Enzyme-Free Detection of Mutations in Cancer DNA Using Synthetic Oligonucleotide Probes and Fluorescence Microscopy

Rapid reliable diagnostics of DNA mutations are highly desirable in research and clinical assays. Current development in this field goes simultaneously in two directions: 1) high-throughput methods, and 2) portable assays. Non-enzymatic approaches are attractive for both types of methods since they would allow rapid and relatively inexpensive detection of nucleic acids. Modern fluorescence microscopy is having a huge impact on detection of biomolecules at previously unachievable resolution. However, no straightforward methods to detect DNA in a non-enzymatic way using fluorescence microscopy and nucleic acid analogues have been proposed so far.Here we report a novel enzyme-free approach to efficiently detect cancer mutations. This assay includes gene-specific target enrichment followed by annealing to oligonucleotides containing locked nucleic acids (LNAs) and finally, detection by fluorescence microscopy. The LNA containing probes display high binding affinity and specificity to DNA containing mutations, which allows for the detection of mutation abundance with an intercalating EvaGreen dye. We used a second probe, which increases the overall number of base pairs in order to produce a higher fluorescence signal by incorporating more dye molecules. Indeed we show here that using EvaGreen dye and LNA probes, genomic DNA containing BRAF V600E mutation could be detected by fluorescence microscopy at low femtomolar concentrations. Notably, this was at least 1000-fold above the potential detection limit.Overall, the novel assay we describe could become a new approach to rapid, reliable and enzyme-free diagnostics of cancer or other associated DNA targets. Importantly, stoichiometry of wild type and mutant targets is conserved in our assay, which allows for an accurate estimation of mutant abundance when the detection limit requirement is met. Using fluorescence microscopy, this approach presents the opportunity to detect DNA at single-molecule resolution and directly in the biological sample of choice

Putative β-Barrel Outer Membrane Proteins of the Bovine Digital Dermatitis-Associated Treponemes: Identification, Functional Characterization, and Immunogenicity

Bovine digital dermatitis (BDD), an infectious disease of the bovine foot with a predominant treponemal etiology, is a leading cause of lameness in dairy and beef herds worldwide. BDD is poorly responsive to antimicrobial therapy and exhibits a relapsing clinical course; an effective vaccine is therefore urgently sought. Using a reverse vaccinology approach, the present study surveyed the genomes of the three BDD-associated Treponema phylogroups for putative β-barrel outer membrane proteins and considered their potential as vaccine candidates. Selection criteria included the presence of a signal peptidase I cleavage site, a predicted β-barrel fold, and cross-phylogroup homology. Four candidate genes were overexpressed in Escherichia coli BL21(DE3), refolded, and purified. Consistent with their classification as β-barrel OMPs, circular-dichroism spectroscopy revealed the adoption of a predominantly β-sheet secondary structure. These recombinant proteins, when screened for their ability to adhere to immobilized extracellular matrix (ECM) components, exhibited a diverse range of ligand specificities. All four proteins specifically and dose dependently adhered to bovine fibrinogen. One recombinant protein was identified as a candidate diagnostic antigen (disease specificity, 75%). Finally, when adjuvanted with aluminum hydroxide and administered to BDD-naive calves using a prime-boost vaccination protocol, these proteins were immunogenic, eliciting specific IgG antibodies. In summary, we present the description of four putative treponemal β-barrel OMPs that exhibit the characteristics of multispecific adhesins. The observed interactions with fibrinogen may be critical to host colonization and it is hypothesized that vaccination-induced antibody blockade of these interactions will impede treponemal virulence and thus be of therapeutic value

Incidental placental choriocarcinoma in a term pregnancy: a case report

<p>Abstract</p> <p>Introduction</p> <p>Gestational choriocarcinoma occurs in 1 in 40,000 pregnancies. Of all forms of gestational choriocarcinoma, placental choriocarcinoma is the most rare. Maternal choriocarcinoma is usually diagnosed in symptomatic patients with metastases. The incidental finding of a choriocarcinoma confined to the placenta with no evidence of dissemination to the mother, or infant is the least common scenario.</p> <p>Case presentation</p> <p>The patient is an 18 year-old Gravida 1 Para 1 African American female who delivered a viable 3641 g female infant at 39 weeks gestation. Her pregnancy course was complicated by gestational hypertension during the third trimester. Her placenta revealed intraplacental choriocarcinoma. She was then followed closely by the Gynecologic Oncology service with a weekly serum beta human chorionic gonadotropin value. Beta human chorionic gonadotropin values dropped from 3070 mIU/ml to less than 2 mIU/ml two months post partum. No chemotherapy was initiated. Metastasis was ruled out by chest x-ray and whole body computed tomography scan. To date, both mother and baby are well.</p> <p>Conclusion</p> <p>Due to the potential fatal outcome of placental choriocarcinoma, careful evaluation of both mother and infant after the diagnosis is made is important. The incidence of placental choriocarcinoma may actually be higher than expected since it is not routine practice to send placentas for pathological evaluation after a normal spontaneous delivery. The obstetrician, pathologist, and pediatrician should have an increased awareness of placental choriocarcinoma and its manifestations.</p

External sources of clean technology: evidence from the clean development mechanism

New technology is fundamental to sustainable development. However, inventors from industrialized countries often refuse technology transfer because they worry about reverse-engineering. When can clean technology transfer succeed? We develop a formal model of the political economy of North–South technology transfer. According to the model, technology transfer is possible if (1) the technology in focus has limited global commercial potential or (2) the host developing country does not have the capacity to absorb new technologies for commercial use. If both conditions fail, inventors from industrialized countries worry about the adverse competitiveness effects of reverse-engineering, so technology transfer fails. Data analysis of technology transfer in 4,894 projects implemented under the Kyoto Protocol’s Clean Development Mechanism during the 2004–2010 period provides evidence in support of the model

Planet Hunters VII. Discovery of a New Low-Mass, Low-Density Planet (PH3 c) Orbiting Kepler-289 with Mass Measurements of Two Additional Planets (PH3 b and d)

We report the discovery of one newly confirmed planet ($P=66.06$ days, $R_{\rm{P}}=2.68\pm0.17R_\oplus$) and mass determinations of two previously validated Kepler planets, Kepler-289 b ($P=34.55$ days, $R_{\rm{P}}=2.15\pm0.10R_\oplus$) and Kepler-289-c ($P=125.85$ days, $R_{\rm{P}}=11.59\pm0.10R_\oplus$), through their transit timing variations (TTVs). We also exclude the possibility that these three planets reside in a $1:2:4$ Laplace resonance. The outer planet has very deep ($\sim1.3$), high signal-to-noise transits, which puts extremely tight constraints on its host star's stellar properties via Kepler's Third Law. The star PH3 is a young ($\sim1$ Gyr as determined by isochrones and gyrochronology), Sun-like star with $M_*=1.08\pm0.02M_\odot$, $R_*=1.00\pm0.02R_\odot$, and $T_{\rm{eff}}=5990\pm38$ K. The middle planet's large TTV amplitude ($\sim5$ hours) resulted either in non-detections or inaccurate detections in previous searches. A strong chopping signal, a shorter period sinusoid in the TTVs, allows us to break the mass-eccentricity degeneracy and uniquely determine the masses of the inner, middle, and outer planets to be $M=7.3\pm6.8M_\oplus$, $4.0\pm0.9M_\oplus$, and $M=132\pm17M_\oplus$, which we designate PH3 b, c, and d, respectively. Furthermore, the middle planet, PH3 c, has a relatively low density, $\rho=1.2\pm0.3$ g/cm$^3$ for a planet of its mass, requiring a substantial H/He atmosphere of $2.1^{+0.8}_{-0.3}$ by mass, and joins a growing population of low-mass, low-density planets.Comment: 21 pages, 10 figures, 5 tables, accepted into Ap

Planet Hunters. V. A Confirmed Jupiter-Size Planet in the Habitable Zone and 42 Planet Candidates from the Kepler Archive Data

We report the latest Planet Hunter results, including PH2 b, a Jupiter-size (R_PL = 10.12 \pm 0.56 R_E) planet orbiting in the habitable zone of a solar-type star. PH2 b was elevated from candidate status when a series of false positive tests yielded a 99.9% confidence level that transit events detected around the star KIC 12735740 had a planetary origin. Planet Hunter volunteers have also discovered 42 new planet candidates in the Kepler public archive data, of which 33 have at least three transits recorded. Most of these transit candidates have orbital periods longer than 100 days and 20 are potentially located in the habitable zones of their host stars. Nine candidates were detected with only two transit events and the prospective periods are longer than 400 days. The photometric models suggest that these objects have radii that range between Neptune to Jupiter. These detections nearly double the number of gas giant planet candidates orbiting at habitable zone distances. We conducted spectroscopic observations for nine of the brighter targets to improve the stellar parameters and we obtained adaptive optics imaging for four of the stars to search for blended background or foreground stars that could confuse our photometric modeling. We present an iterative analysis method to derive the stellar and planet properties and uncertainties by combining the available spectroscopic parameters, stellar evolution models, and transiting light curve parameters, weighted by the measurement errors. Planet Hunters is a citizen science project that crowd-sources the assessment of NASA Kepler light curves. The discovery of these 43 planet candidates demonstrates the success of citizen scientists at identifying planet candidates, even in longer period orbits with only two or three transit events.Comment: 35 pages, 11 figures, 6 tables, accepted and published on ApJ ApJ, 776, 1

Planet Hunters X: Searching for Nearby Neighbors of 75 Planet and Eclipsing Binary Candidates from the K2 Kepler Extended Mission

We present high-resolution observations of a sample of 75 K2 targets from Campaigns 1-3 using speckle interferometry on the Southern Astrophysical Research (SOAR) telescope and adaptive optics (AO) imaging at the Keck II telescope. The median SOAR $I$-band and Keck $K_s$-band detection limits at 1" were $\Delta m_{I}=4.4$~mag and $\Delta m_{K_s}=6.1$~mag, respectively. This sample includes 37 stars likely to host planets, 32 targets likely to be eclipsing binaries (EBs), and 6 other targets previously labeled as likely planetary false positives. We find nine likely physically bound companion stars within 3" of three candidate transiting exoplanet host stars and six likely EBs. Six of the nine detected companions are new discoveries; one of the six, EPIC 206061524, is associated with a planet candidate. Among the EB candidates, companions were only found near the shortest period ones ($P<3$ days), which is in line with previous results showing high multiplicity near short-period binary stars. This high-resolution data, including both the detected companions and the limits on potential unseen companions, will be useful in future planet vetting and stellar multiplicity rate studies for planets and binaries.Comment: Accepted in A

Characterization of subsurface media from locations up- and down-gradient of a uranium-contaminated aquifer.

The processing of sediment to accurately characterize the spatially-resolved depth profiles of geophysical and geochemical properties along with signatures of microbial density and activity remains a challenge especially in complex contaminated areas. This study processed cores from two sediment boreholes from background and contaminated core sediments and surrounding groundwater. Fresh core sediments were compared by depth to capture the changes in sediment structure, sediment minerals, biomass, and pore water geochemistry in terms of major and trace elements including pollutants, cations, anions, and organic acids. Soil porewater samples were matched to groundwater level, flow rate, and preferential flows and compared to homogenized groundwater-only samples from neighboring monitoring wells. Groundwater analysis of nearby wells only revealed high sulfate and nitrate concentrations while the same analysis using sediment pore water samples with depth was able to suggest areas high in sulfate- and nitrate-reducing bacteria based on their decreased concentration and production of reduced by-products that could not be seen in the groundwater samples. Positive correlations among porewater content, total organic carbon, trace metals and clay minerals revealed a more complicated relationship among contaminant, sediment texture, groundwater table, and biomass. The fluctuating capillary interface had high concentrations of Fe and Mn-oxides combined with trace elements including U, Th, Sr, Ba, Cu, and Co. This suggests the mobility of potentially hazardous elements, sediment structure, and biogeochemical factors are all linked together to impact microbial communities, emphasizing that solid interfaces play an important role in determining the abundance of bacteria in the sediments

China and the South : objectives, actors and interactions

China has stepped up its engagement of developing countries through both bilateral interactions and the establishment of more formal multilateral fora. To achieve this, China's leaders have established a clear state policy designed to secure much needed resources and to establish an identity as a new and different type of ‘great power’. China has also become a proactive provider of development aid which, when combined with other forms of economic engagement, have raised concerns in the West that China is undermining the promotion of a liberal global order. But we need to take care in considering who, or what, is driving Chinese policy. The institutional weakness of the Ministry of Foreign Affairs means that commercial and financial agencies have dominated the agenda; major state owned enterprises and increasingly large numbers of small, often private enterprises are pursuing their own commercial agendas overseas, which are often not controlled by Beijing. The result is a patchwork of different types of relationships with developing countries, more often driven by the commercial concerns of economic actors than by a coherent diplomatic strategy

The adhesive properties of pyridine-terminated self-assembled monolayers

The atomic force microscopy (AFM) adhesion force behaviour and contact angle titration behaviour of self-assembled monolayers (SAMs) presenting surface pyridine and substituted pyridine moieties has been investigated as a function of pH and electrolyte concentration. The pKas of the pyridine moieties were modified through the incorporation of fluorine, chlorine and bromine substituents in the pyridyl ring. Contact angle titration and AFM adhesion force measurements were performed using aqueous phosphate buffered saline solutions over the pH range 3–9, and at concentrations of 150 mM and 0.1 mM. AFM adhesion force measurements were performed using a clean Si3N4 pyramidal-tipped AFM cantilever