Use of a LightCycler gyrA mutation assay for rapid identification of mutations conferring decreased susceptibility to ciprofloxacin in multiresistant Salmonella enterica serotype typhimurium DT104 isolates

A LightCycler-based PCR-hybridization gyrA mutation assay (GAMA) was developed to rapidly detect gyrA point mutations in multiresistant (MR) Salmonella enterica serotype Typhimurium DT104 with decreased susceptibility to ciprofloxacin (MIC, 0.25 to 1.0 mg/liter). Ninety-two isolates (49 human, 43 animal) were tested with three individual oligonucleotide probes directed against an Asp-87-to-Asn (GAC --> AAC) mutation, an Asp-87-to-Gly (GAC --> GGC) mutation, and a Ser-83-to-Phe (TCC --> TTC) mutation. Strains homologous to the probes could be distinguished from strains that had different mutations by their probe-target melting temperatures. Thirty-seven human and 30 animal isolates had an Asp-87-to-Asn substitution, 6 human and 6 animal isolates had a Ser-83-to-Phe substitution, and 5 human and 2 animal isolates had an Asp-87-to-Gly substitution. The remaining six strains all had mismatches with the three probes and therefore different gyrA mutations. The sequencing of gyrA from these six isolates showed that one human strain and two animal strains had an Asp-87-to-Tyr (GAC --> TAC) substitution and two animal strains had a Ser-83-to-Tyr (TCC --> TAC) substitution. One animal strain had no gyrA mutation, suggesting that this isolate had a different mechanism of resistance. Fifty-eight of the strains tested were indistinguishable by several different typing methods including antibiograms, pulsed-field gel gel electrophoresis, and plasmid profiling, although they could be further subdivided according to gyrA mutation. This study confirmed that MR DT104 with decreased susceptibility to ciprofloxacin from humans and food animals in England and Wales may have arisen independently against a background of clonal spread of MR DT104.

Commonly used farm disinfectants can select for mutant Salmonella enterica serovar Typhimurium with decreased susceptibility to biocides and antibiotics without compromising virulence

Objectives: To determine if one passage of Salmonella enterica serovar Typhimurium in the presence of farm disinfectants selected for mutants with decreased susceptibility to disinfectants and/or antibiotics. Methods: Eight Salmonella Typhimurium strains including field isolates and laboratory mutants were exposed to either a tar oil phenol (PFD) disinfectant, an oxidizing compound disinfectant (OXC), an aldehyde based disinfectant (ABD) or a dairy sterilizer disinfectant (based on quaternary ammonium biocide) in agar. The susceptibility of mutants obtained after disinfectant exposure to antibiotics and disinfectants was determined as was the accumulation of norfloxacin. The proteome of SL1344 after exposure to PFD and OXC was analysed using two-dimensional liquid chromatography mass spectrometry. Results: Strains with either acrB or tolC inactivated were more susceptible to most disinfectants than other strains. The majority (3/5) of mutants recovered after disinfectant exposure required statistically significantly longer exposure times to disinfectants than their parent strains to generate a 5 log kill. Small decreases in antibiotic susceptibility were observed but no mutants were multiply antibiotic-resistant (MAR). Notably exposure to ABD decreased susceptibility to ciprofloxacin in some strains. Mutants with increased disinfectant tolerance were able to survive and persist in chicks as well as in parent strains. Analysis of proteomes revealed significantly increased expression of the AcrAB-TolC efflux system after PFD exposure. Conclusions: Data presented demonstrate that efflux pumps are required for intrinsic resistance to some disinfectants and that exposure to disinfectants can induce expression of the AcrAB-TolC efflux system, but that single exposure was insufficient to select for MAR strains.

Demand response — a different form of distributed storage?

Reduced flexibility of low carbon generation could pose new challenges for future energy systems. Both demand response and distributed storage may have a role to play in supporting future system balancing. This paper reviews how these technically different, but functionally similar approaches compare and compete with one another. Household survey data is used to test the effectiveness of price signals to deliver demand responses for appliances with a high degree of agency. The underlying unit of storage for different demand response options is discussed, with particular focus on the ability to enhance demand side flexibility in the residential sector. We conclude that a broad range of options, with different modes of storage, may need to be considered, if residential demand flexibility is to be maximised.

Concentrations of free amino acids and sugars in nine potato varieties: effects of storage and relationship with acrylamide formation

Acrylamide forms during cooking and processing predominately from the reaction of free asparagine and reducing sugars in the Maillard reaction. The identification of low free asparagine and reducing sugar varieties of crops is therefore an important target. In this study, nine varieties of potato (French fry varieties Maris Piper (from two suppliers), Pentland Dell, King Edward, Daisy, and Markies; and chipping varieties Lady Claire, Lady Rosetta, Saturna, and Hermes) grown in the United Kingdom in 2009 were analyzed at monthly intervals through storage from November 2009 to July 2010. Acrylamide formation was measured in heated flour and chips fried in oil. Analysis of variance revealed significant interactions between varieties nested within type (French fry and chipping) and storage time for most free amino acids, glucose, fructose, and acrylamide formation. Acrylamide formed in chips correlated significantly with acrylamide formed in flour and with chip color. There were significant correlations between glucose or total reducing sugar concentration and acrylamide formation in both variety types, but with fructose the correlation was much stronger for chipping than for French fry varieties. Conversely, there were significant correlations with acrylamide formation for both total free amino acid and free asparagine concentration in the French fry but not chipping varieties. The study showed the potential of variety selection for preventing unacceptable levels of acrylamide formation in potato products and the variety-dependent effect of long-term storage on acrylamide risk. It also highlighted the complex relationship between precursor concentration and acrylamide risk in potatoes.

Commodity futures prices: more evidence on forecast power, risk premia and the theory of storage

In this paper, we examine the temporal stability of the evidence for two commodity futures pricing theories. We investigate whether the forecast power of commodity futures can be attributed to the extent to which they exhibit seasonality and we also consider whether there are time varying parameters or structural breaks in these pricing relationships. Compared to previous studies, we find stronger evidence of seasonality in the basis, which supports the theory of storage. The power of the basis to forecast subsequent price changes is also strengthened, while results on the presence of a risk premium are inconclusive. In addition, we show that the forecasting power of commodity futures cannot be attributed to the extent to which they exhibit seasonality. We find that in most cases where structural breaks occur, only changes in the intercepts and not the slopes are detected, illustrating that the forecast power of the basis is stable over different economic environments.

A novel alternative to cryopreservation for the short-term storage of stem cells for use in cell therapy using alginate encapsulation

Efficient transport of stem/progenitor cells without affecting their survival and function is a key factor in any practical cell-based therapy. However, the current approach using liquid nitrogen for the transfer of stem cells requires a short delivery time window is technically challenging and financially expensive. The present study aims to use semipermeable alginate hydrogels (crosslinked by strontium) to encapsulate, store, and release stem cells, to replace the conventional cryopreservation method for the transport of therapeutic cells within world-wide distribution time frame. Human mesenchymal stem cell (hMSC) and mouse embryonic stem cells (mESCs) were successfully stored inside alginate hydrogels for 5 days under ambient conditions in an air-tight environment (sealed cryovial). Cell viability, of the cells extracted from alginate gel, gave 74% (mESC) and 80% (hMSC) survival rates, which compared favorably to cryopreservation. More importantly, the subsequent proliferation rate and detection of common stem cell markers (both in mRNA and protein level) from hMSCs and mESCs retrieved from alginate hydrogels were also comparable to (if not better than) results gained following cryopreservation. In conclusion, this new and simple application of alginate hydrogel encapsulation may offer a cheap and robust alternative to cryopreservation for the transport and storage of stem cells for both clinical and research purposes.

Sulphate and sulphurous acid alter the relative susceptibility of wheat to Phaeosphaeria nodorum and Mycosphaerella graminicola

The relative abundances of DNA of Mycosphaerella graminicola and Phaeosphaeria nodorum in archived wheat samples are closely correlated with UK anthropogenic emissions of oxidized sulphur over the last 160 years. To test whether this could be a causal relationship, possible modes of action of sulphur on the two fungi were examined. Mycelial growth of the two fungi in solutions of sulphurous acid was similar. Sulphurous acid at pH 4 reduced percentage germination of P. nodorum conidia more strongly than M. graminicola conidia. In spray inoculations of wheat cv. Squarehead’s Master, Cappelle Desprez and Riband with water or sulphurous acid (pH 4), the ratio of leaves infected by P. nodorum to leaves infected by M. graminicola was increased by factors of 2.5, 2.1 and 0.6, respectively at pH 4. The same three cultivars of wheat were grown in sand and vermiculite and fertilized with nutrient solution containing 2.5 or 0.5 mM sulphate. Both pathogens infected less frequently at 2.5 mM sulphate, by a factor of about 2. The severity of infection by M. graminicola was reduced on all three cultivars by a factor of about 4-5 at 2.5mM sulphate, but severity of P. nodorum was reduced only by a factor of about 2. Both elevated free sulphate concentrations in soil and sulphite in rainwater could therefore increase the prevalence of P. nodorum relative to M. graminicola, which is consistent with the historical changes in abundance

The neural bases of the short-term storage of verbal information are anatomically variable across individuals

What are the precise brain regions supporting the short-term retention of verbal information? A previous functional magnetic resonance imaging (fMRI) study suggested that they may be topographically variable across individuals, occurring, in most, in regions posterior to prefrontal cortex (PFC), and that detection of these regions may be best suited to a single-subject (SS) approach to fMRI analysis (Feredoes and Postle, 2007). In contrast, other studies using spatially normalized group-averaged (SNGA) analyses have localized storage-related activity to PFC. To evaluate the necessity of the regions identified by these two methods, we applied repetitive transcranial magnetic stimulation (rTMS) to SS- and SNGA-identified regions throughout the retention period of a delayed letter-recognition task. Results indicated that rTMS targeting SS analysis-identified regions of left perisylvian and sensorimotor cortex impaired performance, whereas rTMS targeting the SNGA-identified region of left caudal PFC had no effect on performance. Our results support the view that the short-term retention of verbal information can be supported by regions associated with acoustic, lexical, phonological, and speech-based representation of information. They also suggest that the brain bases of some cognitive functions may be better detected by SS than by SNGA approaches to fMRI data analysis.

A case study to investigate the life cycle carbon emissions and carbon storage capacity of a cross laminated timber, multi-storey residential building

Forests are a store of carbon and an eco-system that continually removes carbon dioxide from the atmosphere. If they are sustainably managed, the carbon store can be maintained at a constant level, while the trees removed and converted to timber products can form an additional long term carbon store. The total carbon store in the forest and associated ‘wood chain’ therefore increases over time, given appropriate management. This increasing carbon store can be further enhanced with afforestation. The UK’s forest area has increased continually since the early 1900s, although the rate of increase has declined since its peak in the late 1980s, and it is a similar picture in the rest of Europe. The increased sustainable use of timber in construction is a key market incentive for afforestation, which can make a significant contribution to reducing carbon emissions. The case study presented in this paper demonstrates the carbon benefits of a Cross Laminated Timber (CLT) solution for a multi-storey residential building in comparison with a more conventional reinforced concrete solution. The embodied carbon of the building up to completion of construction is considered, together with the stored carbon during the life of the building and the impact of different end of life scenarios. The results of the study show that the total stored carbon in the CLT structural frame is 1215tCO2 (30tCO2 per housing unit). The choice of treatment at end of life has a significant effect on the whole life embodied carbon of the CLT frame, which ranges from -1017 tCO2e for re-use to +153tCO2e for incinerate without energy recovery. All end of life scenarios considered result in lower total CO2e emissions for the CLT frame building compared with the reinforced concrete frame solution.

Proteolysis of milk heated at high temperatures by native enzymes analysed by trinitrobenzene sulphonic acid (TNBS) method

Native enzymes play a significant role in proteolysis of milk during storage. This is significant for heat resistant native enzymes. Plasmin is one of the most heat resistant enzymes found in milk. It has been reported to survive several heat treatments, causing spoilage during storage. The aim of this study was to assess susceptibility of high temperature heated milk to proteolysis by native enzymes. The trinitrobenzene sulphonic acid (TNBS) method was used for this purpose. Raw milk was heated at 110, 120, 130,142°C for 2 s and 85°C for 15 s and milk processed at low temperature (85°C /15s) was selected to mimic pasteurisation. TNBS method confirmed that raw milk and milk processed at 85°C /15s were the most proteolysed, whereas treatment of milk at high temperatures (110, 120, 130 and 142°C for 2 s) inactivated the native enzymes. It may thus be concluded that high temperature processing positively affects proteolysis by lowering its susceptibility to spoilage during storage.

Physiological, biochemical and transcriptional analysis of onion bulbs during storage

Abstract: During the transition from endo-dormancy to eco-dormancy and subsequent growth, the onion bulb undergoes the transition from sink organ to source, to sustain cell division in the meristematic tissue. The mechanisms controlling these processes are not fully understood. Here, a detailed analysis of whole onion bulb physiological, biochemical and transcriptional changes in response to sprouting is reported, enabling a better knowledge of the mechanisms regulating post-harvest onion sprout development. Biochemical and physiological analyses were conducted on different cultivars ('Wellington', 'Sherpa' and 'Red Baron') grown at different sites over 3 years, cured at different temperatures (20, 24 and 28 degrees C) and stored under different regimes (1, 3, 6 and 6 1 degrees C). In addition, the first onion oligonucleotide microarray was developed to determine differential gene expression in onion during curing and storage, so that transcriptional changes could support biochemical and physiological analyses. There were greater transcriptional differences between samples at harvest and before sprouting than between the samples taken before and after sprouting, with some significant changes occurring during the relatively short curing period. These changes are likely to represent the transition from endo-dormancy to sprout suppression, and suggest that endo-dormancy is a relatively short period ending just after curing. Principal component analysis of biochemical and physiological data identified the ratio of monosaccharides (fructose and glucose) to disaccharide (sucrose), along with the concentration of zeatin riboside, as important factors in discriminating between sprouting and pre-sprouting bulbs. These detailed analyses provide novel insights into key regulatory triggers for sprout dormancy release in onion bulbs and provide the potential for the development of biochemical or transcriptional markers for sprout initiation. Evidence presented herein also suggests there is no detrimental effect on bulb storage life and quality caused by curing at 20 degrees C, producing a considerable saving in energy and costs.

Trafficking of storage proteins in developing grain of wheat

The processing properties of the wheat flour are largely determined by the structures and interactions of the grain storage proteins (also called gluten proteins) which form a continuous visco-elastic network in dough. Wheat gluten proteins are classically divided into two groups, the monomeric gliadins and the polymeric glutenins, with the latter being further classified into low molecular weight (LMW) and high molecular weight (HMW) subunits. The synthesis, folding and deposition of the gluten proteins take place within the endomembrane system of the plant cell. However, determination of the precise routes of trafficking and deposition of individual gluten proteins in developing wheat grain has been limited in the past by the difficulty of developing monospecific antibodies. To overcome this limitation, a single gluten protein (a LMW subunit) was expressed in transgenic wheat with a C-terminal epitope tag, allowing the protein to be located in the cells of the developing grain using highly specific antibodies. This approach was also combined with the use of wider specificity antibodies to compare the trafficking and deposition of different gluten protein groups within the same endosperm cells. These studies are in agreement with previous suggestions that two trafficking pathways occur in wheat, with the proteins either being transported via the Golgi apparatus into the vacuole or accumulating directly within the lumen of the ER. They also suggest that the same individual protein could be trafficked by either pathway, possibly depending on the stage of development, and that segregation of gluten proteins both between and within protein bodies may occur.

Candidate genes for obesity-susceptibility show enriched association within a large genome-wide association study for BMI

Before the advent of genome-wide association studies (GWASs), hundreds of candidate genes for obesity-susceptibility had been identified through a variety of approaches. We examined whether those obesity candidate genes are enriched for associations with body mass index (BMI) compared with non-candidate genes by using data from a large-scale GWAS. A thorough literature search identified 547 candidate genes for obesity-susceptibility based on evidence from animal studies, Mendelian syndromes, linkage studies, genetic association studies and expression studies. Genomic regions were defined to include the genes ±10 kb of flanking sequence around candidate and non-candidate genes. We used summary statistics publicly available from the discovery stage of the genome-wide meta-analysis for BMI performed by the genetic investigation of anthropometric traits consortium in 123 564 individuals. Hypergeometric, rank tail-strength and gene-set enrichment analysis tests were used to test for the enrichment of association in candidate compared with non-candidate genes. The hypergeometric test of enrichment was not significant at the 5% P-value quantile (P = 0.35), but was nominally significant at the 25% quantile (P = 0.015). The rank tail-strength and gene-set enrichment tests were nominally significant for the full set of genes and borderline significant for the subset without SNPs at P < 10(-7). Taken together, the observed evidence for enrichment suggests that the candidate gene approach retains some value. However, the degree of enrichment is small despite the extensive number of candidate genes and the large sample size. Studies that focus on candidate genes have only slightly increased chances of detecting associations, and are likely to miss many true effects in non-candidate genes, at least for obesity-related traits.

Role of genetic polymorphism peroxisome proliferator-activated receptor-gamma2 Pro12Ala on ethnic susceptibility to diabetes in South-Asian and Caucasian subjects: Evidence for heterogeneity

OBJECTIVE: To determine whether the peroxisome proliferator-activated receptor (PPAR)-gamma Pro12ala polymorphism modulates susceptibility to diabetes in South Asians. RESEARCH DESIGN AND METHODS: South Asians (n = 697) and Caucasians (n = 457) living in Dallas/Forth Worth, Texas, and South Asians living in Chennai, India (n = 1,619), were enrolled for this study. PPAR-gamma Pro12Ala was determined using restriction fragment-length polymorphism. Insulin responsiveness to an oral glucose tolerance test (OGTT) was measured in nondiabetic subjects. RESULTS: The Caucasian diabetic subjects had significantly lower prevalence of PPAR-gamma 12Ala when compared with the Caucasian nondiabetic subjects (20 vs. 9%, P = 0.006). However, there were no significant differences between diabetic and nondiabetic subjects with reference to the Pro12Ala polymorphism among the South Asians living in Dallas (20 vs. 23%) and in India (19 vs. 19.3%). Although Caucasians carrying PPAR-gamma Pro12Ala had lower plasma insulin levels at 2 h of OGTT than the wild-type (Pro/Pro) carriers (76 +/- 68 and 54 +/- 33 microU/ml, respectively, P = 0.01), no differences in either fasting or 2-h plasma insulin concentrations were found between South Asians carrying the PPAR-gamma Pro12Ala polymorphism and those with the wild-type genotype at either Chennai or Dallas. CONCLUSIONS: Although further replication studies are necessary to test the validity of the described genotype-phenotype relationship, our study supports the hypothesis that the PPAR-gamma Pro12Ala polymorphism is protective against diabetes in Caucasians but not in South Asians.

Enhanced three-factor security protocol for consumer USB mass storage devices

The Universal Serial Bus (USB) is an extremely popular interface standard for computer peripheral connections and is widely used in consumer Mass Storage Devices (MSDs). While current consumer USB MSDs provide relatively high transmission speed and are convenient to carry, the use of USB MSDs has been prohibited in many commercial and everyday environments primarily due to security concerns. Security protocols have been previously proposed and a recent approach for the USB MSDs is to utilize multi-factor authentication. This paper proposes significant enhancements to the three-factor control protocol that now makes it secure under many types of attacks including the password guessing attack, the denial-of-service attack, and the replay attack. The proposed solution is presented with a rigorous security analysis and practical computational cost analysis to demonstrate the usefulness of this new security protocol for consumer USB MSDs.