Natural antisense transcripts (NATs) exist ubiquitously in mammalian genomes and play roles in the regulation of gene expression. However, both the existence of bidirectional antisense RNA regulation and the possibility of protein-coding genes that function as antisense RNAs remain speculative. Here, we found that the protein-coding gene, deoxyhypusine synthase (DHPS), as the NAT of WDR83, concordantly regulated the expression of WDR83 mRNA and protein. Conversely, WDR83 also regulated DHPS by antisense pairing in a concordant manner. WDR83 and DHPS were capable of forming an RNA duplex at overlapping 3′ untranslated regions and this duplex increased their mutual stability, which was required for the bidirectional regulation. As a pair of protein-coding cis-sense/antisense transcripts, WDR83 and DHPS were upregulated simultaneously and correlated positively in gastric cancer (GC), driving GC pathophysiology by promoting cell proliferation. Furthermore, the positive relationship between WDR83 and DHPS was also observed in other cancers. The bidirectional regulatory relationship between WDR83 and DHPS not only enriches our understanding of antisense regulation, but also provides a more complete understanding of their functions in tumor development.
bidirectional regulation; natural antisense transcript; gastric cancer
Y294002 (LY) is a potent inhibitor of phosphatidylinositol 3-kinases (PI3Ks); however, biological applications of LY are limited by its poor solubility and pharmacokinetic profile. This study aimed at developing LY-loaded surfactant-free poly(lactic-co-glycolic acid) (PLGA) nanoparticles (SF-LY NPs) to improve the therapeutic efficacy of LY.
Materials and methods
Cellular viability was measured by MTT assay. The subcellular distribution of NPs was studied using an ultraviolet-visible spectrophotometer and confocal microscope. The expression of cell-death-associated proteins was determined using Western blotting and the in vivo activity of SF-LY NPs was tested in a xenograft animal model.
SF-LY NPs enhanced the intracellular level of LY, induced sustained suppression of AKT, and induced marked cancer cell death. In addition, SF-LY NPs tended to accumulate in the endoplasmic reticulum (ER) and induce pronounced ER stress. Finally, SF-LY NPs exhibited a prominent antitumor effect in vivo.
The surfactant-free formulation of PLGA is critical to the promising anticancer activity of SF-LY NPs.
LY294002; AKT; surfactant-free poly(lactic-co-glycolic acid); endoplasmic reticulum stress
Osteomyelitis therapy is a long-term and inconvenient procedure for a patient. Antibiotic-loaded bone cements are both a complementary and alternative treatment option to intravenous antibiotic therapy for the treatment of osteomyelitis. In the current study, the biphasic calcium phosphate cement (CPC), called α-TCP/HAP (α-tricalcium phosphate/hydroxyapatite) biphasic cement, was prepared as an antibiotics carrier for osteomyelitis. The developed biphasic cement with a microstructure of α-TCP surrounding the HAP has a fast setting time which will fulfill the clinical demand. The X-ray diffraction and Fourier transform infrared spectrometry analyses showed the final phase to be HAP, the basic bone mineral, after setting for a period of time. Scanning electron microscopy revealed a porous structure with particle sizes of a few micrometers. The addition of gentamicin in α-TCP/HAP would delay the transition of α-TCP but would not change the final-phase HAP. The gentamicin-loaded α-TCP/HAP supplies high doses of the antibiotic during the initial 24 hours when they are soaked in phosphate buffer solution (PBS). Thereafter, a slower drug release is produced, supplying minimum inhibitory concentration until the end of the experiment (30 days). Studies of growth inhibition of Staphylococcus aureus and Pseudomonas aeruginosa in culture indicated that gentamicin released after 30 days from α-TCP/HAP biphasic cement retained antibacterial activity.
AIM: To investigate the 10-year results of treating low rectal cancer by a single surgeon in one institution.
METHODS: From Oct 1998 to Feb 2009, we prospectively followed a total of 62 patients with cT2-4 low rectal cancer with lower tumor margins measuring at 3 to 6 cm above the anal verge. All patients received neoadjuvant chemoradiation (CRT) for 6 wk. Among them, 85% of the patients received 225 mg/m2/d 5-fluorouracil using a portable infusion pump. The whole pelvis received a total dose of 45 Gy of irradiation in 25 fractions over 5 wk. The interval from CRT completion to surgical intervention was planned to be approximately 6-8 wk. Total mesorectal excision (TME) and routine defunctioning stoma construction were performed by one surgeon. The distal resection margin, circumferential resection margin, tumor regression grade (TRG) and other parameters were recorded. We used TRG to evaluate the tumor response after neoadjuvant CRT. We evaluated anal function outcomes using the Memorial Sloan-Kettering Cancer Center anal function scores after closure of the defunctioning stoma.
RESULTS: The median distance from the lower margin of rectal cancer to the anal verge was 5 cm: 6 cm in 9 patients, 5 cm in 32 patients, 4 cm in 10 patients, and 3 cm in 11 patients. Before receiving neoadjuvant CRT, 45 patients (72.6%) had a cT3-4 tumor, and 21 (33.9%) patients had a cN1-2 lymph node status. After CRT, 30 patients (48.4%) had a greater than 50% clinical reduction in tumor size. The final pathology reports revealed that 33 patients (53.2%) had a ypT3-4 tumor and 12 (19.4%) patients had ypN1-2 lymph node involvement. All patients completed the entire course of neoadjuvant CRT. Most patients developed only Grade 1-2 toxicities during CRT. Thirteen patients (21%) achieved a pathologic complete response. Few post-operative complications occurred. Nearly 90% of the defunctioning stomas were closed within 6 mo. The local recurrence rate was 3.2%. Pathologic lymph node involvement was the only prognostic factor predicting disease recurrence (36.5% vs 76.5%, P = 0.006). Nearly 90% of patients recovered sphincter function within 2 year after closure of the defunctioning stoma.
CONCLUSION: Neoadjuvant CRT followed by TME, combined with routine defunctioning stoma construction and high-volume surgeon experience, can provide excellent surgical quality and good local disease control.
Rectal cancer; Neoadjuvant chemoradiation; Total mesorectal excision; Pathologic complete response; Defunctioning stoma
The peptide hormone gastrin binds two ferric ions with high affinity, and iron binding is essential for the biological activity of non-amidated gastrins in vitro and in vivo. Bi3+ ions also bind to glycine-extended gastrin17 (Ggly), but inhibit Ggly-induced cell proliferation and migration in gastrointestinal cell lines in vitro. The aims of the present study were firstly, to establish the mechanism by which Bi3+ ions inhibit the binding of Fe3+ ions to Ggly, and secondly, to test the effect of Bi3+ ions on the activity of non-amidated gastrins in vivo. The interaction between Bi3+ ions, Fe3+ ions and Ggly was investigated by ultraviolet spectroscopy. The effect of Bi3+ ions on colorectal mucosal proliferation was measured in three animal models. In vitro in the presence of Bi3+ ions the affinity of Fe3+ ions for Ggly was substantially reduced; the data was better fitted by a mixed, rather than a competitive, inhibition model. In rats treated with Ggly alone proliferation in the rectal mucosa was increased by 318%, but was reduced to control values (p < 0.001) in animals receiving oral bismuth plus Ggly. Proliferation in the colonic mucosa of mice overexpressing Ggly or progastrin was significantly greater than in wild-type mice, but was no greater than control (p < 0.01) in animals receiving oral bismuth. Thus a reduction in the binding of Fe3+ ions to Ggly and progastrin in the presence of Bi3+ ions is a likely explanation for the ability of oral bismuth to block the biological activity of non-amidated gastrins in vivo.
Bismuth ions; colorectal mucosa; ferric ions; glycine-extended gastrin; proliferation; progastrin
Chronic hepatitis B is characterized by an impaired immune response to hepatitis B virus (HBV). Telbivudine treatment has significantly improved the clinical outcome of chronic HBV infection. However, the underlying mechanism behind the antiviral response of patients treated with nucleoside analogs remains unclear. To gather more evidence about the mechanism responsible for the weak immune response, in this study we analyzed the effects on HBV viral load of treatment with the nucleoside analogue telbivudine and the percentage of Tregs, programmed death-1 (PD-1)/programmed death-ligand 1 (PD-L1) expression, and related cytokine production. Peripheral blood mononuclear cells (PBMCs) and serum of 28 patients with chronic hepatitis B were collected at baseline, and 3 mo and 6 mo after therapy was begun. In parallel with the decline in viral load and serum ALT normalization, we found a decline in circulating CD4+CD25high Tregs, PD-L1 on CD4+ T cells, and IL-9 production. The expression of PD-1 on CD4+ T cells and the production of IFN-γ did not increase during therapy. Our findings suggest that the antiviral effect of the nucleoside analogs may be attributable not only to their direct effect on virus suppression, but also to their immunoregulatory capabilities.
Conventional prenatal screening tests, such as maternal serum tests and ultrasound scan, have limited resolution and accuracy.
We developed an advanced noninvasive prenatal diagnosis method based on massively parallel sequencing. The Noninvasive Fetal Trisomy (NIFTY) test, combines an optimized Student’s t-test with a locally weighted polynomial regression and binary hypotheses. We applied the NIFTY test to 903 pregnancies and compared the diagnostic results with those of full karyotyping.
16 of 16 trisomy 21, 12 of 12 trisomy 18, two of two trisomy 13, three of four 45, X, one of one XYY and two of two XXY abnormalities were correctly identified. But one false positive case of trisomy 18 and one false negative case of 45, X were observed. The test performed with 100% sensitivity and 99.9% specificity for autosomal aneuploidies and 85.7% sensitivity and 99.9% specificity for sex chromosomal aneuploidies. Compared with three previously reported z-score approaches with/without GC-bias removal and with internal control, the NIFTY test was more accurate and robust for the detection of both autosomal and sex chromosomal aneuploidies in fetuses.
Our study demonstrates a powerful and reliable methodology for noninvasive prenatal diagnosis.
Noninvasive Fetal Trisomy (NIFTY) test; Massively parallel sequencing; Autosomal aneuploidies; Sex chromosomal aneuploidies
Ribavirin significantly enhances the antiviral response of interferon-α (IFN-α) against Hepatitis C virus (HCV), but the underlying mechanisms remain poorly understood. Recently, p53 has been identified as an important factor involving the suppression of HCV replication in hepatocytes. We, therefore, decided to investigate whether and how ribavirin inhibits the replication of HCV by promoting the activity of p53.
HepG2 and HCV replicons (JFH1/HepG2) were utilized to study the relationship between ribavirin and p53. The effect of ribavirin on cell cycles was analyzed by flow cytometry. The activation of p53 and the signaling pathways were determined using immunoblotting. By knocking down ERK1/ERK2 and p53 utilizing RNA interference strategy, we further assessed the role of ERK1/2 and p53 in the suppression of HCV replication by ribavirin in a HCV replicon system.
Using HepG2 and HCV replicons, we demonstrated that ribavirin caused the cell cycle arrest at G1 phase and stabilized and activated p53, which was associated with the antiviral activity of ribavirin. Compared to either ribavirin or IFN-α alone, ribavirin plus IFN-α resulted in greater p53 activation and HCV suppression. We further identified ERK1/2 that linked ribavirin signals to p53 activation. More importantly, knockdown of ERK1/2 and p53 partially mitigated the inhibitory effects of ribavirin on the HCV replication, indicating that ERK1/2-p53 pathway was involved in the anti-HCV effects of ribavirin.
Ribavirin stimulates ERK1/2 and subsequently promotes p53 activity which at least partly contributes to the enhanced antiviral response of IFN-α plus ribavirin against HCV.
Previous research has shown variation in the effects of patient factors, including hepatic necroinflammatory activity, on liver stiffness measurement (LSM). This prospective study attempts to identify explanatory factors for LSM in patients with chronic hepatitis C (CHC) using acoustic radiation force impulse (ARFI) technology.
A cohort of 127 Taiwanese patients with CHC underwent ARFI LSM and immediate percutaneous liver biopsy. This study compares the concurrent diagnostic performances of LSM and FibroTest using receiver operating characteristic (ROC) curves. Three multiple linear regression models were used to evaluate the significance of concurrent patient factors in explaining LSM.
To classify METAVIR fibrosis (F) stages, the areas under ROC curves (AUCs) were ARFI LSM, 0.847 (95% confidence interval (CI), 0.779-0.914) and FibroTest, 0.823 (95% CI, 0.748-0.898), for F1 versus F2-4; ARFI LSM, 0.902 (95% CI, 0.835-0.970) and FibroTest, 0.812 (95% CI, 0.735-0.888), for F1-2 versus F3-4; ARFI LSM, 0.831 (95% CI, 0.723-0.939) and FibroTest, 0.757 (95% CI, 0.648-0.865), for F1-3 versus F4. After adjusting for other demographic and biological covariates, biochemical and histological necroinflammatory factors consistently explained LSM. Factors included serum alanine aminotransferase (ALT)/upper limit of normal (ULN) categories (model R2 = 0.661, adjusted R2 = 0.629), ActiTest A scores (R2 = 0.662, adjusted R2 = 0.636), and METAVIR activity (A) grades (R2 = 0.651, adjusted R2 = 0.620). METAVIR F stages, body mass index, and platelet count were also independently associated with LSM. Necroinflammatory degrees, including ALT/ULN, ActiTest A scores, and METAVIR A grades, explained the false positivity of liver fibrosis staging using ARFI LSM.
The degree of hepatic necroinflammatory activity independently and significantly exaggerated liver fibrosis evaluation using ARFI LSM. However, comparisons with concurrent FibroTest indicate that ARFI LSM may be a promising alternative, or adjunctive single indicator, for liver fibrosis evaluation in patients with CHC.
Liver fibrosis; Cirrhosis; Acoustic radiation force impulse; FibroTest; ActiTest; Chronic hepatitis C
The association between human leukocyte antigen (HLA) genes (located in the Major Histocompatibility Complex [MHC] region of chromosome 6p21) and NPC has been known for some time. Recently, two genome-wide association studies (GWAS) conducted in Taiwan and China confirmed that the strongest evidence for NPC association was mapped to the MHC region. It is still unclear, however, whether these findings reflect direct associations with Human Leukocyte Antigen (HLA) genes and/or to other genes in this gene-rich region.
To better understand genetic associations for NPC within the MHC region of chromosome 6, we conducted an evaluation that pooled two previously conducted NPC case-control studies in Taiwan (N = 591 cases and N = 521 controls). PCR-based genotyping was performed for 12 significant SNPs identified within 6p21 in the Taiwan NPC GWAS and for the HLA-A gene (exons 2 and 3).
After confirming homogeneity between the two studies, pooled odds ratios (OR) and 95% confidence intervals (CI) were estimated by logistic regression. We found that HLA-A (p-trend = 0.0006) and rs29232 (within the GABBR1 gene; p-trend = 0.005) were independent risk factors for NPC after adjustment for age, gender, study and each other. NPC risk was highest among individuals who were homozygous for the HLA-A*0207 risk allele and carriers of the rs29232 risk allele (A).
Our study suggests that most of the SNPs significantly associated with NPC from GWAS reflect previously identified HLA-A associations. An independent effect of rs29232 (GABBR1), however, remained, suggesting that additional genes within this region might be associated with NPC risk.
Effective cancer chemotherapy remains an important issue in cancer treatment, and signal transducer and activator of transcription-3 (Stat3) activation leads to cellular resistance of anticancer agents. Polymers are ideal vectors to carry both chemotherapeutics and small interfering ribonucleic acid (siRNA) to enhance antitumor efficacy. In this paper, poly(lactic-co-glycolic acid) (PLGA) nanoparticles loaded with paclitaxel and Stat3 siRNA were successfully synthesized, and their applications in cancer cells were investigated.
Firstly, paclitaxel was enclosed by PLGA nanoparticles through solvent evaporation. They were then coated with cationic polyethylenimine polymer (PLGA-PEI-TAX), enabling it to carry Stat3 siRNA on its surface through electrostatic interactions (PLGA-PEI-TAX-S3SI). The size, zeta potential, deliver efficacy, and release profile of the PLGA nanocomplexes were characterized in vitro. The cellular uptake, intracellular nanoparticle trajectory, and subsequent cellular events were evaluated after treatment with various PLGA nanocomplexes in human lung cancer A549 cells and A549-derived paclitaxel-resistant A549/T12 cell lines with α-tubulin mutation.
A549 and A549/T12 cells contain constitutively activated Stat3, and silencing Stat3 by siRNA made both cancer cells more sensitive to paclitaxel. Therefore, PLGA-PEI-TAX-S3SI was synthesized to test its therapeutic role in A549 and A549/T12 cells. Transmission electron microscopy showed the size of PLGA-PEI-TAX-S3SI to be around 250 nm. PLGA-PEI nanoparticles were nontoxic. PLGA-PEI-TAX was taken up by A549 and A549/T12 cells more than free paclitaxel, and they induced more condensed microtubule bundles and had higher cytotoxicity in these cancer cells. Moreover, the yellowish fluorescence observed in the cytoplasm of the cancer cells indicates that the PLGA-PEI nanoparticles were still simultaneously delivering Oregon Green paclitaxel and cyanine-5-labeled Stat3 siRNA 3 hours after treatment. Furthermore, after the cancer cells were incubated with the synthesized PLGA nanocomplexes, PLGA-PEI-TAX-S3SI suppressed Stat3 expression and induced more cellular apoptosis in A549 and A549/T12 cells compared with PLGA-PEI-TAX.
The PLGA-PEI-TAX-S3SI complex provides a new therapeutic strategy to control cancer cell growth.
PLGA; nanoparticle; paclitaxel; siRNA; simultaneous drug delivery
Hepatitis C virus (HCV) reorganizes intracellular membranes to establish sites of replication. How viral and cellular proteins target, bind, and rearrange specific membranes into the replication factory remains a mystery. We used a lentivirus-based RNA interference (RNAi) screening approach to identify the potential cellular factors that are involved in HCV replication. A protein with membrane-deforming activity, proline-serine-threonine phosphatase-interacting protein 2 (PSTPIP2), was identified as a potential factor. Knockdown of PSTPIP2 in HCV subgenomic replicon-harboring and HCV-infected cells was associated with the reduction of HCV protein and RNA expression. PSTPIP2 was localized predominantly in detergent-resistant membranes (DRMs), which contain the RNA replication complex. PSTPIP2 knockdown caused a significant reduction of the formation of HCV- and NS4B-induced membranous webs. A PSTPIP2 mutant defective in inducing membrane curvature failed to support HCV replication, confirming that the membrane-deforming ability of PSTPIP2 is essential for HCV replication. Taking these results together, we suggest that PSTPIP2 facilitates membrane alterations and is a key player in the formation of the membranous web, which is the site of the HCV replication complex.
Copy number variations (CNVs), a major source of human genetic polymorphism, have been suggested to have an important role in genetic susceptibility to common diseases such as cancer, immune diseases and neurological disorders. Nasopharyngeal carcinoma (NPC) is a multifactorial tumor closely associated with genetic background and with a male preponderance over female (3:1). Previous genome-wide association studies have identified single-nucleotide polymorphisms (SNPs) that are associated with NPC susceptibility. Here, we sought to explore the possible association of CNVs with NPC predisposition. Utilizing genome-wide SNP-based arrays and five CNV-prediction algorithms, we identified eight regions with CNV that were significantly overrepresented in NPC patients compared with healthy controls. These CNVs included six deletions (on chromosomes 3, 6, 7, 8 and 19), and two duplications (on chromosomes 7 and 12). Among them, the CNV located at chromosome 6p21.3, with single-copy deletion of the MICA and HCP5 genes, showed the highest association with NPC. Interestingly, it was more specifically associated with an increased NPC risk among males. This gender-specific association was replicated in an independent case–control sample using a self-established deletion-specific polymerase chain reaction strategy. To the best of our knowledge, this is the first study to explore the role of constitutional CNVs in NPC, using a genome-wide platform. Moreover, we identified eight novel candidate regions with CNV that merit future investigation, and our results suggest that similar to neuroblastoma and prostate cancer, genetic structural variations might contribute to NPC predisposition.
Antigen-specific immunotherapy (SIT) has been widely practiced in treating allergic diseases such as asthma. However, this therapy may induce a series of allergic adverse events during treatment. Peptide immunotherapy (PIT) was explored to overcome these disadvantages. We confirmed that multiple antigen peptides (MAPs) do not cause autoimmune responses, which led to the presumption that MAPs intervention could alleviate allergic airway inflammation without inducing adverse effects.
In this study, synthesized OVA323-339MAP octamers were subcutaneously injected into ovalbumin (OVA)-sensitized and -challenged Balb/c mice to observe its effect on allergic airway inflammation, Th2 immune response, and immune regulating function. It was confirmed that OVA sensitization and challenge led to significant peritracheal inflammatory, cell infiltration, and intensive Th2 response. Treatment of OVA323-339MAP octomers in the airway inflammation mice model increased CD4+CD25+Foxp3+ T regulatory (Treg) cells and their regulatory function in peripheral blood, mediastinal draining lymph nodes, and the spleen. Furthermore, OVA323-339MAP increased IL-10 levels in bronchial alveolar lavage fluid (BALF); up-regulated the expression of IL-10, membrane-bound TGF-β1, as well as Foxp3 in lung tissues; and up-regulated programmed death-1 (PD-1) and cytotoxic T lymphocyte associated antigen 4 (CTLA-4) on the surface of Treg cells. These results were further correlated with the decreased OVA specific immunoglobulin E (sIgE) level and the infiltration of inflammatory cells such as eosinophils and lymphocytes in BALF. However, OVA323-339 peptide monomers did not show any of the mentioned effects in the same animal model.
Our study indicates that OVA323-339MAP had significant therapeutic effects on mice allergic airway inflammation by regulating the balance of Th1/Th2 response through Treg cells in vivo.
Allergic airway inflammation; Specific immunotherapy; Multiple antigen peptide
For Hepatitis C virus (HCV), initiation of translation is cap-independently mediated by its internal ribosome entry site (IRES). Unlike other IRES-containing viruses that shut off host cap-dependent translation, translation of HCV coexists with that of the host. How HCV IRES-mediated translation is regulated in the infected cells remains unclear. Here, we show that the intracellular level of 40S ribosomal subunit plays a key role in facilitating HCV translation over host translation. In a loss-of-function screen, we identified small subunit ribosomal protein 6 (RPS6) as an indispensable host factor for HCV propagation. Knockdown of RPS6 selectively repressed HCV IRES-mediated translation, but not general translation. Such preferential suppression of HCV translation correlated well with the reduction of the abundance of 40S ribosomal subunit following knockdown of RPS6 or other RPS genes. In contrast, reduction of the amount of ribosomal proteins of the 60S subunit did not produce similar effects. Among the components of general translation machineries, only knockdowns of RPS genes caused inhibitory effects on HCV translation, pointing out the unique role of 40S subunit abundance in HCV translation. This work demonstrates an unconventional notion that the translation initiation of HCV and host possess different susceptibility toward reduction of 40S ribosomal subunit, and provides a model of selective modulation of IRES-mediated translation through manipulating the level of 40S subunit.
Hepatitis C virus (HCV) infection causes chronic liver diseases that threaten ∼2% of the world population. There is no effective vaccine, and the current standard therapy, the combination of interferon and ribavirin, is effective to less than 50% of genotype-1 infected patients. While antivirals targeting at specific HCV proteins might ultimately lose their effectiveness due to the emergence of resistance-associated mutations, an alternative strategy that explores the genetic stability of host factors indispensable for HCV replication may provide better therapeutic targets for anti-HCV medicine. Here, we employed a loss-of-function screen method to identify such potential targets and uncovered a potential novel anti-HCV mechanism by manipulating the biogenesis of 40S ribosomal subunit. We showed that inhibiting 40S ribosome biogenesis can selectively suppress HCV translation and thus effectively inhibit HCV replication. In contrast to the conventional thinking, the 40S ribosomal subunit can differentially affect different translational modes, and HCV translation is more sensitive to the amounts of 40S ribosomal subunit as compared to general translation in host cell. Since HCV is known to evade anti-viral effects including translational suppression elicited by interferon, our findings may help design a therapeutic strategy to supplement interferon-based therapy and minimize mutation-associated drug resistance problem.
Autophagy is an evolutionarily conserved catabolic process that maintains cellular homeostasis under stress conditions such as starvation and pathogen infection. Macrophage migration inhibitory factor (MIF) is a multifunctional cytokine that plays important roles in inflammation and tumorigenesis. Cytokines such as IL-1β and TNF-α that are induced by MIF have been shown to be involved in the induction of autophagy. However, the actual role of MIF in autophagy remains unclear. Here, we have demonstrated that incubation of human hepatoma cell line HuH-7 cells with recombinant MIF (rMIF) induced reactive oxygen species (ROS) production and autophagy formation, including LC3-II expression, LC3 punctae formation, autophagic flux, and mitochondria membrane potential loss. The autophagy induced by rMIF was inhibited in the presence of MIF inhibitor, ISO-1 as well as ROS scavenger N-acetyl-L-cysteine (NAC). In addition, serum starvation-induced MIF release and autophagy of HuH-7 cells were partly blocked in the presence of NAC. Moreover, diminished MIF expression by shRNA transfection or inhibition of MIF by ISO-1 decreased serum starvation-induced autophagy of HuH-7 cells. Taken together, these data suggest that cell autophagy was induced by MIF under stress conditions such as inflammation and starvation through ROS generation.
In the title hydrated molecular salt, C6H16N2
2+·2Br−·2H2O, the complete 1,4-dimethylpiperazine-1,4-diium dication is generated by crystallographic inversion symmetry and both exocyclic C—N bonds are in equatorial orientations. In the crystal, the components are linked by N—H⋯O and O—H⋯Br hydrogen bonds, generating chains propagating in .
In the title hydrated molecular salt, C6H16N2
−·2H2O, the complete 1,4-dimethylpiperazine-1,4-diium dication is generated by crystallographic inversion symmetry and both C—N bonds are in equatorial orientations. In the crystal, the components are linked by O—H⋯F and N—H⋯O hydrogen bonds but there are no direct links between cations and anions.
In the title molecule, C10H11NO5, the methyl C atom deviates by 0.830 (6) Å from the mean plane of the remaining non-H atoms. In the crystal, weak C—H⋯O hydrogen bonds link the molecules into layers parallel to the bc plane.
Autophagy has been shown to facilitate replication or production of hepatitis C virus (HCV); nevertheless, how HCV induces autophagy remains unclear. Here, we demonstrate that HCV nonstructural protein 4B (NS4B) alone can induce autophagy signaling; amino acid residues 1 to 190 of NS4B are sufficient for this induction. Further studies showed that the phosphorylation levels of S6K and 4E-BP1 were not altered, suggesting that the mTOR/S6 kinase pathway and mTOR/4E-BP1 pathway did not contribute to NS4B- or HCV-induced autophagy. Inhibition of Rab5 function by silencing Rab5 or overexpressing dominant-negative Rab5 mutant (S34N) resulted in significant reduction of NS4B- or HCV-induced autophagic vesicle formation. Moreover, the autophagy induction was impaired by inhibition of class III phosphoinositide 3-kinase (PI 3-kinase) Vps34 function. Finally, the coimmunoprecipitation assay indicated that NS4B formed a complex with Rab5 and Vps34, supporting the notion that Rab5 and Vps34 are involved in NS4B-induced autophagy. Taken together, these results not only reveal a novel role of NS4B in autophagy but also offer a clue to the mechanism of HCV-induced autophagy.
Calcium independent group VIA phospholipase A2 (iPLA2β) is up-regulated in vascular smooth muscle cells in some diseases, but whether the up-regulated iPLA2β affects vascular morphology and blood pressure is unknown. The current study addresses this question by evaluating the basal- and angiotensin II infusion-induced vascular remodeling and hypertension in smooth muscle specific iPLA2β transgenic (iPLA2β -Tg) mice.
Method and Results
Blood pressure was monitored by radiotelemetry and vascular remodeling was assessed by morphologic analysis. We found that the angiotensin II-induced increase in diastolic pressure was significantly higher in iPLA2β-Tg than iPLA2β-Wt mice, whereas, the basal blood pressure was not significantly different. The media thickness and media∶lumen ratio of the mesenteric arteries were significantly increased in angiotensin II-infused iPLA2β-Tg mice. Analysis revealed no difference in vascular smooth muscle cell proliferation. In contrast, adenovirus-mediated iPLA2β overexpression in cultured vascular smooth muscle cells promoted angiotensin II-induced [3H]-leucine incorporation, indicating enhanced hypertrophy. Moreover, angiotensin II infusion-induced c-Jun phosphorylation in vascular smooth muscle cells overexpressing iPLA2β to higher levels, which was abolished by inhibition of 12/15 lipoxygenase. In addition, we found that angiotensin II up-regulated the endogenous iPLA2β protein in-vitro and in-vivo.
The present study reports that iPLA2β up-regulation exacerbates angiotensin II-induced vascular smooth muscle cell hypertrophy, vascular remodeling and hypertension via the 12/15 lipoxygenase and c-Jun pathways.
The aim of this study was to investigate the effects of subconjunctivally administered mesenchymal stem cells (MSCs) on corneal wound healing in the acute stage of an alkali burn. A corneal alkali burn model was generated by placing a piece of 3-mm diameter filter paper soaked in NaOH on the right eye of 48 Sprague-Dawley female rats. 24 rats were administered a subconjunctival injection of a suspension of 2×106 MSCs in 0.1 ml phosphate-buffered saline (PBS) on day 0 and day 3 after the corneal alkali burn. The other 24 rats were administered a subconjunctival injection of an equal amount of PBS as a control. Deficiencies of the corneal epithelium and the area of corneal neovascularization (CNV) were evaluated on days 3 and 7 after the corneal alkali burn. Infiltrated CD68+ cells were detected by immunofluorescence staining. The mRNA expression levels of macrophage inflammatory protein-1 alpha (MIP-1α), tumor necrosis factor-alpha (TNF-α), monocyte chemotactic protein-1 (MCP-1) and vascular endothelial growth factor (VEGF) were analyzed using real-time polymerase chain reaction (real-time PCR). In addition, VEGF protein levels were analyzed using an enzyme-linked immunosorbent assay (ELISA). MSCs significantly enhanced the recovery of the corneal epithelium and decreased the CNV area compared with the control group. On day 7, the quantity of infiltrated CD68+ cells was significantly lower in the MSC group and the mRNA levels of MIP-1α, TNF-α, and VEGF and the protein levels of VEGF were also down-regulated. However, the expression of MCP-1 was not different between the two groups. Our results suggest that subconjunctival injection of MSCs significantly accelerates corneal wound healing, attenuates inflammation and reduces CNV in alkaline-burned corneas; these effects were found to be related to a reduction of infiltrated CD68+ cells and the down-regulation of MIP-1α, TNF-α and VEGF.
VP2 of chicken anemia virus (CAV) is a dual-specificity phosphatase required for virus infection, assembly and replication. The functions of the nuclear localization signal (NLS) and nuclear export signal (NES) of VP2 in the cell, however, are poorly understood. Our study identified the presence of a NLS in VP2 and showed that the protein interacted significantly with mini-chromosome maintenance protein 3 (MCM3) in the cell.
An arginine-lysine rich NLS could be predicted by software and spanned from amino acids 133 to 138 of VP2. The critical amino acids residues between positions 136 and 138, and either residue 133 or 134 are important for nuclear import in mammalian cells based on systematic mutagenesis. A NES is also predicted in VP2; however the results suggest that no functional NES is present and that this protein is CRM1 independent. It was also shown that VP2 is a chromatin binding protein and, notably, using a co-immunoprecipitation assay, it was found that VP2 association with MCM3 and that this interaction does not require DSP activity.
VP2 contains a NLS that span from amino acids 133 to 138. VP2 is a CRM1 independent protein during nuclear export and associates with MCM3 in cells.
AIM: To study the metabolic profiling of serum samples from compensated and decompensated cirrhosis patients.
METHODS: A pilot metabolic profiling study was conducted using three groups: compensated cirrhosis patients (n = 30), decompensated cirrhosis patients (n = 30) and healthy controls (n = 30). A 1H nuclear magnetic resonance (NMR)-based metabonomics approach was used to obtain the serum metabolic profiles of the samples. The acquired data were processed by multivariate principal component analysis and orthogonal partial least-squares discriminant analysis (OPLS-DA).
RESULTS: The OPLS-DA model was capable of distinguishing between decompensated and compensated cirrhosis patients, with an R2Y of 0.784 and a Q2Y of 0.598. Twelve metabolites, such as pyruvate, phenylalanine and succinate, were identified as the most influential factors for the difference between the two groups. The validation of the diagnosis prediction showed that the accuracy of the OPLS-DA model was 85% (17/20).
CONCLUSION: 1H NMR spectra combined with pattern recognition analysis techniques offer a new way to diagnose compensated and decompensated cirrhosis in the future.
Liver cirrhosis; Metabolic profiling; Orthogonal partial least-squares discriminant analysis
The aim of this study was to investigate whether serotonin (5-hydroxytryptamine, 5-HT) can modulate Na+/K+ pump in rat hippocampal CA1 pyramidal neurons.
5-HT (0.1, 1 mM) showed Na+/K+ pump current (Ip) densities of 0.40 ± 0.04, 0.34 ± 0.03 pA/pF contrast to 0.63 ± 0.04 pA/pF of the control of 0.5 mM strophanthidin (Str), demonstrating 5-HT-induced inhibition of Ip in a dose-dependent manner in hippocampal CA1 pyramidal neurons. The effect was partly attenuated by ondasetron, a 5-HT3 receptor (5-HT3R) antagonist, not by WAY100635, a 5-HT1AR antagonist, while 1-(3-Chlorophenyl) biguanide hydrochloride (m-CPBG), a 5-HT3R specific agonist, mimicked the effect of 5-HT on Ip.
5-HT inhibits neuronal Na+/K+ pump activity via 5-HT3R in rat hippocampal CA1 pyramidal neurons. This discloses novel mechanisms for the function of 5-HT in learning and memory, which may be a useful target to benefit these patients with cognitive disorder.