Outer Epithelial Cells Research Articles

Morphological and Molecular Changes during Limb Regeneration of the Exopalaemon carinicauda.

With the increase in breeding density of Exopalaemon carinicauda, appendage breakage may occur, which seriously affects survival and economic benefits. To study the limb regeneration process of E. carinicauda, we induced autotomy of the pereopods. After a period of time, wound swelling disappeared, the pigment gradually accumulated, and a tawny film subsequently formed in the wound. The healing period of the wound occurred 24 h after autotomy, and the blastema formation stage occurred 48 h after autotomy. After 4 days of cutting, the limb buds began to differentiate, grow, and expand rapidly, and this process lasted approximately 15 days. Microscopic observations revealed significant changes in the type and number of associated cells including outer epithelial cells, granulocytes, embryonic cells, columnar epidermal cells, elongated cells, and blastoma cells, during the process from limb fracture to regeneration. A comparative transcriptome analysis identified 1415 genes differentially expressed between the J0h (0 h post autotomy) and J18h (18 h post autotomy), and 3952 and 4366 differentially expressed genes for J0 and J14d (14 days post autotomy) and J18h and J14d, respectively. Some of these genes may be related to muscle growth or molting, as indicated by the presence of troponin C, chitinase, actin, innexin, and cathepsin L. As a functional gene involved in epidermal formation, the mRNA expression level of the innexin inx2 in the pereopod of E. carinicauda changed significantly in the experimental groups (p < 0.05). The results of this study contribute to existing knowledge of regeneration mechanisms in crustaceans.

Reduced expression of Ankyrin-G and E-cadherin in duodenal mucosal biopsy of subjects with celiac disease

Confirmatory diagnosis of celiac disease (CD) include histopathology of duodenal biopsy and tissue trans-glutaminase-IgA. Identification of tissue-specific histological markers is warranted to improve the diagnosis. A genetic study in CD identified the association of ankyrin-G that connects E-cadherin with β2-spectrin in epithelial cells of the duodenal tissue. We attempted to investigate the differential expression of ankyrin-G, E-cadherin and β2-spectrin in duodenal biopsy of CD subjects compared to non-CD controls. Duodenal tissue was collected from 83 study participants, of which 50 were CD, and 33 were non-CD controls. Whole RNA was isolated from 32 CD and 23 non-CD controls from available tissues, and differential mRNA expression was measured using real-time PCR. Tissue sections from 18 CD cases and 10 non-CD controls were immunostained using monoclonal antibodies. Tissue immunohistochemistry were evaluated for differential expression and pattern of expression. RT-PCR revealed significantly reduced expression of ankyrin-G (fold change=0.63; p=0.03) and E-cadherin (fold change=0.50; p=0.02) among CD subjects compared to non-CD controls. Tissue immunohistochemistry confirmed the reduced expression of ankyrin-G and E-cadherin in CD. Differential expression is grossly limited within the outer columnar epithelial cell layer. Expression fold change of E-cadherin was seen to partially correlate with the serum tTG level (r=0.4; p=0.04). In CD, reduced expression of two key cytoskeletal proteins (ankyrin-G and E-cadherin) in duodenum mucosa was observed, which indicates its implication in disease biology and could be tested as a tissue-specific biomarker for CD. Functional studies may unravel the specific contribution of these proteins in CD pathophysiology.

Microtubule-dependent apical polarization of basement membrane matrix mRNAs in mouse epithelial cells

The basement membrane (BM) demarcating epithelial tissues undergoes rapid expansion to accommodate tissue growth and morphogenesis during embryonic development. To facilitate the secretion of bulky BM proteins, their mRNAs are polarized basally in the follicle epithelial cells of the Drosophila egg chamber to position their sites of production close to their deposition. In contrast, we observed the apical rather than basal polarization of all major BM mRNAs in the outer epithelial cells adjacent to the BM of mouse embryonic salivary glands using single-molecule RNA fluorescence in situ hybridization (smFISH). Moreover, electron microscopy and immunofluorescence revealed apical polarization of both the endoplasmic reticulum (ER) and Golgi apparatus, indicating that the site of BM component production was opposite to the site of deposition. At the apical side, BM mRNAs colocalized with ER, suggesting they may be co-translationally tethered. After microtubule inhibition, the BM mRNAs and ER became uniformly distributed rather than apically polarized, but they remained unchanged after inhibiting myosin II, ROCK, or F-actin, or after enzymatic disruption of the BM. Because Rab6 is generally required for Golgi-to-plasma membrane trafficking of BM components, we used lentivirus to express an mScarlet-tagged Rab6a in salivary gland epithelial cultures to visualize vesicle trafficking dynamics. We observed extensive bidirectional vesicle movements between Golgi at the apical side and the basal plasma membrane adjacent to the BM. Moreover, we showed that these vesicle movements depend on the microtubule motor kinesin-1 because very few vesicles remained motile after treatment with kinesore to compete for cargo-binding sites on kinesin-1. Overall, our work highlights the diverse strategies that different organisms use to secrete bulky matrix proteins: while Drosophila follicle epithelial cells strategically place their sites of BM protein production close to their deposition, mouse embryonic epithelial cells place their sites of production at the opposite end. Instead of spatial proximity, they use the microtubule cytoskeleton to mediate this organization as well as for the apical-to-basal transport of BM proteins.

Pleiotropic Roles of Scavenger Receptors in Circadian Retinal Phagocytosis: A New Function for Lysosomal SR-B2/LIMP-2 at the RPE Cell Surface.

The retinal phagocytic machinery resembles the one used by macrophages to clear apoptotic cells. However, in the retina, the permanent contact between photoreceptor outer segments (POS) and retinal pigment epithelial (RPE) cells requires a tight control of this circadian machinery. In addition to the known receptors synchronizing POS internalization, several others are expressed by RPE cells. Notably, scavenger receptor CD36 has been shown to intervene in the internalization speed. We thus investigated members of the scavenger receptor family class A SR-AI and MARCO and class B CD36, SR-BI and SR-B2/LIMP-2 using immunoblotting, immunohisto- and immunocytochemistry, lipid raft flotation gradients, phagocytosis assays after siRNA/antibody inhibition, RT-qPCR and western blot analysis along the light:dark cycle. All receptors were expressed by RPE cell lines and tissues and colocalized with POS, except SR-BI. All receptors were associated with lipid rafts, and even more upon POS challenge. SR-B2/LIMP-2 inhibition suggested a role in the control of the internalization speed similar to CD36. In vivo, MARCO and CD36 displayed rhythmic gene and protein expression patterns concomitant with the phagocytic peak. Taken together, our results indicate that CD36 and SR-B2/LIMP-2 play a direct regulatory role in POS phagocytosis dynamics, while the others such as MARCO might participate in POS clearance by RPE cells either as co-receptors or via an indirect process.

Deficiency in Lyst function leads to accumulation of secreted proteases and reduced retinal adhesion.

Chediak–Higashi syndrome, caused by mutations in the Lysosome Trafficking Regulator (Lyst) gene, is a recessive hypopigmentation disorder characterized by albinism, neuropathies, neurodegeneration, and defective immune responses, with enlargement of lysosomes and lysosome-related organelles. Although recent studies have suggested that Lyst mutations impair the regulation of sizes of lysosome and lysosome-related organelle, the underlying pathogenic mechanism of Chediak–Higashi syndrome is still unclear. Here we show striking evidence that deficiency in LYST protein function leads to accumulation of photoreceptor outer segment phagosomes in retinal pigment epithelial cells, and reduces adhesion between photoreceptor outer segment and retinal pigment epithelial cells in a mouse model of Chediak–Higashi syndrome. In addition, we observe elevated levels of cathepsins, matrix metallopeptidase (MMP) 3 and oxidative stress markers in the retinal pigment epithelium of Lyst mutants. Previous reports showed that impaired degradation of photoreceptor outer segment phagosomes causes elevated oxidative stress, which could consequently lead to increases of cysteine cathepsins and MMPs in the extracellular matrix. Taken together, we conclude that the loss of LYST function causes accumulation of phagosomes in the retinal pigment epithelium and elevation of several extracellular matrix-remodeling proteases through oxidative stress, which may, in turn, reduce retinal adhesion. Our work reveals previously unreported pathogenic events in the retinal pigment epithelium caused by Lyst deficiency. The same pathogenic events may be conserved in other professional phagocytic cells, such as macrophages in the immune system, contributing to overall Chediak–Higashi syndrome pathology.

Mantle tissue in the pearl oyster Pinctada fucata secretes immune components via vesicle transportation

Molluscan bivalves secrete shell matrices into the extrapallial space (EPS) to guide the precipitation of rigid shells. Meanwhile, immune components are present in the EPS and shell matrices, which are pivotal in resistant to invaded pathogens, thus ensuring the shell formation process. However, the origin of these components remains unclear. In this study, we revealed numerous vesicles were secreted from the outer mantle epithelial cells by using light and electron microscopes. The secreted vesicles were isolated by gradient centrifugation and confirmed by transmission electron microscopy. Proteomics analysis showed that the secreted vesicles were composed of cytoplasmic and immune components, most of which do not have signal peptides, indicating that they were secreted by a non-classical pathway. Moreover, real-time PCR revealed that some immune components were highly expressed in the mantle tissue, compared to the hemocytes. FTIR analysis verified the presence of lipids in the shell matrices, indicating that the vesicles have integrated into the shell layers. Taken together, our results suggested that mantle epithelial cells secreted some important immune components into the EPS via secreted vesicle transportation, thus cooperating with the hemocytes to play a vital role in immunity during shell formation.

Single cell transcriptomics of the developing zebrafish lens and identification of putative controllers of lens development

The vertebrate lens is a valuable model system for investigating the gene expression changes that coordinate tissue differentiation due to its inclusion of two spatially separated cell types, the outer epithelial cells and the deeper denucleated fiber cells that they support. Zebrafish are a useful model system for studying lens development given the organ's rapid development in the first several days of life in an accessible, transparent embryo. While we have strong foundational knowledge of the diverse lens crystallin proteins and the basic gene regulatory networks controlling lens development, no study has detailed gene expression in a vertebrate lens at single cell resolution. Here we report an atlas of lens gene expression in zebrafish embryos and larvae at single cell resolution through five days of development, identifying a number of novel putative regulators of lens development. Our data address open questions about the temperospatial expression of α-crystallins during lens development that will support future studies of their function and provide the first detailed view of β- and γ-crystallin expression in and outside the lens. We describe divergent expression in transcription factor genes that occur as paralog pairs in the zebrafish. Finally, we examine the expression dynamics of cytoskeletal, membrane associated, RNA-binding, and transcription factor genes, identifying a number of novel patterns. Overall these data provide a foundation for identifying and characterizing lens developmental regulatory mechanisms and revealing targets for future functional studies with potential therapeutic impact.

Novel globular C1q domain-containing protein (PmC1qDC-1) participates in shell formation and responses to pathogen-associated molecular patterns stimulation in Pinctada fucata martensii

The C1q protein, which contains the globular C1q (gC1q) domain, is involved in the innate immune response, and is found abundantly in the shell, and it participates in the shell formation. In this study, a novel gC1q domain-containing gene was identified from Pinctada fucata martensii (P. f. martensii) and designated as PmC1qDC-1. The full-length sequence of PmC1qDC-1 was 902 bp with a 534 bp open reading frame (ORF), encoding a polypeptide of 177 amino acids. Quantitative real-time PCR (qRT-PCR) result showed that PmC1qDC-1 was widely expressed in all tested tissues, including shell formation-associated tissue and immune-related tissue. PmC1qDC-1 expression was significantly high in the blastula and gastrula and especially among the juvenile stage, which is the most important stage of dissoconch shell formation. PmC1qDC-1 expression was located in the outer epithelial cells of mantle pallial and mantle edge and irregular crystal tablets were observed in the nacre upon knockdown of PmC1qDC-1 expression at mantle pallial. Moreover, the recombined protein PmC1qDC-1 increased the rate of calcium carbonate precipitation. Besides, PmC1qDC-1 expression was significantly up-regulated in the mantle pallial at 6 h and was significantly up-regulated in the mantle edge at 12 h and 24 h after shell notching. The expression level of PmC1qDC-1 in mantle edge was significantly up-regulated at 48 h after LPS stimulation and was significantly up-regulated at 12 h, 24 h and 48 h after poly I:C stimulation. Moreover, PmC1qDC-1 expression was significantly up-regulated in hemocytes at 6 h after lipopolysaccharide (LPS) and poly I:C challenge. These findings suggest that PmC1qDC-1 plays a crucial role both in the shell formation and the innate immune response in pearl oysters, providing new clues for understanding the shell formation and defense mechanism in mollusk.

Dental Roots: Formation, Lengthening and Malformations of Roots

The Hertwig’s Epithelial Root Sheath (HERS) includes two layers: the Outer Enamel Epithelial Root Sheath and the Inner Enamel Epithelium Epithelium (IEE and OEE). They contribute both to the root formation. The inner columnar epithelial cells (IEE) of the dental papilla are formed by cells located near the dental papilla. They are at the origin of odontoblasts expressing FGF-4, -8 and -9 and at later stages BMP-2 and BMP7. The Outer Epithelial cells (OEE) express SHH, Msx2, enamel matrix proteins, paxillin, and Pax-6. When the cells of the HERS dissociate, intercellular spaces enlarge. Cells migrate from the epithelial sac, and underwent phenotypic inter conversion into cementoblasts and later cementocytes. The Hertwigs enamel epithelium contributes to cementum formation. Epithelial rest of Malassez is remnants of the Hertwig’s root sheath. They are implicated in cementogenesis. Root lengthening and dentin thickening are involved in root elongation and dentin thickening to the detriment of the pulp chamber that is gradually reduced in volume. Apexogenesis and apexification contribute to root formation. Defects and abnormal root formation implicate missing teeth(hypodontia), orteeth in excess (supernumary teeth or hyperdontia) or mis-shapped structures. Genetic defects (Nfic, Ptc, Dkk1, Osx, Smad4, and Wls) have been identified. Premature arrests of root formation are due to apical infection, radiation, chemotherapy, as well as genes alterations. Roots malformations include root dilacerations which are abnormal curvature of the root and sharp bend of the crown or root axis. Taurodontism and other misshaped root structures are also frequently seen in man. Two main forms have been recognized: 1) the CLCN7 encoding a chloride channel, and 2) the second related to a defective PLG gene (encoding plasminogen). Altogether, theses defects contribute to major endodontic difficulties.

Shell-adductor muscle attachment and Ca2+ transport in the bivalves Ostrea stentina and Anomia ephippium

Among bivalve muscles, the adductors are particularly important for animal survival because they control valve closure. Most studies have addressed the type and morphology of this muscle in bivalves but few have focused on the mechanism that anchors it to the shell myostracum layer. Moreover, the possible calcium transport mechanism through the adductor muscle cells to the myostracum shell layer, which is necessary for bivalve biomineralisation, has never been addressed. Our results indicate that the muscle cell-shell attachment is mediated by the outer mantle epithelial cell layer, here termed tendon cells. These cells are modified at the muscle scar zone by the presence of actin cytoskeletal bundles, which anchor cells to the extracellular matrix via focal adhesion (or focal contact) junctions at the basal side and to extrapallial matrix at the apical side, both rich in collagen. From apical focal adhesions, bundles of collagen-rich fibres cross the extrapallial space and penetrate the myostracum shell layer. The latter constitutes one of the strongest anchoring structures among invertebrates. Numerous vesicles protrude from the tendon cells into the extrapallial space. TEM-EDX analysis reveals the presence of Ca2+ inside some of these vesicles both in tendon cells and in the extrapallial space. This suggests a potential mechanism for calcium transport from cells to the myostracum. Statement of SignificanceThe interfaces between bivalve shells and muscular attachments are unique and of special interest as adhesive functional biomaterials, being one of the strongest invertebrate anchoring structures. We present an updated ultrastructural model of the adductor muscle-shell attachment. Muscle cells connect with the shell through epithelial `tendon cells`, which have a cytoskeleton of actin microfilaments that connect to the extracellular matrix via focal adhesions. Collagen-rich fibres arise from apical focal adhesions, cross the nanometric extrapallial space and penetrate the myostracum where they form an organic network. Calcium is present inside vesicles that are released into the extrapallial space. The lack of direct cellular control on secretion restricts the myostracal microstructure to prismatic aragonitic similar to its inorganic counterpart.

InVivo Bioengineering of Fluorescent Conductive Protein-Dye Microfibers.

SummaryEngineering protein-based biomaterials is extremely challenging in bioelectronics, medicine, and materials science, as mechanical, electrical, and optical properties need to be merged to biocompatibility and resistance to biodegradation. An effective strategy is the engineering of physiological processes in situ, by addition of new properties to endogenous components. Here we show that a green fluorescent semiconducting thiophene dye, DTTO, promotes, in vivo, the biogenesis of fluorescent conductive protein microfibers via metabolic pathways. By challenging the simple freshwater polyp Hydra vulgaris with DTTO, we demonstrate the stable incorporation of the dye into supramolecular protein-dye co-assembled microfibers without signs of toxicity. An integrated multilevel analysis including morphological, optical, spectroscopical, and electrical characterization shows electrical conductivity of biofibers, opening the door to new opportunities for augmenting electronic functionalities within living tissue, which may be exploited for the regulation of cell and animal physiology, or in pathological contexts to enhance bioelectrical signaling.

Local synthesis of hepcidin in the anterior segment of the eye: A novel observation with physiological and pathological implications

PurposeThe avascular cornea, trabecular meshwork (TM), and lens obtain iron, an essential biometal, from the aqueous humor (AH). The mechanism by which this exchange is regulated, however, is unclear. Recently we reported that non-pigmented ciliary epithelial cells express ferroportin (Fpn) (Ashok, 2018b), an iron export protein modulated by hepcidin, the master regulator of iron homeostasis secreted mainly by the liver. Here, we explored whether ciliary epithelial and other cells in the anterior segment synthesize hepcidin, suggesting local regulation of iron exchange at this site. MethodsHuman and bovine eyes were dissected to isolate the ciliary body (CB), corneal endothelial (CE), TM, lens epithelial (LE), and outer epithelial cell layer of the iris. Total mRNA and protein lysates were processed to evaluate the synthesis and expression of hepcidin, the iron regulatory peptide hormone, Fpn, the only known iron export protein, ceruloplasmin (Cp), a ferroxidase necessary for iron export, transferrin receptor (TfR), a major iron uptake protein, and ferritin, a major iron storage protein. A combination of techniques including reverse transcription polymerase chain reaction (RT-PCR) of total mRNA, Western blotting of protein lysates, and immunofluorescence of fixed tissue sections were used to accomplish these goals. ResultsRT-PCR of isolated tissue samples revealed hepcidin-specific mRNA in the CB, TM, CE, and LE of the bovine eye. Western blotting of protein lysates from these tissues showed reactivity for hepcidin, Fpn, ferritin, and TfR. Western blotting and immunohistochemistry of similar tissues isolated from cadaveric human eyes showed expression of hepcidin, Fpn, and Cp in these samples. Notably, Fpn and Cp were expressed on the basolateral membrane of non-pigmented ciliary epithelial cells, facing the AH. ConclusionsSynthesis and expression of hepcidin and Fpn in the ciliary epithelium suggests local regulation of iron transport from choroidal plexus in the ciliary body to the AH across the blood-aqueous barrier. Expression of hepcidin and Fpn in CE, TM, and LE cells indicates additional regulation of iron exchange between the AH and cornea, TM, and lens, suggesting autonomous regulation of iron homeostasis in the anterior segment. Physiological and pathological implications of these observations are discussed.

The Characteristics and Expression Profile of Transferrin in the Accessory Nidamental Gland of the Bigfin Reef Squid during Bacteria Transmission

The accessory nidamental gland (ANG) is a female reproductive organ found in most squid and cuttlefish that contains a consortium of bacteria. These symbiotic bacteria are transmitted from the marine environment and selected by the host through an unknown mechanism. In animals, a common antimicrobial mechanism of innate immunity is iron sequestration, which is based on the development of transferrin (TF)-like proteins. To understand this mechanism of host-microbe interaction, we attempted to characterize the role of transferrin in bigfin reef squid (Sepioteuthis lessoniana) during bacterial transmission. qPCR analysis showed that Tf was exclusively expressed in the outer layer of ANG,and this was confirmed by in situ hybridization, which showed that Tf was localized in the outer epithelial cell layer of the ANG. Western blot analysis indicated that TF is a soluble glycoprotein. Immunohistochemical staining also showed that TF is localized in the outer epithelial cell layer of the ANG and that it is mainly expressed in the outer layer during ANG growth. These results suggest that robust Tf mRNA and TF protein expression in the outer layer of the ANG plays an important role in microbe selection by the host during bacterial transmission.

Morphological development of lily (Lilium ‘Siberia’) floral organs

Effects of flower buds of oriental lily “Siberia”, Asian Lily “Desert Storm” and “Shuttle Red” on the growth and development during different developmental stages of plants were investigated. The results showed that the plant height of different lily was not different in different periods, and the growth increased rapidly during seeding stage to squaring period while slowly in the squaring period to full-bloom stage. The size of flower organs was different, the "Siberia" was significantly larger than "desert storm" and "shuttle red", and the development of ovary and anther was earlier. The intraepidermal of epidermal cells of lily petals were smaller than those of outer epidermal cells. The number of intraepidermal cells is much more higher than the number of outer epithelial cells. The infiltration degree of lily petals was related to the growth of intraepidermal and outer epidermal cells of petals. The growth rate of intraepidermal cells in "Siberia" was significantly higher than that in outer epidermal. The growth rate of intraepidermal cells in "desert storm" and "shuttle red" was slightly higher than that of outer epidermal, which proved that the uneven growth of petals of intraepidermal and outer epidermal cells leads to lily petals’ bent, thereby results in the formation of different flower types.

Gill epithelium of an angler catfish, Chaca chaca (Siluriformes, Chacidae): Enzyme and glycoprotein histochemistry.

A series of histochemical techniques have been employed to localize alkaline phosphatase, acid phosphatase, non-specific esterase, catalase and peroxidase; and to visualize and characterize glycoprotein (GPs) moieties in the epithelium of gill arch, gill filaments and secondary lamellae of an angler catfish Chaca chaca. The epithelium of gill arch and gill filament shows strong alkaline phosphatase activity in the deeper layer epithelial cells; strong non-specific esterase activities in the outer layer epithelial cells; and weak acid phosphatase activity throughout the epithelium. The activity of these enzymes in the secondary lamellae is weak. The catalase and peroxidase show strong activities in the blood cells of the secondary lamellae. Various classes of GPs have been identified and characterized in the mucous secretions of the gill epithelium of C.chaca. These include-GPs with oxidizable vicinal diols, GPs with sialic acid residues without O-acyl substitution and GPs with O-sulphate esters. The functional significance of different enzymes in gill epithelium and the GPs in the mucus secreted on the surface has been discussed with the physiology of the gills in relation to the characteristic habit and habitat of the fish.

Progenitor cells expressing nestin, a neural crest stem cell marker, differentiate into outer root sheath keratinocytes.

Nestin, which was originally described as a neural crest stem cell marker, is known to be expressed in bulge follicle cells of human, canine and murine anagen hairs. However, the capacity of nestin-expressing cells to differentiate into the components of the hair follicle or the epidermis has been insufficiently investigated. To determine whether nestin-expressing cells are capable of differentiating into keratinocytes. A double-transgenic mouse line Nes-Cre/CAG-CAT-EGFP, in which enhanced green fluorescent protein (EGFP) is expressed upon Cre-based recombination driven by the nestin promoter. The tissue distribution of EGFP+ and nestin+ cells in the skin of the mouse line was analysed by immunofluorescence and immunohistochemical analyses. EGFP+ cells were recognized in the outer epithelial cell layers of anagen and telogen hair follicles, but rarely seen in the interfollicular epidermis. The EGFP+ cells in the outer layers of the hair follicles coexpressed keratin 14, a marker of the outer root sheath (ORS) keratinocytes, but not trichohyalin granules, an inner root sheath keratinocyte cell marker. Immunostaining for nestin failed to detect its expression in the majority of hair follicle epithelial cells, suggesting that the EGFP+ cells in the ORS were derived from nestin-expressing progenitor cells that had become further committed along the epithelial cell lineage, where nestin is no longer expressed. These results suggest that progenitor cells that differentiate into ORS keratinocytes are distinct from those for other hair follicle or epidermal components and provide implications for regenerative medicine and the molecular classification of hair follicle tumours.

Gene expression profiles at different stages for formation of pearl sac and pearl in the pearl oyster Pinctada fucata

BackgroundThe most critical step in the pearl formation during aquaculture is issued to the proliferation and differentiation of outer epithelial cells of mantle graft into pearl sac. This pearl sac secretes various matrix proteins to produce pearls by a complex physiological process which has not been well-understood yet. Here, we aimed to unravel the genes involved in the development of pearl sac and pearl, and the sequential expression patterns of different shell matrix proteins secreted from the pearl sac during pearl formation by pearl oyster Pinctada fucata using high-throughput transcriptome profiling.ResultsPrincipal component analysis (PCA) showed clearly different gene expression profiles between earlier (before 1 week) and later stages (1 week to 3 months) of grafting. Immune-related genes were highly expressed between 0 h – 24 h (donor dependent) and 48 h – 1 w (host dependent), and in the course of wound healing process pearl sac was developed by two weeks of graft transplantation. Moreover, for the first time, we identified some stem cell marker genes including ABCG2, SOX2, MEF2A, HES1, MET, NRP1, ESR1, STAT6, PAX2, FZD1 and PROM1 that were expressed differentially during the formation of pearl sac. The expression profiling of 192 biomineralization-related genes demonstrated that most of the shell matrix proteins (SMPs) involved in prismatic layer formation were first up-regulated and then gradually down-regulated indicating their involvement in the development of pearl sac and the onset of pearl mineralization. Most of the nacreous layer forming SMPs were up-regulated at 2 weeks after the maturation of pearl sac. Nacrein, MSI7 and shematrin involved in both layer formation were highly expressed during 0 h – 24 h, down-regulated up to 1 week and then up-regulated again after accomplishment of pearl sac formation.ConclusionsUsing an RNA-seq approach we unraveled the expression pattern of the key genes involved in the development of pearl sac and pearl as a result of host immune response after grafting. These findings provide valuable information in understanding the molecular mechanism of pearl formation and immune response in P. fucata.

Recent Advances of Shell Matrix Proteins and Cellular Orchestration in Marine Molluscan Shell Biomineralization

Biomineralization refers to the dynamic physiological processes whereby living organisms elaborate mineralized tissues. The existence of extremely abundant molluscan species implies the diversity of mineralized tissues, since the majority of them (Conchifera) produce shells that vary in size and shape. Over the past decades, great progress has been made on the study of the cellular biology of shell biomineralization. The construction of the molluscan shell is the archetype of a biologically controlled mineralization which requires specialized cellular machinery. It has been so far demonstrated that the cells involved in shell formation come from two different tissues: 1) outer mantle epithelial cells (OME) secrete the organic matrix, among which shell matrix proteins (SMPs) determine mineralogical and crystallographic properties of shell; and 2) circulating hemocytes which take part in the deposition of intracellular biominerals and deliver them to the mineralization sites. Mounts of novel SMPs have been identified by using molecular biology techniques (gene cloning, in situ hybridization, immunohistochemistry et al.) coupled with high-throughput sequencing data (genome, proteome, secretome and transcriptome) , and their corresponding functions during shell formation have also been confirmed. The cellular activity of OME and hemocytes during shell formation are significantly increased during shell regeneration process. A potential cellular basis model for molluscan shell formation is proposed. The shell matrix proteins, mostly secreted from OME, and a few secreted from hemocytes or other organs, are either directly delivered to the mineralization site via exosome or classical secretory pathway, or firstly transported to the hemolymph, and then engulfed by hemocytes (mainly granulocytes), which will disintegrate and release shell proteins and CaCO3 crystals at mineralization front. Besides, OME and hemocytes may be involved in the nucleation and remodeling process of CaCO3 mineralization. These cells and cell products work co-operatively to produce an organo-mineral shell, which is composed of various biomineral ultra-structures and macromolecular organic components.

Characterization of buffalo native pregnancy-associated glycoprotein: mass spectrometry-based glycan composition analysis, sugar-binding characteristics and proteolytic activity assay

Pregnancy-associated glycoproteins constitute a large family of extensively glycosylated secretory proteins which are specifically expressed in outer epithelial cell layer (chorion/trophectoderm) of placenta. The present study reports purification and characterization of buffalo pregnancy-associated glycoprotein (BuPAG) from foetal cotyledons during early pregnancy (< 3 months). Native BuPAG was purified by anion-exchange and Vicia villosa affinity chromatography. Three distinct bands were observed in SDS-PAGE and Western blot with higher molecular weight range of ~ 55–70 kDa in comparison to the calculated one (~ 40 kDa) which may be attributed to the presence of glycosylation in BuPAG. Glycan analysis of purified BuPAG by high-resolution mass spectrometry (ESI-qTof) revealed the presence of nine N-glycan structures and three O-glycan structures. Interaction analysis of various sugars with purified BuPAG using fluorescence quenching assay showed decrease in fluorescence intensity without any shift in emission maxima. BuPAG exhibited proteolytic activity within pH range of 2.0–5.5 with highest activity at pH 3.0. In the presence of inhibitor pepstatin A, proteolytic activity reduced by 84%. The glycan structures and sugar-binding characteristics of BuPAG are identified for the first time in this study. The activity of BuPAGs as active peptidases indicates their possible role in various molecular pathways required for sustenance of pregnancy.

Visualization of the penetration modifying mechanism of laurocapram by Mass Spectrometry Imaging in buccal drug delivery

The aim of the study was to visualize the penetration modifying effect of laurocapram on the delivery of diazepam and codeine across buccal mucosa by MALDI Mass Spectrometry Imaging (MALDI-MSI). A qualitative ex vivo study was carried out by mounting porcine buccal mucosa in Ussing chamber sliders and applying a pre-treatment of phosphate buffered saline (PBS) or a 50% (v/v) laurocapram:ethanol solution apically before incubation for 1 or 3 h with a 0.1 M diazepam or 0.1 M codeine solution. MALDI-MSI analysis was performed on vertical cryo-sections of porcine buccal mucosa. The analysis provided detailed images of the localisation of the drugs, laurocapram and endogenous lipids in the epithelium and connective tissue. While diazepam in the absence of laurocapram was distributed with a steady concentration gradient through the connective tissue, indicating passive diffusion, pre-treatment with laurocapram fundamentally altered the penetration of diazepam through the buccal mucosa. In the presence of laurocapram, the distribution of diazepam was restricted to areas where laurocapram itself was present, in particular in the outer epithelial cell layers and in certain islands in the connective tissue. In contrast, the penetration of codeine was unaffected by the presence of laurocapram in similar experiments.The co-localization of laurocapram and diazepam indicates a reservoir effect, which has previously been found in diffusion experiments in Ussing chambers. The major difference in the penetration of codeine and diazepam through the buccal mucosa in presence of laurocapram was explained by the physicochemical properties of the drugs. Codeine is characterized by being more hydrophilic than diazepam and was partly charged under the given experimental conditions.