Apical Processes Research Articles

Glucose Transporters are differentially regulated in rat intestinal epithelial crypt cells (IEC6) grown in high and low glucose medium

BackgroundGlucose dependent insulinotropic peptide (GIP) is a hormone that regulates both insulin release and nutrient transport. When GIP is secreted in large amounts, it contributes to diabetes and obesity. It is a cyclic process in which GIP increases transport of glucose, which in turn stimulates additional GIP secretion. High blood glucose decreases Na+‐glucose cotransporter‐1 (SGLT‐1) mediated glucose absorption and increases apical glucose transporter‐2 (GLUT2) mediated glucose absorption. In contrast, normal blood glucose maintains glucose absorption through SGLT1, while GLUT2 is not expressed on the apical membranes. Although SGLT1 is the primary apical glucose absorptive process in normal, GLUT2 has been localized on the apical membranes of obese human and mouse intestine. However, signaling mechanisms of how GIP regulates glucose transporters in the presence of normal and high extracellular glucose concentrations is not known.HypothesisExtracellular glucose concentration alters the glucose transporters on the apical membrane of differentiated epithelial cells.MethodsIntestinal epithelial crypt cell‐6 (IEC6 cells) were grown 3–5 days post confluent on snap‐wells using standard DMEM media supplemented with 5% FBS and either normal (0.45 g/L) or high (4.5 g/L) glucose, respectively. Mucosal to serosal (m‐s) glucose fluxes were measured across IEC6 monolayers mounted under voltage clamp conditions in Ussing chambers. Short circuit currents (Isc) were measured to distinguish SGLT1 (i.e. electrogenic) and GLUT2 (i.e. electroneutral) mediated glucose absorption. Apical membranes were isolated from IEC6 cell monolayers and the apical proteins were isolated using biotinylation techniques. Western blot analyses were then performed using SGLT1 and GLUT2 antibodies.ResultsGlucose absorption is substantially increased, while electrogenic (i.e. Isc) and GIP‐stimulated glucose absorption are reduced in IEC6 monolayers grown in medium with high glucose. In contrast, significantly higher Isc and lower level of glucose absorption are present in IEC6 monolayers grown in normal glucose medium. The absorption of glucose is inhibited by a cAMP inhibitor inhibits glucose absorption in IEC6 monolayers grown in both normal and high glucose medium. In contrast, inhibition of EPAC inhibits the glucose absorption in IEC6 monolayers grown in high, but not in low glucose medium. Although GIP stimulated glucose absorption in IE6 monolayers grown in both normal and high glucose medium, the GIP‐stimulated glucose absorption (flux) is higher in IEC6 monolayers grown in high glucose medium. Western blot analyses revealed that GLUT2, but not SGLT1 specific protein is expressed on the apical membrane of IEC6 monolayers grown in high glucose medium.ConclusionsThe EPAC regulated GLUT2, but not SGLT1 is the major glucose transporter present in IEC6 monolayers grown in high extracellular glucose environment. The PKA regulated SGLT1 is the major glucose transporter present in IEC6 monolayers grown in normal extracellular glucose environment.Support or Funding InformationThis research is supported by Montana IMBREThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Description of the second- and third instar larvae of Platynectes (s. str.) decemnotatus (Aubé, 1838) (Coleoptera: Dytiscidae: Agabinae: Platynectini).

The second- and third instar larvae of Platynectes (s. str.) decemnotatus (Aubé, 1838) are described and illustrated in detail for the first time, with special emphasis on morphometry and chaetotaxy. Larvae of P. decemnotatus can be distinguished from most other Agabinae by having secondary setae on the urogomphus and share with the other known species described in detail the presence of a ventroapical spinula on antennomere 3 and the absence of an occipital suture, natatory dorsal setae on tibia and tarsus and natatory setae on urogomphus. Platynectes decemnotatus larvae differ from larvae of Agabus Leach, 1817, Hydrotrupes Sharp, 1882 (currently in Hydrotrupini), Ilybiosoma Crotch, 1873, Ilybius Erichson, 1832 and the previously described Platynectes species in having a one-segmented urogomphus, a character previously observed only in larvae of Agabinus Crotch, 1873. The second- and third instar larvae of P. decemnotatus differ from those of P. (Agametrus) curtulus (Régimbart, 1899) in having the apical lateroventral process of antennomere 3 protruding (not protruding in P. curtulus). The third-instar larva of P. decemnotatus can also be distinguished from that of P. (Gueorguievtes) decempunctatus (Fabricius, 1775) by the absence of secondary dorsal setae on the tibia.

Современные представления о качестве эндодонтического лечения

The aim of the study is to analyze the factors that prevent the improvement of endodontic treatment. Materials and methods: we considered possible prospects for long-term endodontic treatment and prediction of results. Results of the study: these literature data show that intra- and extracorporeal factors influence the prediction of endodon treatment. Incomplete purification of pulp space after preparation, stability, ability of microflora to maintain apical inflammatory process, synthesis of intra- and extraradiclar biofilm, inactivation of medicines in root canal requires search of alternative irrigation agents and disinfection modes. In addition to the conventional preparation, the use of iodine preparations and physical factors in both primary and repeated endodontic treatment is promising. At the modern stage of endodontia, the size of the periapical lesion is not the main factor in deciding on conservative endodontic treatment or surgical treatment of the lesion. Due to the availability of tools, equipment and treatment techniques, endodontic intervention should ideally end successfully. But in the analysis of treatment results, a number of publications note that an unfavourable outcome is even in the case of «well-treated channels». Intracorneal factors include the complexity of endodontic anatomy, infection, diversity of microflora in the root canal system, its resistance and ability to organize into biofilm. Extracorporeal reasons include extradicular infection, «true» cysts, the presence of endo-periodontal lesions, root resorption, the reaction of periapical tissues to the foreign body (endogenous or exogenous origin) and yatrogenic factors (arising during preparation, irrigation of the root canal), toxic and irritating properties of the used drugs. Several of these etiological factors often lead to the development of the inflammatory process in the periodontal. Each can influence the outcome of endodontic treatment. The failure of conservative treatment is still recommended to be considered as the development of an infectious process.

Morphology of GNAT3-immunoreactive chemosensory cells in the rat larynx.

The upper airways play important roles in respiratory defensive reflexes. Although solitary chemosensory cells and chemosensory cell clusters have been reported in the laryngeal mucosa of mammalian species, the distribution and cellular morphology of chemosensory cells remain unclear. In the present study, the distribution and morphology of solitary chemosensory cells and chemosensory cell clusters were examined by immunofluorescence for GNAT3 on whole-mount preparations of the rat laryngeal mucosa. Electrophysiological experiments were performed to analyze the respiratory reflexes evoked by bitter stimuli to the laryngeal cavity. In the whole area of the laryngeal mucosa, the numbers of GNAT3-immunoreactive solitary chemosensory cells and chemosensory clusters were 421.0±20.3 and 62.7±6.9, respectively. GNAT3-immunoreactive solitary chemosensory cells were mainly distributed in the mucosa overlying epiglottic and arytenoid cartilage, and chemosensory clusters were mainly distributed on the edge of the epiglottis and aryepiglottic fold. GNAT3-immunoreactive solitary chemosensory cells were slender with elongated processes or had a flask-like/columnar shape. The number of GNAT3-immunoreactive cells in chemosensory clusters was 6.1±0.4, ranging between 2 and 14 cells. GNAT3-immunoreactive cells in the cluster were variform and the tips of apical processes gathered at one point at the surface of the epithelium. The tips of apical cytoplasmic processes in solitary chemosensory cells and cells in the cluster were immunoreactive for espin, and faced the laryngeal cavity. Physiological experiments showed that the application of 10mm quinine hydrochloride to the laryngeal cavity decreased respiratory frequency. The present results revealed the chemosensory field of the larynx and the morphological characteristics of the laryngeal chemosensory system for respiratory depression.

The small GTPase RAB28 is required for phagocytosis of cone outer segments by the murine retinal pigmented epithelium

RAB28, a member of the RAS oncogene family, is a ubiquitous, farnesylated, small GTPase of unknown function present in photoreceptors and the retinal pigmented epithelium (RPE). Nonsense mutations of the human RAB28 gene cause recessive cone-rod dystrophy 18 (CRD18), characterized by macular hyperpigmentation, progressive loss of visual acuity, RPE atrophy, and severely attenuated cone and rod electroretinography (ERG) responses. In an attempt to elucidate the disease-causing mechanism, we generated Rab28-/- mice by deleting exon 3 and truncating RAB28 after exon 2. We found that Rab28-/- mice recapitulate features of the human dystrophy (i.e. they exhibited reduced cone and rod ERG responses and progressive retina degeneration). Cones of Rab28-/- mice extended their outer segments (OSs) to the RPE apical processes and formed enlarged, balloon-like distal tips before undergoing degeneration. The visual pigment content of WT and Rab28-/- cones was comparable before the onset of degeneration. Cone phagosomes were almost absent in Rab28-/- mice, whereas rod phagosomes displayed normal levels. A protein-protein interaction screen identified several RAB28-interacting proteins, including the prenyl-binding protein phosphodiesterase 6 δ-subunit (PDE6D) and voltage-gated potassium channel subfamily J member 13 (KCNJ13) present in the RPE apical processes. Of note, the loss of PDE6D prevented delivery of RAB28 to OSs. Taken together, these findings reveal that RAB28 is required for shedding and phagocytosis of cone OS discs.

Differential nickel-induced responses of olfactory sensory neuron populations in zebrafish

The olfactory epithelium of fish includes three main types of olfactory sensory neurons (OSNs). Whereas ciliated (cOSNs) and microvillous olfactory sensory neurons (mOSNs) are common to all vertebrates, a third, smaller group, the crypt cells, is exclusive for fish. Dissolved pollutants reach OSNs, thus resulting in impairment of the olfactory function with possible neurobehavioral damages, and nickel represents a diffuse olfactory toxicant. We studied the effects of three sublethal Ni2+ concentrations on the different OSN populations of zebrafish that is a widely used biological model. We applied image analysis with cell count and quantification of histochemically-detected markers of the different types of OSNs. The present study shows clear evidence of a differential responses of OSN populations to treatments. Densitometric values for Gα olf, a marker of cOSNs, decreased compared to control and showed a concentration-dependent effect in the ventral half of the olfactory rosette. The densitometric analysis of TRPC2, a marker of mOSNs, revealed a statistically significant reduction compared to control, smaller than the decrease for Gα olf and without concentration-dependent effects. After exposure, olfactory epithelium stained with anti-calretinin, a marker of c- and mOSNs, revealed a decrease in thickness while the sensory area appeared unchanged. The thickness reduction together with increased densitometric values for HuC/D, a marker of mature and immature neurons, suggests that the decrements in Gα olf and TRPC2 immunostaining may depend on cell death. However, reductions in the number of apical processes and of antigen expression could be a further explanation. We hypothesize that cOSNs are more sensitive than mOSNs to Ni2+ exposure. Difference between subpopulations of OSNs or differences in water flux throughout the olfactory cavity could account for the greater susceptibility of the OSNs located in the ventral half of the olfactory rosette. Cell count of anti-TrkA immunopositive cells reveals that Ni2+ exposure does not affect crypt cells. The results of this immunohistochemical study are not in line with those obtained by electro-olfactogram.

Correlation of spectral domain optical coherence tomography with histology and electron microscopy in the porcine retina

Spectral domain optical coherence tomography (SD-OCT) is used as a non-invasive tool for retinal morphological assessment in vivo. Information on the correlation of SD-OCT with retinal histology in the porcine retina, a model resembling the human retina, is limited. Herein, we correlated the hypo- and hyper-reflective bands on SD-OCT with histology of the lamellar architecture and cellular constituents of the porcine retina. SD-OCT images were acquired with the Heidelberg Spectralis HRA + OCT. Histological analysis was performed using epoxy resin embedded tissue and transmission electron microscopy. Photomicrographs from the histologic sections were linearly scaled to correct for tissue shrinkage and correlated with SD-OCT images. SD-OCT images correlated well with histomorphometric data. A hyper-reflective band in the mid-to-outer inner nuclear layer correlated with the presence of abundant mitochondria in horizontal cell processes and adjacent bipolar cells. A concentration of cone nuclei corresponded to a relative hypo-reflective band in the outer portion of the outer nuclear layer. The presence of 3 hyper-reflective bands in the outer retina corresponded to: 1) the external limiting membrane; 2) the cone and rod ellipsoid zones; and 3) the interdigitation zone of photoreceptor outer segments/retinal pigment epithelium (RPE) apical cell processes and the RPE. These correlative and normative SD-OCT data may be employed to characterize and assess the in vivo histologic changes in retinal vascular and degenerative diseases and the responses to novel therapeutic interventions in this large animal model.

Life history of Subulina octona (Brugüière) (Gastropoda: Pulmonata: Subulinidae) based on four-year laboratory observations and a comparative histological analysis of egg-retaining and ovoviviparous subulinids

ABSTRACTIn the present study, we aimed to characterize the life history of the land snail Subulina octona integrating information on life-history traits and morphology. We also compared the histology of the free-oviduct and spermoviduct of ovoviviparous and egg-retaining species of subulinids. We considered as ovoviviparous the species in which the complete embryonic development as well as egg hatching occurs inside the parent’s body and, at the end of this process, the parent releases juveniles instead of eggs. We considered as egg-retaining the species in which a great part of the embryonic development takes place inside the parent’s body and the eggs laid contain well-developed embryos. The results showed that the free-oviduct of both ovoviviparous and egg-retaining species shows a histological arrangement that confers greater strength to its walls and is probably related to egg retention. The wall of the spermoviduct is formed by pseudostratified columnar epithelium, with cell apical processes (probably cilia), and by underlying secretory cells. In gravid egg-retaining snails, the eggshells appear in close contact with the secretory cells of the spermoviduct. This fact suggests that these cells play a role in eggshell formation. The present study is the first account for histological features of S. octona, Allopeas gracile and Allopeas micra. The life history of S. octona is a combination of long lifespan, early sexual maturity, indeterminate growth and egg retention. Egg retention limits the number of young that can be produced in one reproductive event. However, the reproductive strategy adopted by S. octona, associating egg retention and K-strategism, compensates this cost of retaining eggs, because the reproductive success may be enhanced as a result of the higher survival of juveniles and the possibility of performing several reproductive events during the year.

Resistance to vertical root fracture of apicoected teeth using different devices during two root canal irrigation procedures.

The aim of the present work was to measure the fracture resistance of endodontically treated teeth that were apicoected with different procedures. Seventy-two extracted human maxillary anterior teeth were included in this study. The specimens were randomly assigned to three main groups according to the apical surgery procedures and then two subgroups according to the irrigation protocols during root canal treatment and total of six groups were obtained (n = 12). Group 1: served as a control and apical surgery process was not performed in this group. Group 2: apical surgery process was performed with tungsten carbide fissure bur Group 3: apical surgery process was performed with Er:YAG laser. Subgroup a: In this group, the specimens were irrigated with %5 NaOCl. Subgroup b: 15% EDTA solution was filled into the root canal and then agitated using a 1.5W/100Hz diode laser. The specimens were filled and mounted in acrylic resin blocks and compression strength test was performed. Statistical analysis was performed using two-way ANOVA. The statistical analysis revealed that there were no statistical significant differences between apical surgery procedures (groups 1, 2, and 3) (p < 0.05). Apical resection procedures did not affect the fracture resistance Significant differences were determined between the subgroups (p < 0.05). Agitation of the EDTA with the diode laser reduced the fracture resistance of the specimens. The different canal irrigation techniques altered resistance to fracture; however, apical surgery procedures did not altered the resistance to fracture when compared with the control group.

High glucose induces Glucose transporter‐2 (GLUT2), but not Na‐glucose cotransporter‐1 (SGLT1) mediated glucose absorption

BackgroundGlucose dependent insulinotropic peptide (GIP) is a hormone that has a cyclic pathway in the stimulation of glucose transport. GIP increases transport of glucose, which in turn stimulates GIP secretion. This cycle increases the jejunal absorption of glucose, which stimulates insulin release. High glucose results in excess GIP secretion that causes unusually high glucose absorption and high blood glucose that stimulates insulin release that causes insulin insensitivity. Presence of high blood glucose and insulin lead to obesity and Type‐2 diabetes. Although SGLT1 is the primary apical glucose absorptive process, GLUT2 has been localized on the intestinal apical membranes of obese human and mouse models. However, signaling mechanisms of how GIP regulates glucose transporters in the presence of normal versus high levels of extracellular glucose concentrations is not known.HypothesisExtracellular glucose concentration that alters cellular GIP level would regulate glucose transporters on to the apical membrane of differentiated cells.MethodsIntestinal epithelial crypt cell‐6 (IEC6 cells) where grown 5‐days post confluent on transwell snapwells using standard DMEM media supplemented with 5% FBS and either 4.5 or 0.45 g/liter of glucose for high and low glucose, respectively. Mucosal to serosal (m‐s) glucose fluxes were measured across IEC6 cell layers that were mounted under voltage clamp conditions in Ussing chambers. Short circuit currents were measured to distinguish the SGLT1 (i.e. electrogenic) and GLUT2 (i.e. electroneutral) mediated glucose absorption. Apical membranes were isolated from IEC6 cell monolayers using a biotinylation technique. Western blot analyses were performed using SGLT1 and GLUT2 antibodies.ResultsGlucose absorption is substantially higher in IEC6 cells that were grown in high glucose medium compared to that grown in low glucose medium. In contrast to increased rate of glucose absorption, Isc is not significantly altered as an indication that high glucose increased GLUT2 mediated, but not SGLT1 mediated glucose absorption in IEC6 cells. Western blot analyses revealed that GLUT2, but not SGLT1 specific protein expression is increased on the apical membrane of IEC6 cells grown in the high glucose medium.ConclusionsHigh extracellular glucose environment stimulates GLUT2, but not SGLT1 expression and GLUT2 mediated glucose absorption in intestinal epithelial cells.SpeculationWe speculate that GLUT2 might be regulated similarly in in the villus cells of mouse intestine.Support or Funding InformationIMBRE grant #G110‐18‐W6619This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A Golgi lipid signaling pathway that controls neural stem cell polarity in the developing neocortex

The Golgi apparatus is a central regulator of membrane trafficking and cell polarity in mammalian cells. I will discuss our recent work in a murine embryonic neural stem cell (NSC) model demonstrating that a Golgi phosphatidylinositol 4‐phosphate (PI4P)‐dependent pathway promotes loading of the Golgi system into NSC apical processes. The physiological significance of this mechanism is demonstrated by defects in apical loading of the Golgi system resulting in cell‐autonomous disruption in NSC polarity and, when imposed on a tissue‐wide scale, catastrophic failure in development of the neocortex. This pathway requires the action of a pair of phosphatidylinositol transfer proteins (PITPNA and (PITPNB) to promote PI4P production on Golgi membranes, and the actions of downstream effector proteins that respond to PI4P signaling. These effectors include GOLPH3, the nonconventional myosin Myo18A, and the ceramide transfer protein CERT. Finally, we find the PI4P‐dependent regulation of Golgi apical positioning is transmitted to the differentiated progeny of the affected NSCs and influences later steps in development of the embryonic mouse neocortex. Taken together, we have identified a novel mechanism by which PITPs, PI4P, and Golgi apparatus position regulate cell polarity and stem cell biology in the developing mammalian forebrain.Support or Funding InformationThis research was funded by NIH grant R01‐GM112591 and grant BE‐0017 from the Robert A. Welch Foundation.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A Golgi Lipid Signaling Pathway Controls Apical Golgi Distribution and Cell Polarity during Neurogenesis

Phosphatidylinositol (PtdIns) transfer proteins (PITPs) stimulate PtdIns-4-P synthesis and signaling in eukaryotic cells, but to what biological outcomes such signaling circuits are coupled remains unclear. Herein, we show that two highly related StART-like PITPs, PITPNA and PITPNB, act in a redundant fashion to support development of the embryonic mammalian neocortex. PITPNA/PITPNB do so by driving PtdIns-4-P-dependent recruitment of GOLPH3, and likely ceramide transferprotein (CERT), to Golgi membranes with GOLPH3 recruitment serving to promote MYO18A- and F-actin-directed loading of the Golgi network to apical processes of neural stem cells (NSCs). We propose the primary role for PITP/PtdIns-4-P/GOLPH3/CERT signaling in NSC Golgi is not in regulating bulk membrane trafficking but in optimizing apically directed membrane trafficking and/or apical membrane signaling during neurogenesis.

New species of the leafhopper genera Sophonia and Stenotortor (Hemiptera: Cicadellidae: Evacanthinae) from the Oriental region.

Three new species, Sophonia picta sp. nov., Sophonia similis sp. nov. (both from Indonesia: Kalimantan Tengah) and Stenotortor acuta sp. nov. (from India: Karnataka), are described and illustrated. S. picta closely resembles S. malayana (Baker) externally but differs in having a round carmine red spot on the crown and a black round spot on the apical third of the forewing. Though the aedeagus of S. malayana and S. picta are similar, S. picta has a ventral pair of aedeagal processes with subequal branches compared to a lateral pair with unequal branches in S. malayana. S. similis closely resembles S. sandakanensis (Baker) externally but differs in having a round black spot on the apical third of the forewing and a short apical process on the aedeagal shaft. Sophonia sandakanenesis Baker is redescribed. A revised key to the species of Sophonia from Singapore, Borneo and the Philippines is also provided. Stenotortor acuta sp. nov. resembles both S. inocarpi Baker and S. subhimalya Viraktamath Wesley but differs in a having more acutely pointed head. Illustrations of the three earlier known species of Stenotortor and also the new species are provided along with a key for their identification.

Olfactory receptor neuronal dendrites become mostly intra-sustentacularly enwrapped upon maturity.

The mammalian olfactory epithelium (OE) sustains persistent neurogenesis even in the adult. Sustentacular cells therein play both epithelial and neuroglial roles, although their relation with olfactory receptor neurons (ORNs) and their function in ORN maturation remain insufficiently understood. Sustentacular wrapping of ORN dendrites has been long known but always considered a minor presence, as opposed to the supposedly unwrapped majority of ORN dendrites at inter-sustentacular borderlines. Using immunofluorescence, confocal and immuno-electron microscopy, the current study examined cytoarchitectonic organization and maturation of ORN dendrites at the rat OE apical layer. Contrary to common belief, the observations here on tangential histological sections of the OE apical junctional belt layer showed on average 53.93% sustentacular cell-enwrapped, 18.46% partially wrapped (in the vertical grooves on the sides of sustentacular apices) and 27.61% unwrapped ORN dendrites (at the borderlines between sustentacular cells). The enwrapped dendrites were found within the confines of sustentacular apices but linked to the sides of the latter each by a mesentery (mesodendrite) of sustentacular plasma membranes and autotypic cell junctions. Up to six dendrites were seen in one sustentacular apical process. As marked by high and low immunoreactivity for class III beta-tubulin, respectively, immature and mature ORN dendrites accounted on average for 12.46 and 87.54% of the total ORN dendrites at the OE apical layer. By correlative analysis of the maturity level and wrapping status, most immature ORN dendrites were found unwrapped (immature unwrapped=9.71% of the total dendrites), and practically no immature dendrites appeared enwrapped. In contrast, mature ORN dendrites comprised all the enwrapped (mature enwrapped=53.93% of the total), most of the partially wrapped (mature partially wrapped=15.71% of the total) and a portion of the unwrapped ORN dendrites (mature unwrapped=17.9% of the total dendrites). Based on the current findings and previous data by other researchers, it is concluded that immature ORN dendrites emerge vertically from the OE apical surface between sustentacular cell apices. A large majority of the newly emerged dendrites then undergo sideways migration, sustentacular enwrapment and further maturation. Only a small minority of the newly emerged dendrites reach maturity and remain unwrapped. These divergent maturational courses imply structural or functional differences between the enwrapped and unwrapped mature ORN dendrites.

Primary Cilium Mediated Retinal Pigment Epithelium Maturation is Retarded in Ciliopathy Patient Cells

Primary cilia are sensory organelles that protrude from the cell membrane. Cilia defects cause ciliopathy disorders with retinal degeneration as a prominent phenotype. Here, we demonstrate that the retinal pigment epithelium (RPE), essential for photoreceptor development and function, requires a functional primary cilium for complete maturation, and RPE maturation defects in ciliopathies precede photoreceptor development. Pharmacologically enhanced ciliogenesis in wildtype induced pluripotent stem cells (iPSCs)-RPE leads to fully-mature and functional cells. Whereas, ciliopathy patient-derived iPSCs-RPE and wildtype iPSC-RPE with a knockdown of ciliary-trafficking protein remain immature, with defective apical processes, reduced functionality, and reduced adult-specific gene expression. Primary cilia proteins regulate RPE maturation by simultaneously suppressing canonical-WNT and activating PKC-δ pathways. A similar cilia-dependent maturation pathway exists in lung epithelium. Our results provide novel insights into ciliopathy-induced retinal degeneration, demonstrate a developmental role for primary cilia in epithelial maturation, and provide a method to mature iPSC-epithelial cells for clinical applications.

Primary Cilium-Mediated Retinal Pigment Epithelium Maturation Is Disrupted in Ciliopathy Patient Cells

SUMMARYPrimary cilia are sensory organelles that protrude from the cell membrane. Defects in the primary cilium cause ciliopathy disorders, with retinal degeneration as a prominent phenotype. Here, we demonstrate that the retinal pigment epithelium (RPE), essential for photoreceptor development and function, requires a functional primary cilium for complete maturation and that RPE maturation defects in ciliopathies precede photoreceptor degeneration. Pharmacologically enhanced ciliogenesis in wild-type induced pluripotent stem cells (iPSC)-RPE leads to fully mature and functional cells. In contrast, ciliopathy patient-derived iPSC-RPE and iPSC-RPE with a knockdown of ciliary-trafficking protein remain immature, with defective apical processes, reduced functionality, and reduced adult-specific gene expression. Proteins of the primary cilium regulate RPE maturation by simultaneously suppressing canonical WNT and activating PKCδ pathways. A similar cilium-dependent maturation pathway exists in lung epithelium. Our results provide insights into ciliopathy-induced retinal degeneration, demonstrate a developmental role for primary cilia in epithelial maturation, and provide a method to mature iPSC epithelial cells for clinical applications.

HtrA1 Mediated Intracellular Effects on Tubulin Using a Polarized RPE Disease Model

Age-related macular degeneration (AMD) is the leading cause of irreversible vision loss. The protein HtrA1 is enriched in retinal pigment epithelial (RPE) cells isolated from AMD patients and in drusen deposits. However, it is poorly understood how increased levels of HtrA1 affect the physiological function of the RPE at the intracellular level. Here, we developed hfRPE (human fetal retinal pigment epithelial) cell culture model where cells fully differentiated into a polarized functional monolayer. In this model, we fine-tuned the cellular levels of HtrA1 by targeted overexpression. Our data show that HtrA1 enzymatic activity leads to intracellular degradation of tubulin with a corresponding reduction in the number of microtubules, and consequently to an altered mechanical cell phenotype. HtrA1 overexpression further leads to impaired apical processes and decreased phagocytosis, an essential function for photoreceptor survival. These cellular alterations correlate with the AMD phenotype and thus highlight HtrA1 as an intracellular target for therapeutic interventions towards AMD treatment.

Immunohistochemical characterization of brush cells in the rat larynx.

The immunohistochemical characteristics of brush cells in the laryngeal mucosa were examined using immunohistochemistry for various immunohistochemical cell markers including villin at the light and electron microscopic levels. Cells that were immunoreactive to villin were barrel-shaped with thick cytoplasmic processes extending toward the lumen of the laryngeal cavity. Immunoelectron microscopic observations revealed thick and short microvilli with long rootlets of microfilaments. Numerous small clear vesicles and small finger-like cytoplasmic processes were observed in the apical process and lateral membrane, respectively. Double immunofluorescence showed villin-immunoreactive cells were not immunoreactive for the markers of solitary chemosensory cells, GNAT3 and phospholipase C, β2-subunit (PLCβ2), or for that of neuroendocrine cells, synaptosome-associated protein 25kD. Furthermore, immunoreactivities for cytokeratin 18 (CK18) and doublecortin like-kinase 1 in the perinuclear cytoplasm of villin-immunoreactive cells. However, some CK18-immunoreactive cells were immunoreactive to GNAT3 but not to villin. Regarding sensory innervation, only a few intraepithelial nerve endings with P2X3, SP, or CGRP immunoreactivity attached to villin-immunoreactive cells. In the present study, brush cells in the rat laryngeal mucosa were classified by immunoreactivity for villin, and were independent of other non-ciliated epithelial cells such as solitary chemosensory cells and neuroendocrine cells.

Comparison of multiple transcriptomes exposes unified and divergent features of quiescent and activated skeletal muscle stem cells

BackgroundSkeletal muscle satellite (stem) cells are quiescent in adult mice and can undergo multiple rounds of proliferation and self-renewal following muscle injury. Several labs have profiled transcripts of myogenic cells during the developmental and adult myogenesis with the aim of identifying quiescent markers. Here, we focused on the quiescent cell state and generated new transcriptome profiles that include subfractionations of adult satellite cell populations, and an artificially induced prenatal quiescent state, to identify core signatures for quiescent and proliferating.MethodsComparison of available data offered challenges related to the inherent diversity of datasets and biological conditions. We developed a standardized workflow to homogenize the normalization, filtering, and quality control steps for the analysis of gene expression profiles allowing the identification up- and down-regulated genes and the subsequent gene set enrichment analysis. To share the analytical pipeline of this work, we developed Sherpa, an interactive Shiny server that allows multi-scale comparisons for extraction of desired gene sets from the analyzed datasets. This tool is adaptable to cell populations in other contexts and tissues.ResultsA multi-scale analysis comprising eight datasets of quiescent satellite cells had 207 and 542 genes commonly up- and down-regulated, respectively. Shared up-regulated gene sets include an over-representation of the TNFα pathway via NFKβ signaling, Il6-Jak-Stat3 signaling, and the apical surface processes, while shared down-regulated gene sets exhibited an over-representation of Myc and E2F targets and genes associated to the G2M checkpoint and oxidative phosphorylation. However, virtually all datasets contained genes that are associated with activation or cell cycle entry, such as the immediate early stress response genes Fos and Jun. An empirical examination of fixed and isolated satellite cells showed that these and other genes were absent in vivo, but activated during procedural isolation of cells.ConclusionsThrough the systematic comparison and individual analysis of diverse transcriptomic profiles, we identified genes that were consistently differentially expressed among the different datasets and shared underlying biological processes key to the quiescent cell state. Our findings provide impetus to define and distinguish transcripts associated with true in vivo quiescence from those that are first responding genes due to disruption of the stem cell niche.

STARGARDT DISEASE: Beyond Flecks and Atrophy.

To identify changes in the outer retina in areas without atrophy or flecks of Stargardt disease (STGD) using spectral-domain optical coherence tomography. Twenty-three STGD patients and 26 control subjects were assessed for outer retina (from the outer border of Bruch membrane [BrM] to the inner border of the inner segment ellipsoid zone [EZ]), BrM-retinal pigment epithelium apex, the EZ thickness, and apical process interdigitation zone. Patients with STGD had increased BrM-EZ thickness in areas without apparent disease versus control subjects at 1,000, 1,500, 2,000, and 2,500 μm superior and 1,500 μm, 2,000 μm, and 2,500 μm inferior to the fovea (P < 0.05 to P < 0.001), greatest difference (3.4 μm) at 2,500 μm superiorly. The BrM-retinal pigment epithelium segment showed larger fractional contribution of 0.48 to 0.51 to the overall BrM-EZ thickness compared with 0.35 to 0.42 in control subjects. The thickness of EZ and the interspace between the retinal pigment epithelium apex and EZ were smaller in the STGD patients (P < 0.05 to P < 0.001). Patients with STGD displayed an interrupted interdigitation zone in 16 (84.2%) of 19 eyes versus 6 (23.1%) of 26 eyes of the control subjects (P < 0.001). The BrM-EZ segment of the outer retina of STGD patients lacked the typical normal trilaminar pattern. Subtle changes are present within the BrM-EZ segment of the outer retina of STGD patients in areas that are devoid of atrophy and flecks. These findings suggest that pathologic changes in STGD are more widespread than that seen by clinical examination.