BACKGROUND

Quercetin exerts antiproliferative effects on gastric cancer. However, its mechanisms of action on gastric cancer have not been comprehensively revealed.

OBJECTIVE

We investigated the mechanisms of action of quercetin against gastric cancer cells.

METHODS

Human NCI-N87 gastric cancer cells were treated with 15 μM quercetin or dimethyl sulfoxide (as a control) for 48 h. DNA isolated from cells was sequenced on a HiSeq 2500, and the data were used to identify differentially expressed genes (DEGs) between groups. Then, enrichment analyses were performed for DEGs and a protein-protein interaction (PPI) network was constructed. Finally, the transcription factors (TFs)-DEGs regulatory network was visualized by Cytoscape software.

RESULTS

A total of <em>12</em>1 DEGs were identified in the quercetin group. In the PPI network, Fos proto-oncogene (FOS, degree = <em>12</em>), aryl hydrocarbon receptor (AHR, degree = <em>12</em>), Jun proto-oncogene (JUN, degree = 11), and cytochrome P450 family 1 subfamily A member 1 (CYP1A1, degree = 11) with higher degrees highly interconnected with other proteins. Of the 5 TF-DEGs, early growth response 1 (EGR1), FOS like 1 (FOSL1), FOS, and JUN were upregulated, while AHR was downregulated. Moreover, FOSL1, JUN, and <em>Wnt</em> family member 7B (WNT7B) were enriched in the <em>Wnt</em> signaling pathway.

CONCLUSIONS

CYP1A1 highly interconnected with AHR in the PPI network. Therefore, FOS, AHR, JUN, CYP1A1, EGR1, FOSL1, and WNT7B might be targets of quercetin in gastric cancer.

Long noncoding RNAs (lncRNAs) have been reported to be involved in several neurological pathogenesis conditions including cerebral ischemia. In the current study, the functions of lncRNA EFNA3 on hypoxia-injured rat adrenal pheochromocytoma (PC-<em>12</em>) cells and the underlying molecular mechanism were studied. The expression of lncRNA EFNA3 was silenced by short hairpin RNA transfection, after which the cells were subjected with hypoxia. Cell viability, migration, invasion, and apoptosis were, respectively, determined by trypan blue staining, Transwell assay, annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) double-staining, and Western blot analysis. The cross regulation between lncRNA EFNA3 and miR-101a, as well as between miR-101a and Rho associated coiled-coil containing protein kinase 2 (ROCK2) were detected by performing quantitative real-time polymerase chain reaction, RNA pull-down, RNA immunoprecipitation, luciferase activity assay, and Western blot analysis. Studies showed that lncRNA EFNA3 was highly expressed in response to hypoxia. Deletion of lncRNA EFNA3 significantly aggravated hypoxia-induced injury in PC-<em>12</em> cells, as the impairment of cell viability, migration, and invasion, and the inducement of apoptosis. LncRNA EFNA3 worked as a sponging molecule for miR-101a and miR-101a suppression-protected PC-<em>12</em> cells against hypoxia-induced injury even when lncRNA EFNA3 was silenced. ROCK2 was a target gene of miR-101a. ROCK2 overexpression exhibited neuroprotective activities. Besides, ROCK2 overexpression activated <em>Wnt</em>/β-catenin pathway whereas it deactivated JAK/STAT pathway upon hypoxia. Our study suggests that deletion of lncRNA EFNA3 aggravates hypoxia-induced injury in PC-<em>12</em> cells by upregulating miR-101a, which further targets ROCK2.

Mice with spontaneous coat mutations are ideal animal models for studying skin development and tumorigenesis. In this study, skin hair growth cycle abnormalities were examined in NIH hairless mice 42 days after birth (P42) by using hematoxylin-eosin (H&E) staining. To examine the gene expression patterns in the skin of mutant mice, the dorsal skin of P42 female NIH mice and NIH hairless mice was sequenced by RNA-Seq, and 5,068 differentially expressed genes (DEGs) were identified (false discovery rate [FDR] ≥ 2, P < 0.05). A pathway analysis showed that basal cell carcinoma, the cell cycle and the Hippo, Hedgehog and <em>Wnt</em> signaling pathways were up-regulated in NIH hairless mice. Previous studies have shown that these pathways are closely associated with cell proliferation, cell cycle, organ size and cancer development. In contrast, signal transduction, bacterial and parasitic infection, and receptor-mediated pathways, including calcium signaling, were down-regulated in NIH hairless mice. A gene interaction network analysis was performed to identify genes related to hair follicle development. To verify the reliability of the RNA-Seq results, we used q-PCR to analyze <em>12</em> key genes identified from the gene interaction network analysis, including eight down-regulated and four up-regulated genes, and the results confirmed the reliability of the RNA-Seq results. Finally, we constructed the differential gene expression profiles of mutant mice by RNA-Seq. NIH hairless mice exhibited abnormalities in hair development and immune-related pathways. Pik3r1 and Pik3r3 were identified as key genes, laying the foundation for additional in-depth studies of hairless mice.

Obtaining a comprehensive understanding of the gene regulatory networks, or gene cascades, involved in cell fate determination and cell lineage segregation in Caenorhabditis elegans is a long-standing challenge. Although RNA-sequencing (RNA-Seq) is a promising technique to resolve these questions, the bioinformatics tools to identify associated gene cascades from RNA-Seq data remain inadequate. To overcome these limitations, we developed Gene Cascade Finder (GCF) as a novel tool for building gene cascades by comparison of mutant and wild-type RNA-Seq data along with integrated information of protein-protein interactions, expression timing, and domains. Application of GCF to RNA-Seq data confirmed that SPN-4 and MEX-3 regulate the canonical <em>Wnt</em> pathway during embryonic development. Moreover, lin-35, hsp-3, and gpa-<em>12</em> were found to be involved in MEX-1-dependent neurogenesis, and MEX-3 was found to control the gene cascade promoting neurogenesis through lin-35 and apl-1. Thus, GCF could be a useful tool for building gene cascades from RNA-Seq data.

The aim of this study was to confirm the previously identified link between BAMBI and human obesity by means of a genetic and functional analysis. We performed both a mutation analysis, using high-resolution melting curve analysis, and a genetic association study, including 8 common tagSNPs in the BAMBI gene region. Three of the identified genetic variants (R151W, H201R, and C229R) were evaluated for their <em>Wnt</em> signaling enhancing capacity in a <em>Wnt</em> luciferase reporter assay. Mutation screening of the BAMBI coding region and exon-intron boundaries on our population of 677 obese children and adolescents and 529 lean control subjects resulted in the identification of 18 variants, 10 of which were not previously reported and <em>12</em> of which were exclusively found in obese individuals. The difference in variant frequency, not taking into account common polymorphisms, between obese (3.1 %) and lean (0.9 %) subjects was statistically significant (p = 0.004). Our <em>Wnt</em> luciferase assay, using WT and mutant BAMBI constructs, showed a significantly reduced activity for all of the investigated variants. Logistic and linear regression analysis on our Caucasian population of 1022 obese individuals and 606 lean controls, did not identify associations with obesity parameters (p values >0.05). We found several rare genetic variations, which represent the first naturally occurring missense variants of BAMBI in obese patients. Three variants (R151W, H201R, and C229R) were shown to reduce <em>Wnt</em> signaling enhancing capacity of BAMBI and we believe this result should encourage further study of this gene in other obese populations. In addition, we did not find evidence for the involvement of BAMBI common variation in human obesity in our population.

Mitotane is the only drug approved for the therapy of adrenocortical carcinoma (ACC). Its clinical use is limited by the occurrence of relapse during therapy. To investigate the underlying mechanisms in vitro, we here generated mitotane-resistant cell lines. After long-term pulsed treatment of HAC-15 human adrenocortical carcinoma cells with 70 µM mitotane, we isolated monoclonal cell populations of treated cells and controls and assessed their respective mitotane sensitivities by MTT assay. We performed exome sequencing and electron microscopy, conducted gene expression microarray analysis and determined intracellular lipid concentrations in the presence and absence of mitotane. Clonal cell lines established after pulsed treatment were resistant to mitotane (IC50s of 102.2±7.3 µM (N=<em>12</em>) versus 39.4±6.2 µM (N=6) in controls (biological replicates, mean±SD, p=0.0001). Unlike nonresistant clones, resistant clones maintained normal mitochondrial and nucleolar morphology during mitotane treatment. Resistant clones largely shared structural and single nucleotide variants, suggesting a common cell of origin. Resistance depended in part on extracellular lipoproteins and was associated with alterations in intracellular lipid homeostasis, including levels of free cholesterol, as well as decreased steroid production. By gene expression analysis, resistant cells showed profound alterations in pathways including steroid metabolism and transport, apoptosis, cell growth and <em>Wnt</em> signaling. These studies establish an in vitro model of mitotane resistance in ACC and point to underlying molecular mechanisms. They may enable future studies to overcome resistance in vitro and improve ACC treatment in vivo.

Nuclear YAP1 plays a critical role in regulation of stem cell proliferation, tissue regeneration, and organ size in many types of epithelia. Due to rapid turnover of most epithelial cell types, the cytoplasmic function of YAP1 in epithelial cells has not been well studied. The retinal pigment epithelium (RPE) is a highly polarized epithelial cell type maintained at a senescence state, and offers an ideal cell model to study the active role of YAP1 in maintenance of the adult epithelial phenotype. Here, we show that the cytoplasmic function of YAP1 is essential to maintain adult RPE differentiation. Knockout of Yap1 in the adult mouse RPE caused cell depolarization and tight junction breakdown, and led to inhibition of RPE65 expression, diminishment of RPE pigments, and retraction of microvilli and basal infoldings. These changes in RPE further prompted the loss of adjacent photoreceptor outer segments and photoreceptor death, which eventually led to decline of visual function in older mice between 6 and <em>12</em> months of age. Furthermore, nuclear β-catenin and its activity were significantly increased in mutant RPE. These results suggest that YAP1 plays an important role in active inhibition of <em>Wnt</em>/β-catenin signaling, and is essential for downregulation of β-catenin nuclear activity and prevention of dedifferentiation of adult RPE.

The goal of this study was to clarify the protective role of the <em>Wnt</em>/β-catenin pathway agonist SKL2001 in a rat model of Caerulein-induced acute pancreatitis. AR42J cells and rats were divided into 4 groups: control, Caerulein, SKL2001 + Caerulein, and SKL2001 + control. Cell apoptosis was examined using flow cytometry. Hematoxylin-eosin staining was performed to observe pathological changes in pancreatic and small intestinal tissues. Inflammatory cytokines were detected by enzyme-linked immunosorbent assay (ELISA), while genes related to the <em>Wnt</em>/β-catenin pathway were quantified using quantitative real-time PCR. In vitro results showed that Caerulein promoted cell necrosis, inhibited the <em>Wnt</em>/β-catenin pathway, and increased the level of inflammatory cytokines. However, SKL2001 reduced cell necrosis and inflammatory cytokines and activated the <em>Wnt</em>/β-catenin pathway. Additionally, in vivo results demonstrated the accumulation of fluid (i.e., edema), hemorrhage, inflammation and necrosis of the pancreatic acini occurred 6 h after the final Caerulein induction, with the damage reaching a maximal level <em>12</em> h after the final Caerulein induction; meanwhile, the <em>Wnt</em>/β-catenin pathway was evidently inhibited with an enhanced level of inflammatory cytokines. The aforementioned damage was further aggravated <em>12</em> h later. Nevertheless, the pancreatic and small intestinal tissue damages were alleviated in Caerulein-induced rats treated with SKL2001. In conclusion, activation of the <em>Wnt</em>/β-catenin pathway could inhibit Caerulein-induced cell apoptosis and inflammatory cytokine release, thus improving pancreatic and intestinal damage in rats with acute pancreatitis.

Embryonic stem cells (ESCs) are commonly used for the analysis of gene function in embryonic development and provide valuable models for human diseases. In recent years, ESCs have also become an attractive tool for toxicological testing, in particular for the identification of teratogenic compounds. We have recently described a Bmp-reporter ESC line as a new tool to identify teratogenic compounds and to characterize the molecular mechanisms mediating embryonic toxicity. Here we describe the use of a <em>Wnt</em>/β-Catenin-reporter ESC line isolated from a previously described mouse line that carries the LacZ reporter gene under the control of a β-Catenin responsive promoter. The reporter ESC line stably differentiates into cardiomyocytes within <em>12</em> days. The reporter was endogenously induced between day 3-5 of differentiation reminiscent of its expression in vivo, in which strong LacZ activity is detected around gastrulation. Subsequently its expression becomes restricted to mesodermal cells and cells undergoing an epithelial to mesenchymal transition. The <em>Wnt</em>/β-Catenin-dependent expression of the reporter protein allowed quantification of dose- and time-dependent effects of teratogenic chemicals. In particular, valproic acid reduced reporter activity on day 7 whereas retinoic acid induced reporter activity on day 5 at concentrations comparable to the ones inhibiting the formation of functional cardiomyocytes, the classical read-out of the embryonic stem cell test (EST). In addition, we were also able to show distinct effects of teratogenic chemicals on the <em>Wnt</em>/β-Catenin-reporter compared with the previously described Bmp-reporter ESCs. Thus, different reporter cell lines provide complementary tools for the identification and analysis of potentially teratogenic compounds.

The aims of the present study were to elucidate the regulatory effect of exogenous Tribbles homologue 3 (TRB3) expression on the <em>Wnt</em>/β‑catenin signaling pathway and epithelial‑mesenchymal transition (EMT) in transforming growth factor‑β1 (TGF‑β1)‑induced mouse alveolar epithelial cells (MLE‑<em>12</em>) and investigate the underlying regulatory mechanisms. TRB3 expression was upregulated and downregulated using gene overexpression and RNA interference techniques, respectively. TGF‑β1‑stimulated MLE‑<em>12</em> cells were examined for EMT and activation condition of the <em>Wnt</em>/β‑catenin signaling pathway using Cell Counting Kit‑8, flow cytometry, western blotting, reverse transcription‑quantitative PCR, ELISA and immunofluorescence techniques. During TGF‑β1‑induced EMT, TRB3 expression was found to be significantly upregulated (P<0.05). In the TRB3 overexpression group, upregulated expression of β‑catenin and EMT‑related genes and proteins was observed (P<0.05), and an increase in fibrosis‑related factors in the cell culture supernatant was detected (P<0.05); however, the results were the opposite in the TRB3 downregulated group (P<0.05). TRB3 may be involved in the regulation of EMT in TGF‑β1‑induced MLE‑<em>12</em> cells through the <em>Wnt</em>/β‑catenin signaling pathway.

Primary glandular bladder tumours (bladder adenocarcinoma [BAC], urachal adenocarcinoma [UAC], urothelial carcinoma with glandular differentiation [UCg]) are rare malignancies with histological resemblance to colorectal adenocarcinoma (CORAD) in the majority of this subgroup. Definite case numbers are very low, molecular data are limited and the pathogenesis remains poorly understood. Therefore, this study was designed to complement current knowledge by in depth analysis of BAC (n = <em>12</em>), UAC (n = 13), UCg (n = 11) and non-invasive glandular lesions (n = 19). In BAC, in addition to known alterations in TP53, <em>Wnt</em>, MAP kinase and MTOR pathway, mutations in SMAD4, ARID1A and BRAF were identified. Compared to published data on muscle invasive bladder cancer (BLCA) and CORAD, UCg exhibited frequent "urothelial" like alterations while BAC and UAC were characterised by a more "colorectal" like mutational pattern. Immunohistochemically, there was no evidence of DNA mismatch repair deficiency or PD-L1 tumour cell positivity in any sample. Depending on the used antibody 0-45% of BAC, 0-30% of UCg and 0% UAC cases exhibited PD-L1 expressing tumour associated immune cells. A single BAC (9%, 1/11) showed evidence of ARID1A protein loss, and two cases of UCg (20%, 2/10) showed loss of SMARCA1 and PBRM1, respectively. Taken together, our data suggest at least in part involvement of similar pathways driving tumourigenesis of adenocarcinomas like BAC, UAC and CORAD independent of their tissue origin. Alterations of TERT and FBXW7 in single cases of intestinal metaplasia further point towards a possible precancerous character in line with previous reports.

Zebrafish lateral line neuromasts are composed of central hair cells surrounded by supporting cells. Cisplatin is a common anticancer drug, with hair cell disruption being a frequent side effect of this drug. In our study, we observed complete functional hair cell loss after six hours of cisplatin insult in neuromasts, as demonstrated by anti-parvalbumin 3 immunofluorescence staining or YO-PRO1 vital dye staining. Time course analysis of neuromast hair cell regeneration showed that regenerated hair cells first appeared between <em>12</em> and 24h after damage, and the abundance of these cells increased stepwise with recovery time. After 72h, 90% of the hair cells were regenerated, and after 84h, the number of regenerated hair cells was comparable to the number of neuromast hair cells before treatment. The expression pattern of slc17a8 also showed that hair cells were regenerated after cisplatin exposure. Meanwhile, peripheral supporting cells moved toward the center of the neuromasts, as shown by the in situ expression pattern of sox21a. Increased hair cell progenitor formation was also observed, as demonstrated by the in situ expression pattern of atoh1a. Furthermore, we detected increased expression of wnt2, wnt3a, and ctnnb1 in sorted supporting cells from the sqet10 transgenic line, which labels neuromast supporting cells specifically. In situ hybridization analysis also showed decreased expression of dkk1a and dkk2. Regenerated hair cells were inhibited by early inhibition of <em>Wnt</em>/β-catenin signaling. Taken together, the results presented here showed that <em>Wnt</em>/β-catenin signaling was activated in supporting cells during cisplatin exposure earlier than expected. Our results also indicated that supporting cells enabled hair cell regeneration via <em>Wnt</em>/β-catenin signaling during cisplatin exposure.

<AbstractText>Studies demonstrate the impact of diet and physical activity on epigenetic biomarkers, specifically DNA methylation. However, no intervention studies have examined the combined impact of dietary and activity changes on the blood epigenome. The objective of this study was to examine the impact of the Make Better Choices 2 (MBC2) healthy diet and activity intervention on patterns of epigenome-wide DNA methylation. The MBC2 study was a 9-month randomized controlled trial among adults aged 18-65 with non-optimal levels of health behaviors. The study compared three <em>12</em>-week interventions to (1) simultaneously increase exercise and fruit/vegetable intake, while decreasing sedentary leisure screen time; (2) sequentially increase fruit/vegetable intake and decrease leisure screen time first, then increase exercise; (3) increase sleep and decrease stress (control). We collected blood samples at baseline, 3 and 9 months, and measured DNA methylation using the Illumina EPIC (850 k) BeadChip. We examined region-based differential methylation patterns using linear regression models with the false discovery rate of 0.05. We also conducted pathway analysis using gene ontology (GO), KEGG, and IPA canonical pathway databases.</AbstractText><AbstractText>We found no differences between the MBC2 population (n = 340) and the subsample with DNA methylation measured (n = 68) on baseline characteristics or the impact of the intervention on behavior change. We identified no differentially methylated regions at baseline between the control versus intervention groups. At 3 versus 9 months, we identified 154 and 298 differentially methylated regions, respectively, between controls compared to pooled samples from sequential and simultaneous groups. In the GO database, we identified two gene ontology terms related to hemophilic cell adhesion and cell-cell adhesion. In IPA analysis, we found pathways related to carcinogenesis including PI3K/AKT, <em>Wnt</em>/β-catenin, sonic hedgehog, and p53 signaling. We observed an overlap between 3 and 9 months, including the GDP-L-fucose biosynthesis I, methylmalonyl metabolism, and estrogen-mediated cell cycle regulation pathways.</AbstractText><AbstractText>The results demonstrate that the MBC2 diet and physical activity intervention impacts patterns of DNA methylation in gene regions related to cell cycle regulation and carcinogenesis. Future studies will examine DNA methylation as a biomarker to identify populations that may particularly benefit from incorporating health behavior change into plans for precision prevention.</AbstractText>

Sodium valproate (SV) is an antiepileptic drug that is widely used in the treatment of different seizure disorders. The topical SV has a hair regenerative potential through activating the <em>Wnt</em>/β-catenin pathway and anagen phase induction. The aim of the current investigation was to fabricate nanospanlastics of SV for improving its dermal delivery by providing prolonged drug effect and increasing its permeability for treatment of androgenic alopecia (AGA). SV-loaded nanospanlastics were formulated according to 2³ factorial design by ethanol injection method using a non-ionic surfactant (Span 60) and edge activators (EAs), such as Tween 80 and Cremophor RH 40, to explore the influence of different independent variables on entrapment efficiency (EE%) and percentage drug released after <em>12</em> h (Q_<em>12</em>h) in order to choose the optimized formula using Design-Expert software. The optimized formula (F8) appeared as spherical deformable vesicles with EE% of 90.32 ± 2.18% and Q_<em>12</em>h of 90.27 ± 1.98%. F8 exhibited significant improvement of ex vivo permeation than free SV. The clinical study exhibited no comparable difference between F8 and marketed minoxidil lotion. However, F8 demonstrates less adverse effects than minoxidil lotion. Nanospanlastics could be a safe and effective method for improving the topical delivery of SV in the management of AGA.

Keywords: androgenic alopecia; edge activators; nanospanlastics; sodium valproate; topical delivery.

Endometrial carcinoma is one of the most common cancers in women. A limited number of endometrial carcinoma cell lines are available for studies of signal transduction pathways and experimental therapeutics in vitro. However, these cell lines have not been comprehensively characterized. In this study, we used genome-wide microarray-based comparative genomic hybridization (aCGH) technology to characterize five of the more commonly used endometrial cancer cell lines. We detected DNA copy-number gains in chromosomal regions 2q, 3p, 3q, 5q, 7p, 17q, and 19q in all five cell lines. Other common sites of copy-number gains, which were detected in four of five cell lines, included segments of chromosomes 1, 6, 8, 9, 11, <em>12</em>, and 16. In all five cell lines, we found DNA copy-number losses in regions 3p, 10p, 10q, 11q, 11p, 14q, 15q, 18p, and 21q. Other common sites of genetic aberrations included segments of chromosomes 1, 2, 4, 5, 6, 16, 20, and 22. The genes involved in the copy-number alterations included the oncogenes PIK3CA (3q26.3), K-ras (<em>12</em>p<em>12</em>.1), R-ras (19q13.3-qter), Raf-1 (3p25), EGFR (7p<em>12</em>), Akt1 (14q32.32), and Akt2 (19q13.1-q13.2). A pathway analysis showed that genes in the PI3K and <em>Wnt</em> pathways are commonly affected. Our characterization of genomic alterations in these five commonly used endometrial cancer cell lines provides valuable genomic information for research that focuses on these key oncogenic pathways in endometrial cancer.

Many high-grade serous carcinomas (HGSCs) likely originate in the distal region of the Fallopian tube's epithelium (TE) before metastasizing to the ovary. Unfortunately, molecular mechanisms promoting malignancy in the distal TE are obfuscated, largely due to limited primary human TE gene expression data. Here we report an in depth bioinformatic characterization of 34 primary TE mRNA-seq samples. These samples were prepared from proximal and distal TE regions of <em>12</em> normal Fallopian tubes. Samples were segregated based on their aldehyde dehydrogenase (ALDH) activity. Distal cells form organoids with higher frequency and larger size during serial organoid formation assays when compared to proximal cells. Consistent with enrichment for stem/progenitor cells, ALDH+ cells have greater <em>WNT</em> signaling. Comparative evaluation of proximal and distal TE cell population's shows heightened inflammatory signaling in distal differentiated (ALDH-) TE. Furthermore, comparisons of proximal and distal TE cell populations finds that the distal ALDH+ TE cells exhibit pronounced expression of gene sets characteristic of HGSC sub-types. Overall, our study indicates increased organoid forming capacity, <em>WNT</em>/inflammatory signaling, and HGSC signatures underlie differences between distal and proximal regions of the human TE. These findings provide the basis for further mechanistic studies of distal TE susceptibility to the malignant transformation.

Cadherins, a group of molecules typically associated with planar cell polarity and <em>Wnt</em> signalling, have been little reported outside of the animal kingdom. Here, we identify a new family of cadherins in the Stramenopiles, termed Nonagonal after their 9 transmembrane passes, which contrast to the one or seven passes found in other known cadherin families. Manual curation and experimental validation reveal two subclasses of nonagonal cadherins, depending on the number of uninterrupted extracellular cadherin (EC) modules presented. Firstly, shorter mono-exonic, unimodular, protein models, with 3 to <em>12</em> EC domains occur as duplicate paralogs in the saprotrophic Labyrinthulomycetes Aurantiochytrium limanicum and Schizochytrium aggregatum, the gastrointestinal Blastocystis hominis (Blastocystae) and as a single copy gene in the autotrophic Pelagophyte Aureococcus anophagefferens. Larger, single copy, multi-exonal, tri-modular protein models, with up to 72 EC domain in total, are found in the Oomycete genera Albugo, Phytophthora, Pythium and Eurychasma. No homolog was found in the closely related autotrophic Phaeophyceae (brown algae) or Bacillariophyceae (diatoms), nor in several genera of plant and animal pathogenic oomycetes (Aphanomyces, Saprolegnia and Hyaloperonospora). This potential absence was further investigated by synteny analysis of the genome regions flanking the cadherin gene models, which are found to be highly variable. Novel to this new cadherin family is the presence of intercalated laminin and putative carbohydrate binding in tri-modular oomycete cadherins and at the N-terminus of thraustochytrid proteins. As we were unable to detect any homologs of proteins involved in signalling pathways where other cadherin families are involved, we present a conceptual hypothesis on the function of nonagonal cadherin based around the presence of putative carbohydrate binding domains.

Canine adenovirus type 1 (CAdV-1) is the etiologic agent of fox encephalitis, and a virus strain from fox encephalitis is isolated and related researches are conducted. In this experiment, the results showed that the F1301 strain was confirmed to be the CAdV-1. The whole genome of the CAdV-1 F1301 strain isolated from fox was 30535 bp and had higher homology to the other reported CAdV-1 strains. After 0 h, <em>12</em> h, and 36 h of CAdV-1 infection, the difference gene of the 592 LncRNA and 1<em>12</em>15 mRNA were involved in cell responses to CAdV-1 infection through the PI3K-AKT, <em>Wnt</em>, Herpes simplex, Hepatitis C, and Epstein-Barr virus infection pathway in MDCK. The results indicate that the biological characterization of the CAdV-1 and the MDCK cell-CAdV-1 interaction are clarified. This article is protected by copyright. All rights reserved.

Keywords: Biological characterization; Canine adenovirus 1; MDCK cell; Transcriptome analysis.

As the only known mammalian organ that can fully and annually regenerate, deer antler has significant advantages over lower-order animal models when investigating the control of stem cell-based organ regeneration. Antler regeneration is known to be initiated and maintained by neural crest-derived stem cells in different states of activation. Antler stem cells can therefore be used as a model to study proteins and pathways involved in the maintenance of a stem cell niche and their activation and differentiation during organ formation. In this study, the MSC markers CD73, CD90 and CD105 were examined within the antler tip. Label-free quantification was performed to investigate the protein profiles of antler stem cells under different stages of activation and included: dormant pedicle periosteum (DPP), antler growth center (GC), post-active stem cells from mid-beam antler periosteum (MAP), and deer facial periosteum (FP) as a control (n = 3 per group). PEAKS and IPA software were used to analyze the proteomic data. Our research confirmed the central role of stem cell activation in the development of this mammalian organ by localizing the MSC markers within the antler growth center. Label-free quantification revealed that the greatest number of unique proteins (eighty-seven) was found in the growth center. There were only <em>12</em> proteins found with expression levels that significantly differed between DPP and FP. Protein profiles of these two groups indicated that antler stem cells may use similar mechanisms to maintain dormancy within a stem cell niche. The number of significantly regulated proteins between DPP, MAP and GC was 153. Among them, the majority were upregulated in the growth center. Activation of antler stem cells was associated with many biological processes and signaling pathways such as Hippo and canonical <em>Wnt</em> signaling. This work identifies the key pathways, molecular/cellular functions and upstream regulators involved in mammal organ regeneration. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the iProX partner repository1 with the dataset identifier PXD016824.

Colorectal cancer (CRC) is the third most common cancer in the world. Oplopanax elatus is widely used in traditional medicine. However, little is known about its pharmacological effects and bioactive compounds. We evaluated the effects of the polyyne-enriched extract from O. elatus (PEO) on the progression of colon carcinogenesis in ApcMin/+ mice. In addition, these effects were also investigated in HCT116 and SW480 cells. After PEO oral administration (0.2% diet) for <em>12</em> weeks, PEO significantly improved body weight changes and reduced the tumor burden and tumor multiplicity compared with the untreated mice. Meanwhile, western blot and immunohistochemistry results showed PEO significantly reduced the expression of β-catenin and cyclinD1 in both small intestine and the colon tissues compared with the untreated mice. In addition, PEO treatment significant decreased the cell viability in both HCT116 and SW480 cell lines. It also decreased the levels of β-catenin, cyclinD1, c-myc and p-GSK-3β in HCT116 and SW480 cells at 25 μM. These results indicate that PEO may have potential value in prevention of colon cancer by down-regulating <em>Wnt</em>-related protein.

MicroRNAs regulate bone homeostasis and circulating microRNAs have been proposed as novel bone biomarkers. The effect of anti-osteoporotic treatment on circulating microRNAs has not been described in detail. Therefore, we performed a comprehensive analysis of microRNA serum levels in OVX and SHAM rats over <em>12</em> weeks of anti-resorptive or osteo-anabolic treatment. Forty-two Sprague Dawley rats underwent SHAM surgery (n=10) or ovariectomy (n=32). After 8 weeks, OVX rats were randomized to anti-resorptive treatment with zoledronate (n=11), osteo-anabolic treatment with teriparatide (n=11), or vehicle treatment (n=10). Serum samples were collected at weeks 8, <em>12</em>, 16, and 20 after surgery. 91 microRNAs were analyzed by RT-qPCR in serum samples collected at week 20. Based on the results 29 microRNAs were selected for longitudinal analysis at all four study time points. Changes in bone mineral density and microstructure were followed up by in-vivo micro-CT and ex-vivo nano-CT. Ovariectomy resulted in the loss of trabecular bone, which was reversed by osteo-anabolic and anti-resorptive treatment. Differential expression analysis identified 11 circulating miRNAs that were significantly regulated after treatment. For example, miR-107 and miR-31-5p increased in vehicle-treated OVX animals while decreasing during teriparatide treatment. Additional miRNAs were identified that showed significant correlations to bone microstructure or bone miRNA expression, including miR-203a-3p, which exhibited a significant negative correlation to vertebral and tibial BV/TV (%). Longitudinal analysis confirmed 8 microRNAs with significant changes in serum over time that were prevented by teriparatide and zoledronate treatment (miR-34a-5p, miR-31-5p, miR-30d-3p, miR-378a-5p) or teriparatide treatment only (miR-375-3p, miR-183-5p, miR-203a-3p, miR-203b-3p). Gene target network analysis identified <em>WNT</em> and Notch signaling as the main signaling pathways controlled by these miRNAs. Thus, ovariectomy results in time-dependent de-regulation of circulating miRNAs compared to SHAM animals. Anti-osteoporotic treatments can rescue this effect, showing that bone-related miRNAs might act as novel biomarkers for treatment monitoring.

Keywords: bone microstructure; miRNAs; osteoporosis; postmenopausal osteoporosis; teriparatide; zoledronic acid.

<AbstractText>Dengue caused by dengue virus (DENV) serotypes -1 to -4 is the most important mosquito-borne viral disease in the tropical and sub-tropical countries worldwide. Yet many of the pathophysiological mechanisms of host responses during DENV infection remain largely unknown and incompletely understood.</AbstractText><AbstractText>Using a mouse model, the miRNA expressions in liver during DENV-1 infection was investigated using high throughput miRNA sequencing. The differential expressions of miRNAs were then validated by qPCR, followed by target genes prediction. The identified miRNA targets were subjected to gene ontology (GO) annotation and pathway enrichment analysis to elucidate the potential biological pathways and molecular mechanisms associated with DENV-1 infection.</AbstractText><p><div><b>Results</b></div>A total of 224 and 372 miRNAs out of 433 known mouse miRNAs were detected in the livers of DENV-1-infected and uninfected mice, respectively; of these, 207 miRNAs were present in both libraries. The miR-148a-3p and miR-<em>12</em>2-5p were the two most abundant miRNAs in both groups. Thirty-one miRNAs were found to have at least 2-fold change in upregulation or downregulation, in which seven miRNAs were upregulated and 24 miRNAs were downregulated in the DENV-1-infected mouse livers. The miR-1a-3p was found to be the most downregulated miRNA in the DENV-1-infected mouse livers, with a significant fold change of 0.10. To validate the miRNA sequencing result, the expression pattern of <em>12</em> miRNAs, which were highly differentially expressed or most abundant, were assessed by qPCR and nine of them correlated positively with the one observed in deep sequencing. <i>In silico</i> functional analysis revealed that the adaptive immune responses involving TGF-beta, MAPK, PI3K-Akt, Rap1, <em>Wnt</em> and Ras signalling pathways were modulated collectively by 23 highly differentially expressed miRNAs during DENV-1 infection.</p><p><div><b>Conclusion</b></div>This study provides the first insight into the global miRNA expressions of mouse livers in response to DENV-1 infection <i>in vivo</i> and the possible roles of miRNAs in modulating the adaptive immune responses during DENV-1 infection.</p>

Bone marrow-derived mesenchymal stem cells differentiate into neurons under the induction of Schwann cells. However, key microRNAs and related pathways for differentiation remain unclear. This study screened and identified differentially expressed microRNAs in bone marrow-derived mesenchymal stem cells induced by Schwann cell-conditioned medium, and explored targets and related pathways involved in their differentiation into neuronal-like cells. Primary bone marrow-derived mesenchymal stem cells were isolated from femoral and tibial bones, while primary Schwann cells were isolated from bilateral saphenous nerves. Bone marrow-derived mesenchymal stem cells were cultured in unconditioned (control group) and Schwann cell-conditioned medium (bone marrow-derived mesenchymal stem cell + Schwann cell group). Neuronal differentiation of bone marrow-derived mesenchymal stem cells induced by Schwann cell-conditioned medium was observed by time-lapse imaging. Upon induction, the morphology of bone marrow-derived mesenchymal stem cells changed into a neural shape with neurites. Results of quantitative reverse transcription-polymerase chain reaction revealed that nestin mRNA expression was upregulated from 1 to 3 days and downregulated from 3 to 7 days in the bone marrow-derived mesenchymal stem cell + Schwann cell group. Compared with the control group, microtubule-associated protein 2 mRNA expression gradually increased from 1 to 7 days in the bone marrow-derived mesenchymal stem cell + Schwann cell group. After 7 days of induction, microRNA analysis identified 83 significantly differentially expressed microRNAs between the two groups. Gene Ontology analysis indicated enrichment of microRNA target genes for neuronal projection development, regulation of axonogenesis, and positive regulation of cell proliferation. Kyoto Encyclopedia of Genes and Genomes pathway analysis demonstrated that Hippo, <em>Wnt</em>, transforming growth factor-beta, and Hedgehog signaling pathways were potentially associated with neural differentiation of bone marrow-derived mesenchymal stem cells. This study, which carried out successful microRNA analysis of neuronal-like cells differentiated from bone marrow-derived mesenchymal stem cells by Schwann cell induction, revealed key microRNAs and pathways involved in neural differentiation of bone marrow-derived mesenchymal stem cells. All protocols were approved by the Animal Ethics Committee of Institute of Radiation Medicine, Chinese Academy of Medical Sciences on March <em>12</em>, 2017 (approval number: DWLI-20170311).

<b>Purpose</b>: Type 2 Diabetes mellitus (DM) is a major health problem and its ocular complications like orbital infections, cataract and diabetic retinopathy cause blindness. Meibomian gland (MG) dysfunction and dry eye disease are also important ocular complications of type 2 DM but not enough research has been conducted on these complications. Our hypothesis suggests type 2 DM can alter significant gene expressions of MG. In our study, MGs of leptin-deficient spontaneous diabetic and non-diabetic mice were extracted, and gene expression profiles were analyzed with microarray technology. <b>Methods</b>: Mice were divided into two groups; nine Lep <i>b/ob</i> spontaneous diabetic mice as type 2 DM group and nine non-diabetic Balb/c mice as controls. Blood glucose levels, tearfilm break-up time and fluorescein scores were measured in both two groups for <em>12</em> weeks. MGs were dissected and RNAs were isolated for microarray gene expression analysis. We filtered probes with standard deviation of more than 0.1 and we used 40452 of 45281 probes for processing. We performed fold change analysis and identified which genes are affected, and we analyzed the impact of genes on proteins, pathways and gene ontologies by using various databases. <b>Results</b>: We observed 172 up-regulated and 118 down-regulated genes in type 2 diabetic mice when compared to non-diabetic mice. Interestingly, expression of collagen type I, integrin beta-I binding protein-I, pyruvate dehydrogenase kinase, TNF receptor genes up-regulated with DM; on the other hand, IL-33, cholecystokinin, plasminogen activator, IL-1 and serine peptidase inhibitor genes down-regulated significantly. Also, we have seen a significant decrease in <em>WNT</em> signaling and pentose phosphate pathways-related genes. <b>Conclusion</b>: Our data show these changes in gene expression caused by endocrine and immune mechanisms of type 2 DM which result disrupted homeostasis of epithelial cells of MG. Increased expressions of apoptosis and inflammation-related genes and their effects on related pathways have proven that MGs were negatively affected by type-2 DM.