Apical Region Of Cells Research Articles

Expression of SARS-CoV-2 entry molecules ACE2, NRP1, TMPRSS2, and FURIN in the reproductive tissues of male macaques.

Coronavirus disease 2019 (COVID-19) reportedly affects male reproductive function by causing spermatogenesis dysfunction and suppressing testosterone secretion. However, the relationship between COVID-19 and impaired reproductive function, such as whether these effects on reproductive function are a direct effect of severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) infection in male reproductive organs or an indirect effect of high fever, is not known. Here, we examined whether the cell entry molecules of SARS-CoV-2, namely, ACE2, NRP1, TMPRSS2, and FURIN, are expressed in the male reproductive organs using the testes and accessory gonads of macaques during the breeding season. RT-PCR expression analysis showed that the testes alone expressed all four molecules. Immunohistochemical staining of testis tissue sections revealed that ACE2 is expressed in Leydig cells and the apical region of Sertoli cells, whereas NRP1 is expressed in the cell bodies surrounding the Leydig and Sertoli cell nuclei. FURIN is mainly expressed in Leydig cells, secondary spermatocytes, and spermatids. However, TMPRSS2 immunopositive cells were not observed. Therefore, it was not possible to observe cells expressing all four molecules in the gonads and accessory gonads of male primates. These results suggest that SARS-CoV-2 is unlikely to directly affect spermatogenesis in primates or proliferate in cells of the seminiferous tubules and undergo release into the semen through the previously known ACE2-mediated infection route. However, the expression of three molecules, including ACE2, was observed in Leydig cells, suggesting that testosterone synthesis and secretion may be affected when primates, including humans, are infected with SARS-CoV-2.

Cellular evidence and spatial distribution of endosomal biosynthesis and autophagy in intestinal immune barrier cells of crucian carp (Carassiuscarassius)

Crucian carp (Carassius carassius) is an important aquatic economic animal, and the immune barrier function of its intestine has been a focus of research into oral vaccines and drugs. However, the histological structures of the intestinal barrier and its adjacent areas have not been clearly established, and little subcellular evidence is available to elucidate the spatial distribution of intracellular biological processes. In this study, the spatial distribution of autophagy and endosome formation in the intestinal epithelial cells (IECs) of crucian carp were analyzed. These two biological activities are closely related to intestinal homeostasis, immunity, and cell communication. Periodic acid-Schiff (PAS) and Masson's trichrome staining were employed to elucidate the distinctive histological framework of the Crucian carp's myoid cell network, which resides within the subepithelial layer and is characterized by gap junctions. Transmission electron microscopy (TEM), immunohistochemistry (IHC), and immunofluorescence (IF) were used to detect the structural and functional aspects of the IEC in different intestinal segments. TEM and immunohistochemical analyses captured the biogenesis and maturation of early and late endosomes as well as multivesicular bodies (MVBs), as well as the initiation and progression of autophagy, including macroautophagy and mitophagy. The endosome and MVBs-specific marker CD63 and autophagy-related protein LC3 were highly expressed in IECs and were correlated with autophagy and endosome biosynthesis in the apical and basal regions of individual cells, and differed between different intestinal segments. In summary, this study elucidated the ubiquity and morphological characteristics of autophagy and endosome formation across different intestinal segments of crucian carp. A unique myoid cell network beneath the intestinal epithelium in crucian carp was also identified, expanding the histological understanding of this animal's intestinal tract.

Knickkopf (LmKnk) is required for chitin organization in the foregut of Locusta migratoria.

The foregut, located at the front of the digestive tract, serves a vital role in insects by storing and grinding food into small particles. The innermost layer of the foregut known as the chitinous intima, comes into direct contact with the food and acts as a protective barrier against abrasive particles. Knickkopf (Knk) is required for chitin organization in the chitinous exoskeleton, tracheae and wings. Despite its significance, little is known about the biological function of Knk in the foregut. In this study, we found that LmKnk was stably expressed in the foregut, and highly expressed before molting in Locusta migratoria. To ascertain the biological function of LmKnk in the foregut, we synthesized specific double-stranded LmKnk (dsLmKnk) and injected it into locusts. Our findings showed a significant decrease in the foregut size, along with reduced food intake and accumulation of residues in the foregut after dsLmKnk injection. Morphological observations revealed that newly formed intima became thinner and lacked chitin lamella. Furthermore, fluorescence immunohistochemistry revealed that LmKnk was located in the apical region of new intima and epithelial cells. Taken together, this study provides insights into the biological function of LmKnk in the foregut, and identifies the potential target gene for exploring biological pest management strategies.

Uncovering nanotoxicity of a water-soluble and red-fluorescent [70]fullerene nanomaterial

Engineered fullerene materials have attracted the attention of researchers in the biomedical sciences, especially when their synthetic methodology is developed to endow them with significant levels of water-solubility and bioavailability. In this study, we synthesized and characterized a water-soluble and red-fluorescent [70]fullerene nanomaterial, which fluoresced at 693 nm with a quantum yield of 0.065 and a large Stokes shift (around 300 nm). The fullerene nanomaterial generated mainly singlet oxygen after illumination with blue LED light, while superoxide anion radical production was minimal. The transmission electron microscopy as well as fluorescent studies of Drosophila melanogaster revealed that prepared [70]fullerene nanoparticles had better bioavailability than pristine [70]fullerene nanoparticles. The designed nanomaterials were observed in the apical, perinuclear, and basal regions of digestive cells, as well as the basal lamina of the digestive system's epithelium, with no damage to cell organelles and no activation of degenerative processes and cell death. Our findings provide a new perspective for understanding the in vivo behavior of fullerene nanomaterials and their future application in bioimaging and light-activated nanotherapeutics.

The Structure of the Female Genital System of the Diving Beetle Scarodytes halensis (Fabricius, 1787) (Hydroporinae, Dytiscidae), and the Organization of the Spermatheca and the Spermathecal Gland Complex

The fine structure of the female reproductive organs of the diving beetle Scarodytes halensis has been described, with particular attention to the complex organization of the spermatheca and the spermathecal gland. These organs are fused in a single structure whose epithelium is involved in a quite different activity. The secretory cells of the spermathecal gland have a large extracellular cistern with secretions; duct-forming cells, by their efferent duct, transport the secretions up to the apical cell region where they are discharged into the gland lumen. On the contrary, the spermatheca, filled with sperm, has a quite simple epithelium, apparently not involved in secretory activity. The ultrastructure of the spermatheca is almost identical to that described in a closely related species Stictonectes optatus. Sc. halensis has a long spermathecal duct connecting the bursa copulatrix to the spermatheca-spermathecal gland complex. This duct has a thick outer layer of muscle cells. Through muscle contractions, sperm can be pushed forwarding up to the complex of the two organs. A short fertilization duct allows sperm to reach the common oviduct where eggs will be fertilized. The different organization of the genital systems of Sc. halensis and S. optatus might be related to a different reproductive strategy of the two species.

Elastic properties of the inner-ear tip link explored using all-atom molecular dynamics simulations.

Vertebrate sound and balance perception relies on a process known as mechanotransduction, or the transformation of mechanical sensory information such as sound and head movements into electrochemical signals. Mechanical force sensation occurs in the apical region of inner-ear hair cells, where tensile forces applied to a protein complex called the tip link causes activation of nearby ion channels through a force-gated mechanism. The tip link is a filamentous complex formed by cadherin-23 (CDH23) and protocadherin-15 (PCDH15), proteins with large ectodomains that likely assemble as heterotetrameric structures in vivo. While the identity of the putative mechanotransduction ion channel has been revealed, it remains unclear how the tip link propagates force to it at the molecular level. Studying the mechanical properties of the tip link in situ has been challenging, while in vitro and in silico studies dealing with this large complex have been inconclusive. Using existing and new x-ray crystal structures combined with AlphaFold2 predictions for subdomains of the CDH23 and PCDH15 ectodomains, we constructed an atomistic model of the entire extracellular tip link for in silico force application. Here, we present all-atom equilibrium and steered molecular dynamics simulations of the entire inner-ear tip link ectodomain with predictions of strength and structural domains relevant for its mechanical response. We conclude that the presence of dimerization and membrane adjacent domains (MAD) account for rigid and elastic responses of the tip link responsible for modulation of force propagation. With these results, we begin to understand how the tip-link complex may convey force to ion channels and how deafness mutations on the CDH23 and PCDH15 ectodomains affect their function.

Comparative morphology and ultrastructure of the labial gland among castes of Camponotus japonicus (Hymenoptera: Formicidae)

The labial gland is a major exocrine gland in the thoracic cavity of ants, which is connected to the mouth area and opens at the base of the labium. The labial gland in the various castes and males of Camponotus japonicus were examined using dissection, light microscopy, scanning and transmission electron microscopy. Our findings reveal clear caste differences in the appearance of the labial gland, and suggest a relationship between gland structure and its behavioral role in queens, males and workers: queens possess the largest reservoir sac; males have the smallest; workers have abundant secretion droplets in the gland cells of the tubules; epithelial thickness of gland cells varies considerably between workers and reproductive ants. The apical cell region has a high density of microvilli and polymorphic mitochondria, whereas the central cell region is rich in rough endoplasmic reticulum (RER) which means its main secretions are proteinaceous compounds. We believe that the labial gland has a different function in the castes of C. japonicus: the gland of workers and queens may be used to feed the larvae; workers participate in trophallaxis in the nest, while the males are not involved in these activities. Calculations of the relative size of the labial gland for each caste and males indicated that minor workers have the relative highest developed labial gland, supporting a role in trophallaxis. In addition, we found a phenomenon of enlarged labial gland in minor workers, which accounted for almost 22%, but the reason for this is unknown.

C. elegans BLMP-1 controls apical epidermal cell morphology by repressing expression of mannosyltransferase bus-8 and molting signal mlt-8

Skin epidermis secretes apical extracellular matrix (aECM) as a protective barrier from the external environment. The aECM is highly dynamic and constantly undergoes remodeling during animal development. How aECM dynamics is temporally regulated during development, and whether and how its mis-regulation may impact epidermal cell morphology or function remains to be fully elucidated. Here, we report that the conserved Zn-finger transcription factor BLMP-1/Blimp1, which regulates epidermal development in C. elegans, controls apical cell shape of the epidermis by downregulation of aECM remodeling. Loss of blmp-1 causes upregulation of genes essential for molting, including bus-8 and mlt-8, in adult, leading to an abnormal shape in the apical region of adult epidermal cells. The apical epidermal morphological defect is suppressed by reduction of bus-8 or mlt-8. BUS-8 is a key mannosyltransferase, which functions in glycosylation of N-linked glycoproteins; MLT-8 has a ganglioside GM2 lipid-binding domain and is implicated in signaling during molting, a process where the old cuticle is shed and synthesized anew. Overexpression of bus-8 or mlt-8 induces an apical epidermal cell defect as observed in blmp-1 mutants. MLT-8::GFP fusion protein is localized to lysosomes and secreted to aECM. BUS-8 is important for MLT-8 stability and lysosomal targeting, which may be regulated by BUS-8-mediated glycosylation of MLT-8 and function as a molting signaling cue in aECM remodeling. We propose that BLMP-1 represses MLT-8 expression and glycosylation in the epidermis to prevent inappropriate aECM remodeling, which is essential for maintenance of apical epidermal cell morphology during larva-to-adult transition.

Neurulation in the posterior region of zebrafish, Danio rerio embryos.

The neural tube of amniotes is formed through different mechanisms that take place in the anterior and posterior regions and involve neural plate folding or mesenchymal condensation followed by its cavitation. Meanwhile, in teleost trunk region, the neural plate forms the neural keel, while the lumen develops later. However, the data on neurulation and other morphogenetic processes in the posterior body region in Teleostei remain fragmentary. We proposed that there could be variations in the morphogenetic processes, such as cell shape changes and cell rearrangements, in the posterior region compared to the anterior one at the different stages. Here, we performed morphological and histochemical analyses of morphogenetic processes with an emphasis on neurulation in the zebrafish tail bud (TB) and posterior region. To analyze the posterior expression of sox2 and tbxta we performed whole mount in situ hybridization. We showed that the TB cells of variable shapes and orientation are tightly packed, and the neural and notochord primordia develop first. The shape of the neural primordium undergoes numerous changes as a result of cell rearrangements leading to the development of the neural rod. At the prim-6 stage, the cells of the neural primordium directly form the neural rod. The neuroepithelial cells undergo sequential shape changes. At the stage of the neural rod formation, the apical regions of triangular neuroepithelial cells of the floor plate are enriched in F-actin. The neurocoel development onset is above the apical poles of neuroepithelial cells. The expression domains of sox2 and tbxta become more restricted during the development.

A mathematical model of intricate calcium dynamics and modulation of calcium signalling by mitochondria in pancreatic acinar cells

• The model is divided into two sub domains: the apical cytosolic sub domain, and the mitochondrial sub domain. However, these two sub domains are connected with the Ca2+. • The proposed model is Class II model. It includes the dynamics of IP3, we take into account the Ca2+ modulation of IP3 generation through PLC, and Ca2+ activated IP3 removal by IP3K. • The biphasic regulation of mitochondrial Ca2+ uptake by apical Ca2+ concentration, and the rapid mode of Ca2+ uptake through mitochondria are considered. • In this model the three types of mechanism by Ca2+ have been well considered. Firstly, the time dependent gating of IPR by Ca2+. Secondly, the Ca2+ enhances the production of IP3. Subsequently, due to the 3- kinase stimulated by Ca2+, degradation of IP3. Thirdly, the gating of uniporter by Ca2+: i.e. Ca2+ activated CU uptake, and subsequently it inactivates the uniporter for further up taking Ca2+. Thus, a [Ca2+] concentration is required for the activation and the inactivation of CU. • How these two sub domain: apical sub domain and the mitochondrial sub domain combine together into a well- mixed model. • The bifurcation analysis is performed to identify the oscillatory structures depending on the different agonist strength to produce complex dynamics in the apical region of the cell. The complex bifurcation points like hopf, period doubling, torus, saddle node bifurcation are studied. The pancreatic acinar cells display wide varieties of Ca 2+ oscillations within the cell. The study of these calcium signals/oscillations are of great interest because they are physiologically important. The Ca 2+ release from the endoplasmic reticulum via inositol trisphosphate receptors (IPR) and the ryanodine receptors (RyR) creates a high cytosolic Ca 2+ signal in the acinar cell. The pancreatic acinar cells are highly polarized cells, and the Ca 2+ response is quite extensive in the apical (peri-granular) region than the basal region of the cell. The Ca 2+ signals which originated in the apical region control the exocytosis process and triggered peri-granular mitochondria (Mito) to uptake Ca 2+ . The peri-granular mitochondria play a critical role in confining cytosolic Ca 2+ elevation by acting as a Ca 2+ buffer barrier. They also participate in shaping the Ca 2+ response by restricting the Ca 2+ signals into the granular region of the acinar cells. They sequestered Ca 2+ very rapidly. Then they release Ca 2+ very slowly to provide Ca 2+ supply to cause Ca 2+ oscillations in the apical region. Thus, herein we proposed a Class II compartmental mathematical model which systematically simulates the complex Ca 2+ oscillations in the apical region, and the peri-granular mitochondria matrix of the pancreatic acinar cells. The model assumes the functional coupling between the IPR and the RyR through the mechanism of calcium induced calcium release (CICR). In the proposed compartmental model of Ca 2+ dynamics, the Ca 2+ dependent production and degradations of (1,4,5) inositol-trisphosphate (IP 3 ) along with the apical parameters, apical endoplasmic reticulum (ER), and the peri-granular mitochondria are considered. The bifurcation analysis of the model is performed which explores the intricate dynamic behaviour of the apical region of the acinar cells, and hence derive the complex Ca 2+ oscillations in both the apical region and the peri-granular mitochondria. The model predicts the different patterns of Ca 2+ oscillation from the regular to sinusoidal to raised-baseline to sustained spikes in the apical region of the acinar cells.

Accumulation of milk increases the width of tight junctions in the epithelium of mouse mammary alveoli

The study of the molecular mechanisms of maintaining the integrity of the epithelium during mechanical stress remains a relevant problem in the physiology of tissue barriers. A methodical approach has been applied which makes it possible to reproduce mechanical pressure on the apical region of cells in vivo and to study the participation and role of tight junctions in maintaining the integrity of the epithelial structure. Mammary gland tissue specimens from lactating control mice and animals after a 20-h interruption of suckling were prepared and the width of the tight junction of the secretory epithelium was analyzed. At the ultrastructural level, it was shown that accumulation of milk caused a significant increase in the width of the tight junction between epithelial cells. In the control group, the width of this structure was 2.1 ± 0.1 µm; in the experimental group — 4.2 ± 0.1 μm. The marked increase in the width of tight junctions between epithelial cells is in accordance with an observed increase in the level of claudin-1 and -3 in the secretory epithelium and can be interpreted as adaptive changes aimed at maintaining the structure of the alveoli.

The Spherocrystals in the Tubule Epithelial Cells and Ultrastructure of the Malpighian Tubules of Adult Isophya nervosa (Orthoptera, Tettigoniidae)

The excretory system in insects consists of Malpighian tubules (MTs) which are responsible for osmoregulation. The functions of the MTs are the removal of the last products of metabolism and the transfer of the toxic compounds into the hindgut. The MTs of the insects vary structurally. In this study, the MTs of Isophya nervosa Ramme, 1951, which is a species that belongs to Orthoptera order, were investigated by light and electron microscopes. Adult individuals of Isophya nervosa were collected in Kızılcahamam, Ankara in 2017 and 2018. Extracted MTs were fixed in Formaldehyde for light microscopy, in glutaraldehyde for electron microscopes. They were examined and photographed after dehydration, blocking, sectioning and staining processes were completed. This species has a great number of MTs. One end of the MTs in this species is attached to the ileum and the other closed end is free in hemolymph. The outer surface of the cells is surrounded by muscle layers and trachea. There are microvilli on the apical region of cells. The lumen is covered with a monolayer cuboidal epithelium. Numerous concentrically layered spherocrystals in different shapes were observed in the cells. When the chemical compositions of these spherocrystals were analyzed by SEM-EDX system, it was seen that the contents of them were different ions such as iron (Fe), carbon (C), phosphorus (P), magnesium (Mg), sulfur (S), calcium (Ca), and manganese (Mn).

Organic secretion in ductal cells of the sublingual salivary gland of ferret: Histochemical observations

The presence of organic secretion in ductal cells of the sublingual salivary gland of ferret has been questioned, which prompted the present investigation. Paraffin or cryostat sections from aldehyde fixed or quenched sublingual glands of this species were tested for some amino acid residues, mucosubstances, oxidative and hydrolytic enzymes, and lectins. The glands showed inconspicuous ducts of simple appearances on routine histology. The histochemical procedures, however, revealed a granulated substance in the apical (periluminal) region of ductal cells, which contained tryptophan, disulphides, neutral mucosubstances, αFuc and GalNAc, and showed chloroacetate esterase activity. Occurrence of the substance varied between different ducts of the same gland and/or cells of the same duct. The ductal cells also showed diffuse peroxidase and acid phosphatase, and Golgi-like thiamine pyrophosphatase activities. Acetylcholinesterase-positive nerve fibres embraced the ducts. The results support a particular localisation of protein-bound amino acid residues and enzymatic catalytic activities indicative of organic secretion, possibly tissue kallikrein, in sublingual ductal cells of ferret.

P023 Identification of regulated mechanistic pathways and genes to protect intestinal barrier injury by larazotide acetate in an IBD microenvironment

Abstract Background Inflammatory bowel disease (IBD) is due to a combination of factors, including genetics, mucosal barrier dysfunction and dysregulated immune responses. Recently, it has been appreciated that IBD is associated with profound tissue anoxia. Tight junctions (TJs) located at the apical lateral region of adjacent intestinal epithelial cells are largely responsible for regulating the intestinal mucosal barrier. Larazotide acetate (LA, Innovate Biopharmaceuticals, Inc., Raleigh, NC) is a synthetic, eight amino acid peptide that is known to act as a TJ regulator capable of closing ‘leaky’ interepithelial junctions. Presently, LA is being studied in Phase 3 clinical trials for the treatment of celiac disease. Based on prior work in our lab, we hypothesised that LA would protect the TJ barrier in an anoxic injury IBD model associated with upregulation of TJ-associated signalling pathways. Methods C2BBe1 (Caco-2 brush border expressing) monolayers were treated apically with LA and were subjected to anoxia for 2 h followed by reoxygenation with 21% O2. Barrier function was assessed by measuring transepithelial electrical resistance (TEER) during anoxic injury and recovery. TJ proteins and cytoskeleton protein F-actin were assessed by western blotting and immunofluorescence microscopy. Then, next-generation RNA sequencing was employed to assess cellular regulatory pathways. Results Pre-treatment of anoxic injured C2BBe1cells with 10 mM LA significantly increased TEER as compared with untreated anoxic injured cells. The TJ protein occludin and ZO-1 were disrupted in anoxia-injured monolayer. Alternatively, treatment with 10 mM LA prevented disruption of TJ proteins during anoxic injury. Gene ontology annotation revealed a number of critical signalling pathways that were differentially expressed in cells treated with LA, including biological processes involved in establishment of cell polarity, molecular functions that regulate junctional structures, and cellular components associated with epithelial repair (cell leading edge, ruffle and apical junctional complex). Furthermore, Ras/Rho GTPase binding and protein serine/threonine kinase activity were differentially expressed in cells treated with LA. Additionally, Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis revealed enrichment of target genes for ‘cell cycle,’ ‘adherens junction’ and ‘Wnt signalling pathways’. Conclusion The results of the present study provide novel insights into the molecular mechanism of action of LA on the protection of TJ integrity in anoxic injury, an IBD microenvironment and the potential for a more broad use in important digestive diseases such as IBD.

Systemic Analysis of Tissue Cells Potentially Vulnerable to Sars-Cov-2 Infection by the Protein-Proofed Single-Cell Rna Profiling of Ace2, Tmprss2 and Furin Proteases

Single-cell RNA profiling of ACE2, the SARS-CoV-2 receptor, had proposed multiple tissue cells as the potential targets of SARS-CoV-2, the novel coronavirus causing the COVID-19 pandemic. However, most were not echoed by the patients’ clinical manifestations, largely due to the lack of protein expression information of ACE2 and co-factors. Here, we incorporated the protein information to analyse the expression of ACE2, together with TMPRSS2 and Furin, two proteases assisting SARS-CoV-2 infection, at single cell level in situ, which we called protein-proofed single-cell RNA (pscRNA) profiling. Systemic analysis across 36 tissues revealed a rank list of candidate cells potentially vulnerable to SARS-CoV-2. The top targets are lung AT2 cells and macrophages, then cardiomyocytes and adrenal gland stromal cells, followed by stromal cells in testis, ovary and thyroid. Whereas, the polarized kidney proximal tubule cells, liver cholangiocytes and intestinal enterocytes are less likely to be the primary SARS-CoV-2 targets as ACE2 localizes at the apical region of cells, where the viruses may not readily reach. These findings are in concert with the clinical characteristics of prominent lung symptoms, frequent heart injury, and uncommon intestinal symptoms and acute kidney injury. Together, we provide a comprehensive view on the potential SARS-CoV-2 targets by pscRNA profiling, and propose that, in addition to acute respiratory distress syndrome, attentions should also be paid to the potential injuries in cardiovascular, endocrine and reproductive systems during the treatment of COVID-19 patients.

Effects of Anticholinergic Drugs Used for the Therapy of Overactive Bladder on P-Glycoprotein Activity.

We evaluated the effects of anticholinergic drugs principally used for the therapy of overactive bladder (OAB) on the activity of P-glycoprotein, an efflux transport protein, in Caco-2 cells. The time-dependent changes in the fluorescence of residual rhodamine 123, a P-glycoprotein activity marker, in the apical region of Caco-2 cells were measured in the presence of anticholinergic drugs using time-lapse confocal laser scanning microscopy. The effect of anticholinergic drugs on human P-glycoprotein ATPase activity was also measured. The fluorescence of residual rhodamine 123 in untreated Caco-2 cells decreased over time. The gradual decrease in the fluorescence was significantly inhibited by treatment with cyclosporine A, darifenacin, and trospium. In contrast, oxybutynin, N-desethyl-oxybutynin (DEOB), propiverine, and its active metabolites (M-1, M-2), imidafenacin, solifenacin, or tolterodine had little effect on the efflux of rhodamine 123. P-Glycoprotein ATPase activity was increased by darifenacin. Darifenacin and trospium reduced the rhodamine 123 transfer across the apical cell membrane. These data suggest that darifenacin and trospium interact with P-glycoprotein. Additionally, darifenacin influenced P-glycoprotein ATPase activity. These results suggest that darifenacin may be a substrate of P-glycoprotein. This study is the first paper to test simultaneously the effects of 10 anticholinergic drugs used currently for the therapy of OAB, on the P-glycoprotein.

Abstract 1950: Differential expression of MUC16 mucin (CA125) depending on the architecture of the tumors developed in an orthotopic mouse model of high-grade serous ovarian cancer

Abstract The histopathology of high-grade serous ovarian cancer (HGSOC) in orthotopic mouse models has received limited attention. Using immunosuppressed mice, we generated intraperitoneal HGSOC disease that includes formation of ascites and/or pleural effusions, together with a myriad of intra-abdominal growths. We studied 3 HGSOC cell lines evolved from the same patient along disease progression—a platinum-sensitive, PEO1; a platinum-resistant after further relapse, PEO4; and another at the end of life, PEO6. We found that the animals were prone to develop the disease sooner when implanted with cells obtained from the most advanced disease. Using these models, we characterized the histopathology of the masses preferably developed in the stomach-spleen-pancreas region enclosing the omentum. Other sites of tumor formation we found where the lower pelvic cavity, the peritoneal wall, the liver base, the diaphragm, the lungs, the uterus, and the ovaries. The architecture of the tumors was more often represented by slit-like fenestrations, yet certain zones displayed papillary, glandular, or solid structures sometimes including infiltrating lymphocytes. When we studied the expression of antigen CA125, we found that its pattern of staining was highly heterogeneous. In PEO1/4/6 cells maintained in culture CA125 was expressed in all cell types, yet with a major abundance aligned with disease evolution. In vivo, within the tumors, the expression of CA125 was mostly limited to the apical region of cells facing slit-like spaces generated in between solid sheets of tumor cells surrounded by fibrovascular stroma. When tumor cells were located deep within homogeneous solid masses devoid of slit-like fenestrations, they usually did not express CA125. In contrast, when cells were found gathered as multicellular structures free-floating in open spaces surrounding target organs—e.g. within the bursa around the ovaries—CA125 was highly expressed. This is coincident with the high expression of CA125 observed in vitro in cells spontaneously forming multicellular aggregates in suspension. It is likely that HGSOC cells express CA125 to face or invade into open spaces. In summary, this work reveals tissue heterogeneity and polarity in the expression of CA125 in solid tumors of HGSOC developed in immunosuppressed mice. This selected pattern of expression may be related to the oncogenic functions of CA125 (a.k.a. MUC16, a glycoprotein), such as protecting cells from immunological attacks, or promoting metastases by binding to mesothelin produced by mesothelial cells that line the peritoneal cavity. Citation Format: Alicia A. Goyeneche, Michael A. Lisio, Zu-hua Gao, Carlos M. Telleria. Differential expression of MUC16 mucin (CA125) depending on the architecture of the tumors developed in an orthotopic mouse model of high-grade serous ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1950.

Characterization of potential TRPP2 regulating proteins in early Xenopus embryos.

Transient receptor potential cation channel-2 (TRPP2) is a nonspecific Ca2+ -dependent cation channel with versatile functions including control of extracellular calcium entry at the plasma membrane, release of intracellular calcium ([Ca2+ ]i) from internal stores of endoplasmic reticulum, and calcium-dependent mechanosensation in the primary cilium. In early Xenopus embryos, TRPP2 is expressed in cilia of the gastrocoel roof plate (GRP) involved in the establishment of left-right asymmetry, and in nonciliated kidney field (KF) cells, where it plays a central role in early specification of nephron tubule cells dependent on [Ca2+ ]i signaling. Identification of proteins binding to TRPP2 in embryo cells can provide interesting clues about the mechanisms involved in its regulation during these various processes. Using mass spectrometry, we have therefore characterized proteins from late gastrula/early neurula stage embryos coimmunoprecipitating with TRPP2. Binding of three of these proteins, golgin A2, protein kinase-D1, and disheveled-2 has been confirmed by immunoblotting analysis of TRPP2-coprecipitated proteins. Expression analysis of the genes, respectively, encoding these proteins, golga2, prkd1, and dvl2 indicates that they are likely to play a role in these two regions. Golga2 and prkd1 are expressed at later stage in the developing pronephric tubule where golgin A2 and protein kinase-D1 might also interact with TRPP2. Colocalization experiments using exogenously expressed fluorescent versions of TRPP2 and dvl2 in GRP and KF reveal that these two proteins are generally not coexpressed, and only colocalized in discrete region of cells. This was observed in KF cells, but does not appear to occur in the apical ciliated region of GRP cells.

Structure of the alimentary tract in the Atlantic mudskipper Periophthalmus barbarus (Gobiidae: Oxudercinae): anatomical, histological and ultrastructural studies

The alimentary tract of oxudercine gobies is characterized by a lack of an anatomically distinct stomach, owing to which they are classified as stomachless. Since the environment, food requirements, and feeding habits have a significant impact on the anatomy of the alimentary tract of fish, it was assumed that predominantly carnivorous, semi-terrestrial mudskippers would have a stomach. In order to verify this hypothesis, anatomical, histological, histochemical and ultrastructural analysis of the alimentary tract of the Atlantic mudskipper Periophthalmus barbarus was performed. The results revealed that despite a lack of clear anatomical distinction within the alimentary tract, there were four well-distinguished sections visible at the histological level: oesophagus, stomach, intestine, and rectum. The division was enhanced by the presence of a pyloric sphincter and an ileorectal valve. The stomach contained tubular glands composed of oxynticopeptic cells. Gland cells had pepsinogen granules and a well-developed tubulovesicular network of smooth membranes, which indicates the secretion of gastric juice. The presence of neutral mucus in the apical region of surface epithelial cells as protection against hydrochloric acid as well as the presence of active pepsin also confirm gastric function. However, low pepsin activity seems to implies low protein digestion. The results of this study indicate that the Atlantic mudskipper P. barbarus has a functional stomach.

Spatial heterogeneity of glycogen and its metabolizing enzymes in Aspergillus nidulans hyphal tip cells

Glycogen is a homopolymer of glucose and a ubiquitous cellular-storage carbon. This study investigated which Aspergillus nidulans genes are involved in glycogen metabolism. Gene disruptants of predicted glycogen synthase (gsyA) and glycogenin (glgA) genes accumulated less cellular glycogen than the wild type strain, indicating that GsyA and GlgA synthesize glycogen similarly to other eukaryotes. Meanwhile, gene disruption of gphA encoding glycogen phosphorylase increased the amount of glycogen to a higher degree than wild type during the stationary phase that accompanies carbon-source limitation. GFP-tagged GsyA and GphA were distributed in the cytosol and formed punctate and filamentous structures, respectively. Carbon starvation resulted in elongated GphA-GFP filaments and increased numbers of filaments. These structures were more frequently located in the basal regions of tip cells and adjacent cells than in the apical regions of tip cells. Cellular glycogen visualized by incorporation of a fluorescent glucose analog accumulated in cytoplasmic puncta that were more prevalent in the basal regions, a pattern similar to that seen for GsyA. The colocalization of glycogen and GsyA at punctate structures in tip and sub-apical cells likely represents the cellular machinery for synthesizing glycogen. More frequent colocalization in the basal, rather than tip cell apical regions indicated that tip cells have differentiated subcellular regions for glycogen synthesis. Our findings regarding glycogen, GsyA and GphA distribution evoke the spatial heterogeneity of glycogen metabolism in fungal hyphae.