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Quantitative determination of nucleic acids related to human health and safety has attracted a great interest. Electrochemical sandwich-type biosensor with simple operation and low price shows high sensitivity and specificity with dual recognition mechanism and has been widely used for nucleic acid detection. In this chapter, we highlight the advancements of electrochemical sandwich assay for nucleic acids in recent decade. We first introduced the importance of nucleic acid detection and the principles of design an electrochemical nucleic acid sandwich assay and then summarized the advancements of this strategy based on the types of reporter tags, including redox molecules, enzymes, and nanoparticles. Finally, we discussed the challenges in the development of electrochemical nucleic acid sandwich assay to apply for clinical diagnostics, in cells and in vivo.

The aim of this study was to assess the associations of six single nucleotide polymorphisms (SNPs) of three genes (DRD3, COMT, and SCL6A4) with type 2 diabetes mellitus (T2DM) in Southern Chinese.Five hundred ninety-five cases with T2DM and 725 healthy controls of Han origin were recruited from six hospitals in Guangdong Province, Southern China. Fasting serum concentrations of markers of interest (total cholesterol, triglyceride, plasma glucose, etc.) were measured in hospitals. SNP genotyping was performed using a custom-by-design 2-×48-Plex SNPscan™ kit (Genesky Biotechnologies Inc., Shanghai, China). Single-point SNP analysis, haplotype analysis, and SNP-SNP interactions were carried out.SNP rs4646312 in COMT achieved statistical significance in both allelic association and genotypic association and even after adjusting covariates (odds ratio [OR]=1.26; 95% confidence interval [CI], 1.04-1.53; P=0.021). Two haplotypes consisting of rs4646312 and rs4680 were also significantly associated with T2DM, of which C-G was a protective haplotype for T2DM (OR=0.83; 95% CI, 0.70-0.98; P=0.029), whereas T-A was a risk one (OR=1.23, 95% CI, 1.03-1.46; P=0.022). Interaction analysis identified a significant epistatic effect between rs4680 in COMT and rs2066713 in SCL6A4 after adjusting for covariates (OR=3.59, 95% CI, 1.72-7.48; P=0.001 for dominant-dominant model). However, only the interaction between rs4680 and rs2066713 was significant, and haplotype T-A showed a marginally increased risk after Bonferroni correction.The genetic polymorphisms in COMT and SCL6A4 confer significant effects in joint actions to T2DM in Southern Chinese.

Cancer cells preferentially use aerobic glycolysis to meet their increased energetic and biosynthetic demands, a phenomenon known as the Warburg effect. Its underlying mechanism is not fully understood. RRAD, a small GTPase, is a potential tumor suppressor in lung cancer. RRAD expression is frequently down-regulated in lung cancer, which is associated with tumor progression and poor prognosis. Recently, RRAD was reported to repress the Warburg effect, indicating that down-regulation of RRAD expression is an important mechanism contributing to the Warburg effect in lung cancer. However, the mechanism by which RRAD inhibits the Warburg effect remains unclear. Here, we found that RRAD negatively regulates the NF-κB signaling to inhibit the GLUT1 translocation and the Warburg effect in lung cancer cells. Mechanically, RRAD directly binds to the p65 subunit of the NF-κB complex and inhibits the nuclear translocation of p65, which in turn negatively regulates the NF-κB signaling to inhibit GLUT1 translocation and the Warburg effect. Blocking the NF-κB signaling largely abolishes the inhibitory effects of RRAD on the translocation of GLUT1 to the plasma membrane and the Warburg effect. Taken together, our results revealed a novel mechanism by which RRAD negatively regulates the Warburg effect in lung cancer cells.

This communication describes a robust wet-chemical synthetic strategy for the preparation of monodispersed circular and triangular gold nanorings. The localized surface plasmon resonance of the nanorings can be tuned by controlling the outer diameter and ridge thickness of the nanorings.

TRAF-binding domain (Trabid), one of deubiquitination enzymes, was recently reported to activate Wnt/ β-catenin signaling pathway. However, the role of Trabid in tumors including hepatocellular carcinoma (HCC) and the underlying mechanisms controlling its activity remain poorly understood. Here, we report that Trabid is significantly downregulated in HCC tumor samples and cell lines compared with normal controls and that its expression level is negatively correlated with HCC pathological grading, recurrence, and metastasis. The reintroduction of Trabid expression in tumor cells significantly decreases HCC progression as well as pulmonary metastasis. The effect of Trabid on HCC development occurs at least partially through regulation of Twist1 activity. Mechanistically, Trabid forms a complex with Twist1 and specifically cleaves RNF8-induced K63-linked poly-ubiquitin chains from Twist1, which enhances the association of Twist1 with β-TrCP1 and allows for subsequent K48-linked ubiquitination of Twist1. Knockdown of Trabid increases K63-linked ubiquitination, but abrogates K48-linked ubiquitination and degradation of Twist1, thus enhancing HCC growth and metastasis. Interestingly, Twist1 negatively regulates the promoter activity of Trabid, indicating that a double-negative feedback loop exists. Our findings also identify an essential role for activation of Trabid by AKT-mediated phosphorylation at Ser78/Thr117 in negatively regulating Twist1 signaling, which further provides insights into the mechanisms by which Trabid regulates Twist1 ubiquitination. Our results reveal that Trabid is a previously unrecognized inhibitor of HCC progression and metastasis, which sheds light on new strategies for HCC treatment.

Physalin D is known to show extensive bioactivities. However, no excretion study has elucidated the excretion of physalin D and its metabolites. This study investigates the excretion of physalin D and its metabolites in rats. Metabolites in rat urine and feces were separated and identified by liquid chromatography with triple quadrupole time-of-flight mass spectrometry. Furthermore, a validated high-performance liquid chromatography with tandem mass spectrometry method was developed to quantify physalin D, physalin D glucuronide, and physalin D sulfate in rat feces and urine after the intragastric administration of physalin D. The analyte showed good linearity over a wide concentration range (r > 0.995), and the lower limit of quantification was 0.0532 μg/mL and 0.226 μg/g for urine and feces, respectively. Nine metabolites, including five phase I and four phase II metabolites, were identified and clarified after dosing in vivo. Only 4.0% of the gavaged dose, including physalin D and its phase II metabolites, was excreted in urine, whereas 10.8% was found in feces in the unchanged form. The results indicate that the extensive and rapid metabolism may be the main factors leading to the short half-life of physalin D. These results can provide a basis for further studies on the structural modification and pharmacology of physalin D.

The purpose of this study was to validate the applicability of our proposed disease-specific questionnaire to Cantonese coronary heart disease (CHD) patients. During the investigation from August 2010 to March 2012, 1000 Cantonese inpatients were recruited. The reliability of the scale was judged by the internal consistency, and the content and construct validity were assessed by using Pearson correlation and confirmatory factor analysis, respectively. Results showed that the Cronbach’s α coefficient for the whole scale and most domains/facets were larger than .70 (.59 to .93). Most items had moderate to strong Pearson correlations with their respective facets ( r > 0.50). Confirmatory factor analysis showed that the indices for goodness of fit were nearly acceptable. Overall, the QLICD-CHD scale has adequate psychometric properties when applied to Cantonese CHD patients.

// Juan Liu 1,4 , Cen Zhang 1,4 , Meihua Lin 1 , Wei Zhu 1,2 , Yingjian Liang 1 , Xuehui Hong 1,2 ,Yuhan Zhao 1,2 , Ken H. Young 3 , Wenwei Hu 1,2 , Zhaohui Feng 1 1 Department of Radiation Oncology, 2 Department of Pediatrics, Rutgers Cancer Institute of New Jersey, Rutgers University, New Brunswick, NJ, USA 3 Department of Hematopathology, University of Texas MD Anderson Cancer Center, Houston , TX , USA 4 These two authors contributed equally to this work Correspondence: Wenwei Hu, email: // Zhaohui Feng, email: // Keywords :p53; GLS2; tumor suppression; PI3K/AKT; Hepatocellular carcinoma Received : February 15, 2014 Accepted : March 24, 2014 Published : March 26, 2014 Abstract The tumor suppressor p53 and its signaling pathway play a critical role in tumor prevention. As a direct p53 target gene, the role of glutaminase 2 (GLS2) in tumorigenesis is unclear. In this study, we found that GLS2 expression is significantly decreased in majority of human hepatocellular carcinoma (HCC). Restoration of GLS2 expression in HCC cells inhibits the anchorage-independent growth of cells and reduces the growth of HCC xenograft tumors. Interestingly, we found that GLS2 negatively regulates the PI3K/AKT signaling, which is frequently activated in HCC. Blocking the PI3K/AKT signaling in HCC cells largely abolishes the inhibitory effect of GLS2 on the anchorage-independent cell growth and xenograft tumor growth. The GLS2 promoter is hypermethylated in majority of HCC samples. CpG methylation of GLS2 promoter inhibits GLS2 transcription, whereas reducing the methylation of GLS2 promoter induces GLS2 expression. Taken together, our results demonstrate that GLS2 plays an important role in tumor suppression in HCC, and the negative regulation of PI3K/AKT signaling contributes greatly to this function of GLS2. Furthermore, hypermethylation of GLS2 promoter is an important mechanism contributing to the decreased GLS2 expression in HCC.

Until recently, the underlying genetic mechanisms for coronary artery disease (CAD) have been largely unknown, with just a list of genes identified accounting for very little of the disease in the population. Hence, a systematic dissection of the sophisticated interplays between these individual disease genes and their functional involvements becomes essential. Here, we presented a novel knowledge-based approach to identify the functional modules for CAD. First, we selected 266 disease genes in CADgene database as the initial seed genes, and used PPI knowledge as a guide to expand these genes into a CAD-specific gene network. Then, we used Newman's algorithm to decompose the primary network into 14 compact modules with high modularity. By analysis of these modules, we further identified 114 hub genes, all either directly or indirectly associated with CAD. Finally, by functional analysis of these modules, we revealed several novel pathogenic mechanisms for CAD (for examples, some yet rarely concerned like peptide YY receptor activity, Fc gamma R-mediated phagocytosis and actin cytoskeleton regulation etc.).

Because of the short size and low abundance of microRNAs, it is challenging to develop fast, inexpensive, and simple biosensors to detect them. In this work, we have demonstrated a new generation (the third generation) of E-DNA sensor for the sensitive and specific detection of microRNAs. Our third generation of E-DNA sensor can sensitively detect microRNA target (microRNA-141) as low as 1 fM. The excellent specificity has been demonstrated by its differential ability to the highly similar microRNA analogues. In our design, the use of DNA tetrahedron ensures the stem-loop structure in well controlled density with improved reactivity. The regulation of the thermodynamic stability of the stem-loop structure decreases the background signal and increases the specificity as well. The enzymes attached bring the electrocatalytic signal to amplify the detection. The combination of these effects improves the sensitivity of the E-DNA sensor and makes it suitable to the microRNA detection. Finally, our third generation of E-DNA sensor is generalizable to the detection of other micro RNA targets (for example, microRNA-21).

The situation of infectious diseases and biothreats all over the world remains serious. The effective identification of such diseases plays a very important role. In recent years, gold nanoparticles have been widely used in biosensor design to improve the performance for the detection of infectious diseases and biothreats. Here, recent advances of gold‐nanoparticle‐based biosensors in this field are summarized., Gold‐nanoparticle‐based biosensors play an important role in the identification of infectious diseases and biothreats. Gold nanoparticles can be used as signal producers and signal amplifiers to improve the performance of biosensors. Based on this approach, colorimetric, electrochemical, and fluorescent biosensors with high sensitivity and specificity are designed to fight infectious diseases and biothreats. WILEY-VCH

The pharmacokinetics (PKs) of febuxostat varies among individuals, while the main causes are still unknown. We investigated whether the polymorphisms of UGT1A1 and UGT1A3 played an important role in the disposition of the drug after oral administration of febuxostat tablet in Chinese subjects. A total of 42 healthy subjects were from two previous independent clinical bioequivalence (BE) trials of febuxostat, in which the same reference formulation (ULORIC® tablet, 80 mg) was taken, and thus the PK data were combined for the evaluation of pharmacogenomic effect on febuxostat PKs. Our study clearly indicated that the area under the plasma concentration-time curve (AUC) in the heterozygote and homozygote of UGT1A1*6 (c.211G > A, rs4148323) was significantly higher than that in the wild-type. Meanwhile, the clearance (CL/F) exhibited a significant reduction by 22.2%. Interestingly, UGT1A1*28, in perfect linkage disequilibrium (LD) with UGT1A3*2a, significantly increased its clearance. These results indicate that UGT1A1*6 was an important factor influencing the drug disposition, thus providing a probable explanation for interindividual variation of febuxostat PKs in Chinese subjects. In addition, by considering of the different allele distribution of UGT1A1*6 and *28 in Eastern and Western populations, these findings might further interpret the ethnic difference of febuxostat PKs.

The present study aimed to investigate the therapeutic efficacy and clinical value of recombinant human adenovirus-p53 (rAd-p53) perfusion via the pancreatic artery for the treatment of mid-late stage pancreatic cancer. rAd-p53 (2×1012 virus particles) in 6 ml normal saline was pushed (intravenous bolus) into the gastroduodenal and superior pancreaticoduodenal arteries via interventional superselection, with the catheter retained for subsequent drug administration at a 3-day interval for 4 cycles. Tumor changes in all patients were observed to evaluate tumor response by computed tomography (CT) at 2, 8 and 16 weeks post-treatment. The following improvements were noted in the 23-patient cohort: A total of 73.9% (17/23) of patients demonstrated significant tumor shrinkage (>20%); the symptoms of abdominal and back pain were relieved in 15 patients; the survival time was >12 months in 1 patient and >6 months in 14 patients; the patient's general condition, including appetite, was improved in 13 patients; body weight was increased in 9 patients; jaundice was attenuated in 12 patients; and ascites subsided in 10 patients. However, the therapeutic outcome was poor in 2 patients whose tumors size did not show significant change after treatment as detected by CT. These 2 patients succumbed within 6 months. In conclusion, rAd-p53 perfusion via the pancreatic artery is a safe and minimally invasive option for the treatment of mid-late stage pancreatic cancer.

This review article discussed application of ionic liquids as matrices (ILMs) for mass spectrometry (MS). ILMs were applied for matrix assisted laser desorption/ionization, (ILMALDI-MS), electrospray ionization (ILMs-ESI-MS) and desorption corona beam ionization (DCBI-MS). Ionic liquids matrices provided several advantages such as low vapor pressure, have high stability for storage and under vacuum, extremely high sensitivity and showed low background or interferences. They are non-explosive, non-flammable and are thermally stable. The materials are promising for real measurements and require further investigations to improve the current performance. The combination between the conventional matrices and organic bases leaded to high ionization performance. The proton transfer efficiency of ILMs is higher than conventional matrices as the proton take place from the salt form. They were applied for many analytes such as biomarker, protein, peptides, polymers (synthetic and nature), small organic compounds and pharmaceuticals drugs.

p63 and p73, two p53 family members, play crucial roles in development and tumor suppression. p63 and p73 have multiple isoforms, which have similar or distinct biological functions. Transactivation (TA) isoforms of p63 and p73 have high similarity with p53 and often have biological functions similar to p53. p53 plays an important role in nucleotide excision repair (NER) through transcriptional regulation of target genes involved in NER, including DDB2, XPC and GADD45. To investigate whether TAp63 and TAp73 play a similar role in NER, Saos2 cells with inducible expression of specific isoforms of TAp63 and TAp73, including TAp63α/β/γ and TAp73α/β/γ isoforms, were employed. Overexpression of TAp63γ significantly enhances NER of ultraviolet (UV)-induced DNA damage, including cyclobutane pyrimidine dimers (CPDs) and 6–4 photoproducts, and enhances cell survival after UV irradiation in Soas2 cells. The enhancement of NER of UV-induced DNA damage by TAp63γ was also confirmed in H1299 cells with overexpression of TAp63γ. Consistently, knockdown of endogenous TAp63 decreases NER of UV-induced DNA damage in H1299 cells. TAp63α/β and TAp73α/β/γ isoforms do not have a clear effect on NER in Saos2 or H1299 cells. TAp63γ overexpression clearly induces the expression of DDB2, XPC and GADD45 at both RNA and protein levels. Furthermore, luciferase reporter assays show that TAp63γ transcriptionally activates DDB2, XPC and GADD45 genes through the regulation of the p53 binding elements in these genes. These results demonstrate that TAp63γ enhances NER to remove UV-induced DNA damage and maintain genomic stability through transcriptional induction of a set of NER proteins, which provides an additional important mechanism that contributes to the function of TAp63 in tumor suppression.

Aim This study was intended to cross-culturally adapt and evaluate the Chinese version of the Audit of Diabetes Dependent Quality of Life (ADDQoL) – the Chinese Normal Audit of Diabetes-Dependent Quality of Life (CN-ADDQoL) in Mainland, China. Methods The standard procedure for cross-culture adaptation was used to develop the Chinese version CN-ADDQoL. After the linguistic validation, the validity and reliability of CN-ADDQoL questionnaire were evaluated based on a sample of 697 Type 2 diabetes patients. The Cronbach's α coefficient, correlation analysis and the structural equation model (SEM) were applied, respectively. Results We developed 19 items for the CN-ADDQoL questionnaire. The estimated Cronbach's α coefficient was 0.941, indicating an excellent internal consistency of the scale. All items had high performance in the structural validity evaluation, with most factor loading values being larger than 0.40 (varied from 0.44 to 0.88). Conclusions The Chinese version CN-ADDQoL has maintained its original psychometric properties and achieved adequate reliability and validity. Therefore, it could be efficiently used to evaluate the current trend of diabetes self-management education programs and multinational clinical research trials.

Regulation of energy metabolism is a novel function of p53 in tumor suppression. Parkin (PARK2) , a Parkinson disease-associated gene, is a potential tumor suppressor whose expression is frequently diminished in tumors. Here Parkin was identified as a p53 target gene that is an important mediator of p53's function in regulating energy metabolism. The human and mouse Parkin genes contain functional p53 responsive elements, and p53 increases the transcription of Parkin in both humans and mice. Parkin contributes to the function of p53 in glucose metabolism; Parkin deficiency activates glycolysis and reduces mitochondrial respiration, leading to the Warburg effect. Restoration of Parkin expression reverses the Warburg effect in cells. Thus, Parkin deficiency is a novel mechanism for the Warburg effect in tumors. Parkin also contributes to the function of p53 in antioxidant defense. Furthermore, Parkin deficiency sensitizes mice to γ-irradiation-induced tumorigenesis, which provides further direct evidence to support a role of Parkin in tumor suppression. Our results suggest that as a novel component in the p53 pathway, Parkin contributes to the functions of p53 in regulating energy metabolism, especially the Warburg effect, and antioxidant defense, and thus the function of p53 in tumor suppression.

p53 was discovered 30 years ago. Extensive studies have been done on p53 since then, which makes p53 one of the most extensively studied genes. p53 has long been recognized as a key tumor suppressor. Cell cycle arrest, apoptosis and senescence have been traditionally recognized as the main functions of p53 in tumor suppression. Recently, some novel functions of p53 have been identified, including the regulation of energy metabolism, antioxidant defense, and microRNA expression and maturation, which all contribute to the role of p53 in tumor suppression. Furthermore, the contribution of p53 to normal biologic processes (e.g. reproduction and aging) and some other aspects of diseases (e.g. neurodegenerative diseases) is only now being appreciated. Here we will review recent advances in the study of some new functions of p53.

All-trans retinoic acid (ATRA) has been successfully used in differentiation therapy for acute promyelocytic leukemia (APL) in the clinic. ATRA-induced differentiation of leukemia cells is accompanied by a G0/G1 arrest, yet how ATRA couples cell cycle arrest to differentiation remains largely unknown. Here we observed that the ubiquitin-proteasome pathway (UPP) was activated upon ATRA treatment in the human acute myeloid leukemia cell lines, NB4 and HL-60, as represented by the accumulation of ubiquitinated proteins, the up-regulation of ubiquitin mRNA and increased 20S proteasome activity. Interestingly, we found that complete inhibition of proteasome activity suppressed ATRA-induced proliferation/differentiation (P/D) transition in both cell lines. Furthermore, we demonstrate that the exact protein contributing to this phenomenon is different in these two cell lines. Cyclin-dependent kinase 2 (CDK2) and Cyclin E were degraded by the UPP; they accumulated significantly after complete inhibition of the proteasome in ATRA-treated NB4 and HL-60 cells, respectively. These findings suggested that the UPP might be indispensable in the ATRA-induced G0/G1 arrest and differentiation of leukemia cells. The exact protein degraded by the UPP to promote the myeloid maturation program set in motion by the retinoid may be cell type dependent.

All-trans retinoic acid (ATRA) is nowadays considered to be the sole efficient agent for differentiation-based therapy in leukemia; however, the mechanisms of ATRA's biological effects remain largely unknown. Here we first reported that ATRA-induced myeloid leukemia differentiation was accompanied with the increased level of ubiquitin-protein conjugates and the upregulation of proteasome activity. To explore the functional role of the activated proteasome in retinoic acid (RA) signaling, the effects of proteasome inhibitors on RA-induced cell differentiation were determined. Our results demonstrated that inhibition of ATRA-elevated proteasome activity obviously promoted the myeloid maturation program triggered by ATRA, suggesting that the overactivated proteasome is not beneficial for ATRA's effects. Further studies demonstrated that the synergistic differentiating effects of ATRA and proteasome inhibitors might be associated with the protection of retinoic acid receptor alpha (RARα) from degradation by the ubiquitin-proteasome pathway (UPP). Moreover, the accumulated RARα was able to enhance the transcription of its target gene, which might also contribute to the enhanced differentiation of leukemia cells. Together, by linking the UPP to ATRA-dependent signaling, our data provide a novel insight into studying the mechanisms of ATRA-elicited cellular effects and imply the possibility of combination of ATRA and proteasome inhibitors in leukemia therapy.

All-trans-retinoic acid (ATRA) is a morphogenetic signalling molecule derived from vitamin A and is used clinically to target acute promyelocytic leukemia by inducing differentiation of immature blood cells. Retinoid signals are mediated by retinoic acid (RA) receptors (RARs) and retinoid X receptors (RXRs). Retinoic acid receptors consist of RARalpha, RARbeta and RARgamma isotypes. Among these components, RARalpha is preferentially bound to ATRA, which is used to treat acute T-lymphoblastic leukemia, yet the conditions and mechanisms remain unknown. In this study, we have demonstrated that, in human acute T-lymphoblastic leukemia Molt3 cells, inhibition of RA-induced proliferation results from massive cell death characterised by apoptosis. The effect of ATRA:RARalpha binding on apoptosis in Molt3 cells has been investigated. Consequently, it has been shown that, in RA-treated Molt3 cells, upregulation of p21 due to RA accompanies caspase 3/PARP activation which precedes the occurrence of apoptosis.

Summary We performed a genomic study combining single-cell mRNA differential display and RNA subtractive hybridization to elucidate CD8 T-cell quiescence/ignorance. By comparing actively maintained quiescent CD8 T cells from liver tumour tumour-infiltrating lymphocytes (TILs) with quiescent T cells at the single-cell level, we identified differentially expressed candidate genes by high-throughput screening and comparative analysis of expressed sequence tags (ESTs). While genes for the T-cell receptor, tumour necrosis factor (TNF) receptor, TNF-related apoptpsis inducing ligand (TRAIL) and perforin were down-regulated, key genes such as Tob

The study was carried out to examine the association between apolipoprotein B (ApoB) EcoRI polymorphism (E- vs. E+) (rs1042031) and coronary heart disease (CHD) risk by systematically analyzing multiple independent studies. The Hardy-Weinberg equilibrium (HWE) test was applied to assess genotype frequency distribution in healthy controls. The quality of the studies was assessed using the Newcastle-Ottawa scale (NOS). Power analysis was performed with Power and Precision V4 software. A fixed effect model was used because no deviation from homogeneity was found. Publication bias was quantified and examined with Begg’s funnel plot test and Egger’s linear regression method. The meta-analysis was performed by Stata 12.0 software. A total of 21 eligible association studies were merged in this meta-analysis and the pooled sample consisted of 2994 CHD patients and 3258 healthy controls. No significant publication bias and heterogeneity were observed in these studies. The pooled odds ratio (OR) and 95% confidence interval (CI) of E− vs. E+ were 1.18 (1.06–1.32). The pooled OR (95% CI) of E+ E− + E− E− vs. E+ E+ was 1.18 (1.04–1.34). This meta-analysis indicated that ApoB EcoRI confers a moderate risk for CHD and the E− allele at this locus might be a susceptibility allele for the development of CHD.

Recent studies have shown that the phosphorylation and dephosphorylation of ULK1 and ATG13 are related to autophagy activity. Although ATG16L1 is absolutely required for autophagy induction by affecting the formation of autophagosomes, the post-translational modification of ATG16L1 remains elusive. Here, we explored the regulatory mechanism and role of ATG16L1 phosphorylation for autophagy induction in cardiomyocytes. We showed that ATG16L1 was a phosphoprotein, because phosphorylation of ATG16L1 was detected in rat cardiomyocytes during hypoxia/reoxygenation (H/R). We not only demonstrated that CSNK2 (casein kinase 2) phosphorylated ATG16L1, but also identified the highly conserved Ser139 as the critical phosphorylation residue for CSNK2. We further established that ATG16L1 associated with the ATG12-ATG5 complex in a Ser139 phosphorylation-dependent manner. In agreement with this finding, CSNK2 inhibitor disrupted the ATG12-ATG5-ATG16L1 complex. Importantly, phosphorylation of ATG16L1 on Ser139 was responsible for H/R-induced autophagy in cardiomyocytes, which protects cardiomyocytes from apoptosis. Conversely, we determined that wild-type PPP1 (protein phosphatase 1), but not the inactive mutant, associated with ATG16L1 and antagonized CSNK2-mediated phosphorylation of ATG16L1. Interestingly, one RVxF consensus site for PPP1 binding in the C-terminal tail of ATG16L1 was identified; mutation of this site disrupted its association with ATG16L1. Notably, CSNK2 also associated with PPP1, but ATG16L1 depletion impaired the interaction between CSNK2 and PPP1. Collectively, these data identify ATG16L1 as a bona fide physiological CSNK2 and PPP1 substrate, which reveals a novel molecular link from CSNK2 to activation of the autophagy-specific ATG12-ATG5-ATG16L1 complex and autophagy induction.

Self-assembled DNA nanostructures with precise sizes allow a programmable "soft lithography" approach to engineer the interface of electrochemical DNA sensors. By using millimeter-sized gold electrodes modified with several types of tetrahedral DNA nanostructures (TDNs) of different sizes, both the kinetics and thermodynamics of DNA hybridization were profoundly affected. Because each DNA probe is anchored on an individual TDN, its lateral spacing and interactions are finely tuned by the TDN size. By simply varying the size of the TDNs, the hybridization time was decreased and the hybridization efficiency was increased. More significantly, the detection limit for DNA detection was tuned over four orders of magnitude with differentially nanostructured electrodes, and achieved attomolar sensitivity with polymeric enzyme amplification.

// Cen Zhang 1,* , Juan Liu 1,* , Rui Wu 1 , Yingjian Liang 1 , Meihua Lin 1 , Jia Liu 1 , Chang S. Chan 2 , Wenwei Hu 1 and Zhaohui Feng 1 1 Department of Radiation Oncology, Rutgers Cancer Institute of New Jersey, Rutgers, State University of New Jersey, New Brunswick, USA 2 Department of Medicine, Rutgers Cancer Institute of New Jersey, Rutgers, State University of New Jersey, New Brunswick, USA * These two authors contributed equally to this work Correspondence: Wenwei Hu, email: // Zhaohui Feng, email: // Keywords : p53, glycolysis, RRAD, hypoxia, lung cancer Received : May 17, 2014 Accepted : June 24, 2014 Published : June 26, 2014 Abstract Cancer cells display enhanced glycolysis to meet their energetic and biosynthetic demands even under normal oxygen concentrations. Recent studies have revealed that tumor suppressor p53 represses glycolysis under normoxia as a novel mechanism for tumor suppression. As the common microenvironmental stress for tumors, hypoxia drives the metabolic switch from the oxidative phosphorylation to glycolysis, which is crucial for survival and proliferation of cancer cells under hypoxia. The p53’s role and mechanism in regulating glycolysis under hypoxia is poorly understood. Here, we found that p53 represses hypoxia-stimulated glycolysis in cancer cells through RRAD, a newly-identified p53 target. RRAD expression is frequently decreased in lung cancer. Ectopic expression of RRAD greatly reduces glycolysis whereas knockdown of RRAD promotes glycolysis in lung cancer cells. Furthermore, RRAD represses glycolysis mainly through inhibition of GLUT1 translocation to the plasma membrane. Under hypoxic conditions, p53 induces RRAD, which in turn inhibits the translocation of GLUT1 and represses glycolysis in lung cancer cells. Blocking RRAD by siRNA greatly abolishes p53’s function in repressing glycolysis under hypoxia. Taken together, our results revealed an important role and mechanism of p53 in antagonizing the stimulating effect of hypoxia on glycolysis, which contributes to p53’s function in tumor suppression.

Sensitive detection of cancer cells plays a critically important role in the early detection of cancer and cancer metastasis. However, because circulating tumor cells are extremely rare in peripheral blood, the detection of cancer cells with high analytical sensitivity and specificity remains challenging. Here, we have demonstrated a simple, sensitive and specific detection of cancer cells with the detection sensitivity of four cancer cells, which is lower than the cutoff value with respect to correlation with survival outcomes as well as predictive of metastatic disease in clinical diagnostics. We re-engineered the hybridization chain reaction (HCR) to multibranched HCR (mHCR) that can produce long products with multiple biotins for signal amplification and multiple branched arms for multivalent binding. The capturing gold surface is modified with DNA tetrahedral probes, which provide superior hybridization conditions for the multivalent binding. The synergetic effect of mHCR amplification and multivalent binding lead to the high sensitivity of our detection platform.

Contamination of heavy metal ions in an aquatic environment poses a serious threat to human health. More seriously, heavy metal ions are usually present in the environment in a mixture, and the synergetic toxicity of multiple heavy metal ions is revealed (Aragay et al. Chem. Rev. 2011, 111, 3433; Chu et al. Aquat. Toxicol. 2002, 61, 53). Unfortunately, most of the existing methods based on DNA sequences are focusing on the detection of one type of metal ions. Simple and multiplexed detection of multiple metal ions has been poorly investigated and remains challenging. Here, we re-engineered the DNA sequences for Pb(2+), Hg(2+), and Ag(+), through which the binding of multiple metal ions initiated the self-assembly of these DNA sequences. On the basis of our rationally designed multicolor fluorescent labeling of the DNA sequences, cascade fluorescence resonance energy transfer (FRET) occurred. As a result, a fingerprint fluorescent spectrum was produced to indicate the presence of a single type of metal ions or multiple metal ions. The major advantages of our cascade FRET fingerprint technology include the following: (1) the "mix and read" detection mode in homogeneous solution is simple without the need of complicated instruments; (2) only single excitation is required to provide the cascade FRET fingerprint spectrum; (3) multiplexed detection capability can be realized intuitively and sensitively.

To evaluate the association between the two single nucleotide polymorphisms located in catechol-O-methyltransferase (COMT) gene and type 2 diabetes mellitus (T2DM)in Han population in Guangdong province.Two tagSNPs (rs4646312 and rs4680) were picked out from COMT gene. Using the SNPscan(TM) Kit, SNP genotyping was then performed, in two cohorts, including 595 cases and 725 controls. Finally, Chi-square test, logistic regression model and other methods were employed for statistical analysis.The frequencies of TT, CT and CC of rs4646312 appeared to be 304(51.1%), 234(39.3%)and 57 (8.6%) in cases, 323 (44.6%), 319 (44.0%) and 83(11.4%)in controls, respectively. The frequencies of GG,GA and AA of rs4680 were 311(52.4%), 236 (39.8%) and 46(7.8%)in cases, 417(57.7%), 265 (36.6%) and 41 (5.7%) in controls, respectively.showed that SNP rs4646312 was significantly associated with T2DM both in allelic association analysis (P = 0.020,OR = 1.26, 95%CI:1.04-1.53)and in recessive model (P = 0.022, OR = 1.35, 95% CI:1.05-1.74)after adjustment for sex,BMI and TG. The association between rs4680 and T2DM was not significant, but BMI was remarkably different among the three genotypes of rs4680 after controlling for other factors.SNP rs4646312 of COMT gene was associated with the increased risk of T2DM in Han population in Guangdong province. However, rs4680 was not significantly associated with T2DM.

There remains a great challenge in the sensitive detection of microRNA because of the short length and low abundance of microRNAs in cells. Here, we have demonstrated an ultrasensitive detection platform for microRNA by combining the tetrahedral DNA nanostructure probes and hybridization chain reaction (HCR) amplification. The detection limits for DNA and microRNA are 100 aM and 10 aM (corresponding to 600 microRNAs in a 100 μL sample), respectively. Compared to the widely used supersandwich amplification, the detection limits are improved by 3 orders of magnitude. The uncontrolled surface immobilization and consumption of target molecules that limit the amplification efficiency of supersandwich are eliminated in our platform. Taking advantage of DNA nanotechnology, we employed three-dimensional tetrahedral DNA nanostructure as the scaffold to immobilize DNA recognition probes to increase the reactivity and accessibility, while DNA nanowire tentacles are used for efficient signal amplification by capturing multiple catalytic enzymes in a highly ordered way. The synergetic effect of DNA tetrahedron and nanowire tentacles have proven to greatly improve sensitivity for both DNA and microRNA detection.

Nanomechanical switching of functional three-dimensional (3D) DNA nanostructures is crucial for nanobiotechnological applications such as nanorobotics or self-regulating sensor and actuator devices. Here, DNA tetrahedral nanostructures self-assembled onto gold electrodes were shown to undergo the electronically addressable nanoswitching due to their mechanical reconfiguration upon external chemical stimuli. That enables construction of robust surface-tethered electronic nanodevices based on 3D DNA tetrahedra. One edge of the tetrahedron contained a partially self-complementary region with a stem-loop hairpin structure, reconfigurable upon hybridization to a complementary DNA (stimulus DNA) sequence. A non-intercalative ferrocene (Fc) redox label was attached to the reconfigurable tetrahedron edge in such a way that reconfiguration of this edge changed the distance between the electrode and Fc.

Dasatinib (BMS-354825) is a FDA-approved multitargeted kinase inhibitor of BCR/ABL and Src kinases. It is now used in the treatment of chronic myelogenous leukemia (CML) with resistance or intolerance to prior therapies, including imatinib. Here we report a novel effect of dasatinib on inducing the differentiation of acute myeloid leukemia (AML) cells through MEK/ERK-dependent activation of signal transducer and activator of transcription 1 (STAT1). We found that dasatinib could induce the differentiation of AML cells as demonstrated by the expression of differentiation marker CD11b, G0/G1 phase arrest and decreased ratio of nucleus to cytoplasm. Of note, dasatinib induced robust phosphorylation of STAT1 both at Tyr701 and Ser727 as well as the redistribution of STAT1 from the cytoplasm to the nucleus, thus leading to the transcription of STAT1-targeted genes. Knocking down STAT1 expression by shRNA significantly attenuated dasatinib-induced differentiation, indicating an important role of STAT1 in myeloid maturation. We further found that dasatinib-induced activation of STAT1 was regulated by the MEK/ERK kinases. The phosporylation of MEK and ERK occurred rapidly upon dasatinib treatment and increased progressively as differentiation was induced. MEK inhibitors PD98059 and U0216 not only inhibited the phosphorylation of STAT1, but also abrogated dasatinib-induced myeloid differentiation, suggesting that MEK/ERK dependent phosphorylation of STAT1 might be indispensable for the differentiating effect of dasatinib in AML cells. Taken together, our study suggests that STAT1 is an important mediator in dasatinib-induced differentiation of AML cells, whose activation requires the activation of MEK/ERK cascades.

Tumour cells primarily utilize aerobic glycolysis for energy production, a phenomenon known as the Warburg effect. Its mechanism is not well understood. The tumour suppressor gene p53 is frequently mutated in tumours. Many tumour-associated mutant p53 (mutp53) proteins not only lose tumour suppressive function but also gain new oncogenic functions that are independent of wild-type p53, defined as mutp53 gain of function (GOF). Here we show that tumour-associated mutp53 stimulates the Warburg effect in cultured cells and mutp53 knockin mice as a new mutp53 GOF. Mutp53 stimulates the Warburg effect through promoting GLUT1 translocation to the plasma membrane, which is mediated by activated RhoA and its downstream effector ROCK. Inhibition of RhoA/ROCK/GLUT1 signalling largely abolishes mutp53 GOF in stimulating the Warburg effect. Furthermore, inhibition of glycolysis in tumour cells greatly compromises mutp53 GOF in promoting tumorigenesis. Thus, our results reveal a new mutp53 GOF and a mechanism for controlling the Warburg effect.

Genome-wide analysis of gene-gene interactions has been recognized as a powerful avenue to identify the missing genetic components that can not be detected by using current single-point association analysis. Recently, several model-free methods (e.g. the commonly used information based metrics and several logistic regression-based metrics) were developed for detecting non-linear dependence between genetic loci, but they are potentially at the risk of inflated false positive error, in particular when the main effects at one or both loci are salient. In this study, we proposed two conditional entropy-based metrics to challenge this limitation. Extensive simulations demonstrated that the two proposed metrics, provided the disease is rare, could maintain consistently correct false positive rate. In the scenarios for a common disease, our proposed metrics achieved better or comparable control of false positive error, compared to four previously proposed model-free metrics. In terms of power, our methods outperformed several competing metrics in a range of common disease models. Furthermore, in real data analyses, both metrics succeeded in detecting interactions and were competitive with the originally reported results or the logistic regression approaches. In conclusion, the proposed conditional entropy-based metrics are promising as alternatives to current model-based approaches for detecting genuine epistatic effects.

MicroRNAs (miRNAs) have been identified as promising cancer biomarkers due to their stable presence in serum. As an alternative to PCR-based homogenous assays, surface-based electrochemical biosensors offer great opportunities for low-cost, point-of-care tests (POCTs) of disease-associated miRNAs. Nevertheless, the sensitivity of miRNA sensors is often limited by mass transport and crowding effects at the water-electrode interface. To address such challenges, we herein report a DNA nanostructure-based interfacial engineering approach to enhance binding recognition at the gold electrode surface and drastically improve the detection sensitivity. By employing this novel strategy, we can directly detect as few as attomolar (

Epidemiological studies strongly suggest that chronic psychological stress promotes tumorigenesis. However, its direct link in vivo and the underlying mechanisms that cause this remain unclear. This study provides direct evidence that chronic stress promotes tumorigenesis in vivo; chronic restraint, a well-established mouse model to induce chronic stress, greatly promotes ionizing radiation (IR)-induced tumorigenesis in p53 +/− mice. The tumor suppressor protein p53 plays a central role in tumor prevention. Loss or attenuation of p53 function contriubutes greatly to tumorigenesis. We found that chronic restraint decreases the levels and function of p53 in mice, and furthermore, promotes the growth of human xenograft tumors in a largely p53-dependent manner. Our results show that glucocorticoids elevated during chronic restraint mediate the effect of chronic restraint on p53 through the induction of serum- and glucocorticoid-induced protein kinase (SGK1), which in turn increases MDM2 activity and decreases p53 function. Taken together, this study demonstrates that chronic stress promotes tumorigenesis in mice, and the attenuation of p53 function is an important part of the underlying mechanism, which can be mediated by glucocortcoids elevated during chronic restraint.

BACKGROUND: The rapid advance in large-scale SNP-chip technologies offers us great opportunities in elucidating the genetic basis of complex diseases. Methods for large-scale interactions analysis have been under development from several sources. Due to several difficult issues (e.g., sparseness of data in high dimensions and low replication or validation rate), development of fast, powerful and robust methods for detecting various forms of gene-gene interactions continues to be a challenging task. METHODOLOGY/PRINCIPAL FINDINGS: In this article, we have developed an evolution-based method to search for genome-wide epistasis in a case-control design. From an evolutionary perspective, we view that human diseases originate from ancient mutations and consider that the underlying genetic variants play a role in differentiating human population into the healthy and the diseased. Based on this concept, traditional evolutionary measure, fixation index (Fst) for two unlinked loci, which measures the genetic distance between populations, should be able to reveal the responsible genetic interplays for disease traits. To validate our proposal, we first investigated the theoretical distribution of Fst by using extensive simulations. Then, we explored its power for detecting gene-gene interactions via SNP markers, and compared it with the conventional Pearson Chi-square test, mutual information based test and linkage disequilibrium based test under several disease models. The proposed evolution-based method outperformed these compared methods in dominant and additive models, no matter what the disease allele frequencies were. However, its performance was relatively poor in a recessive model. Finally, we applied the proposed evolution-based method to analysis of a published dataset. Our results showed that the P value of the Fst -based statistic is smaller than those obtained by the LD-based statistic or Poisson regression models. CONCLUSIONS/SIGNIFICANCE: With rapidly growing large-scale genetic association studies, the proposed evolution-based method can be a promising tool in the identification of epistatic effects.

p53 plays a critical role in tumor suppression. As a transcription factor, in response to stress signals, p53 regulates its target genes and initiates stress responses, including cell cycle arrest, apoptosis, and/or senescence, to exert its function in tumor suppression. Emerging evidence has suggested that p53 is also an important but complex player in the regulation of aging and longevity in worms, flies, mice, and humans. Whereas p53 accelerates the aging process and shortens life span in some contexts, p53 can also extend life span in some other contexts. Thus, p53 appears to regulate aging and longevity in a context-dependent manner. Here, the authors review some recent advances in the study of the role of p53 in the regulation of aging and longevity in both invertebrate and vertebrate models. Furthermore, they discuss the potential mechanisms by which p53 regulates aging and longevity, including the p53 regulation of insulin/TOR signaling, stem/progenitor cells, and reactive oxygen species.

All-trans retinoic acid (ATRA) is nowadays considered to be the sole efficient agent for differentiation-based therapy in leukemia; however, the mechanisms of ATRA's biological effects remain largely unknown. Here we first reported that ATRA-induced myeloid leukemia differentiation was accompanied with the increased level of ubiquitin-protein conjugates and the upregulation of proteasome activity. To explore the functional role of the activated proteasome in retinoic acid (RA) signaling, the effects of proteasome inhibitors on RA-induced cell differentiation were determined. Our results demonstrated that inhibition of ATRA-elevated proteasome activity obviously promoted the myeloid maturation program triggered by ATRA, suggesting that the overactivated proteasome is not beneficial for ATRA's effects. Further studies demonstrated that the synergistic differentiating effects of ATRA and proteasome inhibitors might be associated with the protection of retinoic acid receptor alpha (RARα) from degradation by the ubiquitin-proteasome pathway (UPP). Moreover, the accumulated RARα was able to enhance the transcription of its target gene, which might also contribute to the enhanced differentiation of leukemia cells. Together, by linking the UPP to ATRA-dependent signaling, our data provide a novel insight into studying the mechanisms of ATRA-elicited cellular effects and imply the possibility of combination of ATRA and proteasome inhibitors in leukemia therapy.

Neuroblastoma (NB), the most frequent solid tumor of early childhood, is diagnosed as a disseminated disease in60% of cases, and several lines of evidence support the resistance to apoptosis as a prerequisite for NB progression, and new treatment modalities or potent drugs are further needed. Bortezomib owns a substantial cytotoxicity through regulating degradation of protein associated with cell cycle control and tumor growth. The involvement of bortezomib in neuroblastoma is largely unkown. The aim of this study was to investigate the effects and mechanisms of bortezomib on human neuroblastoma CHP126 cells. Our results indicated that bortezomib inhibits proliferation of neuroblastoma cells in a time- and dose- dependent manner, and the concentration that caused 50% inhibition of CHP126 cells growth was 11.25 nM. Furthermore, bortezomib-induced proliferation inhibition results from massive cell death characterized by apoptosis. Besides, the NFkappaB pathway was not involved in bortezomib treatment in neuroblastoma CHP126 cells, bortezomib-driven apoptotic events were associated with promoting p21 and Bax expression and down-regulating Bcl-2 expression. Ultimately, caspase-3 was activated and the cleavage of PARP was induced. Above all, our data revealed that bortezomib triggered apoptosis by enhancing the caspase 3 activation and/or modulating the Bax/Bcl-2 balance, and also provided preliminary data for further researches of bortezomib on pediatric neuroblastoma.

We performed a genomic study combining single-cell mRNA differential display and RNA subtractive hybridization to elucidate CD8 T-cell quiescence/ignorance. By comparing actively maintained quiescent CD8 T cells from liver tumour tumour-infiltrating lymphocytes (TILs) with quiescent T cells at the single-cell level, we identified differentially expressed candidate genes by high-throughput screening and comparative analysis of expressed sequence tags (ESTs). While genes for the T-cell receptor, tumour necrosis factor (TNF) receptor, TNF-related apoptosis inducing ligand (TRAIL) and perforin were down-regulated, key genes such as Tob, transforming growth factor (TGF)-beta, lung Krüpple-like factor (LKLF), Sno-A, Ski, Myc, Ets-2 repressor factor (ERF) and RE1-silencing transcription factor (REST/NRSF) complex were highly expressed in the quiescent TIL CD8 cells. Real-time polymerase chain reaction (PCR) further confirmed these results. A regulation model is proposed for actively maintained quiescence in CD8 T cells, including three components: up-regulation of the TGF-beta pathway, a shift in the MYC web and inhibition of the cell cycle.

Tumor suppressor p53 plays a crucial role in preventing tumor formation. p53 responds to stress signals and transcriptionally regulates its target genes to start various cellular responses, thus preventing tumor initiation and/or progression. p53 is the most frequently-mutated gene in tumors; approximately 50% of human tumors harbor inactivating mutations in p53. The p53 protein is under tight regulation in cells. Loss or the attenuation of p53 function affects cancer risk and tumor progression. Epidemiological studies strongly suggest that chronic psychological stress has negative influences on the onset, progression and mortality of cancers. Studies employing chronic restraint or social isolation in mice, two well-established chronic stress models, have shown that chronic stress enhances tumor growth and metastasis in xenograft tumor assays. However, the direct evidence that chronic stress promotes tumorigenesis in vivo is still lacking, and furthermore, the molecular mechanisms by which chronic stress promotes tumorigenesis remain unclear. To investigate the impact of chronic stress upon tumorigenesis in vivo, we established a mouse model that combines chronic restraint and ionizing radiation (IR)-induced tumorigenesis. IR induces tumorigenesis in both mice and humans. p53+/- mice are susceptible to IR-induced tumorigenesis; a single dose of 4 Gy IR results in the development of tumors, mainly lymphomas, in p53+/- mice. By employing this mouse model system, we found that in p53+/- C57BL6/J mice, chronic restraint stress greatly promoted IR-induced tumorigenesis; chronic restraint reduced IR-induced tumor latency from ∼49 weeks to ∼38 weeks of median survival age (p=0.004). This result provides direct evidence that chronic stress enhances tumorigenesis in vivo. Furthermore, chronic restraint decreased p53 protein levels and function in mice, and promoted the growth of human xenograft tumors in a largely p53-dependent manner. Chronic restraint greatly promotes the growth rate of HCT116 p53+/+ tumors (increased by 3.77-fold) in mice; and has significantly less pronounced promoting effect on the growth of HCT116 p53-/- tumors (increased by 1.52-fold) (p Citation Format: Cen Zhang, Meihua Lin, Yuhan Zhao, Tongsen Zheng, Jiabei Wang, Arnold Levine, Zhaohui Feng, Wenwei Hu. Chronic stress promotes tumorigenesis through attenuation of p53 function . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 784. doi:10.1158/1538-7445.AM2013-784

Tumor suppressor p53 is frequently mutated in tumors. Mutant p53 (Mutp53) proteins often gain new activities in promoting tumorigenesis, defined as gain-of-function (GOF). Mutp53 often accumulates at high levels in tumors, which promotes mutp53 GOF in tumorigenesis. The mechanism of mutp53 accumulation is poorly understood. Here we find that MDM2 isoforms promote mutp53 accumulation in tumors. MDM2 isoform B (MDM2-B), the MDM2 isoform most frequently over-expressed in human tumors, interacts with full-length MDM2 to inhibit MDM2-mediated mutp53 degradation, promoting mutp53 accumulation and GOF in tumorigenesis. Furthermore, MDM2-B over-expression correlates with mutp53 accumulation in human tumors. In mutp53 knock-in mice, a MDM2 isoform similar to human MDM2-B is over-expressed in the majority of tumors, which promotes mutp53 accumulation and tumorigenesis. Thus, over-expression of MDM2 isoforms promotes mutp53 accumulation in tumors, contributing to mutp53 GOF in tumorigenesis. Furthermore, promoting mutp53 accumulation and GOF is an important mechanism by which MDM2 isoforms promote tumorigenesis.