Binary recombinase systems for high-resolution conditional mutagenesis.

Conditional mutagenesis using Cre recombinase expressed from tissue specific promoters facilitates analyses of gene function and cell lineage tracing. Here, we describe two novel dual-promoter-driven conditional mutagenesis systems designed for greater accuracy and optimal efficiency of recombination. Co-Driver employs a recombinase cascade of Dre and Dre-respondent Cre, which processes loxP-flanked alleles only when both recombinases are expressed in a predetermined temporal sequence. This unique property makes Co-Driver ideal for sequential lineage tracing studies aimed at unraveling the relationships between cellular precursors and mature cell types. Co-InCre was designed for highly efficient intersectional conditional transgenesis. It relies on highly active trans-splicing inteins and promoters with simultaneous transcriptional activity to reconstitute Cre recombinase from two inactive precursor fragments. By generating native Cre, Co-InCre attains recombination rates that exceed all other binary SSR systems evaluated in this study. Both Co-Driver and Co-InCre significantly extend the utility of existing Cre-responsive alleles.

Stable isotope response to lake eutrophication: Calibration of a high-resolution lacustrine sequence from Baldeggersee, Switzerland

Stable isotope analyses of discrete seasonal layers from a 108-yr annually laminated freeze-core from Baldeg-gersee, a small, eutrophic lake in central Switzerland, provide information on the climatological and environmental factors, including lake eutrophication, that control oxygen and carbon isotopic composition of epilimnic biologically induced calcite precipitate. During the last 100 yr, Baldeggersee has undergone major increases in productivity and eutrophication in response to nutrient loading from agriculture and industrialization in the lake's watershed. Calibration of the isotopic signal in Baldeggersee to historical limnological data quantitatively links evidence of isotopic depletion in the sedimented calcite to trophic state of the lake. δ18O values from the spring/summer “light” sediment layers steadily diverged to more depleted values in response to historical eutrophication: measured δ18O values were up to 21.5‰ more negative than calculated equilibrium δ18O values. Evidence for 13C depletion in the calcite, relative to equilibrium values, is more difficult to ascertain because of an overall dominance of isotopic enrichment in the dissolved inorganic pool as productivity in Baldeggersee increases. A positive association exists between the degree of oxygen-18 depletion and the calcite crystal size. Thus, large amorphous calcite grains can be used as a proxy for recognizing apparent isotopic nonequilibrium in sediment sequences from highly productive lacustrine environments from all geologic time scales. In contrast to the light layers, the oxygen isotopic composition of the calcite in the late summer/fall “dark” sediment layers is unaffected by the apparent isotope nonequilibrium. Oxygen and carbon isotope values from the dark laminae in the Baldeggersee sediment therefore provide environmental and climatological proxies that can be calibrated with known environmental and regional climate data for the last century.

Characterization of the Abydos region through OSIRIS high-resolution images in support of CIVA measurements

Context. On 12 November 2014, the European mission Rosetta delivered the Philae lander on the nucleus of comet 67P /Churyumov-Gerasimenko (67P). After the first touchdown, the lander bounced three times before finally landing at a site named Abydos. Aims. We provide a morphologically detailed analysis of the Abydos landing site to support Philae's measurements and to give context for the interpretation of the images coming from the Comet Infrared and Visible Analyser (CIVA) camera system onboard the lander. Methods. We used images acquired by the OSIRIS Narrow Angle Camera (NAC) on 6 December 2014 to perform the analysis of the Abydos landing site, which provided the geomorphological map, the gravitational slope map, the size-frequency distribution of the boulders. We also computed the albedo and spectral reddening maps. Results. The morphological analysis of the region could suggest that Philae is located on a primordial terrain. The Abydos site is surrounded by two layered and fractured outcrops and presents a 0.02 km(2) talus deposit rich in boulders. The boulder size frequency distribution gives a cumulative power-law index of 4.0 + 0.3/0.4, which is correlated with gravitational events triggered by sublimation and /or thermal fracturing causing regressive erosion. The average value of the albedo is 5.8% at lambda(1) = 480.7 nm and 7.4% at lambda(2) = 649.2 nm, which is similar to the global albedos derived by OSIRIS and CIVA, respectively.

A Multiplex Real-Time PCR with High Resolution Melting Analysis for the Characterization of Antimicrobial Resistance in Neisseria gonorrhoeae.

Resistance to antibiotics used against Neisseria gonorrhoeae infections is a major public health concern. Antimicrobial resistance (AMR) testing relies on time-consuming culture-based methods. Development of rapid molecular tests for detecting AMR determinants could provide valuable tools for surveillance, epidemiological studies and to inform individual case management. We developed a fast (<1.5 hrs) SYBR-green based real-time PCR method with high resolution melting (HRM) analysis. One triplex and three duplex reactions included two sequences for N. gonorrhoeae identification and seven determinants of resistance to extended-spectrum cephalosporins (ESCs), azithromycin, ciprofloxacin, and spectinomycin. The method was validated by testing 39 previously fully-characterized N. gonorrhoeae strains, 19 commensal Neisseria spp., and an additional panel of 193 gonococcal isolates. Results were compared with culture-based AMR determination. The assay correctly identified N. gonorrhoeae and the presence or absence of the seven AMR determinants. There was some cross-reactivity with non-gonococcal Neisseria species and the detection limit was 10(3)-10(4) gDNA copies/reaction. Overall, the platform accurately detected resistance to ciprofloxacin (sensitivity and specificity, 100%), ceftriaxone (sensitivity 100%, specificity 90%), cefixime (sensitivity 92%, specificity 94%), azithromycin and spectinomycin (both sensitivity and specificity, 100%). In conclusion, our methodology accurately detects mutations generating resistance to antibiotics used to treat gonorrhea. Low assay sensitivity prevents direct diagnostic testing of clinical specimens but this method can be used to screen collections of gonococcal isolates for AMR more quickly than with current culture-based AMR testing.

Epileptogenic developmental venous anomaly: insights from simultaneous EEG/fMRI

Developmental venous anomalies (DVAs) are associated with epileptic seizures; however, the role of DVA in the epileptogenesis is still not established. Simultaneous interictal electroencephalogram/functional magnetic resonance imaging (EEG/fMRI) recordings provide supplementary information to electroclinical data about the epileptic generators, and thus aid in the differentiation of clinically equivocal epilepsy syndromes. The main objective of our study was to characterize the epileptic network in a patient with DVA and epilepsy by simultaneous EEG/fMRI recordings. A 17-year-old woman with recently emerging generalized tonic-clonic seizures, and atypical generalized discharges, was investigated using simultaneous EEG/fMRI at the university hospital. Previous high-resolution MRI showed no structural abnormalities, except a DVA in the right frontal operculum. Interictal EEG recordings showed atypical generalized discharges, corresponding to positive focal blood oxygen level dependent (BOLD) correlates in the right frontal operculum, a region drained by the DVA. Additionally, widespread cortical bilateral negative BOLD correlates in the frontal and parietal lobes were delineated, resembling a generalized epileptic network. The EEG/fMRI recordings support a right frontal lobe epilepsy, originating in the vicinity of the DVA, propagating rapidly to both frontal and parietal lobes, as expressed on the scalp EEG by secondary bilateral synchrony. The DVA may be causative of focal epilepsies in cases where no concomitant epileptogenic lesions can be detected. Advanced imaging techniques, such as simultaneous EEG/fMRI, may thus aid in the differentiation of clinically equivocal epilepsy syndromes.

Examining the gateway to the limbic system with diffusion tensor imaging: the perforant pathway in dementia

Current treatments for Alzheimer's disease (AD) are only able to slow the progression of mental deterioration, making early and reliable diagnosis an essential part of any promising therapeutic strategy. In the initial stages of AD, the first neuropathological alterations occur in the perforant pathway (PP), a large neuronal fiber tract located at the entrance to the limbic system. However, to date, there is no sensitive diagnostic tool for performing in vivo assessments of this structure. In the present bimodal magnetic resonance imaging (MRI) study, we examined 10 elderly controls, 10 subjects suffering from mild cognitive impairment (MCI), and 10 AD patients in order to evaluate the sensitivity of diffusion tensor imaging (DTI), a new MRI technique, for detecting changes in the PP. Furthermore, the diagnostic explanatory power of DTI data of the PP should be compared to high-resolution MRI volumetry and intervoxel coherences (COH) of the hippocampus and the entorhinal cortex, two limbic regions also involved in the pathophysiology of early AD. DTI revealed a marked decrease in COH values in the PP region of MCI (right side: 26%, left side: 29%, as compared to controls) and AD patients (right side: 37%, left side: 43%, as compared to controls). Reductions in COH values of the PP region were significantly correlated with cognitive impairment. DTI data of the PP zone were the only parameter differing significantly between control subjects and MCI patients, while the volumetric measures and the COH values of the hippocampus and the entorhinal cortex did not. DTI of medial temporal brain regions is a promising non-invasive tool for the in vivo diagnosis of the early/preclinical stages of AD.

A 20-channel receive-only mouse array coil for a 3 T clinical MRI system

A 20-channel phased-array coil for MRI of mice has been designed, constructed, and validated with bench measurements and high-resolution accelerated imaging. The technical challenges of designing a small, high density array have been overcome using individual small-diameter coil elements arranged on a cylinder in a hexagonal overlapping design with adjacent low impedance preamplifiers to further decouple the array elements. Signal-to-noise ratio (SNR) and noise amplification in accelerated imaging were simulated and quantitatively evaluated in phantoms and in vivo mouse images. Comparison between the 20-channel mouse array and a length-matched quadrature driven small animal birdcage coil showed an SNR increase at the periphery and in the center of the phantom of 3- and 1.3-fold, respectively. Comparison with a shorter but SNR-optimized birdcage coil (aspect ratio 1:1 and only half mouse coverage) showed an SNR gain of twofold at the edge of the phantom and similar SNR in the center. G-factor measurements indicate that the coil is well suited to acquire highly accelerated images.

MRI monitoring of cartilage repair in the knee: a review

Various treatment options for deep cartilage defects are presently available. The efficacy of bone marrow stimulation with microfracture, of mosaicplasty and of various autologous chondrocyte implantation (ACI) techniques has been subject to numerous studies recently. Magnetic resonance imaging (MRI) has gained a major role in the assessment of cartilage repair. The introduction of high-field MRI to clinical routine makes high resolution and three-dimensional imaging readily available. New quantitative MRI techniques that directly visualize the molecular structure of cartilage may further advance our understanding of cartilage repair. The clinical evaluation of cartilage repair tissue is a complex issue, and MR imaging will become increasingly important both in research and in clinical routine. This article reviews the clinical aspects of microfracture, mosaicplasty, and ACI and reports the recent technical advances that have improved MRI of cartilage. Morphological evaluation methods are recommended for each of the respective techniques. Finally, an overview of T2 mapping and delayed gadolinium-enhanced MR imaging of cartilage in cartilage repair is provided.

Electron microscopy of high pressure frozen samples: bridging the gap between cellular ultrastructure and atomic resolution

Transmission electron microscopy has provided most of what is known about the ultrastructural organization of tissues, cells, and organelles. Due to tremendous advances in crystallography and magnetic resonance imaging, almost any protein can now be modeled at atomic resolution. To fully understand the workings of biological "nanomachines" it is necessary to obtain images of intact macromolecular assemblies in situ. Although the resolution power of electron microscopes is on the atomic scale, in biological samples artifacts introduced by aldehyde fixation, dehydration and staining, but also section thickness reduces it to some nanometers. Cryofixation by high pressure freezing circumvents many of the artifacts since it allows vitrifying biological samples of about 200 mum in thickness and immobilizes complex macromolecular assemblies in their native state in situ. To exploit the perfect structural preservation of frozen hydrated sections, sophisticated instruments are needed, e.g., high voltage electron microscopes equipped with precise goniometers that work at low temperature and digital cameras of high sensitivity and pixel number. With them, it is possible to generate high resolution tomograms, i.e., 3D views of subcellular structures. This review describes theory and applications of the high pressure cryofixation methodology and compares its results with those of conventional procedures. Moreover, recent findings will be discussed showing that molecular models of proteins can be fitted into depicted organellar ultrastructure of images of frozen hydrated sections. High pressure freezing of tissue is the base which may lead to precise models of macromolecular assemblies in situ, and thus to a better understanding of the function of complex cellular structures.

The mean anatomical shape of the tibial plateau at the knee arthroplasty resection level: an investigation using MRI

Clinical assessments after Total Knee Arthroplasty (TKA) show persisting pain after implantation in over 20% of patients. Impingement of soft tissue around the knee, due to imprecise geometry of the tibial implant, can be one reason for persisting ailment. Two hundred and thirty seven MRI scans were evaluated using an active contour detection algorithm (snake) to obtain a high-resolution mean anatomical shape of the tibial plateau. Differences between female and male, older and younger (40) and left and right averaged shapes were determined. The shapes obtained were asymmetric throughout. Absolute differences between the subgroups fell short of inter-individual variations represented by calculated one-sigma confidence intervals. Our results indicate that a differentiation in TKA tibial plateau design by gender, age, or side is of minor relevance.