OBJECTIVE

We examined preadipocyte differentiation in obese and nonobese individuals and the effect of cytokines and wingless-type MMTV (mouse mammary tumor virus) integration site family, member 3A (Wnt3a) protein on preadipocyte differentiation and phenotype.

METHODS

Abdominal subcutaneous adipose tissue biopsies were obtained from a total of 51 donors with varying BMI. After isolation of the adipose and stromalvascular cells, inflammatory cells (CD14- and CD45-positive cells) were removed by immune magnetic separation. CD133-positive cells, containing early progenitor cells, were also isolated and quantified. The CD14- and CD45-negative preadipocytes were cultured with tumor necrosis factor (TNF)-alpha, interleukin (IL)-6, resistin, or Wnt3a with or without a differentiation cocktail.

RESULTS

The number of preadipocytes able to differentiate to adipose cells was negatively correlated with both BMI and adipocyte cell size of the donors, whereas the number of CD133-positive cells was positively correlated with BMI, suggesting an impaired differentiation of preadipocytes in obesity. Cultured preadipocytes, like freshly isolated mature adipocytes, from obese individuals had an increased expression of mitogen-activated protein 4 kinase 4 (MAP4K4), which is known to inhibit peroxisome proliferator-activated receptor-gamma induction. TNF-alpha, but not IL-6 or resistin, increased Wnt10b, completely inhibited the normal differentiation of the preadipocytes, and instead induced a proinflammatory and macrophage-like phenotype of the cells.

CONCLUSIONS

The apparent number of preadipocytes in the abdominal subcutaneous tissue that can undergo differentiation is reduced in obesity with enlarged fat cells, possibly because of increased MAP4K4 levels. TNF-alpha promoted a macrophage-like phenotype of the preadipocytes, including several macrophage markers. These results document the plasticity of human preadipocytes and the inverse relationship between lipid storage and proinflammatory capacity.

Recurrent chromosomal alterations are found in roughly 20% of all uterine fibroids but in the majority cytogenetic changes are lacking. Recently, mutations of the gene mediator subcomplex 12 (MED12) have been detected in a majority of fibroids but no information is available whether or not they co-occur with cytogenetic sub<em>type</em>s as, e.g., rearrangements of the genes encoding high mobility group AT-hook (HMGA) proteins. In a total of 80 cytogenetically characterized fibroids from 50 patients, we were not only able to confirm the frequent occurrence of MED12 mutations but also to stratify two mutually exclusive pathways of leiomyomagenesis with either rearrangements of HMGA2 reflected by clonal chromosome abnormalities affecting 12q1<em>4</em>~15 or by mutations affecting exon 2 of MED12. On average the latter mutations were associated with a significantly smaller tumor size. However, G>A transitions of nucleotides c.130 or c.131 correlate with a significantly larger size of the fibroids compared to other MED12 mutations thus explaining the high prevalence of the former mutations among clinically detectable fibroids. Interestingly, fibroids with MED12 mutations expressed significantly higher levels of the gene encoding <em>wingless</em>-<em>type</em> <em>MMTV</em> <em>integration</em> <em>site</em> <em>family</em>, <em>member</em> <em>4</em> (WNT<em>4</em>). Based on these findings and data from the literature, we hypothesize that estrogen and the mutated MED12 cooperate in activating the Wnt pathway which in turn activates β-catenin known to cause leiomyoma-like lesions in a mouse model. The occurrence of a "fibroid-<em>type</em> mutation" in a rare histologic sub<em>type</em> of endometrial polyps suggests that this mechanism is not confined to uterine leiomyomas.

BACKGROUND

During embryonic development, beta-catenin is central both to the transcriptional activation of Wnt [wingless-type MMTV (murine-mammary-tumour virus) integration site family] target genes and as a mediator of cell-cell adhesion. Signals that regulate its levels and subcellular localization are critical. One mechanism of Wnt signalling results in stabilization of beta-catenin protein, which leads to its translocation into the nucleus, where it interacts with TCF (T-cell factor, HMG box) and activates transcription of target genes. Less well understood are mechanisms of Wnt signalling that do not involve beta-catenin stabilization and result in inhibition of beta-catenin-mediated transcription.

RESULTS

Here, we show that a member of the Wnt protein family, Wnt4 (Wnt, member 4), regulates the subcellular localization of beta-catenin, redirecting it to the cell membrane. Unique among Wnts, this action does not affect the stability of beta-catenin but does prohibit its involvement in TCF gene transactivation.

CONCLUSIONS

This novel mechanism suggests that Wnt4 acts as a switch between the two modes of beta-catenin function, transcriptional activation and cell-cell adhesion.

<em>Type</em> I and/or <em>type</em> II interferons (IFNs) are important in establishing uterine receptivity to implantation in mammals. Gene expression effected by IFNs may be induced, stimulated or inhibited, but most are IFN-stimulated genes (ISGs). Effects of IFNs range from pregnancy recognition signaling in ruminants by IFN tau (IFNT) to effects on cellular functions of the uterus and uterine vasculature. For most, if not all, actions of IFNs on the uterus, progesterone (P(<em>4</em>)) is permissive to ISG expression, with genes being induced by IFN or induced by P(<em>4</em>) and stimulated by IFN. Uterine receptivity to implantation is P(<em>4</em>)-dependent; however, implantation events are preceded by loss of expression of progesterone (PGR) and estrogen (ESR1) receptors by uterine epithelia. Thus, P<em>4</em> likely stimulates PGR-positive stromal cells to express one or more progestamedins, e.g., fibroblast growth factors-7 and -10, and/or hepatocyte growth factor, that act via their respective receptors on uterine epithelia and trophectoderm to regulate expression of ISGs. FGF10 appears to be the most important progestamedin in sheep uteri during pregnancy. Sequential effects of P(<em>4</em>) to induce and IFNs to stimulate gene expression suggest that P(<em>4</em>) and IFNs activate complimentary cell signaling pathways to modulate expression of genes for attachment of trophectoderm to uterine lumenal and superficial glandular epithelia (LE/sGE), modify pheno<em>type</em> of uterine stromal cells, silence PGR and ESR1 genes, signal pregnancy recognition, suppress genes for immune recognition, alter membrane permeability to enhance conceptus-maternal exchange of factors, increase endometrial vascularity and activate genes for transport of nutrients into the uterine lumen. In ewes, IFNT abrogrates the uterine luteolytic mechanism and stimulates expression of classical ISGs by GE and stromal cells, whereas LE/sGE express P(<em>4</em>)-induced and IFNT-stimulated genes important for uterine receptivity to implantation and conceptus development. These include <em>wingless</em>-<em>type</em> <em>MMTV</em> (mouse mammary tumor virus) <em>integration</em> <em>site</em> <em>family</em> <em>member</em> 7A (WNT7A) induced by IFNT, as well as galectin, proteases, protease inhibitors, transporters for glucose and amino acids, gastrin releasing polypeptide, insulin-like growth factor binding protein 1 and a hypoxia inducible factor. The specific functions of IFNs and ISGs induced in primates, pigs and other mammals during pregnancy are not known, but likely are important in establishment of pregnancy. Understanding the roles of IFNs and ISGs in uterine receptivity for implantation is necessary to develop strategies to enhance reproductive health and fertility in humans and domestic animals. The magnitude of the LH surge was reduced in cows receiving endotoxin.

OBJECTIVE

Wingless-type MMTV integration site family (Wnt)/β-catenin signaling plays an essential role in cellular differentiation and matrix formation during skeletal development. However, little is known about its role in tooth-root formation. In a previous study, we found excessive formation of dentin and cementum in mice with constitutive β-catenin stabilization in the dental mesenchyme. In the present study we analyzed the molar roots of these mice to investigate the role of Wnt/β-catenin signaling in root formation in more detail.

METHODS

We generated OC-Cre:Catnb(+/lox(ex3)) mice by intercrossing Catnb(+/lox(ex3)) and OC-Cre mice, and we analyzed their mandibular molars using radiography, histomorphometry and immunohistochemistry.

RESULTS

OC-Cre:Catnb(+/lox(ex3)) mice showed impaired root formation. At the beginning of root formation in mutant molars, dental papilla cells did not show normal differentiation into odontoblasts; rather, they were prematurely differentiated and had a disorganized arrangement. Interestingly, SMAD family member 4 was upregulated in premature odontoblasts. In 4-wk-old mutant mice, molar roots were about half the length of those in their wild-type littermates. In contrast to excessively formed dentin in crown, root dentin was thin and hypomineralized in mutant mice. Biglycan and dentin sialophosphoprotein were downregulated in root dentin of mutant mice, whereas dentin matrix protein 1 and Dickkopf-related protein 1 were upregulated. Additionally, ectonucleotide pyrophosphatase/phosphodiesterase 1 was significantly downregulated in the cementoblasts of mutant molars. Finally, in the cementum of mutant mice, bone sialoprotein was downregulated but Dickkopf-related protein 2 was upregulated.

CONCLUSIONS

These results suggest that temporospatial regulation of Wnt/β-catenin signaling plays an important role in cell differentiation and matrix formation during root and cementum formation.

OBJECTIVE

To investigate cell biological changes in calcified aortas of rats that experienced chronic renal failure.

RESULTS

Vascular smooth muscle cells have the potential to transdifferentiate to either chondrocytes or osteoblasts, depending on the molecular pathways that are stimulated. Uremia-related medial calcification was induced by feeding rats an adenine low-protein diet for <em>4</em> weeks. Aortic calcification was evaluated biochemically and histochemically and with in vivo micro-computed tomographic scanning. Immunohistochemistry and RT-PCR were applied to analyze the time-dependent aortic expression of molecules involved in the segregation between the chondrocyte versus osteoblast differentiation pathway. After <em>4</em> weeks, 85% of the uremic rats had developed distinct aortic medial calcification, which increased to severely calcified lesions during further follow-up. The calcification process was accompanied by a significant time-dependent increase in the expression of the chondrocyte-specific markers sex determining region Y-box 9 (sox9), collagen II, and aggrecan and a nonsignificant trend toward enhanced core binding factor alpha 1 (cbfa1), and collagen I. The expression of the osteoblast marker osterix and both lipoprotein receptor-related protein 6 and beta-catenin, molecules of the <em>wingless</em>-<em>type</em> <em>MMTV</em> <em>integration</em> <em>site</em> <em>family</em> <em>member</em> (Wnt)/beta-catenin pathway induced during osteoblast differentiation, was suppressed.

CONCLUSIONS

In the aorta of uremic rats, medial smooth muscle cells acquire a chondrocyte rather than osteoblast phenotype during the calcification process.

Disruptions of beta-catenin and the canonical Wnt pathway are well documented in cancer. However, little is known of the non-canonical branch of the Wnt pathway. In this study, we investigate the transcript level patterns of genes in the Wnt pathway in squamous cell lung cancer using reverse-transcriptase (RT)-PCR. It was found that over half of the samples examined exhibited dysregulated gene expression of multiple components of the non-canonical branch of the WNT pathway. In the cases where beta catenin (CTNNB1) was not over-expressed, we identified strong relationships of expression between <em>wingless</em>-<em>type</em> <em>MMTV</em> <em>integration</em> <em>site</em> <em>family</em> <em>member</em> 5A (WNT5A)/ frizzled homolog 2 (FZD2), frizzled homolog 3 (FZD3) / dishevelled 2 (DVL2), and low density lipoprotein receptor-related protein 5 (LRP5)/ secreted frizzled-related protein <em>4</em> (SFRP<em>4</em>). This is one of the first studies to demonstrate expression of genes in the non-canonical pathway in normal lung tissue and its disruption in lung squamous cell carcinoma. These findings suggest that the non-canonical pathway may have a more prominent role in lung cancer than previously reported.

BACKGROUND

Aberrant activation of the canonical Wingless-type MMTV integration site family (WNT)/β-catenin signaling pathway is critical for gliomas.

METHODS

In 74 gliomas of different histological grade and in 24 glioblastoma cell lines, protein expression of WNT member 3a (WNT3a), β-catenin and transcription factor 4 (TCF4) was investigated by immunohistochemistry, western blotting, immunofluorescence and immunocytochemistry. In tumors and cell lines, WNT3A expression was assessed at the mRNA level by quantitative real-time polymerase chain reaction.

RESULTS

WNT3a was overexpressed at the protein and mRNA levels in malignant astrocytic tumors and cell lines. Cytoplasmic expression of β-catenin was detected in high-grade gliomas and cell lines, with evidence of nuclear translocation on fractionated protein extracts. Activating mutations in the β-catenin encoding gene (CTNNB1) were excluded by direct sequencing. TCF4 was statistically correlated with Ki-67/MIB-1 and cyclin D1 labeling indices.

CONCLUSIONS

Expression of WNT3a, cytoplasmic β-catenin and TCF4 was significantly associated with the histological malignancy grade and with a worse prognosis for patients with glioma.

We have shown that the uniaxial cyclic tensile strain of magnitude 10% promotes and enhances osteogenesis of human mesenchymal stem cells (hMSC) and human adipose-derived stem cells (hASC) from normal, nonosteoporotic donors. In the present study, MSC from osteoporotic donors were analyzed for changes in mRNA expression in response to 10% uniaxial tensile strain to identify potential mechanisms underlying the use of this mechanical loading paradigm for prevention and treatment of osteoporosis. Human MSC isolated from three female, postmenopausal osteoporotic donors were analyzed for their responses to mechanical loading using microarray analysis of over <em>4</em>7,000 gene probes. Human MSC were seeded in three-dimensional collagen <em>type</em> I constructs to mimic the organic extracellular matrix of bone and 10% uniaxial cyclic tensile strain was applied to promote osteogenesis. Seventy-nine genes were shown to be regulated within hMSC from osteoporotic donors in response to 10% cyclic tensile strain. Upregulation of six genes were further confirmed with real-time RT-PCR: jun D proto-oncogene (JUND) and plasminogen activator, urokinase receptor (PLAUR), two genes identified as potential key molecules from network analysis; phosphoinositide-3-kinase, catalytic, delta polypeptide (PIK3CD) and <em>wingless</em>-<em>type</em> <em>MMTV</em> <em>integration</em> <em>site</em> <em>family</em>, <em>member</em> 5B (WNT5B), two genes with known importance in bone biology; and, PDZ and LIM domain <em>4</em> (PDLIM<em>4</em>) and vascular endothelial growth factor A (VEGFA), two genes that we have previously shown are significantly regulated in hASC in response to this mechanical stimulus. Function analysis indicated that 10% cyclic tensile strain induced expression of genes associated with cell movement, cell proliferation, and tissue development, including development in musculoskeletal and cardiovascular systems. Our results demonstrate that hMSC from aged, osteoporotic donors are capable of enhanced osteogenic differentiation in response to 10% cyclic tensile strain with significant increases in the expression of genes associated with enhanced cell proliferation, musculoskeletal development, and angiogenesis. Surprisingly, cyclic tensile strain of magnitude 10% not only enhanced osteogenesis in hMSC from osteoporotic donors, but also enhanced expression of angiogenic factors. Better understanding and methodologies to promote osteogenesis in hMSC from elderly, osteoporotic donors may greatly facilitate achieving long-term success in bone regeneration and functional bone tissue engineering for this ever-growing patient population.

<em>Wingless</em>-<em>type</em> <em>MMTV</em> <em>integration</em> <em>site</em> <em>family</em> <em>member</em> (WNT) signaling and WNT-inhibitors have been implicated in regulation of adipogenesis, insulin resistance, pancreatic function, and inflammation. Our goal was to determine serum proteins involved in WNT signaling (WNT5 and WISP2) and WNT inhibition (SFRP<em>4</em> and SFRP5) as they relate to obesity, serum adipokines, insulin resistance, insulin secretion, and inflammation in humans. Study population comprised 57 insulin resistant women with polycystic ovary syndrome (PCOS) and 27 reference women. In a cross-sectional study, blood samples were obtained at fasting, during oral, and frequently sampled intravenous glucose tolerance tests. Serum WNT5, WISP2, and SFRP<em>4</em> concentrations did not differ between PCOS vs. reference women. Serum WNT5 correlated inversely with weight both in PCOS and reference women, and correlated directly with insulin response during oral glucose tolerance test in PCOS women. Serum WISP2 correlated directly with fatty acid binding protein <em>4</em>. Serum SFRP5 did not differ between obese (n=32) vs. nonobese (n=25) PCOS women, but reference women had lower SFRP5 (p<5×10(-6) as compared to both PCOS groups). Serum SFRP5 correlated inversely with IL-1β, TNF-α, cholesterol, and apoprotein B. These findings demonstrated that WNT5 correlated inversely with adiposity and directly with insulin response, and the WNT-inhibitor SFRP5 may be anti-inflammatory. Better understanding of the role of WNT signaling in obesity, insulin resistance, insulin secretion, lipoprotein metabolism, and inflammation is important for prevention and treatment of metabolic syndrome, diabetes and cardiovascular disease.

Proliferation and fusion of myoblasts is a well-orchestrated process occurring during muscle development and regeneration. Although myoblasts are known to originate from muscle satellite cells, the molecular mechanisms that coordinate their commitment toward differentiation are poorly understood. Here, we present a novel role for the transcription factor Forkhead box protein C2 (Foxc2) in regulating proliferation and preventing premature differentiation of activated muscle satellite cells. We demonstrate that Foxc2 expression is upregulated early in activated mouse muscle satellite cells and then diminishes during myogenesis. In undifferentiated C2C12 myoblasts, downregulation of endogenous Foxc2 expression leads to a decrease in proliferation, whereas forced expression of FOXC2 sustains proliferation and prevents differentiation into myotubes. We also show that FOXC2 induces Wnt signaling by direct interaction with the Wnt<em>4</em> (<em>wingless</em>-<em>type</em> <em>MMTV</em> <em>integration</em> <em>site</em> <em>family</em> <em>member</em>-<em>4</em>) promoter region. The resulting elevated expression of bone morphogenetic protein-<em>4</em> (Bmp<em>4</em>) and RhoA-GTP proteins inhibits the proper myoblast alignment and fusion required for myotube formation. Interestingly, continuous forced expression of FOXC2 alters the commitment of C2C12 myoblasts toward osteogenic differentiation, which is consistent with FOXC2 expression observed in patients with myositis ossificans, an abnormal bone growth within muscle tissue. In summary, our results suggest that (a) Foxc2 regulates the proliferation of multipotent muscle satellite cells; (b) downregulation of Foxc2 is critical for myogenesis to progress; and (c) sustained Foxc2 expression in myoblast cells suppresses myogenesis and alters their lineage commitment toward osteogenesis by inducing the Wnt<em>4</em> and Bmp<em>4</em> signaling pathways.

Asthma is a chronic disorder characterized by airway inflammation, reversible bronchial obstruction, hyper-responsiveness and remodelling. Data from human in vitro studies and experimental in vivo models of asthma has implicated interleukin (IL)-13 in the asthma pheno<em>type</em> suggesting that a therapeutic agent against it could be effective in treating asthma. The role of biomarkers is becoming increasingly important in the clinical development of therapeutics. Here we describe the use of the GeneChip((R)) DNA microarray technology platform to explore and identify potential response to therapy biomarkers that are associated with the biology of IL-13. Peripheral blood mononuclear cells (PBMCs) from eight healthy donors were cultured in the presence of IL-13, IL-<em>4</em>, an anti-IL-13 monoclonal antibody (mAb) or an iso<em>type</em> control mAb, and RNA from the treated cells was subjected to microarray analysis. The results revealed a number of genes, such as CCL17 (TARC), CCL22 (MDC), CCL23 (MPIF-1), CCL26 (eotaxin 3) and WNT5A (human <em>wingless</em>-<em>type</em> <em>MMTV</em> <em>integration</em> <em>site</em> <em>family</em> <em>member</em> 5A), that showed increased expression in the IL-13 and IL-<em>4</em> treatment groups. Real-time polymerase chain reaction (PCR) subsequently confirmed these results. A follow-up study in PBMCs from five additional healthy donors showed that the neutralization of IL-13 completely blocked IL-13-induced TARC, MDC and eotaxin 3 production at the protein level. These data suggest that TARC, MDC, eotaxin 3, CCL23 and WNT5A if validated could serve as potential biomarkers for anti-IL-13 therapeutics.

The idea that the female sexual development happens by default was born in the middle of the last century after Jost performed his innovative experiments to study the bases of differentiation of the reproductive tract and found that the female reproductive tract develops even in the absence of any gonad. The term default (passive) attributed to the whole female developmental pathway, therefore, established itself, even if it was not originally so intended. However, recent developments have demonstrated that ovarian development is an active process. <em>Wingless</em> <em>type</em> <em>MMTV</em> <em>integration</em> <em>site</em> <em>family</em>, <em>member</em> <em>4</em> (WNT<em>4</em>), one of a few factors with a demonstrated function in the ovarian-determination pathway, has been found to be involved in sexual differentiation by suppressing male sexual differentiation, promoting Müllerian duct differentiation, and maintaining oocyte health. WNT<em>4</em> expression in the ovary seems to be regulated by R-spondin 1 (RSPO1), a thrombospondin <em>family</em> <em>member</em> protein. The role and interactions of WNT<em>4</em>, RSPO1, and other factors such as forkhead transcription factor 2 in ovarian development and function will be discussed.

WNTs (<em>wingless</em>-<em>type</em> <em>MMTV</em> <em>integration</em> <em>site</em> <em>family</em>, <em>member</em>) are morphogenes considered as important factors taking part in uterus developmental processes and implantation. β-catenin is a downstream effector of WNTs action within the cell as well as, through E-cadherin, affecting epithelial organization and function. This study was conducted to investigate WNT<em>4</em>, WNT5A, WNT7A, β-catenin (CTNNB1) and E-cadherin (CDH1) gene expression and protein localization in the endometrium during the periimplantation period. Furthermore, the effect of 17β-estradiol (E(2)) and progesterone (P(<em>4</em>)) on WNTs, CTNNB1 and CDH1 gene expression in the porcine endometrium in vitro was examined. WNT<em>4</em> protein was localized in the luminal and glandular epithelium as well as in the basal lamina of the uterine mucosa. WNT5A protein was detected only in the luminal epithelium. WNT7A, β-catenin and E-cadherin protein were identified both in the luminal and glandular epithelial cells, however, WNT7A protein immunoreactivity varied during respective days of estrous cycle and/or pregnancy. Despite unchanged expression of WNT<em>4</em> mRNA in the endometrium of cyclic and early pregnant pigs, the negative influence of E(2) on WNT<em>4</em> gene during in vitro experiment was observed. WNT<em>4</em> and CDH1 gene expression was negatively correlated with blood plasma E(2) and P(<em>4</em>) level in uterine luminal flushings (ULFs) on Day 12 of pregnancy. Expression of WNT5A gene was up-regulated in the endometrium on Day 9 of pregnancy when compared to the respective day of the estrous cycle. A significant decrease of WNT7A gene expression and increase of CDH1 mRNA amount was detected on Day 12 of pregnancy. Overall, the results show the spatial localization of WNT<em>4</em>, WNT5A, WNT7A, β-catenin and E-cadherin proteins in porcine endometrium during periimplantation period of pregnancy and indicate significant changes of WNT5A, WNT7A and CDH1 gene expression before implantation in the pig.

<AbstractText>Endometriosis is an estrogen-dependent gynecological disorder that affects at least 10% of women of reproductive age. It may lead to infertility and non-specific symptoms such as chronic pelvic pain. Endometriosis screening and diagnosis are difficult and time-consuming. Late diagnosis (with a delay ranging from 3.3 to 10.7 years) is a major problem and may contribute to disease progression and a worse response to treatment once initiated. Efficient screening tests might reduce this diagnostic delay. As endometriosis is presumed to be a complex disease with several genetic and non-genetic pathogenic factors, many researchers have sought to identify polymorphisms that predispose to this condition.</AbstractText><AbstractText>We performed a systematic review and meta-analysis of the most regularly reported polymorphisms in order to identify those that might predispose to endometriosis and might thus be of value in screening.</AbstractText><AbstractText>The MEDLINE database was searched for English-language publications on DNA polymorphisms in endometriosis, with no date restriction. The PubTator text mining tool was used to extract gene names from the selected publications' abstracts. We only selected polymorphisms reported by at least three studies, having applied strict inclusion and exclusion criteria to their control populations. No stratification based on ethnicity was performed. All steps were carried out according to PRISMA guidelines.</AbstractText><AbstractText>The initial selection of 395 publications cited 2<em>4</em>2 different genes. Sixty-two genes (corresponding to 265 different polymorphisms) were cited at least in three publications. After the application of our other selection criteria (an original case-control study of endometriosis, a reported association between endometriosis and at least one polymorphism, data on women of reproductive age and a diagnosis of endometriosis in the cases established by surgery and/or MRI and confirmed by histology), 28 polymorphisms were eligible for meta-analysis. Only five of the 28 polymorphisms were found to be significantly associated with endometriosis: interferon gamma (IFNG) (CA) repeat, glutathione S-transferase mu 1 (GSTM1) null geno<em>type</em>, glutathione S-transferase pi 1 (GSTP1) rs1695 and <em>wingless</em>-<em>type</em> <em>MMTV</em> <em>integration</em> <em>site</em> <em>family</em> <em>member</em> <em>4</em> (WNT<em>4</em>) rs16826658 and rs2235529. Six others showed a significant trend towards an association: progesterone receptor (PGR) PROGINS, interCellular adhesion molecule 1 (ICAM1) rs1799969, aryl-hydrocarbon receptor repressor (AHRR) rs2292596, cytochrome <em>family</em> 17 sub<em>family</em> A polypeptide 1 (CYP17A1) rs7<em>4</em>3572, CYP2C19 rs<em>4</em>2<em>4</em><em>4</em>285 and peroxisome proliferator-activated receptor gamma (PPARG) rs1801282), and 12 showed a significant trend towards the lack of an association: tumor necrosis factor (TNF) rs179996<em>4</em>, interleukin 6 (IL6) rs1800796, transforming growth factor beta 1 (TGFB1) rs1800<em>4</em>69, estrogen receptor 1 (ESR1) rs223<em>4</em>693, PGR rs10895068, FSH receptor (FSHR) rs6166, ICAM1 rs5<em>4</em>98, CYP1A1 rs<em>4</em>6<em>4</em>6903, CYP19A1 rs100<em>4</em>6, tumor protein 53 (TP53) rs10<em>4</em>2522, X-ray repair complementing defective repair in Chinese hamster cells 1 (XRCC1) rs25<em>4</em>87 and serpin peptidase inhibitor clade E <em>member</em> 1 (SERPINE1) rs1799889; however, for the 18 polymorphisms identified in the latter two groups, further studies of the potential association with the endometriosis risk are needed. The remaining five of the 28 polymorphisms were not associated with endometriosis: glutathione S-transferase theta 1 (GSTT1) null geno<em>type</em>, vascular endothelial growth factor alpha (VEGFA) rs6999<em>4</em>7, rs833061, rs2010963 and rs3025039.</AbstractText><AbstractText>By carefully taking account of how the control populations were defined, we identified polymorphisms that might be candidates for use in endometriosis screening and polymorphisms not associated with endometriosis. This might constitute the first step towards identifying polymorphism combinations that predispose to endometriosis (IFNG (CA) repeat, GSTM1 null geno<em>type</em>, GSTP1 rs1695, WNT<em>4</em> rs16826658 and WNT<em>4</em> rs2235529) in a large cohort of patients with well-defined inclusion criteria. In turn, these results might improve the diagnosis of endometriosis in primary care. Lastly, our present findings may enable a better understanding of endometriosis and improve the management of patients with this disease.</AbstractText>

Adipose metabolism is an essential contributor to the efficiency of milk production, and metabolism is controlled by several mechanisms, including gene expression of critical proteins; therefore, the objective of this study was to determine how lactational state and the genetic merit of dairy cattle affects adipose tissue (AT) metabolism and mRNA expression of genes known to control metabolism. Animals of high (HGM) and low genetic merit (LGM) were fed to requirements, and weekly dry matter intake, milk production, blood glucose, and nonesterified fatty acids were measured. Subcutaneous AT biopsies were collected at -21, 7, 28 and 56 d in milk (DIM). The mRNA expression of genes coding for lipogenic enzymes [phosphoenolpyruvate carboxykinase 1 (soluble) (PCK1), fatty acid synthase (FASN), diacylglycerol O-acyltransferase 2 (DGAT2), and stearoyl-coenzyme A desaturase (SCD)], transcription regulators [peroxisome proliferator-activated receptor γ (PPARG), thyroid hormone responsive (THRSP), <em>wingless</em>-<em>type</em> <em>MMTV</em> <em>integration</em> <em>site</em> <em>family</em>, <em>member</em> 10B (WNT10B), sterol regulatory element binding transcription factor 1 (SREBF1), and adiponectin (ADIPOQ)], lipolytic enzymes [hormone-sensitive lipase (LIPE), patatin-like phospholipase domain containing 2 (PNPLA2), monoglyceride lipase (MGLL), adrenoceptor β-2 (ADRB2), adipose differentiation-related protein (ADFP), and α-β-hydrolase domain containing 5 (ABHD5)], and genes controlling the sensing of intracellular energy [phosphodiesterase 3A (PDE3A); PDE3B; protein kinase, AMP-activated, α-1 catalytic subunit (PRKAA1); PRKAA2; and growth hormone receptor (GHR)] was measured. Dry matter intake, blood glucose, and nonesterified fatty acid concentrations did not differ between genetic merit groups. Milk production was greater for HGM cows from 6 to 8 wk postpartum. As expected, the rates of lipogenesis decreased in early lactation, whereas stimulated lipolysis increased. At 7 DIM, lipogenesis in HGM cows increased as a function of substrate availability (0.5, 1, 2, 3, <em>4</em>, or 8mM acetic acid), whereas the response in LGM cows was much less pronounced. However, the lipogenic response at 28 DIM reversed and rates were greater in tissue from LGM than HGM cows. Peak lipolytic response, regardless of DIM, was observed at the lowest dose of isoproterenol (10(-8)M), and -21 d tissue had a greater lipolysis rate than tissue at 7, 28, and 56 d. In HGM compared with LGM cows, stimulated lipolysis at 7 and 28 DIM was greater but peaked at 10(-7)M isoproterenol, suggesting differences in tissue responsiveness due to genetic merit. Regardless of genetic merit, the expression of lipogenic genes decreased markedly in early lactation, whereas those controlling lipolysis stayed similar or decreased slightly. Cows of HGM had lower expression of lipogenic genes after parturition and through 56 DIM. In contrast, the expression of most of the lipolytic enzymes, receptors and proteins was similar in all cows pre- and postpartum. These results confirm that gene transcription is a major control mechanism for AT lipogenesis during early lactation, but that control of lipolysis is likely primarily by posttranslational mechanisms.

The prevalence of obesity is increasing all over the world. Although it has been shown that natural substances influence fat metabolism, little is known about the effect on cellular and molecular mechanisms in human. In this in vitro study, the activity of Rosmarinus officinalis (RO) standardized extract in modulating human primary visceral preadipocytes differentiation, lipolysis, and apoptosis was investigated. Moreover, gene expression of key adipogenesis modulators and microRNAs-seq were evaluated. Preadipocytes treated with RO extract significantly reduced triglyceride incorporation during maturation in a dose-dependent manner without affecting cell viability. In addition, RO extract stimulated lipolytic activity in differentiating preadipocytes and mature adipocytes in treated cells compared to controls. Differentiating preadipocytes incubated in the presence of RO extract showed a decreased expression of cell cycle genes such as cyclin D1, cyclin-dependent kinase <em>4</em>, cyclin-dependent kinase inhibitor 1A (p21, Cip1) and an increased expression of GATA binding protein 3, <em>wingless</em>-<em>type</em> <em>MMTV</em> <em>integration</em> <em>site</em> <em>family</em>, <em>member</em> 3A mRNA levels. Recent studies have demonstrated that some phytochemicals alter the expression of specific genes and microRNAs that play a fundamental role in the pathogenesis of obesity and related diseases. Interestingly, genes modulated in RO-treated cells were found to be validated miRNAs targets, such as let-7f-1, miR-17, and miR-1<em>4</em>3. The results indicated that RO extract modulates human adipocyte differentiation and significantly interferes with adipogenesis and lipid metabolism, supporting its interest as dietary supplement.

The <em>Wingless</em>-<em>type</em> <em>MMTV</em> <em>integration</em> <em>site</em> <em>family</em> <em>member</em> 2b (Wnt2b) has been found to be a principal mediator of liver development and regeneration. However, the significance of Wnt2b in the pathogenesis of fibrosis-related liver diseases remains undefined. Here, we report that Wnt2b was highly expressed in the fibrotic liver tissues, exhibiting protective effects against activation of hepatic stellate cells (HSCs) and fibrosis progression. We identified a negative regulation of Wnt2b on the toll-like receptor <em>4</em> (TLR<em>4</em>) activation-mediated pro-fibrogenic effects. Wnt2b was shown not only to directly suppress LPS-induced HSCs activation, but also to inhibit TLR<em>4</em>-enhanced the sensitivity of HSCs to transforming growth factor beta (TGF-β). Mechanistic study showed that Wnt2b suppresses TLR<em>4</em> signaling through inhibiting the expression of TLR<em>4</em> as well as the activation of NF-κB and MAPKs. These findings provided new insights into the pathophysiology of liver fibrosis by characterizing Wnt2b as a novel endogenous suppressor of TLR<em>4</em> signaling, maintaining tissue homeostasis during the early stage of hepatic fibrosis-associated liver diseases.

OBJECTIVE

To combine mathematical modeling of salivary gene expression microarray data and systems biology annotation with reverse-transcription quantitative polymerase chain reaction amplification to identify (phase I) and validate (phase II) salivary biomarker analysis for the prediction of oral feeding readiness in preterm infants.

METHODS

Comparative whole-transcriptome microarray analysis from 12 preterm newborns pre- and postoral feeding success was used for computational modeling and systems biology analysis to identify potential salivary transcripts associated with oral feeding success (phase I). Selected gene expression biomarkers (15 from computational modeling; 6 evidence-based; and 3 reference) were evaluated by reverse-transcription quantitative polymerase chain reaction amplification on <em>4</em>00 salivary samples from successful (n = 200) and unsuccessful (n = 200) oral feeders (phase II). Genes, alone and in combination, were evaluated by a multivariate analysis controlling for sex and postconceptional age (PCA) to determine the probability that newborns achieved successful oral feeding.

RESULTS

Advancing PCA (P < .001) and female sex (P = .05) positively predicted an infant's ability to feed orally. A combination of 5 genes, neuropeptide Y2 receptor (hunger signaling), adneosine-monophosphate-activated protein kinase (energy homeostasis), plexin A1 (olfactory neurogenesis), nephronophthisis <em>4</em> (visual behavior), and <em>wingless</em>-<em>type</em> <em>MMTV</em> <em>integration</em> <em>site</em> <em>family</em>, <em>member</em> 3 (facial development), in addition to PCA and sex, demonstrated good accuracy for determining feeding success (area under the receiver operator characteristic curve = 0.78).

CONCLUSIONS

We have identified objective and biologically relevant salivary biomarkers that noninvasively assess a newborn's developing brain, sensory, and facial development as they relate to oral feeding success. Understanding the mechanisms that underlie the development of oral feeding readiness through translational and computational methods may improve clinical decision making while decreasing morbidities and health care costs.

The long-term stimulation of hyperglycemia greatly increases the incidence of vascular restenosis (RS) after angioplasty. Neointimal hyperplasia after vascular injury is the pathological cause of RS, but its mechanism has not been elucidated. MicroRNA-2<em>4</em> (miR-2<em>4</em>) has low expression in the injured carotid arteries of diabetic rats. However, the role of miR-2<em>4</em> in the vascular system is unknown. In this study, we explore whether over-expression of miR-2<em>4</em> could attenuate neointimal formation in streptozotocin (STZ)-induced diabetic rats. Adenovirus (Ad-miR-2<em>4</em>-GFP) was used to deliver the miR-2<em>4</em> gene to injured carotid arteries in diabetic rats. The level of neointimal hyperplasia was examined by hematoxylin-eosin (HE) staining. Vascular smooth muscle cell (VSMC) proliferation in the neointima was evaluated by immunostaining for proliferating cell nuclear antigen (PCNA). The mRNA levels of miR-2<em>4</em>, PCNA, <em>wingless</em>-<em>type</em> <em>MMTV</em> <em>integration</em> <em>site</em> <em>family</em> <em>member</em> <em>4</em> (Wnt<em>4</em>), disheveled-1 (Dvl-1), β-catenin and cell cycle-associated molecules (Cyclin D1, p21) were determined by Quantitative Real-Time PCR (qRT-PCR). PCNA, Wnt<em>4</em>, Dvl-1, β-catenin, Cyclin D1 and p21 protein levels were measured by Western blotting analysis. STZ administration decreased plasma insulin and increased fasting blood glucose in Sprague-Dawley (SD) rats. The expression of miR-2<em>4</em> was decreased in the carotid artery after a balloon injury in diabetic rats, and adenoviral transfection (Ad-miR-2<em>4</em>-GFP) increased the expression of miR-2<em>4</em>. Over-expression of miR-2<em>4</em> suppressed VSMC proliferation and neointimal hyperplasia in diabetic rats at 1<em>4</em> days. Furthermore, compared with Sham group, the mRNA and protein levels of PCNA, Wnt<em>4</em>, Dvl-1, β-catenin, and Cyclin D1 were strikingly up-regulated in the carotid arteries of diabetic rats after a balloon injury. Interestingly, up-regulation of miR-2<em>4</em> significantly reduced the mRNA and protein levels of these above molecules. In contrast, the change trend in p21 mRNA and protein levels was oppo<em>site</em> after a balloon injury. However, over-expression of miR-2<em>4</em> after gene delivery increased the mRNA and protein levels of p21. We conclude that over-expression of miR-2<em>4</em> could attenuate VSMC proliferation and neointimal hyperplasia after vascular injuries in diabetic rats. This result is possibly related to the regulation of the expression of Cyclin D1 and p21 through the Wnt<em>4</em>/Dvl-1/β-catenin signaling pathway.

Gastrectomy (GX) is thought to result in osteomalacia due to deficiencies in Vitamin D and Ca. Using a GX rat model, we showed that GX induced high turnover of bone with hyperosteoidosis, prominent increase of mineralization and increased mRNA expression of both osteoclast-derived tartrate-resistant acid phosphatase 5b and osteocalcin. The increased 1, 25(OH)2D3 level and unchanged PTH and calcitonin levels suggested that conventional bone and Ca metabolic pathways were not involved or changed in compensation. Thus, GX-induced bone pathology was different from a typical osteomalacia. Gene expression profiles through microarray analysis and data mining using Ingenuity Pathway Analysis indicated that 612 genes were up-regulated and 1,097 genes were down-regulated in the GX bone. These genes were related functionally to connective tissue development, skeletal and muscular system development and function, Ca signaling and the role of osteoblasts, osteoclasts and chondrocytes. Network analysis indicated 9 genes (Aldehyde dehydrogenase 1 <em>family</em>, <em>member</em> A1; Aquaporin 9; Interleukin 1 receptor accessory protein; Very low density lipoprotein receptor; Periostin, osteoblast specific factor; Aggrecan; Gremlin 1; Angiopoietin-like <em>4</em>; <em>Wingless</em>-<em>type</em> <em>MMTV</em> <em>integration</em> <em>site</em> <em>family</em>, <em>member</em> 10B) were hubs connected with tissue development and immunological diseases. These results suggest that chronic systemic inflammation might underlie the GX-induced pathological changes in bone.

miR-18a is a member of primary transcript called miR-17-92a (C13orf25 or MIR17HG) which also contains five other miRNAs: miR-17, miR-19a, miR-20a, miR-19b and miR-92a. This cluster as a whole shows specific characteristics, where miR-18a seems to be unique. In contrast to the other members, the expression of miR-18a is additionally controlled and probably functions as its own internal controller of the cluster. miR-18a regulates many genes involved in proliferation, cell cycle, apoptosis, response to different kinds of stress, autophagy and differentiation. The disturbances of miR-18a expression are observed in cancer as well as in different diseases or pathological states. The miR-17-92a cluster is commonly described as oncogenic and it is known as 'oncomiR-1', but this statement is a simplification because miR-18a can act both as an oncogene and a suppressor. In this review we summarize the current knowledge about miR-18a focusing on its regulation, role in cancer biology and utility as a potential biomarker.

Keywords: 5-FU, 5-fluorouracyl; ACVR2A, activin A receptor type 2A; AKT, AKT serine/threonine kinase; AR, androgen receptor; ATG7, autophagy related 7; ATM, ATM serine/threonine kinase; BAX, BCL2 associated Xapoptosis regulator; BCL2, BCL2 apoptosis regulator; BCL2L10, BCL2 like 10; BDNF, brain derived neurotrophic factor; BLCA, bladder urothelial carcinoma; BRCA, breast cancer; Biomarker; Bp, base pair; C-myc (MYCBP), MYC binding protein; CASC2, cancer susceptibility 2; CD133 (PROM1), prominin 1; CDC42, cell division cycle 42; CDKN1, Bcyclin dependent kinase inhibitor 1B; COAD, colon adenocarcinoma; Cancer; Circulating miRNA; DDR, DNA damage repair; E2F family (E2F1, E2F2, E2F3), E2F transcription factors; EBV, Epstein-Barr virus; EMT, epithelial-to-mesenchymal transition; ER, estrogen receptor; ERBB (EGFR), epidermal growth factor receptor; ESCA, esophageal carcinoma; FENDRR, FOXF1 adjacent non-coding developmental regulatory RNA; FER1L4, fer-1 like family member 4 (pseudogene); GAS5, growth arrest–specific 5; HIF-1α (HIF1A), hypoxia inducible factor 1 subunit alpha; HNRNPA1, heterogeneous nuclear ribonucleoprotein A1; HNSC, head and neck squamous cell carcinoma; HRR, homologous recombination-based DNA repair; IFN-γ (IFNG), interferon gamma; IGF1, insulin like growth factor 1; IL6, interleukin 6; IPMK, inositol phosphate multikinase; KIRC, clear cell kidney carcinoma; KIRP, kidney renal papillary cell carcinoma; KRAS, KRAS proto-oncogene, GTPase; LIHC, liver hepatocellular carcinoma; LMP1, latent membrane protein 1; LUAD, lung adenocarcinoma; LUSC, lung squamous cell carcinoma; Liquid biopsy; MAPK, mitogen-activated protein kinase; MCM7, minichromosome maintenance complex component 7; MET, mesenchymal-to-epithelial transition; MTOR, mechanistic target of rapamycin kinase; N-myc (MYCN), MYCN proto-oncogene, bHLH transcription factor; NF-κB, nuclear factor kappa-light-chain-enhancer of activated B cells; NOTCH2, notch receptor 2; Oncogene; PAAD, pancreatic adenocarcinoma; PERK (EIF2AK3), eukaryotic translation initiation factor 2 alpha kinase 3; PI3K (PIK3CA), phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha; PIAS3, protein inhibitor of activated STAT 3; PRAD, prostate adenocarcinoma; RISC, RNA-induced silencing complex; SMAD2, SMAD family member 2; SMG1, SMG1 nonsense mediated mRNA decay associated PI3K related kinase; SNHG1, small nucleolar RNA host gene 1; SOCS5, suppressor of cytokine signaling 5; STAD, stomach adenocarcinoma; STAT3, signal transducer and activator of transcription 3; STK4, serine/threonine kinase 4; Suppressor; TCGA; TCGA, The Cancer Genome Atlas; TGF-β (TGFB1), transforming growth factor beta 1; TGFBR2, transforming growth factor beta receptor 2; THCA, papillary thyroid carcinoma; TNM, Classification of Malignant Tumors: T - tumor / N - lymph nodes / M – metastasis; TP53, tumor protein p53; TP53TG1, TP53 target 1; TRIAP1, p53-regulating inhibitor of apoptosis gene; TSC1, TSC complex subunit 1; UCA1, urothelial cancer associated 1; UCEC, uterine corpus endometrial carcinoma; UTR, untranslated region; WDFY3-AS2, WDFY3 antisense RNA 2; WEE1, WEE1 G2 checkpoint kinase; WNT family, Wingless-type MMTV integration site family/Wnt family ligands; ZEB1/ZEB2, zinc finger E-box binding homeobox 1 and 2; ceRNA, competitive endogenous RNA; cncRNA, protein coding and non-coding RNA; lncRNA, long-non coding RNA; miR-17-92a; miR-18a; miRNA.

Esophageal squamous cell carcinoma (ESCC) is a predominant <em>type</em> of esophageal cancer, which is a malignant tumor originating from the esophageal mucosa or gland and is aggressive with poor prognosis. Identification of new gene expression patterns would be helpful for providing new targets for the early detection and treatment of ESCC patients. In the present study, we employed cDNA array technology to compare gene expression profiles between ESCC tissues and adjacent normal epithelial tissues from ESCC patients. There was at least a <em>4</em>-fold change in the expression levels of 72 genes that were significantly increased and 107 genes that were decreased in ESCC compared with normal esophageal epithelium. Among them, genes known to be involved in ESCC were found, including matrix metalloproteinases, transcription factors SOX-<em>4</em> and SOX-17, the <em>Wingless</em>-<em>type</em> <em>MMTV</em> <em>integration</em> <em>site</em> <em>family</em> <em>member</em> 2, and cell cycle regulators. Moreover, we have newly identified the two genes that are down-regulated in ESCC: monoamine oxidase A, an enzyme that catalyzes monoamines oxidation and 15-hydroxyprostaglandin dehydrogenase [NAD+], a prostaglandin-synthesizing enzyme that physiologically antagonizes COX-2. Likewise, we found the three genes that are up-regulated in ESCC: CD7, a cell surface glycoprotein <em>member</em> of the immunoglobulin super<em>family</em>, LIM-domain kinase 1, a small sub<em>family</em> with an unique combination of two N-terminal LIM motifs and a C-terminal protein kinase domain, and TTK protein kinase, a previously unidentified <em>member</em> of the kinase <em>family</em>. These newly identified genes may be involved in the progression of the tumor and/or represent properties specific to ESCC.

Cervical cancer is the second most commonly diagnosed <em>type</em> of cancer and the third leading cause of cancer-associated mortality in women. The current study aimed to determine the genes associated with cervical cancer development. Microarray data (GSE559<em>4</em>0 and GSE<em>4</em>6306) were downloaded from Gene Expression Omnibus. Overlapping genes between the differentially expressed genes (DEGs) in GSE559<em>4</em>0 (identified by Limma package) and the differentially methylated genes were screened. Gene Ontology (GO) enrichment analysis was subsequently performed for these genes using the ToppGene database. In GSE559<em>4</em>0, 91 downregulated and 151 upregulated DEGs were identified. In GSE<em>4</em>6306, 561 overlapping differentially methylated genes were obtained through the differential methylation analysis at the CpG <em>site</em> level, CpG island level and gene level. A total of 5 overlapping genes [dipeptidyl peptidase <em>4</em> (DPP<em>4</em>); endothelin 3 (EDN3); fibroblast growth factor 1<em>4</em> (FGF1<em>4</em>); tachykinin, precursor 1 (TAC1); and <em>wingless</em>-<em>type</em> <em>MMTV</em> <em>integration</em> <em>site</em> <em>family</em>, <em>member</em> 16 (WNT16)] between the 561 overlapping differentially methylated genes and the 2<em>4</em>2 DEGs were identified, which were downregulated and hypermethylated simultaneously in cervical cancer samples. Enriched GO terms were receptor binding (involving DPP<em>4</em>, EDN3, FGF1<em>4</em>, TAC1 and WNT16), ameboidal-<em>type</em> cell migration (DPP<em>4</em>, EDN3 and TAC1), mitogen-activated protein kinase cascade (FGF1<em>4</em>, EDN3 and WNT16) and cell proliferation (EDN3, WNT16, DPP<em>4</em> and TAC1). These results indicate that DPP<em>4</em>, EDN3, FGF1<em>4</em>, TAC1 and WNT16 may be involved in the pathogenesis of cervical cancer.