We report a new approach to probing DNA-protein interactions by combining optical tweezers with a high-throughput DNA curtains technique. Here we determine the forces required to remove the individual lipid-anchored DNA molecules from the bilayer. We demonstrate that DNA anchored to the bilayer through a single biotin-streptavidin linkage withstands ~20 pN before being pulled free from the bilayer, whereas molecules anchored to the bilayer through multiple attachment points can withstand ≥65 pN; access to this higher force regime is sufficient to probe the responses of protein-DNA interactions to force changes. As a proof-of-principle, we concurrently visualized DNA-bound fluorescently-tagged RNA polymerase while simultaneously stretching the DNA molecules. This work presents a step towards a powerful experimental platform that will enable concurrent visualization of DNA curtains while applying defined forces through optical tweezers.
DNA curtains; optical trap; single-molecule; combination; DNA pulling; rupture force
Ferric uptake regulator (Fur) is a global regulator that controls bacterial iron homeostasis. In this study, a fur deletion mutant of the deep-sea bacterium Shewanella piezotolerans WP3 was constructed. Physiological studies revealed that the growth rate of this mutant under aerobic conditions was only slightly lower than that of wild type (WT), but severe growth defects were observed under anaerobic conditions when different electron acceptors (EAs) were provided. Comparative transcriptomic analysis demonstrated that Fur is involved not only in classical iron homeostasis but also in anaerobic respiration. Fur exerted pleiotropic effects on the regulation of anaerobic respiration by controlling anaerobic electron transport, the heme biosynthesis system, and the cytochrome c maturation system. Biochemical assays demonstrated that levels of c-type cytochromes were lower in the fur mutant, consistent with the transcriptional profiling. Transcriptomic analysis and electrophoretic mobility shift assays revealed a primary regulation network for Fur in WP3. These results suggest that Fur may act as a sensor for anoxic conditions to trigger and influence the anaerobic respiratory system.
The “Notch-sparing” γ-secretase inhibitor (GSI) BMS-708,163 (Avagacestat) is currently in phase II clinical trials for Alzheimer’s disease. Unlike previously failed GSIs, BMS-708,163 is considered to be a promising drug candidate due to its reported Notch-sparing activity for the inhibition of Aβ production over Notch cleavage. We now report that BMS-708,163 binds directly to PS1-NTF, and that binding can be competed by other pan-GSIs, but not by γ-secretase modulators (GSMs). Furthermore, BMS-708,163 blocks the binding of four different active site-directed GSI photoaffinity probes. We therefore report that this compound acts as a non-selective γ-secretase inhibitor.
Aminoacyl-tRNA synthetases (AARSs) catalyze aminoacylation of tRNAs in the cytoplasm. Surprisingly, AARSs also have critical extracellular and nuclear functions. Evolutionary pressure for new functions might be manifested by splice variants that skip only an internal catalytic domain (CD) and link non-catalytic N- and C-terminal polypeptides. Using disease-associated histidyl-tRNA synthetase (HisRS) as an example, we discovered an expressed 171 amino acid protein (HisRSΔCD) that deleted the entire CD, and joined an N-terminal WHEP to the C-terminal anticodon-binding domain (ABD). X-ray crystallographic and 3-D NMR methods revealed the first structures of human HisRS and HisRSΔCD. In contrast to homodimeric HisRS, HisRSΔCD is monomeric, where rupture of the ABD’s packing with CD resulted in a dumbbell-like structure of flexibly linked WHEP and ABD domains. In addition, the ABD of HisRSΔCD presents a new local conformation. This natural internally deleted HisRS suggests evolutionary pressure to reshape AARS tertiary and quaternary structures for repurposing.
Clinical application of small interfering RNA (siRNA) requires safe and efficient delivery in vivo. Here, we report the design and synthesis of lipid nanoparticles (LNPs) for siRNA delivery based on cationic lipids with multiple tertiary amines and hydrophobic linoleyl chains. LNPs incorporating the lipid containing tris(2-aminoethyl)amine (TREN) and 3 linoleyl chain, termed TRENL3, were found to have exceptionally high siRNA transfection efficacy that was markedly superior to lipofectamine, a commercial transfection agent. In addition, inclusion of polyunsaturated fatty acids, such as linoleic acid and linolenic acids in the formulation further enhanced the siRNA delivery efficiency. TRENL3 LNPs were further shown to transported siRNA into the cytosol primarily via macropinocytosis rather than clathrin-mediated endocytosis. The new LNPs have demonstrated preferential uptake by the liver and hepatocellular carcinoma in mice, thereby leading to high siRNA gene silencing activity. These data suggest potential therapeutic applications of TRENL3 mediated delivery of siRNA for liver diseases.
Cationic lipids; Lipid nanoparticles; Small interfering RNA; hepatocellular carcinoma
Obesity and β-cell dysfunction due to oxidative stress impact the pathogenesis of type 2 diabetes mellitus. We co-cultured 3T3L1 adipocytes and islet cells in the presence or absence of the antioxidant α-lipoic acid (LA) and assayed the effects of the adipocytes and LA on the secretion of insulin by the islet cells and on the activities of factors involved in secretion and oxidative stress. At low glucose concentrations (2.8 mmol/l), the presence of adipocytes (co-culture) increased insulin secretion compared with islet cells cultured alone (control) and this increase was diminished by LA (co-culture plus LA). At high glucose concentrations (22 mmol/l), insulin secretion levels were similar for all islet groups, resulting in a restoration of the stimulation index in the presence of LA. The mRNA levels of the glucose-stimulated insulin secretion (GSIS) genes glucokinase, glucose transporter 2 and Kir6.2 were downregulated under co-culture and co-culture plus LA conditions. Protein and tyrosine phosphorylation levels of insulin receptor-β and insulin receptor substrate-1 were decreased under co-culture conditions and were restored by LA treatment. Cellular malondialdehyde levels increased in the co-cultured islets and this increase was blocked by LA. The mRNA levels of superoxide dismutase and catalase were reduced under co-culture conditions and these reductions were eliminated by the addition of LA. In conclusion, 3T3L1 adipocytes disturb insulin secretion and induce islet dysfunction. The effects may be mediated by multiple pathways, which include downregulation of GSIS gene expression, suppression of islet cell insulin signaling and the induction of oxidative stress. LA may protect islet cells via activation of islet cell insulin signaling and the mRNA expression of antioxidant enzymes.
α-lipoic acid; 3T3L1 adipocytes; islet cells; oxidative stress; co-culture
Adeno-associated virus serotype 9 (AAV9) vectors provide efficient and uniform gene expression to normal myocardium following systemic administration with kinetics that approach steady state within 2–3 weeks. However, due to the delayed onset of gene expression, AAV vectors have not previously been administered intravenously after reperfusion for post-infarct gene therapy applications. Here, we evaluate the therapeutic potential of post-MI gene delivery using intravenous AAV9.
AAV9 vectors expressing firefly luciferase, eGFP, or extracellular superoxide dismutase genes from the cTnT promoter (AcTnTLuc, AcTnTeGFP, AcTnTEcSOD) were employed. AcTnTLuc was administered intravenously at 10 minutes and at 1, 2 and 3 days post-ischemia/reperfusion (IR), and the kinetics of luciferase expression were assessed with bioluminescence imaging. AcTnTeGFP was used to evaluate the distribution of eGFP expression. High-resolution echocardiography was used to evaluate the effects of AcTnTEcSOD on left ventricular (LV) remodeling when injected 10 min post-IR.
Compared to sham animals, luciferase expression at 2 days after vector administration was elevated by 4-, 24-, 210- and 213-fold in groups injected at 10 min, 1 day, 2 days and 3 days post-IR, respectively. Expression of cTnT-driven eGFP was strongest in cardiomyocytes bordering the infarct zone. In the efficacy study of EcSOD, post-infarct LV end-systolic and end-diastolic volumes at days 14 and 28 were significantly smaller in the EcSOD group compared to control.
Systemic administration of AAV9 vectors after IR both elevates and accelerates gene expression that preferentially targets cardiomyocytes in the border zone with pharmacodynamics suitable for the attenuation of LV remodeling.
AAV; Post myocardial infarction gene delivery; Cardiac gene therapy; ischemia and reperfusion injury; LV remodeling
Objective: Anesthesia has been shown to suppress immune function, which can negatively affect the treatment of patients with various tumors. Here, we assessed two different anesthesia methods, general versus combined regional/general, in treatment of benign ovarian tumor by laparoscopic therapy. Methods: Out of 160 patients with benign ovarian tumors treated by laparoscopic therapy, 80 received general anesthesia combined with thoracic epidural anesthesia during surgery, and 80 received general anesthesia only. Venous blood samples were obtained at the following time points: before induction of anesthesia (T0), 2 hours after anesthesia, during operation, 3 days (d) after operation, 5 d after operation, and 7 d after operation. Percentages of CD3+, CD4+, and CD4+/CD8+ T lymphocytes were determined at these time points by flow cytometry to assess immune function. Results: For both groups, percentages of CD3+, CD4+, and CD4+/CD8+ T cells decreased significantly from T0 to 2 hr after anesthesia (P < 0.05). These percentages decreased again during surgery. However, T cell percentages in patients receiving combined anesthesia returned to normal levels 5 d after surgery, and those receiving only intravenous anesthesia returned to normal by 7 d after surgery. There were no significant differences in CD3+, CD4+, or CD4+/CD8+ T cell percentages between the two anesthesia groups at T0 and 7 d. However, significant differences in these percentages were observed between the two groups at all other time points. Interestingly, the decrease observed within the combined group were less dramatic than those observed within the intravenous-only group (P < 0.05). Conclusions: These findings indicate that, while any anesthesia may suppress immune function of patients treated by laparoscopic therapy, the effect of general anesthesia combined with thoracic epidural anesthesia on immune function was less than that produced by general anesthesia alone.
Epidural anesthesia; general anesthesia; ovarian tumor; laparoscopy; immune function
Hybrid weakness (HW) is an important postzygotic isolation which occurs in both intra- and inter-specific crosses. In this study, we described a novel low temperature-dependent intrasubspecific hybrid weakness in the F1 plants derived from the cross between two indica rice varieties Taifeng A and V1134. HW plants showed growth retardation, reduced panicle number and pale green leaves with chlorotic spots. Cytological assay showed that there were reduced cell numbers, larger intercellular spaces, thicker cell walls, and abnormal development of chloroplast and mitochondria in the mature leaves from HW F1 plants in comparison with that from both of the parental lines. Genetic analysis revealed that HW was controlled by two complementary dominant genes Hw3 from V1134 and Hw4 from Taifeng A. Hw3 was mapped in a 136 kb interval between the markers Indel1118 and Indel1117 on chromosome 11, and Hw4 was mapped in the region of about 15 cM between RM182 and RM505 on chromosome 7, respectively. RT-PCR analysis revealed that only LOC_Os11g44310, encoding a putative calmodulin-binding protein (OsCaMBP), differentially expressed among Taifeng A, V1134 and their HW F1. No recombinant was detected using the markers designed based on the sequence of LOC_Os11g44310 in the BC1F2 (Taifeng A//Taifeng A/V1134) population. Hence, LOC_Os11g44310 was probably the candidate gene of Hw3. Gene amplification suggested that LOC_Os11g44310 was present in V1134 and absent in Taifeng A. BLAST search revealed that LOC_Os11g44310 had one copy in the japonica genomic sequence of Nipponbare, and no homologous sequence in the indica reference sequence of 9311. Our results indicate that Hw3 is a novel gene for inducing hybrid weakness in rice.
The energy transfer mechanism between luminescent centers (LCs) and Er3+ in erbium-doped silicon-rich oxide (SROEr) films prepared by electron beam evaporation is investigated. Intense photoluminescence of the LCs (weak oxygen bonds, neutral oxygen vacancies, and Si=O states) within the active matrixes is obtained. Fast energy transfer from Si=O states to Er3+ takes advantage in the SROEr film and enhances the light emission from Er3+. The introduction of Si nanoclusters, which induces the Si=O states and facilitates the photon absorption of the Si=O states, is essential to obtain intense photoluminescence from both Si=O states and Er3+.
Luminescence centers; Silicon nanoclusters; Erbium ion; Energy transfer; Silicon-rich oxide
Parasite infections often result in a switch of the human body’s predominant immune reaction from T-helper 1 (Th1)-type to Th2-type. Hence, parasite infections are widely expected to accelerate the progression of human immunodeficiency virus (HIV) infections to acquired immunodeficiency syndrome (AIDS). In the People’s Republic of China, both parasitic diseases and AIDS are epidemic in certain rural areas, and co-infections are relatively common. However, no population-based studies have yet investigated the frequency of HIV and parasite co-infections, and its effects on immune responses. We studied (1) the immune status of an HIV-infected population, and (2) the effect of co-infection of HIV and intestinal parasites on selected parameters of the human immune system.
A total of 309 HIV-infected individuals were recruited and compared to an age-matched and sex-matched control group of 315 local HIV-negative individuals. Questionnaires were administered to all participants to obtain information on sociodemographic characteristics, sanitation habits, family income, and recent clinical manifestations. Two consecutive stool samples and 10 ml samples of venous blood were also collected from each individual for the diagnosis of parasite infections and quantitative measurements of selected cytokines and CD4+ T-lymphocytes, respectively.
During the study period, 79 HIV-infected individuals were not under highly active antiretroviral therapy (HAART) and were thus included in our analysis; the prevalence of intestinal helminth infections was 6.3% and that of protozoa was 22.8%. The most common protozoan infections were Blastocystis hominis (B. hominis) (13.9%) and Cryptosporidium spp. (10.1%). The prevalence of Cryptosporidium spp. in HIV-infected individuals was significantly higher than that in HIV negative individuals (P < 0.05). Compared to the non-co-infected population, no significant difference was found for any of the measured immunological indicators (P > 0.05). However, the following trends were observed: IFN-γ levels were lower, but the IL-4 level was higher, in the population co-infected with HIV and helminths. In the population co-infected with HIV and B. hominis, the IL-2 level was higher. The population co-infected with HIV and Cryptosporidium spp. had markedly lower CD4+ T-lymphocyte counts.
According to the immunologic profile, co-infection with helminths is disadvantageous to HIV-infected individuals. It was associated with a shift in the Th1/Th2 balance in the same direction as that caused by the virus itself, which might indicate an acceleration of the progress from an HIV infection to AIDS. Co-infection with Cryptosporidium spp. was not associated with a significant change in immune factors but co-infection with Cryptosporidium spp. was associated with a reduced level of CD4 + T-lymphocytes, confirming the opportunistic nature of such infections. Co-infection with B. hominis, on the other hand, was associated with an antagonistic shift in the immunological profile compared to an HIV infection.
HIV; Intestinal parasite; Co-infection; Immunological profile; People’s Republic of China
Gene expression, DNA replication, and genome maintenance are all initiated by proteins that must recognize specific targets from among a vast excess of nonspecific DNA. For example, to initiate transcription, E. coli RNA polymerase must locate promoter sequences, which comprise <2% of the bacterial genome. This search problem remains one of the least understood aspects of gene expression, largely due to the transient nature of search intermediates. Here we visualize RNAP in real time as it searches for promoters, and we develop a theoretical framework for analyzing target searches at the submicroscopic scale based upon single–molecule target association rates. Contrary to long–held assumptions, we demonstrate that the promoter search is dominated by three–dimensional diffusion at both the microscopic and submicroscopic scales in vitro, which has direct implications for understanding how promoters are located within physiological settings.
Background: Matrix metalloproteinase 10 (MMP10) plays an important role in ischemic stroke and has a close relationship with some stroke risk factors. The aim of this study was to investigate the relationship between two single nucleotide polymorphisms (SNP) in the exon regions of the MMP10 gene and atherothrombotic cerebral infarction risk. Methods: Five hundred and thirty-seven hospital-based patients who had suffered first atherothrombotic cerebral infarction and 580 unrelated healthy controls were enrolled. Demographic and clinical features of the subjects were recorded, and two polymorphisms, rs17435959 (G>C), rs17293607 (C>T) were chosen to be genotyped by real-time polymerase chain reaction-restriction TaqMan probes using the ABI 7300 TaqMan platform. Results: There were several clinical parameters, such as blood pressure, fasting blood glucose, total cholesterol, homocysteine, as well as carotid plaque and smoking, but not average age and sex ratios that showed significant differences between patients and control subjects. For rs17435959, there was no significant difference between the ischemic stroke group and the healthy control group in genotype frequency (OR=1.295, P=0.187, 95% CI (0.882-1.899)) or allele frequency (OR=1.267, P=0.202, 95% CI (0.881-1.823)). Moreover, in smoking, none smoking, having carotid plaque, no carotid plaque, male or female subtypes, there was significant difference between patients and control subjects in genotype frequencies or allele frequencies. The minor allele frequency of rs17293607 was 0.92%, prohibiting further study of this allele. Conclusions: These findings suggest that the rs17435959 SNP may not associated with atherothrombotic cerebral infarction risk. We also found that rs17293607 is not polymorphic in our study population.
Matrix metalloproteinase 10; polymorphism; genetic; genetic predisposition to disease; atherothrombotic cerebral infarction
Nutritional evaluation is important for patients with esophageal cancer, but the impact of undernutrition on outcome of those patients is not well elucidated. Our aim is to assess the impact of baseline nutritional status on overall survival (OS) in Chinese patients with esophageal squamous cell carcinoma (ESCC) and to detect a most appropriate indicator for nutritional evaluation.
502 patients from Southern China diagnosed as ESCC in Sun Yat-Sen University Cancer Center were included. A series of nutritional indicators were introduced to evaluate the baseline nutritional status. Kaplan-Meier method was used to estimate the 5-year OS and the log-rank test was used to determine the survival differences. Cox proportional hazards model was used in the univariate and multivariate analyses of OS.
With a median follow up time of 30 months, the median OS for the entire patient group was 37.3 months with the 5-year OS rate of 43.0%. Only performance status, AJCC 6th stage and body mass index (BMI) were the independent prognostic factors in multivariate analysis of OS. The median OS for patients with BMI less than 18.5, patients with BMI within 18.5-24.9 and patients with BMI more than 24.9 were 19.2, 43.2 and 51.6 months, respectively, with the 5-year OS rates of 25.2%, 46.1% and 48.1% (P<0.001). Patients with BMI <18.5 tended to present with a more advanced stage disease and a poorer tumor grade.
Baseline nutritional status is predictive of OS in Chinese patients with ESCC. BMI is a steady indicator for nutritional evaluation and a sensitive prognostic parameter for ESCC patients. Treatment optimization in ESCC patients with low BMI should integrate the modalities and individual nutritional support.
Nutritional parameters; body mass index (BMI); prognostic value; esophageal squamous cell carcinoma
Although the platinum regimen is adopted widely nowadays in spite of the excessive side effects, there is still no international standard for palliative chemotherapy of advanced gastric cancer. This meta-analysis assessed the efficacy and tolerability of platinum versus non–platinum chemotherapy as first-line palliative treatment in patients with inoperable, advanced gastric cancer.
Randomized phase II and III clinical trials on first-line palliative chemotherapy in inoperable, advanced gastric cancer were identified by electronic searches of PubMed, Embase, and the Cochrane Controlled Trial Register, and hand searches of relevant abstract books and reference lists. Response rates, overall survival, and toxicity were analyzed. Depending on whether new-generation agents (S-1, taxanes and irinotecan) were utilized, the non–platinum regimens were divided into two subgroup.
Compared to non-platinum regimens containing new-generation agents, the use of platinum-based regimens was associated with better response (risk ratio (RR) = 1.94, 95%CI[1.48, 2.55], p<0.001), an increase of overall survival (hazard ratio (HR) = 0.85, 95%CI[0.78, 0.92], p<0.001), a higher risk of hematological and non-hematological toxicity. No statistically significant increase in response (RR = 1.03, 95%CI [0.85, 1.24], p = 0.76) or overall survival (HR = 1.07, 95%CI [0.88, 1.30], p = 0.49) was found when platinum therapies were compared to new-generation agent based combination regimens. The toxicity of platinum-based regimens was significantly higher for hematologic toxicity, nausea and vomiting, and neurotoxicity, but not for diarrhea and toxic death rate.
New-generation agent based combination regimens achieved similar response rate and overall survival as platinum-based therapy that had generally higher side effects. S-1, taxanes and irinotecan seemed to be valid options for patients with inoperable, advanced gastric cancer as first-line chemotherapy.
Previous studies have revealed that traditional real-time quantitative PCR (qPCR) underestimates adeno-associated virus (AAV) titer. Because the inverted terminal repeat (ITR) exists in all AAV vectors, the only remaining element from the wild genome could form special configurations to interfere with qPCR titration. To solve this problem, a modified and universal qPCR method was tested and established.
In this work, there was a great variation in titration of ssAAV2-EGFP (Enhanced Green Fluorescence Protein) and scAAV2-EGFP genome by traditional qPCR. For ssAAV2-EGFP, the highest titer was found by using the targeting EGFP primers and the lowest titer was measured by those targeting bovine growth hormone polyA element (pBGH) primers.
Experimental data were reverse for ssAAV2-EGFP and scAAV2-EGFP. Here we report an improved and universal SmaI qPCR method, based on cleaving all ITRs in AAV2 genome by SmaI with several advantages: (1) impact of all ITRs in ssAAV2 and scAAV2 was dismissed; (2) titers increased remarkably, up to 7-fold, especially for scAAV2; (3) the variation of titers was reduced when different primers were applied. A similar phenomenon was also observed in other ssAAV2 and scAAV2 products when the range of titration was at 3×107 to 7×109 V.G/μl in this study.
This modified qPCR strategy can increase rAAV’ titer and reduce titration variance, possibly become a universal method for titrating AAV vectors.
SmaI; treatment; adeno-associated virus; titration; qPCR
Rehmannia glutinosa, a traditional Chinese medicine herb, is unable to grow normally in a soil where the same species has recently been cultivated. The biological basis of this so called “replanting disease” is unknown, but it may involve the action of microRNAs (miRNAs), which are known to be important regulators of plant growth and development. High throughput Solexa/Illumina sequencing was used to generate a transcript library of the R. glutinosa transcriptome and degradome in order to identify possible miRNAs and their targets implicated in the replanting disease. A total of 87,665 unigenes and 589 miRNA families (17 of which have not been identified in plants to date) was identified from the libraries made from a first year (FP) and a second year (SP) crop. A comparison between the FP and SP miRNAs showed that the abundance of eight of the novel and 295 of the known miRNA families differed between the FP and SP plants. Sequencing of the degradome sampled from FP and SP plants led to the identification of 165 transcript targets of 85 of the differentially abundant miRNA families. The interaction of some of these miRNAs with their target(s) is likely to form an important part of the molecular basis of the replanting disease of R. glutinosa.
Formaldehyde can induce misfolding and aggregation of Tau protein and β amyloid protein, which are characteristic pathological features of Alzheimer’s disease (AD). An increase in endogenous formaldehyde concentration in the brain is closely related to dementia in aging people. Therefore, the discovery of effective drugs to counteract the adverse impact of formaldehyde on neuronal cells is beneficial for the development of appropriate treatments for age-associated cognitive decline.
In this study, we assessed the neuroprotective properties of TongLuoJiuNao (TLJN), a traditional Chinese medicine preparation, against formaldehyde stress in human neuroblastoma cells (SH-SY5Y cell line). The effect of TLJN and its main ingredients (geniposide and ginsenoside Rg1) on cell viability, apoptosis, intracellular antioxidant activity and the expression of apoptotic-related genes in the presence of formaldehyde were monitored.
Cell counting studies showed that in the presence of TLJN, the viability of formaldehyde-treated SH-SY5Y cells significantly recovered. Laser scanning confocal microscopy revealed that the morphology of formaldehyde-injured cells was rescued by TLJN and geniposide, an effective ingredient of TLJN. Moreover, the inhibitory effect of geniposide on formaldehyde-induced apoptosis was dose-dependent. The activity of intracellular antioxidants (superoxide dismutase and glutathione peroxidase) increased, as did mRNA and protein levels of the antiapoptotic gene Bcl-2 after the addition of geniposide. In contrast, the expression of the apoptotic-related gene - P53, apoptotic executer - caspase 3 and apoptotic initiator - caspase 9 were downregulated after geniposide treatment.
Our results indicate that geniposide can protect SH-SY5Y cells against formaldehyde stress through modulating the expression of Bcl-2, P53, caspase 3 and caspase 9, and by increasing the activity of intracellular superoxide dismutase and glutathione peroxidase.
Formaldehyde impairment; Geniposide; Neuroprotection
Angiogenesis is closely related to the growth, invasion and metastasis of tumors, also considered as the key target of anticancer therapy. Scutellaria barbata D. Don (S. barbata), a traditional Chinese medicine, is being used to treat various diseases, including cancer. However, the antitumor molecular mechanism of S. barbata was still unclear. This study aimed to investigate the inhibitory effects of the total flavones in S. barbata (TF-SB) on angiogenesis.
Human umbilical vein endothelial cells (HUVECs) were treated with various concentrations of TF-SB. Cell viability was examined using the MTT assay. The scratch assay was used to detect the migration of HUVECs after treatment with TF-SB. The ability of HUVECs to form network structures in vitro was demonstrated using the tube formation assay. The chick embryo chorioallantoic membrane assay was performed to detect the in vivo anti-angiogenic effect. The expression of VEGF was measured by the enzyme-linked immunosorbent.
Results showed that TF-SB inhibited the proliferation and migration of HUVECs in a dose- dependent manner. Simultaneously, TF-SB significantly suppressed HUVEC angiogenesis in vitro and in vivo. Furthermore, VEGF was downregulated in both HUVECs and MHCC97-H cells after TF-SB treatment.
TF-SB could suppress the process of angiogenesis in vitro and in vivo. TF-SB potentially suppresses angiogenesis in HUVECs by regulating VEGF. These findings suggested that TF-SB may serve as a potent anti-angiogenic agent.
Scutellaria Barbata; Angiogenesis; Hepatocellular Carcinoma; Human Umbilical Vein Endothelial Cells
Odontogenesis is the result of the reciprocal interactions between epithelial–mesenchymal cells leading to terminally differentiated odontoblasts. This process from dental papilla mesenchymal cells to odontoblasts is regulated by a complex signaling pathway. When isolated from the developing tooth germs, odontoblasts quickly lose their potential to maintain the odontoblast-specific phenotype. Therefore, generation of an odontoblast-like cell line would be a good surrogate model for studying the dental mesenchymal cell differentiation into odontoblasts and the molecular events of dentin formation. In this study, immortalized dental papilla mesenchymal cell lines were generated from the first mouse mandibular molars at postnatal day 3 using pSV40. These transformed cells were characterized by RT-PCR, immunohistochemistry, Western blot, and analyzed for alkaline phosphatase activity and mineralization nodule formation. One of these immortalized cell lines, iMDP-3, displayed a high proliferation rate, but retained the genotypic and phenotypic characteristics similar to primary cells as determined by expression of tooth-specific markers and demonstrated the ability to differentiate and form mineralized nodules. Furthermore, iMDP-3 cells had high transfection efficiency as well as were inducible and responded to BMP2 stimulation. We conclude that the establishment of the stable murine dental papilla mesenchymal cell line might be used for studying the mechanisms of dental cell differentiation and dentin formation.
Cell differentiation; Dental papilla mesenchymal cells; Odontoblasts; Immortalization; SV-40 T antigen
Recently, we have demonstrated that trichosanthin (TCS), a promising agent for the treatment of cervical adenocarcinoma, inhibited HeLa cell proliferation through the PKC/MAPK/CREB signal pathway. Furthermore, TCS down-regulated Bcl-2 expression was abrogated by a decoy oligonucleotide (OGN) to the cyclic AMP-responsive element (CRE). The decoy OGN blocked the binding of CRE-binding protein (CREB) to Bcl-2. These results suggested that CRE-mediated gene expression may play a pivotal role in HeLa cell proliferation. However, little is known about the effect of TCS on cell cycle arrests, particularly, whether the genes involved in cell cycle were regulated by CRE. Our present study shows that the arrests of S, G1 and G2/M phases were accompanied by the significant down-regulation of cyclin A, D1 and CDK 2, 4 in HeLa cells, cyclin D1, E and CDK 2, 4 in Caski and C33a cells, and cyclin A, B1, E and CDK 2 in SW1990 cells. However, the cell cycle arrests were reversed via the significant up-regulation of cyclin A and D1, by the combined treatment of TCS and CRE. In conclusion, these data demonstrate for the first time that specific cell cycle arrests in cancer cells can be induced by TCS by inhibiting the binding of CREB to CRE on genes related to cell proliferation.
The purpose of this study was to propose a novel subclassification of pT4 gastric cancers according to the width of serosal changes and to investigate the validity and clinical utility of this subclassification as a predictor of prognosis.
A total of 780 pT4 stage gastric cancer patients classified according to the 7th American Joint Committee on Cancer (AJCC) staging system were reviewed. Clinicopathologic features were compared between patients with narrow serosal changes (nSE), wide serosal changes (wSE) and invasions of adjacent structures (SI). Prognostic factors were evaluated by univariate and multivariate analyses. The 7th AJCC and novel pT4 subclassification were compared for prognostic performance using the linear trend chi-square test, likelihood ratio chi-square test, and Akaike information criterion (AIC) in the Cox regression analysis.
The appropriate serosa infiltrate cutoff value was 8 cm. Most of the evaluated clinicopathologic features significantly differed between nSE and SI cancers. Only 3 factors were significantly different between wSE and SI cancers. The 5-year survival rates for patients with the novel pT4a and pT4b cancers were 47.2% and 14.52%, respectively, while they were 41.66% and 16.34% for the 7th AJCC pT4a and pT4b cancers, respectively. The novel pT4 subclassification had better discriminatory ability, monotonicity of gradients, and homogeneity and had smaller AIC values compared with the 7th AJCC pT4.
It is reasonable to subclassify pT4 to pT4a (nSE) and pT4b (wSE/SI) because the novel pT4 subclassification had more potential to identify the different prognoses for patients with gastric cancer.
Endoscopic resection of gastric subepithelial tumors (SETs) carries a high risk of perforation, particularly for tumors located at the gastric fundus and originating from the muscularis propria. Based on our experience with endoscopic submucosal dissection (ESD) and a novel endoscopic device, namely the ‘Resolution clip’ for the endoscopic closure of iatrogenic upper gastrointestinal (upper GI) perforations, we evaluated the clinical feasibility and safety of ESD for gastric fundus subepithelial tumors originating from the muscularis propria. In this prospective study, 11 consecutive patients who presented with gastric SETs ≤3 cm in diameter were enrolled. Regardless of whether perforation occurred, the gastric wall defect was closed with clips. The patients were followed up after the surgery. Endoscopic resection was successfully performed in 10 patients; however, in one patient a pure endoscopic approach was impossible as the lesion was severely adhered to surrounding tissue, and a switch to laparoscopic wedge resection was necessary. The mean resected tumor size was 18.8×17.2 mm and the mean surgery time of the 10 patients with ESD was 81 min (range 45–130 min). Histological diagnosis was gastrointestinal stromal tumor (GIST) in eight lesions [very low risk according to the National Institutes of Health (NIH) risk classification] and leiomyoma in three lesions. Perforation occurred in 3/10 patients. Gastric closure with the Resolution clips was performed successfully in all cases. Early post-ESD bleeding (EPEB) occurred in one patient. Basic ferric sulfate solution was sprayed during the upper GI endoscopy examination and the bleeding stopped. No complications occurred and the follow-up was unremarkable. In this early study, ESD using the Resolution clip was demonstrated to be a feasible and minimally invasive treatment for gastric fundus subepithelial tumors originating from the muscularis propria.
endoscopic submuscosal dissection; gastric fundus; muscularis propria
Repressor activator protein 1 (RAP1) is the most highly conserved telomere protein. It is involved in protecting chromosome ends in fission yeast, promoting gene silencing in Saccharomyces cerevisiae while in Kluyveromyces lactis it is required to repress homology directed recombination (HDR) at telomeres. Since mammalian RAP1 requires TRF2 for stable expression, its role in telomere function has remained obscure. To understand how RAP1 plays such diverse functions at telomeres, we solved the crystal or solution structures of the C-terminal RCT domains of RAP1 from multiple organisms in complex with their respective protein-binding partners. Our comparative structural analysis establishes the RCT domain of RAP1 as an evolutionarily conserved protein-protein interaction module. In mammalian and fission yeast cells, this module interacts with TRF2 and Taz1, respectively, targeting RAP1 to chromosome ends for telomere end protection. While RAP1 repress NHEJ at fission yeast telomeres, at mammalian telomeres it is required to repress HDR. In contrast, S. cerevisiae RAP1 utilizes the RCT domain to recruit Sir3 to telomeres to mediate gene silencing. Together, our results reveal that depending on the organism, the evolutionarily conserved RAP1 RCT motif plays diverse functional roles at telomeres.
IFN-α exhibits either direct antiviral effects or distinct immunomodulatory properties, which was identified as a ‘natural immune adjuvant’ for both the innate and the adaptive immune responses. Here we have investigated the effects of IFN-α as an adjuvant on the generation of T follicular helper (Tfh) cells and antigen-specific antibody responses. The data showed that adenoviral vectors co-expressing FMDV VP1 proteins and porcine IFN-α potently enhanced the generation of Tfh cells, the secretion of IL-21 protein and the expression of Bcl-6 mRNA, compared with adenoviral vectors sole expressing VP1 alone. Additionally, IFN-α substantial increased the number of germinal-center (GC) B cells and formation of GCs. Furthermore, IFN-α enhanced the antibody response, as shown by increased production of all IgG and subclasses of IgG1 and IgG2a. Thus, our results revealed the potent adjuvant activity of IFN-α which enhanced the generation of Tfh cells and regulated the humoral immunity by promoting germinal-center reactions and antibody responses.