Threshold Cycle Number Research Articles

Respiratory syncytial virus induces leukotriene C4 synthase expression in bronchial epithelial cells

Respiratory syncytial virus (RSV) results in acute wheezing in infants and is frequently associated with recurrent wheezing. Although RSV-induced wheezing clinically resembles that of asthma, corticosteroids are not equivalently effective in RSV-associated wheezing. The study sought to determine the mechanisms of RSV-induced wheezing by establishing an in vitro model of RSV-infected human bronchial epithelial cells (16-HBEC). Leukotriene C4 synthase (LTC4 S) messenger RNA (mRNA) expression in 16-HBEC was detected using fluorescence quantitative polymerase chain reaction, and the relative level of LTC4 S mRNA was expressed as quotient cycle threshold (qCt) based on the threshold cycle number value compared with that of β-actin. Cysteinyl leukotrienes (CysLT) in culture supernatant were measured by enzyme-linked immunosorbent assay. RSV-infected 16-HBEC was incubated with gradient concentration of budesonide (BUD) to assess its effects on LTC4 S expression and CysLT secretion. RSV infection resulted in increased LTC4 S mRNA expression between 48 and 96 h post-infection. High level of CysLT was detected in the supernatant of RSV-infected 16-HBEC. BUD at concentrations of 10(-10) to 10(-5) mol/L did not significantly alter LTC4 S mRNA expression. RSV infection upregulated LTC4 S expression in HBEC leading to increased CysLT secretion. Such induction was not attenuated by BUD, suggesting that CysLT might contribute to the pathogenesis of RSV-induced wheezing.

Elevated Levels of Plasma Mitochondrial DNA DAMPs Are Linked to Clinical Outcome in Severely Injured Human Subjects

Our objective was to execute a prospective cohort study to determine relationships between plasma mtDNA DAMP levels and the occurrence of systemic inflammatory response syndrome (SIRS), multiple organ dysfunction syndrome (MODS), and mortality. Mitochondrial DNA damage-associated molecular patterns (DAMPs) accumulate in the circulation after severe injury. Observations in animal models demonstrate that mtDNA DAMPs contribute to organ dysfunction; however, the link between plasma mtDNA DAMPs and outcome in severely injured human subjects has not been established. DNA was isolated from plasma samples taken from severely injured patients at hospital days 0, 1, and 2. Real-time PCR was used to quantify selected ≈200 base pair sequences of mtDNA within the COX1, ND1, and ND6 genes, as well as from the D-Loop transcriptional regulatory region. MODS was defined as a Denver Multiple Organ Failure score of 4 or greater. MtDNA DAMPs were quantified as PCR threshold cycle number. Lower threshold cycles indicate increased mtDNA DAMP content. Patients with SIRS had significantly increased mtDNA DAMP levels in all 4 sequences examined (32.14 ± 0.90 vs 29.00 ± 1.15 for COX1, 31.90 ± 0.47 vs 30.16 ± 1.42 for ND1, 32.40 ± 0.61 vs 28.94 ± 1.13 for ND6, and 33.12 ± 0.83 vs 28.30 ± 1.14 for D-Loop). Patients who developed MODS also had elevated mtDNA DAMP levels compared with those who did not (32.57 ± 0.74 vs 27.12 ± 0.66 for COX1, 32.45 ± 0.65 vs 28.20 ± 0.73 for ND1, 32.52 ± 0.56 vs 27.60 ± 0.79 for ND6, and 32.85 ± 0.75 vs 27.86 ± 1.27 for D-Loop). Patients with above-median mtDNA DAMP levels had a significantly elevated relative risk for mortality. Four patients died secondary to severe MODS. These findings comprise the first observational evidence that plasma mtDNA DAMPs is associated with the evolution of SIRS, MODS, and mortality in severely injured human subjects.

Bioinformatics and molecular biology for the quantification of closely related bacteria

Molecular biological methods for mixed culture analysis outshine conventional culture-based techniques in terms of better sensitivity and reliability. The majority of these methods exploit the 16S rRNA sequences of the community DNA, which often fall short for the analysis of closely related microorganisms. This research details the development and validation of a comprehensive methodology to differentiate and quantitatively characterize two Pseudomonas species in a mixed culture. A bioinformatics tool based on whole-genome polymorphism comparison was used to identify marker sequences to differentiate the two bacteria using quantitative real-time PCR. The quantification of the two species was achieved through a correlation of the genomic DNA versus cell number (genomic DNA purification) and threshold cycle number versus genomic DNA (real-time PCR). Several factors including the limitation of genomic DNA purification, effects of substrate concentrations and growth phase on cellular DNA, and choice of simplex or duplex reaction for real-time PCR were considered and evaluated. The developed method was experimentally validated against synthetically constructed consortia.

The Auxiliary Subunit KChIP2 Is an Essential Regulator of Homeostatic Excitability

The necessity for, or redundancy of, distinctive KChIP proteins is not known. Deletion of KChIP2 leads to increased susceptibility to epilepsy and to a reduction in IA and increased excitability in pyramidal hippocampal neurons. KChIP2 is essential for homeostasis in hippocampal neurons. Mutations in K(A) channel auxiliary subunits may be loci for epilepsy. The somatodendritic IA (A-type) K(+) current underlies neuronal excitability, and loss of IA has been associated with the development of epilepsy. Whether any one of the four auxiliary potassium channel interacting proteins (KChIPs), KChIP1-KChIP4, in specific neuronal populations is critical for IA is not known. Here we show that KChIP2, which is abundantly expressed in hippocampal pyramidal cells, is essential for IA regulation in hippocampal neurons and that deletion of Kchip2 affects susceptibility to limbic seizures. The specific effects of Kchip2 deletion on IA recorded from isolated hippocampal pyramidal neurons were a reduction in amplitude and shift in the V½ for steady-state inactivation to hyperpolarized potentials when compared with WT neurons. Consistent with the relative loss of IA, hippocampal neurons from Kchip2(-/-) mice showed increased excitability. WT cultured neurons fired only occasional single action potentials, but the average spontaneous firing rate (spikes/s) was almost 10-fold greater in Kchip2(-/-) neurons. In slice preparations, spontaneous firing was detected in CA1 pyramidal neurons from Kchip2(-/-) mice but not from WT. Additionally, when seizures were induced by kindling, the number of stimulations required to evoke an initial class 4 or 5 seizure was decreased, and the average duration of electrographic seizures was longer in Kchip2(-/-) mice compared with WT controls. Together, these data demonstrate that the KChIP2 is essential for physiologic IA modulation and homeostatic stability and that there is a lack of functional redundancy among the different KChIPs in hippocampal neurons.

Quantitative detection of Proteus species by real-time polymerase chain reaction using SYBR Green I

A SYBR Green real-time polymerase chain reaction (PCR) method for rapid detection of Proteus species was developed and evaluated. Of 322 clinical and food samples tested, 75 samples were positive for Proteus species by using conventional PCR and real-time PCR assays. The results were consistent with standard culture methods and the Vitek auto-microbe system, indicating a 100 % specificity obtained by both PCR assays. For the real-time PCR method, the minimum detectable level was 10 colony forming units (CFU) /ml, which was a 103 multiple higher than the conventional PCR method. Correlation coefficients of standard curves which were constructed using the threshold cycle (Ct) versus copy numbers of Proteus showed good linearity (R 2 = 0.997). In conclusion, several significant advantages such as higher sensitivity and rapidness were observed by using the SYBR Green real-time PCR method for identifying Proteus species.

A method for correcting standard-based real-time PCR DNA quantitation when the standard’s polymerase reaction efficiency is significantly different from that of the unknown’s

Standard-based real-time or quantitative polymerase chain reaction quantitation of an unknown sample's DNA concentration (i.e., [DNA](unk)) assumes that the concentration dependence of the standard and unknown reactions (related to reaction efficiency, E) are equivalent. In our work with background food-borne organisms which can interfere with pathogen detection, we have found that it is generally possible to achieve an acceptable E (1 ± 0.05) for standard solutions by optimizing the PCR conditions, template purity, primer sequence, and amplicon lengths. However, this is frequently not true for the solutions containing unknown amounts of target DNA inasmuch as cell extracts are more chemically complex than the standards which have been amplified (2(30)-fold) as well as undergone a purification process. When significant differences in E occur, it is not possible to accurately estimate unknown target DNA concentration from the standard solution's slope and intercept (from threshold cycle number, or C(T), versus Log[DNA] data). What is needed is a standard-mediated intercept which can be specifically coupled with an unknown solution's PCR concentration dependence. In this work, we develop a simple mathematical procedure to generate a new standard curve with a slope (∂C(T)/∂Log[Dilution](unk)) derived from at least three dilutions of the unknown target DNA solution ([DNA](unk)) and an intercept calculated from the unknown's C(T)s, DNA concentrations interpolated from the standard curve (i.e., the traditional estimate of [DNA](unk)), and ∂C(T)/∂Log[Dilution](unk). We were able to achieve this due to our discovery of the predictable way in which the observed and ideal C(T) versus Log[DNA] slopes and intercepts deviate from one another. This "correction" in the standard-based [DNA](unk) determination is typically 20-60% when the difference in the standard and unknown E is >0.1.

Single-machine-based predictive maintenance model considering intelligent machinery prognostics

As an increasing number of manufacturers are beginning to realize the importance of maintaining throughput, many maintenance models have been developed to enable machines to achieve near-zero downtime. However, previous maintenance models usually ignore machine’s deterioration process. Therefore, this paper develops a novel data-driven machinery prognostic approach for machine performance assessment and prediction. With this prognostic information, a predictive maintenance model is proposed for a repairable deteriorating machine. As machine performance can be assessed, once it reaches the maintenance threshold, a maintenance operation is performed to restore the machine. Moreover, an operational cost is introduced to meet real manufacturing process. In this predictive maintenance model, the optimal maintenance threshold and maintenance cycle number are obtained with the aim to minimize the long-term average cost. Finally, a case study is presented. The computational results show the efficiency of this proposed predictive maintenance model.

Quantitative Real-time Polymerase Chain Reaction for Enteropathogenic Escherichia coli: A Tool for Investigation of Asymptomatic Versus Symptomatic Infections

Enteropathogenic Escherichia coli (EPEC) strains are pediatric pathogens commonly isolated from both healthy and sick children with diarrhea in areas of endemicity. The aim of this study was to compare the bacterial load of EPEC isolated from stool samples from children with and without diarrhea to determine whether bacterial load might be a useful tool for further study of this phenomenon. EPEC was detected by polymerase chain reaction (PCR) of colonies isolated on MacConkey plates from 53 diarrheal and 90 healthy children aged <2 years. DNA was isolated from stool samples by cetyltrimethylammonium bromide extraction. To standardize quantification by quantitative real-time PCR (qRT-PCR), the correlation between fluorescence threshold cycle and copy number of the intimin gene of EPEC E2348/69 was determined. The detection limit of qRT-PCR was 5 bacteria/mg stool. The geometric mean load in diarrhea was 299 bacteria/mg (95% confidence interval [CI], 77-1164 bacteria/mg), compared with 29 bacteria/mg (95% CI, 10-87 bacteria/mg) in control subjects (P = .016). Bacterial load was significantly higher in children with diarrhea than in control subjects among children <12 months of age (178 vs 5 bacteria/mg; P = .006) and among children with EPEC as the sole pathogen (463 vs 24 bacteria/mg; P = .006). EPEC load measured by qRT-PCR is higher in diarrheal than in healthy children. qRT-PCR may be useful to study the relationship between disease and colonization in settings of endemicity.

Interleukin-6 (IL-6) Regulates Claudin-2 Expression and Tight Junction Permeability in Intestinal Epithelium

In inflammatory bowel diseases (IBD), intestinal barrier function is impaired as a result of deteriorations in epithelial tight junction (TJ) structure. IL-6, a pleiotropic cytokine, is elevated in IBD patients, although the role of IL-6 in barrier function remains unknown. We present evidence that IL-6 increases TJ permeability by stimulating the expression of channel-forming claudin-2, which is required for increased caudal-related homeobox (Cdx) 2 through the MEK/ERK and PI3K pathways in intestinal epithelial cells. IL-6 increases the cation-selective TJ permeability without any changes to uncharged dextran flux or cell viability in Caco-2 cells. IL-6 markedly induces claudin-2 expression, which is associated with increased TJ permeability. The colonic mucosa of mice injected with IL-6 also exhibits an increase in claudin-2 expression. The claudin-2 expression and TJ permeability induced by IL-6 are sensitive to the inhibition of gp130, MEK, and PI3K. Furthermore, expression of WT-MEK1 induces claudin-2 expression in Caco-2 cells. Claudin-2 promoter activity is increased by IL-6 in a MEK/ERK and PI3K-dependent manner, and deletion of Cdx binding sites in the promoter sequence results in a loss of IL-6-induced promoter activity. IL-6 increases Cdx2 protein expression, which is suppressed by the inhibition of MEK and PI3K. These observations may reveal an important mechanism by which IL-6 can undermine the integrity of the intestinal barrier.

225 GENE MUTATION AND ALTERED EXPRESSION OF NBS1 IN HBV ASSOCIATED HEPATOCELLULAR CARCINOMAS

Background and Aims: The active metabolite of vitamin D3, 1,25-dihydroxyvitamin D33) inhibits cell growth and induces cell differentiation and apoptosis in numerous tumors, such as colon, breast and prostate cancers. The role of 1,25-D3 in the development of the hepatocellular carcinoma has not yet been established. The aim of the present study was to examine the gene expression of the vitamin D inactivating CYP24A1 as well as the activating 1-alpha-hydroxylase (CYP27B1) enzyme after 1,25-D3 treatment in hepatocellular carcinoma cell lines (1,25-D). Methods: HepG2 and HUHneo (Dept. of Molecular Virology, Univ. of Heidelberg) cells were treated with 4nM concentrations of 1,25-D3 in Optimem medium and were incubated in 24-well plates in two parallels for treatment duration of 30, 60, 120 minutes, 5, 8, 10, 12, 14, 24, 26, 28 hours. Total RNA was isolated from each sample with Roche High Pure Total RNA Isolation kit. Five hundred ng of total RNA was reverse-transcribed to cDNA. The expression differences of selected genes were analyzed by Taqman probe-based quantitative real-time PCR. Relative quantification was carried out from collected data (threshold cycle numbers) by Applied Biosystems 7500 System SDS software 1.3. Results: CYP24A1 mRNA expression significantly (p < 0.001) increased in response to 1,25-D3 administration in a timedependent manner in both cell lines. In HepG2 cell line the CYP24A1 mRNA expression showed 5300-fold elevation reaching the maximum value at 8 hours. In the HUHneo cells the increase was 152-fold of the baseline level, and the curve reached the maximum at 14 hours. There was no change in CYP27B1 gene activity of the cells treated with 1,25-D3. The activating effect of vitamin D3 on CYP24 mRNA expression was at 28 hours still detectable. Conclusions: Our novel data raise the possibility that the significant increase in the CYP24A1 gene expression of hepatocellular carcinoma cell lines following vitamin D administration may stimulate the inactivation of 1,25-D3, thus “protecting” the tumor cells against the anti-cancer effects of 1,25-D3. This is the first report on the role of vitamin D3 on the CYP24A1 mRNA transcription in hepatocellular carcinoma cells in vitro.

Estrogen Receptor Expression in Posterior Tibial Tendon Dysfunction: A Pilot Study

The pathophysiology of posterior tibial tendon dysfunction (PTTD) is poorly understood. It has been theorized that changes in hormone physiology may be a factor influencing tendon health. Estrogen's influence on the fibroblast has been studied in other musculoskeletal tissues. Gender differences in anterior cruciate ligament (ACL) injuries have been studied and it has been discovered that the Estrogen receptor (ER) as well as Progesterone receptor (PR) are expressed in the ACL. Eight patients with PTTD requiring surgery were enrolled in our pilot study. The mean patient age was 52.4 (range, 18 to 73) years. There were five female and three male patients. Tendon samples were harvested from diseased PTT. Tendon samples harvested from healthy PTT and healthy flexor digitorum longus (FDL) tendon were used as controls. Tendon samples were processed using specific protocols for total RNA isolation from hypocellular, dense connective tissues. ERα and ERβ transcripts were quantified using real time RT-PCR. Quantitative values were obtained from the threshold cycle (Ct) number at which the increase in fluorescent signal associated with an exponential increase of PCR products can be detected. Transcripts of both ERα and ERβ were reproducibly detected in RNA samples isolated from our tendon samples. There was no difference in receptor expression between diseased and control tendon samples. There was no difference in receptor expression between male and female patients. We found that the tenocyte of the PTT and FDL tendons express ERα and ERβ. Normal and diseased tendons of both male and female patients expressed both estrogen receptors. Identifying ERα and ERβ gene expression in the fibroblast was an initial step in discovering whether tenocytes are targets for estrogen function. Estrogen receptors were identified indirectly by measuring receptor gene expression but we were unable to show a significant difference between diseased and control tendons.

Effects of the recipe for activating blood circulation and nourishing qi on expression of mitochondrial ribosomal protein L51 in CVB3 infection model in rat cardiac myocytes

Objective To study the effects of the recipe for activating blood circulation and nourishing qi on expression of mitochondrial ribosomal protein L51 (MRPL51) in CVB3 infection model in rat cardiac myocytes, to reveal the pathogenesis of CVB3 myocarditis in the genetic level, to explore the therapeutic mechanism of the recipe for activating blood circulation and nourishing qi on CVB3 myocarditis, and to confirm the validity of the recipe for activating blood circulation and nourishing qi on CVB3 myocarditis. Methods After establishing CVB3 infection model and treatment model with recipe for activating blood circulation and nourishing qi by culturing neonatal rat myocardial cells, a modified suppression subtractive hybridization (SSH) was used to isolate differentially expressed genes between two model groups. These results were further verified by fluorescence RT-PCR. Results The results of SSH showed that gene expression of the treatment group was lower than that of the CVB3 infection group. The results of fluorescent RT-PCR which agreed with that of SSH displayed the threshold cycle number (Ct) in the treatment group was higher than the virus group. Conclusion Up-regulation of MRPL51 might be one of the pathogenesis of CVB3 myocarditis. The recipe for activating blood circulation and nourishing qi could treat viral myocarditis by regulating the expression of MRPL51. Key words: Viral myocarditis ; Recipe for activating blood circulation and nourishing qi ; Suppression subtractive hybridization (SSH) ; Mitochondrial ribosomal protein L51 (MRPL51) ;

Notch and Transforming Growth Factor-β (TGFβ) Signaling Pathways Cooperatively Regulate Vascular Smooth Muscle Cell Differentiation

Notch and transforming growth factor-beta (TGFbeta) play pivotal roles during vascular development and the pathogenesis of vascular disease. The interaction of these two pathways is not fully understood. The present study utilized primary human smooth muscle cells (SMC) to examine molecular cross-talk between TGFbeta1 and Notch signaling on contractile gene expression. Activation of Notch signaling using Notch intracellular domain or Jagged1 ligand induced smooth muscle alpha-actin (SM actin), smooth muscle myosin heavy chain, and calponin1, and the expression of Notch downstream effectors hairy-related transcription factors. Similarly, TGFbeta1 treatment of human aortic smooth muscle cells induced SM actin, calponin1, and smooth muscle protein 22-alpha (SM22alpha) in a dose- and time-dependent manner. Hairy-related transcription factor proteins, which antagonize Notch activity, also suppressed the TGFbeta1-induced increase in SMC markers, suggesting a general mechanism of inhibition. We found that Notch and TGFbeta1 cooperatively activate SMC marker transcripts and protein through parallel signaling axes. Although the intracellular domain of Notch4 interacted with phosphoSmad2/3 in SMC, this interaction was not observed with Notch1 or Notch2. However, we found that CBF1 co-immunoprecipitated with phosphoSmad2/3, suggesting a mechanism to link canonical Notch signaling to phosphoSmad activity. Indeed, the combination of Notch activation and TGFbeta1 treatment led to synergistic activation of a TGFbeta-responsive promoter. This increase corresponded to increased levels of phosphoSmad2/3 interaction at Smad consensus binding sites within the SM actin, calponin1, and SM22alpha promoters. Thus, Notch and TGFbeta coordinately induce a molecular and functional contractile phenotype by co-regulation of Smad activity at SMC promoters.

Serious overestimation in quantitative PCR by circular (supercoiled) plasmid standard: microalgal pcna as the model gene.

Quantitative real-time PCR (qPCR) has become a gold standard for the quantification of nucleic acids and microorganism abundances, in which plasmid DNA carrying the target genes are most commonly used as the standard. A recent study showed that supercoiled circular confirmation of DNA appeared to suppress PCR amplification. However, to what extent to which different structural types of DNA (circular versus linear) used as the standard may affect the quantification accuracy has not been evaluated. In this study, we quantitatively compared qPCR accuracies based on circular plasmid (mostly in supercoiled form) and linear DNA standards (linearized plasmid DNA or PCR amplicons), using proliferating cell nuclear gene (pcna), the ubiquitous eukaryotic gene, in five marine microalgae as a model gene. We observed that PCR using circular plasmids as template gave 2.65-4.38 more of the threshold cycle number than did equimolar linear standards. While the documented genome sequence of the diatom Thalassiosira pseudonana shows a single copy of pcna, qPCR using the circular plasmid as standard yielded an estimate of 7.77 copies of pcna per genome whereas that using the linear standard gave 1.02 copies per genome. We conclude that circular plasmid DNA is unsuitable as a standard, and linear DNA should be used instead, in absolute qPCR. The serious overestimation by the circular plasmid standard is likely due to the undetected lower efficiency of its amplification in the early stage of PCR when the supercoiled plasmid is the dominant template.

A rapid real-time PCR assay for the detection of HIV-1 proviral DNA using double-stranded primer

In this study, a rapid real-time PCR assay to detect HIV-1 proviral DNA in whole blood was developed using a novel double-stranded primer that does not require a target-specific fluorescent probe or intercalating dye systems. Co-amplification of a human gene RNase P served as the internal control to monitor the efficiency of the DNA extraction and PCR amplification. The HIV-1 amplification efficiency was 100% and could amplify 1 copy of HIV-1 DNA 64% of the time and all attempts to amplify 4 copies were successful in less than 51 min. All 22 HIV-1 sero-positive and 20 sero-negative whole blood specimens tested were classified correctly by this assay. In addition, 22 cultured PBMC specimens infected with various HIV-1 subtypes or CRF (A = 2, AC = 1, B = 4, C = 3, D = 3, AE = 2, F = 1, BF = 2, G = 4) were amplified equally well with a similar threshold cycle (C t) number (22.9 ± 1.2). The high amplification efficiency and short PCR cycles were in part due to the short target sequence amplified by eliminating the probe-binding sequence between the primers. This assay may be useful as an alternative confirmation test in a variety of HIV testing venues.

Mutations in LTBP4 Cause a Syndrome of Impaired Pulmonary, Gastrointestinal, Genitourinary, Musculoskeletal, and Dermal Development

We report recessive mutations in the gene for the latent transforming growth factor-beta binding protein 4 (LTBP4) in four unrelated patients with a human syndrome disrupting pulmonary, gastrointestinal, urinary, musculoskeletal, craniofacial, and dermal development. All patients had severe respiratory distress, with cystic and atelectatic changes in the lungs complicated by tracheomalacia and diaphragmatic hernia. Three of the four patients died of respiratory failure. Cardiovascular lesions were mild, limited to pulmonary artery stenosis and patent foramen ovale. Gastrointestinal malformations included diverticulosis, enlargement, tortuosity, and stenosis at various levels of the intestinal tract. The urinary tract was affected by diverticulosis and hydronephrosis. Joint laxity and low muscle tone contributed to musculoskeletal problems compounded by postnatal growth delay. Craniofacial features included microretrognathia, flat midface, receding forehead, and wide fontanelles. All patients had cutis laxa. Four of the five identified LTBP4 mutations led to premature termination of translation and destabilization of the LTBP4 mRNA. Impaired synthesis and lack of deposition of LTBP4 into the extracellular matrix (ECM) caused increased transforming growth factor-beta (TGF-beta) activity in cultured fibroblasts and defective elastic fiber assembly in all tissues affected by the disease. These molecular defects were associated with blocked alveolarization and airway collapse in the lung. Our results show that coupling of TGF-beta signaling and ECM assembly is essential for proper development and is achieved in multiple human organ systems by multifunctional proteins such as LTBP4.

Lymphocytic mitochondrial DNA deletions, biochemical folate status and hepatocellular carcinoma susceptibility in a case–control study

Mitochondrial (mt) DNA deletions and low folate status, proposed characteristics of carcinogenesis, in relation to human hepatocellular carcinoma (HCC) susceptibility are not clearly understood. We hypothesised that low folate status may modify frequencies of mtDNA deletions in humans, both of which could predispose individuals to HCC development. Biochemical folate status of serum and lymphocytes, and frequencies of mtDNA deletions in lymphocytes were determined in ninety HCC cases and ninety cancer-free healthy controls, individually matched by age and sex. The data revealed that HCC patients had lower levels of serum folate (P = 0.0002), lymphocytic folate (P = 0.040) and accumulated higher frequency of lymphocytic mtDNA deletions (P < 0.0001) than the controls. In the total studied subjects, frequencies of lymphocytic mtDNA deletions were associated with hepatitic B infection (P = 0.004) and HCC incidents (P = 0.001), and were correlated with serum folate (r - 0.155; P = 0.041), lymphocyte folate (r - 0.314; P = 0.0001), levels of glutamate-oxaloacetate transaminase (GOT) (r 0.206; P = 0.006), glutamate-pyruvate transaminase (GPT) (r 0.163; P = 0.037) and alpha-fetal protein concentrations (r 0.212; P = 0.005). After adjustment for age, sex, lifestyle and one-carbon metabolite factors, individuals with low blood folate ( < 11.5 nmol/l) or high mtDNA deletions (Delta threshold cycle number (Ct)>5.3) had increased risks for HCC (OR 7.7, 95 % CI 1.9, 29.9, P = 0.003; OR 5.4; 95 % CI 1.7, 16.8, P = 0.003, respectively). When combined with folate deficiency (serum folate < 14 nmol/l), the OR of HCC in individuals with high levels of lymphocytic mtDNA deletions was enhanced (OR 13.3; 95 % CI 1.45, 122; P = 0.008). Further controlling for GOT and GPT levels, however, negated those effects on HCC risk. Taken together, the data suggest that biochemical folate status and liver injuries are important modulators to lymphocytic mtDNA deletions. The mt genetic instability that results from a high rate of mtDNA deletions and/or low folate status increased the risk for HCC, which is mediated by clinical hepatic lesions.

Transcriptional Regulation of the Novel Toll-like Receptor Tlr13

Little has been known about Tlr13 (Toll-like receptor 13), a novel member of the Toll-like receptor family. To elucidate the molecular basis of murine Tlr13 gene expression, the activity of the Tlr13 gene promoter was characterized. Reporter gene analysis and electrophoretic mobility shift assays demonstrated that Tlr13 gene transcription was regulated through three cis-acting elements that interacted with the Ets2, Sp1, and PU.1 transcription factors. Furthermore, our work suggests that these transcription factors may cooperate, culminating in maximal transcription of the Tlr13 gene. In contrast, NF-kappaB appeared to act as an inhibitor of Tlr13 transcription. Overexpression of Ets2 caused a strong increase in the transcriptional activity of the Tlr13 promoter; however, overexpression of NF-kappaB p65 dramatically inhibited it. Additionally, interferon-beta is capable of acting Tlr13 transcription, but the activated signaling of lipopolysaccharide/TLR4 and peptidoglycan/TLR2 strongly inhibited the Tlr13 gene promoter. Thus, these findings reveal the mechanism of Tlr13 gene regulation, thereby providing insight into the function of Tlr13 in the immune response to pathogen.

Activation-induced Degradation of FLIPL Is Mediated via the Phosphatidylinositol 3-Kinase/Akt Signaling Pathway in Macrophages

Cellular FLIP (Flice-like inhibitory protein) is critical for the protection against death receptor-mediated cell apoptosis. In macrophages, FLIP long (FLIP(L)) and FLIP short (FLIP(S)) mRNA was induced by tumor necrosis factor (TNF) alpha, mediated through NF-kappaB. However, we observed TNFalpha reduced the protein level of FLIP(L), but not FLIP(S), at 1 and 2 h. Similar results were observed with lipopolysaccharide. The reduction of FLIP(L) by TNFalpha was not mediated by caspase 8, or through JNK or Itch, but was suppressed by inhibition of the phosphatidylinositol 3-kinase/Akt pathway employing chemical inhibitors, a dominant negative Akt-1, or Akt-1 small interfering RNA. The reduction of FLIP(L) resulted in the short term induction of caspase 8-like activity, which augmented NF-kappaB activation. A co-immunoprecipitation assay demonstrated that Akt-1 physically interacts with FLIP(L). Moreover, TNFalpha enhanced FLIP(L) serine phosphorylation, which was increased by activated Akt-1. Serine 273, a putative Akt-1 phosphorylation site in FLIP(L), was critical for the activation-induced reduction of FLIP(L). Thus, these observations document a novel mechanism where by TNFalpha facilitates the reduction of FLIP(L) protein, which is dependent on the phosphatidylinositol 3-kinase/Akt signaling.

A qRT-PCR-based method for the measurement ofrrnoperon copy number

To develop a convenient and accurate method for estimating the rrn operon copy number (Y(rrn)) in cells of pure prokaryotic cultures based on quantitative real-time polymerase chain reaction (qRT-PCR). Using Escherichia coli, the Y(rrn) of which is known to be 7, as a reference, the rrn concentrations of target species and E. coli in sample solutions were measured based on their respective threshold cycle numbers (C(t)), whereas the cell concentrations of both species were measured by microscopic counting after staining. The Y(rrn) of the target species was then calculated from the initial cell concentrations and the rrn concentrations of the target species and E. coli. Using this method, the Y(rrn) values of four species, i.e. Xanthomonas campestris, Staphylococcus aureus, Aeromonas hydrophila and Pseudomonas fluorescens, were estimated as 1.80, 4.73, 8.58 and 5.13, respectively, comparable to their respective known values of 2, 5, 10, and 5, resulting in an average deviation of 8%. The whole cell qRT-PCR based methods were convenient, accurate and reproducible in quantification of rrn copy number of prokaryotic cells. qTR-PCR is a fast and reliable DNA quantification approach. Compared with previous qTR-PCR based methods measuring rrn copy number, the present method avoided the prerequisite for the information on genome size and GC content of target bacteria or a gene with known copy number, thus should be more widely applicable.