Glycosylases and AP-cleaving enzymes as a general tool for probe-directed cleavage of ssDNA targets

The current arsenal of molecular tools for site-directed cleavage of single-stranded DNA (ssDNA) is limited. Here, we describe a method for targeted DNA cleavage that requires only the presence of an A nucleotide at the target position. The procedure involves hybridization of a complementary oligonucleotide probe to the target sequence. The probe is designed to create a deliberate G:A mismatch at the desired position of cleavage. The DNA repair enzyme MutY glycosylase recognizes the mismatch structure and selectively removes the mispaired A from the duplex to create an abasic site in the target strand. Addition of an AP-endonuclease, such as Endonuclease IV, subsequently cleaves the backbone dividing the DNA strand into two fragments. With an appropriate choice of an AP-cleaving enzyme, the 3′- and 5′-ends of the cleaved DNA are suitable to take part in subsequent enzymatic reactions such as priming for polymerization or joining by DNA ligation. We define suitable standard reaction conditions for glycosylase/AP-cleaving enzyme (G/AP) cleavage, and demonstrate the use of the method in an improved scheme for in situ detection using target-primed rolling-circle amplification of padlock probes

A dual-tag microarray platform for high-performance nucleic acid and protein analyses

DNA microarrays serve to monitor a wide range of molecular events, but emerging applications like measurements of weakly expressed genes or of proteins and their interaction patterns will require enhanced performance to improve specificity of detection and dynamic range. To further extend the utility of DNA microarray-based approaches we present a high-performance tag microarray procedure that enables probe-based analysis of as little as 100 target cDNA molecules, and with a linear dynamic range close to 105. Furthermore, the protocol radically decreases the risk of cross-hybridization on microarrays compared to current approaches, and it also allows for quantification by single-molecule analysis and real-time on-chip monitoring of rolling-circle amplification. We provide proof of concept for microarray-based measurement of both mRNA molecules and of proteins, converted to tag DNA sequences by padlock and proximity probe ligation, respectively

Searching for a Stochastic Background of Gravitational Waves with LIGO

The Laser Interferometer Gravitational-wave Observatory (LIGO) has performed the fourth science run, S4, with significantly improved interferometer sensitivities with respect to previous runs. Using data acquired during this science run, we place a limit on the amplitude of a stochastic background of gravitational waves. For a frequency independent spectrum, the new limit is $\Omega_{\rm GW} < 6.5 \times 10^{-5}$. This is currently the most sensitive result in the frequency range 51-150 Hz, with a factor of 13 improvement over the previous LIGO result. We discuss complementarity of the new result with other constraints on a stochastic background of gravitational waves, and we investigate implications of the new result for different models of this background.Comment: 37 pages, 16 figure

Tumor-specific HMG-CoA reductase expression in primary premenopausal breast cancer predicts response to tamoxifen

ABSTRACT: INTRODUCTION: We previously reported an association between tumor-specific 3-hydroxy-3-methylglutharyl-coenzyme A reductase (HMG-CoAR) expression and a good prognosis in breast cancer. Here, the predictive value of HMG-CoAR expression in relation to tamoxifen response was examined. METHODS: HMG-CoAR protein and RNA expression was analyzed in a cell line model of tamoxifen resistance using western blotting and PCR. HMG-CoAR mRNA expression was examined in 155 tamoxifen-treated breast tumors obtained from a previously published gene expression study (Cohort I). HMG-CoAR protein expression was examined in 422 stage II premenopausal breast cancer patients, who had previously participated in a randomized control trial comparing 2 years of tamoxifen with no systemic adjuvant treatment (Cohort II). Kaplan-Meier analysis and Cox proportional hazards modeling were used to estimate the risk of recurrence-free survival (RFS) and the effect of HMG-CoAR expression on tamoxifen response. RESULTS: HMG-CoAR protein and RNA expression were decreased in tamoxifen-resistant MCF7-LCC9 cells compared with their tamoxifen-sensitive parental cell line. HMG-CoAR mRNA expression was decreased in tumors that recurred following tamoxifen treatment (P < 0.001) and was an independent predictor of RFS in Cohort I (hazard ratio = 0.63, P = 0.009). In Cohort II, adjuvant tamoxifen increased RFS in HMG-CoAR-positive tumors (P = 0.008). Multivariate Cox regression analysis demonstrated that HMG-CoAR was an independent predictor of improved RFS in Cohort II (hazard ratio = 0.67, P = 0.010), and subset analysis revealed that this was maintained in estrogen receptor (ER)-positive patients (hazard ratio = 0.65, P = 0.029). Multivariate interaction analysis demonstrated a difference in tamoxifen efficacy relative to HMG-CoAR expression (P = 0.05). Analysis of tamoxifen response revealed that patients with ER-positive/HMG-CoAR tumors had a significant response to tamoxifen (P = 0.010) as well as patients with ER-positive or HMG-CoAR-positive tumors (P = 0.035). Stratification according to ER and HMG-CoAR status demonstrated that ER-positive/HMG-CoAR-positive tumors had an improved RFS compared with ER-positive/HMG-CoAR-negative tumors in the treatment arm (P = 0.033); this effect was lost in the control arm (P = 0.138), however, suggesting that HMG-CoAR predicts tamoxifen response. CONCLUSIONS: HMG-CoAR expression is a predictor of response to tamoxifen in both ER-positive and ER-negative disease. Premenopausal patients with tumors that express ER or HMG-CoAR respond to adjuvant tamoxifen

Rapid Identification of Bio-Molecules Applied for Detection of Biosecurity Agents Using Rolling Circle Amplification

Detection and identification of pathogens in environmental samples for biosecurity applications are challenging due to the strict requirements on specificity, sensitivity and time. We have developed a concept for quick, specific and sensitive pathogen identification in environmental samples. Target identification is realized by padlock- and proximity probing, and reacted probes are amplified by RCA (rolling-circle amplification). The individual RCA products are labeled by fluorescence and enumerated by an instrument, developed for sensitive and rapid digital analysis. The concept is demonstrated by identification of simili biowarfare agents for bacteria (Escherichia coli and Pantoea agglomerans) and spores (Bacillus atrophaeus) released in field

The glutathione redox system is essential to prevent ferroptosis caused by impaired lipid metabolism in clear cell renal cell carcinoma

The publisher's final edited version of this article is available at Oncogene. 2018 Oct;37(40):5435-5450. doi: 10.1038/s41388-018-0315-z. Epub 2018 Jun 5. Non-commercial use onlyThis study was funded by Cancer Research UK, the German Research Foundation (FOR2314) and the German Cancer Aid (111917)

DETECTION OF SOLAR-LIKE OSCILLATIONS, OBSERVATIONAL CONSTRAINTS, AND STELLAR MODELS FOR θ CYG, THE BRIGHTEST STAR OBSERVED BY THE KEPLER MISSION

θ Cygni is an F3 spectral type magnitude V = 4.48 main-sequence star that was the brightest star observed by the original Kepler spacecraft mission. Short-cadence (58.8 s) photometric data using a custom aperture were first obtained during Quarter 6 (2010 June–September) and subsequently in Quarters 8 and 12–17. We present analyses of solar-like oscillations based on Q6 and Q8 data, identifying angular degree l = 0, 1, and 2 modes with frequencies of 1000–2700 μHz, a large frequency separation of 83.9 ± 0.4 μHz, and maximum oscillation amplitude at frequency νmax = 1829 ± 54 μHz. We also present analyses of new ground-based spectroscopic observations, which, combined with interferometric angular diameter measurements, give Teff = 6697 ± 78 K, radius 1.49 ± 0.03 Re, [Fe/H] = −0.02 ± 0.06 dex, and log g = 4.23 ± 0.03. We calculate stellar models matching these constraints using the Yale Rotating Evolution Code and the Asteroseismic Modeling Portal. The best-fit models have masses of 1.35–1.39 Me and ages of 1.0–1.6 Gyr. θ Cyg’s Teff and log g place it cooler than the red edge of the γ Doradus instability region established from pre-Kepler ground-based observations, but just at the red edge derived from pulsation modeling. The pulsation models show γ Dor gravity modes driven by the 1 convective blocking mechanism, with frequencies of 1–3 cycles per day (11 to 33 μHz). However, gravity modes were not seen in Kepler data; one signal at 1.776 cycles per day (20.56 μHz) may be attributable to a faint, possibly background, binary