Fibronectin promotes nasopharyngeal cancer cell motility and proliferation.

Nasopharyngeal cancer (NPC) is an Epstein-Barr virus (EBV)-associated carcinoma. Fibronectin is regarded as a prognosticator in NPC and its involvement in cell motility has been reported in EBV infection and viral latent membrane protein 1 (LMP1) overexpression NPC cell lines. However, its malignant potential in NPC cell lines without harbouring the EBV genome has not been investigated. We investigatd and compared among four NPC cell lines, and the results revealed a positive association between fibronectin levels and NPC cell motility as well as proliferation. Studies of antibody neutralization, exogenous addition, overexpression, and RNA interference confirmed a migration role of fibronectin in NPC cells involving integrin α5, Src, Rac1, and Cdc42, implying a mesenchymal-like cell movement. Furthermore, hypoxia-inducible factor-1α (HIF-1α) and transforming growth factor-ß1 (TGF-ß1) were identified as alternative activators of fibronectin expression and NPC cell migration. Besides cell migration, studies of RNA interference also showed a stimulatory effect of fibronectin in NPC cell proliferation. Mechanistic studies further revealed a subsequent reduction of HIF-1α, TGF-ß1, cyclin D1, ß-catenin, vimentin, and Slug together with decreased Src and Akt phosphorylation after fibronectin knockdown. Parallel studies in a xenograft tumor mice model further showed that tumor growth correlated well with elevation of circulating fibronectin and activation of the identified intracellular signaling molecules. The results of our study highlight a role of fibronectin in NPC cell motility and proliferation in concerted action with HIF-1α and TGF-ß1 possibly through linking molecules Src and Akt. Fibronectin overexpression and autoantibody are known to have potential prognostic value in patients with NPC. Our findings shed light on the biochemical and molecular mechanisms underlying the pathogenic role of fibronectin in this disease.

Histone-modifying genes as biomarkers in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the world's fifth most common cancer and second leading cause of cancer-related death in Taiwan. Over 600,000 HCC patients die each year worldwide despite recent advances in surgical techniques and medical treatments. Epigenetic regulations including DNA methylation and histone modification control gene expressions and play important roles during tumorigenesis. This study evaluates association between histone-modifying genes and prognosis of HCC to ferret out new diagnostic markers. We collected 50 paired HCC and adjacent non-cancerous tissues from Taiwanese patients for survey by RT-qPCR and tissue microarray-based immunohistochemistry (TMA-based IHC) staining. RT-qPCR data showed four of twenty-four genes over eightfold up-regulated in tumor tissues: e.g., histone phosphorylation gene-ARK2, methylation genes-G9a, SUV39H2, and EZH2 (n=50, all p<0.0001). Results of TMA-based IHC staining showed proteins of ARK2, EZH2, G9a, and SUV39H2 also overexpressed in tumor tissues. Staining intensity of SUV39H2 correlated with HCV infection (p=0.025). We further restricted the analysis only in tumor tissues, we found EZH2 staining intensity associated with tumor stage (p=0.016) and survival (p=0.007); SUV39H2 intensity associated with tumor stage (p=0.044). Our findings indicate overexpression of histone-modifying genes EZH2 and SUV39H2 associated with prognosis of HCC cases. EZH2 expression can serve as a novel prognostic biomarker during HCC progression among Taiwanese.

[Comparison of blockage effect of axillary brachial plexus block between ultrasound guidance alone and ultrasound guidance plus neurostimulation].

OBJECTIVE: To compare the blockage effect of multiple injection axillary brachial plexus blockage between ultrasound guidance alone and ultrasound guidance plus neurostimulation. METHODS: Upon the approval of institutional ethical committee, a total of 166 patients underwent hand, forearm and distal arm operations under axillary brachial plexus blockage from January to May in 2011 at Department of Hand Surgery, Beijing Jishuitan Hospital. They were randomly allocated to receive either ultrasound guidance plus nerve stimulation (group US-NS, n = 83) or ultrasound guidance alone (group US, n = 83) for nerve localization. Ten milliliter ropivacaine 0.5% was administered separately on each nerve. Blockage time, needle frequency, success rate, onset of sensory and motor blocks, procedure-related complications (vascular puncture, acute nerve injury and tourniquet pain, etc.) and patient satisfaction were recorded. RESULTS: The procedure time was shorter in US group than in US-NS group [(3.3 ± 1.5) vs (5.3 ± 2.0) min, P < 0.01]. The median (range) number of needle frequency of group US was lower than that of group US-NS [(4.3 ± 0.7) vs (5.6 ± 1.4) temps, P < 0.01]. No intergroup difference existed in success rate. The onset of complete block was shorter in group US-NS than that in group US [(10.6 ± 6.4) vs (12.7 ± 6.9) min, P < 0.05]. Vascular puncture proportion was higher in group US-NS than group US (16/83 vs 1/83, P < 0.01). There was no occurrence of tourniquet pain. Patient acceptance was similar in two groups. CONCLUSION: Multiple injection axillary blockage with ultrasound guidance yield similar success rates whether or not combined with nerve stimulation guidance. There appears to be a lower incidence rates of complications for ultrasound guidance alone versus ultrasound guidance plus neurostimulation.

[Spatiotemporal variation of typical red soil eroded landscape pattern: a case study in Changting County of Fujian Province].

Based on the 1988, 2000, and 2007 remote sensing images of a typical red soil eroded region (Changting County, Fujian Province) and the digital elevation model (DEM), the eroded landscape types were worked out, and the changes of the eroded landscape pattern in the region from 1988 to 2007 were analyzed with the spatial mathematics model. In 1988-2007, different eroded landscape types in the region had the characteristics of inter-transfer, mainly manifested in the transfer from seriously eroded to lightly eroded types but still existed small amount of the transference from lightly eroded to seriously eroded types. Little change was observed in the controid of the eroded landscape. In the County, Hetian Town was all along the eroded center. During the study period, the landscape pattern index showed a tendency of low heterogeneity, low fragmentation, and high regularization at landscape level, but an overall improvement and expansion of lightly eroded and easy-to-tackle patches as well as the partial improvement and fragmentation of seriously eroded and difficult-to-tackle patches at patch level.

Small molecule amiloride modulates oncogenic RNA alternative splicing to devitalize human cancer cells.

Alternative splicing involves differential exon selection of a gene transcript to generate mRNA and protein isoforms with structural and functional diversity. Abnormal alternative splicing has been shown to be associated with malignant phenotypes of cancer cells, such as chemo-resistance and invasive activity. Screening small molecules and drugs for modulating RNA splicing in human hepatocellular carcinoma cell line Huh-7, we discovered that amiloride, distinct from four pH-affecting amiloride analogues, could "normalize" the splicing of BCL-X, HIPK3 and RON/MISTR1 transcripts. Our proteomic analyses of amiloride-treated cells detected hypo-phosphorylation of splicing factor SF2/ASF, and decreased levels of SRp20 and two un-identified SR proteins. We further observed decreased phosphorylation of AKT, ERK1/2 and PP1, and increased phosphorylation of p38 and JNK, suggesting that amiloride treatment down-regulates kinases and up-regulates phosphatases in the signal pathways known to affect splicing factor protein phosphorylation. These amiloride effects of "normalized" oncogenic RNA splicing and splicing factor hypo-phosphorylation were both abrogated by pre-treatment with a PP1 inhibitor. Global exon array of amiloride-treated Huh-7 cells detected splicing pattern changes involving 584 exons in 551 gene transcripts, many of which encode proteins playing key roles in ion transport, cellular matrix formation, cytoskeleton remodeling, and genome maintenance. Cellular functional analyses revealed subsequent invasion and migration defects, cell cycle disruption, cytokinesis impairment, and lethal DNA degradation in amiloride-treated Huh-7 cells. Other human solid tumor and leukemic cells, but not a few normal cells, showed similar amiloride-altered RNA splicing with devitalized consequence. This study thus provides mechanistic underpinnings for exploiting small molecule modulation of RNA splicing for cancer therapeutics.

[Varying concentration of ropivacaine with stimulating catheters on sciatic nerve blocking for postoperative analgesia: comparison of efficacy].

OBJECTIVE: To determine the most suitable concentration of ropivacaine on sciatic nerve blocks with stimulating catheters. METHODS: Sixty adult patients undergoing surgeries involving the foot and ankle were randomly divided into 3 groups (n=20): The patients in group A were given 0.1% ropivacaine, the patients in groups B and C received 0.125% and 0.15% ropivacaine respectively. We combined ultrasound and nerve stimulators for popliteal sciatic nerve blocks, and then placed stimulating catheters for postoperative analgesia. VAS and adverse effects were monitored 6 h, 12 h, 24 h and 48 h after surgery. RESULTS: The difference of VAS was of significance in statistics 6 h and 12 h postoperation. VAS in Group A at postoperative 6 h and 12 h was significantly higher than that in Groups B and C(P<0.05). The incidence of motor disturbance and limbs numbness in Group C was significantly higher than that in Groups A and B (P<0.05). There was no significant difference in the satisfaction of the three groups (P>0.05). CONCLUSION: The most suitable concentration of ropivacaine on sciatic nerve blocks with stimulating catheters is 0.125%.

Extracellular pH change modulates the exon 7 splicing in SMN2 mRNA.

Spinal muscular atrophy (SMA) is caused by homozygous deletions/mutations of SMN1 gene. All SMA patients carry a nearly identical SMN2 gene. A nucleotide change in SMN2 results in exon 7 exclusion in the majority of SMN2 mRNA, thus producing low level of SMN protein. Extracellular pH change has been shown to modulate alternative splicing of several pre-mRNAs. In this study, we showed that extracellular pH change can also modulate SMN2 exon 7 splicing in SMA cells. Low extracellular pH enhances SMN2 exon 7 skipping, whereas high extracellular pH promotes its inclusion. Low extracellular pH also reduces SMN protein expression but increases hnRNP A1 expression. In addition, we tested whether intracellular pH-modulating genes could be the modifier of SMA in a SMA discordant family and found that the mRNA levels of ATP6V1B2 gene are significantly higher in two affected siblings than the unaffected one. In conclusion, our results suggest that extracellular pH change modulates SMN2 exon 7 splicing through regulation of hnRNP A1 expression in SMA cells.

5-(N-ethyl-N-isopropyl)-amiloride enhances SMN2 exon 7 inclusion and protein expression in spinal muscular atrophy cells.

OBJECTIVE: Spinal muscular atrophy (SMA) is a common inherited neuromuscular disorder caused by homozygous loss of function of the survival motor neuron 1 (SMN1) gene. All SMA patients carry at least one copy of a nearly identical SMN2 gene. However, a critical nucleotide change in SMN2 results in alternative splicing and exclusion of exon 7 in the majority of SMN2 messenger RNA (mRNA), thus producing a low level of functional SMN protein. Increasing SMN protein production by promoting SMN2 exon 7 inclusion could be a therapeutic approach for SMA. It has been shown that cellular pH microenvironment can modulate pre-mRNA alternative splicing in vivo. In this study, we tested whether inhibitors of the Na+/H+ exchanger can modulate the exon 7 splicing of SMN2 mRNA METHODS: We treated SMA lymphoid cell lines with Na+/H+ exchanger inhibitors and then measured SMN2 exon 7 splicing by reverse transcriptase polymerase chain reaction and SMN protein production by Western blotting and immunofluorescence RESULTS: We found that treatment with an Na+/H+ exchanger inhibitor, 5-(N-ethyl-N-isopropyl)-amiloride (EIPA), significantly enhances SMN2 exon 7 inclusion and SMN protein production in SMA cells. In addition, EIPA increases the number of nuclear gems in SMA cells. We further explored the underlying mechanism, and our results suggest that EIPA may promote SMN2 exon 7 inclusion through upregulation of the splicing factor SRp20 in the nucleus INTERPRETATION: Our finding that EIPA, an inhibitor of the Na+/H+ exchanger, can increase SMN protein expression in SMA cells provides a new direction for the development of drugs for SMA treatment. However, further translational studies are needed to determine whether this finding is applicable for SMA treatment or just a proof of cellular pH effect on SMN splicing.

Wild-type-like viral replication potential of human immunodeficiency virus type 1 envelope mutants lacking palmitoylation signals.

Palmitoylation of the cytoplasmic domain of the human immunodeficiency type virus type 1 (HIV-1) envelope (Env) transmembrane protein, gp41, has been implicated in Env targeting to detergent-resistant lipid rafts, Env incorporation into the virus, and viral infectivity. In contrast, we provide evidence here to show that HIV-1 infectivity, Env targeting to lipid rafts, and Env incorporation into the virus are independent of cytoplasmic tail palmitoylation. The T-cell (T)-tropic HXB2-based virus, which utilizes CXCR4 as the entry coreceptor, carrying a Cys-to-Ser mutation at residue 764 or 837 or at both replicated with wild-type (WT) virus replication kinetics in CD4+ T cells. The properties of Env expression, precursor processing, cell surface expression, and Env incorporation of these three mutant viruses were normal compared to those of the WT virus. These three mutant Env proteins all effectively mediated one-cycle virus infection. When the Cys residues were replaced by Ala residues, all single and double mutants still retained the phenotypes of infectivity, Env incorporation, and lipid raft localization of the WT Env. When Cys-to-Ala substitutions were introduced into the macrophage (M)-tropic ConB virus, which utilizes CCR5 as the coreceptor, these mutations did not affect the replication potential, Env phenotypes, lipid raft targeting, or Env assembly into the virus of the WT Env. These T- and M-tropic mutants also productively replicated in human primary CD4+ T cells. Moreover, mutations at both Cys residues significantly reduced the level of palmitoylation of the Env. Our results together support the notion that palmitoylation of the cytoplasmic tail of the HIV-1 Env is not essential for the HIV-1 virus life cycle.

Effect of extension of the cytoplasmic domain of human immunodeficiency type 1 virus transmembrane protein gp41 on virus replication.

The biological significance of the presence of a long cytoplasmic domain in the envelope (Env) transmembrane protein gp41 of human immunodeficiency virus type 1 (HIV-1) is still not fully understood. Here we examined the effects of cytoplasmic tail elongation on virus replication and characterized the role of the C-terminal cytoplasmic tail in interactions with the Gag protein. Extensions with six and nine His residues but not with fewer than six His residues were found to severely inhibit virus replication through decreased Env electrophoretic mobility and reduced Env incorporation compared to the wild-type virus. These two mutants also exhibited distinct N glycosylation and reduced cell surface expression. An extension of six other residues had no deleterious effect on infectivity, even though some mutants showed reduced Env incorporation into the virus and/or decreased cell surface expression. We further show that these elongated cytoplasmic tails in a format of the glutathione S-transferase fusion protein still interacted effectively with the Gag protein. In addition, the immediate C terminus of the cytoplasmic tail was not directly involved in interactions with Gag, but the region containing the last 13 to 43 residues from the C terminus was critical for Env-Gag interactions. Taken together, our results demonstrate that HIV-1 Env can tolerate extension at its C terminus to a certain degree without loss of virus infectivity and Env-Gag interactions. However, extended elongation in the cytoplasmic tail may impair virus infectivity, Env cell surface expression, and Env incorporation into the virus.