Although loss of p53 function and activation of canonical Wnt signaling cascades are frequently coupled in cancer, the links between these two pathways remain unclear. We report here that p53 transactivates miRNA-34 (miR-34), which suppresses the transcriptional activity of β-catenin-T-cell factor/lymphoid enhancer factor (TCF/LEF) complexes by targeting the untranslated regions (UTRs) of a set of highly-conserved targets in a network of Wnt pathway-regulated genes. Loss of p53 function increases canonical Wnt signaling through miR-34-specific interactions with target UTRs, whereas miR-34 depletion relieves p53-mediated Wnt repression. Further, gene expression signatures reflecting the status of β-catenin-TCF/LEF transcriptional activity in breast cancer and pediatric neuroblastoma patients are closely associated with p53 and miR-34 functional status. Loss of p53 or miR-34 contributed to neoplastic progression by triggering the Wnt-dependent, tissue-invasive activity of colorectal cancer cells. Further, during development, miR-34 interactions with the β-catenin UTR determine Xenopus body axis polarity and Wnt-dependent gene patterning. These data provide insight into the mechanisms by which a p53-miR-34 network restrains canonical Wnt signaling cascades in developing organisms and human cancer.

Background

Soybean (Glycine max L.) is one of the most important oil crops in the world. It is desirable to increase oil yields from soybean, and so this has been a major goal of oilseed engineering. However, it is still uncertain how many genes and which genes are involved in lipid biosynthesis.

Results

Here, we evaluated changes in gene expression over the course of seed development using Illumina (formerly Solexa) RNA-sequencing. Tissues at 15 days after flowering (DAF) served as the control, and a total of 11592, 16594, and 16255 differentially expressed unigenes were identified at 35, 55, and 65 DAF, respectively. Gene Ontology analyses detected 113 co-expressed unigenes associated with lipid biosynthesis. Of these, 15 showed significant changes in expression levels (log2fold values ≥ 1) during seed development. Pathway analysis revealed 24 co-expressed transcripts involved in lipid biosynthesis and fatty acid biosynthesis pathways. We selected 12 differentially expressed genes and analyzed their expressions using qRT-PCR. The results were consistent with those obtained from Solexa sequencing.

Conclusion

These results provide a comprehensive molecular biology background for research on soybean seed development, particularly with respect to the process of oil accumulation. All of the genes identified in our research have significance for breeding soybeans with increased oil contents.

A novel metabolomic method based on gas chromatography/mass spectrometry (GC-MS) was applied to determine the metabolites in the serum of piglets in response to weaning and dietary L-glutamine (Gln) supplementation. Thirty-six 21-d-old piglets were randomly assigned into three groups. One group continued to suckle from the sows (suckling group), whereas the other two groups were weaned and their diets were supplemented with 1% (w/w) Gln or isonitrogenous L-alanine, respectively, representing Gln group or control group. Serum samples were collected to characterize metabolites after a 7-d treatment. Results showed that twenty metabolites were down-regulated significantly (P<0.05) in control piglets compared with suckling ones. These data demonstrated that early weaning causes a wide range of metabolic changes across arginine and proline metabolism, aminosugar and nucleotide metabolism, galactose metabolism, glycerophospholipid metabolism, biosynthesis of unsaturated fatty acid, and fatty acid metabolism. Dietary Gln supplementation increased the levels of creatinine,D-xylose, 2-hydroxybutyric acid, palmitelaidic acid, and α-L-galactofuranose (P<0.05) in early weaned piglets, and were involved in the arginine and proline metabolism, carbohydrate metabolism, and fatty acid metabolism. A leave-one-out cross-validation of random forest analysis indicated that creatinine was the most important metabolite among the three groups. Notably, the concentration of creatinine in control piglets was decreased (P=0.00001) compared to the suckling piglets, and increased (P=0.0003) in Gln-supplemented piglets. A correlation network for weaned and suckling piglets revealed that early weaning changed the metabolic pathways, leading to the abnormality of carbohydrate metabolism, amino acid metabolism, and lipid metabolism, which could be partially improved by dietary Gln supplementation. These findings provide fresh insight into the complex metabolic changes in response to early weaning and dietary Gln supplementation in piglets.

FAK promotes the epithelial–mesenchymal transition in mouse embryonic cells by regulating the transcription factor Snail1.

Mouse embryonic cells isolated from focal adhesion kinase (FAK)–null animals at embryonic day 7.5 display multiple defects in focal adhesion remodeling, microtubule dynamics, mechanotransduction, proliferation, directional motility, and invasion. To date, the ability of FAK to modulate cell function has been ascribed largely to its control of posttranscriptional signaling cascades in this embryonic cell population. In this paper, we demonstrate that FAK unexpectedly exerts control over an epithelial–mesenchymal transition (EMT) program that commits embryonic FAK-null cells to an epithelial status highlighted by the expression of E-cadherin, desmoplakin, and cytokeratins. FAK rescue reestablished the mesenchymal characteristics of FAK-null embryonic cells to generate committed mouse embryonic fibroblasts via an extracellular signal–related kinase– and Akt-dependent signaling cascade that triggered Snail1 gene expression and Snail1 protein stabilization. These findings indentify FAK as a novel regulator of Snail1-dependent EMT in embryonic cells and suggest that multiple defects in FAK−/− cell behavior can be attributed to an inappropriate commitment of these cells to an epithelial, rather than fibroblastic, phenotype.

Expression of the essential EMT inducer Snail1 is inhibited by miR-34 through a p53-dependent regulatory pathway.

Snail1 is a zinc finger transcriptional repressor whose pathological expression has been linked to cancer cell epithelial–mesenchymal transition (EMT) programs and the induction of tissue-invasive activity, but pro-oncogenic events capable of regulating Snail1 activity remain largely uncharacterized. Herein, we demonstrate that p53 loss-of-function or mutation promotes cancer cell EMT by de-repressing Snail1 protein expression and activity. In the absence of wild-type p53 function, Snail1-dependent EMT is activated in colon, breast, and lung carcinoma cells as a consequence of a decrease in miRNA-34 levels, which suppress Snail1 activity by binding to highly conserved 3′ untranslated regions in Snail1 itself as well as those of key Snail1 regulatory molecules, including β-catenin, LEF1, and Axin2. Although p53 activity can impact cell cycle regulation, apoptosis, and DNA repair pathways, the EMT and invasion programs initiated by p53 loss of function or mutation are completely dependent on Snail1 expression. These results identify a new link between p53, miR-34, and Snail1 in the regulation of cancer cell EMT programs.

Objective. To evaluate diagnostic utility of Dishevelled-3 (DVL-3) mRNA and δ-catenin mRNA expression in pleural effusions of patients with lung cancer. Methods. DVL-3 mRNA and δ-catenin mRNA levels were assessed by performing RT-PCR on pleural effusion specimens from patients with lung cancer (n = 75) and with lung benign disease (n = 51). Results. The expressions of DVL-3 mRNA and δ-catenin mRNA were significantly higher in malignant than in benign lung disease (P < 0.01) and were obviously higher than cytology in adenocarcinoma (P < 0.01). In single use, DVL-3 mRNA had the highest specificity (94.1%) and PPV (95.7%), whereas δ-catenin mRNA had the highest sensitivity (92.0%) and NPV (88.5%). When combinations of markers were evaluated together, DVL-3 mRNA and δ-catenin mRNA gave a high-diagnostic performance: sensitivity of 100.0%, NPV of 100.0%, and accuracy of 96.0%, respectively. Conclusion. As molecular markers of detecting pleural micrometastasis, DVL-3 mRNA and δ-catenin mRNA are helpful to diagnose the cancer cells in pleural effusions of patients with lung cancer.

AIM

To study the optical property and biocompatibility of a tissue engineering cornea.

METHODS

: The cross-linker of N-(3-Dimethylaminoropyl)-N'ethylcarbodiimide hydrochloride (EDC)/N-Hydroxysuccinimide (NHS) was mixed with Type I collagen at 10% (weight/volume). The final solution was molded to the shape of a corneal contact lens. The collagen concentrations of 10%, 12.5%, 15%, 17.5% and 20% artificial corneas were tested by UV/vis-spectroscopy for their transparency compared with normal rat cornea. 10-0 sutures were knotted on the edges of substitute to measure the corneal buttons's mechanical properties. Normal rat corneal tissue primary culture on the collagen scaffold was observed in 4 weeks. Histopathologic examinations were performed after 4 weeks of in vitro culturing.

RESULTS

The collagen scaffold appearance was similar to that of soft contact lens. With the increase of collagen concentration, the transparency of artificial corneal buttons was diminished, but the toughness of the scaffold was enhanced. The scaffold transparency in the 10% concentration collagen group resembled normal rat cornea. To knot and embed the scaffold under the microscope, 20% concentration collagen group was more effective during implantation than lower concentrations of collagen group. In the first 3 weeks, corneal cell proliferation was highly active. The shapes of cells that grew on the substitute had no significant difference when compared with the cells before they were moved to the scaffold. However, on the fortieth day, most cells detached from the scaffold and died. Histopathologic examination of the primary culture scaffold revealed well grown corneal cells tightly attached to the scaffold in the former culturing.

CONCLUSION

Collagen scaffold can be molded to the shape of soft contact corneal lens with NHS/EDC. The biological stability and biocompatibility of collagen from animal species may be used as material in preparing to engineer artificial corneal scaffold.

In the title compound, C14H10N4O5, the mol­ecule exists in a trans conformation with respect to the methyl­idene unit. The dihedral angle between the benzene rings is 9.8 (2)°. In the crystal, mol­ecules are linked through N—H⋯O hydrogen bonds to form chains along the c axis.

In the title compound, C14H10FN3O3, the mol­ecule exists in a trans conformation with respect to the methyl­idene unit. The dihedral angle between the benzene rings is 5.1 (2)°. In the crystal, mol­ecules are linked through N—H⋯O hydrogen bonds, forming chains along the c axis.

The title hydrazone mol­ecule, C14H11ClN2O2, has a trans conformation with respect to the methyl­idene unit. The dihedral angle between the two benzene rings is 37.6 (3)°. In the crystal, the presence of O—H⋯O, O—H⋯N and N—H⋯O hydrogen bonds leads to the formation of a three-dimensional network. The title compound crystallized in the chiral ortho­rhom­bic space group P212121 and was refined as an inversion twin [Flack parameter = −0.20 (18)].

In the title compound, C15H12I2N2O4·CH3OH, the hydrazone mol­ecule exists in an E conformation with respect to the C=N bond. The dihedral angle between the rings is 11.9 (2)°. There is one intra­molecular O—H⋯N hydrogen bond in the hydrazone mol­ecule. In the crystal, the hydrazone and methanol mol­ecules are linked through O—H⋯O and N—H⋯O hydrogen bonds and C—H⋯O inter­actions to form two-dimensional networks lying parallel to (001).

The title compound, C14H10N4O5, has an E conformation with respect to the C=N bond. The dihedral angle between the benzene rings is 2.41 (14)°. In the crystal, mol­ecules are linked through N—H⋯O hydrogen bonds to form chains along the c axis. C—H⋯O inter­actions are also present, linking the chains to form a three-dimensional network.

Background

Epidermal growth factor receptor (EGFR) mutation is strongly associated with the therapeutic effect of tyrosine kinase inhibitors (TKIs) in patients with non-small-cell lung cancer (NSCLC). Nevertheless, tumor tissue that needed for mutation analysis is frequently unavailable. Body fluid was considered to be a feasible substitute for the analysis, but arising problems in clinical practice such as relatively lower mutation rate and poor clinical correlation are not yet fully resolved.

Method

In this study, 50 patients (32 pleural fluids and 18 plasmas) with TKIs therapy experience and with direct sequencing results were selected from 220 patients for further analysis. The EGFR mutation status was re-evaluated by Amplification Refractory Mutation System (ARMS), and the clinical outcomes of TKIs were analyzed retrospectively.

Results

As compared with direct sequencing, 16 positive and 23 negative patients were confirmed by ARMS, and the other 11 former negative patients (6 pleural fluids and 5 plasmas) were redefined as positive, with a fairly well clinical outcome (7 PR, 3 SD, and 1 PD). The objective response rate (ORR) of positive patients was significant, 81.3% (direct sequencing) and 72.7% (ARMS) for pleural fluids, and 80% (ARMS) for plasma. Notably, even reclassified by ARMS, the ORR for negative patients was still relatively high, 60% for pleural fluids and 46.2% for plasma.

Conclusions

When using body fluids for EGFR mutation analysis, positive result is consistently a good indicator for TKIs therapy, and the predictive effect was no less than that of tumor tissue, no matter what method was employed. However, even reclassified by ARMS, the correlation between negative results and clinical outcome of TKIs was still unsatisfied. The results indicated that false negative mutation still existed, which may be settled by using method with sensitivity to single DNA molecule or by optimizing the extraction procedure with RNA or CTC to ensure adequate amount of tumor-derived nucleic acid for the test.

AIM

To study the impact of scleral flap position, under which the posterior chamber intraocular lenses (PC-IOL) were sulcus-fixed by trans-scleral suture, on cornea astigmatism.

METHODS

Twenty-six aphakic or cataract eyes were comprised in this prospective noncomparative case series study. Eleven eyes had traumatic cataract removed without sufficient capsular support, 3 had blunt trauma with subluxated traumatic cataract, 8 had undergone vitreoretinal surgery and 4 had congenital cataract removed. The average age was 54 years (range 21-74 years), with 17 men and 7 women. The foldable PC-IOL was fixed in sulcus by trans-scleral suture. The incision for IOL implantation was made 1mm posterior to limbus along the steepest meridian of cornea, while scleral flaps to bury the knots of trans-scleral suture were made along the flattest meridian. All the surgeries were performed by a single doctor (Ma L), and the follow up was at least 13 months (range 13-28 months). The preoperative, 3 months and 1 year postoperative corneal curvature along the steepest and flattest cornea meridian and overall cornea astigmatism were compared.

RESULTS

The curvature along the steepest meridian changed from 44.25±2.22D preoperatively to 44.08±2.16D at 3 months postoperatively, and 43.65±5.23D at 1 year postoperatively (P>0.05); the curvature along the flattest meridian changed from 41.24±2.21D preoperatively to 43.15±3.94D at 3 months postoperatively, and 42.85±5.17D at 1 year postoperatively (P<0.05); and the surgery induced astigmatism (SIA) on cornea was calculated by vector analysis, which was 2.42±2.13D at 3 months postoperatively, and 2.18±3.42D at 1 year postoperatively, the difference was statistically significant (P<0.05).

CONCLUSION

The scleral flap made along the flattest meridian, under which the posterior chamber intraocular lenses (PCIOL) were sulcus-fixed by trans-scleral suture, can steepen the cornea in varying degrees, thus reducing preexisting corneal astigmatism.

There are three independent mol­ecules in the asymmetric unit of the title compound, C19H16N2O3, in which the dihedral angles between the naphthalene ring system and the benzene ring are 7.52 (16), 18.15 (18), and 13.9 (2)°. All the mol­ecules exist in the trans configuration with respect to the methyl­idene units. In each mol­ecule there is one O—H⋯N and one N—H⋯O intra­molecular hydrogen bond. In the crystal, two of the mol­ecules are linked via a bifurcated N—H⋯(O,O) hydrogen bond. All three mol­ecules are further linked via C—H⋯O inter­actions.

There are three independent mol­eculesi n the asymmetric unit of the title compound, C18H16BrN3O3, in which the dihedral angles between the indole and benzene rings are 76.9 (2), 4.9 (2), and 70.9 (2)°. All three mol­ecules exist in a trans configuration with respect to the methyl­idene units. In each mol­ecule, there is one intra­molecular O—H⋯N hydrogen bond. In the crystal, N—H⋯O hydrogen bonds occur.

In the title compound, C15H12BrClN2O2, the dihedral angle between the two substituted aromatic rings is 77.8 (3)°. The mol­ecule exists in a trans conformation with respect to the methyl­idene unit. In the crystal structure, inversion dimers linked by pairs of N—H⋯O hydrogen bonds generate R 2 8(8) loops.

In the title compound, C15H12Cl2N2O3, the dihedral angle between the two substituted aromatic rings is 5.4 (4)°. Intra­molecular O—H⋯N and N—H⋯O hydrogen bonds affect the planarity of the molcular conformation, with a mean deviation from the plane defined by the non-H atoms of 0.062 (2) Å. The mol­ecule exists in a trans configuration with respect to the methyl­idene unit. In the crystal, mol­ecules are linked by N—H⋯O inter­actions.

Background

The esophageal carcinoma related gene 4 (ECRG4) was initially identified and cloned from human normal esophageal epithelium in our laboratory (GenBank accession no.AF325503). ECRG4 has been described as a novel tumor suppressor gene associated with prognosis in esophageal squamous cell carcinoma (ESCC).

Methods

In this study, binding affinity assay in vitro and co-immunoprecipitation experiment in vivo were utilized to verify the physical interaction between ECRG4 and transmembrane protease, serine 11A (TMPRSS11A, also known as ECRG1, GenBank accession no. AF 071882). Then, p21 protein expression, cell cycle and cell proliferation regulations were examined after ECRG4 and ECRG1 co-transfection in ESCC cells.

Results

We revealed for the first time that ECRG4 interacted directly with ECRG1 to inhibit cancer cell proliferation and induce cell cycle G1 phase block in ESCC. Binding affinity and co-immunoprecipitation assays demonstrated that ECRG4 interacted directly with ECRG1 in ESCC cells. Furthermore, the ECRG4 and ECRG1 co-expression remarkably upregulatd p21 protein level by Western blot (P < 0.001), induced cell cycle G1 phase block by flow cytometric analysis (P < 0.001) and suppressed cell proliferation by MTT and BrdU assay (both P < 0.01) in ESCC cells.

Conclusions

ECRG4 interacts directly with ECRG1 to upregulate p21 protein expression, induce cell cycle G1 phase block and inhibit cancer cells proliferation in ESCC.

Epithelial–mesenchymal transition (EMT) is required for mesodermal differentiation during development. The zinc-finger transcription factor, Snail1, can trigger EMT and is sufficient to transcriptionally reprogram epithelial cells toward a mesenchymal phenotype during neoplasia and fibrosis. Whether Snail1 also regulates the behavior of terminally differentiated mesenchymal cells remains unexplored. Using a Snai1 conditional knockout model, we now identify Snail1 as a regulator of normal mesenchymal cell function. Snail1 expression in normal fibroblasts can be induced by agonists known to promote proliferation and invasion in vivo. When challenged within a tissue-like, three-dimensional extracellular matrix, Snail1-deficient fibroblasts exhibit global alterations in gene expression, which include defects in membrane type-1 matrix metalloproteinase (MT1-MMP)-dependent invasive activity. Snail1-deficient fibroblasts explanted atop the live chick chorioallantoic membrane lack tissue-invasive potential and fail to induce angiogenesis. These findings establish key functions for the EMT regulator Snail1 after terminal differentiation of mesenchymal cells.

In the title compound, [Co(C13H8F2O)(C3H9P)3], the cobalt(0) atom is coordinated by three trimethyl­phosphine ligands and a π-coordinated carbonyl group of the 2,6-difluoro­benzo­phenone ligand in a distorted tetra­hedral geometry. The Co—O and Co—C distances are 1.896 (2) and 2.049 (4) Å, respectively.

The title three-dimensional coordination polymer, [Cu2Cl(C6H4N5)]n, is the product of the hydro­thermal reaction of CuCl2·2H2O and 5-(4-pyrid­yl)-1H-tetra­zole (4-Hptz). The two independent CuI ions are coordinated in distorted tetra­hedral and distorted trigonal coordination environments. In the unique 5-(4-pyrid­yl)-1H-tetra­zolate ligand, the dihedral angle between the pyridine and tetra­zole rings is 17.3 (2)°.

The asymmetric unit of the title compound, {[Zn(C7H7O2)2(C10H8N2)]·4H2O}n, contains a highly distorted octa­hedral ZnII metal center strongly coordinated by two N atoms of two 4,4′-bipyridine (4,4′-bipy) ligands and chelated by two 4-methyl­benzoate anions. The crystallographic inversion center and glide plane present at the center of the C—C single bond of 4,4′-bipy, along with the cis coordination motif of the 4,4′-bipy, lead to one-dimensional zigzag chains. There are a large number of water mol­ecules in the crystal structure, which also form one-dimensional chains through O—H⋯O hydrogen bonds.

In the title compound, C13H9ClN4O4, there are two crystallographically independent mol­ecules in the asymmetric unit, which have very similar conformations. The C=N—N angles in each independent mol­ecule are 115.0 (2) and 116.6 (2)°, which are significantly smaller than the ideal value of 120° expected for sp 2-hybridized N atoms. This is probably a consequence of repulsion between the nitro­gen lone pairs and the adjacent N—N bonds. Two bifurcated intra­molecular N—H⋯O hydrogen bonds help to establish the mol­ecular conformation and consolidate the crystal packing.

Membrane type-1 matrix metalloproteinase (MT1-MMP) drives cell invasion through three-dimensional (3-D) extracellular matrix (ECM) barriers dominated by type I collagen or fibrin. Based largely on analyses of its impact on cell function under two-dimensional culture conditions, MT1-MMP is categorized as a multifunctional molecule with 1) a structurally distinct, N-terminal catalytic domain; 2) a C-terminal hemopexin domain that regulates substrate recognition as well as conformation; and 3) a type I transmembrane domain whose cytosolic tail controls protease trafficking and signaling cascades. The MT1-MMP domains that subserve cell trafficking through 3-D ECM barriers in vitro or in vivo, however, remain largely undefined. Herein, we demonstrate that collagen-invasive activity is not confined strictly to the catalytic, hemopexin, transmembrane, or cytosolic domain sequences of MT1-MMP. Indeed, even a secreted collagenase supports invasion when tethered to the cell surface in the absence of the MT1-MMP hemopexin, transmembrane, and cytosolic tail domains. By contrast, the ability of MT1-MMP to support fibrin-invasive activity diverges from collagenolytic potential, and alternatively, it requires the specific participation of MT-MMP catalytic and hemopexin domains. Hence, the tissue-invasive properties of MT1-MMP are unexpectedly embedded within distinct, but parsimonious, sequences that serve to tether the requisite matrix-degradative activity to the surface of migrating cells.