High throughput, high resolution selection of polymorphic microsatellite loci for multiplex analysis

Background Large-scale genetic profiling, mapping and genetic association studies require access to a series of well-characterised and polymorphic microsatellite markers with distinct and broad allele ranges. Selection of complementary microsatellite markers with non-overlapping allele ranges has historically proved to be a bottleneck in the development of multiplex microsatellite assays. The characterisation process for each microsatellite locus can be laborious and costly given the need for numerous, locus-specific fluorescent primers. Results Here, we describe a simple and inexpensive approach to select useful microsatellite markers. The system is based on the pooling of multiple unlabelled PCR amplicons and their subsequent ligation into a standard cloning vector. A second round of amplification utilising generic labelled primers targeting the vector and unlabelled locus-specific primers targeting the microsatellite flanking region yield allelic profiles that are representative of all individuals contained within the pool. Suitability of various DNA pool sizes was then tested for this purpose. DNA template pools containing between 8 and 96 individuals were assessed for the determination of allele ranges of individual microsatellite markers across a broad population. This helped resolve the balance between using pools that are large enough to allow the detection of many alleles against the risk of including too many individuals in a pool such that rare alleles are over-diluted and so do not appear in the pooled microsatellite profile. Pools of DNA from 12 individuals allowed the reliable detection of all alleles present in the pool. Conclusion The use of generic vector-specific fluorescent primers and unlabelled locus-specific primers provides a high resolution, rapid and inexpensive approach for the selection of highly polymorphic microsatellite loci that possess non-overlapping allele ranges for use in large-scale multiplex assays.

2,2-Dialkyl-2H-benzimidazoles, the high energy tautomers of the corresponding 1,2-dialkyl-1H-benzimidazoles. Syntheses and their complexes with Cu(I) and Ag(I)

Reaction of Cu(1,2-phenylenediamine)(2)(ClO4)(2) with neat RR'=O (R = methyl and/or ethyl) (lives Cu(2,2-dialkyl-2H-benzimidazole)ClO4. demetallation of which by the action of aqueous ammonia yields Pure 2,2-dialkyl-2H-benzimidazoles. These are characterised by NMR. hi the X-ray crystal Structure, Ag(2,2-methyl-2H-benzimi-dazolc)NO3 is Found to be a spiral 1D coordination polymer where the 2H-benzimidazole acts as an N,N bridge between two Ag(I) centus. Although 2H-benzimidazoles are very unstable in the free state, they are quite stable in their Cu(I)(1) and Ag(I) complexes. The 1,2-tautomerisation in imidazole and benzimidazole have been Studied by means of transition state calculations at B3LYP/6-3 11 +G(2d,p)* level.

Opportunities and challenges in high pressure processing of foods

Consumers increasingly demand convenience foods of the highest quality in terms of natural flavor and taste, and which are freedom additives and preservatives. This demand has triggered the need for the development of a number of nonthermal approaches to food processing, of which high-pressure technology has proven to be very valuable. A number of recent publications have demonstrated novel and diverse uses of this technology. Its novel features, which include destruction of microorganisms at room temperature or lower, have made the technology commerically attractive. Enzymes forming bacteria can be by the application of pressure-thermal combinations. This review aims to identify the opportunities and challenges associated with this technology. In addition to discussing the effects of high pressure on food components, this review covers the combined effects of high pressure processing with: gamma irradiation, alternating current, ultrasound, and carbon dioxide or anti-microbial treatment. Further, the applications of this technology in various sectors-fruits and vegetables, dairy and meat processing-have been dealt with extensively. The integration of high-pressure with other matured processing operations such as blanching, dehydration, osmotic dehydration, rehyrdration, frying, freezing/thawing and solid-liquid extraction has been shown to open up new processing options. The key challenges identified include: heat transfer problems and resulting non-uniformity in processing, obtaining reliable and reproducible data, for process validation, lack of detailed knowledge about the interaction between high pressure, and a number of food constituents, packaging and statutory issues.

High temperature reduction of metmyoglobin in aqueous muscle extracts

Oxymyoglobin in aqueous extracts of fresh beef longissimus dorsi muscles was initially oxidised to metmyoglobin during heat treatments at temperatures in the range 50-70 degreesC. The metmyoglobin then underwent reduction to a red pigment that was shown spectrally to be identical to oxymyoglobin. The formation of oxymyoglobin involved a heat induced precipitate that when removed from the solution, allowed oxidation to metmyoglobin to occur. However, on re-addition of the precipitate further reduction to oxymyoglobin took place. Dialysis of the muscle extract prior to heating markedly inhibited the reduction but addition of NADH to the dialysate permitted further reduction. The precipitate plus NADH caused oxymyoglobin formation in the presence of metmyoglobin but neither the precipitate nor NADH alone induced this formation. It is concluded that the initial conversion of oxymyoglobin to metmyoglobin on heating fresh beef muscle extracts was reversible and that the reverse reaction depended on the presence of both NADH and a muscle protein.

High frequency variability of the Atlantic meridional overturning circulation

We compare the variability of the Atlantic meridional overturning circulation (AMOC) as simulated by the coupled climate models of the RAPID project, which cover a wide range of resolution and complexity, and observed by the RAPID/MOCHA array at about 26N. We analyse variability on a range of timescales. In models of all resolutions there is substantial variability on timescales of a few days; in most AOGCMs the amplitude of the variability is of somewhat larger magnitude than that observed by the RAPID array, while the amplitude of the simulated annual cycle is similar to observations. A dynamical decomposition shows that in the models, as in observations, the AMOC is predominantly geostrophic (driven by pressure and sea-level gradients), with both geostrophic and Ekman contributions to variability, the latter being exaggerated and the former underrepresented in models. Other ageostrophic terms, neglected in the observational estimate, are small but not negligible. In many RAPID models and in models of the Coupled Model Intercomparison Project Phase 3 (CMIP3), interannual variability of the maximum of the AMOC wherever it lies, which is a commonly used model index, is similar to interannual variability in the AMOC at 26N. Annual volume and heat transport timeseries at the same latitude are well-correlated within 15-45N, indicating the climatic importance of the AMOC. In the RAPID and CMIP3 models, we show that the AMOC is correlated over considerable distances in latitude, but not the whole extent of the north Atlantic; consequently interannual variability of the AMOC at 50N is not well-correlated with the AMOC at 26N.

High frequency variability of the Atlantic meridional overturning circulation

We compare the variability of the Atlantic meridional overturning circulation (AMOC) as simulated by the coupled climate models of the RAPID project, which cover a wide range of resolution and complexity, and observed by the RAPID/MOCHA array at about 26N. We analyse variability on a range of timescales, from five-daily to interannual. In models of all resolutions there is substantial variability on timescales of a few days; in most AOGCMs the amplitude of the variability is of somewhat larger magnitude than that observed by the RAPID array, while the time-mean is within about 10% of the observational estimate. The amplitude of the simulated annual cycle is similar to observations, but the shape of the annual cycle shows a spread among the models. A dynamical decomposition shows that in the models, as in observations, the AMOC is predominantly geostrophic (driven by pressure and sea-level gradients), with both geostrophic and Ekman contributions to variability, the latter being exaggerated and the former underrepresented in models. Other ageostrophic terms, neglected in the observational estimate, are small but not negligible. The time-mean of the western boundary current near the latitude of the RAPID/MOCHA array has a much wider model spread than the AMOC does, indicating large differences among models in the simulation of the wind-driven gyre circulation, and its variability is unrealistically small in the models. In many RAPID models and in models of the Coupled Model Intercomparison Project Phase 3 (CMIP3), interannual variability of the maximum of the AMOC wherever it lies, which is a commonly used model index, is similar to interannual variability in the AMOC at 26N. Annual volume and heat transport timeseries at the same latitude are well-correlated within 15--45N, indicating the climatic importance of the AMOC. In the RAPID and CMIP3 models, we show that the AMOC is correlated over considerable distances in latitude, but not the whole extent of the north Atlantic; consequently interannual variability of the AMOC at 50N, where it is particularly relevant to European climate, is not well-correlated with that of the AMOC at 26N, where it is monitored by the RAPID/MOCHA array.

Characteristics of high-resolution versions of the Met Office unified model for forecasting convection over the United Kingdom

With many operational centers moving toward order 1-km-gridlength models for routine weather forecasting, this paper presents a systematic investigation of the properties of high-resolution versions of the Met Office Unified Model for short-range forecasting of convective rainfall events. The authors describe a suite of configurations of the Met Office Unified Model running with grid lengths of 12, 4, and 1 km and analyze results from these models for a number of convective cases from the summers of 2003, 2004, and 2005. The analysis includes subjective evaluation of the rainfall fields and comparisons of rainfall amounts, initiation, cell statistics, and a scale-selective verification technique. It is shown that the 4- and 1-km-gridlength models often give more realistic-looking precipitation fields because convection is represented explicitly rather than parameterized. However, the 4-km model representation suffers from large convective cells and delayed initiation because the grid length is too long to correctly reproduce the convection explicitly. These problems are not as evident in the 1-km model, although it does suffer from too numerous small cells in some situations. Both the 4- and 1-km models suffer from poor representation at the start of the forecast in the period when the high-resolution detail is spinning up from the lower-resolution (12 km) starting data used. A scale-selective precipitation verification technique implies that for later times in the forecasts (after the spinup period) the 1-km model performs better than the 12- and 4-km models for lower rainfall thresholds. For higher thresholds the 4-km model scores almost as well as the 1-km model, and both do better than the 12-km model.

Scale-selective verification of rainfall accumulations from high-resolution forecasts of convective events

The development of NWP models with grid spacing down to 1 km should produce more realistic forecasts of convective storms. However, greater realism does not necessarily mean more accurate precipitation forecasts. The rapid growth of errors on small scales in conjunction with preexisting errors on larger scales may limit the usefulness of such models. The purpose of this paper is to examine whether improved model resolution alone is able to produce more skillful precipitation forecasts on useful scales, and how the skill varies with spatial scale. A verification method will be described in which skill is determined from a comparison of rainfall forecasts with radar using fractional coverage over different sized areas. The Met Office Unified Model was run with grid spacings of 12, 4, and 1 km for 10 days in which convection occurred during the summers of 2003 and 2004. All forecasts were run from 12-km initial states for a clean comparison. The results show that the 1-km model was the most skillful over all but the smallest scales (approximately <10–15 km). A measure of acceptable skill was defined; this was attained by the 1-km model at scales around 40–70 km, some 10–20 km less than that of the 12-km model. The biggest improvement occurred for heavier, more localized rain, despite it being more difficult to predict. The 4-km model did not improve much on the 12-km model because of the difficulties of representing convection at that resolution, which was accentuated by the spinup from 12-km fields.

High yield production of a soluble bifidobacterial β-galactosidase (BbgIV) inE. coliDH5α with improved catalytic efficiency for the synthesis of prebiotic galactooligosaccharides

The bifidobacterial β-galactosidase (BbgIV) was produced in E. coli DH5α at 37 and 30 °C in a 5 L bioreactor under varied conditions of dissolved oxygen (dO2) and pH. The yield of soluble BbgIV was significantly (P < 0.05) increased once the dO2 dropped to 0–2% and remained at such low values during the exponential phase. Limited dO2 significantly (P < 0.05) increased the plasmid copy number and decreased the cells growth rate. Consequently, the BbgIV yield increased to its maximum (71–75 mg per g dry cell weight), which represented 20–25% of the total soluble proteins in the cells. In addition, the specific activity and catalytic efficiency of BbgIV were significantly (P < 0.05) enhanced under limited dO2 conditions. This was concomitant with a change in the enzyme secondary structure, suggesting a link between the enzyme structure and function. The knowledge generated from this work is very important for producing BbgIV as a biocatalyst for the development of a cost-effective process for the synthesis of prebiotic galactooligosaccharides from lactose.

High resolution simulation of recent Arctic and Antarctic stratospheric chemical ozone loss compared to observations

Simulations of polar ozone losses were performed using the three-dimensional high-resolution (1∘ × 1∘) chemical transport model MIMOSA-CHIM. Three Arctic winters 1999–2000, 2001–2002, 2002–2003 and three Antarctic winters 2001, 2002, and 2003 were considered for the study. The cumulative ozone loss in the Arctic winter 2002–2003 reached around 35% at 475 K inside the vortex, as compared to more than 60% in 1999–2000. During 1999–2000, denitrification induces a maximum of about 23% extra ozone loss at 475 K as compared to 17% in 2002–2003. Unlike these two colder Arctic winters, the 2001–2002 Arctic was warmer and did not experience much ozone loss. Sensitivity tests showed that the chosen resolution of 1∘ × 1∘ provides a better evaluation of ozone loss at the edge of the polar vortex in high solar zenith angle conditions. The simulation results for ozone, ClO, HNO3, N2O, and NO y for winters 1999–2000 and 2002–2003 were compared with measurements on board ER-2 and Geophysica aircraft respectively. Sensitivity tests showed that increasing heating rates calculated by the model by 50% and doubling the PSC (Polar Stratospheric Clouds) particle density (from 5 × 10−3 to 10−2 cm−3) refines the agreement with in situ ozone, N2O and NO y levels. In this configuration, simulated ClO levels are increased and are in better agreement with observations in January but are overestimated by about 20% in March. The use of the Burkholder et al. (1990) Cl2O2 absorption cross-sections slightly increases further ClO levels especially in high solar zenith angle conditions. Comparisons of the modelled ozone values with ozonesonde measurement in the Antarctic winter 2003 and with Polar Ozone and Aerosol Measurement III (POAM III) measurements in the Antarctic winters 2001 and 2002, shows that the simulations underestimate the ozone loss rate at the end of the ozone destruction period. A slightly better agreement is obtained with the use of Burkholder et al. (1990) Cl2O2 absorption cross-sections.

High sensitivity recording of afferent nerve activity using ultra-compliant microchannel electrodes: an acutein vivovalidation

Neuroprostheses interfaced with transected peripheral nerves are technological routes to control robotic limbs as well as convey sensory feedback to patients suffering from traumatic neural injuries or degenerative diseases. To maximize the wealth of data obtained in recordings, interfacing devices are required to have intrafascicular resolution and provide high signal-to-noise ratio (SNR) recordings. In this paper, we focus on a possible building block of a three-dimensional regenerative implant: a polydimethylsiloxane (PDMS) microchannel electrode capable of highly sensitive recordings in vivo. The PDMS 'micro-cuff' consists of a 3.5 mm long (100 µm × 70 µm cross section) microfluidic channel equipped with five evaporated Ti/Au/Ti electrodes of sub-100 nm thickness. Individual electrodes have average impedance of 640 ± 30 kΩ with a phase angle of −58 ± 1 degrees at 1 kHz and survive demanding mechanical handling such as twisting and bending. In proof-of-principle acute implantation experiments in rats, surgically teased afferent nerve strands from the L5 dorsal root were threaded through the microchannel. Tactile stimulation of the skin was reliably monitored with the three inner electrodes in the device, simultaneously recording signal amplitudes of up to 50 µV under saline immersion. The overall SNR was approximately 4. A small but consistent time lag between the signals arriving at the three electrodes was observed and yields a fibre conduction velocity of 30 m s−1. The fidelity of the recordings was verified by placing the same nerve strand in oil and recording activity with hook electrodes. Our results show that PDMS microchannel electrodes open a promising technological path to 3D regenerative interfaces.

High-resolution provenance of desert dust deposited on Mt. Elbrus, Caucasus in 2009–2012 using snow pit and firn core records

The first record of dust deposition events on Mt. Elbrus, Caucasus Mountains derived from a snow pit and a shallow firn core is presented for the 2009–2012 period. A combination of isotopic analysis, SEVIRI red-greenblue composite imagery, MODIS atmospheric optical depth fields derived using the Deep Blue algorithm, air mass trajectories derived using the HYSPLIT model and analyses of meteorological data enabled identification of dust source regions with high temporal (hours) and spatial (ca. 20–100 km) resolution. Seventeen dust deposition events were detected; fourteen occurred in March–June, one in February and two in October. Four events originated in the Sahara, predominantly in northeastern Libya and eastern Algeria. Thirteen events originated in the Middle East, in the Syrian Desert and northern Mesopotamia, from a mixture of natural and anthropogenic sources. Dust transportation from Sahara was associated with vigorous Saharan depressions, strong surface winds in the source region and mid-tropospheric southwesterly flow with daily winds speeds of 20–30 m s−1 at 700 hPa level. Although these events were less frequent than those originating in the Middle East, they resulted in higher dust concentrations in snow. Dust transportation from the Middle East was associated with weaker depressions forming over the source region, high pressure centred over or extending towards the Caspian Sea and a weaker southerly or southeasterly flow towards the Caucasus Mountains with daily wind speeds of 12–18 m s−1 at 700 hPa level. Higher concentrations of nitrates and ammonium characterised dust from the Middle East deposited on Mt. Elbrus in 2009 indicating contribution of anthropogenic sources. The modal values of particle size distributions ranged between 1.98 µm and 4.16 µm. Most samples were characterised by modal values of 2.0– 2.8 µm with an average of 2.6 µm and there was no signifi- cant difference between dust from the Sahara and the Middle East.

High resolution simulation of the variability of surface energy balance fluxes across central London with urban zones for energy partitioning

The parameterization of surface heat-flux variability in urban areas relies on adequate representation of surface characteristics. Given the horizontal resolutions (e.g. ≈0.1–1km) currently used in numerical weather prediction (NWP) models, properties of the urban surface (e.g. vegetated/built surfaces, street-canyon geometries) often have large spatial variability. Here, a new approach based on Urban Zones to characterize Energy partitioning (UZE) is tested within a NWP model (Weather Research and Forecasting model;WRF v3.2.1) for Greater London. The urban land-surface scheme is the Noah/Single-Layer Urban Canopy Model (SLUCM). Detailed surface information (horizontal resolution 1 km)in central London shows that the UZE offers better characterization of surface properties and their variability compared to default WRF-SLUCM input parameters. In situ observations of the surface energy fluxes and near-surface meteorological variables are used to select the radiation and turbulence parameterization schemes and to evaluate the land-surface scheme