Use of clinical variables for preoperative prediction of lymph node metastasis in endometrial cancer.

OBJECTIVE: Endometrial cancer is the most common gynaecological cancer, and most patients are identified during early disease stages. Noninvasive evaluation of lymph node metastasis likely will improve the quality of clinical treatment, for example, by omitting unnecessary lymphadenectomy. METHODS: The study population comprised 611 patients with endometrial cancer who underwent lymphadenectomy at four types of institutions, comprising seven hospitals in total. We systematically assessed the association of 18 preoperative clinical variables with postoperative lymph node metastasis. We then constructed statistical models for preoperative lymph node metastasis prediction and assessed their performance with a previously proposed system, in which the score was determined by counting the number of high-risk variables among the four predefined ones. RESULTS: Of the preoperative 18 variables evaluated, 10 were significantly associated with postoperative lymph node metastasis. A logistic regression model achieved an area under the curve of 0.85 in predicting lymph node metastasis; this value is significantly higher than that from the previous system (area under the curve, 0.74). When we set the false-negative rate to ~1%, the new predictive model increased the rate of true negatives to 21%, compared with 6.8% from the previous one. We also provide a spreadsheet-based tool for further evaluation of its ability to predict lymph node metastasis in endometrial cancer. CONCLUSIONS: Our new lymph node metastasis prediction method, which was based solely on preoperative clinical variables, performed significantly better than the previous method. Although additional evaluation is necessary for its clinical use, our noninvasive system may help improve the clinical treatment of endometrial cancer, complementing minimally invasive sentinel lymph node biopsy.

SEB genotyping: SmartAmp-Eprimer binary code genotyping for complex, highly variable targets applied to HBV.

BACKGROUND: SmartAmp-Eprimer Binary code (SEB) Genotyping is a novel isothermal amplification method for rapid genotyping of any variable target of interest. METHODS: After in silico alignment of a large number of sequences and computational analysis to determine the smallest number of regions to be targeted by SEB Genotyping, SmartAmp primer sets were designed to obtain a binary code of On/Off fluorescence signals, each code corresponding to a unique genotype. RESULTS: Applied to HBV, we selected 4 targets for which fluorescence amplification signals produce a specific binary code unique to each of the 8 main genotypes (A-H) found in patients worldwide. CONCLUSIONS: We present here the proof of concept of a new genotyping method specifically designed for complex and highly variable targets. Applied here to HBV, SEB Genotyping can be adapted to any other pathogen or disease carrying multiple known mutations. Using simple preparation steps, SEB Genotyping provides accurate results quickly and will enable physicians to choose the best adapted treatment for each of their patients.

Significance of RAS mutations in pulmonary metastases of patients with colorectal cancer.

BACKGROUND: RAS/BRAF mutations of colorectal cancer (CRC) play a crucial role in carcinogenesis and cancer progression and need to be considered for the therapeutic strategy choice. We used next-generation-sequencing (NGS) technology to assess RAS/BRAF mutation differences between primary CRC and corresponding pulmonary metastases (PMs). METHODS: We examined the mutation statuses of the KRAS 12/13/61/146, NRAS 12/13/61/146, and BRAF 600 codons in genomic DNA from fresh-frozen or formalin-fixed paraffin-embedded tissues derived from 34 primary lesions and 52 corresponding PMs from 36 patients with CRC. RESULTS: We found RAS mutations in 76% (26/34) of primary CRC lesions and in 86% (31/36) of PMs. While 27% (7/26) of the primary CRC RAS mutations were heterogeneous, all the RAS mutations in PMs were homogeneous. Of the mutations in PMs, 71% (22/31) were KRAS G>A transitions, of which 82% (18/22) were KRAS G12D or G13D. The RAS mutation discordance between primary tumors and PMs was 12.1% (4/33). RAS mutations with the same genotyping were detected in all synchronous and metachronous PMs from 9 patients. We found no BRAF mutations in either primary or pulmonary tissues. CONCLUSION: Our NGS analysis suggests that RAS mutations of PM of patients with CRC are more common than initially thought. The presence of KRAS mutations in CRC specimens, especially G12D or G13D mutations, seems to promote PM formation.

Identification of chromatin marks at TERRA promoter and encoding region.

TERRA is a long non-coding RNA that is essential for telomere integrity. Although it is transcribed from subtelomeres and telomeres, how it is expressed in heterochromatic region is currently unknown. In this study, we focused our analysis on TERRA-encoding region TelBam3.4 and TelBam3.4-like sequences, and determined their transcription start sites, as well as enrichment of RNA polymerase II and histone modifications. We found that H3K4me3 and H3K9me3 are present at TERRA promoters, whereas H3K27ac and H3K9me3 are present at telomeric repeats. Consistently, we show that presence of active histone modifications H3K4me3 and H3K27ac are correlated to TERRA expression. These results mark an important step towards understanding telomere maintenance and transcription.

Effect of thiazole orange doubly labeled thymidine on DNA duplex formation.

Nucleic acid oligonucleotides are widely used in hybridization experiments for specific detection of complementary nucleic acid sequences. For design and application of oligonucleotides, an understanding of their thermodynamic properties is essential. Recently, exciton-controlled hybridization-sensitive fluorescent oligonucleotides (ECHOs) were developed as uniquely labeled DNA oligomers containing commonly one thymidine having two covalently linked thiazole orange dye moieties. The fluorescent signal of an ECHO is strictly hybridization-controlled, where the dye moieties have to intercalate into double-stranded DNA for signal generation. Here we analyzed the hybridization thermodynamics of ECHO/DNA duplexes, and thermodynamic parameters were obtained from melting curves of 64 ECHO/DNA duplexes measured by ultraviolet absorbance and fluorescence. Both methods demonstrated a substantial increase in duplex stability (ΔΔG°(37) ~ -2.6 ± 0.7 kcal mol(-1)) compared to that of DNA/DNA duplexes of the same sequence. With the exception of T·G mismatches, this increased stability was mostly unaffected by other mismatches in the position opposite the labeled nucleotide. A nearest neighbor model was constructed for predicting thermodynamic parameters for duplex stability. Evaluation of the nearest neighbor parameters by cross validation tests showed higher predictive reliability for the fluorescence-based than the absorbance-based parameters. Using our experimental data, a tool for predicting the thermodynamics of formation of ECHO/DNA duplexes was developed that is freely available at http://genome.gsc.riken.jp/echo/thermodynamics/. It provides reliable thermodynamic data for using the unique features of ECHOs in fluorescence-based experiments.

Isolation of SARS-CoV-2 from COVID-19 Patients and an Asymptomatic Individual.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in Wuhan, China, in December 2019. Despite the recent introduction of vaccines against SARS-CoV-2, more effective vaccines and antiviral drugs must be developed. Here, we isolated five SARS-CoV-2 strains from four patients with coronavirus disease (COVID-19) and an asymptomatic individual using pharyngeal swabs, nasopharyngeal swabs, and sputum samples. Cytopathic effects in inoculated Vero cells were observed between days 3 and 7. SARS-CoV-2 infection was confirmed by quantitative reverse-transcription polymerase chain reaction (RT-qPCR) and next-generation sequencing. Phylogenetic analyses of the whole genome sequences showed that the virus isolates from the clinical samples belonged to the Wuhan and European lineages. These findings and the isolated viruses may contribute to the development of diagnostic tools, vaccines, and antiviral drugs for COVID-19.

Scanning single-molecule counting system for Eprobe with highly simple and effective approach.

Here, we report a rapid and ultra-sensitive detection technique for fluorescent molecules called scanning single molecular counting (SSMC). The method uses a fluorescence-based digital measurement system to count single molecules in a solution. In this technique, noise is reduced by conforming the signal shape to the intensity distribution of the excitation light via a circular scan of the confocal region. This simple technique allows the fluorescent molecules to freely diffuse into the solution through the confocal region and be counted one by one and does not require statistical analysis. Using this technique, 28 to 62 aM fluorescent dye was detected through measurement for 600 s. Furthermore, we achieved a good signal-to-noise ratio (S/N = 2326) under the condition of 100 pM target nucleic acid by only mixing a hybridization-sensitive fluorescent probe, called Eprobe, into the target oligonucleotide solution. Combination of SSMC and Eprobe provides a simple, rapid, amplification-free, and high-sensitive target nucleic acid detection system. This method is promising for future applications to detect particularly difficult to design primers for amplification as miRNAs and other short oligo nucleotide biomarkers by only hybridization with high sensitivity.

Development of a Tet-On Inducible Expression System for the Anhydrobiotic Cell Line, Pv11.

The Pv11 cell line established from an African chironomid, Polypedilum vanderplanki, is the only cell line tolerant to complete desiccation. In Pv11 cells, a constitutive expression system for Pv11 cells was previously exploited and several reporter genes were successfully expressed. Here we report the identification of an effective minimal promoter for Pv11 cells and its application to the Tet-On inducible expression system. First, using a luciferase reporter assay, we showed that a 202 bp deletion fragment derived from the constitutively active 121-promoter functions in Pv11 cells as an appropriate minimal promoter with the Tet-On inducible expression system. The AcGFP1 (Aequorea coerulescens green fluorescent protein) was also successfully expressed in Pv11 cells using the inducible system. In addition to these reporter genes, the avian myeloblastosis virus reverse transcriptase α subunit (AMV RTα), which is one of the most widely commercially available RNA-dependent DNA polymerases, was successfully expressed through the inducible expression system and its catalytic activity was verified. These results demonstrate the establishment of an inducible expression system in cells that can be preserved in the dry state and highlight a possible application to the production of large and complex proteins.

Development of a high-throughput method for the systematic identification of human proteins nuclear translocation potential.

BACKGROUND: Important clues to the function of novel and uncharacterized proteins can be obtained by identifying their ability to translocate in the nucleus. In addition, a comprehensive definition of the nuclear proteome undoubtedly represents a key step toward a better understanding of the biology of this organelle. Although several high-throughput experimental methods have been developed to explore the sub-cellular localization of proteins, these methods tend to focus on the predominant localizations of gene products and may fail to provide a complete catalog of proteins that are able to transiently locate into the nucleus. RESULTS: We have developed a method for examining the nuclear localization potential of human gene products at the proteome scale by adapting a mammalian two-hybrid system we have previously developed. Our system is composed of three constructs co-transfected into a mammalian cell line. First, it contains a PCR construct encoding a fusion protein composed of a tested protein, the PDZ-protein TIP-1, and the transactivation domain of TNNC2 (referred to as ACT construct). Second, our system contains a PCR construct encoding a fusion protein composed of the DNA binding domain of GAL4 and the PDZ binding domain of rhotekin (referred to as the BIND construct). Third, a GAL4-responsive luciferase reporter is used to detect the reconstitution of a transcriptionally active BIND-ACT complex through the interaction of TIP-1 and rhotekin, which indicates the ability of the tested protein to translocate into the nucleus. We validated our method in a small-scale feasibility study by comparing it to green fluorescent protein (GFP) fusion-based sub-cellular localization assays, sequence-based computational prediction of protein sub-cellular localization, and current sub-cellular localization data available from the literature for 22 gene products. CONCLUSION: Our reporter-based system can rapidly screen gene products for their ability to be translocated to the nucleus. Large-scale applications of the system presented herein should provide invaluable information for a more complete biological atlas.

Rapid screening assay for KRAS mutations by the modified smart amplification process.

Previously, the smart amplification process version 2 (SMAP-2) was developed to detect mutations from tissue and in crude cell lysates and has been used for rapid diagnosis of specific somatic mutations with single-nucleotide precision. The purpose of this study was to develop a rapid and practical method to detect cancer and metastasis in specimens using the SMAP-2 assay. We developed modified SMAP-2 assays that enabled detection of any change in a single codon using a single assay. Rapid SMAP-2 screening assays are suitable for routine clinical identification of critical amino acid substitutions such as codon 12 mutations in KRAS. Primers bracketing the first two nucleotides of KRAS codon 12 were designed so that all possible alleles would be amplified by the SMAP-2 assay. In combination with the peptide nucleic acid (PNA) with exact homology to the wild-type allele, our assay amplified all mutant alleles except for the wild-type sequence. With this new assay design (termed PNA-clamp SMAP-2), we could detect KRAS mutations within 60 minutes, including sample preparation. We compared results from PNA-clamp SMAP-2 assay, polymerase chain reaction-restriction fragment length polymorphism, and direct sequencing of clinical samples from pancreatic cancer patients and demonstrated perfect concordance. The PNA-clamp SMAP-2 method is a rapid, simple, and highly sensitive detection assay for cancer mutations.

Sustained effect of continuous positive airway pressure on baroreflex sensitivity in congestive heart failure patients with obstructive sleep apnea.

BACKGROUND: Patients with either heart failure or obstructive sleep apnea have a reduced baroreflex sensitivity for heart rate, a sign of poor prognosis. We previously demonstrated that nocturnal application of continuous positive airway pressure to heart failure patients with obstructive sleep apnea increased baroreflex sensitivity acutely, but it is not known whether these effects persist into wakefulness. OBJECTIVE: To determine whether treating obstructive sleep apnea in heart failure patients with continuous positive airway pressure improves baroreflex sensitivity during wakefulness. METHODS: Spontaneous baroreflex sensitivity was assessed during wakefulness in 33 heart failure patients (left ventricular ejection fraction < or = 45%) with obstructive sleep apnea (apnea-hypopnea index > or = 20). Subsequently, baroreflex sensitivity was reassessed 1 month after patients were randomly allocated to nocturnal continuous positive airway pressure treatment or no treatment (control). RESULTS: Compared with the 14 control patients, the 19 continuous positive airway pressure-treated patients experienced a greater increase in baroreflex sensitivity [median, (25%, 75%)] [from 5.4 (2.2, 8.3) to 7.9 (4.4, 9.4) ms/mmHg; P = 0.01] and left ventricular ejection fraction (P < 0.001). In addition, daytime systolic blood pressure and heart rate decreased more in the continuous positive airway pressure group (from 122 +/- 15 to 113 +/- 12 mmHg; P = 0.02, and from 66 +/- 8 to 62 +/- 8 bpm; P < 0.001, respectively) than in the control group. CONCLUSION: Treatment of coexisting obstructive sleep apnea by continuous positive airway pressure in heart failure patients improves baroreflex sensitivity during wakefulness in addition to improving left ventricular ejection fraction and lowering blood pressure and heart rate. These data indicate that the improved autonomic regulation of heart rate in heart failure patients treated for obstructive sleep apnea during sleep persists into wakefulness.

Continuous positive airway pressure increases heart rate variability in heart failure patients with obstructive sleep apnoea.

Patients with heart failure or OSA (obstructive sleep apnoea) have reduced HF-HRV (high-frequency heart rate variability), indicating reduced cardiac vagal modulation, a marker of poor prognosis. CPAP (continuous positive airway pressure) abolishes OSA in patients with heart failure, but effects on daytime HF-HRV have not been determined. We hypothesized that, in patients with heart failure, treatment of coexisting OSA by CPAP would increase morning HF-HRV. In 19 patients with heart failure (left ventricular ejection fraction <45%) and OSA (>/=20 apnoeas and hypopnoeas/h of sleep), HF-HRV was quantified before and 1 month after randomization to a control or CPAP-treated group. In the control group (n=7), there were no changes in HF-HRV over the 1 month study during wakefulness in the morning. In the CPAP-treated group (n=12) HF-HRV increased significantly during wakefulness in the morning [from 2.43+/-0.55 to 2.82+/-0.50 log(ms(2)/Hz); P=0.002] due to an increase in transfer function between changes in lung volume and changes in HF-HRV (92.37+/-96.03 to 219.07+/-177.14 ms/l; P=0.01). In conclusion, treatment of coexisting OSA by nocturnal CPAP in patients with heart failure increases HF-HRV during morning wakefulness, indicating improved vagal modulation of heart rate. This may contribute to improved prognosis.

Eprobe mediated RT-qPCR for the detection of leukemia-associated fusion genes.

The detection and quantification of leukemia-associated fusion gene transcripts play important roles in the diagnosis and follow-up of leukemias. To establish a standardized method without interlaboratory discrepancies, we developed a novel one-step reverse transcription quantitative PCR (RT-qPCR) assay, called "the Eprobe leukemia assay," for major and minor BCR-ABL1, RUNX1-RUNX1T1, and various isoforms of PML-RARA. This assay is comprised of Eprobes that are exciton-controlled hybridization-sensitive fluorescent oligonucleotides. Melting curve analyses were performed on synthetic quantitative standard RNAs with strict quality control. Quantification capacity was evaluated by comparison with TaqMan RT-qPCR using 67 primary leukemia patient samples. The lower limit of detection and the limit of quantification of this assay were less than 31.3 copies/reaction and 62.5 copies/reaction, respectively. This assay correctly detected the fusion genes in samples with 100% sensitivity and specificity. The specificity of the reactions was confirmed by melting curve analyses. The assay detected low-level expression of minor BCR-ABL1 co-expressed with major BCR-ABL1. These results illustrate the feasibility and high accuracy of the Eprobe leukemia assay, even for minimal residual disease monitoring.

Cardiovascular effects of continuous positive airway pressure in patients with heart failure and obstructive sleep apnea.

BACKGROUND: Obstructive sleep apnea subjects the failing heart to adverse hemodynamic and adrenergic loads and may thereby contribute to the progression of heart failure. We hypothesized that treatment of obstructive sleep apnea by continuous positive airway pressure in patients with heart failure would improve left ventricular systolic function. METHODS: Twenty-four patients with a depressed left ventricular ejection fraction (45 percent or less) and obstructive sleep apnea who were receiving optimal medical treatment for heart failure underwent polysomnography. On the following morning, their blood pressure and heart rate were measured by digital photoplethysmography, and left ventricular dimensions and left ventricular ejection fraction were assessed by echocardiography. The subjects were then randomly assigned to receive medical therapy either alone (12 patients) or with the addition of continuous positive airway pressure (12 patients) for one month. The assessment protocol was then repeated. RESULTS: In the control group of patients who received only medical therapy, there were no significant changes in the severity of obstructive sleep apnea, daytime blood pressure, heart rate, left ventricular end-systolic dimension, or left ventricular ejection fraction during the study. In contrast, continuous positive airway pressure markedly reduced obstructive sleep apnea, reduced the daytime systolic blood pressure from a mean (+/-SE) of 126+/-6 mm Hg to 116+/-5 mm Hg (P=0.02), reduced the heart rate from 68+/-3 to 64+/-3 beats per minute (P=0.007), reduced the left ventricular end-systolic dimension from 54.5+/-1.8 to 51.7+/-1.2 mm (P=0.009), and improved the left ventricular ejection fraction from 25.0+/-2.8 to 33.8+/-2.4 percent (P<0.001). CONCLUSION: In medically treated patients with heart failure, treatment of coexisting obstructive sleep apnea by continuous positive airway pressure reduces systolic blood pressure and improves left ventricular systolic function. Obstructive sleep apnea may thus have an adverse effect in heart failure that can be addressed by targeted therapy.

Use of a competitive probe in assay design for genotyping of the UGT1A1 *28 microsatellite polymorphism by the smart amplification process.

A key feature of the smart amplification process version 2 (SMAP-2) is the ability to suppress mismatch amplification by using a unique asymmetric primer design and Thermus aquaticus MutS (Taq MutS). However we report here that use of SMAP-2 for polymorphism determination of the UGT1A1 *28 allele required a further ancillary approach for complete background suppression. The UGT1A1 *28 allele is a microsatellite copy number polymorphism. This is the first reported SMAP-2 assay designed for genotyping genetic variations of microsatellites. We found that by the addition of a primer to the amplification reaction, called a competitive probe (CP), assay specificity could be significantly enhanced. Including sample preparation time and use of a CP-enhanced SMAP-2 assay, we could rapidly detect the UGT1A1 *28 polymorphism within 60 min. To test our method, we compared results from PCR sequencing and the CP-enhanced SMAP-2 assay on 116 human blood samples for UGT1A1 *28 polymorphism and demonstrated perfect concordance. These results illustrate the versatility of SMAP-2 for molecular diagnostics and provide a new approach for enhancing SMAP-2 assay specificity.

Highly sensitive detection of a HER2 12-base pair duplicated insertion mutation in lung cancer using the Eprobe-PCR method.

Somatic mutation in human epidermal growth factor receptor-related 2 gene (HER2) is one of the driver mutations in lung cancer. HER2 mutations are found in about 2% of lung adenocarcinomas (ADCs). Previous reports have been based mainly on diagnostic screening by Sanger sequencing or next-generation sequencing (NGS); however, these methods are time-consuming and complicated. We developed a rapid, simple, sensitive mutation detection assay for detecting HER2 12 base pair-duplicated insertion mutation based on the Eprobe-mediated PCR method (Eprobe-PCR) and validated the sensitivity of this assay system for clinical diagnostics. We examined 635 tumor samples and analyzed HER2 mutations using the Eprobe-PCR method, NGS, and Sanger sequencing. In a serial dilution study, the Eprobe-PCR was able to detect mutant plasmid DNA when its concentration was reduced to 0.1% by mixing with wild-type DNA. We also confirmed amplification of the mutated plasmid DNA with only 10 copies per reaction. In ADCs, Eprobe-PCR detected the HER2 mutation in 2.02% (9/446), while Sanger sequencing detected it in 1.57% (7/446). Eprobe-PCR was able to detect the mutation in two samples that were undetectable by Sanger sequencing. All non-ADC samples were wild-type. There were no discrepancies between frozen and formalin-fixed paraffin-embedded tissues in the nine samples. HER2 mutations detected by NGS data validated the high sensitivity of the method. Therefore, this new technique can lead to precise molecular-targeted therapies.

Inhibition of awake sympathetic nerve activity of heart failure patients with obstructive sleep apnea by nocturnal continuous positive airway pressure.

OBJECTIVES: This study was designed to determine whether reductions in morning systolic blood pressure (BP) elicited by treatment of moderate to severe obstructive sleep apnea (OSA) in heart failure (HF) patients are associated with a reduction in sympathetic vasoconstrictor tone. BACKGROUND: Daytime muscle sympathetic nerve activity (MSNA) is elevated in HF patients with coexisting OSA. In our recent randomized trial in HF, abolition of OSA by continuous positive airway pressure (CPAP) increased left ventricular ejection fraction (LVEF) and lowered morning systolic BP. METHODS: Muscle sympathetic nerve activity, BP, and heart rate (HR) of medically treated HF patients (EF <45%) and OSA (apnea-hypopnea index > or =20/h of sleep) were recorded on the morning after overnight polysomnography, and again one month after patients were randomly allocated nocturnal CPAP treatment or no CPAP (control). RESULTS: In nine control patients, there were no significant changes in the severity of OSA, MSNA, systolic BP, or HR. In contrast, in the 8 CPAP-treated patients, OSA was attenuated, and there were significant reductions in daytime MSNA (from 58 +/- 4 bursts/min to 48 +/- 5 bursts/min; 84 +/- 4 bursts/100 heart beats to 72 +/- 5 bursts/100 heart beats; p < 0.001 and p = 0.003, respectively), systolic BP (from 135 +/- 5 mm Hg to 120 +/- 6 mm Hg, p = 0.03), and HR (from 69 +/- 2 min(-1) to 66 +/- 2 min(-1); p = 0.013). CONCLUSIONS: Treatment of coexisting OSA by CPAP in HF patients lowers daytime MSNA, systolic BP, and HR. Inhibition of increased central sympathetic vasoconstrictor outflow is one mechanism by which nocturnal CPAP reduces awake BP in HF patients with moderate to severe OSA.

Metabolic syndrome in Japanese patients with obstructive sleep apnea syndrome.

We investigated the prevalence of metabolic syndrome in patients with obstructive sleep apnea syndrome (OSAS) referred to a tertiary university-based medical center. A cross-sectional study of patients with a definite diagnosis of OSAS was performed using new diagnostic criteria for metabolic syndrome that were designed for the Japanese population. Clinical features and comorbidities related to metabolic syndrome were compared between 819 patients with OSAS (719 men and 100 women) and 89 control subjects without OSAS. Metabolic syndrome was significantly more common in the patients with OSAS than in the controls (49.5% vs. 22.0% for men, p < 0.01; 32.0% vs. 6.7% for women, p < 0.01). Men with OSAS (apnea-hypopnea index [AHI] > or =5/h) had a higher risk of metabolic syndrome compared with controls (odds ratio [OR]: 3.47; 95% confidence interval [CI]: 1.84-6.53). There was a significantly increased risk of metabolic syndrome in men with moderate OSAS (AHI: 15-29.9/h) (OR: 2.83; 95% CI: 1.42-5.66) and men with severe OSAS (AHI > or =30/h) (OR: 5.09; 95% CI: 2.67-9.71). Women with OSAS (AHI> or =5/h) also had an increased risk of metabolic syndrome (OR: 6.59; 95% CI: 1.47-29.38), and the risk was significantly higher in women with severe OSAS (AHI > or =30/h) (OR 14.00; 95% CI: 2.93-66.82). Risk factors for metabolic syndrome differed by gender: in men, age, body mass index (BMI), and OSAS (AHI > or =15/h) were significantly associated with metabolic syndrome, whereas, in women, BMI was the only risk factor for metabolic syndrome. The increase of metabolic syndrome in Japanese OSAS patients suggests that this patient population is burdened with multiple risk factors for cardiovascular disease.

Edesign: Primer and Enhanced Internal Probe Design Tool for Quantitative PCR Experiments and Genotyping Assays.

Analytical PCR experiments preferably use internal probes for monitoring the amplification reaction and specific detection of the amplicon. Such internal probes have to be designed in close context with the amplification primers, and may require additional considerations for the detection of genetic variations. Here we describe Edesign, a new online and stand-alone tool for designing sets of PCR primers together with an internal probe for conducting quantitative real-time PCR (qPCR) and genotypic experiments. Edesign can be used for selecting standard DNA oligonucleotides like for instance TaqMan probes, but has been further extended with new functions and enhanced design features for Eprobes. Eprobes, with their single thiazole orange-labelled nucleotide, allow for highly sensitive genotypic assays because of their higher DNA binding affinity as compared to standard DNA oligonucleotides. Using new thermodynamic parameters, Edesign considers unique features of Eprobes during primer and probe design for establishing qPCR experiments and genotyping by melting curve analysis. Additional functions in Edesign allow probe design for effective discrimination between wild-type sequences and genetic variations either using standard DNA oligonucleotides or Eprobes. Edesign can be freely accessed online at http://www.dnaform.com/edesign2/, and the source code is available for download.

Prognostic potential of the MDM2 309T>G polymorphism in stage I lung adenocarcinoma.

The MDM2 protein plays an important role in the regulation of cell proliferation and apoptosis via ubiquitination and proteasome-mediated degradation of p53. The genetic polymorphism rs2279744 (c.309T>G) of the MDM2 gene is reportedly associated with susceptibility and/or prognosis in various cancers. In this study, we investigated the risk factors for worse survival in patients with lung adenocarcinoma (AC). We examined the association between c.309T>G and the prognosis of lung cancer by retrospectively reviewing 453 lung cancer patients. We studied both, clinicopathological and genetic characteristics, including the c.309T>G, p53 Arg72Pro, EGFR, KRAS, and p53 mutations. Associations between these factors and survival outcome were analyzed using Cox proportional hazards models. The frequencies of MDM2 polymorphisms were T/T, 20.8%; T/G, 48.6%, and G/G, 30.7%. The overall survival (OS) of AC patients with pathological stage I disease and the MDM2 T/T genotype was significantly shorter than that of those with the T/G or G/G genotypes (P = 0.02). Multivariate analysis revealed that the MDM2 T/T genotype was an independent, significant prognostic factor (hazard ratio [HR] = 2.23; 95% confidence interval [CI]: 1.07-4.65; P = 0.03). The MDM2 T/T genotype was predictive of poorer survival in a Japanese population. Genotyping for this polymorphism might predict the clinical outcomes of stage I AC patients.