[MUC4 Silencing Inhibits TGF-ß1-induced Epithelial-mesenchymal Transition VIA the ERK1/2 Pathway in Human Airway Epithelial NCI-H292 Cells].

MUC4 is a predominant membrane-tethered mucin lubricating and protecting the epithelial surface and playing various biological roles in the renewal and differentiation of epithelial cells, cell signaling, cell adhesion, and carcinogenesis. Interestingly, recent studies have demonstrated that MUC4 expression regulates the epithelial-mesenchymal transition (EMT) of cancer cells in ovarian, pancreatic, and lung cancer. However, the effects of MUC4 expression on EMT in human airway epithelial cells are not yet well known. Here, we describe the effects of transforming growth factor beta 1 (TGF-ß1)-induced MUC4 expression on EMT and evaluate its downstream signaling pathway in human airway epithelial cells. In human airway epithelial NCI-H292 cells, exposure to TGF-ß1 induced expression of MUC4, CDH2, VIM and SNAI1 genes and encoded by them proteins, MUC4, N-cadherin, vimentin and Snail, and reduced the level of CDH1 and its product, E-cadherin. In MUC4-knockdown cells, TGF-ß1-induced expression levels of MUC4, CDH2, VIM and SNAI1 and corresponding proteins were suppressed, but CDH1 and E-cadherin levels were not. In addition, TGF-ß1-induced phosphorylation of extracellular signal regulated kinase 1/2 (ERK1/2) was suppressed, but that of Smad2/3, Akt, and p38 was not. The results of this study suggest that MUC4 silencing inhibits TGF-ß1 -induced EMT via the ERK1/2 pathway, and a possible role of MUC4 in the induction of EMT in human airway epithelial cells.

Immunogenicity and safety of a quadrivalent meningococcal tetanus toxoid-conjugate vaccine administered concomitantly with other paediatric vaccines in toddlers: a phase III randomised study.

Invasive meningococcal disease has high morbidity and mortality, with infants and young children among those at greatest risk. This phase III, open-label, randomised study in toddlers aged 12-23 months evaluated the immunogenicity and safety of meningococcal tetanus toxoid-conjugate vaccine (MenACYW-TT), a tetanus toxoid conjugated vaccine against meningococcal serogroups A, C, W and Y, when coadministered with paediatric vaccines (measles, mumps and rubella [MMR]; varicella [V]; 6-in-1 combination vaccine against diphtheria, tetanus, pertussis, polio, hepatitis B and Haemophilus influenzae type b [DTaP-IPV-HepB-Hib] and pneumococcal conjugate vaccine [PCV13])(NCT03205371). Immunogenicity to each meningococcal serogroup was assessed by serum bactericidal antibody assay using human complement (hSBA). Vaccine safety profiles were described up to 30 days post-vaccination. A total of 1183 participants were enrolled. The proportion with seroprotection (hSBA ≥1:8) to each meningococcal serogroup at Day 30 was comparable between the MenACYW-TT and MenACYW-TT + MMR + V groups (≥92 and ≥96%, respectively), between the MenACYW-TT and MenACYW-TT + DTaP-IPV-HepB-Hib groups (≥90% for both) and between the MenACYW-TT and MenACYW-TT + PCV13 groups (≥91 and ≥84%, respectively). The safety profiles of MenACYW-TT, and MMR + V, DTaP-IPV-HepB-Hib, and PCV13, with or without MenACYW-TT, were generally comparable. Coadministration of MenACYW-TT with paediatric vaccines in toddlers had no clinically relevant effect on the immunogenicity and safety of any of the vaccines.

Fipronil upregulates inflammatory cytokines and MUC5AC expression in human nasal epithelial cells.

BACKGROUND: Airway inflammation and excessive mucin production are pathophysiological characteristics of airway diseases. Fipronil, a pesticide, is being extensively used in agriculture and veterinary medicine worldwide. However, this compound impairs immune function in non-target organisms. The present study aimed to evaluate the effect of fipronil on pro-inflammatory cytokine and mucus production and signalling pathways in human primary nasal METHODOLOGY: The effect of fipronil on pro-inflammatory cytokine and MUC5AC expression and the signalling pathway of fipronil were investigated using real-time PCR, enzyme immunoassays, immunofluorescence, and immunoblot analysis with specific inhibitors and small interfering RNA. RESULTS: Fipronil treatment increased pro-inflammatory cytokine interleukin (IL)-1beta, IL-6, IL-8, and MUC5AC expression in human primary nasal epithelial cells. It also induced phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) mitogenactivated protein kinase (MAPK), p38 MAPK, and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB). MAPK and NF-kB inhibitor treatment significantly inhibited increases in IL-1beta, IL-6, IL-8, and MUC5AC expression. Ex vivo data confirmed that fipronil-induced MUC5AC expression occurs through ERK1/2, p38, and NF-kB signalling pathways in nasal inferior turbinate tissue. CONCLUSIONS: Fipronil induced pro-inflammatory cytokine IL-1beta, IL-6, IL-8, and MUC5AC expression via ERK1/2 MAPK, p38 MAPK, and NF-kB in human primary nasal epithelial cells.

Antimicrobial activities of an oxygenated cyclohexanone derivative isolated from Amphirosellinia nigrospora JS-1675 against various plant pathogenic bacteria and fungi.

AIMS: To evaluate the antimicrobial activities of an active compound isolated from the culture broth of Amphirosellinia nigrospora JS-1675 against various plant pathogenic bacteria and fungi. METHODS AND RESULTS: While screening for bioactive secondary metabolites from endophytic fungi, we found that A. nigrospora JS-1675 showed strong in vitro antibacterial activity against Ralstonia solanacearum. One compound (1) was isolated and identified as (4S, 5S, 6S)-5,6-epoxy-4-hydroxy-3-methoxy-5-methyl-cyclohex-2-en-1-one. Growth of most of the tested phytopathogenic bacteria was inhibited by compound 1 and the ethyl acetate (EtOAc) layer except Pseudomonas syringae pv. lachrymans. Compound 1 also inhibited the mycelial growth of several plant pathogenic fungi. Both compound 1 and the EtOAc layer reduced bacterial leaf spot disease in detached peach leaves. They also suppressed the development of bacterial wilt on tomato seedlings quite effectively. CONCLUSIONS: Amphirosellinia nigrospora JS-1675 showed antimicrobial activity against plant pathogenic bacteria and fungi by producing compound 1. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report on the occurrence of compound 1 in A. nigrospora JS-1675 and its efficacy against plant pathogenic bacteria and fungi. Their strong disease control efficacy against tomato bacterial wilt suggests that this fungus can be used as a microbial bactericide.

Wnt activity is associated with cementum-type transition.

BACKGROUND AND OBJECTIVE: Cellular and acellular cementum and the cells that form them are postulated to have different characteristics, and the relationship between these two tissues is not well understood. Based on the hypothesis that Wnt signaling is involved in the determination of cementum type, we examined Wnt activity along the tooth root and analyzed cementum formation in genetic mutant models. MATERIAL AND METHODS: We generated mutant models with Wnt signaling upregulation (OC Catnblox(ex3)/+ ), downregulation (OC Wlsfl/fl ), and a compound mutant (Enpp1asj/asj ;OC Catnblox(ex3)/+ ) to compare cementum apposition patterns of ectonucleotide diphosphatase/phosphodiesterase (Enpp1) mutant (Enpp1asj/asj ). The analysis of structural morphology and histology was performed with hematoxylin and eosin and immunohistochemical staining and scanning electron microscopy. RESULTS: The cementum type of upper apical region of tooth roots in the molar is altered from the cellular form at the initial stage to the acellular form at the late stage of cementum formation. However, the basal part of this apical region is not altered and retains cellular cementum characters with strong Wnt activity. In the genetic mutant models for Wnt upregulation, cellular cementum is formed at the cervical region instead of acellular cementum. However, Enpp1 mutant mice have clearly different characteristics with cellular-type cementum even with dramatically increased cervical cementum matrix. In addition, we found that acellular-type formation could be altered into cellular-type formation by analyzing Wnt upregulation and compound mutant models. CONCLUSIONS: Cementum type is not determined by its specific location and could be transformed with Wnt activity during cementum formation.

Antibacterial activities of penicillic acid isolated from Aspergillus persii against various plant pathogenic bacteria.

UNLABELLED: The emergence of pathogenic bacterial strains resistant to agrochemicals and the increasing demand for organic foods have led to the discovery of new antibacterial metabolites that can be used either directly or as a lead molecule for development of synthetic bactericides. During the screening of antibacterial fungal cultures, we found that one fungal strain, Aspergillus persii EML-HPB1-11, showed strong in vitro antibacterial activity against Xanthomonas arboricola pv. pruni (Xap) with a minimum inhibitory concentration (MIC) of 10% of fermentation broth filtrate. The active compound was identified as penicillic acid (PA: 3-methoxy-5-methyl-4-oxo-2,5-hexadienoic acid) by mass and NMR spectroscopy. The in vitro antibacterial activity of PA was tested against 12 phytopathogenic bacteria. All of the bacterial pathogens tested were highly inhibited by PA with MIC values of 12·3-111·1 µg ml(-1) . It also effectively suppressed the development of bacterial spot disease in detached peach leaves, showing control values of 82·4 and 94·1% at concentrations of 111·1 and 333·3 µg ml(-1) respectively. This is the first report on the production of PA by A. persii. This study suggests that PA can be used as a lead molecule for development of synthetic bactericides for control of various plant diseases. SIGNIFICANCE AND IMPACT OF THE STUDY: Penicillic acid (PA) produced by the seed-borne fungus Aspergillus persii EML-HPB1-11 showed antibacterial activity against various plant pathogenic bacteria. The compound effectively inhibited the growth of 12 plant pathogenic bacteria and successfully controlled bacterial spot disease on peach leaf. These results suggest that PA can be used as a lead molecule for development of synthetic agrochemicals to control plant bacterial diseases.

Disruption of Tgfbr2 in odontoblasts leads to aberrant pulp calcification.

Transforming growth factor ß (TGF-ß) signaling has been implicated in dentin formation and repair; however, the molecular mechanisms underlying dentin formation remain unclear. To address the role of TGF-ß signaling in dentin formation, we analyzed odontoblast-specific Tgfbr2 conditional knockout mice. The mutant mice had aberrant teeth with thin dysplastic dentin and pulpal obliteration, similar to teeth from human patients with dentinogenesis imperfecta type II and dentin dysplasia. In mutant, the odontoblasts lost their cellular polarity, and matrix secretion was disrupted after mantle dentin formation. As a consequence, the amount of predentin decreased significantly, and an ectopic fibrous matrix was formed below the odontoblast layer. This matrix gradually calcified and obliterated the pulp chamber with increasing age. Immunohistochemistry revealed decreased expression of alkaline phosphatase in mutant odontoblasts. In mutant dentin, Dsp expression was reduced, but Dmp1 expression increased significantly. Collagen type I, biglycan, and Dsp were expressed in the ectopic matrix. These results suggest that loss of responsiveness to TGF-ß in odontoblasts results in impaired matrix formation and pulpal obliteration. Our study indicates that TGF-ß signaling plays an important role in dentin formation and pulp protection. Furthermore, our findings may provide new insight into possible mechanisms underlying human hereditary dentin disorders and reparative dentin formation.

Osterix regulates tooth root formation in a site-specific manner.

Bone and dentin share similar biochemical compositions and physiological properties. Dentin, a major tooth component, is formed by odontoblasts; in contrast, bone is produced by osteoblasts. Osterix (Osx), a zinc finger-containing transcription factor, has been identified as an essential regulator of osteoblast differentiation and bone formation. However, it has been difficult to establish whether Osx functions in odontoblast differentiation and dentin formation. To understand the role of Osx in dentin formation, we analyzed mice in which Osx was subjected to tissue-specific ablation under the control of either the Col1a1 or the OC promoter. Two independent Osx conditional knockout mice exhibited similar molar abnormalities. Although no phenotype was found in the crowns of these teeth, both mutant lines exhibited short molar roots due to impaired root elongation. Furthermore, the interradicular dentin in these mice showed severe hypoplastic features, which were likely caused by disruptions in odontoblast differentiation and dentin formation. These phenotypes were closely related to the temporospatial expression pattern of Osx during tooth development. These findings indicate that Osx is required for root formation by regulating odontoblast differentiation, maturation, and root elongation. Cumulatively, our data strongly indicate that Osx is a site-specific regulator in tooth root formation.

Wntless regulates dentin apposition and root elongation in the mandibular molar.

Wnt signaling plays an essential role in the dental epithelium and mesenchyme during tooth morphogenesis. However, it remains unclear if Wnt ligands, produced from dental mesenchyme, are necessary for odontoblast differentiation and dentin formation. Here, we show that odontoblast-specific disruption of Wntless (Wls), a chaperon protein that regulates Wnt sorting and secretion, leads to severe defects in dentin formation and root elongation. Dentin thickness decreased remarkably and pulp chambers enlarged in the mandibular molars of OC-Cre;Wls(CO/CO) mice. Although the initial odontoblast differentiation was normal in the mutant crown, odontoblasts became cuboidal and dentin thickness was reduced. In immunohistochemistry, Wnt10a, ß-catenin, type I collagen, and dentin sialoprotein were significantly down-regulated in the odontoblasts of mutant crown. In addition, roots were short and root canals were widened. Cell proliferation was reduced in the developing root apex of mutant molars. Furthermore, Wnt10a and Axin2 expression was remarkably decreased in the odontoblasts of mutant roots. Deletion of the Wls gene in odontoblasts appears to reduce canonical Wnt activity, leading to inhibition of odontoblast maturation and root elongation.

Development of an encapsulation-vitrification protocol for Rubia akane (nakai) hairy roots: a comparison with non-encapsulation.

BACKGROUND: A comparison of different cryopreservation techniques should be based on the characteristics of both the methodology and the material in question using an optimized procedure. OBJECTIVE: This study aimed at developing an encapsulation-vitrification procedure for hairy roots of Rubia akane using alternative loading and vitrification solutions, based on the existing optimized droplet-vitrification procedure. MATERIALS AND METHODS: Encapsulated roots were first precultured in liquid medium with 10% sucrose for 3 days, then with 17.5 % sucrose for 1 day, after which they were osmoprotected with solution C6-40 % (20 % glycerol + 20 % sucrose) for 50 min, cryoprotected with solution A3-90 % (37.5 % glycerol + 15 % DMSO + 15 % EG + 22.5 % sucrose, w/v) on ice for 40 min, cooled and warmed in 2 ml cryovials, and unloaded in 35% sucrose solution for 60 min. RESULTS: Through the application of this procedure to aged-clustered roots, up to 97.5 % post-cryopreservation regeneration was observed. In our previous study, droplet-vitrification of hairy roots of R. akane resulted in 83.8 % post-rewarming regeneration following preculture with 10 % sucrose for 2 days and 17.5 % sucrose for 4-5 h, and osmoprotection with solution C4-35 % (17.5 % glycerol + 17.5 % sucrose) for 30 min, and cryoprotection with solution A3-70 % (29.2 % glycerol + 11.7 % DMSO + 11.7% EG + 17.4% sucrose, w/v) on ice for 20 min. In the present study, higher post-cryopreservation regeneration was observed by using a higher concentration of vitrification solution (A3-70 % → A3-90 %, B5-80 % → B1-100 %) and/or a longer cryoprotection duration (A3-70 % at room temperature (RT) for 8 min → 15-30 min, on ice for 20 min → 40-80 min; B5-80 % for 15 min → 30-60 min). CONCLUSION: Even though encapsulation provided some degree of protection from the cytotoxicity of vitrification solutions to cytotoxicity-sensitive R. akane hairy roots, an overall higher post-cryopreservation regrowth was obtained using the droplet-vitrification procedure under optimized conditions. This result implies that this sensitive material was not sufficiently cryoprotected, and thus, rapid cooling and warming using foil strips was more efficient than cryopreservation of encapsulated samples.

Carotid subclinical atherosclerosis is associated with disease activity but not vitamin D in Korean systemic lupus erythematosus.

Atherosclerosis develops early in systemic lupus erythematosus (SLE) patients and is an important cause of mortality. Vitamin D deficiency is found to be associated with cardiovascular disease and autoimmunity. We evaluated the extent of carotid subclinical atherosclerosis and analyzed its correlation with vitamin D in SLE. One hundred and two female patients with SLE and 52 normal controls (NCs) were recruited. The mean carotid intima-media thickness (IMT) of SLE patients was 0.41 ± 0.08 mm, which was higher than that of NCs (0.32 ± 0.08 mm, p = 0.012). In addition, carotid plaques were more frequent and the plaque index was higher in SLE patients than in NCs (0.68 ± 1.39 vs. 0.26 ± 0.87, p = 0.026). Carotid IMT was correlated with age, body mass index, SLE disease activity index, and aspirin use in SLE patients. The plaque index was correlated with renal involvement. Vitamin 25(OH)D3 level was not correlated with carotid IMT, plaque index or disease activity markers. In SLE, the risk of cardiovascular disease is higher than that in NCs, which may be derived from systemic inflammation. It may be not suitable to assess vitamin D as a marker of disease activity or subclinical atherosclerosis in SLE patients.

The benefits of hypothermic machine perfusion are enhanced with Vasosol and α-tocopherol in rodent donation after cardiac death livers.

The use of hypothermic machine perfusion (HMP) has recently been used to show an improvement in both standard and extended criteria donor liver grafts but creating a more dynamic preservation environment that can be supplemented with a variety of additives to aid in cold temperature metabolism and vasodilatation. Increasing the benefits of HMP, we explore the use of α-tocopherol in reducing inflammatory markers and apoptotic pathways to reduce the incidence of preservation injury. We explored the use of a donation after cardiac death (DCD) rodent model to test the additive benefits of α-tocopherol in HMP. The addition of α-tocopherol reduced the level of alanine aminotransferase (ALT) over the course of reperfusion as well, reduced the levels of inflammatory cytokines within a 90 minute reperfusion biopsy. Further benefit was seen with α-tocopherol through the reduction of the level of caspase 3/7 in the circulation, shown to be a result of the reduction of the levels of Cytochrome C mRNA. Liver perfusion with Vasosol® and HMP could benefit further from the addition of α-tocopherol to existing formulations of Vasosol®.

ß-catenin is required in odontoblasts for tooth root formation.

The tooth root is an important part of the tooth that works together with the surrounding periodontium to maintain the tooth in the alveolar socket. The root develops after crown morphogenesis. While the molecular and cellular mechanisms of early tooth development and crown morphogenesis have been extensively studied, little is known about the molecular mechanisms controlling tooth root formation. Here, we show that ß-catenin is strongly expressed in odontoblast-lineage cells and is required for root formation. Tissue-specific inactivation of ß-catenin in developing odontoblasts produced molars lacking roots and aberrantly thin incisors. At the beginning of root formation in the mutant molars, the cervical loop epithelium extended apically to form Hertwig's epithelial root sheath (HERS), but root odontoblast differentiation was disrupted and followed by the loss of some HERS inner layer cells. However, the outer layer of the HERS extended without the root, and the mutant molars finally erupted. The periodontal tissues extensively invaded the dental pulp. These results indicate that there is a cell-autonomous requirement for Wnt/ß-catenin signaling in the dental mesenchyme for root formation.

Excessive Wnt/ß-catenin signaling disturbs tooth-root formation.

BACKGROUND AND OBJECTIVE: Wingless-type MMTV integration site family (Wnt)/ß-catenin signaling plays an essential role in cellular differentiation and matrix formation during skeletal development. However, little is known about its role in tooth-root formation. In a previous study, we found excessive formation of dentin and cementum in mice with constitutive ß-catenin stabilization in the dental mesenchyme. In the present study we analyzed the molar roots of these mice to investigate the role of Wnt/ß-catenin signaling in root formation in more detail. MATERIAL AND METHODS: We generated OC-Cre:Catnb(+/lox(ex3)) mice by intercrossing Catnb(+/lox(ex3)) and OC-Cre mice, and we analyzed their mandibular molars using radiography, histomorphometry and immunohistochemistry. RESULTS: OC-Cre:Catnb(+/lox(ex3)) mice showed impaired root formation. At the beginning of root formation in mutant molars, dental papilla cells did not show normal differentiation into odontoblasts; rather, they were prematurely differentiated and had a disorganized arrangement. Interestingly, SMAD family member 4 was upregulated in premature odontoblasts. In 4-wk-old mutant mice, molar roots were about half the length of those in their wild-type littermates. In contrast to excessively formed dentin in crown, root dentin was thin and hypomineralized in mutant mice. Biglycan and dentin sialophosphoprotein were downregulated in root dentin of mutant mice, whereas dentin matrix protein 1 and Dickkopf-related protein 1 were upregulated. Additionally, ectonucleotide pyrophosphatase/phosphodiesterase 1 was significantly downregulated in the cementoblasts of mutant molars. Finally, in the cementum of mutant mice, bone sialoprotein was downregulated but Dickkopf-related protein 2 was upregulated. CONCLUSION: These results suggest that temporospatial regulation of Wnt/ß-catenin signaling plays an important role in cell differentiation and matrix formation during root and cementum formation.

Development of a droplet-vitrification protocol for cryopreservation of Rubia akane (Nakai) hairy roots using a systematic approach.

A systematic approach using a set of 13 treatments was applied to develop a droplet-vitrification protocol for Rubia akane hairy roots, based on their responses to preculture, loading, dehydration and cooling/rewarming steps. The roots were very sensitive to osmotic stress induced by both preculture in liquid sucrose-enriched medium (up to 0.5 M sucrose) and by dehydration with highly concentrated vitrification solutions (VSs). Loading was necessary before dehydration of explants with VS, and the composition of the loading solution (LS) significantly affected their post-cryopreservation regeneration. Due to high sensitivity of roots to both chemical cytotoxicity and osmotic stress produced by VSs, cryoprotection with alternative VSs, i.e. B5-80 percent (40 percent glycerol + 40 percent sucrose, w/v) at room temperature for 15 min or with A3-70 percent (29.2 percent glycerol + 11.7 percent DMSO + 11.7 percent EG + 17.4 percent sucrose, w/v) at 0 degree C for 20 min ensured the highest post-cryopreservation regeneration. However, when using these solutions, endothermic peaks (enthalpies) with -2.9 and -5.8 J per gram fresh weight, respectively, were recorded by differential scanning calorimetry (DSC) during the rewarming phase. Droplet-vitrification using foil strips showed higher post-cryopreservation regeneration (86 percent) compared with vitrification in cryovials (59 percent), possibly due to the higher cooling and rewarming rates achieved with droplet-vitrification. The developed protocol was applied to hairy roots of five other species with minor modifications in explant type, the duration of the last subculture before explant excision, and the dehydration duration with VS B5-80 percent.

Hypothermic machine preservation reduces molecular markers of ischemia/reperfusion injury in human liver transplantation.

Hypothermic machine perfusion (HMP) is in its infancy in clinical liver transplantation. Potential benefits include diminished preservation injury (PI) and improved graft function. Molecular data to date has been limited to extrapolation of animal studies. We analyzed liver tissue and serum collected during our Phase 1 trial of liver HMP. Grafts preserved with HMP were compared to static cold stored (SCS) transplant controls. Reverse transcription polymerase chain reaction (RT-PCR), immunohistochemistry and transmission electron microscopy (TEM) were performed on liver biopsies. Expression of inflammatory cytokines, adhesion molecules and chemokines, oxidation markers, apoptosis and acute phase proteins and the levels of CD68 positive macrophages in tissue sections were evaluated. RT-PCR of reperfusion biopsy samples in the SCS group showed high expression of inflammatory cytokines, adhesion molecules and chemokines, oxidative markers and acute phase proteins. This upregulation was significantly attenuated in livers that were preserved by HMP. Immunofluorescence showed larger numbers of CD68 positive macrophages in the SCS group when compared to the HMP group. TEM samples also revealed ultrastructural damage in the SCS group that was not seen in the HMP group. HMP significantly reduced proinflammatory cytokine expression, relieving the downstream activation of adhesion molecules and migration of leukocytes, including neutrophils and macrophages when compared to SCS controls.