Cell invasion through basement membrane (BM) during development, immune surveillance, and metastatic cancer remains poorly understood. We have completed the first in vivo screen for regulators of cell invasion through BM, using the simple model of C. elegans anchor cell invasion, and identified 99 genes that promote invasion, including the chaperonin complex cct. Notably, most of these genes have not been previously implicated in cell invasive behavior. We further characterized members of the cct complex and 11 other genes, determining the distinct aspects of the invasive cascade that they regulate, including formation of a specialized invasive cell membrane and its ability to breach the BM. Suggesting a shared genetic program underlies cell invasion, siRNA knockdown of the human orthologs of cct-5 and lit-1, both previously unknown pro-invasive genes, reduced the invasiveness of metastatic carcinoma cells. Our results reveal the genetic underpinnings of cell invasion and provide new potential therapeutic targets to limit this behavior.
Wheat (Triticum aestivum L.) is one of the most important crops cultivated worldwide. Identifying the complete transcriptome of wheat grain could serve as foundation for further study of wheat seed development. However, the relatively large size and the polyploid complexity of the genome have been substantial barriers to molecular genetics and transcriptome analysis of wheat. Alternatively, RNA sequencing has provided some useful information about wheat genes. However, because of the large number of short reads generated by RNA sequencing, factors that are crucial to transcriptome assembly, including software, candidate parameters and assembly strategies, need to be optimized and evaluated for wheat data. In the present study, four cDNA libraries associated with wheat grain development were constructed and sequenced. A total of 14.17 Gb of high-quality reads were obtained and used to assess different assembly strategies. The most successful approach was to filter the reads with Q30 prior to de novo assembly using Trinity, merge the assembled contigs with genes available in wheat cDNA reference data sets, and combine the resulting assembly with an assembly from a reference-based strategy. Using this approach, a relatively accurate and nearly complete transcriptome associated with wheat grain development was obtained, suggesting that this is an effective strategy for generation of a high-quality transcriptome from RNA sequencing data.
Two new species of the genus Pseudolathra Casey from mainland China are described and attributed to their respective species groups, P. cylindrata
sp. n. from Hubei and Sichuan, and P. superficiaria
sp. n. from Yunnan. Pseudolathra pulchella (Kraatz, 1859), P. transversiceps Assing, 2013 and P. bipectinata Assing, 2013 from Yunnan are reported from China for the first time. The history of the exploration of the Chinese fauna of Pseudolathra is summarized.
New species; Pseudolathra; Paederinae; Staphylinidae; China
The objective of this study is to develop a structural nanocomposite of multiple components in the form of core-sheath nanofibres using coaxial electrospinning for the fast dissolving of a poorly water-soluble drug quercetin. Under the selected conditions, core-sheath nanofibres with quercetin and sodium dodecyl sulphate (SDS) distributed in the core and sheath part of nanofibres, respectively, were successfully generated, and the drug content in the nanofibres was able to be controlled simply through manipulating the core fluid flow rates. Field emission scanning electron microscope (FESEM) images demonstrated that the nanofibres prepared from the single sheath fluid and double core/sheath fluids (with core-to-sheath flow rate ratios of 0.4 and 0.7) have linear morphology with a uniform structure and smooth surface. The TEM images clearly demonstrated the core-sheath structures of the produced nanocomposites. Differential scanning calorimetry (DSC) and X-ray diffraction (XRD) results verified that quercetin and SDS were well distributed in the polyvinylpyrrolidone (PVP) matrix in an amorphous state, due to the favourite second-order interactions. In vitro dissolution studies showed that the core-sheath composite nanofibre mats could disintegrate rapidly to release quercetin within 1 min. The study reported here provides an example of the systematic design, preparation, characterization and application of a new type of structural nanocomposite as a fast-disintegrating drug delivery system.
nanocomposites; core-sheath nanofibres; coaxial electrospinning; fast disintegrating; quercetin
Golgi protein 73 (GP73) is a resident Golgi type II transmembrane protein that has been reported to markedly increase in chronic liver disease, particularly in hepatocellular carcinoma (HCC). However, it remains unclear as to whether serum GP73 represents a reliable serum marker for the diagnosis of HCC. The aim of the present study was to evaluate the diagnostic value of serum GP73 in patients with HCC and to determine the diagnostic accuracy of measuring serum GP73 in combination with α-fetoprotein (AFP) and γ-glutamyl transferase isoenzyme II (GGT-II) in HCC. Serum GP73 was detected using a time-resolved fluorescence immunological assay (TRFIA) and enzyme-linked immunosorbent assay (ELISA) in 79 HCC cases, including 16 liver cirrhosis, 30 chronic hepatitis and 28 healthy individuals. The correlation between serum GP73 and tumor size and HCC grading was analyzed and the complementary diagnostic value of serum GP73, AFP and GGT-II was evaluated. TRFIA was established for the detection of serum GP73 and was sensitive and reproducible. The expression levels of serum GP73 were markedly higher in the patients with HCC when compared with those of the individuals with liver cirrhosis and chronic hepatitis or the healthy individuals. According to the receiver operating characteristic (ROC) curve, diagnostic sensitivity and specificity for HCC with a cut-off value of 78.1 ng/l were 73.4 and 79.0%, respectively. However, no correlation was identified among serum GP73 and tumor size or grading, and no correlations were identified among serum GP73, AFP and GGT-II. The diagnostic sensitivities for HCC, as detected by TRFIA of GP73, AFP and GGT-II, were 73.4, 55.6 and 68.4%, respectively, and the specificities were 80.0, 86.7 and 97.1%, respectively. The combined determination of these markers increased the diagnostic sensitivity to 96.3% for HCC. TRFIA functions as a sensitive and replicable assay for the detection of serum GP73. The levels of serum GP73 were significantly higher in the HCC group when compared with the individuals with benign liver diseases. Serum GP73 may serve as a potential independent diagnostic candidate for HCC and the combined determination of serum GP73, AFP and GGT-II may increase the diagnostic efficiency of HCC.
time-resolved fluorescence immunological assay; golgi protein 73; hepatocellular carcinoma
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.
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.
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.
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.
Gene expression; Lipid; RNA-sequencing; Soybean; Unigene
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.
Piglet; Metabolomics; Weaning; Glutamine; Gas chromatography/mass spectrometry
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.
To study the optical property and biocompatibility of a tissue engineering cornea.
: 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.
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.
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.
tissue engineering; collagen cross-linking scaffold; primary culture in vitro; optical property; biocompatibility
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.
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.
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.
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.
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.
Body Fluids; EGFR Mutation; Direct Sequencing; ARMS; TKIs; NSCLC
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.
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.
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).
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.
intraocular lens; suture fixation; astigmatism; lack of posterior capsule
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
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.
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).
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.
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.
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.