associated with closure of the critical period of synaptic plasticity. During postnatal development of the neocortex dendritic spines on pyramidal neurons are initially overproduced; later they are pruned to achieve an appropriate balance of excitatory to inhibitory synapses. Little is understood about how spine pruning is terminated upon maturation. NrCAM (Neuron-glial related cell adhesion molecule) was found to mediate spine pruning as a subunit of the receptor complex for the repellent ligand Semaphorin 3F (Sema3F). As shown here in the postnatal mouse frontal and visual neocortex, Neurocan was localized at both light and electron microscopic level to the cell surface of cortical pyramidal neurons and was adjacent to neuronal processes and dendritic spines. Sema3F-induced spine elimination was inhibited by Neurocan in cortical neuron cultures. Neurocan also blocked Sema3F-induced morphological retraction in COS-7 cells, which was mediated through NrCAM and other subunits of the Sema3F holoreceptor, Neuropilin-2, and PlexinA3. Cell binding and ELISA assays demonstrated an association of Neurocan with NrCAM. Glycosaminoglycan chain interactions of Neurocan were required for inhibition of Sema3F-induced spine elimination, but the C-terminal sushi domain was dispensable. These results describe a novel mechanism wherein Neurocan inhibits NrCAM/Sema3F-induced spine elimination.

NEURORESTORATIVE MULBERRIES POTENTIAL OF ALZHEIMER’S DISEASE IN ANIMAL MODEL

Objective: Alzheimer’s disease (AD) is one of the most common neurodegenerative disorders. The present study aimed to identify the potential neurorestorative of Morus alba and Morus rubra fruits extract on aluminum chloride (AlCl3)-induced AD rats.Methods: Each Morus species extract M. alba and M. rubra at a dose 300 mg/kg body weight (b.wt.) was orally administered, daily for 6 weeks post AlCl3 induction (100 mg/kg b.wt./day/6 weeks, orally). The polyphenols content was determined according to the colorimetric method of Folin–Ciocalteu using gallic acid as a standard. In addition, brain neurotransmitters, total protein content (TPC), and antioxidant biomarkers were determined. Histopathological assessment was performed on the brains of rats.Results: Administration of AlCl3 neurotoxicity induced a significant increment of brain acetylcholinesterase, and nitric oxide, while significant decrease in four monoamines (norepinephrine, epinephrine, 5-hydroxytryptamine serotonin, and dopamin), along with brain tissue ATPase, and TPC were determined. AlCl3-induced changes in peroxidative products, such as protein carbonyl, catalase, and superoxide dismutase activity. AlCl3 induction resulted in significant decrease in antioxidant enzymes, serum level of total antioxidant capacity, and caused histological alteration for brain tissue. Treatment of AD-rats with M. alba and M. rubra (300 mg/kg b.wt./day) causes modulation of most biochemical markers brain function and confirmed by brain histopathological study.Conclusion: Collectively, the present results indicated that M. rubra and M. alba exerts neuroprotection against AlCl3-induced brain pathology. To the best of our knowledge, none of the literature studies have demonstrated the effect of these Morus species against Al-neurotoxicity at biochemical and electron microscopic levels.

Structural and Synaptic Organization of the Adult Reeler Mouse Somatosensory Neocortex: A Comparative Fine-Scale Electron Microscopic Study of Reeler With Wild Type Mice.

The reeler mouse has been widely used to study various aspects of cortico- and synaptogenesis, but also as a model for several neurological and neurodegenerative disorders. In contrast to development, comparably little is known about the neuronal composition and synaptic organization of the adult reeler mouse neocortex, in particular at the fine-scale electron microscopic level, which was investigated here and compared with wild type (WT) mice. In this study, the “barrel field” of the adult reeler and WT mouse somatosensory neocortex is used as a model system. In reeler the characteristic six-layered structure is no longer existent, but replaced by a conglomerate of neurons organized in homologous clusters with maintained morphological identity and heterologous clusters between neurons and/or oligodendrocytes. These clusters are loosely scattered throughout the neocortical mass between the pial surface and the white matter. In contrast to WT, layer 1 (L1), if existent, seems to be diluted into the volume of the neocortical mass with no clear boundary. L1 also contains clusters of migrated or persistent neurons, oligodendro- and astrocytes. As in WT, myelinated and unmyelinated axons were found throughout the neocortical mass, but in reeler they were organized in massive fiber bundles with a high fiber packing density. A prominent and massive thalamocortical projection traverses through the neocortical mass, always accompanied by numerous “active” oligodendrocytes whereas in WT no such projections were found and “silent” oligodendrocytes were restricted to the white matter. In the adult reeler mouse neocortex, synaptic boutons terminate on somata, dendritic shafts, spines of different types and axon initial segments with no signs of structural distortion and/or degeneration, indicating a “normal” postsynaptic innervation pattern of neurons. In addition, synaptic complexes between boutons and their postsynaptic targets are tightly ensheathed by fine astrocytic processes, as in WT. In conclusion, the neuronal clusters may represent a possible alternative organization principle in adult reeler mice “replacing” layer formation. If so, these homologous clusters may represent individual “functional units” where neurons are highly interconnected and may function as the equivalent of neurons integrated in a cortical layer. The structural composition and postsynaptic innervation pattern of neurons by synaptic boutons provide the structural basis for the establishment of a functional although altered cortical network in the adult reeler mouse.

Anatomic Studies of the Miniature Swine Cornea.

The domestic swine eye resembles the human eye both anatomically and physiologically. Xenotransplantation of the swine cornea to humans in need of full keratoplasty shows promise as a potential therapeutic strategy to restore vision in individuals with advanced corneal disease, especially those residing in developing nations. That said, we characterized the morphology of corneas from miniature swine, which are smaller in size, easier to handle, and more cost-effective compared to domestic swine. Eyes (N = 15) were harvested from miniature swine from different age groups: 1 month (N = 3), 2 month (N = 3), 4 month (N = 3), 8 month (N = 3), as well as 24 month old adult domestic swine (N = 3). They were immediately submerged in fixative and processed for histological examination at the light and transmission electron microscopic level. Gross anatomic measurements of the cornea were significantly less (P value ≤ 0.05) in miniature swine versus domestic swine. Corneal strata exhibited morphological characteristics similar to the domestic swine cornea. Adult miniature swine corneas show similar overall corneal thickness at 8 months of age versus domestic swine. Miniature swine exhibit similar corneal morphology with the domestic pig and humans, with the exception of Bowman's layer, which is absent in pigs. Therefore, miniature pigs may be a useful resource of corneal tissue for humans in need of full keratoplasty, as well as serve as a large eye model for ophthalmology residents to develop surgical skills and for development and testing of ocular therapeutic strategies that translate to humans. Anat Rec, 301:1955-1967, 2018. © 2018 Wiley Periodicals, Inc.

Dark Neurons of the Brain

The structure and functional characteristics of dark hyperchromic and hyperchromic shrunken neurons in the brain have been studied at the light and electron microscopic levels in health and various pathologies. Hyperchromic dark neurons are cells with active protein synthesis which, however, die by apoptosis as a result of prolonged and intense exposure to unfavorable factors or because of genetic abnormalities.

Age features of the morpho-functional state of the liver in the distant terms of experimental diabetes mellitus.

Background. Between the most frequent complications of diabetes mellitus are diabetic hepatopathies, which are accompanied by a violation of the functional activity of the liver. Objective. Therefore, the purpose of our study was to establish the morpho-functional changes of the liver of 24 months old rats in the distant terms of streptozotocine diabetes mellitus. Methods. The study was conducted on 24 male Wistar rats that had reached the age of 24 months. The animals were divided into 2 groups: experimental (15 animals) and control (9 animals). For streptozotocine diabetes mellitus modelling, animals of the experimental group were administered intraperitoneally with streptozotocin in a dose of 5 mg per 100 g of body weight. Biochemical, histological (coloring of hematoxylin-eosin, Shabadash, Sudan III) and electron-microscopic methods of research were used. The material was taken on the 42, 56 and 70 days of the streptozotocine diabetes mellitus course. Results. At the 42 day of streptozotocine diabetes mellitus we detect the unevenness of the coloration of the hepatocyte cytoplasm due to the presence of small vacuoles, indicating the development of vacuolic dystrophy. At the 56 day of streptozotocine diabetes mellitus, both on the optical and electron microscopic levels, we find the signs of lipid, vacuolic and balloon dystrophy. In hepatocytes there are numerous lipid drops, which may indicate the development of fatty dystrophy. At the 70 day of streptozotocine diabetes mellitus, a significant polymorphism of morphological changes in hepatocytes is observed. Some of them show the signs of vacuolation, others - balloon dystrophy. We observe that separate hepatocytes are in the state of colic necrosis, others - are in a state of apoptosis. Starting from the 42 day of streptozotocine diabetes mellitus, we observe an increased level of activity of enzymes of the cytolysis of hepatocytes, both in blood serum and in liver homogenates, in comparison with the control group of animals, and the reduction of the Ritis coefficient. Conclusion. Thus, in the distant terms of streptozotocine diabetes mellitus (42-70 days), we observe the morpho-functional changes in the liver, which are manifested: vacuolar and hydropic dystrophies, colic-type necrosis, apoptosis. Such structural changes in the liver lead to a violation of its function, which is confirmed by a significant increase in the level of alanine aminotransferase and aspartate aminotransferase and simultaneous decrease in the Ritis coefficient.

Mycolicibacterium smegmatis, Basonym Mycobacterium smegmatis, Expresses Morphological Phenotypes Much More Similar to Escherichia coli Than Mycobacterium tuberculosis in Quantitative Structome Analysis and CryoTEM Examination.

A series of structome analyses, that is, quantitative and three-dimensional structural analysis of a whole cell at the electron microscopic level, have already been achieved individually in Exophiala dermatitidis, Saccharomyces cerevisiae, Mycobacterium tuberculosis, Myojin spiral bacteria, and Escherichia coli. In these analyses, sample cells were processed through cryo-fixation and rapid freeze-substitution, resulting in the exquisite preservation of ultrastructures on the serial ultrathin sections examined by transmission electron microscopy. In this paper, structome analysis of non pathogenic Mycolicibacterium smegmatis, basonym Mycobacterium smegmatis, was performed. As M. smegmatis has often been used in molecular biological experiments and experimental tuberculosis as a substitute of highly pathogenic M. tuberculosis, it has been a task to compare two species in the same genus, Mycobacterium, by structome analysis. Seven M. smegmatis cells cut into serial ultrathin sections, and, totally, 220 serial ultrathin sections were examined by transmission electron microscopy. Cell profiles were measured, including cell length, diameter of cell and cytoplasm, surface area of outer membrane and plasma membrane, volume of whole cell, periplasm, and cytoplasm, and total ribosome number and density per 0.1 fl cytoplasm. These data are based on direct measurement and enumeration of exquisitely preserved single cell structures in the transmission electron microscopy images, and are not based on the calculation or assumptions from biochemical or molecular biological indirect data. All measurements in M. smegmatis, except cell length, are significantly higher than those of M. tuberculosis. In addition, these data may explain the more rapid growth of M. smegmatis than M. tuberculosis and contribute to the understanding of their structural properties, which are substantially different from M. tuberculosis, relating to the expression of antigenicity, acid-fastness, and the mechanism of drug resistance in relation to the ratio of the targets to the corresponding drugs. In addition, data obtained from cryo-transmission electron microscopy examination were used to support the validity of structome analysis. Finally, our data strongly support the most recent establishment of the novel genus Mycolicibacterium, into which basonym Mycobacterium smegmatis has been classified.

Ultrastructure and phylogeny of a new species of mixotrophic dinoflagellate, Paragymnodinium stigmaticum sp. nov. (Gymnodiniales, Dinophyceae)

:A new dinoflagellate, Paragymnodinium stigmaticum Yokouchi, Onuma & Horiguchi sp. nov., was described from sand samples collected at Sumiyoshi beach, Sado Island, Niigata Prefecture, Japan, based on observations at the light, scanning and transmission electron microscope levels together with molecular characterisation. The cells were 8.5–15.2 μm long and 6.3–12.4 μm wide. The episome and hyposome were subequal and hemispherical; although, the episome could also be conical. The cingulum was wide, well excavated and descended one quarter to a half of its own width. The sulcus was straight and widened slightly as it reached the antapex. A slightly curved sulcal extension-like furrow (SEF) ran from the right end of cingulum toward the apex. Cells possessed five to 10 yellowish brown chloroplasts, a red eyespot and small nematocysts. The dinoflagellate ingested prey cells despite the possession of chloroplasts, indicating a mixotrophic mode of nutrition. Paragymnodinium stigmaticum shared many morphological features and nutritional strategies with a known species of the genus Paragymnodinium, Paragymnodinium shiwhaense, including the possession of nematocysts, small polygonal amphiesmal vesicles and a mixotrophic mode of nutrition. However, it is clearly distinguished from P. shiwhaense by its feeding mechanism, its chloroplast ultrastructure, the presence of an eyespot, the absence of a plate-like structure in the amphiesmal vesicles and a benthic lifestyle (P. shiwhaense is planktonic). A phylogenetic analysis inferred from concatenated small and large subunit ribosomal DNA sequences recovered P. stigmaticum in a robust clade with P. shiwhaense that was included in the clade Gymnodinium sensu stricto. Therefore, it is reasonable to conclude that this dinoflagellate from Japan is a new species in the genus Paragymnodinium.

MORPHOLOGICAL CHARACTERISTICS OF THE ANTERIOR ABDOMINAL WALL TIS-SUES IN CASES OF IMPLANTATION OF POLYPROPYLENE MESH WITH PRF MEMBRANE

According to the experimental study on Vietnamese pigs after implantation of a polypropylene mesh with PRF membrane into retromuscular space, the results of ultrastructural changes in the cells of muscular aponeurotic layer of abdominal wall were presented. It was proved that on the 14th day of the experiment, inflammatory changes in tissues were expressed significantly less than in cases of the isolated implantation of the polypropylene mesh without PRF membrane. Activation of fibroblasts and signs of fiber structures development around the mesh material increased. In the late experimental period (28 days), at the microscopic and electron-microscopic levels the minor manifestations of inflammatory reaction, improved microcirculation in the area of implantation of the mesh with PRF membrane were found that in turn contributed to the increased activity of fibroblasts and development of collagen fibers around the mesh material. The use of PRF plasma membranes stimulated an active development of new capillaries, improved blood flow, accelerated metabolic processes in tissues, and suddenly increased development of collagen, hyaluronic acid that in turn created favourable environment for a complete integration of the polypropylene mesh into the muscular aponeurotic layer of anterior abdominal wall tissue.

Identification and mapping of brain natriuretic peptide in the normal ventricular myocardium of a desert-dwelling mammalian model, the camel (Camelus dromedarius): Immunohistochemical and ultrastructural study.

Brain natriuretic peptide (BNP) is mainly produced in the ventricular myocardium, where it is released into the circulation, producing rapid volume decrease by diuresis, natriuresis, and water shift into the extracellular space, and vasodilation. The dromedary camel, a mammalian model of the desert nomads, lives under unfavorable physiological stresses during thirst, starvation, desiccation, and hot climate, thus has a special demand for water homeostasis. The present studies characterized BNP in the ventricular myocardium of healthy camels, immunohistochemically with a specific antibody, and ultrastructurally identified the endocrine property of the cardiomyocytes and Purkinje fibers. The paranuclear, granular, immunoreactive material was not restricted to the cardiomyocytes, as it was also visible in the Purkinje fibers and their associated nerve varicosities. The intensity of immunoreactive BNP showed a transmural gradient from the subepicardium to the myocardium. Intense immunoreactivity was also noted among the perivascular cardiomyocytes. At the electron microscopic level, specific granules were demonstrated in the paranuclear cytosol of cardiomyocytes and Purkinje fibers. The current study provides the first immunohistochemical localization pattern of BNP in the camel myocardium and suggests a relationship between the intense subepicardial BNP-immunoexpression and a possible translocation of the active hormone to the pericardial fluid for further paracrine actions on the heart and its coronaries.

Perichromatin region: a moveable feast.

The perichromatin region is an elusive zone of the cell nucleus located at the periphery of the condensed chromatin areas. This region is visible at the electron microscope level under special staining treatments, otherwise it is merged with the border of condensed chromatin. In this 200nm-thick area, several fundamental cell processes take place, such as replication, DNA repair and transcription. In addition, RNA processing occurs in the perichromatin region, including 5'-capping and 3'-polyadenylation as well as splicing. Recently, it has become clear that also some epigenetics modifications take place there, such as methylation of DNA and RNA on cytosine and adenosine. In summary, this thin interface between chromatin and the interchromatinic space represents the zone where the majority of the functions of DNA in interphase occur, in a place where there is no steric hindrance of condensed chromatin, the products can easily move away toward their target and the enzymes can freely dock.

Abstract 1276: Differentiation of human colon adenomas in an enteroid culture: A treatment comparison

Abstract Introduction: Previous murine studies have demonstrated that dietary Aquamin®, a calcium-rich, multi-mineral natural product, suppressed colon polyp formation and transition to invasive tumors more effectively than calcium alone when provided over the lifespan of the animals. Methods: In the present study, we compared Aquamin® to calcium for modulation of growth and differentiation in human colon adenomas in enteroid culture. Adenoma growth and differentiation were assessed at the light and electron microscopic levels and by immunostaining. Image quantitation of immunohistochemical markers was performed using Aperio Imagescope to compare these interventions. A proteomic assessment was done to compare protein expression patterns in treated cultures as compared to cultures maintained under control conditions (calcium 0.15 mM). Enteroids established from normal colonic tissue were examined in parallel. Results: Both calcium and Aquamin® provided at 1.5 mM calcium fostered differentiation in the adenoma enteroid cultures as compared to control, but Aquamin® was more effective. Even at a concentration providing only 0.15 mM calcium, Aquamin® induced differentiation in some individual enteroids. Aquamin® was more effective than calcium in inducing upregulation of proteins. There were 35 proteins upregulated with Aquamin as compared to 20 with calcium 1.5mM at 1.8-fold change or above (in 3 colonic adenomas) with less than 2% FDR. Both calcium and Aquamin® induced differences in the expression pattern of proteins known to be involved in differentiation (table). In addition, several keratins and histones, as well as merlin and Olfactomedin-4 were upregulated. Conclusion: These findings support that i) calcium (1.5 mM) has the capacity to modulate growth and differentiation in large human colon adenomas and ii) the additional trace elements provided along with calcium in Aquamin® can have effects on proliferation and differentiation at lower levels than observed with calcium at 1.5mM. Expression of Immunohistochemical Markers in enteroid cultures of Human Adenomas (Fold-Changes)IHC MarkersCalcium 0.15mMAquamin 0.15mMCalcium 1.5mMAquamin 1.5mMKi6710.750.59*0.49*CK2012.13*2.36*2.35*E-Cadherin11.051.57*1.57*NF2 (Merlin)12.44*#1.012.56*#Occludin11.23*1.25*1.29**reflects significance as compared to Ca 0.15mM. #reflects significance as compared to Ca 1.5mM Citation Format: Shannon D. McClintock, Justin A. Colacino, Durga Attili, Michael K. Dame, Areeba H. Rizvi, Aliah Richter, Anusha R. Reddy, Venkatesha Basrur, D. Kim Turgeon, James Varani, Muhammad N. Aslam. Differentiation of human colon adenomas in an enteroid culture: A treatment comparison [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1276.