The aim of the study was to evaluate the effect of preloads differing in fatty acid composition, content, and delivery form on acute behavioral, subjective, and biological outcomes of satiety. Four energy- and volume-matched preloads were tested in normal weight men and women (n = <em>12</em> and 13, respectively), using a random, crossover design. Preloads were semisolid shakes differing in fat source [walnut or safflower (SAFF)], delivery [ground walnuts (<em>WNT</em>) or walnut oil (WOL)] or content [39% fat energy (SAFF, <em>WNT</em>, WOL) or 4% low-fat control (LFC)]. Blood was collected and subjective satiety assessed at 0 (fasting), 15, 30, and 45 min after preload consumption. Lunch (test meal) was provided thereafter. Energy intake at lunch was not affected by preload; however, subjects selected more carbohydrate, fiber-rich foods at the test meal lunch after walnut preloads than after LFC or SAFF preloads. Compared with the LFC preload, appetite satisfaction was significantly greater after SAFF and <em>WNT</em>, but not after WOL. Women were hungrier after SAFF than after WOL, whereas men were less hungry after SAFF and LFC than after WOL or <em>WNT</em>. Plasma cholecystokinin (CCK) concentrations reflected preload fat content and availability, particularly among men; CCK was higher after WOL and SAFF preloads than after LFC or <em>WNT</em> preloads. Plasma insulin was higher after LFC and SAFF preloads, corresponding to hunger suppression in men. Dietary restraint was associated with a blunted CCK response to preloads, whereas insulin was not affected by restraint. The results indicate that test meal energy intake after preloads containing approximately 40% walnut or safflower fat or 4% fat did not differ; however, walnut consumption may promote food patterns consistent with consuming diets higher in fiber.

Disturbed flow has been suggested to contribute to aneurysm susceptibility in bicuspid aortic valve (BAV) patients. Lately, flow has emerged as an important modulator of DNA methylation. Hear we combined global methylation analysis with in vitro studies of flow-sensitive methylation to identify biological processes associated with BAV-aortopathy and the potential contribution of flow. Biopsies from non-dilated and dilated ascending aortas were collected from BAV (n = 21) and tricuspid aortic valve (TAV) patients (n = 23). DNA methylation and gene expression was measured in aortic intima-media tissue samples, and in EA.hy926 and primary aortic endothelial cells (ECs) isolated from BAV and TAV exposed to oscillatory (±<em>12</em> dynes/cm2) or laminar (<em>12</em> dynes/cm2) flow. We show methylation changes related to epithelial-mesenchymal-transition (EMT) in the non-dilated BAV aorta, associated with oscillatory flow related to endocytosis. The results indicate that the flow-response in BAV ECs involves hypomethylation and increased expression of <em>WNT</em>/β-catenin genes, as opposed to an angiogenic profile in TAV ECs. The EMT-signature was exasperated in dilated BAV aortas. Aberrant EMT in BAV aortic walls could contribute to increased aneurysm susceptibility, and may be due to disturbed flow-exposure. Perturbations during the spatiotemporally related embryonic development of ascending aorta and semilunar valves can however not be excluded.

A hallmark of aging-related organ deterioration is a dysregulated immune response characterized by pathologic leukocyte infiltration of affected tissues. Mechanisms and genes involved are as yet unknown. To identify genes associated with aging-related renal infiltration, we analyzed kidneys from aged mice (≥20 strains) for infiltrating leukocytes followed by Haplotype Association Mapping (HAM) analysis. Immunohistochemistry revealed CD45+ cell clusters (predominantly T and B cells) in perivascular areas coinciding with PNAd+ high endothelial venules and podoplanin+ lymph vessels indicative of tertiary lymphoid organs. Cumulative cluster size increased with age (analyzed at 6, <em>12</em> and 20 months). Based on the presence or absence of clusters in male and female mice at 20 months, HAM analysis revealed significant associations with loci on Chr1, Chr2, Chr8 and Chr14 in male mice, and with loci on Chr4, Chr7, Chr13 and Chr14 in female mice. Wisp2 (Chr2) showed the strongest association (P = 5.00×10(-137)) in male mice; Ctnnbip1 (P = 6.42×10(-267)) and Tnfrsf8 (P = 5.42×10(-245)) (both on Chr4) showed the strongest association in female mice. Both Wisp2 and Ctnnbip1 are part of the <em>Wnt</em>-signaling pathway and the encoded proteins were expressed within the tertiary lymphoid organs. In conclusion, this study revealed differential lymphocytic infiltration and tertiary lymphoid organ formation in aged mouse kidneys across different inbred mouse strains. HAM analysis identified candidate genes involved in the <em>Wnt</em>-signaling pathway that may be causally linked to tertiary lymphoid organ formation.

Growth is one of the most economically important traits in the poultry industry. In this study, we identified single-nucleotide polymorphisms (SNPs) and candidate genes associated with growth traits of the Jinghai Yellow chicken. Genome-wide association studies were conducted using the Illumina 60 K SNP Chicken array to genotype 400 Jinghai Yellow chickens. For each bird, the body weights at hatching and at 2, 4, 6, 8, <em>12</em>, 14, and 16 weeks were recorded. The SNPs that were significantly associated with the growth traits were identified using the general linear regression model. The results revealed a total of 18 SNPs that reached Bonferroni genome-wide significance (P < 1.80E-6). Three proximal genes (BTRC, NLK, and NF1) were found to participate in the <em>Wnt</em>-signaling pathway and mitogen-activated protein kinase signaling pathway. Haplotype analysis identified 19 significant haplotypes and identified a region 152.4-156.3M on GGA1 affecting 3 growth traits (BW4, BW14, and BW16). These results may help identify the exact locations of body weight quantitative trait loci on a genome level and indicate variants that can be used for subsequent investigations for Jinghai Yellow chicken.

Specific microRNAs (miRs) and the <em>Wnt</em> signaling pathway play critical roles in regulating bone development and homeostasis. Our previous studies revealed the ability of miR-335-5p to promote osteogenic differentiation by downregulating <em>Wnt</em> antagonist Dickkopf-1 (DKK1). The purpose of this study was to use nano-materials to efficiently deliver miR-335-5p into osteogenic cells for tissue engineering applications. We synthesized and screened a library of <em>12</em> candidate nano-lipidoids，of which L8 was identified as the preferred biodegradable lipidoid for miRNA molecule delivery into cells. We then investigated whether a lipidoid-miRNA formulation of miR-335-5-p (LMF-335) could successfully deliver miR-335-5-p into cells to promote osteogenesis in vitro and calvarial bone healing in vivo. Transfection of C3H10T1/2 cells and bone marrow stromal cells (BMSCs) with LMF-335 led to decreased expression of DKK1 and increased expression of the key osteogenic genes. LMF-335 and LMF-335-transfected BMSCs were then used in combination with silk scaffolds to evaluate healing of critical-size calvarial bone defects in mice. The results revealed significant new bone formation in the defects in LMF-335 groups as compared with control groups. In conclusion, this first report supports the notion that lipidoid delivery of miRNA can be used to induce osteogenic differentiation of stem cells and bone regeneration.

BACKGROUND

<em>Wnt</em>5A, which is a member of the non-transforming <em>Wnt</em> protein family, is implicated in inflammatory processes. It is also highly expressed by ovarian cancer cells. ROR2, which is a member of the Ror-family of receptor tyrosine kinases, acts as a receptor or co-receptor for <em>Wnt</em>5A. The <em>Wnt</em>5A-ROR2 signaling pathway plays essential roles in the migration and invasion of several types of tumor cell and influences their cell polarity. We investigated the modulation of <em>Wnt</em>5A-ROR2 by inflammatory mediators and its involvement in the migration of the human ovarian cancer cell line SKOV-3.

METHODS

SKOV-3 cells were treated with LPS (lipopolysaccharide), LTA (lipoteichoic acid) and recombinant human IL-6 alone or in combination with STAT3 inhibitor (S1155S31-201) or NF-kB inhibitor (BAY11-7082) for 4, 8, 12, 24 and 48 h. The <em>Wnt</em>5A and ROR2 expression levels were determined at the gene and protein levels. Cells were transfected with specific siRNA against <em>Wnt</em>5A in the absence or presence of human anti-ROR2 antibody and cell migration was assessed using transwells.

RESULTS

There was a strong downregulation of <em>Wnt</em>5A expression in the presence of STAT3 or NF-kB inhibitors. Cell stimulation with LTA or IL-6 for 8 h led to significantly increased levels of <em>Wnt</em>5A (5- and 3-fold higher, respectively). LPS, LTA or IL-6 treatment significantly increased ROR2 expression (2-fold after 48 h). LPS- or LTA-induced <em>Wnt</em>5A or ROR2 expression was abrogated in the presence of STAT3 inhibitor (p < 0.001). IL-6-induced <em>Wnt</em>5A expression was abrogated by both STAT3 and NF-kB inhibitors (p < 0.001). Although not significant, IL-6-induced ROR2 expression showed a modest decrease when STAT3 inhibitor was used. Moreover, cell migration was decreased by 80 % in siRNA <em>Wnt</em>5A-transfected cells in the presence of anti-human ROR2 antibody (p < 0.001).

CONCLUSIONS

This study revealed for the first time that inflammatory mediators modulate <em>Wnt</em>5A and ROR2 through NF-kB and STAT3 transcription factors and this may play a role in ovarian cancer cell migration. The results described here provide new insight into the role of the <em>Wnt</em>5A-ROR2 complex in ovarian cancer progression in relation to inflammation.

The U.S. Food and Drug Administration utilizes the presence of filth and extraneous materials as one of the criteria for implementing regulatory actions and assessing adulteration of food products of public health importance. Twenty-two prevalent pest species (also known as the ''Dirty 22'' species) have been considered by this agency as possible vehicles for the spread of foodborne diseases, and the presence of these species is considered an indicator of unsanitary conditions in food processing and storage facilities. In a previous study, we further categorized the Dirty 22 species into four groups: group I includes four cockroach species, group II includes two ant species, group III includes <em>12</em> fly species, and group IV includes four rodent species. Here, we describe the development of three nested PCR primer sets and multilocus genetic characterization by amplifying the small subunit rRNA, elongation factor 1-alpha, and wingless (<em>WNT</em>-1) genes of group II Dirty 22 ant species Monomorium pharaonis and Solenopsis molesta. These novel group II Dirty 22 species-specific nested PCR primer sets can be used when the specimens cannot be identified using conventional microscopic methods. These newly developed assays will provide correct identification of group II Dirty 22 ant species, and the information can be used in the control of foodborne pathogens.

Diabetes mellitus is considered as a risk factor for Alzheimer disease. The aim of the present study was to evaluate the possibility whether treadmill exercise ameliorates Alzheimer disease-associated memory loss in the diabetes mellitus. For this study, the effects of treadmill exercise on short-term memory and spatial learning ability in relation with Wnt signaling pathway were evaluated using the streptozotocin (STZ)-induced diabetic rats. Diabetes was induced by intraperitoneal injection of STZ. Step-down avoidance task and 8-arm radial maze test were performed for the memory function. Immunohistochemistry for 5-bro-mo-2'-deoxyridine (BrdU) and doublecortin (DCX) and Western blot for Wnt weeks. In the present results, short-term memory and spatial learning ability were deteriorated by induction of diabetes. Treadmill exercise improved short-term memory and spatial learning ability in the diabetic rats. The numbers of BrdU-positive and DCX-positive cells in the hippocampal dentate gyrus were decreased by induction of diabetes. Treadmill exercise increased these numbers in the diabetic rats. WntWntWnt signaling pathway in the diabetic rats.

The present study aimed to investigate the effects of the phosphoinositide 3‑kinase (PI3K)/Akt signaling pathway on the <em>Wnt</em>/β‑catenin signaling pathway in rats with focal cerebral ischemia‑reperfusion injury. A total of 96 rat focal cerebral ischemia‑reperfusion models, established according to a modified version of Longa's method, were randomly divided into four groups: Sham‑operated (S), cerebral ischemia‑reperfusion injury (I), cerebral ischemia‑reperfusion + basic fibroblast growth factor (bFGF) post‑processing and, finally, cerebral ischemia‑reperfusion + bFGF post‑processing + PI3K inhibitor LY294002 (LY). Each group consisted of 24 rats and each group was divided into four subgroups according to the indicated reperfusion times of <em>12</em>, 24, 48 and 72 h. The morphological changes of the cortical tissue and the cellular apoptosis were determined using hematoxylin and eosin staining and the terminal deoxynucleotidyl transferase dUTP nick end labeling method, respectively. The expression levels of phosphorylated (p‑)Akt, glycogen synthase kinase‑3β (GSK‑3β) mRNA and β‑catenin in the cortical tissue were detected at different time‑points. The number of apoptotic cells and the expression levels of p‑Akt, GSK‑3β mRNA and β‑catenin in the I and LY groups were significantly higher compared with those in the S group (P<0.05). In the bFGF group, the number of apoptotic cells and the mRNA expression levels of GSK‑3β were significantly decreased, whereas the expression levels of p‑Akt and β‑catenin were significantly increased compared with those in the I and LY groups (P<0.05). In cerebral ischemia‑reperfusion injury, the PI3K/Akt signaling pathway regulated β‑catenin, the main member of the <em>Wnt</em> signaling pathway, via GSK‑3β, providing information to assist in further investigation of the mechanism of β‑catenin in ischemia‑reperfusion injury.

Seasonal or photoperiodically sensitive animals respond to altered day length with changes in physiology (growth, food intake and reproductive status) and behaviour to adapt to predictable yearly changes in the climate. Typically, different species of hamsters, voles and sheep are the most studied animal models of photoperiodism. Although laboratory rats are generally considered nonphotoperiodic, one rat strain, the inbred Fischer 344 (F344) rat, has been shown to be sensitive to the length of daylight exposure by changing its physiological phenotype and reproductive status according to the season. The present study aimed to better understand the nature of the photoperiodic response in the F344 rat. We examined the effects of five different photoperiods on the physiological and neuroendocrine responses. Young male F344 rats were held under light schedules ranging from 8 h of light/day to 16 h of light/day, and then body weight, including fat and lean mass, food intake, testes weights and hypothalamic gene expression were compared. We found that rats held under photoperiods of ≥ <em>12</em> h of light/day showed increased growth and food intake relative to rats held under photoperiods of ≤ 10 h of light/day. Magnetic resonance imaging analysis confirmed that these changes were mainly the result of a change in lean body mass. The same pattern was evident for reproductive status, with higher paired testes weight in photoperiods of ≥ <em>12</em> h of light/day. Accompanying the changes in physiological status were major changes in hypothalamic thyroid hormone (Dio2 and Dio3), retinoic acid (Crabp1 and Stra6) and <em>Wnt</em>/β-Catenin signalling genes (sFrp2 and Mfrp). Our data demonstrate that a photoperiod schedule of <em>12</em> h of light/day is interpreted as a stimulatory photoperiod by the neuroendocrine system of young male F344 rats.

<em>Wnt</em>/β-Catenin signaling plays crucial roles in regenerative processes in eumetazoans. It also acts in regeneration and axial patterning in the simple freshwater polyp Hydra, whose morphallactic regenerative capacity is unparalleled in the animal kingdom. Previous studies have identified β-catenin as an early response gene activated within the first 30min in Hydra head regeneration. Here, we have studied the role of β-Catenin in more detail. First, we show that nuclear β-Catenin signaling is required for head and foot regeneration. Loss of nuclear β-Catenin function blocks head and foot regeneration. Transgenic Hydra tissue, in which β-Catenin is over-expressed, regenerates more heads and feet. In addition, we have identified a set of putative β-Catenin target genes by transcriptional profiling, and these genes exhibit distinct expression patterns in the hypostome, in the tentacles, or in an apical gradient in the body column. All of them are transcriptionally up-regulated in the tips of early head and foot regenerates. In foot regenerates, this is a transient response, and expression starts to disappear after <em>12</em>-36h. ChIP experiments using an anti-HydraTcf antibody show Tcf binding at promoters of these targets. We propose that gene regulatory β-Catenin activity in the pre-patterning phase is generally required as an early regeneration response. When regenerates are blocked with iCRT14, initial local transcriptional activation of β-catenin and the target genes occurs, and all these genes remain upregulated at the site of both head and foot regeneration for the following 2-3 days. This indicates that the initial regulatory network is followed by position-specific programs that inactivate fractions of this network in order to proceed to differentiation of head or foot structures. brachyury1 (hybra1) has previously been described as early response gene in head and foot regeneration. The HyBra1 protein, however, appears in head regenerating tips not earlier than about twelve hours after decapitation, and HyBra1 translation does not occur in iCRT14-treated regenerates. Foot regenerates never show detectable levels of HyBra1 protein at all. These results suggest that translational control mechanisms may play a decisive role in the head- and foot-specific differentiation phase, and HyBra1 is an excellent candidate for such a key regulator of head specification.

Hepatocarcinogenesis comprises of multiple, complex steps that occur after liver injury and usually involve several pathways, including telomere dysfunction, cell cycle, <em>WNT</em>/β-catenin signaling, oxidative stress and mitochondria dysfunction, autophagy, apoptosis, and AKT/mTOR signaling. Following liver injury, gene mutations, accumulation of oxidative stress, and local inflammation lead to cell proliferation, differentiation, apoptosis, and necrosis. The persistence of this vicious cycle in turn leads to further gene mutation and dysregulation of pro- and anti-inflammatory cytokines, such as interleukin (IL)-1β, IL-6, IL-10, IL-<em>12</em>, IL-13, IL-18, and transforming growth factor (TGF)-β, resulting in immune escape by means of the NF-κB and inflammasome signaling pathways. In this review, we summarize studies focusing on the roles of hepatocarcinogenesis and the immune system in liver cancer. In addition, we furnish an overview of recent basic and clinical studies to provide a strong foundation to develop novel anti-carcinogenesis targets for further treatment interventions.

Pediatric high-grade gliomas represent 8-<em>12</em>% of all primary tumors of the nervous system in children. Five-year survival for these pediatric aggressive tumors is poor (15-35%) indicating the need to develop better treatments for pediatric high-grade gliomas. In this work we used SF188 and SJ-GBM2 cell lines to study the function of the ubiquitin carboxyl-terminal esterase L1 (UCHL1), a deubiquitinase de-regulated in several cancers, in pediatric high-grade gliomas. UCHL1 depletion in SF188 and SJ-GBM2 glioma cells was associated with decreased cell proliferation and invasion, along with a reduced ability to grow in soft agar and to form spheres (i.e. self-renewal measure). A 70% reduction in <em>Wnt</em> signaling was also observed in the SF188 and SJ-GBM2 UCHL1 knockdowns (KDs) using a TCF-dependent TOPflash reporter assay. Transcriptome comparisons of UCHL1 KDs versus vector control identified a list of 306 differentially expressed genes (at least 2-fold change; p <0.05) which included genes known to be involved in cancer like ACTA2, POSTN, LIF, FBXL7, FBXW11, GDF15, HEY2, but also potential novel genes such us IGLL5, ABCA4, AQP3, AQP4, CALB1, and ALK. Bioinformatics gene ontology (GO) analysis of these 306 genes revealed significant enrichment in "signal peptides", "extracellular matrix"and "secreted proteins" GO Terms. "Angiogenesis and blood vessel development", "neuron differentiation/development", cell adhesion", and "cell migration" also showed significant enrichment in our GO analysis. Top canonical pathways identified by Ingenuity Pathway Analysis (IPA) included "Clathrin-mediated Endocytosis Signaling" (p = 5.14x10-4), "Virus Entry via Endocytic Pathways" (p = 6.15x 10-4), and "High Mobility Group-Box 1 (HMGB1) Signaling" (p = 6.15x10-4). While FGF2, IL1B, TNF and PDGFB were predicted as top upstream regulators (p < 2x10-16) of the UCHL1 KD-associated transcriptome. Aberrant expression of UCHL1 in pediatric high-grade gliomas may promote cell invasion, transformation, and self-renewal properties, at least in part, by modulating <em>Wnt</em>/Beta catenin activity. UCHL1 might act as an oncogene in glioma within the gene network that imparts stem-like characteristics to these cancer cells.

The role of SRY-related high-mobility-group box (SOX) <em>12</em> in leukaemia progression and haematopoiesis remains elusive. This study aimed to examine the expression and function of SOX<em>12</em> in acute myeloid leukaemia (AML) using human myeloid leukaemia samples and the acute myeloid cell line THP1. Mononuclear cells were isolated from the bone marrow of AML patients and healthy donors. SOX<em>12</em> expression in haematopoietic cells was evaluated by reverse transcription polymerase chain reaction (RT-PCR). SOX<em>12</em> short hairpin RNAs (shRNAs) were transduced into THP1 cells, and gene knockdown was confirmed by quantitative RT-PCR and Western blot analysis. SOX<em>12</em> was preferentially expressed in CD34+ cells in AML patients. The THP1 cells transduced with SOX<em>12</em> shRNAs exhibited significantly reduced SOX<em>12</em> expression and cell proliferation. SOX<em>12</em> knockdown had no effect on apoptosis, but it induced cell cycle arrest at G1 phase and reduced the number of colonies. The transduced THP1 and primary AML cells were reconstituted in non-obese diabetic-severe combined immunodeficient (NOD/SCID) mice, and their numbers were significantly reduced 6-<em>12</em> weeks after transplantation. The mRNA and protein levels of β-catenin were significantly diminished following SOX<em>12</em> knockdown, accompanied by a decrease in TCF/<em>Wnt</em> activity. SOX<em>12</em> may be involved in leukaemia progression by regulating the expression of β-catenin and then interfering with TCF/<em>Wnt</em> pathway, which may be a target for AML.

Solid-pseudopapillary neoplasms are rare, but are distinctive pancreatic tumors of low-malignant potential. While the histogenesis of these tumors is unclear, they are often associated with gain-of-function mutations in the catenin (cadherin-associated protein), beta 1 (88 kDa), or CTNNB1 gene, resulting in nuclear accumulation of CTNNB1. CTNNB1 is a central component of the <em>Wnt</em> signaling pathway and mediates gene expression through the lymphoid enhancer-binding factor 1 (LEF1) /T-cell factor transcription complex. Although LEF1 has a pivotal role in the transactivation of <em>Wnt</em>/CTNNB1 responsive genes, the status of LEF1 in solid-pseudopapillary neoplasms and other pancreatic tumors has not been examined. We analyzed both LEF1 and CTNNB1 in a large cohort of pancreatic tumors (n=155). In all cases of solid-pseudopapillary neoplasms including surgical resections (n=27) and cytologic samples (n=8) had strong and diffuse nuclear labeling for both LEF1 and CTNNB1. The surrounding uninvolved pancreatic parenchyma was devoid of any LEF1 staining. All resection and cytologic specimens from well-differentiated pancreatic neuroendocrine tumors (n=44; n=29, respectively), high-grade pancreatic neuroendocrine carcinomas (n=2; n=1), pancreatic ductal adenocarcinomas (n=25; n=<em>12</em>), and acinar cell carcinomas (n=9; n=2) studied were negative for both nuclear LEF1 and CTNNB1. However, nuclear LEF1 and CTNNB1 were detected in all four resected pancreatoblastomas (no cytologic specimens were available for immunolabeling), but primarily centered around and within squamoid corpuscles. In summary, abnormal CTNNB1 accumulation was accompanied by nuclear LEF1 overexpression in both solid-pseudopapillary neoplasms and pancreatoblastomas. But, in contrast to pancreatoblastomas, a diffuse, nuclear labeling was observed in solid-pseudopapillary neoplasms and further implicates the CTNNB1/LEF1 transcriptional complex in the development of solid-pseudopapillary neoplasms. In addition, as part of an immunohistochemical panel, LEF1 can be a useful ancillary stain in the diagnosis of solid-pseudopapillary neoplasms.

Treatment of osseous defects remains a formidable clinical challenge. Porous titanium alloys (pTi) have been emerging as ideal endosseous implants due to the excellent biocompatibility and structural properties, whereas inadequate osseointegration poses risks for unreliable long-term implant stability. Substantial evidence indicates that pulsed electromagnetic fields (PEMF), as a safe noninvasive method, inhibit osteopenia/osteoporosis experimentally and clinically. We herein investigated the efficiency and potential mechanisms of PEMF on osteogenesis and osseointegration of pTi in vitro and in vivo. We demonstrate that PEMF enhanced cellular attachment and proliferation, and induced well-organized cytoskeleton for in vitro osteoblasts seeded in pTi. PEMF promoted gene expressions in Runx2, OSX, COL-1 and Wnt/β-catenin signaling. PEMF-stimulated group exhibited higher Runx2, Wnt-week PEMF promoted osteogenesis, bone ingrowth and bone formation rate of pTi in rabbit femoral bone defect. PEMF promoted femoral gene expressions of Runx2, BMP2, OCN and Wnt/β-catenin signaling. Together, we demonstrate that PEMF improve osteogenesis and osseointegration of pTi by promoting skeletal anabolic activities through a Wnt/β-catenin signaling-associated mechanism. PEMF might become a promising biophysical modality for enhancing the repair efficiency and quality of pTi in bone defect.

Wnt/β-catenin signaling is known to play an important role in colorectal cancer (CRC). Niclosamide, a salicylamide derivative used in the treatment of tapeworm infections, targets the Wnt/β-catenin pathway. The objective of this study was to investigate niclosamide as a therapeutic agent against CRC.

The antiproliferative effects of 1, 3, 10, and 50 μM concentrations of niclosamide on human (SW480 and SW620) and rodent (CC531) CRC cell lines were determined by MTS assay and direct cell count. The lymphoid enhancer-binding factor 1/transcription factor (LEF/TCF) reporter assay monitored the activity of Wnt signaling. Immunofluorescence staining demonstrated the expression pattern of active β-catenin. Gene expression of canonical and noncanonical Wnt signaling components was analyzed using qRT-PCR. Western blot analysis was performed with antibodies detecting nuclear localization of β-catenin and c-jun.

Cell proliferation in CRC cell lines was blocked dose dependently after 12 and 24 h of incubation. The Wnt promoter activity of LEF/TCF significantly decreased with niclosamide concentrations of 10 and 50 μM after 12 h of incubation. Active β-catenin did not shift from the nuclear to the cytosolic pool. However, canonical target genes (met, MMP7, and cyclin D1) as well as the coactivating factor Bcl9 were downregulated, whereas the noncanonical key player c-jun was clearly activated.

Niclosamide treatment is associated with an inhibitory effect on CRC development and reduced Wnt activity. It may exert its effect by interfering with the nuclear β-catenin-Bcl9-LEF/TCF triple-complex and by upregulation of c-jun representing noncanonical Wnt/JNK signaling. Thus, our findings warrant further research into this substance as a treatment option for patients with advanced CRC.

<AbstractText>Pekin duck is an important animal model for its ability for fat synthesis and deposition. However, transcriptional dynamic regulation of adipose differentiation driven by complex signal cascades remains largely unexplored in this model. This study aimed to explore adipogenic transcriptional dynamics before (proliferation) and after (differentiation) initial preadipocyte differentiation in ducks.</AbstractText><AbstractText>Exogenous oleic acid alone successfully induced duck subcutaneous preadipocyte differentiation. We explored 36 mRNA-seq libraries in order to study transcriptome dynamics during proliferation and differentiation processes at 6 time points. Using robust statistical analysis, we identified 845, 652, 359, 2401 and 1933 genes differentially expressed between -48 h and 0 h, 0 h and <em>12</em> h, <em>12</em> h and 24 h, 24 h and 48 h, 48 h and 72 h, respectively (FDR < 0.05, FC > 1.5). At the proliferation stage, proliferation related pathways and basic cellular and metabolic processes were inhibited, while regulatory factors that initiate differentiation enter the ready-to-activate state, which provides a precondition for initiating adipose differentiation. According to weighted gene co-expression network analysis, pathways positively related to adipogenic differentiation are significantly activated at the differentiation stage, while <em>WNT</em>, FOXO and other pathways that inhibit preadipocyte differentiation are negatively regulated. Moreover, we identified and classified more than 100 transcription factors that showed significant changes during differentiation, and found novel transcription factors that were not reported to be related to preadipoctye differentiation. Finally, we manually assembled a proposed regulation network model of subcutaneous preadipocyte differentiation base on the expression data, and suggested that E2F1 may serve as an important link between the processes of duck subcutaneous preadipocyte proliferation and differentiation.</AbstractText><AbstractText>For the first time we comprehensively analyzed the transcriptome dynamics of duck subcutaneous preadipocyte proliferation and differentiation. The current study provides a solid basis for understanding the synthesis and deposition of subcutaneous fat in ducks. Furthermore, the information generated will allow future investigations of specific genes involved in particular stages of duck adipogenesis.</AbstractText>

Periarticular bone loss in rheumatoid arthritis (RA) is considered to be mainly related to synovial inflammation. However, strong bone loss has also described at the time of arthritis onset. Recently, a paradoxical exacerbation of joint damage was described when blocking sclerostin in various arthritis models. Thus, we aimed to determine kinetics of bone loss and its mechanisms in the adjuvant induced arthritis (AIA) rat model of RA. AIA was induced (n = 35) or not (n = 35) at day 0. In addition to well-known arthritis at day <em>12</em>, we showed with 3D-imaging and histomorphometry that bone microstructural alterations occurred early from day 8 post-induction, characterized by cortical porosity and trabecular bone loss. Active osteoclastic surfaces were increased from day 8 with RANKL upregulation. More surprisingly SOST and DKK1 were overexpressed from day 6 and followed by a dramatic decrease in bone formation from day 8. At the time of arthritis onset, SOST and DKK1 returned to control values, but frizzled related protein 1 (SFRP1), proinflammatory cytokines, and MMPs started to increase. Bone alterations before arthritis onset reinforce the hypothesis of an early bone involvement in arthritis. Kinetics of osteocyte markers expression should be considered to refine <em>Wnt</em> inhibitor treatment strategies.

The highly conserved <em>wnt</em> gene family has roles in developmental processes ranging from axis formation to cell fate determination. The polychaete Platynereis dumerilii has retained <em>12</em> of the 13 ancient <em>wnt</em> subfamilies and is a good model system to study the roles of the <em>wnt</em> ligands in spiralian development. While it has been shown that Platynereis uses a global beta-catenin-mediated binary cell fate specification module in development, the early roles of the <em>12</em> <em>wnt</em> genes present in Platynereis are unknown. Transcriptional profiling by RNA-Seq during early development and whole-mount in situ hybridization of embryo and larval stages were used to determine the temporal and spatial regulation of the <em>wnt</em> complement in Platynereis. None of the <em>12</em> <em>wnt</em> transcripts were maternally provided at significant levels. In pregastrula embryos, zygotic <em>wnt</em>A, <em>wnt</em>4, and <em>wnt</em>5 transcripts exhibited distinctive patterns of differential gene expression. In contrast, in trochophore larvae, all <em>12</em> <em>wnt</em> ligands were expressed and each had a distinct expression pattern. While three <em>wnt</em> ligands were expressed in early development, none were expressed in the right place for a widespread role in beta-catenin-mediated binary specification in early Platynereis development. However, the expression patterns of the <em>wnt</em> ligands suggest the presence of numerous <em>wnt</em> signaling centers, with the most prominent being a bias for staggered posterior <em>wnt</em> expression in trochophore larvae. The similarity to <em>wnt</em> expression domains in cnidarians around the blastopore and the tail organizer in chordates supports a hypothesis of a common evolutionary origin of posterior organizing centers.

Non-ampullary duodenal adenoma with activation of <em>Wnt</em>/β-catenin signalling is common in familial adenomatous polyposis (FAP) patients, whereas sporadic non-ampullary adenoma is uncommon. The adenoma-carcinoma sequence similar to colon cancer is associated with duodenal tumors in FAP, but not always in sporadic tumors. We obtained 37 non-ampullary duodenal tumors, including 25 adenomas and <em>12</em> adenocarcinomas, were obtained from biopsies and endoscopic resections. We performed immunohistochemistry for β-catenin, the hallmark of <em>Wnt</em> activation, and aldehyde dehydrogenase 1 (ALDH1), a putative cancer stem cell marker. In non-ampullary lesions, abnormal nuclear localization of β-catenin was observed in 21 (84.0%) of 25 adenomas and 4 (33.3%) of <em>12</em> adenocarcinomas. In the proximal duodenum, nuclear β-catenin was less frequent in both adenomas and adenocarcinomas. Gastric duodenal metaplasia (GDM) was observed only in the proximal duodenum. All adenomas with GDM were the gastric foveolar and pyloric gland types, and showed only membranous β-catenin. The intestinal-type adenomas had nuclear β-catenin in the proximal and distal duodenum. ALDH1-positive cells were more frequent in adenocarcinomas than adenomas. Nuclear β-catenin accumulation frequently occurred in ALDH1-positive cells in adenoma, but not in adenocarcinoma. In the non-ampullary proximal duodenum, <em>Wnt</em>/β-catenin pathway activation was more closely associated with adenomas than adenocarcinomas, and while it might cooperate with ALDH1 in adenoma, it does not in adenocarcinoma. The pathogenesis thus may differ between sporadic adenoma and adenocarcinoma of non-ampullary duodenal lesions, especially in the proximal and distal duodenum.

With more than 1.4 billion overweight or obese adults worldwide, obesity and progression of the metabolic syndrome are major health and economic challenges. To address mechanisms of obesity, adipose tissue-derived mesenchymal stem cells (ADSCs) are being studied to detail the molecular mechanisms involved in adipogenic differentiation. Activation of the <em>Wnt</em> signalling pathway has inhibited adipogenesis from precursor cells. In our study, we examined this anti-adipogenic effect in further detail stimulating <em>Wnt</em> with lithium chloride (LiCl) and 6-bromo indirubin 3'oxime (BIO). We also examined the effect of <em>Wnt</em> inhibition using secreted frizzled-related protein 4 (sFRP4), which we have previously shown to be pro-apoptotic, anti-angiogenic, and anti-tumorigenic. <em>Wnt</em> stimulation in LiCl and BIO-treated ADSCs resulted in a significant reduction (2.7-fold and <em>12</em>-fold respectively) in lipid accumulation as measured by Oil red O staining while <em>Wnt</em> inhibition with sFRP4 induced a 1.5-fold increase in lipid accumulation. Furthermore, there was significant 1.2-fold increase in peroxisome proliferator-activated receptor gamma (PPARγ) and CCAAT/enhancer binding protein alpha (C/EBPα), and 1.3-fold increase in acetyl CoA carboxylase protein levels. In contrast, the expression of adipogenic proteins (PPARγ, C/EBPα, and acetyl CoA carboxylase) were decreased significantly with LiCl (by 1.6, 2.6, and 1.9-fold respectively) and BIO (by 7, 17, and 5.6-fold respectively) treatments. These investigations demonstrate interplay between <em>Wnt</em> antagonism and <em>Wnt</em> activation during adipogenesis and indicate pathways for therapeutic intervention to control this process.

In the present study, we aimed to investigate the effects of different states of oxidative stress on fetal rat alveolar type II epithelial cells (AEC IIs) cultured in vitro as well as reactive oxygen species (ROS)-induced changes in the <em>Wnt</em> signaling pathway expression. Primary AEC II cultures were randomly divided into three oxidative damage groups: a high oxygen fraction (>0.95) group (95% O2), a low oxygen fraction (0.4) group (40% O2) and a room air group (21% O2). Each group was exposed to the different states of oxidative stress for <em>12</em>, 24 and 48 h. Cell morphological changes were observed using an inverted microscope, the cell survival rate was determined by the MTT assay, and the apoptosis rate was determined using flow cytometry. <em>Wnt</em>5α gene expression was determined using reverse transcriptase‑polymerase chain reaction (RT-PCR) and changes in non-phosphorylated β-catenin protein in the cell nucleus were determined using western blot analysis. Compared with the room air group, the survival and apoptosis rates of the low oxygen fraction (0.4) group were not significantly different after <em>12</em> and 24 h, while significant differences were observed after 48 h of exposure. However, a significant difference was detected in the high oxygen fraction (>0.95) group at all three time-points. After <em>12</em> h, ROS concentration in the high oxygen fraction (>0.95) group was significantly higher compared with the room air group, and continued to rise after 24 h of exposure. Following <em>12</em> h of exposure, the expression of the <em>Wnt</em> signaling pathway in the high volume oxygen fraction (>0.95) group was significantly higher, whereas it decreased after 24 h of exposure. The groups exposed to room air showed no significant difference. In conclusion, with an increase in the time of exposure to oxidative stress, oxidative damage was evident after 48 h even with stimulation with low concentrations of oxygen (40%). The <em>Wnt</em> signaling pathway as an early regulatory factor is involved in hyperoxia lung injury, which is prematurely activated by ROS.

Folate coenzymes are involved in biochemical reactions of one-carbon transfer, and deficiency of this vitamin impairs cellular proliferation, migration, and survival in many cell types. Here, the effect of folate restriction on mammary cancer was evaluated using three distinct breast cancer subtypes differing in their aggressiveness and metastatic potential: noninvasive basal-like (E-<em>Wnt</em>), invasive but minimally metastatic claudin-low (M-<em>Wnt</em>), and highly metastatic claudin-low (metM-<em>Wnt</em>liver) cell lines, each derived from the same pool of MMTV-<em>Wnt</em>-1 transgenic mouse mammary tumors. NMR-based metabolomics was used to quantitate 41 major metabolites in cells grown in folate-free medium versus standard medium. Each cell line demonstrated metabolic reprogramming when grown in folate-free medium. In E-<em>Wnt</em>, M-<em>Wnt</em>, and metM-<em>Wnt</em>liver cells, <em>12</em>, 29, and 25 metabolites, respectively, were significantly different (P < 0.05 and at least 1.5-fold change). The levels of eight metabolites (aspartate, ATP, creatine, creatine phosphate, formate, serine, taurine and β-alanine) were changed in each folate-restricted cell line. Increased glucose, decreased lactate, and inhibition of glycolysis, cellular proliferation, migration, and invasion occurred in M-<em>Wnt</em> and metM-<em>Wnt</em>liver cells (but not E-<em>Wnt</em> cells) grown in folate-free versus standard medium. These effects were accompanied by altered levels of several folate-metabolizing enzymes, indicating that the observed metabolic reprogramming may result from both decreased folate availability and altered folate metabolism. These findings reveal that folate restriction results in metabolic and bioenergetic changes and a less aggressive cancer cell phenotype.

Metabolic reprogramming driven by folate restriction represents a therapeutic target for reducing the burden of breast cancer. Mol Cancer Res; 15(2); 189-200. ©2016 AACR.