A series of tetracyclic nitrofuran isoxazoline antituberculosis agents was designed and synthesized to improve the pharmacokinetic properties of an initial lead compound, which had potent antituberculosis activity but suffered from poor solubility, high protein binding and rapid metabolism. In this study, structural modifications were carried on the outer phenyl and piperidine rings to introduce solubilizing and metabolically blocking functional groups. The compounds generated were evaluated for their in vitro antitubercular activity, bacterial spectrum of activity, solubility, permeability, microsomal stability and protein binding. Pharmacokinetic profiles for the most promising candidates were then determined. Compounds with phenyl morpholine and pyridyl morpholine outer rings were found to be the most potent antituberculosis agents in the series. These compounds retained a narrow antibacterial spectrum of activity, with weak anti-gram positive and no gram negative activity, as well as good activity against non-replicating M. tuberculosis in a low oxygen model. Overall, the addition of solubilizing and metabolically blocked outer rings did improve solubility and decrease protein binding as designed. However, the metabolic stability for compounds in this series was generally lower than desired. The best three compounds selected for in vivo pharmacokinetic testing all showed high oral bioavailability, with one notable compound showing a significantly longer half-life and good tolerability supporting its further advancement.
Tuberculosis; antibiotic; nitrofuran; isoxazoline; nitroaromatic
Estrogen deficiency is associated with increased incidence of cardiovascular diseases. But merely estrogen supplementary treatment can induce many severe complications such as breast cancer. The present study was designed to elucidate molecular mechanisms underlying increased susceptibility of arrhythmogenesis during myocardial infarction with estrogen deprivation, which provides us a new target to cure cardiac disease accompanied with estrogen deprivation. We successfully established a rat model of myocardial ischemia (MI) accompanied with estrogen deprivation by coronary artery ligation and ovariectomy (OVX). Vulnerability and mortality of ventricular arrhythmias increased in estrogen deficiency rats compared to non estrogen deficiency rats when suffered MI, which was associated with down-regulation of microRNA-151-5p (miR-151-5p). Luciferase Reporter Assay demonstrated that miR-151-5p can bind to the 3′-UTR of FXYD1 (coding gene of phospholemman, PLM) and inhibit its expression. We found that the expression of PLM was increased in (OVX+MI) group compared with MI group. More changes such as down-regulation of Kir2.1/IK1, calcium overload had emerged in (OVX+MI) group compared to MI group merely. Transfection of miR-151-5p into primary cultured myocytes decreased PLM levels and [Ca2+]i, however, increased Kir2.1 levels. These effects were abolished by the antisense oligonucleotides against miR-151-5p. Co-immunoprecipitation and immunofluorescent experiments confirmed the co-localization between Kir2.1 and PLM in rat ventricular tissue. We conclude that the increased ventricular arrhythmias vulnerability in response to acute myocardial ischemia in rat is critically dependent upon down-regulation of miR-151-5p. These findings support the proposal that miR-151-5p could be a potential therapeutic target for the prevention of ischemic arrhythmias in the subjects with estrogen deficiency.
To explore the protective effects of aminoguanidine (AG) on retinal apoptosis in mice with oxygen-induced retinopathy (OIR).
A total of 80 C57BL/6J mice, aged 7 days, were randomly divided into four groups: normal, high oxygen, high oxygen saline and high oxygen treated with AG. In the normal group, mice were housed in normoxic conditions from postnatal day P7 to P17. Mice in the other 3 groups were placed under hyperoxic conditions (75±2%O2) in an oxygen-regulated chamber for 5 days and subsequently placed in normoxic conditions for 5 days. Mice in the AG group were treated once daily, from P12 to P17, with AG hemisulfate (100mg/kg body weight, intraperitoneally) dissolved in physiological saline. An equivalent amount of 0.9% physiological saline was administered, as above, to mice in the high oxygen saline group. Ten mice were randomly selected from each group on P14 and on P17, euthanized and the retinas examined. Apoptotic cells in the retina were detected using the terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL) method. The expression of nitric oxide synthase (iNOS) in the retina was detected by immunohistochemistry and changes in rod cells were observed using electron microscopy.
TUNEL-positive cells and iNOS immunoreactive neurons were present in the inner nuclear and ganglion cell retinal layers of mice in the high oxygen group. The number of TUNEL-positive cells was significantly greater in the high oxygen group compared with the normal group (t=-20.81, P14d <0.05; t=-15.05, P17d <0.05). However, the number of TUNEL-positive cells in the AG treatment group was significantly lower (t=-13.21, P14d<0.05; t=-6.61, P17d <0.05) compared with the high oxygen group. The expression of iNOS was significantly higher in the high oxygen group compared with the normal group (t=-21.95, P14d<0.05; t=-17.30, P17d<0.05). However, the expression of iNOS in the AG treatment group was significantly lower (t=-12.17, P14d<0.05; t=-10.30, P17d<0.05) compared with the high oxygen group. The outer segments of the rods were disorganized and short in the high oxygen group. Rod morphology appeared to be slightly improved in the AG group.
AG may protect retinal neurons in OIR by inhibiting apoptosis. The mechanism may be related to iNOS.
aminoguanidine; retinopathy of prematurity; apoptosis; inhibitor of nitric oxide synthase
In China during March 4–April 28, 2013, avian influenza A(H7N9) virus testing was performed on 20,739 specimens from patients with influenza-like illness in 10 provinces with confirmed human cases: 6 (0.03%) were positive, and increased numbers of unsubtypeable influenza-positive specimens were not seen. Careful monitoring and rapid characterization of influenza A(H7N9) and other influenza viruses remain critical.
influenza; avian influenza; avian influenza A(H7N9) virus; H7N9; subtype H7N9; China; influenza-like illness; surveillance; viruses
Multipotent human dental follicle cells (HDFCs) have been intensively studied in periodontal regeneration research, yet the role of Notch1 in HDFCs has not been fully understood. The aim of the current study is to explore the role of Notch1 signaling in HDFCs self-renewal and proliferation. HDFCs were obtained from the extracted wisdom teeth from adolescent patients. Regulation of Notch1 signaling in the HDFCs was achieved by overexpressing the exogenous intracellular domain of Notch1 (ICN1) or silencing Notch1 by shRNA. The regulatory effects of Notch1 on HDFC proliferation, cell cycle distribution and the expression of cell cycle regulators were investigated through various molecular technologies, including plasmid construction, retrovirus preparation and infection, qRT-PCR, western blot, RBP-Jk luciferase reporter and cell proliferation assay. Our data clearly show that constitutively activation of Notch1 stimulates the HDFCs proliferation while inhibition of the Notch1 suppresses their proliferation in vitro. In addition, the HDFCs proliferation is associated with the increased expression of cell cycle regulators, e.g. cyclin D1, cyclin D2, cyclin D3, cyclin E1, CDK2, CDK4, CDK6, and SKP2 and the decreased expression of p27 kip1. Moreover, our data show that the G1/S phase transition (indicating proliferation) and telomerase activity (indicating self-renewal) can be enhanced by overexpression of ICN1 but halted by inhibition of Notch1. Together, the current study provides evidence for the first time that Notch1 signaling regulates the proliferation and self-renewal capacity of HDFCs through modulation of the G1/S phase transition and the telomerase activity.
2-Zinc-glycoprotein 1 (AZGP1) is a multidisciplinary protein that participates in many important functions in the human body, including fertilization, immunoregulation and lipid mobilization. Recently, it has been shown that AZGP1 is also involved in carcinogenesis and tumor differentiation. In this study, we investigated the expression levels and prognostic value of AZGP1 in primary gastric cancers.
Methods and Results
We examined the expression of AZGP1 in 35 paired cancerous and matched adjacent noncancerous gastric mucosa tissues by real-time quantitative RT-PCR (qRT-PCR) and western blotting. Furthermore, we analyzed AZGP1 expression in 248 patients who underwent resection procedures between 2005 and 2007 using immunohistochemistry. The relationships between the AZGP1 expression levels, the clinicopathological factors, and patient survival were investigated. AZGP1 expression was significantly reduced at both the mRNA (P = 0.023) and protein levels (P = 0.019) in tumor tissue samples, compared with expression in matched adjacent non-tumor tissue samples. The immunohistochemical staining data showed that AZGP1 expression was significantly decreased in 52.8% (131/248) of gastric adenocarcinoma cases. Clinicopathological analysis showed that the reduced expression of AZGP1 was significantly correlated with tumor location (P = 0.011), histological grade (P = 0.005) and T stage (P = 0.008). Kaplan–Meier survival curves revealed that the reduced expression of AZGP1 was associated with a poor prognosis in gastric adenocarcinoma patients (P = 0.009). Multivariate Cox analysis identified AZGP1 expression was an independent prognostic factor for overall survival of gastric adenocarcinoma patients (HR = 1.681, 95% CI = 1.134–2.494, P = 0.011).
Our study suggests that AZGP1 might serve as a candidate tumor suppressor and a potential prognostic biomarker in gastric carcinogenesis.
Cronobacter spp. is an emerging pathogen that causes meningitis, sepsis, bacteremia, and necrotizing enterocolitis in neonates and children. The present study developed an assay integrating real-time PCR and high resolution melting (HRM) analysis targeting the OmpA gene for the specific detection and rapid identification of Cronobacter spp. (formerly Enterobacter sakazakii) in powdered infant formula. Eleven Cronobacter field isolates and 25 reference strains were examined using one pair of primers, having the accuracy of 100% in reference to conventional methods. The assay was proved to be highly sensitive with a detection limit of 102 CFU/ml without pre-enrichment, and highly concordant (100%) when compared with ISO-IDF 22964 in 89 actual samples. The method performed for Cronobacter spp. detection was less than 24 h, drastically shortened, compared to several days using standard culturing method, it is probe-free and reduces a risk of PCR carryover. Moreover, all Cronobacter strains examined in this study were genotyped into two species according to their HRM profiles. The established method should provide a molecular tool for direct detection and simultaneous genotyping of Cronobacter spp. in powdered infant formula.
Sensitive and specific detection of liver cirrhosis is an urgent need for optimal individualized management of disease activity. Substantial studies have identified circulation miRNAs as biomarkers for diverse diseases including chronic liver diseases. In this study, we investigated the plasma miRNA signature to serve as a potential diagnostic biomarker for silent liver cirrhosis.
A genome-wide miRNA microarray was first performed in 80 plasma specimens. Six candidate miRNAs were selected and then trained in CHB-related cirrhosis and controls by qPCR. A classifier, miR-106b and miR-181b, was validated finally in two independent cohorts including CHB-related silent cirrhosis and controls, as well as non−CHB-related cirrhosis and controls as validation sets, respectively.
A profile of 2 miRNAs (miR-106b and miR-181b) was identified as liver cirrhosis biomarkers irrespective of etiology. The classifier constructed by the two miRNAs provided a high diagnostic accuracy for cirrhosis (AUC = 0.882 for CHB-related cirrhosis in the training set, 0.774 for CHB-related silent cirrhosis in one validation set, and 0.915 for non−CHB-related cirrhosis in another validation set).
Our study demonstrated that the combined detection of miR-106b and miR-181b has a considerable clinical value to diagnose patients with liver cirrhosis, especially those at early stage.
Myeloperoxidase (MPO) is an endogenous oxidant enzyme that produces reactive oxygen species (ROS) and may be involved in lung carcinogenesis. The MPO−463G>A polymorphism influences MPO transcription and has been associated with lung cancer susceptibility. However, the association between the MPO−463G>A polymorphism and lung cancer risk remains controversial.
To investigate the effect of this polymorphism on lung cancer susceptibility, we performed a meta-analysis based on 22 published case–control studies including 7,520 patients with lung cancer and 8,600 controls. Odds ratios (ORs) with 95% confidence intervals (CIs) were used to assess the strength of the association.
Overall, there was no evidence for significant association between MPO−463G>A polymorphism and lung cancer susceptibility (for AA versus GG: OR = 0.91, 95%CI = 0.67–1.24; for GA versus GG: OR = 0.87, 95% CI = 0.78–0.98; for AA/GA versus GG: OR = 0.90, 95% CI = 0.80–1.01; for AA versus GA/GG: OR = 0.96, 95% CI = 0.72–1.28). In the stratified analyses by ethnicity, source of controls and smoking status, we also did not find any significant association between them.
In summary, this meta-analysis suggests MPO−463G>A polymorphism may not be a risk factor for developing lung cancer. However, further prospective well-designed population-based studies with larger sample size are expected to validate the results.
Previous molecular genetic studies of physiology and pigmentation of sheep skin have focused primarily on a limited number of genes and proteins. To identify additional genes that may play important roles in coat color regulation, Illumina sequencing technology was used to catalog global gene expression profiles in skin of sheep with white versus black coat color.
There were 90,006 and 74,533 unigenes assembled from the reads obtained from white and black sheep skin, respectively. Genes encoding for the ribosomal proteins and keratin associated proteins were most highly expressed. A total of 2,235 known genes were differentially expressed in black versus white sheep skin, with 479 genes up-regulated and 1,756 genes down-regulated. A total of 845 novel genes were differentially expressed in black versus white sheep skin, consisting of 107 genes which were up-regulated (including 2 highly expressed genes exclusively expressed in black sheep skin) and 738 genes that were down-regulated. There was also a total of 49 known coat color genes expressed in sheep skin, from which 13 genes showed higher expression in black sheep skin. Many of these up-regulated genes, such as DCT, MATP, TYR and TYRP1, are members of the components of melanosomes and their precursor ontology category.
The white and black sheep skin transcriptome profiles obtained provide a valuable resource for future research to understand the network of gene expression controlling skin physiology and melanogenesis in sheep.
Sheep skin; Transcriptome; Gene expression; Pigmentation; Melanogenesis
We report here the complete genome sequence of a novel duck hepatitis A virus type 3 (DHAV-3) isolated from a dead Cherry Valley duckling in eastern China. The whole genomic nucleotide sequence and polyprotein amino acid sequence of the virus had higher homology with those of Chinese DHAV-3 isolates, medium homology with those of Korean DHAV-3 isolates, and the lowest homology with those of Vietnamese isolate DN2. The result indicated that the genetic evolution of DHAV-3 isolates had obvious geographical features.
The known functions of telomerase in tumor cells include replenishing telomeric DNA and maintaining cell immortality. We have previously shown the existence of a negative correlation between human telomerase reverse transcriptase (hTERT) and radiosensitivity in tumor cells. Here we set out to elucidate the molecular mechanisms underlying regulation by telomerase of radiosensitivity in MCF-7 cells. Toward this aim, yeast two-hybrid (Y2H) screening of a human laryngeal squamous cell carcinoma radioresistant (Hep2R) cDNA library was first performed to search for potential hTERT interacting proteins. We identified ubiquitin-conjugating enzyme E2D3 (UBE2D3) as a principle hTERT-interacting protein and validated this association biochemically. ShRNA-mediated inhibition of UBE2D3 expression attenuated MCF-7 radiosensitivity, and induced the accumulation of hTERT and cyclin D1 in these cells. Moreover, down-regulation of UBE2D3 increased hTERT activity and cell proliferation, accelerating G1 to S phase transition in MCF-7 cells. Collectively these findings suggest that UBE2D3 participates in the process of hTERT-mediated radiosensitivity in human breast cancer MCF-7 cells by regulating hTERT and cyclin D1.
Seedling establishment is a critical phase in the life of plants when they are the most vulnerable to environment. Growth arrest at post-germinative stage under stress is the major adaptive strategy to help germinating seedlings to survive a spectrum of stressful conditions. ABA signaling is the key pathway to control stress-induced developmental arrest. However, mechanisms controlling the phase transition under abiotic stress are not fully understood. Here, we described miR172b as a new key regulator controlling transition of germinating seedlings from heterotrophic to autotrophic growth under osmotic stress in Arabidopsis. We showed that miR172b and its target SNZ were co-expressed during early seedling development. Expression of miR172b and SNZ was low after radicle emergence and sharply increased at the checkpoint to autotrophic development under normal conditions. Interestingly, activation of miR172b and SNZ was completely abolished by ABA and osmotic stress. miR172b overexpression and snz-1 exhibited increased sensitivity to ABA and osmotic stress during specific post-germinative stage, and resulted in higher expression of ABI3, ABI5 and downstream genes, such as Em6 and RAB18, than wild type under ABA treatment. Our results revealed that miR172b is a critical regulator specifically controlling cotyledon greening during post-germinative growth by directly targeting SNZ under ABA treatment and osmotic stress.
Limited data are available on the ectomycorrhizae-induced changes in surface structure and composition of soil colloids, the most active portion in soil matrix, although such data may benefit the understanding of mycorrhizal-aided soil improvements. By using ectomycorrhizae (Gomphidius viscidus) and soil colloids from dark brown forest soil (a good loam) and saline-alkali soil (heavily degraded soil), we tried to approach the changes here. For the good loam either from the surface or deep soils, the fungus treatment induced physical absorption of covering materials on colloid surface with nonsignificant increases in soil particle size (P > 0.05). These increased the amount of variable functional groups (O–H stretching and bending, C–H stretching, C=O stretching, etc.) by 3–26% and the crystallinity of variable soil minerals (kaolinite, hydromica, and quartz) by 40–300%. However, the fungus treatment of saline-alkali soil obviously differed from the dark brown forest soil. There were 12–35% decreases in most functional groups, 15–55% decreases in crystallinity of most soil minerals but general increases in their grain size, and significant increases in soil particle size (P < 0.05). These different responses sharply decreased element ratios (C : O, C : N, and C : Si) in soil colloids from saline-alkali soil, moving them close to those of the good loam of dark brown forest soil.
To investigate the ability of rESAT6 to identify different mycobacteria-sensitized guinea pigs and its safety in preclinical and phase I clinical study.
Guinea pigs were sensitized with different Mycobacteria. After sensitization, all animals were intradermally injected with rESAT6 and either PPD or PPD-B. At 24 h after the injection, the erythema of the injection sites were measured using a double-blind method. For the preclinical safety study, different doses of rESAT6 and BSA were given 3 times intramuscularly to guinea pigs. On day 14 after the final immunization, the guinea pigs were intravenously injected with the same reagents in the hind legs and the allergic reactions were observed. A single-center, randomized, open phase I clinical trial was employed. The skin test was conducted in 32 healthy volunteers aged 19–65 years with 0.1 μg, 0.5 μg, and 1 μg rESAT6. Physical examination and laboratory tests were performed before and after the skin test and adverse reactions were monitored. The volunteers’ local and systemic adverse reactions and adverse events were recorded for 7 days.
Positive PPD or PPD-B skin tests were observed in all Mycobacteria-sensitized guinea pigs; the diameters of erythema were all >10 mm. The rESAT6 protein induced a positive skin test result in the guinea pigs sensitized with MTB, M. bovis, M. africanum and M. kansasii; the diameters of erythema were 14.7±2.0, 9.3±3.8, 18.7±2.4, and 14.8±4.2 mm, respectively. A negative skin test result was detected in BCG-vaccinated and other NTM-sensitized guinea pigs. The rESAT6 caused no allergic symptoms, but many allergic reactions, such as cough, dyspnea, and even death, were observed in the guinea pigs who were administered BSA. During the phase I clinical trial, no adverse reactions were found in the 0.1 μg rESAT6 group, but in the 0.5 μg rESAT6 group 2 volunteers reported pain and 1 reported itching, and in the 1 μg rESAT6 group there was 1 case of pain, 1 case of itching, and 1 case of blister. No other local or systemic adverse reactions or events were reported.
The rESAT6 can differentiate effectively among MTB infection, BCG vaccination, and NTM infection and is safe in healthy volunteers.
phase I clinical trial; recombinant protein; skin test; latent M. tuberculosis infection
We investigated the effects of changing extracellular K+ concentrations on block of the weak inward-rectifier K+ channel Kir1.1b (ROMK2) by the three intracellular cations Mg2+, Na+, and TEA+. Single-channel currents were monitored in inside-out patches made from Xenopus laevis oocytes expressing the channels. With 110 mM K+ in the inside (cytoplasmic) solution and 11 mM K+ in the outside (extracellular) solution, these three cations blocked K+ currents with a range of apparent affinities (Ki (0) = 1.6 mM for Mg2+, 160 mM for Na+, and 1.8 mM for TEA+) but with similar voltage dependence (zδ = 0.58 for Mg2+, 0.71 for Na+, and 0.61 for TEA+) despite having different valences. When external K+ was increased to 110 mM, the apparent affinity of all three blockers was decreased approximately threefold with no significant change in the voltage dependence of block. The possibility that the transmembrane cavity is the site of block was explored by making mutations at the N152 residue, a position previously shown to affect rectification in Kir channels. N152D increased the affinity for block by Mg2+ but not for Na+ or TEA+. In contrast, the N152Y mutation increased the affinity for block by TEA+ but not for Na+ or Mg2+. Replacing the C terminus of the channel with that of the strong inward-rectifier Kir2.1 increased the affinity of block by Mg2+ but had a small effect on that by Na+. TEA+ block was enhanced and had a larger voltage dependence. We used an eight-state kinetic model to simulate these results. The effects of voltage and external K+ could be explained by a model in which the blockers occupy a site, presumably in the transmembrane cavity, at a position that is largely unaffected by changes in the electric field. The effects of voltage and extracellular K+ are explained by shifts in the occupancy of sites within the selectivity filter by K+ ions.
In this paper, a human electrocardiogram (ECG) identification system based on ensemble empirical mode decomposition (EEMD) is designed. A robust preprocessing method comprising noise elimination, heartbeat normalization and quality measurement is proposed to eliminate the effects of noise and heart rate variability. The system is independent of the heart rate. The ECG signal is decomposed into a number of intrinsic mode functions (IMFs) and Welch spectral analysis is used to extract the significant heartbeat signal features. Principal component analysis is used reduce the dimensionality of the feature space, and the K-nearest neighbors (K-NN) method is applied as the classifier tool. The proposed human ECG identification system was tested on standard MIT-BIH ECG databases: the ST change database, the long-term ST database, and the PTB database. The system achieved an identification accuracy of 95% for 90 subjects, demonstrating the effectiveness of the proposed method in terms of accuracy and robustness.
biometrics; ECG Identification System; ensemble empirical mode decomposition; k-nearest neighbors
Background. The efficacy and tolerability of peginterferon α-2a and peginterferon α-2b in chronic hepatitis C (CHC) patients remain controversial. Methods. PubMed, Ovid, and Cochrane libraries were electronically searched until August 30, 2012. Studies that met the inclusion criteria were systematically evaluated by two reviewers independently. Results. The overall sustained virologic response (SVR) rate of the peginterferon α-2a group was significantly higher than that of the peginterferon α-2b group (46.7% versus 42.4%, P value = 0.01). The same tendency was observed for naïve, genotype 1/4, and genotype 2/3 patients. The early virologic response (EVR) and end-of-treatment response (ETR) rates were significantly higher in the peginterferon α-2a group than in the peginterferon α-2b group (56.1% versus 49.8%, P < 0.0001; 67.9% versus 56.6%, P < 0.00001, resp.). Peginterferon α-2a had a significantly lower discontinuation rate than peginterferon α-2b (27.9% versus 33.9%, P < 0.0001) in naïve patients. In both naïve CHC and hepatitis C virus genotype 1 patients, peginterferon α-2a had a higher relapse rate than peginterferon α-2b. Conclusions. Peginterferon α-2a has superior efficacy with higher EVR, ETR, and SVR than peginterferon α-2b for CHC patients, both plus ribavirin. Peginterferon α-2a might obtain a similar or even lower discontinuation rate than peginterferon α-2b. However, peginterferon α-2a had a higher relapse rate than peginterferon α-2b.
We previously identified a series of methylsulfonylnitrobenzoates (MSNB's) that block the interaction of the thyroid hormone receptor with its coactivators. MSNB's inhibits coactivator binding through irreversibly modifying cysteine 298 of thyroid hormone receptor (TR). Although MSNB's have better pharmacological features than our first generation inhibitors (β-aminoketones) they contain a potentially unstable ester linkage. Here we report the bioisosteric replacement of the ester linkage with a thiazole moiety, yielding sulfonylnitrophenylthiazoles (SNPT's). An array of SNPT's representing optimal side chains from the MSNB series was constructed using parallel chemistry and evaluated to test their antagonism of the TR-coactivator interaction. Selected active compounds were evaluated in secondary confirmatory assays including regulation of thyroid response element driven transcription in reporter constructs and native genes. In addition the selected SNPT's shown to be selective for TR relative to other nuclear hormone receptor (NR).
Melanoma is a malignant tumor with high invasive and metastatic properties. Though radiation is the major therapy for melanoma, its radio-resistance has been shown to severely influence the clinical outcome. So it is imperative to enhance the sensitivity of uveal melanoma cells to radiotherapy. Previously, we found that the cell cycle of 92-1 uveal melanoma cells was suspended and remained unchanged for up to 5 days after exposure to 10 Gy of X-rays, which might be relevant to the high radio-sensitivity of 92-1 cells. To further investigate the cell cycle suspension-associated proteins, we employed two analyses with stable isotope labeling with amino acids in cell culture technology and two-dimensional liquid chromatography tandem mass spectrometry. Cells were incubated for 15 h or 48 h after irradiation with 10 Gy of X-rays. We identified a total of 737 proteins at 15 h (Group A) and 530 proteins at 48 h post-irradiation (Group B). The gene ontology biological pathway was used to obtain a systems level view of proteome changes in 92-1cells under cell cycle suspension. We further selected the significantly changed proteins for investigation of their potential contribution to cell cycle suspension, growth arrest and cell senescence. These proteins are involved in the cell cycle, stress response, glycolysis and the tricarboxylic acid cycle, etc. Our study expected to reveal potential marker proteins associated with cell suspension induced by irradiation, which might contribute to understanding the mechanism beyond the cell cycle suspension.
radiation; uveal melanoma; 2D-LC-MS/MS; GO biological pathway
Corneal transparency depends on a unique extracellular matrix secreted by stromal keratocytes, mesenchymal cells of neural crest lineage. Derivation of keratocytes from human embryonic stem (hES) cells could elucidate the keratocyte developmental pathway and open a potential for cell-based therapy for corneal blindness. This study seeks to identify conditions inducing differentiation of pluripotent hES cells to the keratocyte lineage. Neural differentiation of hES cell line WA01(H1) was induced by co-culture with mouse PA6 fibroblasts. After 6 days of co-culture, hES cells expressing cell-surface NGFR protein (CD271, p75NTR) were isolated by immunoaffinity adsorption, and cultured as a monolayer for one week. Keratocyte phenotype was induced by substratum-independent pellet culture in serum-free medium containing ascorbate. Gene expression, examined by quantitative RT-PCR, found hES cells co-cultured with PA6 cells for 6 days to upregulate expression of neural crest genes including NGFR, SNAI1, NTRK3, SOX9, and MSX1. Isolated NGFR-expressing cells were free of PA6 feeder cells. After expansion as a monolayer, mRNAs typifying adult stromal stem cells were detected, including BMI1, KIT, NES, NOTCH1, and SIX2. When these cells were cultured as substratum-free pellets keratocyte markers AQP1, B3GNT7, PTDGS, and ALDH3A1 were upregulated. mRNA for keratocan (KERA), a cornea-specific proteoglycan, was upregulated more than 10,000 fold. Culture medium from pellets contained high molecular weight keratocan modified with keratan sulfate, a unique molecular component of corneal stroma. These results show hES cells can be induced to differentiate into keratocytes in vitro. Pluripotent stem cells, therefore, may provide a renewable source of material for development of treatment of corneal stromal opacities.
This study attempts to discuss the relationship between land use spatial distribution structure and energy-related carbon emission intensity in Guangdong during 1996–2008. We quantized the spatial distribution structure of five land use types including agricultural land, industrial land, residential and commercial land, traffic land, and other land through applying spatial Lorenz curve and Gini coefficient. Then the corresponding energy-related carbon emissions in each type of land were calculated in the study period. Through building the reasonable regression models, we found that the concentration degree of industrial land is negatively correlated with carbon emission intensity in the long term, whereas the concentration degree is positively correlated with carbon emission intensity in agricultural land, residential and commercial land, traffic land, and other land. The results also indicate that land use spatial distribution structure affects carbon emission intensity more intensively than energy efficiency and production efficiency do. These conclusions provide valuable reference to develop comprehensive policies for energy conservation and carbon emission reduction in a new perspective.
To explore the process of pressure ulcer formation, interleukin (IL)-17 expression levels were observed in a mouse model of pressure ulcers. Twenty mice were divided into experimental and control groups (10 mice per group). A mouse model of pressure ulcers was established by inducing ischemia-reperfusion injury on local tissue in the experimental group. Pressure ulcer tissues in the experimental group and normal mouse tissue in the control group were stained using hematoxylin and eosin (H&E) and observed using light microscopy. The protein and mRNA expression levels of IL-17, in mouse pressure ulcer tissues from the experimental group and in the normal tissue from the control group, were determined using real-time PCR and western blot analysis, respectively. The mRNA and protein expression levels of IL-17 were compared between the two groups. H&E staining indicated that striated muscle was arranged orderly and cellular structure was intact in the control group, whilst inflammatory cell infiltration was observed in the muscle tissue of the experimental group. The expression levels of IL-17 mRNA were 0.307±0.058 ng in the experimental group and 0.112±0.042 ng in the control group (P<0.05). The expression levels of the IL-17 protein were 0.434±0.097 ng in the experimental group and 0.181±0.040 ng in the control group (P<0.05). IL-17 expression levels were increased in pressure ulcers, which suggests that IL-17 may be associated with pressure ulcers.
pressure ulcer; animal model; interleukin-17
Adenocarcinoma (AC) and squamous cell carcinoma (SqCC) are two major histological subtypes of lung cancer. Genome-wide association studies (GWAS) have made considerable advances in the understanding of lung cancer susceptibility. Obvious heterogeneity has been observed between different histological subtypes of lung cancer, but genetic determinants in specific to lung SqCC have not been systematically investigated. Here, we performed the GWAS analysis specifically for lung SqCC in 833 SqCC cases and 3,094 controls followed by a two-stage replication in additional 2,223 lung SqCC cases and 6,409 controls from Chinese populations. We found that rs12296850 in SLC17A8-NR1H4 gene region at12q23.1 was significantly associated with risk of lung SqCC at genome-wide significance level [additive model: odds ratio (OR) = 0.78, 95% confidence interval (CI) = 0.72–0.84, P = 1.19×10−10]. Subjects carrying AG or GG genotype had a 26% (OR = 0.74, 95% CI = 0.67–0.81) or 32% (OR = 0.68, 95% CI = 0.56–0.83) decreased risk of lung SqCC, respectively, as compared with AA genotype. However, we did not observe significant association between rs12296850 and risk of lung AC in a total of 4,368 cases with lung AC and 9,486 controls (OR = 0.96, 95% CI = 0.90–1.02, P = 0.173). These results indicate that genetic variations on chromosome 12q23.1 may specifically contribute to lung SqCC susceptibility in Chinese population.
Previous genome-wide association studies (GWAS) strongly suggested the importance of genetic susceptibility for lung cancer. However, the studies specific to different histological subtypes of lung cancer were limited. We performed the GWAS analysis specifically for lung squamous cell carcinoma (SqCC) with 570,009 autosomal SNPs in 833 SqCC cases and 3,094 controls and replicated in additional 2,223 lung SqCC cases and 6,409 controls from Chinese populations (822 SqCC cases and 2,243 controls for the first replication stage and 1,401 SqCC cases and 4,166 controls for the second replication stage). We found a novel association at rs12296850 (SLC17A8-NR1H4) on12q23.1. However, rs12296850 didn't show significant association with risk of lung adenocacinoma (AC) in 4,368 lung AC cases and 9,486 controls. These results indicate that genetic variations on chromosome 12q23.1 may specifically contribute to lung SqCC susceptibility in Chinese population.
The opportunistic pathogen Pseudomonas aeruginosa is a frequent colonizer of the airways of patients suffering from cystic fibrosis (CF). Depending on early treatment regimens, the colonization will, with high probability, develop into chronic infections sooner or later, and it is important to establish under which conditions the switch to chronic infection takes place. In association with a recently established sinus surgery treatment program for CF patients at the Copenhagen CF Center, colonization of the paranasal sinuses with P. aeruginosa has been investigated, paralleled by sampling of sputum from the same patients. On the basis of genotyping and phenotypic characterization including transcription profiling, the diversity of the P. aeruginosa populations in the sinuses and the lower airways was investigated and compared. The observations made from several children show that the paranasal sinuses constitute an important niche for the colonizing bacteria in many patients. The paranasal sinuses often harbor distinct bacterial subpopulations, and in the early colonization phases there seems to be a migration from the sinuses to the lower airways, suggesting that independent adaptation and evolution take place in the sinuses. Importantly, before the onset of chronic lung infection, lineages with mutations conferring a large fitness benefit in CF airways such as mucA and lasR as well as small colony variants and antibiotic-resistant clones are part of the sinus populations. Thus, the paranasal sinuses potentially constitute a protected niche of adapted clones of P. aeruginosa, which can intermittently seed the lungs and pave the way for subsequent chronic lung infections.
adaptive evolution; chronic infection; cystic fibrosis; protected environment