Nasopharyngeal carcinoma (NPC) is a multi-factorial malignancy closely associated with environmental factors, genetic factors and Epstein-Barr virus infection. Human leukocyte antigen (HLA) complex, specially the region near HLA-A locus, was regarded as a major candidate region bearing NPC genetic susceptibility loci in many previous studies including two recent genome-wide association (GWA) studies. To provide further evidence for the NPC susceptibility in the region near HLA-A locus based on other previous studies, we carried out a two-stage hospital-based case control association study including 535 sporadic NPC patients and 525 cancer-free control subjects from Guangdong, a high prevalence area of NPC in China.
38 tag SNPs were initially selected by Heploview from the segment around HLA-A locus (from D6S211 to D6S510) and genotyped on GenomeLab SNPstream platform in 206 cases and 180 controls in the stage 1. Subsequently, the stage 1 significant SNPs and 17 additional SNPs were examined on another platform (Sequenom iPlex Assay) in another independent set of study population including 329 cases and 345 controls.
Totally eight SNPs from the segment from D6S211 to D6S510 within HLA complex were found to be significantly associated with NPC. Two of the most significant SNPs (rs9260734 and rs2517716) located near to HLA-A and HCG9 respectively were in strong LD with some other SNPs of this region reported by two previous GWA studies. Meanwhile, Meanwhile, novel independent susceptibility loci (rs9404952, Pcombined = 6.6 × 10-5, OR combined = 1.45) was found to be close to HLA-G.
Therefore, our present study supports that the segment from D6S211 to D6S510 in HLA complex region might contain NPC susceptibility loci which indeed needs to be fully investigated in the future.
Nasopharyngeal carcinoma; Single nucleotide polymorphism; Human leukocyte antigen (HLA)
The methylotrophic yeast, Pichia pastoris, offers the possibility to generate a high amount of recombinant proteins in a fast and easy way to use expression system. Being a single-celled microorganism, P. pastoris is easy to manipulate and grows rapidly on inexpensive media at high cell densities. A simple and direct method for the selection of high-producing clones can dramatically enhance the whole production process along with significant decrease in production costs.
A visual method for rapid selection of high-producing clones based on mannanase reporter system was developed. The study explained that it was possible to use mannanase activity as a measure of the expression level of the protein of interest. High-producing target protein clones were directly selected based on the size of hydrolysis holes in the selected plate. As an example, the target gene (9elp-hal18) was expressed and purified in Pichia pastoris using this technology.
A novel methodology is proposed for obtaining the high-producing clones of proteins of interest, based on the mannanase reporter system. This system may be adapted to other microorganisms, such as Saccharomyces cerevisiae for the selection of clones.
NPC (nasopharyngeal carcinoma) is a common malignancy in southern China without defined aetiology. Recent studies have shown that TGFBR3 (transforming growth factor type III receptor, also known as betaglycan), exhibits anticancer activities. This study was to investigate the effects of TGFBR3 on NPC growth and the mechanisms for its actions. Effects of TGFBR3 overexpression on cell viability and apoptosis were measured by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide], AO/EB (acridine orange/ethidium bromide) staining and electron microscopy in human NPC CNE-2Z cells. The expression of apoptosis-related proteins, p-Bad, Bad, XIAP (X-linked inhibitor of apoptosis), AIF (apoptosis-inducing factor), Bax and Bcl-2, was determined by Western blot or immunofluorescence analysis. Caspase 3 activity was measured by caspase 3 activity kit and [Ca2+]i (intracellular Ca2+ concentration) was detected by confocal microscopy. Transfection of TGFBR3 containing plasmid DNA at concentrations of 0.5 and 1 μg/ml reduced viability and induced apoptosis in CNE-2Z in concentration- and time-dependent manners. Forced expression of TGFBR3 up-regulated pro-apoptotic Bad and Bax protein, and down-regulated anti-apoptotic p-Bad, Bcl-2 and XIAP protein. Furthermore, transient overexpression of TGFBR3 also enhanced caspase 3 activity, increased [Ca2+]i and facilitated AIF redistribution from the mitochondria to the nucleus in CNE-2Z cells, which is independent of the caspase 3 pathway. These events were associated with TGFBR3-regulated multiple targets involved in CNE-2Z proliferation. Therefore transient overexpression of TGFBR3 may be a novel strategy for NPC prevention and therapy.
apoptosis; CNE-2Z; nasopharyngeal carcinoma (NPC); transforming growth factor type III receptor (TGFBR3); AIF, apoptosis-inducing factor; AO/EB, acridine orange/ethidium bromide; [Ca2+]i, intracellular Ca2+ concentration; DAB, diaminobenzidine; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide; NC, negative control; NPC, nasopharyngeal carcinoma; PBST, PBS containing 0.1% Tween 20; PFA, paraformaldehyde; pNA, p-nitroanilide; TGFBR3, transforming growth factor type III receptor; TRPM, transient receptor potential melastatin; Xiap, X-linked inhibitor of apoptosis
To assess the prevalence of HIV infection and characteristically risk of factors which associated with HIV infection among MSM in Harbin, China.
A face-to-face questionnaire interview was conducted among 463 Men Who Have Sex with Men (MSM) who were recruited by the snowball sampling in Harbin from April, 2011 to July, 2011. The questionnaire mainly included demographics, AIDS knowledge, homosexual behavior and the status of intervention in MSM. Blood specimens were obtained and tested for the diagnoses of HIV, syphilis and hepatitis C virus (HCV). Associations between above exposed factors and HIV infection were analyzed using a univariate analysis and forward stepwise logistic regression.
The prevalence of HIV and syphilis was 9.5 and 14.3%. The awareness rate of AIDS was 86.8%. The rate of unprotected sexual behavior was 57.6% of MSM during the past 6 months. The univariate analysis identified that the age (age≥35 years old), cohabitation, more than 10 years of homosexual behavior and more than 10 homosexual partners were risk factors which associated with the HIV infection, and that protected sex during the past 6 months was a protective factor for the HIV infection. The multivariate analysis identified that the duration of homosexual behavior and commercial sexual behavior were independent risk factors which associated with the HIV infection, and the protected sex during the past 6 months was a protective factor for the HIV infection.
The prevalence of HIV among MSM in Harbin has been rapidly increasing in the past few years. Targeted, tailored, and comprehensive interventions are urgently needed to prevent the HIV infection from MSM.
Hematopoietic stem cells (HSC) are maintained in a tightly regulated bone microenvironment constituted by a rich milieu of cells. Bone cells such as osteoblasts are associated with niche maintenance as regulators of the endosteal microenvironment. Bone remodeling also plays a role in HSC mobilization although it is poorly defined. The effects of zoledronic acid (ZA), a potent bisphosphonate that inhibits bone resorption, were investigated on bone marrow cell populations focusing on HSCs, and the endosteal and vascular niches in bone. ZA treatment significantly increased bone volume and HSCs in both young and adult mice (4 week and 4 month old, respectively). ZA increased vessel numbers with no overall change in vascular volume in bones of young and had no effect on vasculature in adult mice. Since both young and adult mice had increased HSCs and bone mass with differing vasculature responses, this suggests that ZA indirectly supports HSCs via the osteoblastic niche and not the vascular niche. Additionally, gene expression in Lin- cells demonstrated increased expression of self-renewal-related genes Bmi1 and Ink4a suggesting a role of ZA in the modulation of cell commitment and differentiation toward a long-term self-renewing cell. Genes that support the osteoblastic niche, BMP2 and BMP6 were also augmented in ZA treated mice. In conclusion, ZA-induced HSC expansion occurs independent of the vascular niche via indirect modulation of the osteoblastic niche.
HEMATOPOIETIC STEM CELLS; NICHE; BONE VASCULATURE; BLOOD VESSELS; BISPHOSPHONATE; ZOLEDRONIC ACID
Gemcitabine is among the most efficacious and widely used antimetabolite agents. Its molecular targets are ribonucleotide reductase M1 (RRM1) and elongating DNA. Acquired and de novo resistance as a result of RRM1 overexpression are major obstacles to therapeutic efficacy. We deployed a synthetic lethality screen to investigate if knockdown of 87 selected protein kinases by siRNA could overcome RRM1-dependent gemcitabine resistance in high and low RRM1-expressing model systems. The models included genetically RRM1-modified lung and breast cancer cell lines, cell lines with gemcitabine-induced RRM1 overexpression, and a series of naturally gemcitabine-resistant cell lines. Lead molecular targets were validated by determination of differential gemcitabine activity using cell lines with and without target knock down, and by assessing synergistic activity between gemcitabine and an inhibitor of the lead target. CHK1 was identified has the kinase with the most significant and robust interaction, and it was validated using AZD7762, a small-molecule ATP-competitive inhibitor of CHK1 activation. Synergism between CHK1 inhibition and RRM1-dependent gemcitabine efficacy was observed in cells with high RRM1 levels, while antagonism was observed in cells with low RRM1 levels. In addition, four cell lines with natural gemcitabine resistance demonstrated improved gemcitabine efficacy after CHK1 inhibition. In tumor specimens from 187 patients with non-small-cell lung cancer, total CHK1 and RRM1 in situ protein levels were significantly (p = 0.003) and inversely correlated. We conclude that inhibition of CHK1 may have its greatest clinical utility in malignancies where gemcitabine resistance is a result of elevated RRM1 levels. We also conclude that CHK1 inhibition in tumors with low RRM1 levels may be detrimental to gemcitabine efficacy.
Studies have revealed that tumor associated myeloid cells (TAMC) are one of the major sources of IL-10 in tumor-bearing mice. However, the significance of TAMC-derived IL-10 in tumor immunity is poorly understood. Here we show that IL-10 blockade or IL-10-deficiency reduces the capacity of TAMC in suppressing the proliferation of P1A-specific CD8 T cells. In the spleen, IL-10-deficient and wild type (WT) mice bearing large tumor burdens have similar TAMC populations. The tumors from IL-10-deficient mice however, have reduced numbers of TAMC compared with tumors from their WT counterparts. IL-10−/−RAG-2−/− mice also had reduced numbers of TAMC compared with tumors from IL-10+/+RAG-2−/− mice; therefore the reduction of TAMC in IL-10-deficient tumors was not due to adaptive immune response in tumors. Adoptively transferred tumor antigen specific CD8 T cells expanded more efficiently within tumors in IL-10−/−RAG-2−/− mice than in tumors from IL-10+/+RAG-2−/− mice. CTL adoptive transfer therapy prevented tumor evasion in IL-10−/−RAG-2−/− mice more efficiently than in IL-10+/+RAG-2−/− mice. Thus, IL-10 enhances accumulation of myeloid cells in tumors and TAMC-derived IL-10 suppresses activation and expansion of tumor antigen specific T cells.
Neointimal hyperplasia (NIH) in bypass conduits such as veins and prosthetic grafts is an important clinical entity that limits the long-term success of vascular interventions. Although the development of NIH in the conduits shares many of the same features of NIH that develops in native arteries after injury, vascular grafts are exposed to unique circumstances that predispose them to NIH, including surgical trauma related to vein handling, hemodynamic changes creating areas of low flow, and differences in biocompatibility between the conduit and the host environment. Multiple different approaches, including novel surgical techniques and targeted gene therapies, have been developed to target and prevent the causes of NIH. Recently, the PREVENT trials, the first molecular biology trials in vascular surgery aimed at preventing NIH, have failed to produce improved clinical outcomes, highlighting the incomplete knowledge of the pathways leading to NIH in vascular grafts. In this review, we aim to summarize the pathophysiologic pathways that underlie the formation of NIH in both vein and synthetic grafts and discuss current and potential mechanical and molecular approaches under investigation that may limit NIH in vascular grafts.
edifoligide; fistula; gene therapy; heparin; neointimal hyperplasia; patch; vascular graft; vein
Several peripheral proteins that might be useful for detecting the presence of ectopic pregnancy (EP) have been evaluated, but none have been proven entirely useful in the clinic. We investigated the presence and the possible changes in circulating molecules that distinguish between normal intrauterine pregnancy (IUP) and tubal ectopic pregnancy.
Non-pregnant women during the menstrual cycle, women with IUP, and women with tubal EP after informed consent. Serum levels of 17β-estradiol (E2), progesterone (P4), testosterone (T), beta-human chorionic gonadotropin (β-hCG), vascular endothelial growth factor-A (VEGF-A), placental growth factor (PIGF), and a distintegrin and metalloprotease protein 12 (ADAM12) were analyzed. Receiver operating characteristic analysis was used to assess the diagnostic discrimination of EP and gestational age-matched IUP.
E2, P4, PIGF, and ADAM12 levels increased and β-hCG decreased throughout IUP. E2 and VEGF-A levels were significantly different between women with tubal EP and IUP. However, using a serum β-hCG cut-off of less than 1000 mIU/mL, P4 was significantly lower in women with tubal EP compared to IUP. Although E2 was inversely correlated with VEGF-A in women in the early stages of IUP, E2 was not correlated with VEGF-A in women with EP prior to tubal surgery. There were no significant differences in either PIGF or ADAM12 alone between women with tubal EP or IUP. Although no significant correlations were seen between E2 and PIGF or P4 and ADAM12 in women in the early stages of IUP, E2 was positively correlated with PIGF and P4 was positively correlated with ADAM12 in women with EP prior to tubal surgery. Our studies defined associations but not causality.
Individual measurements of serum E2 or VEGF-A levels are strongly related to early pregnancy outcomes for women with IUP and EP, and pregnancy-associated E2 and VEGF-A levels provide diagnostic accuracy for the presence of tubal EP. This study demonstrates that correlation analysis of E2/VEGF-A and E2/PIGF serum levels may be able to distinguish a tubal EP from a normal IUP.
Steroid hormones; VEGF-A; PIGF; ADAM12; Hypoxia; Tubal ectopic pregnancy
This study was designed to investigate the role of serum GP73 for diagnosing significant fibrosis in patients with chronic hepatitis B virus (HBV) infections. Two populations were enrollment. All subjects were patients with chronic HBV infections. First population included 761 patients, who received liver stiffness measurement; the second population included 633 patients, who undertaken liver biopsy, in which 472 patients with nearly normal ALT. All patients received serum GP73 test. The effect of GP73 recombinant protein to HepG2 cells and LX2 cells were observed in vitro. Results showed that serum GP73 concentration is correlated with liver stiffness (r = 0.601). The area under ROC curve is 0.76. The sensitivity and specificity of GP73 for significant fibrosis (≥F2) diagnosis were 62.81%, 80.05% respectively (cut off: 76.6 ng/ml). Serum GP73 concentration was significantly correlated with the grading of fibrosis (r = 0.32, and 0.35, in 633 and 472 patients, respectively.) GP73 had a striking performance for diagnosing S2 in patients with chronic HBV infections. In 472 patients with nearly normal ALT, the sensitivity and specificity of GP73 for S2 diagnosis were 62.5% and 80.0% respectively, where the cut-off was set at 82 ng/ml. GP73 recombinant protein may prompt LX2 cells proliferation at the concentration 10–100 ng/ml. The present results indicated that GP73 may be a marker for diagnosing significant fibrosis in patients with chronic HBV infections, and may be a new contributor to fibrogensis.
There are a number of gas ionization sensors using carbon nanotubes as cathode or anode. Unfortunately, their applications are greatly limited by their multi-valued sensitivity, one output value corresponding to several measured concentration values. Here we describe a triple-electrode structure featuring two electric fields with opposite directions, which enable us to overcome the multi-valued sensitivity problem at 1 atm in a wide range of gas concentrations. We used a carbon nanotube array as the first electrode, and the two electric fields between the upper and the lower interelectrode gaps were designed to extract positive ions generated in the upper gap, hence significantly reduced positive ion bombardment on the nanotube electrode, which allowed us to maintain a high electric field near the nanotube tips, leading to a single-valued sensitivity and a long nanotube life. We have demonstrated detection of various gases and simultaneously monitoring temperature, and a potential for applications.
Biochemical and topographical features of an artificial extracellular matrix (aECM) can direct stem cell fate. However, it is difficult to vary only the biochemical cues without changing nanotopography to study their unique role. We took advantage of two unique features of M13 phage, a non-toxic nanofiber-like virus, to generate a virus-activated aECM with constant ordered ridge/groove nanotopography but displaying different fibronectin-derived peptides (RGD, its synergy site PHSRN, and a combination of RGD and PHSRN). One feature is the self-assembly of phage into a ridge/groove structure, another is the ease of genetically surface-displaying a peptide. We found that the unique ridge/groove nanotopography and the display of RGD and PHSRN could induce the osteoblastic differentiation of mesenchymal stem cells (MSCs) without any osteogenic supplements. The aECM formed through self-assembly and genetic engineering of phage can be used to understand the role of peptide cues in directing stem cell behavior while keeping nanotopography constant.
Lipin-1 is a protein that exhibits dual functions as a phosphatidic acid phosphohydrolase (PAP) enzyme in the triglyceride synthesis pathways and a transcriptional co-regulator. Our previous studies have shown that ethanol causes fatty liver by activation of sterol regulatory element-binding protein 1 (SREBP-1) and inhibition of hepatic AMP-activated kinase (AMPK) in mice. Here, we tested the hypothesis that AMPK-SREBP-1 signaling may be involved in ethanol-mediated up-regulation of lipin-1 gene expression. The effects of ethanol on lipin-1 were investigated in cultured hepatic cells and in the livers of chronic ethanol-fed mice. Ethanol exposure robustly induced activity of a mouse lipin-1 promoter, promoted cytoplasmic localization of lipin-1 and caused excess lipid accumulation both in cultured hepatic cells and in mouse livers. Mechanistic studies showed that ethanol-mediated induction of lipin-1 gene expression was inhibited by a known activator of AMPK or overexpression of a constitutively active form of AMPK. Importantly, overexpression of processed nuclear form of SREBP-1c (nSREBP-1c) abolished the ability of AICAR to suppress ethanol-mediated induction of lipin-1 gene expression level. Chromatin immunoprecipitation (ChIP) assays further revealed that ethanol exposure significantly increased association of acetylated Histone H3 at lysine 9 (Lys9) with the SRE-containing region in the promoter of the lipin-1 gene. In conclusion, ethanol-induced up-regulation of lipin-1 gene expression is mediated through inhibition of AMPK and activation of SREBP-1.
Alcoholic fatty liver; signal transduction; lipid metabolism; acetylation; sumoylation
The accumulation of thermal time usually represents the local heat resources to drive crop growth. Maps of temperature-based agro-meteorological indices are commonly generated by the spatial interpolation of data collected from meteorological stations with coarse geographic continuity. To solve the critical problems of estimating air temperature (T
a) and filling in missing pixels due to cloudy and low-quality images in growing degree days (GDDs) calculation from remotely sensed data, a novel spatio-temporal algorithm for T
a estimation from Terra and Aqua moderate resolution imaging spectroradiometer (MODIS) data was proposed. This is a preliminary study to calculate heat accumulation, expressed in accumulative growing degree days (AGDDs) above 10 °C, from reconstructed T
a based on MODIS land surface temperature (LST) data. The verification results of maximum T
a, minimum T
a, GDD, and AGDD from MODIS-derived data to meteorological calculation were all satisfied with high correlations over 0.01 significant levels. Overall, MODIS-derived AGDD was slightly underestimated with almost 10% relative error. However, the feasibility of employing AGDD anomaly maps to characterize the 2001–2010 spatio-temporal variability of heat accumulation and estimating the 2011 heat accumulation distribution using only MODIS data was finally demonstrated in the current paper. Our study may supply a novel way to calculate AGDD in heat-related study concerning crop growth monitoring, agricultural climatic regionalization, and agro-meteorological disaster detection at the regional scale.
MODIS land surface temperature; Air temperature estimation; Reconstruction; Heat accumulation; Rice growing season; Growing degree day (GDD)
For years, emerging infectious diseases have appeared worldwide and threatened the health of people. The emergence and spread of an infectious-disease outbreak are usually unforeseen, and have the features of suddenness and uncertainty. Timely understanding of basic information in the field, and the collection and analysis of epidemiological information, is helpful in making rapid decisions and responding to an infectious-disease emergency. Therefore, it is necessary to have an unobstructed channel and convenient tool for the collection and analysis of epidemiologic information in the field.
Baseline information for each county in mainland China was collected and a database was established by geo-coding information on a digital map of county boundaries throughout the country. Google Maps was used to display geographic information and to conduct calculations related to maps, and the 3G wireless network was used to transmit information collected in the field to the server. This study established a decision support system for the response to infectious-disease emergencies based on WebGIS and mobile services (DSSRIDE). The DSSRIDE provides functions including data collection, communication and analyses in real time, epidemiological detection, the provision of customized epidemiological questionnaires and guides for handling infectious disease emergencies, and the querying of professional knowledge in the field. These functions of the DSSRIDE could be helpful for epidemiological investigations in the field and the handling of infectious-disease emergencies.
The DSSRIDE provides a geographic information platform based on the Google Maps application programming interface to display information of infectious disease emergencies, and transfers information between workers in the field and decision makers through wireless transmission based on personal computers, mobile phones and personal digital assistants. After a 2-year practice and application in infectious disease emergencies, the DSSRIDE is becoming a useful platform and is a useful tool for investigations in the field carried out by response sections and individuals. The system is suitable for use in developing countries and low-income districts.
Polycystic ovary syndrome (PCOS) presents with a range of clinical complications including hyperandrogenism, polycystic ovaries, chronic oligo/anovulation, infertility, and metabolic alterations related to insulin resistance. Because the mechanism by which this disorder develops is poorly understood, information from experimental models of human disease phenotypes may help to define the mechanisms for the initiation and development of PCOS-related pathological events. The establishment of animal models compatible with human PCOS is challenging, and applying the lessons learned from these models to human PCOS is often complicated. In this mini-review we provide examples of currently available genetic mouse models, their ovarian phenotypes, and their possible relationship to different aspects of human PCOS. Because of the practical and ethical limitations of studying PCOS-related events in humans, our understanding of the mechanisms that contribute to the etiology of human PCOS may be enhanced through further study of these transgenic and knockout mouse models.
PCOS; hemorrhagic cystic follicles; transgenic and knockout mice
The kinetic mechanism of SCS [succinyl-CoA (coenzyme A) synthetase], which participates in the TCA (tricarboxylic acid) cycle, ketone body metabolism and haem biosynthesis, has not been fully characterized. Namely, a representative catalytic mechanism and associated kinetic parameters that can explain data on the enzyme-catalysed reaction kinetics have not been established. To determine an accurate model, a set of putative mechanisms of SCS, proposed by previous researchers, were tested against experimental data (from previous publication) on SCS derived from porcine myocardium. Based on comparisons between model simulation and the experimental data, an ordered ter–ter mechanism with dead-end product inhibition of succinate against succinyl-CoA is determined to be the best candidate mechanism. A thermodynamically constrained set of parameter values is identified for this candidate mechanism.
catalytic mechanism; dead-end binding; enzyme; succinyl-CoA; AIC, Akaike information criterion; SCS, succinyl-CoA synthetase; TCA, tricarboxylic acid
AIM: To investigate the role of inositol-requiring enzyme 1α (IRE1α) in gut development of Xenopus lavies embryos.
METHODS: Xenopus embryos were obtained with in vitro fertilization and cultured in 0.1 × MBSH. One and half nanogram of IRE1α, 1 ng of IRE1α-GR mRNA, 1 ng of IRE1αΔC-GR mRNA, and 50 ng of IRE1α morpholino oligonucleotide (MO) or XBP1(C)MO were injected into four blastomeres at 4-cell stage for scoring the phenotype and marker gene analysis. To rescue the effect of IRE1α MO, 1 ng of IRE1α-GR mRNA was co-injected with 50 ng of MO. For the activation of the GR-fusion proteins, dexamethasone was prepared as 5 mmol/L stock solutions in 100% ethanol and applied to the mRNA injected embryos at desired stages in a concentration of 10 μmol/L in 0.1 × MBSH. Embryos were kept in dexamethasone up to stage 41. Whole-mount in situ hybridization was used to determine specific gene expression, such as IRE1α, IRE1β, Xbra and Xsox17α. IRE1α protein expression during Xenopus embryogenesis was detected by Western blotting.
RESULTS: In the whole-mount in situ hybridization analysis, xenopus IRE1α and IRE1β showed quite different expression pattern during tadpole stage. The relatively higher expression of IRE1α was observed in the pancreas, and significant transcription of IRE1β was found in the liver. IRE1α protein could be detected at all developmental stages analyzed, from stage 1 to stage 42. Gain-of-function assay showed that IRE1α mRNA injected embryos at tailbud stage were nearly normal and the expression of the pan-mesodermal marker gene Xbra and the endodermal gene Xsox17α at stage 10.5 was not significantly changed in embryos injected with IRE1α mRNA as compared to uninjected control embryos. And at tadpole stage, the embryos injected with IRE1α-GR mRNA did not display overt phenotype, such as gut-coiling defect. Loss-of-function assay demonstrated that the IRE1α MO injected embryos were morphologically normal before the tailbud stages. We did not observe a significant change of mesodermal and endodermal marker gene expression, while after stage 40, about 80% of the MO injected embryos exhibited dramatic gut defects in which the guts did not coil, but other structures outside the gastrointestinal tract were relatively normal. To test if the phenotypes were specifically caused by the knockdown of IRE1α, a rescue experiment was performed by co-injection of IRE1α-GR mRMA with IRE1α MO. The data obtained demonstrated that the gut coiling defect was rescued. The deletion mutant of IRE1α was constructed, consisting of the N-terminal part without the C-terminal kinase and RNase domains named IRE1αΔC, to investigate the functional domain of IRE1α. Injection of IRE1αΔC-GR mRNA caused similar morphological alterations with gut malformation by interfering with the function of endogenous xIRE1α. In order to investigate if IRE1α/XBP1 pathway was involved in gut development, 50 ng of XBP1 MO was injected and the results showed that knockdown of XBP1 resulted in similar morphological alterations with gut-coiling defect at tadpole stage.
CONCLUSION: IRE1α is not required for germ layer formation but for gut development in Xenopus lavies and it may function via XBP1-dependent pathway.
Inositol-requiring enzyme 1α; XBP1; Xenopus lavies; Gut; Development
Previously, we have documented that isolated autophagosomes from tumor cells could efficiently cross-prime tumor-reactive naïve T cells and mediate tumor regression in preclinical mouse models. However, the effect of tumor-derived autophagosomes, here we refer as to DRibbles, on B cells has not been studied so far. At present study, we found that DRibbles generated from a murine hepatoma cell line Hep1-6, induced B-cell activation after intravenous injection into mice. B-cell populations were significantly expanded and the production of Hep1-6 tumor-specific antibodies was successfully induced. Moreover, in vitro studies showed that DRibbles could induce more efficient B-cell proliferation and activation, antibody production, and cytokine secretion than whole tumor cell lysates. Notably, we found that B-cell activation required proteins but not DNA in the DRibbles. We further showed that B cells could capture DRibbles and present antigens in the DRibbles to directly induce T cell activation. Furthermore, we found that B-cell activation, antibody production, cytokine secretion and antigen cross-presentation were TLR2-MyD88 pathway dependent. Taken together, the present studies demonstrated that tumor-derived autophagosomes (DRibbles) efficiently induced B cells activation, antibody production, cytokine secretion and antigen cross-presentation mainly depending on their protein component via TLR2/MyD88 dependent manner.
Whether neoadjuvant chemotherapy safely allows close margins in osteosarcoma patients is still unknown. This study investigates the impact of close margins on local recurrence (LR) and overall survival (OS) for osteosarcoma patients treated with neoadjuvant chemotherapy.
We retrospectively reviewed 47 cases of conventional osteosarcoma who were treated at our institution. Patient and treatment factors such as age, gender, MSTS stage, tumour site, surgery type, pathological type, tumour size, surgical margin, tumour necrosis rate, chemotherapy regimens and cycles were recorded. A close margin was defined as tumour present less than 5 mm from the closest resection margin. The average followup was 87.6 months (range, 25–135 months).
Twenty-five patients were alive, 22 patients had died, and eight had LR. Twenty-eight patients had wide margins, seven had positive margins and 12 had close margins. Positive margins had a greater risk of LR (57.1%) than wide margins and close margins. There was no difference in LR (8.3% vs 10.7%) between close margins and wide margins. Margin status was not correlated with OS.
Compared with wide margins, close margins did not lead to increased local recurrence in our study group. Whether close margins, as defined in our study, are just as acceptable as wide margins in terms of patient outcomes for osteosarcoma patients with neoadjuvant chemotherapy needs to be further confirmed in the future.
Recent studies have shown that circulating microRNAs might be useful, novel biomarkers for the diagnosis of acute myocardial infarction. The aims of this study were to evaluate the expression of cardiac-specific miRNAs (miR-1, -133a, -208b, and -499) in patients with acute myocardial infarction and to compare the diagnostic values of these miRNAs with that of cardiac troponin T.
Sixty-seven plasma samples obtained from patients with acute myocardial infarction and 32 plasma specimens collected from healthy volunteers were analyzed in this study. The levels of cardiac-specific miRNAs (miR-1, -133a, -208b, and -499) were measured by quantitative reverse transcription-polymerase chain reaction, and the concentrations of plasma cardiac troponin T were measured using electrochemiluminescence-based methods and an Elecsys 2010 Immunoassay Analyzer.
The levels of plasma miR-1, -133a, -208b, and -499 were significantly higher in acute myocardial infarction patients (all p<0.001) than in healthy volunteers. The expression of the cardiac-specific miRNAs in acute myocardial infarction patients decreased to close to the baseline levels at the time of hospital discharge (all p>0.05). There were no correlations between the levels of the four circulating miRNAs and the clinical characteristics of the study population (all p>0.05). Furthermore, receiver operating characteristic curve analyses showed that the four plasma miRNAs were not superior to cardiac troponin T for the diagnosis of acute myocardial infarction (all p>0.05).
Our results demonstrate that circulating miR-1, -133a, -208b, and -499 may be useful biomarkers in acute myocardial infarction patients but that these miRNAs are not superior to cardiac troponin T for the diagnosis of acute myocardial infarction.
microRNAs; cardiac troponin T; Acute Myocardial Infarction; Circulating Biomarkers
The traditional Chinese medicine bufalin, extracted from toad’s skin, has been demonstrated to exert anticancer activities in various kinds of human cancers. The mechanisms of action lie in its capacity to induce apoptosis, or termed type I programmed cell death (PCD). However, type II PCD, or autophagy, participates in cancer proliferation, progression, and relapse, as well. Recent studies on autophagy seem to be controversial because of the dual roles of autophagy in cancer survival and death. In good agreement with previous studies, we found that 100 nM bufalin induced extensive HepG2 cell apoptosis. However, we also noticed bufalin triggered autophagy and enhanced Beclin-1 expression, LC3-I to LC3-II conversion, as well as decreased p62 expression and mTOR signaling activation in HepG2 cells. Blockage of autophagy by selective inhibitor 3-MA decreased apoptotic ratio in bufalin-treated HepG2 cells, suggesting a proapoptotic role of bufalin-induced autophagy. Furthermore, we investigated the underlying mechanisms of bufalin-induced autophagy. Bufalin treatment dose-dependently promoted AMPK phosphorylation while AMPK inhibition by compound C significantly attenuated bufalin-induced autophagy. Taken together, we report for the first time that bufalin induces HepG2 cells PCD, especially for autophagy, and the mechanism of action is, at least in part, AMPK-mTOR dependent.
hepatocellular carcinoma; bufalin; apoptosis; autophagy; AMPK; mTOR
AIM: To investigate the short-term outcome of laparoscopic total mesorectal excision (TME) in patients with mid and low rectal cancers.
METHODS: A consecutive series of 138 patients with middle and low rectal cancer were randomly assigned to either the laparoscopic TME (LTME) group or the open TME (OTME) group between September 2008 and July 2011 at the Department of Colorectal Cancer of Shanghai Cancer Center, Fudan University and pathological data, as well as surgical technique were reviewed retrospectively. Short-term clinical and oncological outcome were compared in these two groups. Patients were followed in the outpatient clinic 2 wk after the surgery and then every 3 mo in the first year if no adjuvant chemoradiation was indicated. Statistical analysis was performed using SPSS 13.0 software.
RESULTS: Sixty-seven patients were treated with LTME and 71 patients were treated with OTME (sex ratio 1.3:1 vs 1.29:1, age 58.4 ± 13.6 years vs 59.6 ± 9.4 years, respectively). The resection was considered curative in all cases. The sphincter-preserving rate was 65.7% (44/67) vs 60.6% (43/71), P = 0.046; mean blood loss was 86.9 ± 37.6 mL vs 119.1 ± 32.7 mL, P = 0.018; postoperative analgesia was 2.1 ± 0.6 d vs 3.9 ± 1.8 d, P = 0.008; duration of urinary drainage was 4.7 ± 1.8 d vs 6.9 ± 3.4 d, P = 0.016, respectively. The conversion rate was 2.99%. The complication rate, circumferential margin involvement, distal margins and lymph node yield were similar for both procedures. No port site recurrence, anastomotic recurrence or mortality was observed during a median follow-up period of 21 mo (range: 9-56 mo).
CONCLUSION: Laparoscopic TME is safe and feasible, with an oncological adequacy comparable to the open approach. Further studies with more patients and longer follow-up are needed to confirm the present results.
Rectum; Neoplasms; Colorectal surgery; Laparoscopy; Treatment outcome
A motif in a network is a connected graph that occurs significantly more frequently as an induced subgraph than would be expected in a similar randomized network. By virtue of being atypical, it is thought that motifs might play a more important role than arbitrary subgraphs. Recently, a flurry of advances in the study of network motifs has created demand for faster computational means for identifying motifs in increasingly larger networks. Motif detection is typically performed by enumerating subgraphs in an input network and in an ensemble of comparison networks; this poses a significant computational problem. Classifying the subgraphs encountered, for instance, is typically performed using a graph canonical labeling package, such as Nauty, and will typically be called billions of times. In this article, we describe an implementation of a network motif detection package, which we call NetMODE. NetMODE can only perform motif detection for -node subgraphs when , but does so without the use of Nauty. To avoid using Nauty, NetMODE has an initial pretreatment phase, where -node graph data is stored in memory (). For we take a novel approach, which relates to the Reconstruction Conjecture for directed graphs. We find that NetMODE can perform up to around times faster than its predecessors when and up to around times faster when (the exact improvement varies considerably). NetMODE also (a) includes a method for generating comparison graphs uniformly at random, (b) can interface with external packages (e.g. R), and (c) can utilize multi-core architectures. NetMODE is available from netmode.sf.net.
MyD88 is an adapter protein that links toll-like receptors (TLRs) and Interleukin-1 receptors (IL-1Rs) with downstream signaling molecules. The MyD88 has been found to be an essential mediator in the development of osteoarthritis in articular cartilage. However, the role of the MyD88 pathway has yet to be elucidated in the intervertebral disk (IVD). Using in vitro techniques, we analyzed the effect of MyD88 pathway-specific inhibition on the potent inflammatory and catabolic mediator LPS and IL-1 in bovine and human nucleus pulposus (NP) cells by assessing matrix-degrading enzyme expression, including matrix metalloproteases (MMPs) and a disintegrin-like and metalloprotease with thrombospondin motifs (ADAMTS family). We also analyzed inhibition of MyD88 in the regulation of inducible nitric oxide synthase and TLR-2. Finally, we used an ex vivo organ culture model to assess the effects of MyD88 inhibitor (MyD88i) on catabolic factor-induced disk degeneration in mice lumbar disks. In bovine NP cells, MyD88i potently antagonizes LPS- or IL-1-mediated induction of cartilage-degrading enzyme production, including MMP-1, MMP-13, ADAMTS-4, and ADAMTS-5. MyD88i also attenuates the LPS- or IL-1-mediated induction of iNOS and TLR-2 gene expression. Our ex vivo findings reveal inhibition of MyD88 via counteraction of IL-1-mediated proteoglycan depletion. The findings from this study demonstrate the potent anti-inflammatory and anti-catabolic effects of inhibition of MyD88 pathway inhibition on IVD homeostasis, suggesting a potential therapeutic benefit of a MyD88i in degenerative disk disease in the future.
MyD88 inhibitor; Intervertebral disk degeneration; Toll-like receptors; Low back pain; Signaling pathway