XAF1 promotes colorectal cancer metastasis via VCP-RNF114-JUP axis.

Metastasis is the main cause of colorectal cancer (CRC)-related death, and the 5-year relative survival rate for CRC patients with distant metastasis is only 14%. X-linked inhibitor of apoptosis (XIAP)-associated factor 1 (XAF1) is a zinc-rich protein belonging to the interferon (IFN)-induced gene family. Here, we report a metastasis-promoting role of XAF1 in CRC by acting as a novel adaptor of valosin-containing protein (VCP). XAF1 facilitates VCP-mediated deubiquitination of the E3 ligase RING finger protein 114 (RNF114), which promotes K48-linked ubiquitination and subsequent degradation of junction plakoglobin (JUP). The XAF1-VCP-RNF114-JUP axis is critical for the migration and metastasis of CRC cells. Moreover, we observe correlations between the protein levels of XAF1, RNF114, and JUP in clinical samples. Collectively, our findings reveal an oncogenic function of XAF1 in mCRC and suggest that the XAF1-VCP-RNF114-JUP axis is a potential therapeutic target for CRC treatment.

An Intelligence Method for Recognizing Multiple Defects in Rail.

Ultrasonic guided waves are sensitive to many different types of defects and have been studied for defect recognition in rail. However, most fault recognition algorithms need to extract features from the time domain, frequency domain, or time-frequency domain based on experience or professional knowledge. This paper proposes a new method for identifying many different types of rail defects. The segment principal components analysis (S-PCA) is developed to extract characteristics from signals collected by sensors located at different positions. Then, the Support Vector Machine (SVM) model is used to identify different defects depending on the features extracted. Combining simulations and experiments of the rails with different kinds of defects are established to verify the effectiveness of the proposed defect identification techniques, such as crack, corrosion, and transverse crack under the shelling. There are nine channels of the excitation-reception to acquire guided wave detection signals. The results show that the defect classification accuracy rates are 96.29% and 96.15% for combining multiple signals, such as the method of single-point excitation and multi-point reception, or the method of multi-point excitation and reception at a single point.

The unique molecular mechanism of diabetic nephropathy: a bioinformatics analysis of over 250 microarray datasets.

BACKGROUND/AIMS: Diabetic nephropathy (DN) is one of the main causes of end-stage kidney disease worldwide. Emerging studies have suggested that its pathogenesis is distinct from nondiabetic renal diseases in many aspects. However, it still lacks a comprehensive understanding of the unique molecular mechanism of DN. METHODS: A total of 255 Affymetrix U133 microarray datasets (Affymetrix, Santa Calra, CA, USA) of human glomerular and tubulointerstitial tissues were collected. The 22 215 Affymetrix identifiers shared by the Human Genome U133 Plus 2.0 and U133A Array were extracted to facilitate dataset pooling. Next, a linear model was constructed and the empirical Bayes method was used to select the differentially expressed genes (DEGs) of each kidney disease. Based on these DEG sets, the unique DEGs of DN were identified and further analyzed using gene ontology and pathway enrichment analysis. Finally, the protein-protein interaction networks (PINs) were constructed and hub genes were selected to further refine the results. RESULTS: A total of 129 and 1251 unique DEGs were identified in the diabetic glomerulus (upregulated n = 83 and downregulated n = 203) and the diabetic tubulointerstitium (upregulated n = 399 and downregulated n = 874), respectively. Enrichment analysis revealed that the DEGs in the diabetic glomerulus were significantly associated with the extracellular matrix, cell growth, regulation of blood coagulation, cholesterol homeostasis, intrinsic apoptotic signaling pathway and renal filtration cell differentiation. In the diabetic tubulointerstitium, the significantly enriched biological processes and pathways included metabolism, the advanced glycation end products-receptor for advanced glycation end products signaling pathway in diabetic complications, the epidermal growth factor receptor (EGFR) signaling pathway, the FoxO signaling pathway, autophagy and ferroptosis. By constructing PINs, several nodes, such as AGR2, CSNK2A1, EGFR and HSPD1, were identified as hub genes, which might play key roles in regulating the development of DN. CONCLUSIONS: Our study not only reveals the unique molecular mechanism of DN but also provides a valuable resource for biomarker and therapeutic target discovery. Some of our findings are promising and should be explored in future work.

[Inhibitory effect of iguratimod on TNFalpha production and NF-kappaB activity in LPS-stimulated rat alveolar macrophage cell line].

AIM: To investigate the effect of iguratimod (T-614), a non-steroidal anti-inflammatory drug, on TNFalpha mRNA expression and TNFalpha production, and on the activity of nuclear factor-kappaB (NF-kappaB) in the rat alveolar macrophage cell line (NR8383) activated by LPS. METHODS: NR8383 cells were pretreated with T-614 (13.4, 26.7, 53.4 micromol x L(-1)), then were stimulated with LPS. The production of TNFalpha in the supernatant of NR8383 was assayed by enzyme-linked immunosorbent assay (ELISA). The TNFalpha mRNA level was determined by a semi-quantitative PCR assay. Assessment of the NF-kappaB DNA binding activity was performed by an ELISA kit. RESULTS: T-614 inhibited LPS-stimulated mRNA expression and production of TNFalpha in a concentration-dependent manner, as well as the activity of NF-kappaB. The IC50 value of effect of T-614 on TNFalpha level was 26.2 micromol x L(-1). CONCLUSION: The inhibitory effect of T-614 on the production of TNFalpha in LPS-stimulated NR8383 cells may be mediated by suppression of NF-kappaB activity.

RTN4-C gene expression in hepatocellular carcinoma and its influence on SMMC7721 cell growth and apoptosis.

RTN4-C gene is a member of RTNs. To investigate its expression in human hepatocellular carcinoma tissues and study its function on SMMC7721 cells, RT-PCR was conducted to detect its expressions in human hepatocellular carcinoma tissues. RTN4-C-pcDNA3. 1 plasmid was reconstructed and stably transfected into SMMC7721 cells by Lipofect AMINE. Growth curve of SMMC7721 measured by MTT and apoptosis indentified with acridine orange staining were examined to demonstrate the effect of RTN4-C on SMMC7721. Immunocytochemical analysis for mutant p53, c-Fos, Hsp70 proteins were conducted. The results showed that the transfected SMMC7721 cells grew more slowly than control and the average inhibition rate at the 1st, 2nd and 3rd day were 33.8% +/- 0.026, 56.2% +/- 0. 094, 34.8% +/- 0.077 respectively. In transfected SMMC7721 cell line, the apoptosis was strengthened,mutant p53 protein tranferred from nucleus to cytoplasm and c-Fos, Hsp70 proteins were decreased. Our data indicated RTN4-C gene was expressed differently in hepatocellular carcinoma and its paracancerous tissues. By tranferring mutant p53 protein from nucleus to cytoplasm and decreasing c-Fos, Hsp70 protein expression, RTN4-C inhibited SMMC7721 cells growth and promoted its apoptosis.

[Phylogenetic relationship between tufted deer(Elaphodus cephalophus) and Muntiacus deer is revealed by the exon and intron of K+ channel gene].

In this study, partial fragments of potassium ion channel gene were amplified using the genomic DNA of muntjak, reevesi, crinifrons, and Elaphodus cephalophus. The PCR products were ligated to the plasmid of pMD18-T Vector by the method of direct T-A cloning. The positive clones were identified by colony PCR. The sequences of the recombinant clones were determined using M13-47/RV-M universal primers and aligned by the software CLUSTALW. The nucleotide divergences of exon were 0.90%-1.44% among three species of Muntiacus, 0.90%-1.26% between E. cephalophus and each of Muntiacus deer. In the nucleotide of intron there is 0%-1.22% difference among these muntjac deers, and the divergene reached about 1.83% between E. cephalophus and the three species of Muntiacus. Using the software of MEGA to analyse molecular phylogeny, Phylogenetic trees were constructed with neighbor-joining method and maximum parsimony method. The result showed Muntiacus, crinifrons is most closely related to muntjak, with reevesi as their sister species. E. cephalophus is in the other genus.

Down regulation of DRET gene in human hepatocellular carcinoma tissues samples from Qidong area, China.

OBJECTIVE: To analyze the expression of DRET gene in hepatocellular carcinomas (HCCs) tissue specimens in comparison with normal liver tissue to evaluate the relationship between DRET gene and HCC. METHODS: 250 primary HCCs and 50 normal liver tissue samples from Qidong area, China, were studied for DRET mRNA and protein expression with the use of Northern blot analysis, in situ hybridization and immunohistochemistry. RESULTS: By Northern analysis, moderate to strong DRET mRNA expression was present in normal liver samples. In contrast, DRET mRNA expression in tissue samples of primary HCCs was markedly decreased compared with normal controls. Primary HCCs that gave rise to metastasis showed significantly lower DRET mRNA levels than nonmetastasizing HCCs. By in situ hybridization analysis, nonmetastatic HCCs samples didn't differ from controls. In contrast, most of the primary metastasizing HCC showed only faint or moderate DRET mRNA expression. Tissue sections of nonmetastatic HCC exhibited lower DRET immunoreactivity than control samples, but higher labeling index than metastatic HCC samples. CONCLUSIONS: Expression of DRET on mRNA and protein levels in HCC cells is related to HCC metastatic ability.

Spatiotemporal pattern of calmodulin and [Ca2+]i is related to resumption of meiosis in mouse oocytes.

During meiotic maturation, mammalian oocytes undergo a series of morphological and physiological changes that prepare them for fertilization. Calcium-initiated signaling is thought to trigger these processes. In this study, we examine the spatio-temporal pattern of calcium and calmodulin (CaM), its downstream receptor, in order to investigate their association with meiotic maturation. Intracellular free calcium and activated CaM levels were measured using the fluorescent probes Calcium Green-1 and TA-CaM, respectively. The distribution patterns were examined using confocal microscopy. Both calcium and activated CaM showed a dynamic spatiotemporal distribution during meiotic maturation. After release from IBMX buffer, calcium was found to periodically translocate from the perinuclear region to the germinal vesicle (GV) in 90 s intervals. After 90 min, calcium stopped oscillating and became concentrated within the GV. After a further 60 min, the GV broke down and calcium dispersed into the ooplasm, but calcium levels were slightly lower here than in the original nuclear region. Activated CaM also showed a dynamic patterning process similar to calcium. Taking the data from calcium chelation and CaM inhibition together, our results suggest that the dynamic distribution patterns of calcium and activated CaM are crucial for oocyte maturation.

Expression of TN4 gene and its role in human hepatocarcinogenesis from Qidong, a liver cancer risk area.

BACKGROUND: We investigated the expression and role of TN4 in the oncogenesis of human hepatocellular carcinoma (HCC) from Qidong which is a HCC risk area. METHODS: The expression of TN4 in HCC was observed using immunohistochemical staining (IHC). TN4 levels were manipulated in human liver cancer cell SMMC7721, using pcDNA3.1 eukaryotic expression constructs designed to express the complete TN4 cDNA. The biological changes of the cells were observed before and after transfection of TN4 and the change of gene expression was analysed by atlas cDNA expression array. RESULTS: Among 100 pairs of samples of HCC, TN4 down-regulation expression and up-regulation expression positive rate were 81% (81/100), 19% (19/100), respectively (P < 0.01). TN4 protein was mainly localized in cytoplasm and membrane. The positive rate of TN4 were 10% (3/30), 100% (70/70) in lymph node metastasis and no lymph node metastasis, respectively (P < 0.01). The growth rates of the derivative SMMC7721-TN4 cell lines were decreased in comparison with that of normal SMMC7721 cells and pcDNA-SMMC7721. Some gene expression was changed before and after transfection of TN4. At 30 days of post-implantation of SMMC7721-TN4, SMMC7721-pcDNA3, SMMC7721 group produced tumors of (301.9 +/- 143.4) mm(3), (2418.7 +/- 362.8) mm(3), (2317.4 +/- 587.8) mm(3), respectively, (P < 0.01). Tumor inhibiting rate was 82.4% in TN4 transfection group. Sections of tumors were observed for their degree of tissue necrosis and there was higher degree of necrosis in tumors of the TN4-SMMC7721 cell group than those of the SMMC7721, SMMC7721-pcDNA groups. CONCLUSIONS: TN4 may play an important role in the oncogenesis of human HCC, especially in Qidong, the HCC risk area and TN4 could be a candidate tumor suppressor gene for HCC.

[Cloning of ZFY /ZFX gene of the tufted deer and identification of its sex].

According to the human sex differentiation related ZFY and ZFX genes, a pair of primers were designed , and fragments were amplified from the genomic DNA of male or female tufted deer. Subsequently the amplified fragments were cloned into the vector pMD18T and were sequenced. It is found that the sequences of ZFY gene and ZFX gene have 91% homology. Based on the different nucleotides, restriction site of Ava II was found to be specific to ZFX gene. The results show that the combination of PCR with Ava II digestion is a simple and sensitive way to identify the tufted deer sex.

Codon 249 mutation in exon 7 of p53 gene in plasma DNA: maybe a new early diagnostic marker of hepatocellular carcinoma in Qidong risk area, China.

AIM: One of the characteristics of hepatocellular carcinoma (HCC) in Qidong area is the selective mutation resulting in a serine substitution at codon 249 of the p53 gene (1, 20), and it has been identified as a "hotspot" mutation in heptocellular carcinomas occurring in populations exposed to aflatoxin and with high prevalence of hepatitis B virus carriers (2,3,9, 10,16,24). We evaluated in this paper whether this "hotspot" mutation could be detected in cell-free DNA circulating in plasma of patients with hepatocellular carcinoma and cirrhosis in Qidong, China, and tried to illustrate the significance of the detection of this molecular biomarker. METHODS: We collected blood samples from 25 hepatocellular carcinoma patients, 20 cirrhotic patients and 30 healthy controls in Qidong area. DNA was extracted and purified from 200 microl of plasma from each sample. The 249(Ser) p53 mutation was detected by restriction digestion analysis and direct sequencing of exon-7 PCR products. RESULTS: We found in exon 7 of p53 gene G-T transversion at the third base of codon 249 resulting 249(Arg) - 249(Ser) mutation in 10/25 (40 %) hepatocellular carcinoma cases, 4/20 (20 %) cirrhotics, and 2/30 (7 %) healthy controls. The adjusted odds ratio for having the mutation was 22.1(95 % CI, 3.2-91.7) for HCC cases compared to controls. CONCLUSION: These data show that the 249(Ser) p53 mutation in plasma is strongly associated with hepatocellular carcinoma in Qidong patients. We found this mutation was also detected, although it was at a much lower frequency, in plasma DNA of Qidong cirrhotics and healthy controls; We consider that these findings, together with the usual method of HCC diagnosis, will give more information in early diagnosis of HCC, and 249(Ser) p53 mutation should be developed to a new early diagnostic marker for HCC.

The relationship between metallothionein-1F (MT1F) gene and hepatocellular carcinoma.

To investigate the expression of MT1F gene in hepatocellular carcinoma tissue and the growth suppression effect of exogenous introduction of MT1F gene on liver cell line HepG2 and to explore the potential application of MT1F gene in gene therapy of tumor. Eukaryotic expression vector of pCMV-MT1F plasmid was introduced into HepG2 line which expressed no MT1F protein originally with lipofectamine transfection method. The cell growth curve, soft agar colony formation rate and tumorigenicity in SCID mice were examined to demonstrate the growth suppression effect of exogenous MT1F gene on HepG2 cell line. The MT1F mRNA and MT1F protein were also detected in 60 pairs of surgical specimens of hepatocellular carcinoma by in situ hybridization and immunohistochemistry. The transfected HepG2 cell line grew more slowly than control HepG2 as shown by cell growth curves, the soft agar colony formation rate (3.8 percent vs. 7.4 percent, p <.01) and the average growth rate of tumor in SCID mice (30.9 +/- 6.9 vs. 70.3 +/- 5.6, p <.01). The expression level of MT1F mRNA and protein significantly increased in paracancerous tissue, normal tissue than in cancer tissues (75 percent, 70 percent vs. 16.7 percent by ISH and 66.7 percent, 60 percent vs. 10 percent by IHC, p <.01). Exogenous MT1F gene shows the strong effect of growth inhibition on HepG2 cell line. In the liver cancer tissue, MT1F shows down-regulated expression that supports the inhibited function of MT1F in cancer growth and suggests MT1F may have an important role in gene therapy of hepatocellular carcinoma.

[Preliminary study on hens immunized with recombinant human testis prostaglandin D synthase DNA].

OBJECTIVES: In order to identify whether the hens immunized with recombinant human testis prostaglandin D synthase (rhtL-PGDS) DNA can produce anti-L-PGDS antibody. METHODS: The serum were got from the hens immunized with recombinant plasmid pGEX-2T/htL-PGDS DNA (100 micrograms) every 2 weeks for 2 times. The exist of anti-L-PGDS antibody and its titer were tested with agarose dual immunodiffusion and ELISA with rhtL-PGDS as antigen. RESULTS: The serum anti-L-PGDS antibody in hen immunized with pGEX-2T/htL-PGDS DNA were confirmed and its titer tested by ELISA was 1:2,048. CONCLUSIONS: It is feasible to produce anti-L-PGDS antibody by immunizing hens with recombinant pGEX-2T/htL-PGES DNA.

[Advances in sperm capacitation].

Sperm must be capacitated before sperm-ovum fusion. Capacitation was once considered as hyperactivation. But now many investigators thought that capacitation wasn't equal to hyperactivation, and that sperm hyperactivation might be a moiety of capacitation or the result of capacitation. In the present, the methods used to study sperm capacitation include fertilization in vitro, induction of sperm acrosome reaction, FITC-labeled chlortetracycline and plant hemoagglutinin. The studies on sperm capacitation in vitro mainly focused on the inductive substances of sperm capacitation and subsequent results analysis. It could lay foundation for the manifestation of molecular mechanism of sperm capacitation and destination of sperm capacitation in molecular levels.

[Study on the role of calcium signal during mature resumption of isolated mouse germinal vesicles in a cell free system].

A cell-free system including HeLa cell lysate of synchronized metaphase or G2-phase and isolated germinal vesicles (GV) from mouse oocytes was used to study the role of calcium and its downstream mediator during mature resumption. The isolated GVs could resume meiotic maturation in the lysate prepared from M phase HeLa cell, which marked by chromatin condensation. And this process was not affected by calcium chelating agent. But calcium in lysate from G2 phase cells was critical to meiotic maturation. Only in mid-G2 phase cell lysate (released from nocodazole for about 20-23h) chromatin condensation could be induced by calcium. Calcium had no effect on the cell lysate prepared from earlier (18-20h) and later (24h) G2 phase cells. Further studies showed that down stream mediator CaM and CaMKII might also involove in this process. Inhibition the function of CaM and CaMKII could block GVBD and first polar body extrusion of DOs cultured in vitro. The target of calcium signal might be MPF because MPF was existed from mid-G2 phase to metaphase and the tyrosine phosphorylation level of Cdc2 subunit was significantly dephosphorylated in M phase. Our results further confirmed that the resumption of meiosis maturation was promoted in a calcium/CaM depended pathway.

[Association between apolipoprotein E gene polymorphism and the patients with persistent vegetative state in the Chinese].

We studied the relationship between apolipoprotein E gene polymorphism and persistent vegetative state (PVS) to explore the genetics background of PVS, and evaluated the effect of ApoE gene polymorphism on lipid levels in plasma. The ApoE genotype of fifty-six patients with PVS and fifty-three controls were determined by PCR and restriction fragment length polymorphism (PCR-RFLP). Plasma lipid levels were measured by using routine methods. Results demonstrated that there were five genotypes in the two groups: E3/3, E3/4, E2/2, E2/3 and E2/4. The genotype frequencies of ApoE gene in PVS were 21(37.5%), 26(46.4%), 2 (3.6%), 5(8.9%), 2(3.6%) and that in control were 37(69.8%), 7(13.2%), 2(3.8%) 5(9.4%), 2(3.8%) respectively. We compared the genotype frequencies between the two groups and found there was a significantly increase in E3/4(chi 2 = 14.236, P < 0.001) and decrease in E3/3(chi 2 = 5.348, P < 0.05). epsilon 2, epsilon 3 and epsilon 4 allele frequencies of ApoE were 11(9.8%), 73(65.2%), 28(25%) in PVS and were 11 (10.4%), 86(81.1%), 9(8.5%) in control respectively. Allele frequencies, significantly increased in epsilon 4 (chi 2 = 10.533, P < 0.001) and decreased in epsilon 3 (chi 2 = 7.022, P < 0.01). We also found that E3/4, E2/4 genotype and epsilon 4 allele can largely increase total cholesterol (TC) and lower density lipoprotein cholesterol (LDL-C) levels in plasma, and epsilon 2 alleles also can largely increase LDL-C levels inplasma. Our finding indicates that the ApoE gene polymorphism may be in association with the PVS, and may be a factor in the genetic susceptibility to PVS in Chinese; Genotype and alleles of ApoE in PVS can affect the lipid levels in plasma.