Graphitic carbon nitride (g-C3N4) was hybridized with CdS nanoparticles and reduced graphene oxide (RGO) sheets using a facile chemical method, for the application of catalytic photodegradation of Rhodamine B and Congo red dyes under irradiation with UV and visible light. Fourier-transform infrared (FTIR) spectroscopy and X-ray photoemission spectroscopy (XPS) analyses confirmed the formation of pure g-C3N4, as well as g-C3N4/CdS, g-C3N4/RGO, and g-C3N4/CdS/RGO composites. The large surface area of the g-C3N4/CdS/RGO composite (70.42 m(2) g(-1)) resulted in rapid dye adsorption onto the surface of the photocatalyst, leading to effective photodegradation of organic pollutants. The addition of CdS and RGO increased the photocatalytic activity of g-C3N4 by a factor of approximately twenty compared with that of the commercially available TiO2 catalyst under visible light, and the g-C3N4/CdS/RGO composite was found to significantly enhance the catalytic effect compared with pure g-C3N4 and with the g-C3N4/CdS and g-C3N4/RGO composites. The superior photocatalytic activity of the g-C3N4/CdS/RGO composite is attributed to enhanced separation of the photogenerated electron-hole pairs, as well as increased visible-light absorption. The improved transport of photoelectrons was consistent with the results of transient photocurrent measurements. Therefore, g-C3N4/CdS/RGO composites using a facile method are applicable to the development of high-efficiency photocatalytic devices for industrial applications.
Novel psychoactive substances (NPS) are becoming increasingly popular worldwide in recent years, some of which have been reported to cause considerable harm and even fatalities. Currently, simultaneous screening for a comprehensive panel of conventional and novel drugs of abuse is not widely available in most clinical laboratories. The aim of this study was to establish a chromatography/mass spectrometry-based analytical system for the simultaneous detection of conventional drugs of abuse and NPS in urine. Sample preparation entails enzyme digestion and solid phase extraction; analytes were then detected by liquid-chromatography tandem mass spectrometry (LC-MS/MS) with multiple reaction monitoring. Forty-seven conventional drugs (28 parent drugs, 19 metabolites) and 46 NPS analytes (44 parent drugs, two metabolites) are covered by the established method, which has been validated according to international guidelines. The method was then applied to 964 urine samples collected from drug abusers and the results revealed the presence of two NPS - TFMPP and methcathinone - as well as conventional drugs of abuse. To conclude, an LC-MS/MS method has been established that allows the simultaneous detection of over 90 conventional as well as novel psychoactive substances and metabolites in urine samples. The method was successfully applied to authentic specimens revealing the presence of conventional as well as novel drugs of abuse in the local population.
Chronic infection with hepatitis B virus (HBV) is a risk factor for developing hepatocellular carcinoma (HCC). The life cycle of HBV is complex and has been difficult to study because HBV does not infect cultured cells. The HBV regulatory X protein (HBx) controls the level of HBV replication and possesses an HCC cofactor role. Attempts to understand the mechanism(s) that underlie HBx effects on HBV replication and HBV-associated carcinogenesis have led to many reported HBx activities that are likely influenced by the assays used. This review summarizes experimental systems commonly used to study HBx functions, describes limitations of these experimental systems that should be considered, and suggests approaches for ensuring the biological relevance of HBx studies. (Hepatology 2014;).
The dust diseases silicosis and asbestosis were the first occupational diseases to have widespread impact on workers. Knowledge that asbestos and silica were hazardous to health became public several decades after the industry knew of the health concerns. This delay was largely influenced by the interests of Metropolitan Life Insurance Company (MetLife) and other asbestos mining and product manufacturing companies.
Hepatocellular Carcinoma (HCC) is one of the most common cancers in the world and it is often associated with poor prognosis. Liver transplantation and resection are two currently available curative therapies. However, most patients cannot be treated with such therapies due to late diagnosis. This underscores the urgent need to identify potential markers that ensure early diagnosis of HCC. As more evidences are suggesting that epigenetic changes contribute hepatocarcinogenesis, DNA methylation was poised as one promising biomarker. Indeed, genome wide profiling reveals that aberrant methylation is frequent event in HCC. Many studies showed that differentially methylated genes and CpG island methylator phenotype (CIMP) status in HCC were associated with clinicopathological data. Some commonly studied hypermethylated genes include p16, SOCS1, GSTP1 and CDH1. In addition, studies have also revealed that methylation markers could be detected in patient blood samples and associated with poor prognosis of the disease. Undeniably, increasing number of methylation markers are being discovered through high throughput genome wide data in recent years. Proper and systematic validation of these candidate markers in prospective cohort is required so that their actual prognostication and surveillance value could be accurately determined. It is hope that in near future, methylation marker could be translate into clinical use, where patients at risk could be diagnosed early and that the progression of disease could be more correctly assessed.
The underlying cause of predisposition to obesity is complex but one marker is cortisol responsiveness. Selection of sheep for high (HR) or low (LR) cortisol responses to adrenocorticotropin shows that HR are more likely to become obese. Increased propensity to obesity is associated with reduced skeletal muscle thermogenesis. We sought to determine whether metabolic or behavioral responses to stress also contribute to altered propensity to obesity in LR and HR. Animals (n=5-10/group) were exposed to 3 stressors and we measured food intake and thermogenesis (recorded with dataloggers implanted into muscle). Stressors were hypoglycaemia (0.125 units/kg insulin, IV), a barking dog and immune challenge (200 ng/kg lipopolysaccharide--LPS, IV). LR animals showed a greater catabolic state in response to both immune and psychosocial stressors. LPS reduced (P<0.01) food intake in both groups but LR showed a greater (P<0.05) reduction in food intake and a more substantial (P<0.05) rise in muscle temperature. Introduction of the barking dog reduced (P<0.05) food intake in LR only. These metabolic differences coincided with differences in cortisol responsiveness, where HR animals had increased (P<0.05) cortisol in response to both immune and psychosocial stressors. We also assessed behavior in the following paradigms: 1, isolation in the open field test; 2, response to a human intruder; and 3, food competition. LR had greater (P<0.05) activity, reduced fearfulness and displayed a proactive coping style of behavior. Thus we demonstrate that high cortisol responsiveness identifies animals with stress-induced metabolic and behavioral traits that may contribute to susceptibility to obesity.
5-Fluorouracil (5-FU) and its pro-drug Capecitabine have been widely used in treating colorectal cancer. However, not all patients will respond to the drug, hence there is a need to develop reliable early predictive biomarkers for 5-FU response. Here, we report a novel potentially functional Single Nucleotide Polymorphism (pfSNP) approach to identify SNPs that may serve as predictive biomarkers of response to 5-FU in Chinese metastatic colorectal cancer (CRC) patients. 1547 pfSNPs and one variable number tandem repeat (VNTR) in 139 genes in 5-FU drug (both PK and PD pathway) and colorectal cancer disease pathways were examined in 2 groups of CRC patients. Shrinkage of liver metastasis measured by RECIST criteria was used as the clinical end point. Four non-responder-specific pfSNPs were found to account for 37.5% of all non-responders (P<0.0003). Five additional pfSNPs were identified from a multivariate model (AUC under ROC?=?0.875) that was applied for all other pfSNPs, excluding the non-responder-specific pfSNPs. These pfSNPs, which can differentiate the other non-responders from responders, mainly reside in tumor suppressor genes or genes implicated in colorectal cancer risk. Hence, a total of 9 novel SNPs with potential functional significance may be able to distinguish non-responders from responders to 5-FU. These pfSNPs may be useful biomarkers for predicting response to 5-FU.
HIV-1 hijacks and disrupts many processes in the cells it infects in order to suppress antiviral immunity and to facilitate its replication. Resting CD4 T cells are important early targets of HIV-1 infection in which HIV-1 must overcome intrinsic barriers to viral replication. Although resting CD4 T cells are refractory to infection in vitro, local environmental factors within lymphoid and mucosal tissues such as cytokines facilitate viral replication while maintaining the resting state. These factors can be utilized in vitro to study HIV-1 replication in resting CD4 T cells. In vivo, the migration of resting naïve and central memory T cells into lymphoid tissues is dependent upon expression of CD62L (L-selectin), a receptor that is subsequently down-modulated following T cell activation. CD62L gene transcription is maintained in resting T cells by Foxo1 and KLF2, transcription factors that maintain T cell quiescence and which regulate additional cellular processes including survival, migration, and differentiation. Here we report that HIV-1 down-modulates CD62L in productively infected naïve and memory resting CD4 T cells while suppressing Foxo1 activity and the expression of KLF2 mRNA. Partial T cell activation was further evident as an increase in CD69 expression. Several other Foxo1- and KLF2-regulated mRNA were increased or decreased in productively infected CD4 T cells, including IL-7r?, Myc, CCR5, Fam65b, S1P1 (EDG1), CD52, Cyclin D2 and p21CIP1, indicating a profound reprogramming of these cells. The Foxo1 inhibitor AS1842856 accelerated de novo viral gene expression and the sequella of infection, supporting the notion that HIV-1 suppression of Foxo1 activity may be a strategy to promote replication in resting CD4 T cells. As Foxo1 is an investigative cancer therapy target, the development of Foxo1 interventions may assist the quest to specifically suppress or activate HIV-1 replication in vivo.
Hepatocellular Carcinoma (HCC) is one of the leading causes of cancer-associated mortality worldwide. However, the role of epigenetic changes such as aberrant DNA methylation in hepatocarcinogenesis remains largely unclear. In this study, we examined the methylation profiles of 59 HCC patients. Using consensus hierarchical clustering with feature selection, we identified three tumor subgroups based on their methylation profiles and correlated these subgroups with clinicopathological parameters. Interestingly, one tumor subgroup is different from the other 2 subgroups and the methylation profile of this subgroup is the most distinctly different from the non-tumorous liver tissues. Significantly, this subgroup of patients was found to be associated with poor overall as well as disease-free survival. To further understand the pathways modulated by the deregulation of methylation in HCC patients, we integrated data from both the methylation as well as the gene expression profiles of these 59 HCC patients. In these patients, while 4416 CpG sites were differentially methylated between the tumors compared to the adjacent non-tumorous tissues, only 536 of these CpG sites were associated with differences in the expression of their associated genes. Pathway analysis revealed that forty-four percent of the most significant upstream regulators of these 536 genes were involved in inflammation-related NF?B pathway. These data suggest that inflammation via the NF?B pathway play an important role in modulating gene expression of HCC patients through methylation. Overall, our analysis provides an understanding on aberrant methylation profile in HCC patients.
FAT10 is an ubiquitin-like modifier, which has been implicated in immune response and cancer development. In particular, the hypothesis of FAT10 as a mediator of tumorigenesis stems from its ability to associate with a spindle checkpoint protein, Mad2 during mitosis and cause aneuploidy, a hallmark of cancer cells. Furthermore, FAT10 is overexpressed in several carcinomas types, including that of liver and colon. Nevertheless, direct evidence linking FAT10 to cell malignant transformation and progression is lacking. Here, we demonstrate that high FAT10 expression enhanced the proliferative, invasive, migratory and adhesive functions of the transformed cell line, HCT116. These observations were consistently demonstrated in an immortalized, non-tumorigenic liver cell line NeHepLxHT. Importantly FAT10 can induce malignant transformation as evidenced from the anchorage-independent growth as well as in vivo tumor-forming abilities of FAT10-overexpressing NeHepLxHT cells, while in rapidly proliferating HCT116, increased FAT10 further augmented tumor growth. FAT10 was found to activate NF?B which in turn upregulated the chemokine receptors CXCR4 and CXCR7. Importantly, siRNA depletion of CXCR7 and CXCR4 attenuated cell invasion of FAT10-overexpressing cells, indicating that the CXCR4/7 is crucial for the FAT10-dependent malignant phenotypes. Taken together, our data reveals novel functions of FAT10 in malignant transformation and progression, via the NFB-CXCR4/7 pathway.
M-mode and 2-dimensional (2D) echocardiographic imaging are routinely used to quantify left-ventricular (LV) size and function in pediatric patients with dilated cardiomyopathy (DCM). The reproducibility of and correlation between these techniques are unknown. This analysis sought to compare interreader, intrareader, and interacquisition reproducibility of M-mode versus 2D measurements in pediatric DCM patients. The Ventricular Volume Variability study of the Pediatric Heart Network is a multicenter, prospective, observational study assessing the course of chronic DCM in children. Two sonographers performed baseline image acquisitions locally, and two readers performed measurements at the echocardiographic core laboratory. One reader repeated measurements 1 month later. These data were used to assess reproducibility and agreement between M-mode and 2D measurements. One hundred sixty-nine subjects were enrolled. M-mode had similar or greater reproducibility in both intrareader and interreader settings for LV dimensions, shortening fraction (SF), and most wall thicknesses. In contrast, 2D reproducibility was similar or better for nearly all variables in the interacquisition setting but not for SF. Interacquisition variability was approximately twice the intrareader variability. LV dimensions by either modality consistently had high reproducibility and had the highest agreement between modalities. In pediatric DCM patients, variability of linear echocardiographic assessment could be minimized by relying on a single reader and using a consistent method (M-mode or 2D) for serial measurements, preferably M-mode when SF is the primary variable of interest. Except for LV dimensions, M-mode and 2D values should not be used interchangeably due to poor agreement.
Mammalian primordial germ cells (PGCs) are unipotent progenitors of the gametes. Nonetheless, they can give rise directly to pluripotent stem cells in vitro or during teratocarcinogenesis. This conversion is inconsistent, however, and has been difficult to study. Here, we delineate requirements for efficient resetting of pluripotency in culture. We demonstrate that in defined conditions, routinely 20% of PGCs become EG cells. Conversion can occur from the earliest specified PGCs. The entire process can be tracked from single cells. It is driven by leukemia inhibitory factor (LIF) and the downstream transcription factor STAT3. In contrast, LIF signaling is not required during germ cell ontogeny. We surmise that ectopic LIF/STAT3 stimulation reconstructs latent pluripotency and self-renewal. Notably, STAT3 targets are significantly upregulated in germ cell tumors, suggesting that dysregulation of this pathway may underlie teratocarcinogenesis. These findings demonstrate that EG cell formation is a robust experimental system for exploring mechanisms involved in reprogramming and cancer.
Axitinib is an inhibitor of tyrosine kinase vascular endothelin growth factor receptors 1, 2, and 3. The ATP-binding cassette (ABC) and solute carrier (SLC) transport properties of axitinib were determined in selected cellular systems. Axitinib exhibited high passive permeability in all cell lines evaluated (Papp ? 6 × 10(-6) cm/s). Active efflux was observed in Caco-2 cells, and further evaluation in multidrug resistance gene 1 (MDR1) or breast cancer resistance protein (BCRP) transfected Madin-Darby canine kidney cells type 2 (MDCK) cells indicated that axitinib is at most only a weak substrate for P-glycoprotein (P-gp) but not BCRP. Axitinib showed incomplete inhibition of P-gp-mediated transport of digoxin in Caco-2 cells and BCRP transport of topotecan in BCRP-transfected MDCK cells with IC50 values of 3 ?M and 4.4 ?M, respectively. Axitinib (10 mg) did not pose a risk for systemic drug interactions with P-gp or BCRP per regulatory guidance. A potential risk for drug interactions through inhibition of P-gp and BCRP in the gastrointestinal tract was identified because an axitinib dose of 10 mg divided by 250 mL was greater than 10-fold the IC50 for each transporter. However, a GastroPlus simulation that considered the low solubility of axitinib resulted in lower intestinal concentrations and suggested a low potential for gastrointestinal interactions with P-gp and BCRP substrates. Organic anion transporting polypeptide 1B1 (OATP1B1) and OATP1B3 transfected human embryonic kidney 293 (HEK293) cells transported axitinib to a minor extent but uptake into suspended hepatocytes was not inhibited by rifamycin SV suggesting that high passive permeability predominates. Mouse whole-body autoradiography revealed that [(14)C]axitinib-equivalents showed rapid absorption and distribution to all tissues except the brain. This suggests that efflux transport of axitinib may occur at the mouse blood-brain barrier.
Sequence type 398 (ST398) Staphylococcus aureus, frequently carried by livestock, has caused severe human infections and often carries transmissible antibiotic resistance genes. Among methicillin-susceptible S. aureus isolates colonizing Dallas County Jail detainees, 13.2% were ST398, spa type t571, and were genetically similar to human colonization isolates from New York, Chicago, and the Dominican Republic.
In the 2012 Food and Drug Administration (FDA) draft guidance on drug-drug interactions (DDIs), a new molecular entity that inhibits P-glycoprotein (P-gp) may need a clinical DDI study with a P-gp substrate such as digoxin when the maximum concentration of inhibitor at steady state divided by IC?? ([I?]/IC??) is ?0.1 or concentration of inhibitor based on highest approved dose dissolved in 250 ml divide by IC?? ([I?]/IC??) is ?10. In this article, refined criteria are presented, determined by receiver operating characteristic analysis, using IC?? values generated by 23 laboratories. P-gp probe substrates were digoxin for polarized cell-lines and N-methyl quinidine or vinblastine for P-gp overexpressed vesicles. Inhibition of probe substrate transport was evaluated using 15 known P-gp inhibitors. Importantly, the criteria derived in this article take into account variability in IC?? values. Moreover, they are statistically derived based on the highest degree of accuracy in predicting true positive and true negative digoxin DDI results. The refined criteria of [I?]/IC?? ? 0.03 and [I?]/IC?? ? 45 and FDA criteria were applied to a test set of 101 in vitro-in vivo digoxin DDI pairs collated from the literature. The number of false negatives (none predicted but DDI observed) were similar, 10 and 12%, whereas the number of false positives (DDI predicted but not observed) substantially decreased from 51 to 40%, relative to the FDA criteria. On the basis of estimated overall variability in IC?? values, a theoretical 95% confidence interval calculation was developed for single laboratory IC?? values, translating into a range of [I?]/IC?? and [I?]/IC?? values. The extent by which this range falls above the criteria is a measure of risk associated with the decision, attributable to variability in IC?? values.
A P-glycoprotein (P-gp) IC?? working group was established with 23 participating pharmaceutical and contract research laboratories and one academic institution to assess interlaboratory variability in P-gp IC?? determinations. Each laboratory followed its in-house protocol to determine in vitro IC?? values for 16 inhibitors using four different test systems: human colon adenocarcinoma cells (Caco-2; eleven laboratories), Madin-Darby canine kidney cells transfected with MDR1 cDNA (MDCKII-MDR1; six laboratories), and Lilly Laboratories Cells--Porcine Kidney Nr. 1 cells transfected with MDR1 cDNA (LLC-PK1-MDR1; four laboratories), and membrane vesicles containing human P-glycoprotein (P-gp; five laboratories). For cell models, various equations to calculate remaining transport activity (e.g., efflux ratio, unidirectional flux, net-secretory-flux) were also evaluated. The difference in IC?? values for each of the inhibitors across all test systems and equations ranged from a minimum of 20- and 24-fold between lowest and highest IC?? values for sertraline and isradipine, to a maximum of 407- and 796-fold for telmisartan and verapamil, respectively. For telmisartan and verapamil, variability was greatly influenced by data from one laboratory in each case. Excluding these two data sets brings the range in IC?? values for telmisartan and verapamil down to 69- and 159-fold. The efflux ratio-based equation generally resulted in severalfold lower IC?? values compared with unidirectional or net-secretory-flux equations. Statistical analysis indicated that variability in IC?? values was mainly due to interlaboratory variability, rather than an implicit systematic difference between test systems. Potential reasons for variability are discussed and the simplest, most robust experimental design for P-gp IC?? determination proposed. The impact of these findings on drug-drug interaction risk assessment is discussed in the companion article (Ellens et al., 2013) and recommendations are provided.
Food restriction is considered to be a welfare issue in extensively reared animals. However, the effects of food restriction on the affective state, and its physiological regulation, are unknown. In Experiment 1, we aimed to assess the effects of increased plasma concentrations of acyl-ghrelin on judgement bias (an indicator of affective states) by fasting sheep for 24h or by ghrelin administration. In Experiment 2, we aimed to assess the effects of chronic food restriction on judgement bias and attention bias towards a food-related cue. For the judgement bias test, sheep were trained in an arena to approach a positive location cue associated with conspecifics and not approach a negative location cue associated with a dog. Three non-trained, non-reinforced ambiguous location cues were situated between the positive and negative locations. Attention bias towards a food-related cue was assessed by placing an empty food bucket against the wall of the arena halfway between the entry point and the positive location. In Experiment 1, sheep were divided into three treatments; 24h fast, ghrelin administration or control. Judgement bias, locomotor activity and plasma cortisol concentrations were assessed. The ghrelin treated group tended to express a more pessimistic bias compared to the control group (P<0.1), and plasma cortisol concentrations tended to be increased (P<0.1). In Experiment 2, sheep were subjected to a high feeding level (HF) or low feeding level (LF) for 7days. The LF group tended to show a more optimistic judgement bias (P<0.1). When the food-related cue was presented, LF ewes took longer to reach the positive location (P<0.001), spent longer with their head inside the bucket (P<0.001) and more time interacting with the bucket (P<0.01). This study provides preliminary evidence that food restriction alters judgement bias and attention bias towards a food-related cue which may indicate altered affective states of sheep.
ATP-binding cassette (ABC) proteins in the placenta regulate fetal exposure to xenobiotics. We hypothesized that functional polymorphisms in ABC genes influence risk for non-syndromic oral clefts (NSOC). Both family-based and case-control studies were undertaken to evaluate the association of nine potentially functional single-nucleotide polymorphisms within four ABC genes with risk of NSOC. Peripheral blood DNA from a total of 150 NSOC case-parent trios from Singapore and Taiwan were genotyped, as was cord blood DNA from 189 normal Chinese neonates used as controls. In trios, significant association was observed between the ABCB1 single-nucleotide polymorphisms and NSOC (P<0.05). Only ABCB1 rs1128503 retained significant association after Bonferroni correction (odds ratio (OR)=2.04; 95% confidence interval (CI)=1.42-2.98), while rs2032582 and rs1045642 showed nominal significance. Association with rs1128503 was replicated in a case-control analysis comparing NSOC probands with controls (OR=1.58; 95% CI=1.12-2.23). A comparison between the mothers of probands and controls showed no evidence of association, suggesting NSOC risk is determined by fetal and not maternal ABCB1 genotype. The two studies produced a combined OR of 1.79 (95% CI=1.38-2.30). The T-allele at rs1128503 was associated with higher risk. This study thus provides evidence that potentially functional polymorphisms in fetal ABCB1 modulate risk for NSOC, presumably through suboptimal exclusion of xenobiotics at the fetal-maternal interface.
While much is known about the geographic distribution of different clonal types of methicillin-resistant Staphylococcus aureus (MRSA), few studies have assessed the molecular epidemiology of methicillin-susceptible S. aureus (MSSA), despite its continued clinical importance. In each U.S. Census region, reference laboratories collected successive MSSA isolates from patients with invasive or superficial staphylococcal infections for use in the Tigecycline Evaluation and Surveillance Trial. All isolates from the periods of 2004 to 2005 and 2009 to 2010 underwent antimicrobial susceptibility testing and characterization of their staphylococcal protein A (spa) type. Of the 708 isolates analyzed, 274 spa types were identified and divided into 15 genetic clusters. The most common clones were spa t002 (n = 63, 8.9%) and t008 (n = 56, 7.9%). While the distribution of the predominant spa types did not differ by U.S. Census region or time period, spa t008 was nearly twice as common in community skin and soft tissue infections than in nosocomial bloodstream infections (11.1% versus 5.6%, respectively; P = 0.008). Despite such differences, both community and nosocomial settings had diverse staphylococcal clonal types representing all major spa clusters. In contrast to those of MRSA, MSSA infectious isolates show wide genetic diversity without clear geographical or temporal clustering. Notably, the prevalent MSSA strains (spa t002 and spa t008) are analogous to the predominant MRSA clones, further demonstrating the importance of both lineages.
MicroRNA-224 (miR-224) is frequently over-expressed in liver and colorectal cancers. We and others have previously described the role of miR-224 over-expression in cell proliferation in vitro but we have yet to identify the relevant miR-224 direct target. In this study, we further demonstrated that miR-224 up-regulation promotes cell proliferation using both in vitro assays and in vivo tumor growth models. We systematically screened for high confidence miR-224 targets by overlapping in silico predicted targets from multiple algorithms and significantly down-regulated genes in miR-224-expressing cells from whole genome expression microarrays. A total of 72 high confidence miR-224 targets were identified and found to be enriched in various cancer-related processes. SMAD family member 4 (SMAD4) is experimentally validated as the direct cellular target through which miR-224 promotes cell proliferation. The clinical relevance of our experimental observations was supported by a statistically significant inverse correlation between miR-224 and SMAD4 transcript expression in tumor versus paired adjacent non-tumorous tissues from HCC patients (p<0.001, r=?-0.45, R(2)?=0.122). Furthermore, miR-224 up-regulation and SMAD4 down-regulation is significantly associated with poorer patient survival (p<0.05). In summary, miR-224/SMAD4 pathway is a clinically relevant pathway to provide new insights in understanding HCC. (191 words).
We describe a case of an infant with a single ventricle physiology, who presented with spontaneous microbubbles originating from her inferior vena cava. Imaging revealed a persistent patent ductus venosus, leading to a portosystemic shunt, streaming the microbubbles into the heart. We discuss the possible mechanisms for this rare phenomenon in a child.
Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide, characterized by high mortality rate and poor prognosis. Our understanding of the HCC pathology is still very much fragmented and little progress has been made to improve the clinical outcome of HCC patients. While recently discovered microRNA deregulation in HCC has added to the complexity of our understanding of HCC, it has also presented promising novel approaches to understand, diagnose and treat HCC. Here, we highlight one miRNA, miR-224, which has been more consistently reported to be upregulated in HCC than other miRNAs. We will discuss the validated and predicted functional roles of this miRNA in HCC, speculate on the possible mechanism for its upregulation in HCC and explore the potential of miR-224 as an exciting novel biomarker for the early detection of liver malignancies as well as a novel therapeutic target for HCC treatment.
Tumor necrosis factor-alpha (TNF-?) plays important roles in chronic inflammation-associated tumorigenesis but the mechanisms involved remain poorly understood. Previously, we reported that high levels of FAT10 led to chromosomal instability that is mediated by an abbreviated mitotic phase. Here, we show that TNF-? induces FAT10 gene expression through TNF receptor 1 (TNFR1) and activates the NF-?B pathway in HCT116 and SW620 cells. TNF-? treatment also leads to an abbreviated mitotic phase that can be reversed by inhibiting FAT10 expression. This abbreviated mitotic phase is correlated with a TNF-?-induced reduction in the kinetochore localization of MAD2 during prometaphase which, again, can be reversed by inhibiting FAT10 gene expression. There is greater variability of chromosome numbers in HCT116 and SW620 cells treated with TNF-? than in untreated cells, which can be reversed by the introduction of short hairpin RNA (shRNA) against FAT10. The more stable chromosome numbers in HCT116 cells expressing FAT10 shRNA can revert to greater variability with the addition of a mutant FAT10 that is not recognized by the FAT10 shRNA. Upon TNF-? stimulation, higher cell death is observed when FAT10 expression is inhibited by shRNA. These data strongly suggest that FAT10 plays an important role in mediating the function of TNF-? during tumorigenesis by inducing cell cycle deregulation and chromosomal instability, and by inhibiting apoptosis.
Digoxin, an orally administered cardiac glycoside cardiovascular drug, has a narrow therapeutic window. Circulating digoxin levels (maximal concentration of ?1.5 ng/ml) require careful monitoring, and the potential for drug-drug interactions (DDI) is a concern. Increases in digoxin plasma exposure caused by inhibition of P-glycoprotein (P-gp) have been reported. Digoxin has also been described as a substrate of various organic anion-transporting polypeptide (OATP) transporters, posing a risk that inhibition of OATPs may result in a clinically relevant DDI similar to what has been observed for P-gp. Although studies in rats have shown that Oatps contribute to the disposition of digoxin, the role of OATPs in the disposition of digoxin in humans has not been clearly defined. Using two methods, Boehringer Ingelheim, GlaxoSmithKline, Pfizer, and Solvo observed that digoxin is not a substrate of OATP1A2, OATP1B1, OATP1B3, and OATP2B1. However, digoxin inhibited the uptake of probe substrates of OATP1B1 (IC(50) of 47 ?M), OATP1B3 (IC(50) > 8.1 ?M), and OATP2B1 (IC(50) > 300 ?M), but not OATP1A2 in transfected cell lines. It is interesting to note that digoxin is a substrate of a sodium-dependent transporter endogenously expressed in HEK293 cells because uptake of digoxin was significantly greater in cells incubated with sodium-fortified media compared with incubations conducted in media in which sodium was absent. Thus, although digoxin is not a substrate for the human OATP transporters evaluated in this study, in addition to P-gp-mediated efflux, its uptake and pharmacokinetic disposition may be partially facilitated by a sodium-dependent transporter.
Polar bears (Ursus maritimus) are being impacted by climate change and increased exposure to pollutants throughout their northern circumpolar range. In this study, we quantified concentrations of total mercury (THg) in the hair of polar bears from Canadian high- (southern Beaufort Sea, SBS) and sub- (western Hudson Bay, WHB) Arctic populations. Concentrations of THg in polar bears from the SBS population (14.8 ± 6.6 ?g g(-1)) were significantly higher than in polar bears from WHB (4.1 ± 1.0 ?g g(-1)). On the basis of ?(15)N signatures in hair, in conjunction with published ?(15)N signatures in particulate organic matter and sediments, we estimated that the pelagic and benthic food webs in the SBS are ? 4.7 and ? 4.0 trophic levels long, whereas in WHB they are only ? 3.6 and ? 3.3 trophic levels long. Furthermore, the more depleted ?(13)C ratios in hair from SBS polar bears relative to those from WHB suggests that SBS polar bears feed on food webs that are relatively more pelagic (and longer), whereas polar bears from WHB feed on those that are relatively more benthic (and shorter). Food web length and structure accounted for ? 67% of the variation we found in THg concentrations among all polar bears across both populations. The regional difference in polar bear hair THg concentrations was also likely due to regional differences in water-column concentrations of methyl Hg (the toxic form of Hg that biomagnifies through food webs) available for bioaccumulation at the base of the food webs. For example, concentrations of methylated Hg at mid-depths in the marine water column of the northern Canadian Arctic Archipelago were 79.8 ± 37.3 pg L(-1), whereas, in HB, they averaged only 38.3 ± 16.6 pg L(-1). We conclude that a longer food web and higher pelagic concentrations of methylated Hg available to initiate bioaccumulation in the BS resulted in higher concentrations of THg in polar bears from the SBS region compared to those inhabiting the western coast of HB.
Chronic hepatitis B virus (HBV) infection has been identified as a major risk factor in hepatocellular carcinoma (HCC), which is one of the most common cancers worldwide. The pathogenesis of HBV-mediated hepatocarcinogenesis is, however, incompletely understood. Evidence suggests that the HBV X protein (HBx) plays a crucial role in HCC development. HBx is a multifunctional regulator that modulates transcription, signal transduction, cell cycle progression, apoptosis, protein degradation pathways, and genetic stability through interaction with host factors. This review describes the current state of knowledge of the molecular pathogenesis of HBV-induced HCC, with a focus on the role of HBx in hepatocarcinogenesis.
In this study, microstructures of Cu powders coated with octanethiol were analyzed using (scanning) transmission electron microscopy. Moreover, aging process of the octanethiol-coated layer as time passes by was analyzed using the electron energy loss spectroscopy technique. The octanethiol layer coated on the surface of Cu powders was kept until it was exposed to air for around 30 days. As days passes by, the coating layer had been decomposed and then a Cu(2)O layer was formed on the surface of powders.
Permeability is an important property of drug candidates. The Madin-Darby canine kidney cell line (MDCK) permeability assay is widely used and the primary concern of using MDCK cells is the presence of endogenous transporters of nonhuman origin. The canine P-glycoprotein (Pgp) can interfere with permeability and transporter studies, leading to less reliable data. A new cell line, MDCKII-LE (low efflux), has been developed by selecting a subpopulation of low-efflux cells from MDCKII-WT using an iterative fluorescence-activated cell sorting technique with calcein-AM as a Pgp and efflux substrate. MDCKII-LE cells are a subpopulation of MDCKII cells with over 200-fold lower canine Pgp mRNA level and fivefold lower protein level than MDCKII-WT. MDCKII-LE cells showed less functional efflux activity than MDCKII-WT based on efflux ratios. Notably, MDCKII-MDR1 showed about 1.5-fold decreased expression of endogenous canine Pgp, suggesting that using the net flux ratio might not completely cancel out the background endogenous transporter activities. MDCKII-LE cells offer clear advantages over the MDCKII-WT by providing less efflux transporter background signals and minimizing interference from canine Pgp. The MDCKII-LE apparent permeability values well differentiates compounds from high to medium/low human intestinal absorption and can be used for Biopharmaceutical Classification System. The MDCKII-LE permeability assay (4-in-1 cassette dosing) is high throughput with good precision, reproducibility, robustness, and cost-effective.
Throat carriage (42.7%) of Staphylococcus aureus exceeded nasal carriage (35.0%) in 2 New York prisons. Methicillin resistance, primarily due to USA300, was high at both sites; 25% of dually colonized inmates had different strains. Strategies to reduce S. aureus transmission will need to consider the high frequency of throat colonization.
Stochastic and deterministic allele specific gene expression (ASE) might influence single cell phenotype, but the extent and nature of the phenomenon at the onset of early mouse development is unknown. Here we performed single cell RNA-Seq analysis of single blastomeres of mouse embryos, which revealed significant changes in the transcriptome. Importantly, over half of the transcripts with detectable genetic polymorphisms exhibit ASE, most notably, individual blastomeres from the same two-cell embryo show similar pattern of ASE. However, about 6% of them exhibit stochastic expression, indicated by altered expression ratio between the two alleles. Thus, we demonstrate that ASE is both deterministic and stochastic in early blastomeres. Furthermore, we also found that 1,718 genes express two isoforms with different lengths of 3UTRs, with the shorter one on average 5-6 times more abundant in early blastomeres compared to the transcripts in epiblast cells, suggesting that microRNA mediated regulation of gene expression acquires increasing importance as development progresses.
FAT10, also known as diubiquitin, has been implicated in the regulation of diverse cellular processes, including mitosis, immune response, and apoptosis. We seek to identify FAT10-targeted proteins, an essential step in elucidating the physiological function of FAT10. To this end, human FAT10 or its non-conjugatable derivative, FAT10?GG, was overexpressed in HEK293 cells. We observed a number of high molecular weight FAT10 conjugates in cells expressing wild-type FAT10, but not in FAT10?GG. The FAT10 conjugates are inducible by TNF-? and accumulated significantly when cells were treated with proteasome inhibitor, MG132. Among them, tumor suppressor p53 was found to be FATylated. The p53 transcriptional activity was found to be substantially enhanced in FAT10-overexpressing cells. In addition, overexpressing FAT10 in HEK293 cells also reduced the population of p53 which cross reacted with monoclonal anti-p53 antibody, PAB240, known to recognize only the transcriptionally inactive p53. FAT10 in the nucleus was found co-localized with p53 and altered its subcellular compartmentalization. Furthermore, overexpressing FAT10 led to a reduction in the size of promyelocytic leukemia nuclear bodies (PML-NBs) and altered their distribution in the nucleus. Based on these observations, a potential mechanism which correlates FATylation of p53 to its translocation and transcriptional activation is discussed.
Animal welfare research is now starting to measure the cognitive component of affective states in an effort to improve welfare assessments of animals. Twenty-six Romane ewe lambs were trained to a spatial location task previously demonstrated to test for judgement bias in sheep. This required a go/no-go response according to the location of a bucket in a pen, with one location being positively reinforced (with a feed reward) and the other negatively reinforced (with a fan-forced blower). While training in the judgement bias arena continued, half of the sheep (n=13) were subjected to a chronic, intermittent treatment that consisted of stressful events common to production systems. After 3 weeks of treatment, all sheep were tested for biases in judgement by placing the bucket in ambiguous locations between the two learnt reference locations. The emotional reactivity, as characterised by behavioural and physiological responses, of all sheep to events that were unexpected, novel or sudden was then tested. A significant treatment × bucket location interaction was seen on day 3 with Stressed sheep approaching the bucket locations less than Control sheep (p=0.007). This may reflect a bias in judgement, however it is also possibly a treatment-induced difference in learning. Cardiac data did not indicate treatment differences, however the RMSSD of Control sheep in novel and unfamiliar situations was always higher than the Stressed animals. No meaningful treatment differences in emotional reactivity behaviours were evident. This paper provides further evidence that affective states exist and can be measured in animals.
We describe incidence and risk factors for pandemic (H1N1) 2009 virus infection in healthcare personnel during the June-September 2009 epidemic in Singapore. Personnel contributed 3 serologic samples during June-October 2009, with seroconversion defined as a ?4-fold increase in hemagglutination inhibition titers to pandemic (H1N1) 2009. Of 531 participants, 35 showed evidence of seroconversion. Seroconversion rates were highest in nurses (28/290) and lowest in allied health staff (2/116). Significant risk factors on multivariate analysis were being a nurse (adjusted odds ratio [aOR] 4.5, 95% confidence interval [CI] 1.0-19.6) and working in pandemic (H1N1) 2009 isolation wards (aOR 4.5, 95% CI 1.3-15.6). Contact with pandemic (H1N1) 2009-infected colleagues (aOR 2.5, 95% CI 0.9-6.6) and larger household size (aOR 1.2, 95% CI 1.0-1.4) were of borderline significance. Our study suggests that seroconversion was associated with occupational and nonoccupational risk factors.
We have previously shown that a base-paired complex formed by two of the spliceosomal RNA components, U6 and U2 small nuclear RNAs (snRNAs), can catalyze a two-step splicing reaction that depended on an evolutionarily invariant region in U6, the ACAGAGA box. Here we further analyze this RNA-catalyzed reaction and show that while the 5 and 3 splice site substrates are juxtaposed and positioned near the ACAGAGA sequence in U6, the role of the snRNAs in the reaction is beyond mere juxtaposition of the substrates and likely involves the formation of a sophisticated active site. Interestingly, the snRNA-catalyzed reaction is metal dependent, as is the case with other known splicing RNA enzymes, and terbium(III) cleavage reactions indicate metal binding by the U6/U2 complex within the evolutionarily conserved regions of U6. The above results, combined with the structural similarities between U6 and catalytically critical domains in group II self-splicing introns, suggest that the base-paired complex of U6 and U2 snRNAs is a vestigial ribozyme and a likely descendant of a group II-like self-splicing intron.
Genome-wide active DNA demethylation in primordial germ cells (PGCs), which reprograms the epigenome for totipotency, is linked to changes in nuclear architecture, loss of histone modifications, and widespread histone replacement. Here, we show that DNA demethylation in the mouse PGCs is mechanistically linked to the appearance of single-stranded DNA (ssDNA) breaks and the activation of the base excision repair (BER) pathway, as is the case in the zygote where the paternal pronucleus undergoes active DNA demethylation shortly after fertilization. Whereas BER might be triggered by deamination of a methylcytosine (5mC), cumulative evidence indicates other mechanisms in germ cells. We demonstrate that DNA repair through BER represents a core component of genome-wide DNA demethylation in vivo and provides a mechanistic link to the extensive chromatin remodeling in developing PGCs.
Since the fi rst imported case on 26 May 2009, pandemic (H1N1) 2009 has spread from travellers and has resulted in sustained community transmission. Singapore began with a strict containment policy where all suspected and confirmed cases of pandemic (H1N1) 2009 were admitted for testing. We describe here the clinical and laboratory characteristics of the fi rst 50 adult cases with confirmed pandemic (H1N1) 2009.
P-glycoprotein (P-gp) is the most characterized drug transporter in terms of its clinical relevance for pharmacokinetic disposition and interaction with other medicines. Clinically significant P-gp related drug interactions appear restricted to digoxin. P-gp may act as a major barrier to current and effective drug treatment in a number of diseases including cancer, AIDS, Alzheimers and epilepsy due to its expression in tumors, lymphocytes, cell membranes of brain capillaries and the choroid plexus.
The pleiotropic hepatitis B virus (HBV) x protein (HBx), associated with hepatocellular carcinoma (HCC), has been implicated in the deregulation of cellular gene expression at the transcriptional level. To date, it remains unknown if HBx regulates the expression of miRNAs which play important roles in gene-regulation at the post-transcriptional and/or translational level.
Judgement bias has potential as a measure of affective state in animals. The serotonergic system may be one mechanism involved with the formation of negative judgement biases. It was hypothesised that depletion of brain serotonin would induce negative judgement biases in sheep. A dose response trial established that 40 mg/kg of p-Chlorophenylalanine (pCPA) administered to sheep for 3 days did not affect feeding motivation or locomotion required for testing judgement biases. Thirty Merino ewes (10 months old) were trained to an operant task for 3 weeks. Sheep learnt to approach a bucket when it was placed in one corner of the testing facility to receive a feed reward (go response), and not approach it when in the alternate corner (no-go response) to avoid a negative reinforcer (exposure to a dog). Following training, 15 sheep were treated with pCPA (40 mg/kg daily) for an extended duration (5 days). Treated and control sheep were tested for judgement bias following 3 and 5 days of treatment, and again 5 days after cessation of treatment. Testing involved the bucket being presented in ambiguous locations between the two learnt locations, and the response of the sheep (go/no-go) measured their judgement of the bucket locations. Following 5 days of treatment, pCPA-treated sheep approached the most positive ambiguous location significantly less than control sheep, suggesting a pessimistic-like bias (treatment × bucket location interaction F(1,124.6)=49.97, p=0.011). A trend towards a significant interaction was still evident 5 days after the cessation of pCPA treatment (p=0.068), however no significant interaction was seen on day 3 of testing (p=0.867). These results support the suggestion that judgement bias is a cognitive measure of affective state, and that the serotonergic pathway may be involved.
Membrane transporters can be major determinants of the pharmacokinetic, safety and efficacy profiles of drugs. This presents several key questions for drug development, including which transporters are clinically important in drug absorption and disposition, and which in vitro methods are suitable for studying drug interactions with these transporters. In addition, what criteria should trigger follow-up clinical studies, and which clinical studies should be conducted if needed. In this article, we provide the recommendations of the International Transporter Consortium on these issues, and present decision trees that are intended to help guide clinical studies on the currently recognized most important drug transporter interactions. The recommendations are generally intended to support clinical development and filing of a new drug application. Overall, it is advised that the timing of transporter investigations should be driven by efficacy, safety and clinical trial enrolment questions (for example, exclusion and inclusion criteria), as well as a need for further understanding of the absorption, distribution, metabolism and excretion properties of the drug molecule, and information required for drug labelling.
During the transition from the inner cell mass (ICM) cells of blastocysts to pluripotent embryonic stem cells (ESCs) in vitro, a normal developmental program is replaced in cells that acquire a capacity for infinite self-renewal and pluripotency. We explored the underlying mechanism of this switch by using RNA-Seq transcriptome analysis at the resolution of single cells. We detected significant molecular transitions and major changes in transcript variants, which include genes for general metabolism. Furthermore, the expression of repressive epigenetic regulators increased with a concomitant decrease in gene activators that might be necessary to sustain the inherent plasticity of ESCs. Furthermore, we detected changes in microRNAs (miRNAs), with one set that targets early differentiation genes while another set targets pluripotency genes to maintain the unique ESC epigenotype. Such genetic and epigenetic events may contribute to a switch from a normal developmental program in adult cells during the formation of diseased tissues, including cancers.
Testing judgement biases of animals may provide insight into their affective states; however important questions about methodologies need to be answered. This experiment investigated the effect of repeated testing using unreinforced, ambiguous cues on the response of sheep to a go/no-go judgement bias test. Fifteen sheep were trained to differentiate between two locations, reinforced respectively with feed (positive) or with the presentation of a dog (negative). The responses to nine ambiguous locations, positioned between the positively and negatively reinforced locations, were tested repeatedly over 3 weeks. Sheep exhibited a symmetrical gradation in response to ambiguous locations between the positive and negative reinforcers. There was a significant decline (P=0.001) in the total number of approaches to the ambiguous positions over time (weeks). This effect of time suggests that sheep learnt that the ambiguous locations were unrewarded. This result supplies evidence of a limitation identified in current judgement bias methodology, due to repeated testing, which has the potential to provide misleading results.
Members of the ATP-binding-cassette transporter family are implicated in the traffic of drugs/xenobiotics. Several SNPs in these ATP-binding-cassette genes were previously identified to show evidence of recent positive selection. These recent positive selection SNPs may confer functional effects and account for variation in drug response. To facilitate association studies between these SNPs and drug response, we report the development of a homogeneous (realtime exonuclease-mediated allelic discrimination) assay to genotype these SNPs.
Several antihistamine drugs including terfenadine, ebastine, and astemizole have been identified as substrates for CYP2J2. The overall importance of this enzyme in drug metabolism has not been fully explored. In this study, 139 marketed therapeutic agents and compounds were screened as potential CYP2J2 substrates. Eight novel substrates were identified that vary in size and overall topology from relatively rigid structures (amiodarone) to larger complex structures (cyclosporine). The substrates displayed in vitro intrinsic clearance values ranging from 0.06 to 3.98 mul/min/pmol CYP2J2. Substrates identified for CYP2J2 are also metabolized by CYP3A4. Extracted ion chromatograms of metabolites observed for albendazole, amiodarone, astemizole, thioridazine, mesoridazine, and danazol showed marked differences in the regioselectivity of CYP2J2 and CYP3A4. CYP3A4 commonly metabolized compounds at multiple sites, whereas CYP2J2 metabolism was more restrictive and limited, in general, to a single site for large compounds. Although the CYP2J2 active site can accommodate large substrates, it may be more narrow than CYP3A4, limiting metabolism to moieties that can extend closer toward the active heme iron. For albendazole, CYP2J2 forms a unique metabolite compared with CYP3A4. Albendazole and amiodarone were evaluated in various in vitro systems including recombinant CYP2J2 and CYP3A4, pooled human liver microsomes (HLM), and human intestinal microsomes (HIM). The Michaelis-Menten-derived intrinsic clearance of N-desethyl amiodarone was 4.6 greater in HLM than in HIM and 17-fold greater in recombinant CYP3A4 than in recombinant CYP2J2. The resulting data suggest that CYP2J2 may be an unrecognized participant in first-pass metabolism, but its contribution is minor relative to that of CYP3A4.
Preeclampsia (PE) is a leading cause of maternal and fetal mortality and morbidity that occurs only during pregnancy. Pregnancy is the only physiological situation where killer-cell immunoglobulin-like receptors (KIRs) may meet cognate nonself variants of human leukocyte antigen (HLA) allotypes. We previously reported that presence of fetal HLA-G*0106 was significantly associated with risk for PE in multigravid pregnancies. We have now tested the KIR2DL4 receptor gene for association with PE, as well as for its interaction with HLA-G in modulating disease risk, in a case-control study of 83 PE and 240 normotensive pregnancies. No significant association was observed between alleles of KIR2DL4 and PE in both maternal and fetal groups, either among primigravid or multigravid pregnancies. Alleles of KIR2DL4 and HLA-G were then analyzed together to determine whether particular variant ligand-receptor combinations were associated with an increased risk for PE. Gene-gene interaction analyses suggest that the presence of fetal HLA-G*0106 in combination with maternal KIR2DL4*006 is significantly associated with PE risk in multigravid pregnancies (P < .001). These data provide the first preliminary evidence suggesting that although KIR2DL4 itself is not associated with PE, it may modulate the effect of HLA-G*0106 on risk for PE.
Opioid drug response and pain perception differs greatly amongst different individuals. The micro-opioid receptor (MOR) is the main receptor target for important opioid analgesics. As SNPs may contribute to interindividual differences in drug response, in silico signatures of recent positive selection (RPS) were utilized to seek out potentially functional SNPs in the MOR gene in order to facilitate the prioritization of SNPs for evaluation in genetic association studies. Out of over 1000 SNPs at the MOR locus, 184 high-frequency SNPs were interrogated for signatures of RPS. A total of five SNPs (four noncoding and one nonsynonymous coding) demonstrated in silico evidence of RPS. Significantly, the nonsynonymous E1/A118G SNP, which was previously reported to be functionally important, showed in silico evidence of RPS. This reaffirms the feasibility of utilizing in silico signatures of RPS to identify potentially functionally significant SNPs for association studies. Interestingly, the positively selected G allele of this RPS SNP was also predicted to create a novel exon splice enhancer as well as p53 binding sites.
The biosynthesis of serotonin requires aromatic substrates to be bound in the active sites of the enzymes tryptophan hydroxylase and aromatic amino acid decarboxylase. These aromatic substrates are held in place partially by dispersion and induction interactions with the enzymes aromatic amino acid residues. Mutations that decrease substrate binding can result in a decrease in serotonin production and thus can lead to depression and related disorders. We use optimized crystal structures of these two enzymes to examine pair-wise electronic interaction energies between aromatic residues in the active sites and the aromatic ligands. We also perform in silico mutations on the aromatic residues to determine the change in interaction energies as mutations occur. Our second-order Moller-Plessett perturbation theory calculations show that drastic changes in interaction energy can occur and, in light of our previous work, we are able to use these data to offer predictions on the loss of protein function and on the possibility of disease upon mutation. We also examine local and gradient corrected density functional theory methods to evaluate their ability to predict these induction/dispersion-dominated interaction energies. We find that the hybrid B3LYP cannot model these interactions well, whereas the GGA HCTH407 offers largely qualitatively correct results, and the local functional SVWN quantitatively mimics the MP2 results rather well.
The hepatitis B-X (HBx) protein is strongly associated with hepatocellular carcinoma. It is implicated not to directly cause cancer but to play a role in hepatocellular carcinoma as a co-factor. The oncogenic potential of HBx primarily lies in its interaction with transcriptional regulators resulting in aberrant gene expression and deregulated cellular pathways. Utilizing ultraviolet irradiation to simulate a tumor-initiating event, we integrated chip-based chromatin immunoprecipitation (ChIP-chip) with expression microarray profiling and identified 184 gene targets directly deregulated by HBx. One-hundred forty-four transcription factors interacting with HBx were computationally inferred. We experimentally validated that HBx interacts with some of the predicted transcription factors (pTF) as well as the promoters of the deregulated target genes of these pTFs. Significantly, we demonstrated that the pTF interacts with the promoters of the deregulated HBx target genes and that deregulation by HBx of these HBx target genes carrying the pTF consensus sequences can be reversed using pTF small interfering RNAs. The roles of these deregulated direct HBx target genes and their relevance in cancer was inferred via querying against biogroup/cancer-related microarray databases using web-based NextBio(TM) software. Six pathways, including the Jak-STAT pathway, were predicted to be significantly deregulated when HBx binds indirectly to direct target gene promoters. In conclusion, this study represents the first ever demonstration of the utilization of ChIP-chip to identify deregulated direct gene targets from indirect protein-DNA binding as well as transcriptional factors directly interacting with HBx. Increased knowledge of the gene/transcriptional factor targets of HBx will enhance our understanding of the role of HBx in hepatocellular carcinogenesis and facilitate the design of better strategies in combating hepatitis B virus-associated hepatocellular carcinoma.
Oxidation of Hbs leads to the formation of different forms of Fe(III) that are relevant to a range of biochemical and physiological functions. Here we report a combined EPR/x-ray crystallography study performed at acidic pH on six ferric tetrameric Hbs. Five of the Hbs were isolated from the high-Antarctic notothenioid fishes Trematomus bernacchii, Trematomus newnesi, and Gymnodraco acuticeps, and one was isolated from the sub-Antarctic notothenioid Cottoperca gobio. Our EPR analysis reveals that 1), in all of these Hbs, at acidic pH the aquomet form and two hemichromes coexist; and 2), only in the three Hbs that exhibit the Root effect is a significant amount of the pentacoordinate (5C) high-spin Fe(III) form found. The crystal structure at acidic pH of the ferric form of the Root-effect Hb from T. bernacchii is also reported at 1.7 A resolution. This structure reveals a 5C state of the heme iron for both the alpha- and beta-chains within a T quaternary structure. Altogether, the spectroscopic and crystallographic results indicate that the Root effect and hemichrome stability at acidic pH are correlated in tetrameric Hbs. Furthermore, Antarctic fish Hbs exhibit higher peroxidase activity than mammalian and temperate fish Hbs, suggesting that a partial hemichrome state in tetrameric Hbs, unlike in monomeric Hbs, does not remove the need for protection from peroxide attack, in contrast to previous results from monomeric Hbs.
SNPs are known to contribute to variations in drug response and there are more than 14 million polymorphisms spanning the human genome. However, not all of these SNPs are functional. It would be impractical and costly to evaluate every individual SNP for functionality experimentally. Consequently, one of the major challenges for researchers has been to seek out functional SNPs from all the SNPs in the human genome. In silico or bioinformatic methods are economical, less labor intensive, yet powerful approaches to filter out potentially functional SNPs in drug-response genes for further study. This allows researchers to prioritize which SNPs to subsequently evaluate experimentally for drug-response studies, as well as potentially providing insights into possible mechanisms underlying how SNPs may affect drug-response genes.
It is quickly becoming apparent that situating human variation in a pathway context is crucial to understanding its phenotypic significance. Toward this end, we have developed a general method for finding pathways associated with traits that control for pathway size. We have applied this method to a new whole genome survey of coding SNP variation in 187 patients afflicted with Parkinson disease (PD) and 187 controls. We show that our dataset provides an independent replication of the axon guidance association recently reported by Lesnick et al. [PLoS Genet 2007;3:e98], and also indicates that variation in the ubiquitin-mediated proteolysis and T-cell receptor signaling pathways may predict PD susceptibility. Given this result, it is reasonable to hypothesize that pathway associations are more replicable than individual SNP associations in whole genome association studies. However, this hypothesis is complicated by a detailed comparison of our dataset to the second recent PD association study by Fung et al. [Lancet Neurol 2006;5:911-916]. Surprisingly, we find that the axon guidance pathway does not rank at the very top of the Fung dataset after controlling for pathway size. More generally, in comparing the studies, we find that SNP frequencies replicate well despite technologically different assays, but that both SNP and pathway associations are globally uncorrelated across studies. We thus have a situation in which an association between axon guidance pathway variation and PD has been found in 2 out of 3 studies. We conclude by relating this seeming inconsistency to the molecular heterogeneity of PD, and suggest future analyses that may resolve such discrepancies.
MicroRNAs (miRs) are small non-coding RNAs regulating gene expression at the post-transcriptional and/or translational levels. miRs play important roles in diverse biological processes, including development, cell differentiation, proliferation and apoptosis. Recent evidence has shown that miR loci frequently map to cancer-associated genomic regions and deregulated miR expression profiles are associated with many cancer types, implicating miRs in crucial processes that lead to tumourigenesis. Here, we review the current findings about miRs and tumourigenesis, focusing on their involvement in the apoptosis pathway. A significant observation is that greater than one-quarter of all known human miRs were reported to be deregulated in at least one cancer type. The expression of a subset of miRs (e.g. miR-21 and miR-155) was found to be consistently up-regulated, whereas another subset of miRs (e.g.miR-143 and miR-145) was consistently down-regulated across different cancer types suggesting their involvement in regulating common cellular processes whose deregulation may lead to tumourigenesis. Several miRs were implicated to play roles in cell proliferation and apoptosis. Some miRs, such as miR-29b and miR-15-16, influence only the apoptotic pathway, whereas others including let-7/miR-98 and miR-17-92 may play roles in both the apoptotic and cell-proliferation pathways. In conclusion, although our current understanding of the functions of miRs is still fragmentary, taken together, this review highlights the complex and intricate roles that miRs play in the regulation of cellular processes. Perturbation of the expression of miRs may thus lead to tumourigenesis.
Patients with malignant pleural mesothelioma (MPM), who undergo chest instrumentation, may develop seeding at the site of intervention, leading to subcutaneous tumour. This is believed to be reduced by the common practice of prophylactic irradiation to intervention tracts (PIT). However, evidence to support PIT is currently inadequate and contentious.
Chronic hepatitis B virus (HBV) infection is epidemiologically associated with hepatocellular carcinoma (HCC), but its role in HCC remains poorly understood due to technological limitations. In this study, we systematically characterize HBV in HCC patients. HBV sequences were enriched from 48 HCC patients using an oligo-bead-based strategy, pooled together and sequenced using the FLX-Genome-Sequencer. In the tumors, preferential integration of HBV into promoters of genes (P < 0.001) and significant enrichment of integration into chromosome 10 (P < 0.01) were observed. Integration into chromosome 10 was significantly associated with poorly differentiated tumors (P < 0.05). Notably, in the tumors, recurrent integration into the promoter of the human telomerase reverse transcriptase (TERT) gene was found to correlate with increased TERT expression. The preferred region within the HBV genome involved in integration and viral structural alteration is at the 3-end of hepatitis B virus X protein (HBx), where viral replication/transcription initiates. Upon integration, the 3-end of the HBx is often deleted. HBx-human chimeric transcripts, the most common type of chimeric transcripts, can be expressed as chimeric proteins. Sequence variation resulting in non-conservative amino acid substitutions are commonly observed in HBV genome. This study highlights HBV as highly mutable in HCC patients with preferential regions within the host and virus genome for HBV integration/structural alterations.
Mouse primordial germ cells (PGCs) undergo sequential epigenetic changes and genome-wide DNA demethylation to reset the epigenome for totipotency. Here, we demonstrate that erasure of CpG methylation (5mC) in PGCs occurs via conversion to 5-hydroxymethylcytosine (5hmC), driven by high levels of TET1 and TET2. Global conversion to 5hmC initiates asynchronously among PGCs at embryonic day (E) 9.5 to E10.5 and accounts for the unique process of imprint erasure. Mechanistically, 5hmC enrichment is followed by its protracted decline thereafter at a rate consistent with replication-coupled dilution. The conversion to 5hmC is an important component of parallel redundant systems that drive comprehensive reprogramming in PGCs. Nonetheless, we identify rare regulatory elements that escape systematic DNA demethylation in PGCs, providing a potential mechanistic basis for transgenerational epigenetic inheritance.
The key cellular regulator p53 is a common target of viral oncoproteins. However, the mechanism by which p53 transcription regulation is modulated by hepatitis B virus X protein (HBx), a transcription cofactor implicated in hepatitis B virus-associated hepatocellular carcinoma (HCC), is poorly understood. By integrating p53 chromatin immunoprecipitation (ChIP)-on-chip and expression profiling of an HBx-expressing cell culture system, we report that HBx alters p53 binding site selectivity in the regulatory regions of genes, and this is associated with their aberrant expression. Using an HBx-deregulated gene, p53AIP1, as a model, we show that HBx aberrantly increases p53AIP1 expression by conferring p53 selectivity for a more conserved binding site in its regulatory region. We further demonstrate that HBx-deregulated increased p53AIP1 expression is relevant in HCC livers and define a functional role for p53AIP1 in mediating HBx-induced apoptosis in vitro. Significantly, we provide evidence that specific p53-associated transcription cofactors and coregulators are differentially recruited in the presence of HBx, effecting a PCAF-mediated "p53 Lys320 acetylation switch" that results in altered binding site selection of distinct p53 transcription cassettes. The findings here clarify the role of HBx in modulating p53 transcription regulation and provide a novel mechanistic insight into this deregulation.
Fentanyl-induced emesis (FIE) is a distressing adverse effect in the postoperative setting. The genetic basis of FIE remains largely unknown, therefore, we examined whether it was associated with specific genetic variants of OPRM1, the gene encoding the main receptor target of fentanyl.
TiO2 layers were fabricated using a nano-particle deposition system (NPDS) on transparent conductive oxide (TCO) glass for dye sensitized solar cells (DSSCs). Conventionally, TiO2 paste for working electrodes has been fabricated using paste type methods. The fabricated paste composed of a mixture of nano-sized TiO2 powders, binders and solutions is then painted on TCO glass. After drying, the TiO2 layer on TCO glass is sintered to make a path for electron transfer. TiO2 layers formed by this paste type method require numerous steps, which can be time consuming. In this study, TiO2 powders were sprayed directly on TCO glass using NPDS in order to simplify the fabrication steps. To improve porosity and produce scattering layers, commercial nanocrystalline TiO, powders with different sizes were alternately deposited. Moreover, powders with different sizes were mixed and deposited on the TCO glass. The results indicate that the DSSCs with a TiO2 layer composed of different particle sizes had better cell performance than the cells assembled with single-sized TiO2 particles. Therefore, this study shows that a dry TiO2 coating process is possible for DSSC fabrication to improve its cell efficiencies, and this method can easily be applied on flexible substrates since NPDS is a room-temperature deposition process.
Sub-50 nm copper nanoparticles coated with sub-5 nm 1-octanethiol layer for oxidation inhibition were examined to confirm the 1-octanethiol removal temperature as the sub-50 nm copper nanoparticles are sintered. As a result, 1-octanethiol Self-Assembled Multi-layers (SAMs) on sub-50 nm copper nanoparticles were successfully removed before sintering of copper nanoparticles so that a high density of copper line could be obtained. Finally, the line resistivity was measured and compared to verify the effect of sintering in different atmospheres. As a result, electrical resistivity of the copper pattern sintered in hydrogen atmosphere was measured at 6.96 x 10(-6) ohm-cm whereas that of the copper pattern sintered in mixed gas atmosphere was measured at 2.62 x 10(-5) ohm-cm. Thus, sintering of copper patterns was successfully done to show low electrical resistivity values. Moreover, removal of 1-octanethiol coating after sintering process was confirmed using X-ray photoelectron spectroscopy (XPS) analysis. By showing no sulfur content, XPS results indicate that 1-octanethiol is completely removed. Therefore, the vapor form of 1-octanethiol coating layers can be safely used as an oxidation inhibition layer for low temperature sintering processes and ink-jet applications.
TiO2 powders were deposited on indium tin oxide (ITO) coated polyethylene terephthalate (PET) substrates for application to the photoelectrode of a dye-sensitized solar cell (DSSC). In the conventional DSSC manufacturing process, a semiconductor oxide such as TiO2 powder requires a sintering process at higher temperature than the glass transition temperature (T(g)) of polymers, and thus utilization of flexible polymer substrates in DSSC research has been constrained. To overcome this restriction related to sintering, we used a nanoparticle deposition system (NPDS) that could produce a thin coating layer through a dry-spray method under atmospheric pressure at room temperature. The powder was sprayed through a slit-type nozzle having a 0.4 x 10 mm2 rectangular outlet. In order to determine the deposited TiO2 thickness, five kinds of TiO2 layered specimens were prepared, where the specimens have single and double layer structures. Deposited powders on the ITO coated PET substrates were observed using FE-SEM and a scan profiler The thicker TiO2 photoelectrode with a DSSC having a double layer structure showed higher energy efficiency than the single layer case. The highest fabricated flexible DSSC displayed a short circuit current density J(sc) = 1.99 mA cm(-2), open circuit voltage V(oc) = 0.71 V, and energy efficiency eta = 0.94%. These results demonstrate the possibility of utilizing the dry-spray method to fabricate a TiO2 layer on flexible polymer substrates at room temperature under atmospheric pressure.
High mechanical properties of a tungsten carbide micro-end-mill tool was achieved by extending its tool life by electroplating nano-sized SiC particles (< 100 nm) that had a hardness similar to diamond in a nickel-based material. The co-electroplating method on the surface of the micro-end-mill tool was applied using SiC particles and Ni particles. Organic additives (saccharin and ammonium chloride) were added in a Watts bath to improve the nickel matrix density in the electroplating bath and to smooth the surface of the co-electroplating. The morphology of the coated nano-sized SiC particles and the composition were measured using Scanning Electron Microscope and Energy Dispersive Spectrometer. As the Ni/SiC co-electroplating layer was applied, the hardness and friction coefficient improved by 50%. Nano-sized SiC particles with 7 wt% were deposited on the surface of the micro-end mill while the Ni matrix was smoothed by adding organic additives. The tool life of the Ni/SiC co-electroplating coating on the micro-end mill was at least 25% longer than that of the existing micro-end mills without Ni/SiC co-electroplating. Thus, nano-sized SiC/Ni coating by electroplating significantly improves the mechanical properties of tungsten carbide micro-end mills.
Blimp1 (Prdm1), the key determinant of primordial germ cells (PGCs), plays a combinatorial role with Prdm14 during PGC specification from postimplantation epiblast cells. They together initiate epigenetic reprogramming in early germ cells toward an underlying pluripotent state, which is equivalent to embryonic stem cells (ESCs). Whereas Prdm14 alone can promote reprogramming and is important for the propagation of the pluripotent state, it is not known whether Blimp1 is similarly involved. By using a genetic approach, we demonstrate that Blimp1 is dispensable for the derivation and maintenance of ESCs and postimplantation epiblast stem cells (epiSCs). Notably, Blimp1 is also dispensable for reprogramming epiSCs to ESCs. Thus, although Blimp1 is obligatory for PGC specification, it is not required for the reversion of epiSCs to ESCs and for their maintenance thereafter. This study suggests that reprogramming, including that of somatic cells to ESCs, may not entail an obligatory route through a Blimp1-positive PGC-like state.
Extranasal colonization is increasingly recognized as an important reservoir for Staphylococcus aureus among high-risk populations. We conducted a cross-sectional study of multiple body site colonization among 173 randomly selected STD clinic patients in Baltimore, Maryland. Staphylococcal carriage at extranasal sites, including the oropharynx, groin, rectum, and genitals, was common among study subjects. The USA300 clone was particularly associated with multiple sites of colonization compared with non-USA300 strains (p = .01). Given their high burden of multi-site colonization and confluence of established staphylococcal risk factors, STD clinic patients may represent a community-based reservoir for S. aureus and be well suited for innovative infection control initiatives.
Copper nanoparticles were coated with 1-octanethiol self-assembled monolayers (SAMs) using the dry-coating method for oxidation prevention. In this study, thicknesses of 1-octanethiol SAMs were successfully controlled, and the stability of SAMs as a passivation layer on copper nanoparticles was examined. Thicknesses of 1-octanethiol SAMs varied with vacuum levels and coating cycles. Under low-vacuum conditions, the thickness was 10 nm, regardless of the coating conditions. In contrast, various thicknesses resulted under ultra-high vacuum (UHV) and ranged from 4 nm to 10 nm. SAMs that were nearly a monolayer thick (4 nm) resulted from two coating cycles of 1.5 min, and the oxidation inhibition period was 15 days. Thus, the dry-coating method successfully controlled the thicknesses of SAMs with satisfactory oxidation inhibition properties under ultra-high vacuum.
Single nucleotide polymorphisms (SNPs) are the commonest genetic variant in the human genome and have been associated with inter-individual differences in drug response. Finding the causative SNPs underlying variations in drug response has been a cornerstone of personalized medicine. However, as there are over 19 million SNPs, the task of finding causative SNPs underlying differences in drug response using in vitro and in vivo methods can be intimidating. SNP related web resources can be invaluable in the search for SNPs relevant to drug response phenotypes as they represent relatively cheaper yet efficient ways of prioritizing relevant SNPs for further study. These resources serve as repositories of SNP information or contain in silico tools that can predict the functionality of a SNP. More sophisticated resources integrate the information repository function with the predictive function to create a one stop SNP resource for researchers. SNP related web resources can also aid researchers in planning and analyzing different types of genetic association studies by aiding in selecting SNPs for genotyping in these studies. The focus of this mini review is to outline the SNP related web resources that are available to researchers and how these resources may aid researchers studying SNP-drug response phenotype associations. Through efficient utilization of SNP related web resources, researchers will hopefully be able accelerate the pace of SNP related research in pharmacogenomics by identifying high risk SNP variants contributing to drug response as well as developing novel therapeutic targets based on understanding how SNPs alter drug response pathways.
MicroRNA-224 (miR-224) is one of the most commonly up-regulated microRNAs in hepatocellular carcinoma (HCC), which affects crucial cellular processes such as apoptosis and cell proliferation. In this study, we aim to elucidate the molecular mechanism that leads to the overexpression of miR-224 in HCC. We examined the transcript expression of miR-224 and neighboring miR-452 and genes on chromosome Xq28 in tumor and paired adjacent nontumorous tissues from 100 patients with HCC and found that miR-224 is coordinately up-regulated with its neighboring microRNA (miRNA) and genes. This coordinated up-regulation of miRNAs and genes at the Xq28 locus can be mimicked in nontransformed immortalized human liver cells by the introduction of histone deacetylase (HDAC) inhibitors, which resulted in a corresponding increase in histone H3 acetylation in this region. This miR-224-residing locus in Xq28 is reciprocally regulated by HDAC1, HDAC3, and histone acetylase protein, E1A binding protein p300 (EP300). Notably, in HCC tumors that significantly overexpress microRNA-224, EP300 is also overexpressed and displays increased binding to the Xq28 locus. In transformed HCC cells, high miR-224 expression can be attenuated through the inhibition of EP300, using either siRNA or the specific drug C646. In summary, overexpression of EP300 may account, in part, for the up-regulation of miR-224 expression in patients with HCC.
Little is known about the clonality of Staphylococcus epidermidis in the United States, although it is the predominant pathogen in infections involving prosthetic materials, including ventricular assist devices (VADs).
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