BIOLOGICAL PECULIARITIES OF ADIPOSE TISSUE-DERIVED MESENCHYMAL STEM CELLS AT DIFFERENT PASSAGES OF CULTIVATION

The studies were conducted on 2–3-months-old males of C57BL/6 mice weighing 20–24 g. Obtaining and operating with adipose tissue-derived mesenchymal stem cell (MSC) culture was performed in a sterile laminar box under conditions of asepsis and antiseptics. The adipose tissue-derived MSC of the 2, 4, 7 and 12 passages were analyzed. Morphometric analysis was performed using a light microscopy. Morphometric parameters such as cell and nucleus area or nuclear-cytoplasmic ratio were calculated using the Axiovision light microscope (Carl Zeiss, Germany) and Image J 1.45 software. Trypan blue dye used for investigation of the viability of MSC. The morphological characteristics of adipose tissue-derived MSC during the process of cultivation changes: at the first passages of cultivation, the cells are spindle-shaped with two, at least three, long cytoplasmic processes, which are located bipolar. Near the nucleus, the Golgi complex is clearly visible – a sign of active cells. At later passages, cells have a small cytoplasmic processes and the bipolar arrangement of processes changes by stellar arrangement. Golgi complex is also clearly visualized. The indicator of the nuclear-cytoplasmic ratio in MSC from adipose tissue is significantly reduced at the 7th passage to 0.2189 ± 0.0122 (P < 0.01), and at the 12th passage to 0.1111 ± 0.0086 (P < 0.001) compared to the 2nd passage. The coefficient of proliferation of adipose tissue-derived MSC is significantly reduced at 12th passage. The viability of MSC from adipose tissue with an increasing of a number of passages significantly reduces and at the 12th passage of cultivation reaches 84.67 ± 1.36 (P < 0.05). The content of apoptotic cells that exhibited sensitivity to serum-free cultivation significantly increased at the 7th and 12th passages and was 21.33 ± 1.36 (P < 0.05) and 23.67 ± 0.97% (P < 0.05), respectively.

Dynamics of pathomorphological changes in the structural organization of the intervertebral disc at the end of the seventh and fourteenth day of experimental opioid exposure at the ultrastructural level

In general, the modern literature pays attention to the issues of spine pathology and intervertebral discs. A significant percentage of vertebral disorders - scoliosis, osteochondrosis, spinal disc herniation, etc., occur as a result of exposure to various factors and manifest in changes of the intervertebral discs. The aim of our work was to study at the ultrastructural level the features of pathomorphological manifestations in the structural components of the intervertebral disc at the end of the seventh and fourteenth days of experimental opioid exposure. Materials and methods of research. The objects of the study were 32 sexually mature, white, male rats, weighing 92 - 103 g, aged 4.5 months. Animals were injected with nalbupine intramuscularly once daily (at 10-11 a.m.) for 14 days. The initial dose of nalbuphine was 8 mg/kg during the first week, 15 mg/kg during the second week. It created the conditions of chronic opioid exposure. Before sampling, the animals were withdrawn from the experiment using dibutyl ether. Intervertebral discs of rats were used as a material for ultrastructural study. Ultrastructural specimens were prepared according to the accepted methods. The results of the study. As a result of the sampling after 7 days of opioid exposure we found inhomogeneous osmiophilicity and compaction of the nucleus pulposus matrix in which intensively accumulated osmiophilic grains of glycogen proteoglycans, increased the number of collagen fibers, some of them were heterogeneous. It was also noted the development of moderate degenerative changes in some notochondral cells, which was accompanied by increased vacuolization of the cytoplasm by inhomogeneous compaction of the nucleus and an increase of heterochromatin there. After 14 days necrotic changes in the cells of the nucleus pulposus, as well as the destruction of collagen fibers of the annulus fibrosus were found. In particular, an increase in the amount of heterochromatin in the nucleui of notochondral cells, which was accompanied by a decrease in the volume of the nucleui and inhomogeneous swelling of the cytoplasm. Active fibroblasts were often visualized in the annulus fibrosus. Intense osmiophilicity and thickening of collagen fibers of the annulus fibrosus were observed in some areas of the fibrous ring. Focal destruction of collagen fibers was also noted. In the areas of destruction the fibrils of collagen fibers disintegrated into an inhomogeneous fine-grained stratified mass and were located loosely. Conclusions. At the end of the first week we found that the cytoplasmic processes of chondrocytes decreased in volume, shortened, underwent fragmentation and destruction, some of them detached from the surface of the plasmolemma. At the end of the second week signs of opioid exposure progressed and manifested by an increase in the destruction of cytoplasmic processes in chondrocytes. Also focal destruction of collagen fibers was noted.

Cx43 mediates changes in myofibroblast contraction and collagen release in human amniotic membrane defects after trauma

The wound healing capacity of the fetal membranes after spontaneous or iatrogenic membrane rupture is unclear. We examined the healing mechanisms in amniotic membrane (AM) defects after trauma. Traumatised human AM defects were cultured for 4 days. Markers for nuclear (DAPI), cell type (vimentin, αSMA) and healing (Cx43, TGFβ1, collagen) were examined by immunofluorescence (IMF) confocal microscopy, Second Harmonic Generation (SHG) imaging and RT-qPCR. After trauma, AMCs and myofibroblasts migrated to the AM wound edge. Within four days, αSMA expressing myofibroblasts showed abundant Cx43 localized in the cytoplasmic processes. The highly contractile spindle-shaped myofibroblasts were present in the defect site and released collagen. In contrast, AMCs expressed vimentin and formed Cx43 plaques between cells found in the outer edges of the wound. Whilst AMCs were absent in the defect site, αSMA expressing myofibroblasts continued to elongate and polarize the collagen fibres. Both TGFβ1 and Cx43 gene expression were significantly increased after trauma. Cx43 has differential effects on AM cell populations that increase cellularity, contraction and potentially migration to the wound edge resulting in collagen polarisation in the AM defect site. Establishing how Cx43 regulates AM cell function could be an approach to repair defects in the membranes after trauma.

Nucleocytoplasmic Communication in Healthy and Diseased Plant Tissues

The double membrane of the nuclear envelope (NE) constitutes a selective compartment barrier that separates nuclear from cytoplasmic processes. Plant viability and responses to a changing environment depend on the spatial communication between both compartments. This communication is based on the bidirectional exchange of proteins and RNAs and is regulated by a sophisticated transport machinery. Macromolecular traffic across the NE depends on nuclear transport receptors (NTRs) that mediate nuclear import (i.e. importins) or export (i.e. exportins), as well as on nuclear pore complexes (NPCs) that are composed of nucleoporin proteins (NUPs) and span the NE. In this review, we provide an overview of plant NPC- and NTR-directed cargo transport and we consider transport independent functions of NPCs and NE-associated proteins in regulating plant developmental processes and responses to environmental stresses.

Telocytes: An Emerging Component of Stem Cell Niche Microenvironment

Telocytes (TCs) are newly identified interstitial cells characterized by thin and long cytoplasmic processes, called telopodes, which exhibit a distinctive moniliform shape and, often, a sinuous trajectory. Telopodes typically organize in intricate networks within the stromal space of most organs, where they communicate with neighboring cells by means of specialized cell-to-cell junctions or shedding extracellular vesicles. Hence, TCs are generally regarded as supporting cells that help in the maintenance of local tissue homeostasis, with an ever-increasing number of studies trying to explore their functions both in physiological and pathological conditions. Notably, TCs appear to be part of stem cell (SC) niches in different organs, including the intestine, skeletal muscle, heart, lung, and skin. Indeed, growing evidence points toward a possible implication of TCs in the regulation of the activity of tissue-resident SCs and in shaping the SC niche microenvironment, thus contributing to tissue renewal and repair. Here, we review how the introduction of TCs into the scientific literature has deepened our knowledge of the stromal architecture focusing on the intestine and skeletal muscle, two organs in which the recently unveiled unique relationship between TCs and SCs is currently in the spotlight as potential target for tissue regenerative purposes.

Reproductive biology, embryonic development and matrotrophy in the phylactolaemate bryozoan Plumatella casmiana

Bryozoa is a phylum of aquatic, colonial suspension-feeders within the Lophotrochozoa. In the Phylactolaemata embryonic development occurs in an internal brood sac on the body wall accompanied by extraembryonic nutrition. Owing to previous contradictive descriptions, many aspects of their sexual reproduction require restudy. Consequently, this study analyses embryogenesis of the freshwater bryozoan Plumatella casmiana by serial sections, 3D reconstruction and transmission electron microscopy. Early embryos cleave and soon develop into blastulae with a small central cavity. The mesoderm forms by delamination starting from the distal side towards the proximal end. In later embryos two polypides form on the posterior side that ultimately will be covered by a ciliated mantle in the larva. Embryos increase in size during development and form temporary cell contacts to the embryo sac. Mesodermal cells of the embryo sac show signs of transcellular transport indicating that embryos are nourished by transferring nutrients from the maternal coelom towards the brood cavity. This study clarifies several details such as mesoderm formation and the onset of bud development. Embryos are connected to their respective embryo sacs by a variety of temporary cytoplasmic processes formed by both tissues during embryogenesis, including a ‘placental’ ring zone. Although ultrastructural data of these cell contacts are not entirely conclusive about their function, we suggest that embryos absorb nutrients via the entire surface. The close opposition of embryos to the embryo sac implies placentation as matrotrophic mode in phylactolaemate bryozoans, with embryo sacs acting as placental analogues.

Surface ultrastructure of the adult and juvenile stages of the trematode Astiotrema impletum (Looss, 1899) Looss 1900 (incertae sedis) from the Nile puffer, Tetraodon lineatus Linnaeus, 1758

Summary A scanning electron microscope study was performed on the surface of the trematode Astiotrema impletum (Looss, 1899) Looss 1900 from the Nile puffer, Tetraodon lineatus Linnaeus, 1758 (Syn. Tetraodon fahaka) for the first time. Adult A. impletum have a markedly large, sub-terminal oral sucker and a small ventral sucker. As with most trematodes, tegumental spines are concentrated anteriorly and are absent just anterior the ventral sucker. Spines have serrated tips on a short, tongue–shaped body. At the level of the ventral sucker the base of each spine exhibits a three bulbous-like structure that can be partially or completely withdrawn into the tegument. Sensory papillae are concentrated around the oral and ventral suckers and genital pores. Papillae are conical or knob-like, either ciliated or non-ciliated; some are protruded and others are embedded inside the tegument. In the juvenile stage, spines are smaller, less well-developed and tightly packed. The cytoplasmic processes of the posterior quarter of body show brain-like velvety appearance on the adult and small cobblestone-like on the juvenile.

The interplay of microRNAs and transcription factors in autophagy regulation in nonalcoholic fatty liver disease

The autophagy-lysosomal degradation system has an important role in maintaining liver homeostasis by removing unnecessary intracellular components. Impaired autophagy has been linked to nonalcoholic fatty liver disease (NAFLD), which includes hepatitis, steatosis, fibrosis, and cirrhosis. Thus, gaining an understanding of the mechanisms that regulate autophagy and how autophagy contributes to the development and progression of NAFLD has become the focus of recent studies. Autophagy regulation has been thought to be primarily regulated by cytoplasmic processes; however, recent studies have shown that microRNAs (miRNAs) and transcription factors (TFs) also act as key regulators of autophagy by targeting autophagy-related genes. In this review, we summarize the miRNAs and TFs that regulate the autophagy pathway in NAFLD. We further focus on the transcriptional and posttranscriptional regulation of autophagy and discuss the complex regulatory networks involving these regulators in autophagy. Finally, we highlight the potential of targeting miRNAs and TFs involved in the regulation of autophagy for the treatment of NAFLD.

SANS (USH1G) regulates pre-mRNA splicing by mediating the intra-nuclear transfer of tri-snRNPs from Cajal bodies to nuclear speckles for spliceosome assembly

Splicing is catalyzed by the spliceosome, a compositionally dynamic complex assembled stepwise on pre-mRNA. We reveal the link between splicing machinery components with the intrinsically disordered ciliopathy protein SANS. Pathogenic mutations in SANS/USH1G lead to Usher syndrome – the most common cause of deaf-blindness. SANS functions has been associated with cytoplasmic processes so far. Here, we show SANS localization in Cajal bodies and nuclear speckles. There SANS interacts with components of spliceosomal complexes and the large splicing cofactor SON and PRPFs of the tri-snRNP complex. SANS is required for the release of tri-snRNPs from Cajal bodies and their recruitment to nuclear speckles. SANS depletion alters spliceosome assembly kinetics, leading to stalled complex A formation, which can be chased to spliced products by the addition of tri-snRNPs. SANS deficiency and USH1G mutations affects splicing of genes related to cell proliferation and USH. We provide the first evidence that splicing deregulation may participate at the pathophysiology of Usher syndrome.

Emerging Roles of Long Noncoding RNAs in the Cytoplasmic Milieu

While the important functions of long noncoding RNAs (lncRNAs) in nuclear organization are well documented, their orchestrating and architectural roles in the cytoplasmic environment have long been underestimated. However, recently developed fractionation and proximity labelling approaches have shown that a considerable proportion of cellular lncRNAs is exported into the cytoplasm and associates nonrandomly with proteins in the cytosol and organelles. The functions of these lncRNAs range from the control of translation and mitochondrial metabolism to the anchoring of cellular components on the cytoskeleton and regulation of protein degradation at the proteasome. In the present review, we provide an overview of the functions of lncRNAs in cytoplasmic structures and machineries und discuss their emerging roles in the coordination of the dense intracellular milieu. It is becoming apparent that further research into the functions of these lncRNAs will lead to an improved understanding of the spatiotemporal organization of cytoplasmic processes during homeostasis and disease.