Probing Nucleic Acid Structure with Nickel‐ and Cobalt‐Based Reagents

The use of nickel and cobalt reagents is presented for characterizing the solvent exposure of guanine residues in DNA and RNA. These reagents promote guanine oxidation in the presence of a peracid such as monopersulfate, and the extent of reaction indicates the steric and electronic environment surrounding the N7 and aromatic face of this residue. Since oxidation does not itself perturb target structure or induce strand scission, it is coupled with fragmentation by treatment with piperidine (for smaller polynucleotides) or termination of primer extension (for larger polynucleotides).Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/143767/1/cpnc0604.pd

Dynamics of a DNA mismatch site held in confinement discriminate epigenetic modifications of cytosine

The identification and discrimination of four epigenetic modifications to cytosine in the proposed active demethylation cycle is demonstrated at the single-molecule level, without the need for chemical pretreatment or labeling. The wild-type protein nanopore α-hemolysin is used to capture individual DNA duplexes containing a single cytosine–cytosine mismatch. The mismatch is held at the latch constriction of α-hemolysin, which is used to monitor the kinetics of base-flipping at the mismatch site. Base-flipping and the subsequent interactions between the DNA and the protein are dramatically altered when one of the cytosine bases is replaced with methyl-, hydroxymethyl-, formyl-, or carboxylcytosine. As well as providing a route to single-molecule analysis of important epigenetic markers in DNA, our results provide important insights into how the introduction of biologically relevant, but poorly understood, modifications to cytosine affect the local conformational dynamics of a DNA duplex in a confined environment

Temperature and electrolyte optimization of the α-hemolysin latch sensing zone for detection of base modification in double-stranded DNA

The latch region of the wild-type protein pore α-hemolysin (α-HL) constitutes a sensing zone for individual abasic sites (and furan analogs) in double-stranded DNA (dsDNA). The presence of an abasic site or furan within a DNA duplex, electrophoretically captured in the α-HL vestibule and positioned at the latch region, can be detected based on the current blockage prior to duplex unzipping. We investigated variations in blockage current as a function of temperature (12–35°C) and KCl concentration (0.15–1.0 M) to understand the origin of the current signature and to optimize conditions for identifying the base modification. In 1 M KCl solution, substitution of a furan for a cytosine base in the latch region results in an ∼8 kJ mol−1 decrease in the activation energy for ion transport through the protein pore. This corresponds to a readily measured ∼2 pA increase in current at room temperature. Optimal resolution for detecting the presence of a furan in the latch region is achieved at lower KCl concentrations, where the noise in the measured blockage current is significantly lower. The noise associated with the blockage current also depends on the stability of the duplex (as measured from the melting temperature), where a greater noise in the measured blockage current is observed for less stable duplexes

Energetics of base flipping at a DNA mismatch site confined at the latch constriction of α-hemolysin

Unique, two-state modulating current signatures are observed when a cytosine-cytosine mismatch pair is confined at the 2.4 nm latch constriction of the [small alpha]-hemolysin ([small alpha]HL) nanopore. We have previously speculated that the modulation is due to base flipping at the mismatch site. Base flipping is a biologically significant mechanism in which a single base is rotated out of the DNA helical stack by 180[degree]. It is the mechanism by which enzymes are able to access bases for repair operations without disturbing the global structure of the helix. Here, temperature dependent ion channel recordings of individual double-stranded DNA duplexes inside [small alpha]-HL are used to derive thermodynamic ([capital Delta]H, [capital Delta]S) and kinetic (Ea) parameters for base flipping of a cytosine at an unstable cytosine-cytosine mismatch site. The measured activation energy for flipping a cytosine located at the latch of [small alpha]HL out of the helix (18 +/- 1 kcal mol-1) is comparable to that previously reported for base flipping at mismatch sites from NMR measurements and potential mean force calculations. We propose that the [small alpha]HL nanopore is a useful tool for measuring conformational changes in dsDNA at the single molecule level

Very Early Optical Afterglows of Gamma-Ray Bursts: Evidence for Relative Paucity of Detection

Very early observations with the Swift satellite of gamma-ray burst (GRB) afterglows reveal that the optical component is not detected in a large number of cases. This is in contrast to the bright optical flashes previously discovered in some GRBs (e.g. GRB 990123 and GRB 021211). Comparisons of the X-ray afterglow flux to the optical afterglow flux and prompt gamma-ray fluence is used to quantify the seemingly deficient optical, and in some cases X-ray, light at these early epochs. This comparison reveals that some of these bursts appear to have higher than normal gamma-ray efficiencies. We discuss possible mechanisms and their feasibility for explaining the apparent lack of early optical emission. The mechanisms considered include: foreground extinction, circumburst absorption, Ly-alpha blanketing and absorption due to high redshift, low density environments, rapid temporal decay, and intrinsic weakness of the reverse shock. Of these, foreground extinction, circumburst absorption, and high redshift provide the best explanations for most of the non-detections in our sample. There is tentative evidence of suppression of the strong reverse shock emission. This could be because of a Poynting-flux-dominated flow or a pure non-relativistic hydrodynamical reverse shock.Comment: 22 pages, 5 figures. Accepted for publication in Ap

TBCRC 001: Randomized Phase II Study of Cetuximab in Combination With Carboplatin in Stage IV Triple-Negative Breast Cancer

Epidermal growth factor receptor (EGFR) is a targetable receptor frequently overexpressed in basal-like breast cancer, which comprises most triple-negative breast cancers (TNBCs), the only subtype without established targeted therapy

Estimating the health effects of COVID-19-related immunisation disruptions in 112 countries during 2020-30: a modelling study.

BACKGROUND: There have been declines in global immunisation coverage due to the COVID-19 pandemic. Recovery has begun but is geographically variable. This disruption has led to under-immunised cohorts and interrupted progress in reducing vaccine-preventable disease burden. There have, so far, been few studies of the effects of coverage disruption on vaccine effects. We aimed to quantify the effects of vaccine-coverage disruption on routine and campaign immunisation services, identify cohorts and regions that could particularly benefit from catch-up activities, and establish if losses in effect could be recovered. METHODS: For this modelling study, we used modelling groups from the Vaccine Impact Modelling Consortium from 112 low-income and middle-income countries to estimate vaccine effect for 14 pathogens. One set of modelling estimates used vaccine-coverage data from 1937 to 2021 for a subset of vaccine-preventable, outbreak-prone or priority diseases (ie, measles, rubella, hepatitis B, human papillomavirus [HPV], meningitis A, and yellow fever) to examine mitigation measures, hereafter referred to as recovery runs. The second set of estimates were conducted with vaccine-coverage data from 1937 to 2020, used to calculate effect ratios (ie, the burden averted per dose) for all 14 included vaccines and diseases, hereafter referred to as full runs. Both runs were modelled from Jan 1, 2000, to Dec 31, 2100. Countries were included if they were in the Gavi, the Vaccine Alliance portfolio; had notable burden; or had notable strategic vaccination activities. These countries represented the majority of global vaccine-preventable disease burden. Vaccine coverage was informed by historical estimates from WHO-UNICEF Estimates of National Immunization Coverage and the immunisation repository of WHO for data up to and including 2021. From 2022 onwards, we estimated coverage on the basis of guidance about campaign frequency, non-linear assumptions about the recovery of routine immunisation to pre-disruption magnitude, and 2030 endpoints informed by the WHO Immunization Agenda 2030 aims and expert consultation. We examined three main scenarios: no disruption, baseline recovery, and baseline recovery and catch-up. FINDINGS: We estimated that disruption to measles, rubella, HPV, hepatitis B, meningitis A, and yellow fever vaccination could lead to 49 119 additional deaths (95% credible interval [CrI] 17 248-134 941) during calendar years 2020-30, largely due to measles. For years of vaccination 2020-30 for all 14 pathogens, disruption could lead to a 2·66% (95% CrI 2·52-2·81) reduction in long-term effect from 37 378 194 deaths averted (34 450 249-40 241 202) to 36 410 559 deaths averted (33 515 397-39 241 799). We estimated that catch-up activities could avert 78·9% (40·4-151·4) of excess deaths between calendar years 2023 and 2030 (ie, 18 900 [7037-60 223] of 25 356 [9859-75 073]). INTERPRETATION: Our results highlight the importance of the timing of catch-up activities, considering estimated burden to improve vaccine coverage in affected cohorts. We estimated that mitigation measures for measles and yellow fever were particularly effective at reducing excess burden in the short term. Additionally, the high long-term effect of HPV vaccine as an important cervical-cancer prevention tool warrants continued immunisation efforts after disruption. FUNDING: The Vaccine Impact Modelling Consortium, funded by Gavi, the Vaccine Alliance and the Bill & Melinda Gates Foundation. TRANSLATIONS: For the Arabic, Chinese, French, Portguese and Spanish translations of the abstract see Supplementary Materials section