Hypoplastic right heart syndrome (HRHS) is a rare congenital defect characterized by underdeveloped and malformed structures of the right heart. Familial recurrence of HRHS indicates genetic factors contribute to its etiology. Our study investigates the presence of copy number variants (CNVs) in HRHS cases. We genotyped 42 HRHS cases identified from live births throughout California (2003-2010) using the Illumina HumanOmni2.5-8 array. We identified 14 candidate CNVs in 14 HRHS cases (33%) based on the genes included in the CNVs and their functions. Duplications overlapping part of ERBB4 were identified in two unrelated cases. ERBB4 is a neuregulin receptor with a pivotal role in cardiomyocyte differentiation and heart development. We also described a 7.5 Mb duplication at 16q11-<em>12</em>. Multiple genes in the duplicated region have previously been linked to heart defects and cardiac development, including RPGRIP1L, RBL2, SALL1, and MYLK3. Of the 14 validated CNVs, we identified four CNVs in close proximity to genes linked to the <em>Wnt</em> signaling pathway. This study expands on our previous work supporting the role of genetics in HRHS. We identified CNVs affecting crucial genes and signaling pathways involved in right heart development. ERBB4 and duplication of the 16q11-<em>12</em> region are important areas for future investigation.

The inbred mouse strain RIII has long been known for shedding large amounts of mouse mammary tumor virus (MMTV) particles in milk and for the development of hormone-dependent early mammary tumors at a very high incidence (>90%). We have established one RIII subline (RIII/Sa) that shows a pattern of virus expression and tumor incidence similar to that in RIII mice. In the present study, we analyzed the milk and mammary tumors of RIII/Sa mice for virus characterization by reverse transcriptase PCR (RT-PCR) cloning and sequencing of the open reading frame (ORF) of the MMTV long terminal repeats (LTRs). Our results show that these mice express a mixture of at least three different MMTV strains, two of which, designated here as RIII/Sa MMTV-1 and RIII/Sa MMTV-2, are exogenous. The third virus, RIII/Sa MMTV-3, appears to carry the signature of an endogenous provirus, Mtv-17. Similar studies done with the milk and mammary glands of another subline, RIIIS/J, revealed that they do not express MMTV in their milk. The RIII/Sa and RIIIS/J mice also exhibited differences in their endogenous proviral contents. Twelve spontaneously developed mammary tumors of RIII/Sa mice were examined for possible <em>Wnt</em>-1 and/or int-2/Fgf3 mutations that are usually found to occur in most mouse mammary tumors as a consequence of MMTV proviral integration. This work led to the isolation of one MMTV-<em>Wnt</em>-1 junction fragment and one MMTV-int-2/Fgf3 junction fragment from 2 of the <em>12</em> tumors. Further analyses showed that both junction fragments contained the RIII/Sa MMTV-2-specific LTR ORF, indicating that this virus was involved in the development of both tumors. Whether RIII/Sa MMTV-1 and/or RIII/Sa MMTV-3 plays any role in mammary tumor development in RIII/Sa mice remains to be established. Overall, the present study demonstrates, to our surprise, that (i) RIII/Sa mice express, unlike other native mouse strains, three strains of MMTVs; and (ii) the virions are completely different from the virus expressed by another subline of RIII mice, the BR6 mice.

The regulation of early B cell development and the interaction of hematopoietic precursors with stromal cells in the bone marrow (BM) are controlled by various secreted signaling molecules. Several recent studies showed <em>Wnt</em> signaling involved in B-lymphogenesis through stromal cells. However, the molecules modulated by <em>Wnt</em> signaling in stromal cells regulating B-lymphogenesis have not been identified yet. Interleukin (IL)-7 and CXC chemokine ligand (CXCL) <em>12</em> are known to be express in stromal cells, and both molecules are essential for B-lymphogenesis. In the present study, we examined the role of <em>Wnt</em> signaling in regulating IL-7 and CXCL<em>12</em> expression and in affecting B-lymphogenesis. In mouse stromal ST2 cells, expression of IL-7 and CXCL<em>12</em> mRNA was augmented by noncanonical <em>Wnt</em>5a. When mouse BM-derived cells were cultured on <em>Wnt</em>5a-overexpressing ST2 cells, an increased number of B220+/IgM- B-lymphoid precursor cells was observed. These results show that <em>Wnt</em>5a regulates IL-7 gene expression in stromal cells and suggest the possibility that noncanonical <em>Wnt</em> regulates B-lymphogenesis via IL-7 expression in stromal cells.

OBJECTIVE

Chibby (CBY), a β-catenin binding partner, inhibits Wnt/β-catenin-mediated transcriptional activation by competing with Tcf/Lef factors for β-catenin binding and promoting the export of β-catenin from nucleus to cytoplasm. The regulatory effect of CBY in this signaling pathway suggests its biological importance as a potential tumor suppressor gene. The purposes of this study were to determine whether the expression of CBY was downregulated in human laryngeal squamous cell carcinoma (LSCC) samples, the CpG sites of CBY at the promoter region were methylated in these tumor samples, and reduced expression of CBY was induced by methylation of CBY promoters.

METHODS

CBY expression was investigated by quantitative real-time PCR and immunohistochemistry in samples from 36 LSCC patients. The methylation status of the CBY promoter was detected by methylation-specific PCR.

RESULTS

Compared with normal laryngeal mucosa, the expression of CBY was downregulated in LSCC samples. The reduced CBY expression rate was 58.33% (21/36) at the mRNA and 66.67% (24/36) at the protein level. The promoters of CBY were methylated in 12/36 tumor samples, partially methylated in 5, and unmethylated in 19 samples. The methylation rate including incomplete methylation was 47.22% (17/36) in tumor samples, while no methylation was detected in normal laryngeal squamous epithelium. Compared with the unmethylated group, the expression of CBY was significantly different in the methylated group (p<0.05) but similar in the partially methylated group (p>0.05).

CONCLUSIONS

Our data indicate that CBY expression was downregulated in LSCC, which may be partially caused by methylation of CBY promoters.

The prospective ventral uterus of the hermaphrodite gonad primordium consists of two pairs of sister cells, with each pair consisting of a proximal "α" cell and a distal "β" cell. All four cells initially are competent to become the anchor cell (AC), a unique cell type that acts as the organizer of subsequent uterine and vulval development. However, the β cells soon lose this competence and always become ventral uterine precursor cells (VUs), whereas the α cells maintain their AC competence longer, until lin-<em>12</em>/Notch-mediated interactions between them specify one as the AC and the other as a VU. Here, we investigate this asymmetry in developmental potential and VU fate specification between the α and β sister cells. We find evidence that lin-<em>12</em> activity contributes to the robustness of βVU fate at elevated temperature, that the Caenorhabditis elegans Notch paralog glp-1 is not functionally redundant with lin-<em>12</em> in specifying βVU fate, and that the activity of POP-1, the sole C. elegans TCF ortholog, influences βVU fate. We propose a model for how <em>Wnt</em> and LIN-<em>12</em>/Notch signaling together lead to robust specification of the βVU fate.

BACKGROUND

During development, different signaling pathways interact to specify cell fate by regulating transcription factors necessary for fate specification and morphogenesis. In Caenorhabditis elegans, the EGF-Ras and <em>Wnt</em> signaling pathways have been shown to interact to specify cell fate in three equivalence groups: the vulval precursor cells (VPCs), the hook competence group (HCG) and P11/<em>12</em>. In the VPCs, HCG and P11/<em>12</em> pair, EGF and <em>Wnt</em> signaling positively regulate different Hox genes, each of which also functions during fate specification. In the male, EGF-Ras signaling is required to specify the Bgamma fate within the Bgamma/delta equivalence pair, while Notch signaling is required for Bdelta fate specification. In addition, TGF-beta signaling by dbl-1/dpp controls ceh-13/labial/Hox1 expression in Bgamma.

RESULTS

We show that EGF-Ras signaling is required for Bgamma expression of ceh-13/labial/Hox1. The transcription factors lin-1/ETS and lin-31/Forkhead, functioning downstream of the EGF pathway, as well as sur-2/MED23 (a component of the Mediator complex) also control ceh-13 expression in Bgamma. In addition, our results indicate that lin-44/<em>Wnt</em>, mom-2/<em>Wnt</em> and lin-17/Fz are necessary to maintain the division of Bgamma along a longitudinal axis. We also show that dbl-1/dpp acts either in parallel or downstream of EGF pathway to regulate ceh-13/Hox1 expression in Bgamma. Lastly, we find that a dbl-1/dpp null mutation did not cause any vulval or P<em>12</em> defects and did not enhance vulval and P<em>12</em> defects of reduction-of-function mutations of components of the EGF pathway.

CONCLUSIONS

ceh-13/labial/Hox1 expression in Bgamma is regulated by the EGF pathway and downstream factors lin-1/ETS lin-31/Forkhead and sur-2/MED23. <em>Wnt</em> signaling is required for proper Bgamma division, perhaps to orient the Bgamma mitotic spindle. Our results suggest that dbl-1/dpp is not required for VPC and P<em>12</em> specification, highlighting another difference among these EGF-dependent equivalence groups.

Myricetin is a natural dietary flavonoid compound. We evaluated the efficacy of myricetin against intestinal tumorigenesis in adenomatous polyposis coli multiple intestinal neoplasia (APCMin/+) mice. Myricetin was given orally once a day for <em>12</em> consecutive weeks. APCMin/+ mice fed with myricetin developed fewer and smaller polyps without any adverse effects. Histopathological analysis showed a decreased number of dysplastic cells and degree of dysplasia in each polyp. Immunohistochemical and western blot analysis revealed that myricetin selectively inhibits cell proliferation and induces apoptosis in adenomatous polyps. The effects of myricetin were associated with a modulation the GSK-3β and <em>Wnt</em>/β-catenin pathways. ELISA analysis showed a reduced concentration of pro-inflammatory cytokines IL-6 and PGE2 in blood, which were elevated in APCMin/+ mice. The effect of myricetin treatment was more prominent in the adenomatous polyps originating in the colon. Further studies showed that myricetin downregulates the phosphorylated p38 MAPK/Akt/mTOR signaling pathways, which may be the mechanisms for the inhibition of adenomatous polyps by myricetin. Taken together, our data show that myricetin inhibits intestinal tumorigenesis through a collection of biological activities. Given these results, we suggest that myricetin could be used preventatively to reduce the risk of developing colon cancers.

<em>Wnt</em> signaling is a key developmental pathway that regulates dopaminergic progenitor cell proliferation and differentiation during neuronal development. This makes <em>Wnt</em> signaling an important therapeutic target for neurodegenerative conditions such as Parkinson's disease. <em>Wnt</em> signaling can be modulated using peptides such as UM206, which bind to the <em>Wnt</em> receptor Frizzled. Previous work has demonstrated remote activation of the <em>Wnt</em> pathway through Frizzled using peptide-functionalized magnetic nanoparticles (MNPs) with magnetic field stimulation. Using this technology, <em>Wnt</em> signaling is remotely activated in the neuronal cell line SH-SY5Y, and the phenotypic response to stimulation is assessed. Results indicate β-catenin translocalization and activation of TCF/LEF responsive transcription in response to MNP and magnetic fields, which result in dopaminergic marker expression when synergistically combined with differentiation factors retinoic acid and the phorbol ester phorbol <em>12</em>-myristate 13-acetate. This approach is translated into ex vivo postnatal rat brain slices modeling the developing nigrostriatal pathway. Dopaminergic marker expression is maintained in MNP-labeled SH-SY5Y cells after injection and magnetic stimulation. These results demonstrate the translational value of remote control of signal transduction for controlling neuronal precursor cell behavior and highlight the potential applications for controlled cell differentiation as part of cell therapies for neurodegenerative disease.

Keywords: SH-SY5Y; Wnt signaling; magnetic nanoparticles; neuronal differentiation.

<AbstractText>To explore the action mechanism of electroacupuncture (EA) for knee osteoarthritis (KOA) based on <em>Wnt</em>/beta-catenin (<em>Wnt</em>/β-catenin) signaling pathway.</AbstractText><AbstractText>Ten rats were randomly selected into a sham-operation group among 50 male 2-month-old SD rats, and the KOA model was established in the remaining 40 rats by modified Hulth method. Four weeks after the model establishment, the rats were randomly divided into a model group, an experimental A group, an experimental B group and an experimental C group, 10 rats in each group. The rats in the sham-operation group and model group did not receive any intervention. The rats in the experimental A group were treated with EA at "Neixiyan" (EX-LE 4) and "Dubi"(ST 35) for 15 min. The rats in the experimental B group were treated with EA at "Neixiyan" (EX-LE 4) and "Dubi"(ST 35) for 30 min. The rats in the experimental C group were treated with EA at non-acupoint for 15 min. EA intervention was given once a day, five times a week, and totally <em>12</em>-week treatment was given. After <em>12</em> weeks, the knee cartilage tissues were stained and the morphological changes were observed under light microscopy; the severity of cartilage degeneration was evaluated by modified Mankin's score; the content of interleukin-1β (IL-1β) in synovium tissues was detected by ELISA method; the content of <em>Wnt</em>-4, β-catenin and matrix metalloprotein-13 (MMP-13) in cartilage tissues was detected by Western blot method.</AbstractText><p><div><b>RESULTS</b></div>Compared with the sham-operation group, in the model group the morphology and structure of cartilage were disordered, the number of cells was significantly reduced, the matrix was decontaminated and tidal line was incomplete; the Mankin's score was significantly increased (<i>P</i><0.01), the content of IL-1β in synovium tissues were significantly increased (<i>P</i><0.01), and the expressions of <em>Wnt</em>-4, β-catenin and MMP-13 at protein level were significantly increased (<i>P</i><0.01). Compared with the model group, in the experimental A group and experimental B group the morphology and structure of cartilage were more orderly, the number of cells was increased, the matrix staining was deepened and the tidal line was more complete; Mankin's scores were decreased significantly (<i>P</i><0.01); the contents of IL-1β in synovium tissues were decreased significantly (<i>P</i><0.01), and the expressions of <em>Wnt</em>-4, β-catenin and MMP-13 at protein level were decreased significantly (<i>P</i><0.01). Compared with the model group, no improvement was observed in the experimental C group. Compared with the experimental A group, in the experimental C group the morphology and structure of cartilage were disordered, the number of cells was significantly reduced, the matrix was decontaminated and the tidal line was incomplete; Mankin's score was significantly increased (<i>P</i><0.01), the content of IL-1β in synovium tissues was significantly increased (<i>P</i><0.01), and the expressions of <em>Wnt</em>-4, β-catenin and MMP-13 at protein level were significantly increased (<i>P</i><0.01). The morphological structure of cartilage in the experimental B group was similar to that in the experimental A group, and there was no significant difference in Mankin's score, IL-1β content in synovium tissues and the expressions of <em>Wnt</em>-4, β- catenin and MMP-13 at protein level between the two groups (<i>P</i>>0.05).</p><AbstractText>EA at "Neixiyan" (EX-LE 4) and "Dubi"(ST 35) may reduce the expression of MMP-13 and the production of inflammatory factor IL-1β through <em>Wnt</em>/β-catenin signaling pathway, thus inhibit the degradation of cartilage matrix and the apoptosis of chondrocyte, and improve the morphology and structure of cartilage.</AbstractText>

Diabetes mellitus in pregnancy has been known to affect the embryonic development of various systems, including cardiovascular and nervous systems. However, whether this disease could have a negative impact on embryonic respiratory system remains controversial. In this study, we demonstrated that pregestational diabetes mellitus (PGDM)-induced defects in lung development in mice are mainly characterized by the changes in the morphological structure of the lung. Immunostaining and Western blotting showed that proliferation increased and apoptosis decreased in PGDM. Hyperglycaemia caused pulmonary tissue fibrationas manifested by an increase in Masson staining and decorin expression in PGDM lungs, and the immunofluorescent pro-SPC+ type II pulmonary epithelial cell number was decreased. The alteration of pulmonary epithelial cell differentiation might be due to hyperglycaemia-activated <em>Wnt</em> signalling and suppressed GATA6 expression in PGDM mouse lung tissues and MLE-<em>12</em> cells. The treatment of MLE-<em>12</em> cells with high glucose in the presence/absence of XAV939 or su5402 further proved that hyperglycaemia suppressed the expression of GATA6 and pro-SPC by activating <em>Wnt</em> signalling and induced the expression of decorin, α-SMA and TGF-β by activating Fgf signalling. Therefore, in this study, we revealed that hyperglycemia induced dysfunctional pulmonary cell apoptosis and proliferation, as well as pulmonary myofibroblast hyperplasia, which contributed to the formation of aberrant structure of alveolar walls. Furthermore, the hyperglycaemia also inhibited the differentiation of pulmonary epithelial cells through the canonical <em>Wnt</em> and Fgf signalling, and the alteration of Fgf and <em>Wnt</em> signalling activated TGF-β, which would promote the AECII EMT process.

Recent studies have shown a role for miRNAs in aging and age-related diseases, and the modulation of miRNA expression by diet attracts attention as a new therapeutic strategy. Here, we focused on identifying specific exosomal miRNAs derived from serum of aged rats and the effect of short-term calorie restriction (CR) on their expression. Exosomes from serum of young (7-month), old (22-month), and old-CR Sprague Dawley rats were isolated and characterized by transmission electron microscopy analyses, dynamic light scattering measurements, and Western blotting. A total of <em>12</em> significantly expressed miRNAs in serum exosomes of young and old rats were identified by next generation sequencing. After analysis of qRT-PCR, we found that miR-500-3p and miR-770-3p expression was significantly upregulated by aging and downregulated by CR. Furthermore, receiver operating characteristic (ROC) curve revealed that the selected miRNAs represented high accuracy in discriminating old rats from young rats. Finally, PANTHER analysis predicted selected miRNAs targets genes involved in <em>Wnt</em>/chemokines and cytokines -related inflammatory signaling pathway and function as transcription factor. In conclusion, our results suggest that the expression of serum exosomal miR-500-3p and miR-770-3p was significantly increased with aging, whereas these were decreased by CR, and age-/CR-modulated exosomal miR-500-3p and miR-770-3p could potentially be used as informative biomarkers candidates for aging.

BACKGROUND

Trans-10, cis-<em>12</em> (t10-c<em>12</em>) CLA treatment reduces lipid accumulation in differentiating mouse and human adipocytes, and decreases fat mass in mice, yet the mechanism of action remains unknown.

OBJECTIVE

This study investigated the effect of the cis-9, trans-11 (c9-t11) and t10-c<em>12</em> CLA isomers on the Wnt/β-catenin pathway, which has been reported to inhibit adipogenesis by down-regulating PPARγ.

RESULTS

We observed that t10-c<em>12</em> CLA treatment of 3T3-L1 adipocytes increases the levels of β-catenin and Ser-675 phosphorylated β-catenin due to inhibition of its degradation. These changes in β-catenin were not linked to either the Wnt/β-catenin agonist Wnt10b or other upstream effectors such as SFRP-5. Paradoxically, the presence of higher amounts of β-catenin did not elevate cyclin D1 levels, which is recognized as a critical target gene. Neither of the CLA isomers affected the localization of β-catenin in the cytosol and nucleus as determined by immunofluorescence microscopy. However, subcellular fractionation suggested the level of cytosolic β-catenin was reduced in t10-c<em>12</em> CLA treated cells. Immunoprecipitation revealed that t10-c<em>12</em> CLA increased the interaction of β-catenin and PPARγ.

CONCLUSIONS

t10-c<em>12</em>-CLA inhibits adipocyte differentiation by increasing β-catenin stability in 3T3-L1 adipocytes, thus enhancing sequestration of PPARγ in an inactive complex, which prevents progression of adipogenesis.

Background: The prognosis of adrenocortical carcinoma (ACC) is heterogeneous. Genomic studies have identified ACC subgroups characterized by specific molecular alterations, including features measured at DNA level (somatic mutations, chromosome alterations, DNA methylation), which are closely associated with outcome. The aim of this study was to evaluate intratumor heterogeneity of prognostic molecular markers at the DNA level.

<strong class="sub-title"> Methods: </strong> Two different tissue samples (primary tumor, local recurrence or metastasis) were analyzed in 26 patients who underwent surgery for primary or recurrent ACC. DNA-related biomarkers with prognostic role were investigated in frozen and paraffin-embedded samples. Somatic mutations of p53/Rb and <em>Wnt</em>/β-catenin pathways were assessed using next-generation sequencing (n=26), chromosome alteration profiles were determined using SNP arrays (n=14) and methylation profiles were determined using 4-gene bisulfite pyrosequencing (n=<em>12</em>).

<strong class="sub-title"> Results: </strong> Somatic mutations for ZNRF3, TP53, CTNN1B and CDKN2A were found in 7, 6, 6 and 4 patients respectively, with intratumor heterogeneity in 8/26 patients (31%). Chromosome alteration profiles were 'Noisy' (numerous and anarchic alterations) in 8/14 and 'Chromosomal' (extended patterns of loss of heterozygosity) in 5/14 of the study samples. For these profiles, no intratumor heterogeneity was observed. Methylation profiles were hypermethylated in 5/<em>12</em> and non-hypermethylated in 7/<em>12</em> of the study samples. Intratumor heterogeneity of methylation profiles was observed in 2/<em>12</em> patients (17%).

Conclusions: Intratumor heterogeneity impacts DNA-related molecular markers. While somatic mutation can differ, prognostic DNA methylation and chromosome alteration profile seem rather stable, and might be more robust for the prognostic assessment.

Prohibitin1 (<i>PHB1</i>) is a mitochondrial chaperone with diverse functions that include cell proliferation, apoptosis, and mitochondrial homoeostasis. Liver-specific <i>Phb1</i> knockout (KO) mice develop spontaneous injury and hepatocellular carcinoma (HCC). Our previous work demonstrated that PHB1 negatively regulates the H19-insulin-like growth factor 2 (IGF2)-H19-IGF2 axis signaling pathway and E-box activity in hepatocytes and HCC cells. <i>Phb1</i> KO livers exhibited increased expression of multiple wingless/integrated (<em>WNT</em>) target genes compared to control littermates. Therefore, we hypothesized that PHB1 is a negative regulator of <em>WNT</em>-beta-catenin signaling in the liver. Analysis of livers from <i>Phb1</i> KO mice demonstrated an activation of the <em>WNT</em>-beta-catenin pathway as determined by phosphorylation of glycogen synthase kinase 3 (GSK3)beta^{serine [Ser]9} and protein kinase B (AKT)^Ser473. <i>Phb1</i> KO livers showed increased messenger RNA (mRNA) levels of multiple <em>WNT</em> ligands, with <i>Wnt7a</i> (79-fold), <i>Wnt10a</i> (<em>12</em>-fold), and <i>Wnt16</i> (48-fold) being most highly overexpressed compared to control littermates. Subcellular fractionation of liver cells from <i>Phb1</i> KO mice indicated that hepatocytes are the main source of <em>WNT</em> ligands. Immunostaining and cellular colocalization analysis of <i>Phb1</i> KO livers demonstrated expression of <em>WNT</em>7a, <em>WNT</em>10a, and <em>WNT</em>16 in hepatocytes. Chromatin immunoprecipitation revealed increased binding of transcription factor E2F1 (E2F1) to the <i>Wnt10a</i> promoter in <i>Phb1</i> KO livers and <i><em>WNT</em>9A</i> in HepG2 cells. <i>PHB1</i> silencing in HepG2 cells activated <em>WNT</em> signaling, whereas its overexpression caused inactivation of this pathway. <i>PHB1</i> silencing in HepG2 cells induced the expression of multiple <em>WNT</em> ligands of which <i><em>WNT</em>9A</i> induction was partly regulated through E2F1. <i>Conclusion:</i> PHB1 acts as a negative regulator of <em>WNT</em> signaling, and its down-regulation causes the induction of multiple <em>WNT</em> ligands and downstream activation of canonical <em>WNT</em>-beta-catenin signaling in murine liver and human HCC cells, in part through E2F1.

Glucocorticoids-induced osteonecrosis of femoral head (GIONFH) is difficult to treat due to the pathophysiology remains uncertain. Core decompression and bone grafting are regarded as effective measures for treating early GIONFH. Furthermore, commonly used bone graft materials at nowadays are still unsatisfactory. We generated a novel calcium polyphosphate (CPP) composite scaffold, which contains Li and VEGF-loaded gelatin microspheres (LiCPP/GMs/VEGF). The LiCPP/GMs/VEGF scaffold also demonstrated a porosity of 70.5% ± 2.3% with interconnected porous structures, and pore sizes of 100-200 μm and compressive strength of 3.7 MPa. Additionally, bone marrow mesenchymal stem cells (BMSCs) were seeded on scaffolds in vitro. Further characterization showed that the LiCPP/GMs/VEGF scaffolds were biocompatible and enhanced osteogenesis and angiogenesis in vitro. Using a rabbit model of GIONFH, LiCPP/GMs/VEGF scaffolds were implanted into the bone tunnels of core decompression in the femoral head for <em>12</em> weeks. Radiographic, histological analysis and western blot analysis demonstrated that the LiCPP/GMs/VEGF scaffolds exhibited good biocompatibility, and osteogenic and angiogenic activity in vivo. Besides, the osteogenic and angiogenic factors were increased along with VEGF release and the activation of factors in <em>Wnt</em> signal pathway. In conclusion, the LiCPP/GMs/VEGF scaffold has an effect on improving the osteogenesis and angiogenesis, which benefited to repair the GIONFH.

BACKGROUND

Studies indicated that Mer might be the main contributor to the specific internalization of photoreceptor outer segments (POS) in retinal pigment epithelium (RPE). It is very important to understand the mechanism of POS phagocytosis under the pathway of Mer and its ligands. The objective of this study was to identify changes in gene expression profiles caused by Mer gene knockout (Mer-/-) during phagocytosis of POS in RPE.

METHODS

RPE from both Mer-/- and wild-type (WT) mice were isolated and cultured to the 3rd passage. POS were subjected to culture medium with 20 nmol/L Gas6 and protein S to activate specific mer-mediated phagocytosis. RPE phagocytosis was evaluated by phagocytosis assays and differential gene expression identified by microarray at 3 and <em>12</em> hours; the 0-hour time point served as the control. Three independent samples for each Mer-/- or WT RPE were subjected to the same protocol of microarray. Five genes were confirmed by real-time quantitative PCR (QPCR).

RESULTS

The Mer-/- RPE had less internalized POS than WT RPE after both 3 and <em>12</em> hours in phagocytosis assay. Compared to WT RPE and the 0-hour control, 38 and 45 different known genes were increased and 68 and 59 known genes were decreased in Mer-/- RPE after 3 and <em>12</em> hours, respectively. Abnormal POS phagocytosis in Mer-/- RPE was associated with significant gene expression changes in, for example, signal transduction (WNT, MAPK), phagocytosis (Vav3, Hsd11b1), cytoskeleton components (Myo7a), and metabolism, in a time-specific manner. QPCR results showed Vav3, Hsd11b1, Myo7a, Rtn2 and Itga8 in those independent samples were consistent with microarray.

CONCLUSIONS

Gene expression profiles modulated in a time-specific manner in Mer-/- RPE indicate a possible internalization mechanism for abnormal POS phagocytosis, which gives insight into the mechanism of retinitis pigmentosa caused by the mutation of MerTK in humans.

Traumatic brain injury (TBI) and autism spectrum disorder (ASDs) share several same biochemical mechanisms and symptoms, such as learning memory impairments and communication deficits. Chromodomain helicase DNA binding protein 8 (CHD8), a member of the CHD family of ATP-dependent chromatin remodeling factors, is one of the top risk genetic factors in ASDs and is highly associated with <em>Wnt</em>/β-catenin signaling. Yet, the possible effect of CHD8 on TBI remains poorly understood. In vivo, we found that Chd8 co-localized in neurons, astrocytes, and microglia, but predominantly presented in neurons in the prefrontal cortex, hippocampus, and cortex. Both Chd8 and β-catenin expression peaked at <em>12</em> h and shared the similar change tendency after TBI. Chd8 knockdown inhibited <em>wnt</em> pathway, promoted the activation of apoptosis and autophagy, and caused learning and memory impairments both at normal and TBI condition. In addition, overexpression of Chd8 via 17β-estrogen (E2) treatment enhanced <em>wnt</em> signaling pathway and suppressed TBI-induced apoptosis and autophagic activation. In vitro, a significant increase of Chd8 and β-catenin expression was observed in HT22 cells after lipopolysaccharide (lps) treatment or mechanical injury, respectively. Chd8 knockdown inhibited <em>wnt</em> signaling pathway and increased apoptosis and autophagy activation in lps-stimulated HT22 cells. But activation of <em>wnt</em> signaling inverted the effects of Chd8-siRNA. Our results demonstrated that Chd8 exerted neuroprotection and promoted cognitive recovery through inhibiting apoptosis and autophagy activation following TBI, at least partially by <em>wnt</em> signaling pathway.

BACKGROUND

Radiation-induced skin injury is a dose-limiting complication of radiotherapy. To investigate this problem and to develop a framework for making decisions on treatment and dose prescription, a murine model of radiation-induced skin injury was developed.

METHODS

The dorsal skin of the mice was isolated, and irradiation was applied at single doses of 15, 30, and 50 Gy. The mice were followed for <em>12</em> weeks with serial photography and laser Doppler analysis. Sequential skin biopsy samples were obtained and subjected to a histological analysis, immunostaining against transforming growth factor beta (TGF-β), and Western blotting with <em>Wnt</em>-3 and β-catenin. Increases in the levels of TGF-β, <em>Wnt</em>, and β-catenin were detected after irradiation.

RESULTS

All tested radiation doses caused progressive dermal thickening and fibrosis. The cause of this process, however, may not be radiation alone, as the natural course of wound healing may elicit a similar response. The latent appearance of molecular and histological markers that induce fibrosis in the 15 Gy group without causing apparent gross skin injuries indicates that 15 Gy is an appropriate dose for characterizing the effects of chronic irradiation alone. Thus, this model best mimics the patterns of injury that occur in human subjects.

CONCLUSIONS

This animal model can be used to elucidate the gross and molecular changes that occur in radiation-induced skin injury and provides an effective platform for studying this adverse effect without complicating the process of wound healing.

The <em>Wnt</em>-LPR5 signalling pathway plays an important role in skeletal homeostasis, especially in regulating osteoblastic activity. Activation of this pathway is regulated by various inhibitors, including DKK1. The objective of this study was to evaluate DKK1 levels in patients with ischemic heart disease, the response to atorvastatin and the relationship with bone mass. Twenty-one patients with acute myocardial infarction and twenty-three controls with a mean age of 61 ± 9 years with acute coronary syndrome were included. Patients were allocated to low (10-20mg) and high doses (40-80 mg) of atorvastatin according to baseline levels of cholesterol and triglycerides and the index of vascular risk. Patients were studied at hospital admission (baseline) and at <em>12</em> months of follow up. DKK1 was determined in all patients at baseline and at <em>12</em> months of follow up. Densitometric studies were conducted in the lumbar spine (L2-L4) and the femoral neck and trochanter using an X-ray densitometer. Patients had higher levels of DKK1 than controls, (111 ± 41 nmol/l versus 84 ± 27 nmol/l, p=0.014). Osteoporotic patients had higher levels of DKK1 (137.5 ± 33 nmol/l versus 95.4 ± 36 nmol/l, p=0.021). Analysis of the response to atorvastatin showed reduced DKK1 levels. In conclusion, in patients with acute coronary syndrome, atorvastatin decreases DKK1 levels. This may be a previously unreported mechanism of action of atorvastatin on bone, stimulating the <em>Wnt</em> signalling pathway and increasing bone mass.

Somatic mutations in 3'-untranslated regions (3'UTR) do not alter amino acids and are considered to be silent in cancers. We found that such mutations can promote tumor progression by altering microRNA (miRNA) targeting efficiency and consequently affecting miRNA⁻mRNA interactions. We identified 67,159 somatic mutations located in the 3'UTRs of messenger RNAs (mRNAs) which can alter miRNA⁻mRNA interactions (functional somatic mutations, funcMutations), and 69.3% of these funcMutations (the degree of energy change>> <em>12</em> kcal/mol) were identified to significantly promote loss of miRNA-mRNA binding. By integrating mRNA expression profiles of 21 cancer types, we found that the expression of target genes was positively correlated with the loss of absolute affinity level and negatively correlated with the gain of absolute affinity level. Functional enrichment analysis revealed that genes carrying funcMutations were significantly enriched in the MAPK and <em>WNT</em> signaling pathways, and analysis of regulatory modules identified eighteen miRNA modules involved with similar cellular functions. Our findings elucidate a complex relationship between miRNA, mRNA, and mutations, and suggest that 3'UTR mutations may play an important role in tumor development.

Objective: To explore effect of alprostadil on wound healing of scalded rats and the mechanism. Methods: According to random number table method, forty-eight Sprague Dawley rats were divided into sham scald group, simple scald group, lithium chloride group, and alprostadil group, with <em>12</em> rats in each group. Rats in sham injury group were sham injured on the back, and rats in the other three groups were inflicted with 30% total body surface area deep partial thickness scald on the back.Immediately after scald, rats in sham scald group and simple scald group were injected with 1 mL saline through caudal vein, and rats in lithium chloride group and alprostadil group were injected respectively with 1 mL lithium chloride and alprostadil through caudal vein. Saline, lithium chloride, and alprostadil were injected once in a day and lasted for 14 days. General wound appearance and wound healing rate on post scald day (PSD) 7, 10, 14 were observed and calculated. Expressions of protein and mRNA of <em>Wnt</em>1 and β-catenin on PSD 14 were detected. Data were processed with analysis of variance of factorial design, one-way analysis of variance, Student Newman Keuls q test, t test, and Bonferroni correction. Results: (1) On PSD 7, wounds of scalded rats in each group formed dry eschar and had little exudation. On PSD 10, wounds of rats in simple scald group were covered with eschar, with little exudation, and wounds of rats in lithium chloride group were covered with eschar, and partial wounds healed under the eschar. On PSD 10, partial eschar of rats in alprostadil group desquamated; partial wounds healed; newly burned skin was ruddy. On PSD 14, partial wounds of rats in simple scald group were healed under eschar with little exudation. On PSD 14, most of the eschar of rats in lithium chloride group were desquamated with patial wounds healed and little exudation. On PSD 14, wounds of rats in alprostadil group were basically healed with vigorously growing hair on the back. (2) On PSD 7, the wound healing rates of rats in simple scald group, lithium chloride group, and alprostadil group were close (F=0.41, P>0.05). On PSD 10 and 14, wound healing rate of rats in lithium chloride group and alprostadil group were significantly higher than that in simple scald group (q=5.73, 17.45, 26.30, 11.28, P<0.05), and wound healing rate of rats in alprostadil group was significantly higher than that in lithium chloride group (q=32.03, 28.73, P<0.05). (3) On PSD 14, the mRNA expressions of <em>Wnt</em>1 and β-catenin of rats in lithium chloride group and alprostadil group were significantly higher than those in simple scald group (q=65.40, 19.16, 66.79, 18.41, P<0.05), and the mRNA expressions of <em>Wnt</em>1 and β-catenin of rats in simple scald group was significantly higher than those in sham scald group (t=14.86, 4.46, P<0.05). (4) On PSD 14, the protein expressions of <em>Wnt</em>1 and β-catenin of rats in lithium chloride group and alprostadil group were 0.98±0.05, 0.98±0.06, 0.97±0.06, and 1.00±0.06, which were significantly higher than 0.49±0.04 and 0.66±0.04 of rats in simple scald group (q=34.62, 22.38, 33.61, 23.47, P<0.05). On PSD 14, the protein expressions of <em>Wnt</em>1 and β-catenin of rats in simple scald group was significantly higher than 0.29±0.03 and 0.31±0.03 of rats in sham scald group (q=14.73, 23.88, P<0.05). Conclusions: Alprostadil can accelerate wound healing through activating <em>Wnt</em>/β-catenin signal pathway and upregulating the expressions of <em>Wnt</em>1 and β-catenin.

Previously, we showed that cortical mineralization is coordinately adjusted to mechanically offset external bone size differences between A/J (narrow) and C57BL/6J (wide) mouse femora to achieve whole bone strength equivalence at adulthood. The identity of the genes and their interactions that are responsible for establishing this homeostatic state (ie, canalization) remain unknown. We hypothesize that these inbred strains, whose interindividual differences in bone structure and material properties mimic that observed among humans, achieve functional homeostasis by differentially adjusting key molecular pathways regulating external bone size and mineralization throughout growth. The cortices of A/J and C57BL/6J male mouse femora were phenotyped and gene expression levels were assessed across growth (ie, ages 2, 4, 6, 8, <em>12</em>, 16 weeks). A difference in total cross-sectional area (p < 0.01) and cortical tissue mineral density were apparent between mouse strains by age 2 weeks and maintained at adulthood (p < 0.01). These phenotypic dissimilarities corresponded to gene expression level differences among key regulatory pathways throughout growth. A/J mice had a 1.55- to 7.65-fold greater expression among genes inhibitory to <em>Wnt</em> pathway induction, whereas genes involved in cortical mineralization were largely upregulated 1.50- to 3.77-fold to compensate for their narrow diaphysis. Additionally, both mouse strains showed an upregulation among <em>Wnt</em> pathway antagonists corresponding to the onset of adult ambulation (ie, increased physiological loads). This contrasts with other studies showing an increase in <em>Wnt</em> pathway activation after functionally isolated, experimental in vivo loading regimens. A/J and C57BL/6J long bones provide a model to develop a systems-based approach to identify individual genes and the gene-gene interactions that contribute to trait differences between the strains while being involved in the process by which these traits are coordinately adjusted to establish similar levels of mechanical function, thus providing insight into the process of canalization. © 2017 American Society for Bone and Mineral Research.

Platelets are produced upon profound reorganization of mature megakaryocytes (MK) leading to proplatelet elongation and release into the blood stream, a process termed thrombopoiesis. This highly dynamic process requires microtubules (MT) reorganization by mechanisms that are still incompletely understood. Adenomatous polyposis coli (APC) is a microtubule plus-end tracking protein involved in the regulation of MT in a number of cell systems and its inactivation has been reported to alter hematopoiesis. The aim of our study was to investigate the role of APC in megakaryopoiesis and the final steps of platelet formation. Down-regulation of APC in cultured human MK by RNA interference increased endomitosis and the proportion of cells able to extend proplatelets (68.8% (shAPC1) and 52.5% (shAPC2) vs 28.1% in the control). Similarly an increased ploidy and amplification of the proplatelet network were observed in MK differentiated from Lin- cells of mice with APC-deficiency in the MK lineage. In accordance, these mice exhibited increased platelet counts when compared to wild type mice (1,323 ± 111 vs 919 ± 52 platelets/µL; n = <em>12</em> p 0.0033**). Their platelets had a normal size, ultrastructure and number of microtubules coils and their main functions were also preserved. Loss of APC resulted in lower levels of acetylated tubulin and decreased activation of the <em>Wnt</em> signaling pathway. Thus, APC appears as an important regulator of proplatelet formation and overall thrombopoiesis.

MicroRNAs are small non-coding RNAs that function as regulators of gene expression. The altered expression of microRNAs influences the pathogenesis of Alzheimer's disease. Many researchers have focused on studies based on the relatively distinctive etiology of familial Alzheimer's disease due to the absence of risk factors in the pathogenesis of sporadic Alzheimer's disease. Although there is a limitation in Alzheimer's disease studies, both Alzheimer's disease types have a common risk factor-aging. No study to date has examined the aging factor in Alzheimer's disease animal models with microRNAs. To investigate the effect of aging on the changes in microRNA expressions in the Alzheimer's disease animal model, we selected 37 hippocampal microRNAs whose expression in <em>12</em>- and 18-month aged mice changed significantly using microRNA microarray. On the basis of bioinformatics databases, 30 hippocampal microRNAs and their putative targets of PSEN1/PSEN2 double knockout mice were included in 28 pathways such as the <em>wnt</em> signaling pathway and ubiquitin-mediated proteolysis pathway. Cortical microRNAs and its putative targets involved in pathological aging were included in only four pathways such as the heparin sulfate biosynthesis. The altered expressions of these hippocampal microRNAs were associated to the imbalance between neurotoxic and neuroprotective functions and seemed to affect neurodegeneration in PSEN1/PSEN2 double knockout mice more severely than in wild-type mice. This microRNA profiling suggests that microRNAs play potential roles in the normal aging process, as well as in the Alzheimer's disease process.