TGF-ß1 Triggers Salivary Hypofunction via Attenuating Protein Secretion and AQP5 Expression in Human Submandibular Gland Cells.

Aging-related salivary gland degeneration usually causes poor oral health. Periductal fibrosis frequently occurs in the submandibular gland of the elderly. Transforming growth factor ß1 (TGF-ß1) is the primary driving factor for fibrosis, which exhibits an increase in the fibrotic submandibular gland tissue. This study aimed to investigate the effects of TGF-ß1 on the human submandibular gland (HSG) cell secretory function and its influences on aquaporin 5 (AQP5) expressions and distribution. We found that TGF-ß1 reduces the protein secretion amount of HSG and leads to the abundance alteration of 151 secretory proteins. Data are available via ProteomeXchange with the identifier PXD043185. The majority of HSG secretory proteins (84.11%) could be matched to the human saliva proteome. Meanwhile, TGF-ß1 enhances the expression of COL4A2, COL5A1, COL7A1, COL1A1, COL2A1, and α-SMA, hinting that TGF-ß1 possesses the potential to drive HSG fibrosis-related events. Besides, TGF-ß1 also attenuates the AQP5 expression and its membrane distribution in HSGs. The percentage for TGF-ß1-induced AQP5 reduction (52.28%) is much greater than that of the TGF-ß1-induced secretory protein concentration reduction (16.53%). Taken together, we concluded that TGF-ß1 triggers salivary hypofunction via attenuating protein secretion and AQP5 expression in HSGs, which may be associated with TGF-ß1-driven fibrosis events in HSGs.

Leukolectin-proteins are synthesized and secreted by lectocytes, a distinct category of fish embryonic mucus cells.

Fish perivitelline fluid (PVF) is a vital extra-embryonic compartment. At hatching, PVF-contents dissolve into the hatching fluid (HF). Analysis of Atlantic salmon HF reveals nearly a hundred distinct proteins, most of which were identified by advanced mass-spectrometry. However, one entity with an apparent molecular weight 26 kDa, necessitated identification from its tryptic peptides. Subsequent cloning and sequencing revealed novel leukolectin-proteins. From bioinformatic analysis, leukolectins (LL) belong in the tectonin protein-family, with recognized functions in innate immunity. This study aims to identify LL-expressing cells in diverse fish species, and to characterize the LL-gene in order to predict bio-functions of leukolectins. LL-proteins were detected in HF from several fish species and one invertebrate, using polyclonal LL-specific IgGs. Embryonic LL-immunoreactive cells were numerous in Atlantic salmon, rainbow trout, fewer in Atlantic cod, and rare in Atlantic halibut and Oikopleura dioica. LL-immunoreactive cells were termed lectocytes, which corresponded to peridermal mucuscells stained by PAS, but unstained by eosin. Hence, lectocytes and hatching-gland cells were clearly distinguished. Northern blots revealed two salmon LL-transcripts at mid-embryogenesis. Such transcripts were detected in epithelial cells of the periderm, gills and oral cavity. LL-transcripts predominated in the periderm, while choriolysin-transcripts were dominant in the gills. No co-expression of choriolysins and LL-transcripts was detected. BAC-library screening yielded salmon LL's genestructure with 4 introns, 5 exons, TATA-box, multiple upstream putative transcription-factor bindingsites and polyadenylation site. LL-gene location on chromosome ssa17 was identified in Ssal_v3.1, the 2021version of the salmon genome. In conclusion, larvae from several fish species are outfitted with mucus enriched by LL-proteins. Mucus cells are present in embryos of all fishes, but embryonic lectocyte-numbers are far higher in species with near total larval survival. When (maternal) chorionic first-line immuno-defence is lost at hatching, leukolectin-enriched mucus may provide vital protection for larvae.

Aquaporin expression and cholesterol content in eel swimbladder tissue.

Leakiness of the swimbladder wall of teleost fishes must be prevented to avoid diffusional loss of gases out of the swimbladder. Guanine incrustation as well as high concentrations of cholesterol in swimbladder membranes in midwater and deep-sea fish has been connected to a reduced gas permeability of the swimbladder wall. On the contrary, the swimbladder is filled by diffusion of gases, mainly oxygen and CO2 , from the blood and the gas gland cells into the swimbladder lumen. In swimbladder tissue of the zebrafish and the Japanese eel, aquaporin mRNA has been detected, and the aquaporin protein has been considered important for the diffusion of water, which may accidentally be gulped by physostome fish when taking an air breath. In the present study, the expression of two aquaporin 1 genes (Aqp1aa and Aqp1ab) in the swimbladder tissue of the European eel, a functional physoclist fish, was assessed using immunohistochemistry, and the expression of both genes was detected in endothelial cells of swimbladder capillaries as well as in basolateral membranes of gas gland cells. In addition, Aqp1ab was present in apical membranes of swimbladder gas gland cells. The authors also found high concentrations of cholesterol in these membranes, which were several fold higher than in muscle tissue membranes. In yellow eels the cholesterol concentration exceeded the concentration detected in silver eel swimbladder membranes. The authors suggest that aquaporin 1 in swimbladder gas gland cells and endothelial cells facilitates CO2 diffusion into the blood, enhancing the switch-on of the Root effect, which is essential for the secretion of oxygen into the swimbladder. It may also facilitate CO2 diffusion into the swimbladder lumen along the partial gradient established by CO2 production in gas gland cells. Cholesterol has been shown to reduce the gas permeability of membranes and thus could contribute to the gas tightness of swimbladder membranes, which is essential to avoid diffusional loss of gas out of the swimbladder.

Circadian miR-449c-5p regulates uterine Ca2+ transport during eggshell calcification in chickens.

BACKGROUND: miRNAs regulate circadian patterns by modulating the biological clocks of animals. In our previous study, we found that the clock gene exhibited a cosine expression pattern in the fallopian tube of chicken uterus. Clock-controlled miRNAs are present in mammals and Drosophila; however, whether there are clock-controlled miRNAs in the chicken uterus and, if so, how they regulate egg-laying rhythms is unclear. In this study, we selected 18 layer hens with similar ovipositional rhythmicity (each of three birds were sacrificed for study per 4 h throughout 24 h); their transcriptomes were scanned to identify the circadian miRNAs and to explore regulatory mechanisms within the uterus of chickens. RESULTS: We identified six circadian miRNAs that are mainly associated with several biological processes including ion trans-membrane transportation, response to calcium ion, and enrichment of calcium signaling pathways. Verification of the experimental results revealed that miR-449c-5p exhibited a cosine expression pattern in the chicken uterus. Ca2+-transporting ATPase 4 (ATP2B4) in the plasma membrane is the predicted target gene of circadian miR-449c-5p and is highly enriched in the calcium signaling pathway. We speculated that clock-controlled miR-449c-5p regulated Ca2+ transportation during eggshell calcification in the chicken uterus by targeting ATP2B4. ATP2B4 mRNA and protein were rhythmically expressed in the chicken uterus, and dual-luciferase reporter gene assays confirmed that ATP2B4 was directly targeted by miR-449c-5p. The expression of miR-449c-5p showed an opposite trend to that of ATP2B4 within a 24 h cycle in the chicken uterus; it inhibited mRNA and protein expression of ATP2B4 in the uterine tubular gland cells. In addition, overexpression of ATP2B4 significantly decreased intracellular Ca2+ concentration (P < 0.05), while knockdown of ATP2B4 accelerated intracellular Ca2+ concentrations. We found similar results after ATP2B4 knockdown by miR-449c-5p. Taken together, these results indicate that ATP2B4 promotes uterine Ca2+ trans-epithelial transport. CONCLUSIONS: Clock-controlled miR-449c-5p regulates Ca2+ transport in the chicken uterus by targeting ATP2B4 during eggshell calcification.

Involvement of Pattern Recognition Receptors in the Direct Influence of Bacterial Components and Standard Antiacne Compounds on Human Sebaceous Gland Cells.

INTRODUCTION: Pattern recognition receptors are involved in innate and adaptive immunity by detecting microbial components. Bacteria have been accused to play a role in inflammatory acne. We investigated the potential involvement of Toll-like receptor (TLR)2, TLR4, TLR6, and CD14 in the direct influence of bacterial components and standard antiacne compounds on human sebocytes. METHODS: mRNA and protein expression of TLR2, TLR4, TLR6, and CD14 in SZ95 sebocytes was evaluated by real-time qRT-PCR and immunocytochemistry. The effects of lipopolysaccharides (LPS) and lipoteichoic acid on TLR2, TLR4, and CD14 expression and of cytokine/chemokine secretion by 13-cis-retinoic acid, all-trans-retinoic acid, retinol, and hydrocortisone at the mRNA and protein levels were assessed by real-time qRT-PCR and ELISA and verified by cocultivation with neutralizing antibodies. RESULTS: The constitutive expression of TLR2, TLR4, and CD14 in SZ95 sebocytes was augmented by exposure to LPS. Hydrocortisone induced TLR2, but markedly reduced TLR4 expression. 13-cis-retinoic acid and all-trans-retinoic acid regulated IL-6 release. LPS enhanced and hydrocortisone reduced cytokine and chemokine release. Anti-TLR4 and anti-CD14 mAb blocked LPS-induced IL-8 and IL-6 release. CONCLUSIONS: Microbial components use pattern recognition receptors to directly activate sebocytes to express a wide range of proinflammatory molecules and especially IL-8 and IL-6 in a TLR4- and CD14-specific manner. Retinoids, but mostly corticosteroids, also use this pathway to exhibit anti-inflammatory effects.

A new esophageal gland transcriptome reveals signatures of large scale de novo effector birth in the root lesion nematode Pratylenchus penetrans.

BACKGROUND: The root lesion nematode Pratylenchus penetrans is a migratory plant-parasitic nematode responsible for economically important losses in a wide number of crops. Despite the importance of P. penetrans, the molecular mechanisms employed by this nematode to promote virulence remain largely unknown. RESULTS: Here we generated a new and comprehensive esophageal glands-specific transcriptome library for P. penetrans. In-depth analysis of this transcriptome enabled a robust identification of a catalogue of 30 new candidate effector genes, which were experimentally validated in the esophageal glands by in situ hybridization. We further validated the expression of a multifaceted network of candidate effectors during the interaction with different plants. To advance our understanding of the "effectorome" of P. penetrans, we adopted a phylogenetic approach and compared the expanded effector repertoire of P. penetrans to the genome/transcriptome of other nematode species with similar or contrasting parasitism strategies. Our data allowed us to infer plausible evolutionary histories that shaped the effector repertoire of P. penetrans, as well as other close and distant plant-parasitic nematodes. Two remarkable trends were apparent: 1) large scale effector birth in the Pratylenchidae in general and P. penetrans in particular, and 2) large scale effector death in sedentary (endo) plant-parasitic nematodes. CONCLUSIONS: Our study doubles the number of validated Pratylenchus penetrans effectors reported in the literature. The dramatic effector gene gain in P. penetrans could be related to the remarkable ability of this nematode to parasitize a large number of plants. Our data provide valuable insights into nematode parasitism and contribute towards basic understating of the adaptation of P. penetrans and other root lesion nematodes to specific host plants.

STATAWAARS: a promoter motif associated with spatial expression in the major effector-producing tissues of the plant-parasitic nematode Bursaphelenchus xylophilus.

BACKGROUND: Plant-parasitic nematodes cause severe damage to a wide range of crop and forest species worldwide. The migratory endoparasitic nematode, Bursaphelenchus xylophilus, (pinewood nematode) is a quarantine pathogen that infects pine trees and has a hugely detrimental economic impact on the forestry industry. Under certain environmental conditions large areas of infected trees can be destroyed, leading to damage on an ecological scale. The interactions of B. xylophilus with plants are mediated by secreted effector proteins produced in the pharyngeal gland cells. Identification of effectors is important to understand mechanisms of parasitism and to develop new control measures for the pathogens. RESULTS: Using an approach pioneered in cyst nematodes, we have analysed the promoter regions of a small panel of previously validated pharyngeal gland cell effectors from B. xylophilus to identify an associated putative regulatory promoter motif: STATAWAARS. The presence of STATAWAARS in the promoter region of an uncharacterized gene is a predictor that the corresponding gene encodes a putatively secreted protein, consistent with effector function. Furthermore, we are able to experimentally validate that a subset of STATAWAARS-containing genes are specifically expressed in the pharyngeal glands. Finally, we independently validate the association of STATAWAARS with tissue-specific expression by directly sequencing the mRNA of pharyngeal gland cells. We combine a series of criteria, including STATAWAARS predictions and abundance in the gland cell transcriptome, to generate a comprehensive effector repertoire for B. xylophilus. The genes highlighted by this approach include many previously described effectors and a series of novel "pioneer" effectors. CONCLUSIONS: We provide a major scientific advance in the area of effector regulation. We identify a novel promoter motif (STATAWAARS) associated with expression in the pharyngeal gland cells. Our data, coupled with those from previous studies, suggest that lineage-specific promoter motifs are a theme of effector regulation in the phylum Nematoda.

The hatching process and mechanisms of adaptive hatching acceleration in hourglass treefrogs, Dendropsophus ebraccatus.

Environmentally cued hatching is well documented in anurans, enabling embryos to escape diverse threats. However, knowledge of anuran hatching mechanisms is limited and based largely on aquatic-breeding species without known plasticity in hatching timing. Generally, hatching gland cells produce a hatching enzyme that degrades the vitelline membrane. We investigated hatching and its regulation in terrestrial embryos of hourglass treefrogs, Dendropsophus ebraccatus, which accelerate hatching to escape dehydration. We specifically tested if changes in hatching gland cell development or hatching enzyme gene expression are associated with accelerated hatching. We measured perivitelline chamber size of well-hydrated eggs over development as an indicator of breakdown of the vitelline membrane and found that the size of the perivitelline chamber increased steadily until hatching, suggesting gradual hatching enzyme release and vitelline membrane degradation. Hatching gland cells peaked in abundance and began regression substantially prior to hatching, but we found no developmental differences in the abundance or surface area of hatching gland cells between dry and well-hydrated embryos. Hatching enzyme gene expression also peaked early in development then declined, with no difference between hydration treatments. In D. ebraccatus breakdown of the vitelline membrane appears gradual, mediated by hatching enzyme release starting long before hatching. However, hatching acceleration is not associated with ontogenetic changes in hatching gland cell development or hatching enzyme gene expression. This hatching process contrasts with that of red-eyed treefrogs, Agalychnis callidryas, which appear to release enzyme acutely at hatching, yet both species are capable of hatching to escape acute threats.

The differentiation of amniotic fluid stem cells into sweat glandlike cells is enhanced by the presence of Sonic hedgehog in the conditioned medium.

After patients suffer severe full-thickness burn injuries, the current treatments cannot lead to the complete self-regeneration of the sweat gland structure and function. Therefore, it is important to identify new methods for acquiring sufficient functional sweat gland cells to restore skin function. In this study, we induced CD117+ human amniotic fluid stem (hAFS) cells to differentiate into sweat glandlike (hAFS-SG) cells based on the use of conditioned medium (CM) from the human sweat gland (hSG) cells. Real-time PCR and immunofluorescent staining were used to confirm the expression of the sweat gland-related genes Ectodysplasin-A (EDA), Ectodysplasin-A receptor (EDAR), keratin 8 (K8) and carcino-embryonic antigen (CEA). Transmission electron microscopy analysis shows that microvilli, the cellular structures that are typical for hSG cells, can also be observed on the membrane of the hAFS-SG cells. Our test for the calcium response to acetylcholine (Ach) proved that hAFS-SG cells have the potential to respond to Ach in a manner similar to normal sweat glands. A three-dimensional culture is an effective approach that stimulates the hAFS-SG cells to form tubular structures and drives hAFS-SG cells to mature into higher stage. We also found that epidermal growth factor enhances the efficiency of differentiation and that Sonic hedgehog is an important factor of the CM that influences sweat gland differentiation. Our study provides the basis for further investigations into novel methods of inducing stem cells to differentiate into sweat glandlike cells.

How embryos escape from danger: the mechanism of rapid, plastic hatching in red-eyed treefrogs.

Environmentally cued hatching allows embryos to escape dangers and exploit new opportunities. Such adaptive responses require a flexibly regulated hatching mechanism sufficiently fast to meet relevant challenges. Anurans show widespread, diverse cued hatching responses, but their described hatching mechanisms are slow, and regulation of timing is unknown. Arboreal embryos of red-eyed treefrogs, Agalychnis callidryas, escape from snake attacks and other threats by very rapid premature hatching. We used videography, manipulation of hatching embryos and electron microscopy to investigate their hatching mechanism. High-speed video revealed three stages of the hatching process: pre-rupture shaking and gaping, vitelline membrane rupture near the snout, and muscular thrashing to exit through the hole. Hatching took 6.5-49 s. We hypothesized membrane rupture to be enzymatic, with hatching enzyme released from the snout during shaking. To test this, we displaced hatching embryos to move their snout from its location during shaking. The membrane ruptured at the original snout position and embryos became trapped in collapsed capsules; they either moved repeatedly to relocate the hole or shook again and made a second hole to exit. Electron microscopy revealed that hatching glands are densely concentrated on the snout and absent elsewhere. They are full of vesicles in embryos and release most of their contents rapidly at hatching. Agalychnis callidryas' hatching mechanism contrasts with the slow process described in anurans to date and exemplifies one way in which embryos can achieve rapid, flexibly timed hatching to escape from acute threats. Other amphibians with cued hatching may also have novel hatching mechanisms.

Expression of ligands for Siglec-8 and Siglec-9 in human airways and airway cells.

BACKGROUND: Balanced activation and inhibition of the immune system ensures pathogen clearance while avoiding hyperinflammation. Siglecs, sialic acid-binding proteins found on subsets of immune cells, often inhibit inflammation: Siglec-8 on eosinophils and Siglec-9 on neutrophils engage sialoglycan ligands on airways to diminish ongoing inflammation. The identities of human siglec ligands and their expression during inflammation are largely unknown. OBJECTIVE: The histologic distribution, expression, and molecular characteristics of siglec ligands were explored in healthy and inflamed human upper airways and in a cellular model of airway inflammation. METHODS: Normal and chronically inflamed upper airway tissues were stained for siglec ligands. The ligands were extracted from normal and inflamed tissues and from human Calu-3 cells for quantitative analysis by means of siglec blotting and isolation by means of siglec capture. RESULTS: Siglec-8 ligands were expressed on a subpopulation of submucosal gland cells of human inferior turbinate, whereas Siglec-9 ligands were expressed more broadly (submucosal glands, epithelium, and connective tissue); both were significantly upregulated in patients with chronic rhinosinusitis. Human airway (Calu-3) cells expressed Siglec-9 ligands on mucin 5B (MUC5B) under inflammatory control through the nuclear factor κB pathway, and MUC5B carried sialoglycan ligands of Siglec-9 on human upper airway tissue. CONCLUSION: Inflammation results in upregulation of immune-inhibitory Siglec-8 and Siglec-9 sialoglycan ligands on human airways. Siglec-9 ligands are upregulated through the nuclear factor κB pathway, resulting in their enhanced expression on MUC5B. Siglec sialoglycan ligand expression in inflamed cells and tissues may contribute to the control of airway inflammation.

CEH-28 activates dbl-1 expression and TGF-ß signaling in the C. elegans M4 neuron.

M4 is a multifunctional neuron in the Caenorhabditis elegans pharynx that can both stimulate peristaltic contractions of the muscles in the pharyngeal isthmus and function systemically to regulate an enhanced sensory response under hypoxic conditions. Here we identify a third function for M4 that depends on activation of the TGF-ß family gene dbl-1 by the homeodomain transcription factor CEH-28. dbl-1 is expressed in M4 and a subset of other neurons, and we show CEH-28 specifically activates dbl-1 expression in M4. Characterization of the dbl-1 promoter indicates that CEH-28 targets an M4-specific enhancer within the dbl-1 promoter region, while expression in other neurons is mediated by separate regulatory sequences. Unlike ceh-28 mutants, dbl-1 mutants do not exhibit M4 synaptic and signaling defects. Instead, both ceh-28 and dbl-1 mutants exhibit morphological defects in the g1 gland cells located adjacent to M4 in the pharynx, and these defects can be partially rescued by M4-specific expression of dbl-1 in these mutants. Identical gland cell defects are observed in sma-6 and daf-4 mutants defective in the receptor for DBL-1, but they are not observed in sma-2 and sma-3 mutants lacking the R-Smads functioning downstream of this receptor. Together these results identify a novel neuroendocrine function for M4 and provide evidence for an R-Smad-independent mechanism for DBL-1 signaling in C. elegans.

Upregulation of ITGB6 in primary palmar hyperhidrosis.

BACKGROUND: The regulatory effect of integrin ß6 (ITGB6) on sweat gland cells in primary palmar hyperhidrosis (PPH) remains unclear. OBJECTIVES: This study investigated the involvement of ITGB6 in the pathogenesis of PPH. MATERIAL AND METHODS: Sweat gland tissues were collected from PPH patients and healthy volunteers. The expression levels of ITGB6 in sweat gland tissues were detected with quantitative polymerase chain reaction (qPCR), western blot and immunohistochemical staining. Sweat gland cells were extracted from PPH patients, and identified with immunofluorescence staining of CEA and CK7. The expression of aquaporin 5 (AQP5) and Na-K-Cl cotransporter 1 (NKCC1) in primary sweat gland cells that overexpress ITGB6 were also detected. Through a series of bioinformatic methods, differentially expressed genes in sweat gland tissues were examined and validated via comparing PPH samples and controls. The key proteins and biological functions enriched in PPH were determined using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses. RESULTS: The ITGB6 was upregulated in sweat gland tissues of PPH patients compared to that of healthy volunteers. The CEA and CK7 were positively expressed in sweat gland cells extracted from PPH patients. The overexpression of ITGB6 upregulated AQP5 and NKCC1 protein expression in the sweat gland cells of PPH patients. A total of 562 differentially expressed mRNAs were identified using high-throughput sequencing (394 upregulated, 168 downregulated), which were mainly active in the chemokine and Wnt signaling pathways. After verification with qPCR and western blot, the overexpression of ITGB6 significantly upregulated CXCL3, CXCL5, CXCL10, and CXCL11, and downregulated Wnt2 mRNA and protein expression in sweat gland cells. CONCLUSIONS: The ITGB6 is upregulated in PPH patients. It may be involved in the pathogenesis of PPH by upregulating AQP5, NKCC1, CXCL3, CXCL5, CXCL10, and CXCL11, and downregulating Wnt2 expression in sweat glands.

Gene expression profile during seed development of Bixa orellana accessions varying in bixin pigment.

Diverse morphological, cellular and physiological changes occur during seed maturation in Bixa orellana when the seed tissues form specialized cell glands that produce reddish latex with high bixin amounts. Transcriptomic profiling during seed development in three B. orellana accessions (P12, N4 and N5) with contrasting morphologic characteristics showed enrichment in pathways of triterpenes, sesquiterpenes, and cuticular wax biosynthesis. WGCNA allows groups of all identified genes in six modules the module turquoise, the largest and highly correlated with the bixin content. The high number of genes in this module suggests a diversification of regulatory mechanisms for bixin accumulation with the genes belonging to isoprene, triterpenes and carotene pathways, being more highly correlated with the bixin content. Analysis of key genes of the mevalonate (MVA) and the 2C-methyl-D-erythritol-4-phosphate (MEP) pathways revealed specific activities of orthologs of BoHMGR, BoFFP, BoDXS, and BoHDR. This suggests that isoprenoid production is necessary for compounds included in the reddish latex of developing seeds. The carotenoid-related genes BoPSY2, BoPDS1 and BoZDS displayed a high correlation with bixin production, consistent with the requirement for carotene precursors for apocarotenoid biosynthesis. The BoCCD gene member (BoCCD4-4) and some BoALDH (ALDH2B7.2 and ALDH3I1) and BoMET (BoSABATH1 and BoSABATH8) gene members were highly correlated to bixin in the final seed development stage. This suggested a contributing role for several genes in apocarotenoid production. The results revealed high genetic complexity in the biosynthesis of reddish latex and bixin in specialized seed cell glands in different accessions of B. orellana suggesting gene expression coordination between both metabolite biosynthesis processes.

Effect of Soybean Isoflavones on Proliferation and Related Gene Expression of Sow Mammary Gland Cells In Vitro.

The present study was conducted to investigate the effects of synthetic soybean isoflavones (ISO) on the proliferation and related gene expression of sow mammary gland cells. Cells were cultured with 0 (control), 10, 20, or 30 µM of ISO under incubation conditions. After a 48 h incubation, these ISO-incubated cells proliferated more (p < 0.05) than the control cells. Cyclin E expression was higher (p < 0.05) in the 10 µM ISO and 20 µM ISO treatment groups than in the control group. Cyclin D1 and p21 expressions decreased (p < 0.05) with the 10 µM ISO treatment for 48 h. The relative mRNA abundances of the cells' IG-1R (Insulin-like growth factor-1R), EGFR (Epidermal growth factor receptor), STAT3 (Signal transducer and activator of transcription 3) and AKT (protein kinase B) were enhanced (p < 0.05) by the 20 µM ISO treatment for 24 h and 48 h in the medium. The relative mRNA abundances of κ-casein at 48 h of incubation and ß-casein at 24 h and 48 h of incubation were increased (p < 0.05) by 10 µM of ISO supplementation. It was concluded that ISO improved the proliferation of sow mammary gland cells, possibly by regulating cyclins and function genes expression in the cell proliferation signaling pathway.

Isolation, culture, and identification of ceruminous gland cells.

External auditory canal (EAC) stenosis or atresia usually requires a skin graft to repair, but due to the lack of a graft containing functional glands, postoperative complications such as infection and eczema are common. The aim of this study was to isolate and characterize seed cells for the construction of tissue engineered EAC skin containing ceruminous gland by isolating and cultivating cells of ceruminous gland. In this study, EAC skin samples were harvested from adult goats for ceruminous gland cell isolation. Cell morphology and proliferation rates, expression of CK7, CK8, CK18, and CK19 (glandular cell specific-markers), and secretion of ß-defensin-1, lysozyme, and polysaccharides were evaluated at different passages to verify the presence of ceruminous gland cells and determine whether function and proliferation potential were maintained. Ceruminous glands were successfully isolated and extracted from goat EAC skin. Furthermore, the isolated glandular cells maintained robust proliferation potential, exhibited high expression of CK7, CK8, CK18, and CK19, and vigorously secreted ß-defensin-1, lysozyme, and polysaccharides in this culture system. However, expression of glandular cell specific-markers and secretory function gradually declined with increasing passage number, indicating dedifferentiation of the subcultured ceruminous gland cells after five passages. In conclusion, ceruminous glands were successfully isolated, cultured, and expanded from goat EAC skin using the serumcontaining culture system. Importantly, the isolated glandular cells retained robust proliferation potential and maintained their phenotype and function in early passages (P1-P3), indicating the method's potential application for ceruminous gland regeneration.

TNF-α suppresses sweat gland differentiation of MSCs by reducing FTO-mediated m6A-demethylation of Nanog mRNA.

An effect of inhibition of tumor necrosis factor-α (TNF-α) on differentiation of mesenchymal stromal cells (MSCs) has been demonstrated, but the exact mechanisms that govern MSCs differentiation remain to be further elucidated. Here, we show that TNF-α inhibits the differentiation of MSCs to sweat glands in a specific sweat gland-inducing environment, accompanied with reduced expression of Nanog, a core pluripotency factor. We elucidated that fat mass and obesity-associated protein (FTO)-mediated m6A demethylation is involved in the regulation of MSCs differentiation potential. Exposure of MSCs to TNF-α reduced expression of FTO, which demethylated Nanog mRNA. Reduced expression of FTO increased Nanog mRNA methylation, decreased Nanog mRNA and protein expression, and significantly inhibited MSCs capacity for differentiation to sweat gland cells. Our finding is the first to elucidate the functional importance of m6A modification in MSCs, providing new insights that the microenvironment can regulate the multipotency of MSCs at the post-transcriptional level. Moreover, to maintain differentiation capacity of MSCs by regulating m6A modification suggested a novel potential therapeutic target for stem cell-mediated regenerative medicine.

Three-dimensional Organization of Polytene Chromosomes in Somatic and Germline Tissues of Malaria Mosquitoes.

Spatial organization of chromosome territories and interactions between interphase chromosomes themselves, as well as with the nuclear periphery, play important roles in epigenetic regulation of the genome function. However, the interplay between inter-chromosomal contacts and chromosome-nuclear envelope attachments in an organism's development is not well-understood. To address this question, we conducted microscopic analyses of the three-dimensional chromosome organization in malaria mosquitoes. We employed multi-colored oligonucleotide painting probes, spaced 1 Mb apart along the euchromatin, to quantitatively study chromosome territories in larval salivary gland cells and adult ovarian nurse cells of Anopheles gambiae, An. coluzzii, and An. merus. We found that the X chromosome territory has a significantly smaller volume and is more compact than the autosomal arm territories. The number of inter-chromosomal, and the percentage of the chromosome-nuclear envelope, contacts were conserved among the species within the same cell type. However, the percentage of chromosome regions located at the nuclear periphery was typically higher, while the number of inter-chromosomal contacts was lower, in salivary gland cells than in ovarian nurse cells. The inverse correlation was considerably stronger for the autosomes. Consistent with previous theoretical arguments, our data indicate that, at the genome-wide level, there is an inverse relationship between chromosome-nuclear envelope attachments and chromosome-chromosome interactions, which is a key feature of the cell type-specific nuclear architecture.

In vitro transdifferentiation of adipose tissue-derived stem cells into salivary gland acinar-like cells.

Current clinical approaches to treat irradiation-induced salivary gland hypofunction are ineffective. We previously reported that adipose-derived stem cell (ADSC)-based therapy ameliorates damaged salivary gland function in mice and that the effects were enhanced when the therapy was co-administrated with platelet-rich fibrin (PRF). We examined the feasibility of ADSC transdifferentiation into salivary gland acinar-like cells (SGALCs) and analyzed the potential of PRF to promote the transdifferentiation process in vitro. Salivary gland cells (SGCs) and ADSCs were indirectly co-cultured using Transwell inserts, and increasing concentrations of PRF-conditioned medium were applied to the co-culture system. The expression of α-amylase and AQP-5 were used to evaluate ADSC transdifferentiation. Notably, on day 7, 14, and 21, expression of both α-amylase and AQP-5 were detected in the co-cultured ADSCs. Additionally, PRF increased α-amylase and AQP-5 levels in ADSCs that were co-cultured for 7 days. These data demonstrate that ADSCs have the potential to transdifferentiate into SGALCs and that PRF can promote the transdifferentiation process. Therefore, these data reveal a possible mechanism to treat irradiation-induced salivary gland hypofunction and have translational medicine implications.

Targeting ectodysplasin promotor by CRISPR/dCas9-effector effectively induces the reprogramming of human bone marrow-derived mesenchymal stem cells into sweat gland-like cells.

BACKGROUND: Patients with a deep burn injury are characterized by losing the function of perspiration and being unable to regenerate the sweat glands. Because of their easy accession, multipotency, and lower immunogenicity, bone marrow-derived mesenchymal stem cells (BM-MSCs) represent as an ideal biological source for cell therapy. The aim of this study was to identify whether targeting the promotor of ectodysplasin (EDA) by CRISPR/dCas9-effector (dCas9-E) could induce the BM-MSCs to differentiate into sweat gland-like cells (SGCs). METHODS: Activation of EDA transcription in BM-MSCs was attained by transfection of naive BM-MSCs with the lenti-CRISPR/dCas9-effector and single-guide RNAs (sgRNAs). The impact of dCas9-E BM-MSCs on the formation of SGCs and repair of burn injury was identified and evaluated both in vitro and in a mouse model. RESULTS: After transfection with sgRNA-guided dCas9-E, the BM-MSCs acquired significantly higher transcription and expression of EDA by doxycycline (Dox) induction. Intriguingly, the specific markers (CEA, CK7, CK14, and CK19) of sweat glands were also positive in the transfected BM-MSCs, suggesting that EDA plays a critical role in promoting BM-MSC differentiation into sweat glands. Furthermore, when the dCas9-E BM-MSCs with Dox induction were implanted into a wound in a laboratory animal model, iodine-starch perspiration tests revealed that the treated paws were positive for perspiration, while the paws treated with saline showed a negative manifestation. For the regulatory mechanism, the expression of downstream genes of NF-κB (Shh and cyclin D1) was also enhanced accordingly. CONCLUSIONS: These results suggest that EDA is a pivotal factor for sweat gland regeneration from BM-MSCs and may also offer a new approach for destroyed sweat glands and extensive deep burns.