First Report of Canola Powdery Mildew Caused by Erysiphe polygoni in Argentina.
Canola (Brassica napus) is a developing oleaginous crop grown commercially in the Buenos Aires and Santa Fe provinces of Argentina. During the autumn of 2003, typical signs of powdery mildew were observed on canola plants in experimental field plots in Buenos Aires. Average disease incidence was 42% on 3- to 6-month-old canola cultivars developed in the following countries: Argentina (Eclipse, Impulse Master, Mistral, and Nolza); Australia (Oscar and Rainbow); Canada (Sentry); France (Cadillac, Camberra, and Capitol); and Sweden (Maskot, Sponsor, and Wildcat). The range of incidence on these cultivars was 35 to 93%. Other cultivars exhibited an apparent high level of resistance or escaped disease. These included: Charlton (Argentina); 46CO3, Dunkeld, Insignia, Mystic, Monty, Outback, Rivette, and Surpass 400 (Australia), and Caviar (France). Climatic conditions in Buenos Aires, especially rainfall, from March to May 2003 were apparently favorable for powdery mildew development. On susceptible cultivars, fungal growth was observed on leaves, stems, and pods that resulted in premature senescence of the tissues. The mycelium, with multilobed hausthoria, was white to gray, dense or fine, and in patches or covering the entire adaxial leaf surfaces. Appressoria were lobed and conidiophores were straight. Foot cells were cylindrical, straight, measured 35 to 42 × 7 to 10 μm, and were followed by two cells. Conidia were produced singly, cylindrical to ovoid, and measured 36 to 40 × 18 to 20 μm. The conidial length-to-width ratio was 2.0. No fibrosin bodies were observed in the conidia and conidia germinated at the ends. Cleistothecia were not observed. On the basis of mycelial, conidial, and hausthoria characteristics observed on six leaves for each affected cultivar, the fungus was identified as Erysiphe polygoni DC (1). Pathogenicity was confirmed on 5-week-old canola plants of cvs. Eclipse, Impulse, Master, Mistral, and Maskot by gently pressing (1 min) one adaxial infected leaf with abundant sporulation onto one adaxial healthy leaf. The experiment, which included five inoculated plants and three noninoculated control plants for each cultivar, was conducted in a greenhouse at 22 to 24°C and maintained at 75% relative humidity with no supplemental light. Inoculated and control plants were covered with polyethylene bags for 48 h after inoculation. Powdery mildew developed on all inoculated plants of all cultivars after 12 to14 days. The control plants did not develop disease. The experiment was repeated with similar results. E. polygoni has a worldwide distribution (2); however, the results suggest that this fungus may be a threat to the main cultivars being grown in Argentina (Eclipse, Impulse, Master, Mistral, and Nolza), since high levels of disease incidence, as much as 70%, were observed. Under propitious environments, this pathogen could cause severe yield losses in commercially grown canola in Argentina. To our knowledge, this is the first report of canola powdery mildew caused by E. polygoni in Argentina. References: (1) H. J. Boesewinkel. Rev. Mycol. Tome 41:493, 1977. (2) D. F. Farr et al. Fungi on Plants and Plant Products in the United States. The American Phytopathological Society, St.Paul, MN, 1989.


CAM photosynthesis: the acid test.
There is currently considerable interest in the prospects for bioengineering crassulacean acid metabolism (CAM) photosynthesis - or key elements associated with it, such as increased water-use efficiency - into C3 plants. Resolving how CAM photosynthesis evolved from the ancestral C3 pathway could provide valuable insights into the targets for such bioengineering efforts. It has been proposed that the ability to accumulate organic acids at night may be common among C3 plants, and that the transition to CAM might simply require enhancement of pre-existing fluxes, without the need for changes in circadian or diurnal regulation. We show, in a survey encompassing 40 families of vascular plants, that nocturnal acidification is a feature entirely restricted to CAM species. Although many C3 species can synthesize malate during the light period, we argue that the switch to night-time malic acid accumulation requires a fundamental metabolic reprogramming that couples glycolytic breakdown of storage carbohydrate to the process of net dark CO2 fixation. This central element of the CAM pathway, even when expressed at a low level, represents a biochemical capability not seen in C3 plants, and so is better regarded as a discrete evolutionary innovation than as part of a metabolic continuum between C3 and CAM.


Green and black tea inhibit cytokine-induced IL-8 production and secretion in AGS gastric cancer cells via inhibition of NF-κB activity.
Consumption of tea is associated with a reduced risk for several gastrointestinal cancers. Inflammatory processes, such as secretion of IL-8 from the gastric epithelium in response to chronic chemokine or antigen exposure, serve both as a chemoattractant for white blood cells and a prerequisite for gastric carcinogenesis. In this study, the gastric adenocarcinoma cell line AGS was used to investigate the effect of green tea extract, black tea extract, and epigallocatechin gallate (EGCG), the most abundant catechin in tea, on cytokine-induced inflammation. AGS cells were stimulated with interleukin-1β (IL-1β) to initiate inflammation, followed by exposure to either tea extracts or EGCG. We found that both green and black tea extracts at concentrations of 20 and 2 µM total catechins, respectively, significantly (p < 0.05) inhibited IL-1β-induced IL-8 production and secretion to a similar extent. Treatment of AGS cells with EGCG (8 µM) produced similar reductions in IL-1β-induced IL-8 production and secretion. Inhibition of NF-κB activity was found to be responsible, in part, for these observed effects. Our findings demonstrate that both green and black tea extracts with distinctly different catechin profiles, are capable of disrupting the molecular link between inflammation and carcinogenesis via inhibition of NF-κB activity in AGS cells.


Oral versus intravenous flucytosine in patients with human immunodeficiency virus-associated cryptococcal meningitis.
In a randomized controlled trial of amphotericin B-based therapy for human immunodeficiency virus (HIV)-associated cryptococcal meningitis in Thailand, we also compared the mycological efficacy, toxicity, and pharmacokinetics of oral versus intravenous flucytosine at 100 mg/kg of body weight/day for the initial 2 weeks. Half of 32 patients assigned to the two arms containing flucytosine were randomized to oral and half to intravenous flucytosine. Early fungicidal activity was determined from serial quantitative cultures of cerebrospinal fluid (CSF), and toxicity was assessed by clinical and laboratory monitoring. Flucytosine and fluorouracil concentrations in plasma and CSF were measured by high-performance liquid chromatography. No significant bone marrow or hepatotoxicity was seen, there was no detectable difference in bone marrow toxicity between patients on intravenous and those on oral formulation, and no patients discontinued treatment. In patients receiving intravenous flucytosine, the median 24-h area under the concentration-time curve was significantly higher than in the oral group. Despite this difference, there was no difference in early fungicidal activity between patients on intravenous compared with patients on oral flucytosine. The results suggest that either formulation can be used safely at this dosage in a developing country setting, without drug concentration monitoring. The bioavailability of the oral formulation may be reduced in late-stage HIV-infected patients in Thailand. Concentrations of flucytosine with intravenous formulation at 100 mg/kg/day may be in excess of those required for maximal fungicidal activity.


Antimalarial drug targets in Plasmodium falciparum predicted by stage-specific metabolic network analysis.
Despite enormous efforts to combat malaria the disease still afflicts up to half a billion people each year of which more than one million die. Currently no approved vaccine is available and resistances to antimalarials are widely spread. Hence, new antimalarial drugs are urgently needed. Here, we present a computational analysis of the metabolism of Plasmodium falciparum, the deadliest malaria pathogen. We assembled a compartmentalized metabolic model and predicted life cycle stage specific metabolism with the help of a flux balance approach that integrates gene expression data. Predicted metabolite exchanges between parasite and host were found to be in good accordance with experimental findings when the parasite's metabolic network was embedded into that of its host (erythrocyte). Knock-out simulations identified 307 indispensable metabolic reactions within the parasite. 35 out of 57 experimentally demonstrated essential enzymes were recovered and another 16 enzymes, if additionally the assumption was made that nutrient uptake from the host cell is limited and all reactions catalyzed by the inhibited enzyme are blocked. This predicted set of putative drug targets, shown to be enriched with true targets by a factor of at least 2.75, was further analyzed with respect to homology to human enzymes, functional similarity to therapeutic targets in other organisms and their predicted potency for prophylaxis and disease treatment. The results suggest that the set of essential enzymes predicted by our flux balance approach represents a promising starting point for further drug development.


Genome-wide and fine-resolution association analysis of malaria in West Africa.
We report a genome-wide association (GWA) study of severe malaria in The Gambia. The initial GWA scan included 2,500 children genotyped on the Affymetrix 500K GeneChip, and a replication study included 3,400 children. We used this to examine the performance of GWA methods in Africa. We found considerable population stratification, and also that signals of association at known malaria resistance loci were greatly attenuated owing to weak linkage disequilibrium (LD). To investigate possible solutions to the problem of low LD, we focused on the HbS locus, sequencing this region of the genome in 62 Gambian individuals and then using these data to conduct multipoint imputation in the GWA samples. This increased the signal of association, from P = 4 × 10(-7) to P = 4 × 10(-14), with the peak of the signal located precisely at the HbS causal variant. Our findings provide proof of principle that fine-resolution multipoint imputation, based on population-specific sequencing data, can substantially boost authentic GWA signals and enable fine mapping of causal variants in African populations.


Evaluation of reading speed and contrast sensitivity in dry eye disease.
Visual disturbance is a common symptom reported by patients with dry eye disease (DED). The purpose of this study was to evaluate visual performance, including reading speed and contrast sensitivity, in control and DED subjects. Fifty-two DED patients (mild, n = 17; moderate, n = 22; severe, n = 13; based on corneal staining and the Ocular Surface Disease Index ≥ 20) and 20 control subjects (Ocular Surface Disease Index <13, no corneal staining) took part in this study. The age ranges for the control and DED patients were 18 to 45 years and 19 to 84 years, respectively. Contrast sensitivity was measured using the Holladay Automated Contrast Sensitivity System, and reading speed was determined using the Wilkins Rate of Reading Test. Analysis of covariance was conducted to compare clinical characteristics among subject groups while adjusting for age, sex, and study site. Partial correlation coefficients from linear regression were used to measure the linear relationship between contrast sensitivity and reading speed with DED parameters. The log of the minimum angle of resolution visual acuities and contrast sensitivity were not significantly different across subject groups. The DED patients (134.9 ± 4.95 words per minute) exhibited slower reading speeds than the control subjects (158.3 ± 8.40 words per minute, p = 0.046). As DED severity increased, the reading speed decreased (141.0 ± 7.96 words per minute, 136.8 ± 7.15 words per minute, and 127.0 ± 9.63 words per minute in mild, moderate, and severe groups, respectively). Reading rate was found to correlate weakly with corneal staining based on a partial correlation coefficient (-0.345, p < 0.001) but not with other DED parameters. The reading rate was lower in DED subjects than that in control subjects. As the DED severity increased, the reading rate decreased. This finding is consistent with patient-reported symptoms and provides direct evidence for the impact of DED on reading performance. These findings suggest that reading speed may be used to monitor treatment benefit in DED.


Diagnosis of neglected tropical diseases among patients with persistent digestive disorders (diarrhoea and/or abdominal pain ≥14 days): Pierrea multi-country, prospective, non-experimental case-control study.
Diarrhoea still accounts for considerable mortality and morbidity worldwide. The highest burden is concentrated in tropical areas where populations lack access to clean water, adequate sanitation and hygiene. In contrast to acute diarrhoea (<14 days), the spectrum of pathogens that may give rise to persistent diarrhoea (≥14 days) and persistent abdominal pain is poorly understood. It is conceivable that pathogens causing neglected tropical diseases play a major role, but few studies investigated this issue. Clinical management and diagnostic work-up of persistent digestive disorders in the tropics therefore remain inadequate. Hence, important aspects regarding the pathogenesis, epidemiology, clinical symptomatology and treatment options for patients presenting with persistent diarrhoea and persistent abdominal pain should be investigated in multi-centric clinical studies. This multi-country, prospective, non-experimental case-control study will assess persistent diarrhoea (≥14 days; in individuals aged ≥1 year) and persistent abdominal pain (≥14 days; in children/adolescents aged 1-18 years) in up to 2000 symptomatic patients and 2000 matched controls. Subjects from Côte d'Ivoire, Indonesia, Mali and Nepal will be clinically examined and interviewed using a detailed case report form. Additionally, each participant will provide a stool sample that will be examined using a suite of diagnostic methods (i.e., microscopic techniques, rapid diagnostic tests, stool culture and polymerase chain reaction) for the presence of bacterial and parasitic pathogens. Treatment will be offered to all infected participants and the clinical treatment response will be recorded. Data obtained will be utilised to develop patient-centred clinical algorithms that will be validated in primary health care centres in the four study countries in subsequent studies. Our research will deepen the understanding of the importance of persistent diarrhoea and related digestive disorders in the tropics. A diversity of intestinal pathogens will be assessed for potential associations with persistent diarrhoea and persistent abdominal pain. Different diagnostic methods will be compared, clinical symptoms investigated and diagnosis-treatment algorithms developed for validation in selected primary health care centres. The findings from this study will improve differential diagnosis and evidence-based clinical management of digestive syndromes in the tropics. ClinicalTrials.gov; identifier: NCT02105714 .


Laser Doppler flowmetry to measure changes in cerebral blood flow.
Laser Doppler flowmetry (LDF) is a method by which relative cerebral blood flow (CBF) of the cortex can be measured. Although the method is easy to employ, LDF only measures relative CBF, while absolute CBF cannot be quantified. LDF is useful for investigating CBF changes in a number of different applications including neurovascular and stroke research. This chapter will prepare the reader for rodent experiments using LDF with two preparations. The closed skull preparation can be used to monitor CBF with an intact skull, but in adult rats, thinning of the skull is required to obtain an accurate cortical CBF signal. The open skull preparation requires a craniotomy to expose the surface of the brain and the LDF probe is held close to the surface to measure cerebral perfusion.


Usefulness of thiopurine methyltransferase and thiopurine metabolite analysis in clinical practice in patients with inflammatory bowel diseases.
Thiopurines (TP) are widely used in the management of inflammatory bowel diseases. Side-effects and inefficacy are a major concern as they lead to withdrawal of the drug. Tools investigating TP metabolism are useful to avoid inadequate cessation of TP therapy. TP metabolism is complex and many enzymes are involved. Among them, Thiopurine methyltransferase (TPMT) is the only one routinely measured by pheno- or genotyping. In this review, the rationale for TPMT and thiopurine metabolites, 6-thioguanine nucleotides and 6-methylmercaptopurine, determination in clinical practice is discussed, specifically in case of thiopurine failure and recommendations are given about their interpretation and potential dose optimization of TP drugs.


Assessing the direct effects of deep brain stimulation using embedded axon models.
To better understand the spatial extent of the direct effects of deep brain stimulation (DBS) on neurons, we implemented a geometrically realistic finite element electrical model incorporating anisotropic and inhomogenous conductivities. The model included the subthalamic nucleus (STN), substantia nigra (SN), zona incerta (ZI), fields of Forel H2 (FF), internal capsule (IC) and Medtronic 3387/3389 electrode. To quantify the effects of stimulation, we extended previous studies by using multi-compartment axon models with geometry and orientation consistent with anatomical features of the brain regions of interest. Simulation of axonal firing produced a map of relative changes in axonal activation. Voltage-controlled stimulation, with clinically typical parameters at the dorso-lateral STN, caused axon activation up to 4 mm from the target. This activation occurred within the FF, IC, SN and ZI with current intensities close to the average injected during DBS (3 mA). A sensitivity analysis of model parameters (fiber size, fiber orientation, degree of inhomogeneity, degree of anisotropy, electrode configuration) revealed that the FF and IC were consistently activated. Direct activation of axons outside the STN suggests that other brain regions may be involved in the beneficial effects of DBS when treating Parkinsonian symptoms.


Susceptibility to Friend helper virus leukemias in CXB recombinant inbred mice.
The seven CXB recombinant inbred strains were tested for susceptibility to Friend helper virus (F-MuLV) hematopoietic neoplasms. BALB/c and CXB-H mice develop erythroblastosis after neonatal inoculation with F-MuLV, while C57BL/6 and the six other RI strains develop lymphoma and myelogenous leukemia. This strain distribution pattern is different from that for H-2, Gpd-1 (linked to Fv-1), Fv-2, Rfv-3, and Cv (linked to Rmcf) but the same as that for Bv, the endogenous ecotropic virus of C57BL/6. However, analysis of crosses segregating Bv show that resistance to F-MuLV erythroblastosis is not linked to Bv. Disease-free survival is shortest for BALB/c mice, intermediate for CXB-H and CXB-J, and longest for C57BL/6 and the other RI strains. We conclude: (a) the major C57BL/6 gene for resistance to F-MuLV erythroblastosis is different from previously identified Friend virus restriction loci; (b) latency for F-MuLV leukemias is controlled by more than one gene; and (c) latency and susceptibility to F-MuLV erythroblastosis are not inherited concordantly in the CXB-RI strains.


A prospective randomized controlled trial of tumour chemosensitivity assay directed chemotherapy versus physician's choice in patients with recurrent platinum-resistant ovarian cancer.
The primary aim of this randomized trial was to determine response rate and progression-free survival following chemotherapy in patients with platinum-resistant recurrent ovarian cancer, who had been treated according to an ATP-based tumour chemosensitivity assay in comparison with physician's choice. A total of 180 patients were randomized to assay-directed therapy (n=94) or physician's-choice chemotherapy (n=86). Median follow-up at analysis was 18 months. Response was assessable in 147 patients: 31.5% achieved a partial or complete response in the physician's-choice group compared with 40.5% in the assay-directed group (26 versus 31% by intention-to-treat analysis respectively). Intention-to-treat analysis showed a median progression-free survival of 93 days in the physician's-choice group and 104 days in the assay-directed group (hazard ratio 0.8, 95% confidence interval 0.59-1.10, not significant). No difference was seen in overall survival between the groups, although 12/39 (41%) of patients who crossed over from the physician's-choice arm obtained a response. Increased use of combination therapy was seen in the physician's-choice arm during the study as a result of the observed effects of assay-directed therapy in patients. Patients entering the physician's-choice arm of the study during the first year did significantly worse than those who entered in the subsequent years (hazard ratio 0.44, 95% confidence interval 0.2-0.9, P<0.03). This small randomized clinical trial has documented a trend towards improved response and progression-free survival for assay-directed treatment. Chemosensitivity testing might provide useful information in some patients with ovarian cancer, although a larger trial is required to confirm this. The ATP-based tumour chemosensitivity assay remains an investigational method in this condition.


The swaying mouse as a model of osteogenesis imperfecta caused by WNT1 mutations.
Osteogenesis imperfecta (OI) is a heritable disorder of connective tissue characterized by bone fragility and low bone mass. Recently, our group and others reported that WNT1 recessive mutations cause OI, whereas WNT1 heterozygous mutations cause early onset osteoporosis. These findings support the hypothesis that WNT1 is an important WNT ligand regulating bone formation and bone homeostasis. While these studies provided strong human genetic and in vitro functional data, an in vivo animal model to study the mechanism of WNT1 function in bone is lacking. Here, we show that Swaying (Wnt1(sw/sw)) mice previously reported to carry a spontaneous mutation in Wnt1 share major features of OI including propensity to fractures and severe osteopenia. In addition, biomechanical and biochemical analyses showed that Wnt1(sw/sw) mice exhibit reduced bone strength with altered levels of mineral and collagen in the bone matrix that is also distinct from the type I collagen-related form of OI. Further histomorphometric analyses and gene expression studies demonstrate that the bone phenotype is associated with defects in osteoblast activity and function. Our study thus provides in vivo evidence that WNT1 mutations contribute to bone fragility in OI patients and demonstrates that the Wnt1(sw/sw) mouse is a murine model of OI caused by WNT1 mutations.


Evaluation of the impact of finite-resolution effects on scintillation compensation using two deformable mirrors.
The impact of finite-resolution deformable mirrors and wave-front sensors is evaluated as it applies to fullwave conjugation using two deformable mirrors. The first deformable mirror is fixed conjugate to the pupil, while the second deformable mirror is at a finite range. The control algorithm to determine the mirror commands for the two deformable mirrors is based on a modification of the sequential generalized projection algorithm. The modification of the algorithm allows the incorporation of Gaussian spatial filters into the optimization process to limit the spatial-frequency content applied to the two deformable mirrors. Simulation results are presented for imaging and energy projection scenarios that establish that the optimal spatial filter waist to be applied is equal to the subaperture side length in strong turbulence. The effect of varying the subaperture side length is examined, and it is found that to effect a significant degree of scintillation compensation, the subapertures, and corresponding spacing between actuators, must be much smaller than the coherence length of the input field.


Performance of a statistical model to predict stroke outcome in the context of a large, simple, randomized, controlled trial of feeding.
Statistical models to predict the outcome of stroke patients have several uses. Their utility depends on their predictive accuracy in patients other than those on whom they were developed (ie, external validity). We sought to test the external validity of some recently described models in patients enrolled in the FOOD (Feed Or Ordinary Diet) trial: a large randomized trial evaluating feeding policies in patients with stroke. The predictive variables were collected during a telephone call to randomize the patient a median of 5 days after stroke onset. Patients were followed up 6 months later to establish their survival, functional status, and residence. Charts were plotted to demonstrate the discrimination and calibration of the models. The models performed well in the first 2955 patients enrolled and followed up in the FOOD trial. The area under the receiver operating characteristic curves varied between 0.78 and 0.81 (with 0.5 indicating no discrimination and 1.0 indicating perfect discrimination). The discrimination was marginally better for patients enrolled within the first day of stroke than later. The models tended to provide rather pessimistic predictions in all groups except those predicted to have a high likelihood of surviving free of dependency. As one might predict, the discriminatory power in the selected cohort of trial patients was marginally less good than in previously studied unselected cohorts used to test their external validity. These models provide a well-tested tool for stratification in trials, comparing outcomes in different cohorts and examining the additional predictive power of novel factors.


Evolution of non-kin cooperation: social assortment by cooperative phenotype in guppies.
Cooperation among non-kin constitutes a conundrum for evolutionary biology. Theory suggests that non-kin cooperation can evolve if individuals differ consistently in their cooperative phenotypes and assort socially by these, such that cooperative individuals interact predominantly with one another. However, our knowledge of the role of cooperative phenotypes in the social structuring of real-world animal populations is minimal. In this study, we investigated cooperative phenotypes and their link to social structure in wild Trinidadian guppies (Poecilia reticulata). We first investigated whether wild guppies are repeatable in their individual levels of cooperativeness (i.e. have cooperative phenotypes) and found evidence for this in seven out of eight populations, a result which was mostly driven by females. We then examined the social network structure of one of these populations where the expected fitness impact of cooperative contexts is relatively high, and found assortment by cooperativeness, but not by genetic relatedness. By contrast, and in accordance with our expectations, we did not find assortment by cooperativeness in a population where the expected fitness impact of cooperative contexts is lower. Our results provide empirical support for current theory and suggest that assortment by cooperativeness is important for the evolution and persistence of non-kin cooperation in real-world populations.


Correlative STED and Atomic Force Microscopy on Live Astrocytes Reveals Plasticity of Cytoskeletal Structure and Membrane Physical Properties during Polarized Migration.
The plasticity of the cytoskeleton architecture and membrane properties is important for the establishment of cell polarity, adhesion and migration. Here, we present a method which combines stimulated emission depletion (STED) super-resolution imaging and atomic force microscopy (AFM) to correlate cytoskeletal structural information with membrane physical properties in live astrocytes. Using STED compatible dyes for live cell imaging of the cytoskeleton, and simultaneously mapping the cell surface topology with AFM, we obtain unprecedented detail of highly organized networks of actin and microtubules in astrocytes. Combining mechanical data from AFM with optical imaging of actin and tubulin further reveals links between cytoskeleton organization and membrane properties. Using this methodology we illustrate that scratch-induced migration induces cytoskeleton remodeling. The latter is caused by a polarization of actin and microtubule elements within astroglial cell processes, which correlates strongly with changes in cell stiffness. The method opens new avenues for the dynamic probing of the membrane structural and functional plasticity of living brain cells. It is a powerful tool for providing new insights into mechanisms of cell structural remodeling during physiological or pathological processes, such as brain development or tumorigenesis.


Fluoride content in table salt distributed in Mexico City, Mexico.
The aim of this study was to analyze table salt available in Mexico City's market to identify the fluoride concentrations and to compare these with the Mexican regulations. We analyzed 44 different brands of table salt. All samples were purchased at random in different stores, supermarkets, and groceries from Mexico City's metropolitan area and analyzed in triplicate in three different laboratories (nine determinations per sample) with an Orion 720 A potentiometer and an Orion 9609 BN ion-specific electrode. Fluoride concentration in the samples varied from 0 ppm to 485 ppm. It was found that fluoride concentration varied widely among the analyzed brands. Also, we found that fluoride concentration in 92 percent of the analyzed samples did not match with that printed on the label. Only 6.8 percent of the analyzed samples contained fluoride concentrations that meet Mexican and WHO regulations. The broad variation in the analyzed samples suggests that Mexican Public Health authorities must implement more stringent regulation guidelines and procedures for controlling the distribution of salt and its fluoride concentration for human consumption.


The chemistry of the collagen cross-links. Purification and characterization of cross-linked polymeric peptide material from mature collagen containing unknown amino acids.
A polymeric form of the alpha 1-chain C-terminal peptide alpha 1 CB6 (poly-alpha 1 CB6) was purified from CNBr digests of insoluble bovine tendon type-I-collagen by gel filtration and ion-exchage chromatography. The purified material had a molecular weight of 1.5 x 10(6)-5 x 10(6) on gel filtration and an amino acid content virtually identical with that of monomeric peptide alpha 1 CB6. The material could be adsorbed on affinity gels containing immobilized anti-(alpha 1 CB6-peptide non-helical region) antibodies and was an inhibitor of haemagglutination by the same antibodies of alpha 1 CB6-peptide-coated sheep erythrocytes. Periodate treatment of the material had no effect. Alkali hydrolysates were shown to contain two unknown amino acids, which were purified by gel filtration and ion-exchange chromatography in volatile buffers and are believed to be components of the mature cross-link of collagen.


Nonglycosidic agonists of invariant NKT cells for use as vaccine adjuvants.
Based on the crystal structures of human alpha-GalCer-CD1d and iNKT-alpha-GalCer-CD1d complexes, nonglycosidic analogues of alpha-GalCer were synthesized. They activate iNKT cells resulting in dendritic cell maturation and the priming of antigen-specific T and B cells. Therefore, they are attractive adjuvants in vaccination strategies for cancer and infectious diseases.


Analysis of oxidative modification of proteins.
Proteins are targets of oxidative modification. This unit describes detailed procedures for the analysis of popular indices of protein oxidation including protein carbonyl formation, loss of protein thiols, and nitrotyrosine and dityrosine formation, as well as isoaspartate formation. Procedures are detailed for the analysis of protein carbonyls labeled with 2,4-dinitrophenylhydrazine, tritiated sodium borohydride, and biotin-hydrazide, followed by detection measurements that are based on the distinguishing feature of each labeling chemical. Methods are outlined for the determination of protein cysteine oxidation by quantifying the loss of free protein thiols using radiolabeled [(14)C]-iodoacetamide. Protocols are described for the measurement of protein dityrosine by gas chromatography/mass spectrometry, as are the details for the detection of protein nitrotyrosine by a competitive ELISA approach. Finally, methods are described for the quantification of protein-bound isoaspartate using protein-L-isoaspartyl methyltransferase that converts aberrant L-isoaspartyl residues in peptides and proteins to normal aspartyl residues.


Plasmodium falciparum rhoptry neck protein 5 peptides bind to human red blood cells and inhibit parasite invasion.
Plasmodium falciparum malaria parasite invasion of erythrocytes is an essential step in host infection and the proteins involved in such invasion are the main target in developing an antimalarial vaccine. Secretory organelle-derived proteins (micronemal AMA1 protein and the RON2, 4, and 5 rhoptry neck proteins) have been recently described as components of moving junction complex formation allowing merozoites to move into a newly created parasitophorous vacuole. This study led to identifying RON5 regions involved in binding to human erythrocytes by using a highly robust, sensitive and specific receptor-ligand interaction assay; it is further shown that the RON5 protein remains highly conserved throughout different parasite strains. It is shown that the binding peptide-erythrocyte interaction is saturable and sensitive to chymotrypsin and trypsin. Invasion inhibition assays using erythrocyte binding peptides showed that the RON5-erythrocyte interaction could be critical for merozoite invasion of erythrocytes. This work provides evidence (for the first time) suggesting a fundamental role for RON5 in erythrocyte invasion.


[Influenza associated excess mortality in Argentina: 1992-2002].
To describe the effect of influenza on mortality in Argentina, from 1992 to 2002. In order to fulfill this objective, influenza associated excess mortality was determined by the application of ARIMA method to mortality data for pneumonia and influenza and for all causes. Excess mortality was only detected during subtype A/H3N2 seasons. The model yielded about 31,240 excess mortality for all causes. Pneumonia deaths contributed in about 15%. Approximately 80-95% of pneumonia and influenza excess mortality was restricted to persons > 64 years old. These estimations show that the virus circulation has had an important influence on mortality, increasing the number of deaths, especially in elderly population. The aging of the population reinforces the need of preventing strategies, including vaccination programs with high coverage in elderly population.


Cytoreductive treatment patterns among US veterans with polycythemia vera.
Polycythemia vera (PV) is a myeloproliferative neoplasm associated with increased thrombotic and cardiovascular risk, which are key contributors to patient morbidity and mortality. The Veterans Health Administration (VHA) is the largest integrative health network in the United States. Available data concerning patients with PV in this population are limited. This retrospective observational study evaluated the characteristics, management, and outcomes of patients with PV in the VHA Medical SAS® Dataset (October 1, 2005, to September 30, 2012). Inclusion criteria were ≥ 2 claims for PV (ie, PV diagnostic code was recorded) ≥30 days apart during the identification period, age ≥ 18 years, and continuous health plan enrollment from ≥12 months before the index date until the end of follow-up. All data were analyzed using descriptive statistics. The analysis included 7718 patients (median age, 64 years; male, 98%; white, 64%). The most common comorbidities before the index date were hypertension (72%), dyslipidemia (54%), and diabetes (24%); 33% had a history of smoking. During the follow-up period (median, 4.8 years), most patients did not receive treatment with cytoreductive therapy, including phlebotomy (53%), or antiplatelet agents, such as aspirin (57%). The thrombotic and cardiovascular event rates per 1000 patient-years were 60.5 and 83.8, respectively. Among patients who received cytoreductive treatment, the thrombotic event rate was 48.9 per 1000 patient-years. The overall mortality rate was 51.2 per 1000 patient-years. The notable rates of thrombotic and cardiovascular events observed in this analysis, even among patients receiving cytoreductive treatment, highlight the important unmet clinical needs of patients with PV in the VHA.


Linking two opposites of pregnancy loss: Induced abortion and infertility in Yoruba society, Nigeria.
Involuntary infertility and induced abortion exist on opposite sides of the spectrum: the first being the unwanted loss of childbearing potential while the second is the intentional termination of pregnancy. However, this paper proposes that these two poles of pregnancy loss are in fact related in Yoruba society, Nigeria. This argument is supported by qualitative and quantitative data drawn from an applied research project in communities and health institutions of Lagos State, from 1996 to 1999, where a total of 693 women recounted 1114 personal abortion experiences, and 233 women shared their experiences of fertility problems. Study statistics show that 37% of secondary infertility was most probably the result of induced abortion and that half of women with abortion complications interviewed in a referral hospital will have fertility problems. This paper provides insight into the reasons why single and married women decide to abort, and use unsafe methods, despite awareness of the serious health risks, including infertility. This is paradoxical given that fear of infertility is a major reason why women do not use modern contraceptives when trying to prevent unwanted pregnancy. By analysing the relations between infertility and abortion within the socio-cultural, economic, and services-related structures that influence women's decisions, this paper suggests ways of addressing the problems related to both types of pregnancy loss.


Optic nerve head component responses of the multifocal electroretinogram in MS.
To employ a novel stimulation paradigm in order to elicit multifocal electroretinography (mfERG)-induced optic nerve head component (ONHC) responses, believed to be contingent upon the transformation in electrical transmission properties of retinal ganglion cell axons from membrane to saltatory conduction mechanisms, as they traverse the lamina cribrosa and obtain oligodendrocyte myelin. We further sought to characterize abnormalities in ONHC responses in eyes from patients with multiple sclerosis (MS). In 10 normal subjects and 7 patients with MS (including eyes with and without a history of acute optic neuritis), we utilized a novel mfERG stimulation paradigm that included interleaved global flashes in order to elicit the ONHC responses from 103 retinal patches of pattern-reversal stimulation. The number of abnormal or absent ONHC responses was significantly increased in MS patient eyes compared to normal subject eyes (p < 0.001, by general estimating equation modeling, and accounting for age and within-subject, intereye correlations). Studying the relationship between ONHC abnormalities and alterations in validated structural and functional measures of the visual system may facilitate the ability to dissect and characterize the pathobiological mechanisms that contribute to tissue damage in MS, and may have utility to detect and monitor neuroprotective or restorative effects of novel therapies.


Dendritic Cell Subsets and Effector Function in Idiopathic and Connective Tissue Disease-Associated Pulmonary Arterial Hypertension.
Pulmonary arterial hypertension (PAH) is a cardiopulmonary disease characterized by an incurable condition of the pulmonary vasculature, leading to increased pulmonary vascular resistance, elevated pulmonary arterial pressure resulting in progressive right ventricular failure and ultimately death. PAH has different underlying causes. In approximately 30-40% of the patients no underlying risk factor or cause can be found, so-called idiopathic PAH (IPAH). Patients with an autoimmune connective tissue disease (CTD) can develop PAH [CTD-associated PAH (CTD-PAH)], suggesting a prominent role of immune cell activation in PAH pathophysiology. This is further supported by the presence of tertiary lymphoid organs (TLOs) near pulmonary blood vessels in IPAH and CTD-PAH. TLOs consist of myeloid cells, like monocytes and dendritic cells (DCs), T-cells, and B-cells. Next to their T-cell activating function, DCs are crucial for the preservation of TLOs. Multiple DC subsets can be found in steady state, such as conventional DCs (cDCs), including type 1 cDCs (cDC1s), and type 2 cDCs (cDC2s), AXL+Siglec6+ DCs (AS-DCs), and plasmacytoid DCs (pDCs). Under inflammatory conditions monocytes can differentiate into monocyte-derived-DCs (mo-DCs). DC subset distribution and activation status play an important role in the pathobiology of autoimmune diseases and most likely in the development of IPAH and CTD-PAH. DCs can contribute to pathology by activating T-cells (production of pro-inflammatory cytokines) and B-cells (pathogenic antibody secretion). In this review we therefore describe the latest knowledge about DC subset distribution, activation status, and effector functions, and polymorphisms involved in DC function in IPAH and CTD-PAH to gain a better understanding of PAH pathology.


Potential inhibition of HIV-1 encapsidation by oligoribonucleotide-dendrimer nanoparticle complexes.
Encapsidation, the process during which the genomic RNA of HIV is packaged into viral particles, is an attractive target for antiviral therapy. This study explores a novel nanotechnology-based strategy to inhibit HIV encapsidation by an RNA decoy mechanism. The design of the 16-mer oligoribonucleotide (RNA) decoy is based on the sequence of stem loop 3 (SL3) of the HIV packaging signal (Ψ). Recognition of the packaging signal is essential to the encapsidation process. It is theorized that the decoy RNA, by mimicking the packaging signal, will disrupt HIV packaging if efficiently delivered into lymphocytes by complexation with a carbosilane dendrimer. The aim of the study is to measure the uptake, toxicity, and antiviral activity of the dendrimer-RNA nanocomplex. A dendriplex was formed between cationic carbosilane dendrimers and the RNA decoy. Uptake of the fluorescein-labeled RNA into MT4 lymphocytes was determined by flow cytometry and confocal microscopy. The cytoprotective effect (50% effective concentration [EC50]) and the effect on HIV replication were determined in vitro by the methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay and viral load measurements, respectively. Flow cytometry and confocal imaging demonstrated efficient transfection of lymphocytes. The dendriplex containing the Ψ decoy showed some activity (EC50 =3.20 µM, selectivity index =8.4). However, there was no significant suppression of HIV viral load. Oligoribonucleotide decoys containing SL3 of the packaging sequence are efficiently delivered into lymphocytes by carbosilane dendrimers where they exhibit a modest cytoprotective effect against HIV infection.


Ultrastructural localization of the serotonin transporter in superficial and deep layers of the rat prelimbic prefrontal cortex and its spatial relationship to dopamine terminals.
Dopamine levels within the prefrontal cortex (PFC) can be manipulated by selective inhibitors of the serotonin transporter (SERT). However, the cellular mechanisms underlying these effects are not clear. The present study sought to examine the distribution of immunogold-silver labeling for SERT (SERT-ir) in the rat prelimbic PFC and to describe its ultrastructural spatial relationship to dopamine axons labeled by immunoperoxidase staining for tyrosine hydroxylase (TH-ir). SERT was localized to axonal profiles that ranged in size from fine caliber fibers containing dense SERT-ir, primarily along the membrane, and rarely forming synapses to large, spherical varicosities exhibiting less dense staining, mainly within the cytoplasm, and more commonly forming synapses. Synaptic contacts of SERT profiles were typically asymmetric, axospinous, and more frequent in superficial (38%) than deep (19%) layers. For TH-ir profiles, only 24% were within the same 13.8 microm(2) microenvironment as SERT-ir profiles. Furthermore, TH-ir and SERT-ir profiles were rarely directly apposed to each other or convergent onto common dendritic structures. Instead, these two profiles were typically separated by an average distance of 1.30 microm in the coronal plane, a value that did not vary with the size of SERT-ir axons, the amount of SERT labeling, or the cortical layer examined. These results are consistent with two populations of SERT profiles within the rat prelimbic PFC that may arise from different raphe nuclei or that represent varicose and intervaricose portions of the same axons. Moreover, the functional interactions between cortical serotonin and dopamine systems that may contribute to the therapeutic efficacy of antidepressant drugs are likely to occur over distances greater than 1 microm.


Aggregated and monomeric alpha-synuclein bind to the S6' proteasomal protein and inhibit proteasomal function.
The accumulation of aggregated alpha-synuclein is thought to contribute to the pathophysiology of Parkinson's disease, but the mechanism of toxicity is poorly understood. Recent studies suggest that aggregated proteins cause toxicity by inhibiting the ubiquitin-dependent proteasomal system. In the present study, we explore how alpha-synuclein interacts with the proteasome. The proteasome exists as a 26 S and a 20 S species. The 26 S proteasome is composed of the 19 S cap and the 20 S core. Aggregated alpha-synuclein strongly inhibited the function of the 26 S proteasome. The IC(50) of aggregated alpha-synuclein for ubiquitin-independent 26 S proteasomal activity was 1 nm. Aggregated alpha-synuclein also inhibited 26 S ubiquitin-dependent proteasomal activity at a dose of 500 nm. In contrast, the IC(50) of aggregated alpha-synuclein for 20 S proteasomal activity was > 1 microm. This suggests that aggregated alpha-synuclein selectively interacts with the 19 S cap. Monomeric alpha-synuclein also inhibited proteasomal activity but with lower affinity and less potency. Recombinant monomeric alpha-synuclein inhibited the activity of the 20 S proteasomal core with an IC(50) > 10 microm, exhibited no inhibition of 26 S ubiquitin-dependent proteasomal activity at doses up to 5 microm, and exhibited only partial inhibition (50%) of the 26 S ubiquitin-independent proteasomal activity at doses up to 10 mm. Binding studies demonstrate that both aggregated and monomeric alpha-synuclein selectively bind to the proteasomal protein S6', a subunit of the 19 S cap. These studies suggest that proteasomal inhibition by aggregated alpha-synuclein could be mediated by interaction with S6'.


Excitation wavelength dependence of the photoluminescence quantum yield and decay behavior of CdSe/CdS quantum dot/quantum rods with different aspect ratios.
The excitation wavelength (λexc) dependence of the photoluminescence (PL) quantum yield (ΦPL) and decay behavior (τPL) of a series of CdSe/CdS quantum dot/quantum rods (QDQRs), consisting of the same spherical CdSe core and rod-shaped CdS shells, with aspect ratios ranging from 2 to 20 was characterized. λexc between 400-565 nm were chosen to cover the first excitonic absorption band of the CdSe core material, the onset of absorption of the CdS shell, and the region of predominant shell absorption. A strong λexc dependence of relative and absolutely measured ΦPL and τPL was found particularly for the longer QDQRs with higher aspect ratios. This is attributed to combined contributions from a length-dependent shell-to-core exciton localization efficiency, an increasing number of defect states within the shell for the longest QDQRs, and probably also the presence of absorbing, yet non-emitting shell material. Although the ΦPL values of the QDQRs decrease at shorter wavelength, the extremely high extinction coefficients introduced by the shell outweigh this effect, leading to significantly higher brightness values at wavelengths below the absorption onset of the CdS shell compared with direct excitation of the CdSe cores. Moreover, our results present also an interesting example for the comparability of absolutely measured ΦPL using an integrating sphere setup and ΦPL values measured relative to common ΦPL standards, and underline the need for a correction for particle scattering for QDQRs with high aspect ratios.


Pearls of wisdom: using the single case study or 'gem' to identify strategies for mediating stress and work-life imbalance in healthcare staff.
The growing levels of stress and work-life imbalance reported in contemporary health and social care arenas in the UK can be linked to the neoliberal principles driving performance and intensification in the workforce. These pressures are an area of concern in terms of staff health and wellbeing and the impact of these on the care and compassion of patients/service users. This paper reports on a single case study that was part of a wider interpretative phenomenological analysis (IPA) that aimed to explore the levels of stress and work-life imbalance experienced by occupational therapists working in health and social-care sectors in Wales in the UK. Interpretative phenomenological analysis was used as both an approach and tool of analysis. The use of the single case study and gem as a tool in IPA to pique interest and promote further investigation is also explicated. Identified four key strategies that underpinned an approach to mediating stress and work-life balance for staff in the workplace. These were: a sense of choice and autonomy over workloads and decision-making; congruence with family values; supportive workplace attitudes and expectations of others; and finally, that the ability to reconcile conflicts i.e. integrate or harmonise them with personal values, as opposed to holding a sense of compromise marked by concession or loss, can address cognitive and emotional dissonance. The existing constructs to support the health and wellbeing of staff in health and social-care settings are ineffective. These strategies offer tools to address this and successfully promote a sense of personal integrity and meaning in life. In turn, this can achieve and sustain a more resilient workforce providing the positive energy needed to be caring and compassionate in their practice.


Fidaxomicin for the Treatment of Clostridium Difficile Infection in the Pediatric Population - Not Quite So Soon Yet.
Fidaxomicin is a new narrow spectrum macrocyclic antibiotic. It inhibits bacterial RNA polymerase and eradicates C difficile with minimal effect on normal intestinal flora. The US FDA granted orphan drug designation for all formulations of fidaxomicin for the treatment of C difficile infections in pediatric patients on January 10, 2011. Fidaxomicin has bactericidal activity against C difficile with a prolonged post-antibiotic effect. Even though this medication has an orphan designation for pediatrics, all the available pharmacokinetic and pharmacodynamic data were in subjects ≥ 18 years old. The MIC90 for fidaxomicin against C. difficile varies from 0.0078 to 0.25 mcg/ml. Fidaxomicin is poorly absorbed. The highest peak plasma concentration in patients treated with fidaxomicin was 0.191 mcg/ml. Fecal levels of fidaxomicin after oral administration are extremely high. The average fecal concentrations in C difficile patients were 255.6 mcg/g, 441.7 mcg/g, and 1443.3 mcg/g in the 100, 200, and 400 mg/day groups, respectively. At a dose of 400 mg/day the average fecal concentration was 5700 times higher than the highest MIC90 of fidaxomicin against C difficile. In a phase III clinical trial fidaxomicin 200 mg twice daily was compared with vancomycin 125 mg four times per day orally for 10 days. Only two patients were 18 years old, and no patients younger than 18 years old participated in the study. The rates of clinical cure with fidaxomicin were noninferior to those with vancomycin. Patients who were infected with non-North American Pulsed Field type 1 strains had fewer recurrences in the fidaxomicin group than patients in the vancomycin group. Side effects were similar between both therapies. Most patients experienced mild gastro-intestinal symptoms. Fidaxomicin is a good therapeutic alternative to vancomycin and metronidazole, especially in patients with recurrence of C difficile infection. Patients rarely experience systemic side effects which improves compliance. The dose of fidaxomicin is expected to be 200 mg given orally twice a day for patients 16 years and older. At this point, Optimer Pharmaceuticals Inc has conducted clinical trials in adults only. Additional clinical trials in pediatric patients are needed before therapeutic recommendations can be made in this population.


Minor surgery -- one general practitioner's experiences.
A study was made of 869 personal cases of minor surgery performed in a cottage hospital over the seven years 1974-1980 inclusive. Results showed a wide range of procedures, little waiting time for patients, low infection rates and low referral to consultants subsequently. The cost benefits to the community and hospital services and the job satisfaction for the general practitioner are discussed.


Characteristics of Turkish children with Type 2 diabetes at onset: a multicentre, cross-sectional study.
To describe the baseline clinical and laboratory findings and treatment modalities of 367 children and adolescents diagnosed with Type 2 diabetes in various paediatric endocrinology centres in Turkey. A standard questionnaire regarding clinical and laboratory characteristics at onset was uploaded to an online national database system. Data for 367 children (aged 6-18 years) newly diagnosed with Type 2 diabetes at 37 different paediatric endocrinology centres were analysed. After exclusion of the children with a BMI Z-score < 1 SD, those with genetic syndromes associated with Type 2 diabetes, and those whose C-peptide and/or insulin levels were not available, 227 cases were included in the study. Mean age was 13.8 ± 2.2 (range 6.5-17.8) years, with female preponderance (68%). Family history of Type 2 diabetes was positive in 86% of the children. The mean BMI was 31.3 ± 6.5 kg/m2 (range 18.7-61) and BMI Z-score was 2.4 ± 0.8 (range 1-5). More than half (57%) of the children were identified by an opportunistic diabetes screening due to existing risk markers without typical symptoms of diabetes. Only 13% (n = 29) were treated solely by lifestyle modification, while 40.5% (n = 92) were treated with metformin, 13% (n = 30) were treated with insulin, and 33.5% (n = 76) were treated with a combination of insulin and metformin initially. Mean HbA1C levels of the insulin and combination of insulin and metformin groups were 98 (11.1%) and 102 mmol/mol (11.5%), respectively, and also were significantly higher than the lifestyle modification only and metformin groups mean HbA1C levels (70(8.6%) and 67 mmol/mol (8.3%), respectively). An opportunistic screening of children who are at high risk of Type 2 diabetes is essential, as our data showed that > 50% of the children were asymptomatic at diagnosis. The other important result of our study was the high rate of exclusion from the initial registration (38%), suggesting that accurate diagnosis of Type 2 diabetes in youth is still problematic, even for paediatric endocrinologists.


Further validation of the Six-Spot Step Test as a measure of ambulation in multiple sclerosis.
There is preliminary evidence regarding the validity of the Six-Spot Step Test (SSST) as a promising measure of ambulatory function in persons with multiple sclerosis (MS). To date, this assessment has not been subject to the same rigor and extent of psychometric evaluation as other widely-accepted measures of ambulatory (e.g., timed 25-foot walk (T25FW)). This study aimed to provide additional validity evidence for the SSST in 96 persons with MS, based on construct validity and precision. Construct validity involves examining the pattern of associations between the SSST and other measures, and precision involves comparing SSST performance relative to other valid measures of ambulation for differentiating between levels of disability status, MS clinical course, and fall risk based on balance confidence. All participants completed the SSST, T25FW, Timed Up-and-Go (TUG), six-minute walk, Multiple Sclerosis Walking Scale-12, Late-Life Function and Disability Inventory, Activities-specific Balance Confidence, and Paced Auditory Serial Addition Test. All participants further underwent a neurological examination for generating EDSS scores, and then wore an ActiGraph accelerometer for the waking hours of a 7-day period. SSST performance was strongly associated with other valid measures of ambulation (|ρ| = .65-.90) and disability status (ρ = .73), moderately-to-strongly associated with balance confidence (ρ = .58), and weakly-to-moderately associated with cognitive processing speed and non-ambulatory measures (|ρ| = .35-.39). The SSST demonstrated stronger relative precision in discriminating between levels of disability status, MS clinical course, and fall risk based on balance confidence than the T25FW and TUG. We provide comprehensive validity evidence for the SSST that supports its consideration for inclusion alongside other highly-regarded objective measures of ambulatory function for clinical research and practice in persons with MS.


AAPM Task Group 108: PET and PET/CT shielding requirements.
The shielding of positron emission tomography (PET) and PET/CT (computed tomography) facilities presents special challenges. The 0.511 MeV annihilation photons associated with positron decay are much higher energy than other diagnostic radiations. As a result, barrier shielding may be required in floors and ceilings as well as adjacent walls. Since the patient becomes the radioactive source after the radiopharmaceutical has been administered, one has to consider the entire time that the subject remains in the clinic. In this report we present methods for estimating the shielding requirements for PET and PET/CT facilities. Information about the physical properties of the most commonly used clinical PET radionuclides is summarized, although the report primarily refers to fluorine-18. Typical PET imaging protocols are reviewed and exposure rates from patients are estimated including self-attenuation by body tissues and physical decay of the radionuclide. Examples of barrier calculations are presented for controlled and noncontrolled areas. Shielding for adjacent rooms with scintillation cameras is also discussed. Tables and graphs of estimated transmission factors for lead, steel, and concrete at 0.511 MeV are also included. Meeting the regulatory limits for uncontrolled areas can be an expensive proposition. Careful planning with the equipment vendor, facility architect, and a qualified medical physicist is necessary to produce a cost effective design while maintaining radiation safety standards.


Do people with schizophrenia who have objective cognitive impairment identify cognitive deficits on a self report measure?
Individuals diagnosed with schizophrenia spectrum disorders have significant neuropsychological deficits that have been linked to poor functional outcome. As more treatments are developed and implemented for cognitive impairments, it will be important to consider whether people with schizophrenia have insight into the neuropsychological symptoms of their illness. Just as insight into psychotic symptoms plays a significant role in treatment compliance, it is likely that insight into neuro-cognitive dysfunction will be associated with compliance with cognitive remediation or cognitive enhancing medications. The objective of this study was to evaluate self reported awareness of cognitive deficit in schizophrenia using the MIC-SR, a new scale, and to compare self report of cognitive deficit with actual neuropsychological performance, and with the MIC-SR performance of Healthy Controls. Patients with schizophrenia reported significantly more cognitive problems as occurring "Almost Daily" than did Healthy Controls. However, Patients were most likely to respond that a cognitive problem "Never" occurred, whereas Healthy Controls most often responded "Once a Week or Less". The Total Score on the MIC-SR did not prove useful in differentiating Healthy Controls from Patients. About one quarter of the cognitively impaired patients with schizophrenia showed no awareness of cognitive deficit on the MIC-SR. This suggests that clinicians must use self report judiciously as an indicator of actual cognitive impairment. The MIC-SR captures a range of awareness of cognitive difficulty. The data indicates that there are many patients who would benefit from psycho-education about the impact of schizophrenia on neuro-cognition.


Carnitine-acylcarnitine translocase deficiency in three neonates presenting with rapid deterioration and cardiac arrest.
We report on three Chinese neonates with carnitine-acylcarnitine translocase deficiency. They presented within the first 48 hours of life. Two neonates were found in cardiac arrest; one of them survived after resuscitation. The third neonate suddenly developed cardiorespiratory insufficiency and succumbed eventually. The clustering of three cases in 5 years suggests that carnitine-acylcarnitine translocase deficiency is not rare in our Chinese population. We advocate that investigation for metabolic diseases including carnitine-acylcarnitine translocase deficiency should be performed in cases of sudden infant death and unexplained abrupt clinical deterioration in the early neonatal period. Non-ketotic hypoglycaemia is an early clue. The mainstay of initial treatment is glucose infusion at a rate greater than 7 mg/kg/minute, which inhibits beta-oxidation of fatty acids (the defective enzymatic steps in carnitine-acylcarnitine translocase deficiency) and thus prevents the accumulation of toxic long-chain acylcarnitines.


Commentary: 'Unhealthy diet,' nutrient status, and ADHD symptoms: a confounding role for environmental nitrous oxide exposure - reflections on Rijlaarsdam et al. (2016).
Rijlaarsdam et al. (2016) recently published their findings utilizing a longitudinal design showing that prenatal 'unhealthy diet' was positively associated with IGF2 DNA methylation at birth across both youth cohorts. However, only in the EOP youth was prenatal 'unhealthy diet' positively associated with ADHD symptoms presumably through IGF2 DNA hypermethylation. Rijlaarsdam et al.'s () choice to assess high fat and sugar diet with the Food Frequency Questionnaire (FFQ) may offer some indication as to prenatal nutrient status, as the foods identified by the FFQ in their study are relatively low in free choline. It has been shown that gestational choline deficiency in rats leads to hypermethylation of IGF2. Consistent with the literature describing an association between air pollution and cognitive neurodevelopmental impairment, the author of this commentary has previously proposed through empirical investigation that chronic environmental exposure to the trace levels of the pervasive air pollutant, nitrous oxide (N2 O), may facilitate core features of neurodevelopmental disorders, like ADHD. Impaired acetylcholine synthesis in rats exposed to N2 O has been shown, with a 53% reduction in [1-2H2,2-2H2] choline. Low-dose N2 O exposure is also thought to stimulate central release of opioid peptides, like dynorphin, which play a role in significantly increasing food intake behavior and/or modulating sucrose intake. Taken altogether, these studies present a strong confounder to the interpretation made by Rijlaarsdam et al. () that prenatal 'unhealthy diet' may play a role in the onset of ADHD symptoms in youth with EOP conduct problems through induction of IGF2 DNA hypermethylation. While the 'unhealthy diet' may represent possible maternal nutrient deficiencies during gestation, it is also possible that exposure to air pollutants, particularly N2 O, may not only directly reduce fetal cholinergic status thereby enhancing IGF2 DNA hypermethylation but may also significantly modulate maternal food intake behaviors (i.e. sucrose).


Dyskeratosis Congenita Without Oral Involvement: A Rare Hereditary Disease.
Dyskeratosis congenita (DC) is a rare, progressive, multi-system, inherited disorder of telomere biology, first described in 1906 as the Zinsser-Engman-Cole syndrome. The condition presents with the classic triad of nail dystrophy, reticulate skin pigmentation, and oral leukoplakia. Variable somatic abnormalities may be present; these include pulmonary, gastrointestinal, genitourinary, cerebral, and dental complications. Pancytopenia eventually develops, progressing to bone marrow failure. In our patient, the progression of DC did not follow the "classic triad" typically seen in the condition. Here we report the case of a 27-year-old male who had features consistent with this syndrome but who had a normal oral cavity associated with aplastic anemia for which longstanding nail dystrophy and reticular pigmentation were the dominant clinical features. The patient was advised to avoid smoking and severe exposure to the sun, and to return regularly for follow-up. Because all symptoms of this disorder are not necessarily seen collectively in any one patient, physicians should consider it as a probability in all patients showing signs of reticular hyperpigmentation, especially accompanied by aplastic anemia. Our report emphasizes the fact that not all components of the syndrome need to be present and in all cases of genodermatosis, pleomorphism may be expected with variable clinical manifestations. Even though many variants have been described in literature, sparing of the oral cavity as in our patient is extremely uncommon. Therefore, physicians should be aware of the mucocutaneous manifestation of DC and its diagnosis and refer the patient for better evaluation.


Anterograde transport of surfactant protein C proprotein to distal processing compartments requires PPDY-mediated association with Nedd4 ubiquitin ligases.
Biosynthesis of surfactant protein C (SP-C) by alveolar type 2 cells requires proteolytic processing of a 21-kDa propeptide (proSP-C21) in post-Golgi compartments to yield a 3.7-kDa mature form. Scanning alanine mutagenesis, binding assays, and co-immunoprecipitation were used to characterize the proSP-C targeting domain. Delivery of proSP-C21 to distal processing organelles is dependent upon the NH2-terminal cytoplasmic SP-C propeptide, which contains a conserved PPDY motif. In A549 cells, transfection of EGFP/proSP-C21 constructs containing polyalanine substitution for Glu11-Thr18, 13PPDY16, or 14P,16Y produced endoplasmic reticulum retention of the fusion proteins. Protein-protein interactions of proSP-C with known WW domains were screened using a solid-phase array that revealed binding of the proSP-C NH2 terminus to several WW domains found in the Nedd4 family of E3 ligases. Specificity of the interaction was confirmed by co-immunoprecipitation of proSP-C and Nedd4 or Nedd4-2 in epithelial cell lines. By Western blotting and reverse transcription-PCR, both forms were detected in primary human type 2 cells. Knockdown of Nedd4-2 by small interference RNA transfection of cultured human type 2 cells blocked processing of 35S-labeled proSP-C21. Mutagenesis of potential acceptor sites for ubiquitination in the cytosolic domain of proSP-C (Lys6, Lys34, or both) failed to inhibit trafficking of EGFP/proSP-C21. These results indicate that PPDY-mediated interaction with Nedd4 E3-ligases is required for trafficking of proSP-C. We speculate that the Nedd4/proSP-C tandem is part of a larger protein complex containing a ubiquitinated component that further directs its transport.


Clinical Utility of Preoperative Computed Tomography in Patients With Endometrial Cancer.
The aim of this study was to determine the clinical utility of routine preoperative pelvic and abdominal computed tomography (CT) examinations in patients with endometrial cancer (EC). We retrospectively reviewed records from patients with EC who underwent a preoperative endometrial biopsy and had surgery at our institution from January 1999 through December 2008. In the subset with an abdominal CT scan obtained within 3 months before surgery, we evaluated the clinical utility of the CT scan. Overall, 224 patients (18%) had a preoperative endometrial biopsy and an available CT scan. Gross intra-abdominal disease was observed in 10% and 20% of patients with preoperative diagnosis of endometrioid G3 and type II EC, respectively, whereas less than 5% of patients had a preoperative diagnosis of hyperplasia or low-grade EC. When examining retroperitoneal findings, we observed that a negative CT scan of the pelvis did not exclude the presence of pelvic node metastasis. Alternately, a negative CT scan in the para-aortic area generally reduced the probability of finding para-aortic dissemination but with an overall low sensitivity (42%). However, the sensitivity for para-aortic dissemination was as high as 67% in patients with G3 endometrioid cancer. In the case of negative para-aortic nodes in the CT scan, the risk of para-aortic node metastases decreased from 18.8% to 7.5% in patients with endometrioid G3 EC. Up to 15% of patients with endometrioid G3 cancer had clinically relevant incidental findings that necessitated medical or surgical intervention. In patients with endometrioid G3 and type II EC diagnosed by the preoperative biopsy, CT scans may help guide the operative plan by facilitating preoperative identification of gross intra-abdominal disease and enlarged positive para-aortic nodes that are not detectable during physical examinations. In addition, CT may reveal other clinically relevant incidental findings.


Diagnostic application of PIK3CA mutation analysis in Chinese esophageal cancer patients.
The PIK3CA gene mutation was found to associate with prognosis and might affect molecular targeted therapy in esophageal carcinoma (EC). The aim of this study is to compare different methods for analyzing the PIK3CA gene mutation in EC. Genomic DNA was extracted from 106 surgically resected EC patient tissues. The PIK3CA mutation status (exons 9 and 20) were screened by mutant-enrich liquid chip (ME-Liquidchip), Sanger sequencing, and pyrosequencing. And all samples with mutations were independently reassessed using amplification refractory mutation system (ARMS) methods again. PIK3CA mutation rates were identified as 11.3% (12/106) by ME-Liquidchip. 10 mutations occurred in exon 9 and 2 in exon 20, including G1624A:E542K (n = 4), G1633A:E545K (n = 6) and A3140G:H1047R (n = 2). The results were further verified by ARMS methods. Among these 12 cases characterized for PIK3CA mutation, however, only 7 and 6 cases were identified by Sanger sequencing (6.6%,7/106) and pyrosequencing (5.7%,6/106), respectively. Sanger sequencing and pyrosequencing are less sensitive and are not efficiently applicable to the detection of PIK3CA mutation in EC samples. Choosing between ME-Liquidchip and ARMS will depend on laboratory facilities and expertise. The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/13000_2014_153.


The rich get richer: brain injury elicits hyperconnectivity in core subnetworks.
There remains much unknown about how large-scale neural networks accommodate neurological disruption, such as moderate and severe traumatic brain injury (TBI). A primary goal in this study was to examine the alterations in network topology occurring during the first year of recovery following TBI. To do so we examined 21 individuals with moderate and severe TBI at 3 and 6 months after resolution of posttraumatic amnesia and 15 age- and education-matched healthy adults using functional MRI and graph theoretical analyses. There were two central hypotheses in this study: 1) physical disruption results in increased functional connectivity, or hyperconnectivity, and 2) hyperconnectivity occurs in regions typically observed to be the most highly connected cortical hubs, or the "rich club". The current findings generally support the hyperconnectivity hypothesis showing that during the first year of recovery after TBI, neural networks show increased connectivity, and this change is disproportionately represented in brain regions belonging to the brain's core subnetworks. The selective increases in connectivity observed here are consistent with the preferential attachment model underlying scale-free network development. This study is the largest of its kind and provides the unique opportunity to examine how neural systems adapt to significant neurological disruption during the first year after injury.


Results of endoscopic maxillary mega-antrostomy in recalcitrant maxillary sinusitis.
In patients with chronically diseased maxillary sinuses, poor mucociliary clearance may result from long-standing inflammation or scarring from previous surgery. This subset of patients often has persistent sinus disease despite medical therapy and adequate antrostomy. Endoscopic maxillary mega-antrostomy (EMMA) is a mucosal sparing technique that facilitates mucus clearance and sinus irrigation in terminally dysfunctional maxillary sinuses. EMMA involves extending the antrostomy through the posterior half of the inferior turbinate down to the floor of the nose, creating a significantly enlarged antrostomy. This study describes our results of EMMA in recalcitrant maxillary sinusitis. A retrospective review was performed of patients who underwent EMMA for recalcitrant maxillary sinusitis between July 2005 and September 2007. We studied diagnoses, comorbid factors, clinical efficacy, revision rate, and complications. Twenty-eight patients (average age, 48 years) underwent 42 EMMAs for recalcitrant maxillary sinusitis. Average follow-up was 11 months. All patients had previous maxillary sinus surgery (mean = 2.3). Relevant comorbid factors included prior Caldwell-Luc or maxillofacial surgery (16/42), cystic fibrosis (11/42), asthma (11/42), and IgG deficiency (3/42). The most common symptoms reported were facial pain/pressure and purulent rhinorrhea. At the time of the most recent postoperative examination, 74% of patients reported complete resolution of symptoms while 26% reported partial symptomatic improvement. There were no complications and the revision rate was 0%. Maxillary sinuses that appear to be terminally diseased may be rehabilitated surgically without the need for surgical stripping. EMMA is an effective and safe treatment option for the management of recalcitrant maxillary sinus disease.


Harnessing symbiotic plant-fungus interactions to unleash hidden forces from extreme plant ecosystems.
Global climate change is arguably one of the biggest threats of modern times and has already led to a wide range of impacts on the environment, economy, and society. Owing to past emissions and climate system inertia, global climate change is predicted to continue for decades even if anthropogenic greenhouse gas emissions were to stop immediately. In many regions, such as central Europe and the Mediterranean region, the temperature is likely to rise by 2-5 °C and annual precipitation is predicted to decrease. Expected heat and drought periods followed by floods, and unpredictable growing seasons, are predicted to have detrimental effects on agricultural production systems, causing immense economic losses and food supply problems. To mitigate the risks of climate change, agricultural innovations counteracting these effects need to be embraced and accelerated. To achieve maximum improvement, the required agricultural innovations should not focus only on crops but rather pursue a holistic approach including the entire ecosystem. Over millions of years, plants have evolved in close association with other organisms, particularly soil microbes that have shaped their evolution and contemporary ecology. Many studies have already highlighted beneficial interactions among plants and the communities of microorganisms with which they coexist. Questions arising from these discoveries are whether it will be possible to decipher a common molecular pattern and the underlying biochemical framework of interspecies communication, and whether such knowledge can be used to improve agricultural performance under environmental stress conditions. In this review, we summarize the current knowledge of plant interactions with fungal endosymbionts found in extreme ecosystems. Special attention will be paid to the interaction of plants with the symbiotic root-colonizing endophytic fungus Serendipita indica, which has been developed as a model system for beneficial plant-fungus interactions.


Characterization of echocardiographic measures of cardiac structure and function in healthy octogenarians.
Currently, there is not enough echocardiographic information regarding aging-associated changes in the octogenarian population. We aimed to characterize echocardiographic measures of structure and function among a group of healthy octogenarians. Approximately 350 octogenarians, residing in nursing homes, were screened in Ankara, Turkey. According to inclusion criteria, 40 octogenarians were enrolled. These subjects underwent conventional and tissue Doppler echocardiography according to the guidelines of the American Society of Echocardiography (ASE). The population was also separated into various groups according to gender, body mass index (BMI, <25 vs. 25-29.9), and blood pressure (<80/120 mmHg vs. 80-89/120-139 mmHg). All measurements were indexed by dividing to body surface area (BSA) for standardization. Left ventricular mass (LVM), posterior wall thickness, right ventricular diameter, tricuspid E/A ratio, and septal e'-wave velocity were significantly higher in men, which lost significance after adjusting for BSA. There was no significant difference between groups formed by BMI and blood pressure. Moreover, mild global left and right ventricular dysfunction including a prominent diastolic counterpart, however, with normal ejection fraction was revealed using conventional and tissue Doppler techniques. Finally, we checked our results with the current reference values of the ASE and observed the following differences: ventricular septum, relative wall thickness, LVM, and mass index values were above ASE reference range, posterior wall measurements were close to upper range. On the contrary, left ventricular diameters and volumes were below ASE reference range. We described echocardiographic measures of structure and function in a group of healthy octogenarians.


Waist circumference as screening instrument for cardiovascular disease risk factors in schoolchildren.
To propose cutoff points for waist circumference of schoolchildren for the identification of cardiovascular disease risk factors. This school-based cross-sectional study surveyed 1,413 schoolchildren aged 7 to 12 years old, selected by random cluster sampling. Waist circumference, total cholesterol and systolic and diastolic pressures were measured. Reference values for cardiovascular disease risk factors were developed from measures of total cholesterol and systolic and diastolic blood pressures. The receiver operating characteristic (ROC) curve and bivariate analysis, followed by calculation of sensitivity, specificity and odds ratio, were used in data analysis. The cutoff points had area values under the ROC curve ranging between 0.603 and 0.949, while sensitivity and specificity ranged between 0.500 and 1.00. Among all proposals analyzed, the one presented by this study best balanced sensitivity and specificity values. Bivariate analysis followed by odds ratios calculation indicated that subjects with waist circumference above the proposed cutoff points have increased chances of presenting cardiovascular disease risk factors. The cutoff points proposed in the present study seem to be a valid alternative and better balance sensibility and specificity than other proposals for screening students with increased chances of presenting cardiovascular disease risk factors.


Maternal Circulating Placental Growth Factor and Neonatal Metabolic Health Biomarkers in Small for Gestational Age Infants.
Small for gestational age (SGA) infants are at increased risk of type 2 diabetes in adulthood. It is unknown whether any prenatal biomarkers are helpful for identifying SGA infants with altered metabolic health profile at birth or later life. In a nested study of 162 SGA (birth weight < 10th percentile) and 161 optimal birth weight (25th-75th percentiles) control infants in the 3D (design, develop and discover) birth cohort in Canada, we assessed whether maternal circulating placental growth factor (PlGF), a biomarker of placental function, is associated with metabolic health biomarkers in SGA infants. Main outcomes were cord plasma insulin, proinsulin, insulin-like growth factor-I (IGF-I), leptin, and high-molecular weight (HMW) adiponectin concentrations. Maternal PlGF concentrations at 32-35 weeks of gestation were substantially lower in SGA versus control infants (P < 0.001), so as were cord plasma proinsulin (P = 0.005), IGF-I (P < 0.001), leptin (P < 0.001), and HMW adiponectin (P = 0.002) concentrations. In SGA infants with both low (<25th percentile) and normal maternal PlGF concentrations, cord plasma IGF-I and leptin concentrations were lower than control infants, but the decreases were to a greater extent in SGA infants with low maternal PlGF. Cord blood leptin levels were lower comparing SGA infants with low vs. normal maternal PlGF levels (P = 0.01). SGA infants with low maternal circulating PlGF levels at late gestation were characterized by greater decreases in cord blood IGF-I and leptin concentrations. Maternal circulating PlGF appears to be associated with neonatal metabolic health profile in SGA infants.


The Caenorhabditis elegans hif-1 gene encodes a bHLH-PAS protein that is required for adaptation to hypoxia.
Hypoxia-inducible factor, a heterodimeric transcription complex, regulates cellular and systemic responses to low oxygen levels (hypoxia) during normal mammalian development or tumor progression. Here, we present evidence that a similar complex mediates response to hypoxia in Caenorhabditis elegans. This complex consists of HIF-1 and AHA-1, which are encoded by C. elegans homologs of the hypoxia-inducible factor (HIF) alpha and beta subunits, respectively. hif-1 mutants exhibit no severe defects under standard laboratory conditions, but they are unable to adapt to hypoxia. Although wild-type animals can survive and reproduce in 1% oxygen, the majority of hif-1-defective animals die in these conditions. We show that the expression of an HIF-1:green fluorescent protein fusion protein is induced by hypoxia and is subsequently reduced upon reoxygenation. Both hif-1 and aha-1 are expressed in most cell types, and the gene products can be coimmunoprecipitated. We conclude that the mechanisms of hypoxia signaling are likely conserved among metazoans. Additionally, we find that nuclear localization of AHA-1 is disrupted in an hif-1 mutant. This finding suggests that heterodimerization may be a prerequisite for efficient nuclear translocation of AHA-1.


The role of the glassy dynamics and thermal mixing in the dynamic nuclear polarization and relaxation mechanisms of pyruvic acid.
The temperature dependence of (1)H and (13)C nuclear spin-lattice relaxation rate 1/T1 has been studied in the 1.6-4.2 K temperature range in pure pyruvic acid and in pyruvic acid containing trityl radicals at a concentration of 15 mM. The temperature dependence of 1/T1 is found to follow a quadratic power law for both nuclei in the two samples. Remarkably the same temperature dependence is displayed also by the electron spin-lattice relaxation rate 1/T1e in the sample containing radicals. These results are explained by considering the effect of the structural dynamics on the relaxation rates in pyruvic acid. Dynamic nuclear polarization experiments show that below 4 K the (13)C build up rate scales with 1/T1e, in analogy to (13)C 1/T1 and consistently with a thermal mixing scenario where all the electrons are collectively involved in the dynamic nuclear polarization process and the nuclear spin reservoir is in good thermal contact with the electron spin system.


Age determination of vessel wall hematoma in spontaneous cervical artery dissection: a multi-sequence 3T cardiovascular magnetic resonance study.
Previously proposed classifications for carotid plaque and cerebral parenchymal hemorrhages are used to estimate the age of hematoma according to its signal intensities on T1w and T2w MR images. Using these classifications, we systematically investigated the value of cardiovascular magnetic resonance (CMR) in determining the age of vessel wall hematoma (VWH) in patients with spontaneous cervical artery dissection (sCAD). 35 consecutive patients (mean age 43.6 ± 9.8 years) with sCAD received a cervical multi-sequence 3T CMR with fat-saturated black-blood T1w-, T2w- and TOF images. Age of sCAD was defined as time between onset of symptoms (stroke, TIA or Horner's syndrome) and the CMR scan. VWH were categorized into hyperacute, acute, early subacute, late subacute and chronic based on their signal intensities on T1w- and T2w images. The mean age of sCAD was 2.0, 5.8, 15.7 and 58.7 days in patients with acute, early subacute, late subacute and chronic VWH as classified by CMR (p < 0.001 for trend). Agreement was moderate between VWH types in our study and the previously proposed time scheme of signal evolution for cerebral hemorrhage, Cohen's kappa 0.43 (p < 0.001). There was a strong agreement of CMR VWH classification compared to the time scheme which was proposed for carotid intraplaque hematomas with Cohen's kappa of 0.74 (p < 0.001). Signal intensities of VWH in sCAD vary over time and multi-sequence CMR can help to determine the age of an arterial dissection. Furthermore, findings of this study suggest that the time course of carotid hematomas differs from that of cerebral hematomas.


Efficient moment-based inference of admixture parameters and sources of gene flow.
The recent explosion in available genetic data has led to significant advances in understanding the demographic histories of and relationships among human populations. It is still a challenge, however, to infer reliable parameter values for complicated models involving many populations. Here, we present MixMapper, an efficient, interactive method for constructing phylogenetic trees including admixture events using single nucleotide polymorphism (SNP) genotype data. MixMapper implements a novel two-phase approach to admixture inference using moment statistics, first building an unadmixed scaffold tree and then adding admixed populations by solving systems of equations that express allele frequency divergences in terms of mixture parameters. Importantly, all features of the model, including topology, sources of gene flow, branch lengths, and mixture proportions, are optimized automatically from the data and include estimates of statistical uncertainty. MixMapper also uses a new method to express branch lengths in easily interpretable drift units. We apply MixMapper to recently published data for Human Genome Diversity Cell Line Panel individuals genotyped on a SNP array designed especially for use in population genetics studies, obtaining confident results for 30 populations, 20 of them admixed. Notably, we confirm a signal of ancient admixture in European populations-including previously undetected admixture in Sardinians and Basques-involving a proportion of 20-40% ancient northern Eurasian ancestry.


The Extended "Two-Barrel" Polymerases Superfamily: Structure, Function and Evolution.
DNA and RNA polymerases (DNAP and RNAP) play central roles in genome replication, maintenance and repair, as well as in the expression of genes through their transcription. Multisubunit RNAPs carry out transcription and are represented, without exception, in all cellular life forms as well as in nucleo-cytoplasmic DNA viruses. Since their discovery, multisubunit RNAPs have been the focus of intense structural and functional studies revealing that they all share a well-conserved active-site region called the two-barrel catalytic core. The two-barrel core hosts the polymerase active site, which is located at the interface between two double-psi β-barrel domains that contribute distinct amino acid residues to the active site in an asymmetrical fashion. Recently, sequencing and structural studies have added a surprising variety of DNA and RNA to the two-barrel superfamily, including the archaeal replicative DNAP (PolD), which extends the family to DNA-dependent DNAPs involved in replication. While all these polymerases share a minimal core that must have been present in their common ancestor, the two-barrel polymerase superfamily now encompasses a remarkable diversity of enzymes, including DNA-dependent RNAPs, RNA-dependent RNAPs, and DNA-dependent DNAPs, which participate in critical biological processes such as DNA transcription, DNA replication, and gene silencing. The present review will discuss both common features and differences among the extended two-barrel polymerase superfamily, focusing on the newly discovered members. Comparing their structures provides insights into the molecular mechanisms evolved by the contemporary two-barrel polymerases to accomplish their different biological functions.


Selective activation of mechanosensitive ion channels using magnetic particles.
This study reports the preliminary development of a novel magnetic particle-based technique that permits the application of highly localized mechanical forces directly to specific regions of an ion-channel structure. We demonstrate that this approach can be used to directly and selectively activate a mechanosensitive ion channel of interest, namely TREK-1. It is shown that manipulation of particles targeted against the extended extracellular loop region of TREK-1 leads to changes in whole-cell currents consistent with changes in TREK-1 activity. Responses were absent when particles were coated with RGD (Arg-Gly-Asp) peptide or when magnetic fields were applied in the absence of magnetic particles. It is concluded that changes in whole-cell current are the result of direct force application to the extracellular loop region of TREK-1 and thus these results implicate this region of the channel structure in mechano-gating. It is hypothesized that the extended loop region of TREK-1 may act as a tension spring that acts to regulate sensitivity to mechanical forces, in a nature similar to that described for MscL. The development of a technique that permits the direct manipulation of mechanosensitive ion channels in real time without the need for pharmacological drugs has huge potential benefits not only for basic biological research of ion-channel gating mechanisms, but also potentially as a tool for the treatment of human diseases caused by ion-channel dysfunction.


Quantitation of multiple mycotoxins and cyanogenic glucosides in cassava samples from Tanzania and Rwanda by an LC-MS/MS-based multi-toxin method.
A multi-mycotoxin method based on liquid chromatography/tandem mass spectrometry (LC-MS/MS) was used for a mycotoxin survey in 627 samples of processed cassava collected from different districts across Tanzania and Rwanda after the method performance for this matrix had been determined. Matrix effects as well as extraction efficiencies were found to be similar to most other previously investigated matrices with the exception of distinct matrix effects in the negative ionisation mode for early eluting compounds. Limits of detection were far below the regulatory limits set in the European Union for other types of commodities. Relative standard deviations were generally lower than 10% as determined by replicates spiked on two concentration levels. The sample-to-sample variation of the apparent recoveries was determined for 15 individually spiked samples during three different analytical sequences. The related standard deviation was found to be lower than 15% for most of the investigated compounds, thus confirming the applicability of the method for quantitative analysis. The occurrence of regulated mycotoxins was lower than 10% (with the exception of zearalenone) and the related limits were exceeded only in few samples, which suggests that cassava is a comparatively safe commodity as regards mycotoxins. The most prevalent fungal metabolites were emodin, kojic acid, beauvericin, tryptophol, 3-nitropropionic acid, equisetin, alternariol methylether, monocerin, brevianamide F, tenuazonic acid, zearalenone, chrysophanol, monilifomin, enniatins, apicidin and macrosporin. The related concentrations exceeded 1 mg kg(-1) only in few cases. However, extremely high levels of cyanogenic plant toxins, which had been previously added to the method, were observed in few samples, pointing out the need for improved post-harvest management to decrease the levels of these compounds.


Responses of Mn2+ speciation in Deinococcus radiodurans and Escherichia coli to γ-radiation by advanced paramagnetic resonance methods.
The remarkable ability of bacterium Deinococcus radiodurans to survive extreme doses of γ-rays (12,000 Gy), 20 times greater than Escherichia coli, is undiminished by loss of Mn-dependent superoxide dismutase (SodA). D. radiodurans radiation resistance is attributed to the accumulation of low-molecular-weight (LMW) "antioxidant" Mn(2+)-metabolite complexes that protect essential enzymes from oxidative damage. However, in vivo information about such complexes within D. radiodurans cells is lacking, and the idea that they can supplant reactive-oxygen-species (ROS)-scavenging enzymes remains controversial. In this report, measurements by advanced paramagnetic resonance techniques [electron-spin-echo (ESE)-EPR/electron nuclear double resonance/ESE envelope modulation (ESEEM)] reveal differential details of the in vivo Mn(2+) speciation in D. radiodurans and E. coli cells and their responses to 10 kGy γ-irradiation. The Mn(2+) of D. radiodurans exists predominantly as LMW complexes with nitrogenous metabolites and orthophosphate, with negligible EPR signal from Mn(2+) of SodA. Thus, the extreme radiation resistance of D. radiodurans cells cannot be attributed to SodA. Correspondingly, 10 kGy irradiation causes no change in D. radiodurans Mn(2+) speciation, despite the paucity of holo-SodA. In contrast, the EPR signal of E. coli is dominated by signals from low-symmetry enzyme sites such as that of SodA, with a minority pool of LMW Mn(2+) complexes that show negligible coordination by nitrogenous metabolites. Nonetheless, irradiation of E. coli majorly changes LMW Mn(2+) speciation, with extensive binding of nitrogenous ligands created by irradiation. We infer that E. coli is highly susceptible to radiation-induced ROS because it lacks an adequate supply of LMW Mn antioxidants.


Effects of linoleic and gamma-linolenic acids (efamol evening primrose oil) on fatty acid-binding proteins of rat liver.
We have studied the effects of Efamol evening primrose oil (EPO) on fatty acid-binding proteins (L-FABP) of rat liver. EPO contains 72% cis-linoleic acid and 9% cis-gamma linolenic acid. EPO has been clinically used for treatment of a number of diseases in humans and animals. EPO is also known to lower cholesterol level in humans and animals. Feeding of an EPO supplemented diet to rats (n = 9) for 2 months decreases the oleate binding capacity of purified L-FABP of rat liver whereas the palmitate binding activity was increased by 38%. However, EPO feeding did not alter the L-FABP concentrations significantly as measured by using the fluorescence fatty acid probe, dansylamino undecanoic acid. Endogenous fatty acid analysis of L-FABPs revealed significant qualitative and quantitative changes in fatty acid pattern after EPO feeding. EPO feeding decreased the endogenous palmitate level by 53% and oleate level by 64% in L-FABPs and also EPO feeding decreased the total endogenous fatty acid content from 62 nanomole per mg of protein to 42 nanomole per mg of L-FABP (n = 3).


Labeling characteristics and separation of Ia antigen subunits.
Experiments with biosynthetic incorporation of 3H-amino acids into murine and guinea pig Ia antigens have indicated that these antigens consist of two polypeptide chains of 33,000 and 25,000 daltons, respectively, occasionally linked by disulfide bonds into a 58,000 dalton molecule. In contrast, studies with lactoperoxidase-catalyzed radioiodination have indicated that these Ia antigens consist of only a single chain of 25,000 daltons. We therefore undertook a study to explore the basis of these discrepant results. Since 3H-tyrosine labeled both chains well, the lack of tyrosine residues in the 33,000 dalton chain could not be the explanation for the lack of radioiodination. However, by partially purifying the Ia antigen preparation with Lens culinaris (lentil) lectin affinity chromatography before immunoprecipitation and by increasing the resolution of analysis by using discontinuous-SDS polyacrylamide gel electrophoresis, it was possible to show that the 33,000 dalton chain was in fact radioiodinated, though still poorly so relative to the 25,000 dalton chain, and that a radioiodinated 58,000 dalton molecule could be detected. These experiments suggest that the 25,000 dalton chain is more exposed to the external cellular environment, and thus more readily iodinated by lactoperoxidase. In addition, the studies indicate that the choice of labeling method, purification procedures, and analytical methods must be taken into account when interpreting experimental results.