Antisense oligonucleotide-induced exon skipping restores dystrophin expression in vitro in a canine model of DMD.
Manipulation of pre-mRNA splicing by antisense oligonucleotides (AOs) offers considerable potential for a number of genetic disorders. One of these is Duchenne muscular dystrophy (DMD), where mutations in the dystrophin gene typically result in premature termination of translation that causes a loss of functional protein. AOs can induce exon skipping such that the mutation is by-passed and the reading frame restored, producing an internally deleted protein similar to that found in the milder Becker muscular dystrophy. To date, this approach has been applied to the mdx mouse model in vitro and in vivo and in human myoblast cultures. Here, we report the application of AO-directed exon skipping to induce dystrophin expression in vitro in a canine model of DMD, golden retriever muscular dystrophy (GRMD). The efficacy of 2'-O-methyl phosphorothioate (2OMe), phosphorodiamidate morpholino oligomers (PMOs) and peptide-linked PMOs (PMO-Pep) to induce dystrophin expression was assessed. The 2OMe chemistry was only effective for short-term induction of corrected transcript and could not induce detectable dystrophin protein. The PMO chemistry generally induced limited exon skipping at only high concentrations; however, a low level of dystrophin protein was produced in treated cells. Use of the PMO-Pep, applied here for the first time to a DMD model, was able to induce high and sustained levels of exon skipping and induced the highest level of dystrophin expression with no apparent adverse effects upon the cells. The induction of dystrophin in the GRMD model offers the potential for further testing of AO delivery regimens in a larger animal model of DMD, in preparation for application in human clinical trials.


Montelukast in the treatment of allergic rhinitis: an evidence-based review.
Cysteinyl-leukotrienes (CysLTs) are endogenous mediators of inflammation and play an important role in allergic airway disease by stimulating bronchoconstriction, mucus production, mucosal oedema and inflammation, airway infiltration by eosinophils, and dendritic cell maturation that prepares for future allergic response. Montelukast inhibits these actions by blocking type 1 CysLT receptors found on immunocytes, smooth muscle and endothelium in the respiratory mucosa. Initially developed as a treatment for asthma, montelukast has more recently found use in the treatment of allergic rhinitis (AR). We conducted a systematic review of studies that have evaluated montelukast in the treatment of seasonal AR (SAR) and perennial AR (PAR), with and without concomitant asthma. Primary consideration was given to large, randomised, placebo-controlled, double-blind clinical trials in which AR endpoints were assessed and the use of concurrent treatments for AR was excluded. Eight such studies were found in the literature. The primary endpoint in these was daytime nasal symptom severity represented by a composite score derived from individual self-ratings of nasal congestion, rhinorrhoea, nasal pruritus and sneezing. Secondary endpoints have included these individual nasal symptom scores, additional scores for eye, ear and throat symptoms, the impact of rhinitis on quality of sleep, global evaluations of outcome by patients and physicians, and measures of the severity of concomitant asthma. A general outcome was that patients treated with montelukast had significantly greater improvements in their symptoms of SAR and PAR than did patients who were given a placebo. As monotherapy, montelukast exhibited efficacy similar to that of loratadine, but less than that of the intranasally administered corticosteroid fluticasone propionate. The use of montelukast in combination with antihistamines such as loratadine or cetirizine has generally resulted in greater efficacy than when these agents were used alone, and in some studies has produced results comparable with intranasally applied corticosteroids. In patients with AR comorbid with asthma, montelukast treatment has resulted in significant improvements in both, compared with placebo. Montelukast is well tolerated and has a favourable safety profile; adverse events have occurred at similar frequencies in patients taking either montelukast or placebo. Montelukast provides an effective and well tolerated oral treatment for allergic airway inflammation in patients with SAR or PAR without asthma, and in patients in whom AR is comorbid with asthma.


Prediction of immunogenicity of therapeutic proteins: validity of computational tools.
Most protein therapeutics have the potential to induce undesirable immune responses in patients. Many patients develop anti-therapeutic antibodies, which can affect the safety and efficacy of the therapeutic protein, particularly if the response is neutralizing. There are a variety of factors that influence the immunogenicity of protein therapeutics and, in particular, the presence of B- and T-cell epitopes is considered to be of importance. In silico tools to identify the location of both B- and T-cell epitopes and to assess the potential for immunogenicity have been developed, and such tools provide an alternative to more complex in vitro or in vivo immunogenicity assays. This article reviews computational epitope prediction methods and also the use of manually curated databases containing experimentally derived epitope data. However, due to the complexities of the molecular interactions involved in epitope recognition by the immune system, the heterogeneity of key proteins in human populations and the adaptive nature of the immune response, in silico methods have not yet achieved a level of accuracy that enables them to be used as stand-alone tools for predicting clinical immunogenicity. Computational methods, particularly with regard to T-cell epitopes, only consider a limited number of events in the process of epitope formation and therefore routinely over-predict the number of epitopes within a molecule. Epitope databases such as the Immune Epitope Database (IEDB) and the proprietary T Cell Epitope Database (TCED) have reached a size and level of organization that increases their utility; however, they are not exhaustive. These methods have greatest utility as an adjunct to in vitro assays where they can be used either to reduce the amount and complexity of the in vitro screening, or they can be used as tools to analyze the sequence of the identified epitope in order to locate amino acids critical for its properties.


Clinical classification of pulmonary hypertension.
In 1998, during the Second World Symposium on Pulmonary Hypertension (PH) held in Evian, France, a clinical classification of PH was proposed. The aim of the Evian classification was to individualize different categories sharing similarities in pathophysiological mechanisms, clinical presentation, and therapeutic options. The Evian classification is now well accepted and widely used in clinical practice, especially in specialized centers. In addition, this classification has been used by the U.S. Food and Drug Administration and the European Agency for Drug Evaluation for the labeling of newly approved medications in PH. In 2003, during the Third World Symposium on Pulmonary Arterial Hypertension held in Venice, Italy, it was decided to maintain the general architecture and philosophy of the Evian classification. However, some modifications have been proposed, mainly to abandon the term "primary pulmonary hypertension" and to replace it with "idiopathic pulmonary hypertension"; to reclassify pulmonary veno-occlusive disease and pulmonary capillary hemangiomatosis; to update risk factors and associated conditions for pulmonary arterial hypertension and to propose guidelines in order to improve the classification of congenital systemic-to-pulmonary shunts.


The protozoan, Paramecium primaurelia, as a non-sentient model to test laser light irradiation: The effects of an 808nm infrared laser diode on cellular respiration.
Photobiomodulation (PBM) has been used in clinical practice for more than 40 years. Unfortunately, conflicting literature has led to the labelling of PBM as a complementary or alternative medicine approach. However, past and ongoing clinical and research studies by reputable investigators have re-established the merits of PBM as a genuine medical therapy, and the technique has, in the last decade, seen an exponential increase in the numbers of clinical instruments available, and their applications. This resurgence has led to a clear need for appropriate experimental models to test the burgeoning laser technology being developed for medical applications. In this context, an ethical model that employs the protozoan, Paramecium primaurelia, is proposed. We studied the possibility of using the measure of oxygen consumption to test PBM by irradiation with an infrared or near-infrared laser. The results show that an 808nm infrared laser diode (1W; 64J/cm²) affects cellular respiration in P. primaurelia, inducing, in the irradiated cells, a significantly (p < 0.05) increased oxygen consumption of about 40%. Our findings indicate that Paramecium can be an excellent tool in biological assays involving infrared and near-infrared PBM, as it combines the advantages of in vivo results with the practicality of in vitro testing. This test represents a fast, inexpensive and straightforward assay, which offers an alternative to both traditional in vivo testing and more expensive mammalian cellular cultures.


On the relationship between ontogenetic and static allometry.
Ontogenetic and static allometries describe how a character changes in size when the size of the organism changes during ontogeny and among individuals measured at the same developmental stage, respectively. Understanding the relationship between these two types of allometry is crucial to understanding the evolution of allometry and, more generally, the evolution of shape. However, the effects of ontogenetic allometry on static allometry remain largely unexplored. Here, we first show analytically how individual variation in ontogenetic allometry and body size affect static allometry. Using two longitudinal data sets on ontogenetic and static allometry, we then estimate variances and covariances for the different parameters of the ontogenetic allometry defined in our model and assess their relative contribution to the static allometric slope. The mean ontogenetic allometry is the main parameter that determines the static allometric slope, while the covariance between the ontogenetic allometric slope and body size generates most of the discrepancies between ontogenetic and static allometry. These results suggest that the apparent evolutionary stasis of the static allometric slope is not generated by internal (developmental) constraints but more likely results from external constraints imposed by selection.


Repeated intranasal ketamine for treatment-resistant depression - the way to go? Results from a pilot randomised controlled trial.
Ketamine research in depression has mostly used intravenous, weight-based approaches, which are difficult to translate clinically. Intranasal (IN) ketamine is a promising alternative but no controlled data has been published on the feasibility, safety and potential efficacy of repeated IN ketamine treatments. This randomised, double-blind, placebo-controlled pilot study compared a 4-week course of eight treatments of 100 mg ketamine or 4.5 mg midazolam. Each treatment was given as 10 separate IN sprays, self-administered 5 min apart. The study was stopped early due to poor tolerability after five treatment-resistant depressed participants were included. Feasibility, safety (acute and cumulative), cognitive and efficacy outcomes were assessed. Plasma ketamine and norketamine concentrations were assayed after the first treatment. Significant acute cardiovascular, psychotomimetic and neurological side effects occurred at doses < 100 mg ketamine. No participants were able to self-administer all 10 ketamine sprays due to incoordination; treatment time occasionally had to be extended (>45 min) due to acute side effects. No hepatic, cognitive or urinary changes were observed after the treatment course in either group. There was an approximately two-fold variation in ketamine and norketamine plasma concentrations between ketamine participants. At course end, one participant had remitted in each of the ketamine and midazolam groups. IN ketamine, with the drug formulation and delivery device used, was not a useful treatment approach in this study. Absorption was variable between individuals and acute tolerability was poor, requiring prolonged treatment administration time in some individuals. The drug formulation, the delivery device, the insufflation technique and individual patient factors play an important role in tolerability and efficacy when using IN ketamine for TRD.


Relationship between C-Reactive Protein Level and Diabetic Retinopathy: A Systematic Review and Meta-Analysis.
To date, the relationship between C-reactive protein (CRP) level and diabetic retinopathy (DR) remains controversial. Therefore, a systematic review and meta-analysis was used to reveal the potential relationship between CRP level and DR. A systematic search of PubMed, Embase.com, and Web of Science was performed to identify all comparative studies that compared the CRP level of two groups (case group and control group). We defined that diabetic patients without retinopathy and/or matched healthy persons constituted the control group, and patients with DR were the case group. Two cross sectional studies and twenty case control studies including a total of 3679 participants were identified. After pooling the data from all 22 studies, obvious heterogeneity existed between the studies, so a subgroup analysis and sensitivity analysis were performed. Removing the sensitivity studies, the blood CRP levels in the case group were observed to be higher than those in the control group [SMD = 0.22, 95% confidence interval (CI), 0.11-0.34], and the blood CRP levels in the proliferative diabetic retinopathy (PDR) group were also higher than those in the non-proliferative diabetic retinopathy (NPDR) group [SMD = 0.50, 95% CI, 0.30-0.70]. The results from this current meta-analysis indicate that the CRP level might be used as a biomarker to determine the severity of DR.


Salmonella typhimurium synthesizes cobalamin (vitamin B12) de novo under anaerobic growth conditions.
In this paper, we report that the enteric bacterium Salmonella typhimurium synthesized cobalamin de novo under anaerobic culture conditions. Aerobically, metE mutants of S. typhimurium need either methionine or cobalamin as a nutritional supplement for growth. The growth response to cobalamin depends upon a cobalamin-requiring enzyme, encoded by the gene metH, that catalyzes the same reaction as the metE enzyme. Anaerobically, metE mutants grew without any nutritional supplements; the metH enzyme functioned under these conditions due to the endogenous biosynthesis of cobalamin. This conclusion was confirmed by using a radiochemical assay to measure cobalamin production. Insertion mutants defective in cobalamin biosynthesis (designated cob) were isolated in the three major branches of the cobalamin biosynthetic pathway. Type I mutations blocked the synthesis of cobinamide, type II mutations blocked the synthesis of 5,6-dimethylbenzimidazole, and type III mutations blocked the synthesis of cobalamin from cobinamide and 5,6-dimethylbanzimidazole. Mutants that did not synthesize siroheme (cysG) were blocked in cobalamin synthesis. Genetic mapping experiments showed that the cob mutations are clustered in the region of the S. typhimurium chromosome between supD (40 map units) and his (42 map units). The discovery that S. typhimurium synthesizes cobalamin de novo only under anaerobic conditions raises the possibility that anaerobically grown cells possess a variety of enzymes which are dependent upon cobalamin as a cofactor.


Targeting Survivin Inhibits Renal Cell Carcinoma Progression and Enhances the Activity of Temsirolimus.
Elevated expression of the antiapoptotic factor survivin has been implicated in cancer cell survival and disease progression. However, its specific contribution to renal cell carcinoma (RCC) pathogenesis is not well defined. We investigated the roles of survivin in RCC tumor progression, resistance to mTOR inhibitors, and evaluated the therapeutic activity of the survivin suppressant YM155 in RCC models. Here, we report that survivin expression levels were significantly higher in RCC cell lines compared with normal renal cells. Stable targeted knockdown of survivin completely abrogated the ability of 786-O RCC tumors to grow in mice, thus demonstrating its importance as a regulator of RCC tumorigenesis. We next explored multiple strategies to therapeutically inhibit survivin function in RCC. Treatment with the mTOR inhibitor temsirolimus partially diminished survivin levels and this effect was augmented by the addition of YM155. Further analyses revealed that, in accordance with their combined anti-survivin effects, YM155 significantly improved the anticancer activity of temsirolimus in a panel of RCC cell lines in vitro and in xenograft models in vivo. Similar to pharmacologic inhibition of survivin, shRNA-mediated silencing of survivin expression not only inhibited RCC tumor growth, but also significantly sensitized RCC cells to temsirolimus therapy. Subsequent experiments demonstrated that the effectiveness of this dual survivin/mTOR inhibition strategy was mediated by a potent decrease in survivin levels and corresponding induction of apoptosis. Our findings establish survivin inhibition as a novel approach to improve RCC therapy that warrants further investigation.


Lkb1 inactivation drives lung cancer lineage switching governed by Polycomb Repressive Complex 2.
Adenosquamous lung tumours, which are extremely poor prognosis, may result from cellular plasticity. Here, we demonstrate lineage switching of KRAS+ lung adenocarcinomas (ADC) to squamous cell carcinoma (SCC) through deletion of Lkb1 (Stk11) in autochthonous and transplant models. Chromatin analysis reveals loss of H3K27me3 and gain of H3K27ac and H3K4me3 at squamous lineage genes, including Sox2, ΔNp63 and Ngfr. SCC lesions have higher levels of the H3K27 methyltransferase EZH2 than the ADC lesions, but there is a clear lack of the essential Polycomb Repressive Complex 2 (PRC2) subunit EED in the SCC lesions. The pattern of high EZH2, but low H3K27me3 mark, is also prevalent in human lung SCC and SCC regions within ADSCC tumours. Using FACS-isolated populations, we demonstrate that bronchioalveolar stem cells and club cells are the likely cells-of-origin for SCC transitioned tumours. These findings shed light on the epigenetics and cellular origins of lineage-specific lung tumours.


Locomotion of electrocatalytic nanomotors due to reaction induced charge autoelectrophoresis.
Bimetallic rod-shaped nanomotors swim autonomously in hydrogen peroxide solutions. Here, we present a scaling analysis, computational simulations, and experimental data that show that the nanomotor locomotion is driven by fluid slip around the nanomotor surface due to electrical body forces. The body forces are generated by a coupling of charge density and electric fields induced by electrochemical reactions occurring on the nanomotor surface. We describe the dependence of nanomotor motion on the nanomotor surface potential and reaction-driven flux.


Molecular characterization of the first saltwater crocodilepox virus genome sequences from the world's largest living member of the Crocodylia.
Crocodilepox virus is a large dsDNA virus belonging to the genus Crocodylidpoxvirus, which infects a wide range of host species in the order Crocodylia worldwide. Here, we present genome sequences for a novel saltwater crocodilepox virus, with two subtypes (SwCRV-1 and -2), isolated from the Australian saltwater crocodile. Affected belly skins of juvenile saltwater crocodiles were used to sequence complete viral genomes, and perform electron microscopic analysis that visualized immature and mature virions. Analysis of the SwCRV genomes showed a high degree of sequence similarity to CRV (84.53% and 83.70%, respectively), with the novel SwCRV-1 and -2 complete genome sequences missing 5 and 6 genes respectively when compared to CRV, but containing 45 and 44 predicted unique genes. Similar to CRV, SwCRV also lacks the genes involved in virulence and host range, however, considering the presence of numerous hypothetical and or unique genes in the SwCRV genomes, it is completely reasonable that the genes encoding these functions are present but not recognized. Phylogenetic analysis suggested a monophyletic relationship between SwCRV and CRV, however, SwCRV is quite distinct from other chordopoxvirus genomes. These are the first SwCRV complete genome sequences isolated from saltwater crocodile skin lesions.


TLR stimulation initiates a CD39-based autoregulatory mechanism that limits macrophage inflammatory responses.
Sepsis is a highly fatal disease caused by an initial hyperinflammatory response followed by a state of profound immunosuppression. Although it is well appreciated that the initial production of proinflammatory cytokines by macrophages accompanies the onset of sepsis, it remains unclear what causes the transition to an immunosuppressive state. In this study, we reveal that macrophages themselves are key regulators of this transition and that the surface enzyme CD39 plays a critical role in self-limiting the activation process. We demonstrate that Toll-like receptor (TLR)-stimulated macrophages modulate their activation state by increasing the synthesis and secretion of adenosine triphosphate (ATP). This endogenous ATP is paradoxically immunosuppressive due to its rapid catabolism into adenosine by CD39. Macrophages lacking CD39 are unable to transition to a regulatory state and consequently continue to produce inflammatory cytokines. The importance of this transition is demonstrated in a mouse model of sepsis, where small numbers of CD39-deficient macrophages were sufficient to induce lethal endotoxic shock. Thus, these data implicate CD39 as a key "molecular switch" that allows macrophages to self-limit their activation state. We propose that therapeutics targeting the release and hydrolysis of ATP by macrophages may represent new ways to treat inflammatory diseases.


A multiple-phenotype imputation method for genetic studies.
Genetic association studies have yielded a wealth of biological discoveries. However, these studies have mostly analyzed one trait and one SNP at a time, thus failing to capture the underlying complexity of the data sets. Joint genotype-phenotype analyses of complex, high-dimensional data sets represent an important way to move beyond simple genome-wide association studies (GWAS) with great potential. The move to high-dimensional phenotypes will raise many new statistical problems. Here we address the central issue of missing phenotypes in studies with any level of relatedness between samples. We propose a multiple-phenotype mixed model and use a computationally efficient variational Bayesian algorithm to fit the model. On a variety of simulated and real data sets from a range of organisms and trait types, we show that our method outperforms existing state-of-the-art methods from the statistics and machine learning literature and can boost signals of association.


Measuring population differentiation using GST or D? A simulation study with microsatellite DNA markers under a finite island model and nonequilibrium conditions.
The genetic differentiation of populations is a key parameter in population genetic investigations. Wright's F(ST) (and its relatives such as G(ST) ) has been a standard measure of differentiation. However, the deficiencies of these indexes have been increasingly realized in recent years, leading to some new measures being proposed, such as Jost's D (Molecular Ecology, 2008; 17, 4015). The existence of these new metrics has stimulated considerable debate and induced some confusion on which statistics should be used for estimating population differentiation. Here, we report a simulation study with neutral microsatellite DNA loci under a finite island model to compare the performance of G(ST) and D, particularly under nonequilibrium conditions. Our results suggest that there exist fundamental differences between the two statistics, and neither G(ST) nor D operates satisfactorily in all situations for quantifying differentiation. D is very sensitive to mutation models but G(ST) noticeably less so, which limits D's utility in population parameter estimation and comparisons across genetic markers. Also, the initial heterozygosity of the starting populations has some important effects on both the individual behaviours of G(ST) and D and their relative behaviours in early differentiation, and this effect is much greater for D than G(ST) . In the early stages of differentiation, when initial heterozygosity is relatively low (<0.5, if the number of subpopulations is large), G(ST) increases faster than D; the opposite is true when initial heterozygosity is high. Therefore, the state of the ancestral population appears to have some lasting impacts on population differentiation. In general, G(ST) can measure differentiation fairly well when heterozygosity is low whatever the causes; however, when heterozygosity is high (e.g. as a result of either high mutation rate or high initial heterozygosity) and gene flow is moderate to strong, G(ST) fails to measure differentiation. Interestingly, when population size is not very small (e.g. N ≥ 1000), G(ST) measures differentiation quite linearly with time over a long duration when gene flow is absent or very weak even if mutation rate is not low (e.g. μ = 0.001). In contrast, D, as a differentiation measure, performs rather robustly in all these situations. In practice, both indexes should be calculated and the relative levels of heterozygosities (especially H(S) ) and gene flow taken into account. We suggest that a comparison of the two indexes can generate useful insights into the evolutionary processes that influence population differentiation.


Measurement of mass transfer during dip-pen nanolithography with phospholipids.
Dip-pen nanolithography (DPN) has been extensively used before for patterning surfaces; however a complete understanding of the ink transport mechanisms is still lacking. Moreover, quality control of the fabricated structures is a bottleneck in DPN fabrication, and one aspect of this is the quantification of the ink mass transfer to the substrate during the lithographic process. There is a demand for measuring the exact amount of molecules deposited on a surface by lithographic methods, especially for biological applications. This article demonstrates a quantitative method for measuring the amount of ink transferred onto the substrate in DPN with phospholipids by dynamic force spectroscopy. To achieve this, the harmonic oscillation of a microcantilever in an atomic force microscope is used, obtaining picogram mass sensitivity in the determination of mass deposition.


An experimental model of preretinal neovascularization in the rabbit.
Although progressive retinal neovascularization is a potentially blinding complication of several diseases, there are no good animal models. The authors developed a consistent model of preretinal neovascularization in the rabbit by partially digesting the posterior vitreous with repeated injection and aspiration of 1 IU of hyaluronidase before injection of 250,000 homologous dermal fibroblasts. The evolution of the new preretinal vessels was monitored by indirect ophthalmoscopy, fundus photography, and fluorescein angiography. A grading system was devised using fundus photographs and fluorescein angiograms to describe the progression of new vessel growth and the extent of fluorescein leakage. Ninety-five percent of the eyes had vascular enlargement and hyperemia but no fluorescein leakage by day 1. Fifteen percent of the eyes had clinically evident new preretinal vessels, and 32% had severe fluorescein leakage by day 7. Ninety-five percent of the eyes had definite neovascularization by day 14. Severe fluorescein leakage peaked at day 14 (55% of the eyes) and decreased thereafter. Involution or atrophy of the vessels occurred in all eyes by day 42. This model will be useful for studying the pathogenesis of preretinal neovascularization and evaluating potential treatments for its prevention.


Covariance in diurnal patterns of suicide-related expressions on Twitter and recorded suicide deaths.
Social media data is increasingly used to gain insights into trends in mental health, but prior studies aimed at confirming a link between online expression of suicidal ideation on social media and actual suicide deaths have been inconclusive. Using comprehensive six-year data sets of Twitter posts and suicide deaths in Japan, we examine the diurnal relationship between the proportional incidence of a suicide-related keyword, "kietai" ("I want to disappear"), and suicide deaths with an OLS regression model. We also use co-occurrence analysis to reveal changes in the linguistic context of the suicide-related keyword at different hours of the day. We find a clear diurnal pattern in the use of this suicide-related keyword, peaking between 1am and 5am. This diurnal trend is positively correlated with suicide deaths among younger cohorts (ages 15 to 44), but the correlation is negative among older adults (45 and over). The correlation among young adults strengthens when a delay between tweet incidence and suicide deaths is included. Compared to daytime tweets, nighttime tweets exhibited a stronger relationship between words related to self-disgust and words directly indicating suicidal intent. This study confirms the hypothesised link between online suicidal ideation and suicide death. Despite frequent flippant use of the keyword, the consistent correlation and the diurnal changes in the context of the keyword's usage demonstrate the value of social media data to the study of mental health trends in groups at risk of suicide.


Impact of genotype imputation on the performance of GBLUP and Bayesian methods for genomic prediction.
The aim of this study was to evaluate the impact of genotype imputation on the performance of the GBLUP and Bayesian methods for genomic prediction. A total of 10,309 Holstein bulls were genotyped on the BovineSNP50 BeadChip (50 k). Five low density single nucleotide polymorphism (SNP) panels, containing 6,177, 2,480, 1,536, 768 and 384 SNPs, were simulated from the 50 k panel. A fraction of 0%, 33% and 66% of the animals were randomly selected from the training sets to have low density genotypes which were then imputed into 50 k genotypes. A GBLUP and a Bayesian method were used to predict direct genomic values (DGV) for validation animals using imputed or their actual 50 k genotypes. Traits studied included milk yield, fat percentage, protein percentage and somatic cell score (SCS). Results showed that performance of both GBLUP and Bayesian methods was influenced by imputation errors. For traits affected by a few large QTL, the Bayesian method resulted in greater reductions of accuracy due to imputation errors than GBLUP. Including SNPs with largest effects in the low density panel substantially improved the accuracy of genomic prediction for the Bayesian method. Including genotypes imputed from the 6 k panel achieved almost the same accuracy of genomic prediction as that of using the 50 k panel even when 66% of the training population was genotyped on the 6 k panel. These results justified the application of the 6 k panel for genomic prediction. Imputations from lower density panels were more prone to errors and resulted in lower accuracy of genomic prediction. But for animals that have close relationship to the reference set, genotype imputation may still achieve a relatively high accuracy.


Ultrathin flexible terahertz polarization converter based on metasurfaces.
We present a method to design and fabricate a kind of converters based on flexible metasurfaces which can change the polarization state of an incident terahertz beam. The metasurface consists of a two-dimensional array of rectangular metallic antennas that can abruptly change the phase of the incoming terahertz beam. Experimentally demonstrated half-wave plates generate 0.1 THz beam with a π/2 polarization rotation. By slightly changing the structure of the converter, an elliptically or circularly polarized beam is expected to be obtained. These flexible terahertz converters may have many potential applications in terahertz technology.


Adolescents' and mothers' temperament types and their roles in early adolescents' socioemotional functioning.
The present study examined adolescents' and mothers' temperament types and their roles in the socioemotional functioning of early adolescents. A total of 869 sixth-grade students and 668 mothers participated in the study. The students rated their temperament and socioemotional functioning and the mothers rated their own temperament. Latent profile analyses identified four temperament types among the adolescents (resilient, reserved, average, and mixed) and three types among the mothers (resilient, average, and mixed). The results showed that the adolescents with resilient or reserved temperaments reported significantly fewer conduct problems and emotional symptoms, less hyperactivity, and higher prosociality than adolescents with a mixed temperament type. The most adaptive adolescent-mother temperament matches were between a resilient or reserved adolescent and a resilient or average mother; these adolescents reported the highest levels of socioemotional functioning. Mothers with mixed or average temperaments were related to fewer conduct problems and emotional symptoms and less hyperactivity among adolescents with a mixed temperament, while mothers with a resilient temperament type were beneficial for prosocial behavior among adolescents with a mixed temperament. These findings increase understanding of the role of temperament and the interplay between adolescents' and mothers' temperaments in the development of early adolescents' socioemotional adjustment.


Differential activation of microglia in neurogenic versus non-neurogenic regions of the forebrain.
Proliferation decreases in the neurogenic subventricular zone (SVZ) of mice after aspiration lesions of the cerebral cortex. We hypothesized that microglial activation may contribute to this given microglial activation attenuates neurogenesis in the hippocampus. Using CD45, CD11b, IB4, and IL-6 immunohistochemistry (IHC), BrdU IHC, and fluorescent bead tracking of peripheral monocytes into the brain, we compared microglial activation in the SVZ to non-neurogenic forebrain regions. SVZ microglia exhibited greater constitutive activation and proliferation than did microglia in non-neurogenic regions. In contrast to the SVZ, the dentate gyrus (DG) contained relatively few CD45(+) cells. After aspiration cerebral cortex lesions, microglia became activated in the cerebral cortex, corpus callosum, and striatum. SVZ microglial activation did not increase, and similarly, microglia in the DG were less activated after injury than in adjacent non-neurogenic regions. We next showed that SVZ microglia are not categorically refractory to activation, since deep cortical contusion injuries increased SVZ microglial activation. Macrophages migrate into the brain during development, but it is unclear if this is recapitulated after injury. Infiltration of microbead-labeled macrophages into the brain did not change after injury, but resident SVZ microglia were induced to migrate toward the injury. Our data show that both constitutive and postlesion levels of microglial activation differ between neurogenic and non-neurogenic regions.


Ultrastructural Characterization of Turnip Mosaic Virus-Induced Cellular Rearrangements Reveals Membrane-Bound Viral Particles Accumulating in Vacuoles.
Positive-strand RNA [(+) RNA] viruses remodel cellular membranes to facilitate virus replication and assembly. In the case of turnip mosaic virus (TuMV), the viral membrane protein 6K2 plays an essential role in endomembrane alterations. Although 6K2-induced membrane dynamics have been widely studied by confocal microscopy, the ultrastructure of this remodeling has not been extensively examined. In this study, we investigated the formation of TuMV-induced membrane changes by chemical fixation and high-pressure freezing/freeze substitution (HPF/FS) for transmission electron microscopy at different times of infection. We observed the formation of convoluted membranes connected to rough endoplasmic reticulum (rER) early in the infection process, followed by the production of single-membrane vesicle-like (SMVL) structures at the midstage of infection. Both SMVL and double-membrane vesicle-like structures with electron-dense cores, as well as electron-dense bodies, were found late in the infection process. Immunogold labeling results showed that the vesicle-like structures were 6K2 tagged and suggested that only the SMVL structures were viral RNA replication sites. Electron tomography (ET) was used to regenerate a three-dimensional model of these vesicle-like structures, which showed that they were, in fact, tubules. Late in infection, we observed filamentous particle bundles associated with electron-dense bodies, which suggests that these are sites for viral particle assembly. In addition, TuMV particles were observed to accumulate in the central vacuole as membrane-associated linear arrays. Our work thus unravels the sequential appearance of distinct TuMV-induced membrane structures for viral RNA replication, viral particle assembly, and accumulation. Positive-strand RNA viruses remodel cellular membranes for different stages of the infection process, such as protein translation and processing, viral RNA synthesis, particle assembly, and virus transmission. The ultrastructure of turnip mosaic virus (TuMV)-induced membrane remodeling was investigated over several days of infection. The first change that was observed involved endoplasmic reticulum-connected convoluted membrane accumulation. This was followed by the formation of single-membrane tubules, which were shown to be viral RNA replication sites. Later in the infection process, double-membrane tubular structures were observed and were associated with viral particle bundles. In addition, TuMV particles were observed to accumulate in the central vacuole as membrane-associated linear arrays. This work thus unravels the sequential appearance of distinct TuMV-induced membrane structures for viral RNA replication, viral particle assembly, and accumulation.


Compared to casein, bovine lactoferrin reduces plasma leptin and corticosterone and affects hypothalamic gene expression without altering weight gain or fat mass in high fat diet fed C57/BL6J mice.
Several studies in both humans and rodents have examined the use of lactoferrin as a dietary solution to weight gain and visceral fat accretion and have shown promising results in the short term (up to 7 weeks). This study examined the effects of giving lactoferrin over a longer period of time. For 13 weeks, male C57/BL6J mice were given a diet containing 10 % kJ fat and 20 % kJ casein (LFD) or a diet with 45 % kJ fat and either 20 % kJ casein (HFD) or 20 % kJ lactoferrin (HFD + Lac). Physiological, metabolic, and biochemical parameters were investigated. Gene expression was investigated by Real-Time PCR and microarray. All data was assessed using t-test, ANOVA or ANCOVA. Gene Set Enrichment Analysis was used to interpret microarray data and assess the impact on gene sets with common biological roles. By the end of the trial, HFD + Lac fed mice did not alter energy balance, body composition, bodyweight, or weight gain when compared to the HFD group. Notably, there were no changes in subcutaneous or epididymal adipose leptin mRNA levels between high fat diet groups, however plasma leptin was significantly reduced in the HFD + Lac compared to HFD group (P < 0.05) suggesting reduced leptin secretion. Global microarray analysis of the hypothalamus indicate an overall reduction in gene sets associated with feeding behaviour (P < 0.01) and an up-regulation of gene sets associated with retinol metabolism in the HFD + Lac group compared to the HFD group (P < 0.01). Genes in the latter catergory have been shown to impact on the hypothalamic-pituitary-adrenal axis. Notably, plasma corticosterone levels in the HFD + Lac group were reduced compared to the HFD fed mice (P < 0.05). The data suggests that prolonged feeding of full-length dietary lactoferrin, as part of a high fat diet, does not have a beneficial impact on weight gain when compared to casein. However, its impact on leptin secretion and accompanying changes in hypothalamic gene expression may underlie how this dietary protein alters plasma corticosterone. The lactoferrin fed mouse model could be used to identify leptin and corticosterone regulated genes in the hypothalamus without the confounding effects of body weight change.


Renal Na+ excretion consequent to pharmacogenetic activation of Gq-DREADD in principal cells.
Stimulation of metabotropic Gq-coupled purinergic P2Y2 receptors decreases activity of the epithelial Na+ channel (ENaC) in renal principal cells of the distal nephron. The physiological consequences of P2Y2 receptor signaling disruption in the P2Y2 receptor knockout mouse are decreased Na+ excretion and increased arterial blood pressure. However, because of the global nature of this knockout model, the quantitative contribution of ENaC and distal nephron compared with that of upstream renal vascular and tubular elements to changes in urinary excretion and arterial blood pressure is obscure. Moreover, it is uncertain whether stimulation of P2Y2 receptor inhibition of ENaC is sufficient to drive renal (urinary) Na+ excretion (UNaV). Here, using a pharmacogenetic approach and selective agonism of the P2Y2 receptor, we test the sufficiency of targeted stimulation of Gq signaling in principal cells of the distal nephron and P2Y2 receptors to increase UNaV. Selective stimulation of the P2Y2 receptor with the ligand MRS2768 decreased ENaC activity in freshly isolated tubules (as assessed by patch-clamp electrophysiology) and increased UNaV (as assessed in metabolic cages). Similarly, selective agonism of hM3Dq-designer receptors exclusively activated by designer drugs (DREADD) restrictively expressed in principal cells of the distal nephron with clozapine- N-oxide decreased ENaC activity and, consequently, increased UNaV. Clozapine- N-oxide, when applied to control littermates, failed to affect ENaC and UNaV. This study represents the first use of pharmacogenetic (DREADD) technology in the renal tubule and demonstrated that selective activation of the P2Y2 receptor and Gq signaling in principal cells is sufficient to promote renal salt excretion.


A pharmacodynamic study of the epidermal growth factor receptor tyrosine kinase inhibitor ZD1839 in metastatic colorectal cancer patients.
Epidermal growth factor receptor (EGFR) appears to play an important role in the pathogenesis of colorectal cancer. We have performed a Phase I/II study of the EGFR tyrosine kinase inhibitor ZD1839 in metastatic colorectal cancer patients in which serial biopsies were taken pre- and posttreatment to assess biological activity. Paired biopsies were obtained from colorectal cancer patients before and after treatment. Proliferation and apoptosis were assessed using Ki67 immunohistochemistry and terminal deoxynucleotidyl transferase-mediated nick end labeling assays, respectively. Immunohistochemistry for EGFR, activated EGFR, phosphorylated Akt, phosphorylated ERK, p27(Kip1), and beta-catenin was also performed. Posttreatment samples showed a statistically significant reduction in the cancer cell proliferation index (mean proliferation index pretreatment 31%; posttreatment 21%; P = 0.047). The mean cancer cell apoptosis index also increased from 6 to 12% in posttreatment samples, although this difference did not achieve statistical significance. All pretreatment samples showed strong staining for EGFR. Loss of immunohistochemical staining for activated EGFR, phosphorylated Akt, and phosphorylated ERK in cancer cells was observed in some patients after treatment. p27(Kip1) was absent in the cancer cells of most pretreatment biopsies; two patients showed a marked increase in staining for nuclear p27(Kip1) after treatment with ZD1839. These two patients also showed large increases in apoptotic index. ZD1839 inhibits EGFR signaling and proliferation in the cancer cells of patients with metastatic colorectal cancer. ZD1839 may also induce cancer cell apoptosis in a subset of colorectal cancer patients via up-regulation of p27(Kip1).


Is Action Planning Helpful for Smoking Cessation? Assessing the Effects of Action Planning in a Web-Based Computer-Tailored Intervention.
The aim of this study was to examine the efficacy of a web-assisted computer-tailored smoking cessation intervention, an action planning (AP) intervention in which potential quitters were encouraged to form action plans (e.g., plan a quit date) and execute them (e.g., remove ashtrays). We also investigated whether the AP intervention resulted in more AP and plan execution than a similar, control intervention without the supplementary AP component. In a randomized controlled trial, the AP intervention (N = 977) was compared with the control intervention (N = 1,005) in terms of self-reported continued abstinence (CA) and point prevalence abstinence (PPA) six months after baseline. AP, plan execution, and opinion of the intervention were measured one month after baseline. Complete-case logistic regression analysis showed that the AP intervention had a significant effect on CA (OR = 2.01; CI 1.08-3.84, p = .02), whereas intention-to-treat analysis showed a borderline significant effect (OR = 1.68; CI .96-2.92, p = .07). Sixteen percent of the experimental group achieved CA compared to 10% of the control group. The AP intervention had no effect on PPA. The experimental group also showed significantly more AP and plan execution at one month. Execution of plans was associated with smoking cessation. The effects of the AP intervention on CA, AP, and execution of plans were encouraging. The potential for widespread use of web-based interventions means that even small behavioral effects may have an impact on public health. We recommend that the intervention be intensified and improved.


2-Ethoxy-4,5-diphenyl-1,3-oxazine-6-one activates the Nrf2/HO-1 axis and protects against oxidative stress-induced neuronal death.
Apoptosis or programmed cell death has been suggested as an important mode of neurodegeneration in Alzheimer's disease pathogenesis. The present study explored the neuroprotective effect of 2-ethoxy-4,5-diphenyl-1,3-oxazine-6-one (EDPOO) against H(2)O(2)-induced cell death in rat pheochromocytoma (PC12) cells. We found that H(2)O(2) triggered a range of cellular cascades which leads to cell death, whereas pretreatment of the cells with this oxazine derivative attenuated the extent of apoptosis, as assessed by MTT assay, acridine orange/ethidium bromide staining and caspase-3 expression assay. We further showed that EDPOO exerts its neuroprotective effect by enhancing Hsp-70 level, stabilizing Nrf2 and upregulation of HO-1 and γ-GCS. Moreover, this oxazine derivative regulated cellular redox status via antioxidant enzyme upregulation. The neuroprotective effect of this compound may provide a new potential application for the treatment of neurodegenerative diseases.


Effect of 12-Week Rebound Therapy Exercise on Static Stability of Patients With Spinal Cord Injury.
Background: To resolve the impairments associated with spinal cord injury (SCI), such as decreased balance, patients have been recommended to undergo various therapeutic strategies, including the use of different physical exercise methods. The aim of this study was to evaluate the efficiency of using rebound therapy (exercise on a trampoline) on SCI individuals' static stability. Methods: Sixteen people with SCI (American Spinal Cord Association classification: A = 6, B = 6, C = 2, and D = 2) were randomly assigned to an experimental (rebound exercise) group or a control group. The rebound therapy exercise program, lasting 12 weeks, was performed by means of a modified trampoline. During the said period, the experimental group received rebound therapy exercise for 10 to 30 minutes 3 sessions a week. Standing stability parameters (ie, excursion, velocity, and path length of the center of pressure in mediolateral and anteroposterior plane) were assessed before and after the exercise intervention by Kistler force plate (50 × 60 cm). Data were analyzed by repeated measures analysis of variance. Results: Significant interactions were observed for all 6 dependent variables except excursion of the center of pressure in mediolateral and the path length of center of pressure in anteroposterior plane (P < .01). This means that the control group had no progress, whereas the experimental group made a significant improvement in terms of static stability. Conclusion: The results of this study confirmed that rebound therapy could reinforce the static stability of individuals with SCI during motionless standing. It suggests that rebound exercise is a useful sports rehabilitation method for patients with SCI and other wheelchair-bound individuals.


Should we be concerned about jejunoileal atresia during repair of duodenal atresia?
During repair for duodenal atresia, it has been emphasized that inspection of the small bowel to identify a second atresia is required. The laparoscopic approach for repair of duodenal atresia has been criticized for its limitation to perform this step. Given that duodenal atresia and jejunoileal atresias do not share common embryologic origins, we question the validity of this concern. Therefore, we conducted a multicenter retrospective review of duodenal atresia patients to quantify the incidence of jejunoileal atresia in this population. After institutional review board approval (IRB #07-12-187X), a retrospective review was conducted on all patients who have undergone duodenal atresia repair at seven institutions over the past 7-12 years. Demographics and the presence or absence of a jejunoileal atresia were recorded. Four hundred eight patients with duodenal atresia were identified. The mean gestaational age was 36.3 ± 2.9 weeks, and the mean weight was 2.5 ± 0.8 kg. Mean age at operation was 19 days (range, 1-1314). There was a 28% incidence of trisomy 21. Two patients (0.5%) were identified as having a second intestinal atresia, and both were type IIIb. One patient was diagnosed at the time of duodenal atresia repair; the other was a delayed diagnosis. Both patients did well after repair. In this, the largest series of duodenal atresia patients compiled to date, the rate of a concomitant jejunoileal atresia is less than 1%. This low incidence is not high enough to mandate extensive inspection of the entire bowel in these patients, and a second atresia should not be a concern during laparoscopic repair of duodenal atresia.


Amelioration of dry eye syndrome in db/db mice with diabetes mellitus by treatment with Tibetan Medicine Formula Jikan Mingmu Drops.
Jikan Mingmu Drops (JMD), a traditional Tibetan medicine containing six herbs, has been used to treat dry eye syndrome (DES) in individuals with diabetes mellitus. However, the activity of JMD ameliorates DES with diabetes mellitus has not been previously examined. The aim of the study is to investigate the molecular mechanism of JMD on db/db mice. The main chemical constituents of JMD were analyzed by high-performance liquid chromatography and gas chromatography-mass spectrometry. DES was then induced in db/db mice by applying 0.2% benzalkonium chloride to the ocular surface for 7 days. Eye drops containing JMD (0.25, 0.5, or 1 g/mL) or vehicle subsequently were administered three times daily for another 7 days, and the therapeutic effects were evaluated by phenol red thread tear and sodium fluorescein tests. Conjunctival specimens were subjected to hematoxylin and eosin staining and periodic acid-Schiff staining to examine pathological changes and number of goblet cells. ELISA was performed to assess the levels of various inflammatory cytokines. JMD contains hydroxysafflor yellow A, magnoflorine, jatrorrhizine hydrochloride, palmatine hydrochloride, berberine hydrochloride, gallic acid, ellagic acid, tauroursodeoxycholic acid, camphor, isoborneol, borneol, trans-cinnamic acid, and muscone. JMD treatment significantly increased the tear volume, decreased the corneal fluorescein staining score, restored the morphology and structure of conjunctival epithelial cells, and markedly downregulated the levels of interleukin (IL)-6, IL-17α, IL-1β, tumor necrosis factor-α, and vascular endothelial growth factor in the conjunctiva. Further data showed that these protective effects were accompanied by inhibition of inflammation in a dose-dependent manner. Amelioration of DES in db/db mice with diabetes mellitus by treatment with Tibetan medicine formula JMD maybe related to its anti-inflammatory effects.


Tomographic breathing detection: a method to noninvasively assess in situ respiratory dynamics.
Physiological monitoring is a critical aspect of in vivo experimentation, particularly imaging studies. Physiological monitoring facilitates gated acquisition of imaging data and more robust experimental interpretation but has historically required additional instrumentation that may be cumbersome. As frame rates have increased, imaging methods have been able to capture ever more rapid dynamics, passing the Nyquist sampling rate of most physiological processes and allowing the capture of motion, such as breathing. With this transition, image artifacts have also changed their nature; rather than intraframe motion causing blurring and deteriorating resolution, interframe motion does not affect individual frames and may be recovered as useful information from an image time series. We demonstrate a method that takes advantage of interframe movement for detection of gross physiological motion in real-time image sequences. We further demonstrate the ability of the method, dubbed tomographic breathing detection to quantify the dynamics of respiration, allowing the capture of respiratory information pertinent to anesthetic depth monitoring. Our example uses multispectral optoacoustic tomography, but it will be widely relevant to other technologies.


A high-sensitivity electrochemiluminescence-based ELISA for the measurement of the oxidative stress biomarker, 3-nitrotyrosine, in human blood serum and cells.
The generation of 3-nitrotyrosine, within proteins, is a post-translational modification resulting from oxidative or nitrative stress. It has been suggested that this modification could be used as a biomarker for inflammatory diseases. Despite the superiority of mass spectrometry-based determinations of nitrotyrosine, in a high-throughput clinical setting the measurement of nitrotyrosine by an enzyme-linked immunosorbent assay (ELISA) is likely to be more cost-effective. ELISAs offer an alternative means to detect nitrotyrosine, but many commercially available ELISAs are insufficiently sensitive to detect nitrotyrosine in healthy human serum. Here, we report the development, validation and clinical application of a novel electrochemiluminescence-based ELISA for nitrotyrosine which provides superior sensitivity (e.g. a 50-fold increase in sensitivity compared with one of the tested commercial colorimetric ELISAs). This nitrotyrosine ELISA has the following characteristics: a lower limit of quantitation of 0.04 nM nitrated albumin equivalents; intra- and inter-assay coefficients of variation of 6.5% and 11.3%, respectively; a mean recovery of 106 ± 3% and a mean linearity of 0.998 ± 0.001. Far higher nitration levels were measured in normal human blood cell populations when compared to plasma. Mass spectrometry was used to validate the new ELISA method. The analysis of the same set of chemically modified albumin samples using the ELISA method and mass spectrometry showed good agreement for the relative levels of nitration present in each sample. The assay was applied to serum samples from patients undergoing elective surgery which induces the human inflammatory response. Matched samples were collected before and one day after surgery. An increase in nitration was detected following surgery (median (IQR): 0.59 (0.00-1.34) and 0.97 (0.00-1.70) nitrotyrosine (fmol of nitrated albumin equivalents/mg protein) for pre- and post-surgery respectively. The reported assay is suitable for nitrotyrosine determination in patient serum samples, and may also be applicable as a means to determine oxidative stress in primary and cultured cell populations.


The efficacy of vigorous-intensity exercise as an aid to smoking cessation in adults with elevated anxiety sensitivity: study protocol for a randomized controlled trial.
Although cigarette smoking is a leading cause of death and disability in the United States (US), over 40 million adults in the US currently smoke. Quitting smoking is particularly difficult for smokers with certain types of psychological vulnerability. Researchers have frequently called attention to the relation between smoking and anxiety-related states and disorders, and evidence suggests that panic and related anxiety vulnerability factors, specifically anxiety sensitivity (AS or fear of somatic arousal), negatively impact cessation. Accordingly, there is merit to targeting AS among smokers to improve cessation outcome. Aerobic exercise has emerged as a promising aid for smoking cessation for this high-risk (for relapse) group because exercise can effectively reduce AS and other factors predicting smoking relapse (for example, withdrawal, depressed mood, anxiety), and it has shown initial efficacy for smoking cessation. The current manuscript presents the rationale, study design and procedures, and design considerations of the Smoking Termination Enhancement Project (STEP). STEP is a randomized clinical trial that compares a vigorous-intensity exercise intervention to a health and wellness education intervention as an aid for smoking cessation in adults with elevated AS. One hundred and fifty eligible participants will receive standard treatment (ST) for smoking cessation that includes cognitive behavioral therapy (CBT) and nicotine replacement therapy (NRT). In addition, participants will be randomly assigned to either an exercise intervention (ST+EX) or a health and wellness education intervention (ST+CTRL). Participants in both arms will meet 3 times a week for 15 weeks, receiving CBT once a week for the first 7 weeks, and 3 supervised exercise or health and wellness education sessions (depending on randomization) per week for the full 15-week intervention. Participants will be asked to set a quit date for 6 weeks after the baseline visit, and smoking cessation outcomes as well as putative mediator variables will be measured up to 6 months following the quit date. The primary objective of STEP is to evaluate whether vigorous-intensity exercise can aid smoking cessation in anxiety vulnerable adults. If effective, the use of vigorous-intensity exercise as a component of smoking cessation interventions would have a significant public health impact. Specifically, in addition to improving smoking cessation treatment outcome, exercise is expected to offer benefits to overall health, which may be particularly important for smokers. The study is also designed to test putative mediators of the intervention effects and therefore has the potential to advance the understanding of exercise-anxiety-smoking relations and guide future research on this topic. ClinicalTrials.gov, NCT01065506, http://clinicaltrials.gov/ct2/show/NCT01065506.


Restoration of open reading frame resulting from skipping of an exon with an internal deletion in the COL7A1 gene.
Restoration of open reading frame resulting from exon skipping has been documented as a mechanism of rescuing an mRNA transcript containing mutations. In this study, we describe a mutation in a family with dystrophic epidermolysis bullosa consisting of a 16-bp deletion within exon 87 of the type VII collagen gene (COL7A1) and predicted to lead to a frameshift and downstream premature termination codon. Unexpectedly, mRNA analysis revealed instead that the intraexonic deletion led to skipping of exon 87 and subsequent restoration of the open reading frame. The phenotypes and patterns of inheritance observed in this family arise from three different mutations, all of which affect RNA processing. Restoration of open reading frame by exon skipping represents a previously undescribed mechanism of action of intraexonic deletion mutations.


Novel inflammatory cell infiltration scoring system to investigate healthy and footrot affected ovine interdigital skin.
Ovine footrot is a degenerative disease of sheep feet leading to the separation of hoof-horn from the underlying skin and lameness. This study quantitatively examined histological features of the ovine interdigital skin as well as their relationship with pro-inflammatory cytokine (IL-1β) and virulent Dichelobacter nodosus in footrot. From 55 healthy and 30 footrot ovine feet, parallel biopsies (one fixed for histology) were collected post-slaughter and analysed for lesions and histopathological analysis using haematoxylin and eosin and Periodic Acid-Schiff. Histological lesions were similar in both conditions while inflammatory scores mirror IL-1β expression levels. Increased inflammatory score corresponded with high virulent D. nodosus load and was significant (p < 0.0001) in footrot feet with an inflammatory score of 3 compared to scores 1 and 2. In addition, in contrast to healthy tissues, localisation of eubacterial load extended beyond follicular depths in footrot samples. The novel inflammatory cell infiltration scoring system in this study may be used to grade inflammatory response in the ovine feet and demonstrated an association between severity of inflammatory response and increased virulent D. nodosus load.


Use of Multiple Low Cost Carbon Dioxide Sensors to Measure Exhaled Breath Distribution with Face Mask Type and Wearing Behaviour.
The use of cloth face coverings and face masks has become widespread in light of the COVID-19 pandemic. This paper presents a method of using low cost wirelessly connected carbon dioxide (CO2) sensors to measure the effects of properly and improperly worn face masks on the concentration distribution of exhaled breath around the face. Four types of face masks are used in two indoor environment scenarios. CO2 as a proxy for exhaled breath is being measured with the Sensirion SCD30 CO2 sensor, and data are being transferred wirelessly to a base station. The exhaled CO2 is measured in four directions at various distances from the head of the subject, and interpolated to create spatial heat maps of CO2 concentration. Statistical analysis using the Friedman's analysis of variance (ANOVA) test is carried out to determine the validity of the null hypotheses (i.e., distribution of the CO2 is same) between different experiment conditions. Results suggest CO2 concentrations vary little with the type of mask used; however, improper use of the face mask results in statistically different CO2 spatial distribution of concentration. The use of low cost sensors with a visual interpolation tool could provide an effective method of demonstrating the importance of proper mask wearing to the public.


Mucin-chitosan complexes at the solid-liquid interface: multilayer formation and stability in surfactant solutions.
The adsorption of a biologically important glycoprotein, mucin, and mucin-chitosan complex layer formation on negatively charged surfaces, silica and mica, have been investigated employing ellipsometry, the interferometric surface apparatus, and atomic force microscopy techniques. Particular attention has been paid to the effect of an anionic surfactant sodium, dodecyl sulfate (SDS), with respect to the stability of the adsorption layers. It has been shown that mucin adsorbs on negatively charged surfaces to form highly hydrated layers. Such mucin layers readily associate with surfactants and are easily removed from the surfaces by rinsing with solutions of SDS at concentrations > or =0.2 cmc (1 cmc SDS in 30 mM NaCl is equal to 3.3 mM). The mucin adsorption layer is negatively charged, and we show how a positively charged polyelectrolyte, chitosan, associates with the preadsorbed mucin to form mucin-chitosan complexes that resist desorption by SDS even at SDS concentrations as high as 1 cmc. Thus, a method of mucin layer protection against removal by surfactants is offered. Further, we show how mucin-chitosan multilayers can be formed.


Variation in hospital charges in patients with external ventricular drains: comparison between the intensive care and surgical floor settings.
Placement of an external ventricular drain (EVD) is a common and potentially life-saving neurosurgical procedure, but the economic aspect of EVD management and the relationship to medical expenditure remain poorly studied. Similarly, interinstitutional practice patterns vary significantly. Whereas some institutions require that patients with EVDs be monitored strictly within the intensive care unit (ICU), other institutions opt primarily for management of EVDs on the surgical floor. Therefore, an ICU burden for patients with EVDs may increase a patient's costs of hospitalization. The objective of the current study was to examine the expense differences between the ICU and the general neurosurgical floor for EVD care. The authors performed a retrospective analysis of data from 2 hospitals within a single, large academic institution-the University of Washington Medical Center (UWMC) and Seattle Children's Hospital (SCH). Hospital charges were evaluated according to patients' location at the time of EVD management: SCH ICU, SCH floor, or UWMC ICU. Daily hospital charges from day of EVD insertion to day of removal were included and screened for days that would best represent baseline expenses for EVD care. Independent-samples Kruskal-Wallis analysis was performed to compare daily charges for the 3 settings. Data from a total of 261 hospital days for 23 patients were included in the analysis. Ten patients were cared for in the UWMC ICU and 13 in the SCH ICU and/or on the SCH neurosurgical floor. The median values for total daily hospital charges were $19,824.68 (interquartile range [IQR] $12,889.73-$38,494.81) for SCH ICU care, $8,620.88 (IQR $6,416.76-$11,851.36) for SCH floor care, and $10,002.13 (IQR $8,465.16-$12,123.03) for UWMC ICU care. At SCH, it was significantly more expensive to provide EVD care in the ICU than on the floor (p < 0.001), and the daily hospital charges for the UWMC ICU were significantly greater than for the SCH floor (p = 0.023). No adverse clinical event related to the presence of an EVD was identified in any of the settings. ICU admission solely for EVD care is costly. If safe EVD care can be provided outside of the ICU, it would represent a potential area for significant cost savings. Identifying appropriate patients for EVD care on the floor is multifactorial and requires vigilance in balancing the expenses associated with ICU utilization and optimal patient care.


[A retrospective evaluation of the behaviour of groups under out-patient care at a health center].
To study and analyse the Out-Patient Care Groups (OCGs), and evaluate how they affect use of health resources. An observational, retrospective study. Ciudad Jardín Health Centre, Málaga. 2999 patients with a clinical history opened before 31.12.95, chosen from 5 of the 17 medical lists at the Health Centre, were included. The statistical analysis was performed with the SPSS software package of the Calculation Centre at Málaga University. A descriptive test produced the following results: 33% of the patients were classified in OCG 41 (combination of 2 or 3 out-patient diagnosis groups in people over 34); 19% belonged to groups of stable or unstable chronic illnesses (OCGs 8, 9 and 10); and 9% had acute children's diseases. Then multiple regression constructed a model with the OCGs as independent variable and annual visits, further tests performed and referral to specialists as dependent variables. In this model the OCGs were able to explain 20.3% of resource consumption. In the retrospective study and with a limited sample of 2999 patients, the OCGs are able to explain 20.3% of resource consumption. However, it does seem a valid model for discriminating between normal and over-using patients.


Alpha 7-nicotinic acetylcholine receptor mediates the sensitivity of gastric cancer cells to 5-fluorouracil.
Gastric cancer is the second most common cause of cancer mortality worldwide. Most gastric cancer patients are asymptomatic until the advanced stages, for which current therapeutic treatments are suboptimal. 5-Fluorouracil (5-FU), an antimetabolite agent, is widely used in gastric cancer therapy. However, the presence of drug resistance in gastric cancer patients reduces the cytotoxic activity of 5-FU. In gastric cancer, no research has yet been conducted to analyze the effect of alpha 7-nicotinic acetylcholine receptor (A7-nAChR) on the therapeutic response to 5-FU. In this study, we generated A7-nAChR knockdown (A7-nAChR-KD) AGS cells by a small interfering RNA (siRNA) technique in gastric cancer cells. The anti-proliferative effects of 5-FU were determined by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, a terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) assay, and cell cycle determination. We found that A7-nAChR-KD cells were more resistant to 5-FU treatment compared with the scrambled control cells according to the MTT assay. The apoptotic cell population was increased more in scrambled control cells treated with 5-FU than A7-nAChR-KD cells according to the cell cycle distribution and TUNEL assays. We analyzed expression levels of survival and apoptosis-associated proteins (pAkt, Akt, Mcl-1, Bcl-2, Bad, and Bax) altered by 5-FU treatment. Survival and antiapoptosis signaling (pAkt, Akt, Mcl-1 and Bcl-2) was downregulated, and the proapoptotic proteins (Bad and Bax) were upregulated in 5-FU-treated control cells but expression levels of Bcl-2, Bad, and Bad were not altered in 5-FU-treated A7-nAChR-KD cells. This is consistent with A7-nAChR-KD cells exhibiting more resistance to 5-FU treatment. In our study, we carried out an in vitro study on AGS gastric cancer cell line to elucidate the anticancer efficacy and molecular mechanisms of A7-nAChR silencing on 5-FU-induced cell death. The results clearly showed that depletion of A7-nAChR suppressed the drug sensitivity of gastric cancer cells to 5-FU treatment.


QPatch: the missing link between HTS and ion channel drug discovery.
The conventional patch clamp has long been considered the best approach for studying ion channel function and pharmacology. However, its low throughput has been a major hurdle to overcome for ion channel drug discovery. The recent emergence of higher throughput, automated patch clamp technology begins to break this bottleneck by providing medicinal chemists with high-quality, information-rich data in a more timely fashion. As such, these technologies have the potential to bridge a critical missing link between high-throughput primary screening and meaningful ion channel drug discovery programs. One of these technologies, the QPatch automated patch clamp system developed by Sophion Bioscience, records whole-cell ion channel currents from 16 or 48 individual cells in a parallel fashion. Here, we review the general applicability of the QPatch to studying a wide variety of ion channel types (voltage-/ligand-gated cationic/anionic channels) in various expression systems. The success rate of gigaseals, formation of the whole-cell configuration and usable cells ranged from 40-80%, depending on a number of factors including the cell line used, ion channel expressed, assay development or optimization time and expression level in these studies. We present detailed analyses of the QPatch features and results in case studies in which secondary screening assays were successfully developed for a voltage-gated calcium channel and a ligand-gated TRP channel. The increase in throughput compared to conventional patch clamp with the same cells was approximately 10-fold. We conclude that the QPatch, combining high data quality and speed with user friendliness and suitability for a wide array of ion channels, resides on the cutting edge of automated patch clamp technology and plays a pivotal role in expediting ion channel drug discovery.


Use of the receiver operating characteristic curve to evaluate sensitivity, specificity, and accuracy of methods for detection of peaks in hormone time series.
We utilize the "Receiver Operating Characteristic" to describe the relationship between sensitivity and specificity as the threshold for peak detection is varied systematically, to provide objective comparison of the performance of methods for detection of episodic hormonal secretion. A computer program was used to generate synthetic data with peaks with variable durations, with constant or variable height, shape and/or interpulse interval. This approach was used to compare the CLUSTER and DETECT programs. For both programs, the observed false positive rates estimated using signal-free data were in good agreement with the nominal rates, but in the presence of signal the observed false positive rates were systematically lower. Sensitivity increases with increasing signal/noise ratio, as expected. Program DETECT, using its standard options, provided excellent sensitivity (90-100%) with very low false positive rate under all conditions tested. Its performance could be further improved by the use of a more stringent definition of a peak requiring the presence of "UP" followed by a "DOWN". The CLUSTER program was found to have very poor sensitivity when using the "local variance" option. Use of the true fixed standard deviation or percent coefficient of variation resulted in a modest improvement. Optimal performance of program CLUSTER was obtained by the use of the best of 3 variance models, testing 12 different cluster sizes (from 1x1 to 4x4) and selecting the best among these: under these conditions it can achieve high sensitivity (90-100%) for very low observed false positive rate, such that its performance was comparable to that of DETECT. The methods developed and illustrated here should permit the definitive characterization and validation of the performance of any one method, the objective comparison of the relative performance of two or more methods for analysis of pulsatile hormone levels for episodic hormone secretion, and lead to the improvement of algorithms for peak detection.


Quantum chemical assessment of benzimidazole derivatives as corrosion inhibitors.
The majority of well-known inhibitors are organic compounds containing multiple bonds and heteroatoms, such as O, N or S, which allow adsorption onto the metal surface. These compounds can adsorb onto the metal surface and block active surface sites, reducing the rate of corrosion. A comparative theoretical study of three benzimidazole isomers, benzimidazole (BI), 2-methylbenzimidazole (2-CH3-BI), and 2-mercaptobenzimidazole (2-SH-BI), as corrosion inhibitors was performed using density functional theory (DFT) with the B3LYP functional basis set. Nitro and amino groups were selected for investigation as substituents of the three corrosion inhibitors. Nitration of the corrosion inhibitor molecules led to a decrease in inhibition efficiency, while reduction of the nitro group led to an increase in inhibition efficiency. These aminobenzimidazole isomers represent a significant improvement in the inhibition efficiency of corrosion inhibitor molecules.


High-quality t2-weighted 4-dimensional magnetic resonance imaging for radiation therapy applications.
The purpose of this study was to improve triggering efficiency of the prospective respiratory amplitude-triggered 4-dimensional magnetic resonance imaging (4DMRI) method and to develop a 4DMRI imaging protocol that could offer T2 weighting for better tumor visualization, good spatial coverage and spatial resolution, and respiratory motion sampling within a reasonable amount of time for radiation therapy applications. The respiratory state splitting (RSS) and multi-shot acquisition (MSA) methods were analytically compared and validated in a simulation study by using the respiratory signals from 10 healthy human subjects. The RSS method was more effective in improving triggering efficiency. It was implemented in prospective respiratory amplitude-triggered 4DMRI. 4DMRI image datasets were acquired from 5 healthy human subjects. Liver motion was estimated using the acquired 4DMRI image datasets. The simulation study showed the RSS method was more effective for improving triggering efficiency than the MSA method. The average reductions in 4DMRI acquisition times were 36% and 10% for the RSS and MSA methods, respectively. The human subject study showed that T2-weighted 4DMRI with 10 respiratory states, 60 slices at a spatial resolution of 1.5 × 1.5 × 3.0 mm(3) could be acquired in 9 to 18 minutes, depending on the individual's breath pattern. Based on the acquired 4DMRI image datasets, the ranges of peak-to-peak liver displacements among 5 human subjects were 9.0 to 12.9 mm, 2.5 to 3.9 mm, and 0.5 to 2.3 mm in superior-inferior, anterior-posterior, and left-right directions, respectively. We demonstrated that with the RSS method, it was feasible to acquire high-quality T2-weighted 4DMRI within a reasonable amount of time for radiation therapy applications.


The effect of lactose-conjugated silk biomaterials on the development of fibrogenic fibroblasts.
Surface properties of implanted biomaterials can cause fibrotic tissue reactions by stimulating differentiation of host fibroblasts into contractile myofibroblasts. Silk fibroin (SF) protein has been used as biomaterial in pure and blended form. however, its effect on myofibroblast differentiation remains elusive. We here conjugated SF with lactose using cyanuric chloride as coupling spacer. NMR spectroscopy and the conjugates ability to agglomerate Abrus precatorius agglutinin verified efficient conjugation. Two-dimensional films and three-dimensional scaffolds produced from pure and lactose-conjugated SF solutions were tested as culture substrates for subcutaneous fibroblasts and myofibroblasts. Lactose-conjugated SF substrates mediated higher adhesion, proliferation and viability of fibroblastic cells than pure SF. This SF film composition promotes better attachment of fibroblasts than myofibroblasts. Pro-fibrotic cytokine TGFbeta1 was ineffective in inducing fibroblast-to-myofibroblast differentiation on such substrates. Pre-differentiated myofibroblasts lost their contractile phenotype within a few days of being cultured on lactose-conjugated SF. Myofibroblast differentiation was also suppressed by growth in three-dimensional lactose-conjugated SF scaffolds that, however, support population with fibroblasts. We propose that this biomaterial will promote tissue integration without causing a fibrotic host reaction.


Role of lipoteichoic acid in the phagocyte response to group B streptococcus.
Group B Streptococcus (GBS) cell walls potently activate phagocytes by a largely TLR2-independent mechanism. In contrast, the cell wall component lipoteichoic acid (LTA) from diverse Gram-positive bacterial species has been shown to engage TLR2. In this study we examined the role of LTA from GBS in phagocyte activation and the requirements for TLR-LTA interaction. Using cells from knockout mice and genetic complementation in epithelial cells we found that highly pure LTA from both GBS and Staphylococcus aureus interact with TLR2 and TLR6, but not TLR1, in contrast to previous reports. Furthermore, NF-kappaB activation by LTA required the integrity of two putative PI3K binding domains within TLR2 and was inhibited by wortmannin, indicating an essential role for PI3K in cellular activation by LTA. However, LTA from GBS proved to be a relatively weak stimulus of phagocytes containing approximately 20% of the activity observed with LTA from Staphylococcus aureus. Structural analysis by nuclear magnetic resonance spectrometry revealed important differences between LTA from GBS and S. aureus, specifically differences in glycosyl linkage, in the glycolipid anchor and a lack of N-acetylglucosamine substituents of the glycerophosphate backbone. Furthermore, GBS expressing LTA devoid of d-alanine residues, that are essential within immune activation by LTA, exhibited similar inflammatory potency as GBS with alanylated LTA. In conclusion, LTA from GBS is a TLR2/TLR6 ligand that might contribute to secreted GBS activity, but does not contribute significantly to GBS cell wall mediated macrophage activation.


Solid-state density functional theory investigation of the terahertz spectra of the structural isomers 1,2-dicyanobenzene and 1,3-dicyanobenzene.
The high-resolution waveguide terahertz (THz) time-domain spectra (20-100 cm(-1)) of the two structural isomers 1,2-dicyanobenzene (1,2-DCB) and 1,3-dicyanobenzene (1,3-DCB) have been modeled and assigned using solid-state density functional theory. The THz spectra of these similar molecules are distinctly different in the low-frequency region with the differences being driven by modifications of the crystal packing arrangement between the isomers. Simulations utilizing the hybrid density functionals B3LYP and PBE0 were performed to determine the origins of the observed vibrational features. External lattice vibrations (hindered translations and rotations) are found to dominate these spectra, reinforcing the need for proper solid-state models in the analysis of the THz spectra of organic molecular solids. These calculations were able to account for all of the observed spectral features exhibited by both isomers, even in the case of 1,2-DCB, where the spectrum was found to be the result of two coexisting crystalline polymorphs.


Low rates of streptococcal pharyngitis and high rates of pyoderma in Australian aboriginal communities where acute rheumatic fever is hyperendemic.
Acute rheumatic fever is a major cause of heart disease in Aboriginal Australians. The epidemiology differs from that observed in regions with temperate climates; streptococcal pharyngitis is reportedly rare, and pyoderma is highly prevalent. A link between pyoderma and acute rheumatic fever has been proposed but is yet to be proven. Group C beta-hemolytic streptococci and group G beta-hemolytic streptococci have also been also implicated in the pathogenesis. Monthly, prospective surveillance of selected households was conducted in 3 remote Aboriginal communities. People were questioned about sore throat and pyoderma; swab specimens were obtained from all throats and any pyoderma lesions. Household population density was determined. From data collected during 531 household visits, the childhood incidence of sore throat was calculated to be 8 cases per 100 person-years, with no cases of symptomatic group A beta-hemolytic streptococci pharyngitis. The median point prevalence for throat carriage was 3.7% for group A beta-hemolytic streptococci, 0.7% for group C beta-hemolytic streptococci, and 5.1% for group G beta-hemolytic streptococci. Group A beta-hemolytic streptococci were recovered from the throats of 19.5% of children at some time during the study. There was no seasonal trend or correlation with overcrowding. Almost 40% of children had pyoderma at least once, and the prevalence was greatest during the dry season. In community 1, the prevalence of pyoderma correlated with household crowding. Group C and G beta-hemolytic streptococci were rarely recovered from pyoderma lesions. These data are consistent with the hypothesis that recurrent skin infections immunize against throat colonization and infection. High rates of acute rheumatic fever were not driven by symptomatic group A beta-hemolytic streptococci throat infection. Group G and C beta-hemolytic streptococci were found in the throat but rarely in pyoderma lesions.


Amplification of engrafted hepatocytes by preparative manipulation of the host liver.
Scarcity of donor livers is a major obstacle to the general application of hepatocytes for the development of bioartificial liver assist devices as well as intracorporeal engraftment of hepatocytes for the treatment of inherited metabolic diseases. The number of hepatocytes that can be transplanted into the liver safely in a single sitting also limits the utility of this procedure. These limitations could be addressed by providing preferential proliferative advantage to the transplanted cells. Studies using transgenic mouse recipients or donors have indicated that massive repopulation of the host liver by engrafted hepatocytes requires that the transplanted cells are subjected to a proliferative stimulus to which the host hepatocytes cannot respond. Prevention of host hepatocyte proliferation has been achieved by treatment with a plant alkaloid, retrorsine. Because retrorsine is carcinogenic, we have evaluated preparative irradiation for this purpose. The proliferative stimulus may consist of the loss of hepatic mass (e.g., partial hepatectomy, reperfusion injury or induction of Fas-mediated apoptosis by gene transfer) or administration of stimulants of hepatocellular mitosis (e.g., growth factors or thyroid hormone). Potential applications of these preparative manipulations of the host liver include the treatment of inherited metabolic disorders by transplantation of allogeneic hepatocytes, hepatocyte-mediated ex vivo gene therapy, rescuing liver cancer patients from radiation-induced liver damage, and expansion of human hepatocytes in animal livers.


End-capping of amphiphilic nanotubes with phospholipid vesicles: impact of the phospholipid on the cap formation and vesicle loading under osmotic conditions.
Soft amphiphilic nanotubes are capped with vesicles comprised of either overall neutral, zwitterionic phospholipids, or those that carry a net charge. The phase transition temperature of the zwitterionic phospholipids plays a crucial role in the phase separation that leads to the end-capped nanotubes. The cationic vesicle caps can be loaded into the nanotubes via osmosis whereas the anionic vesicle caps are stable under hyper-osmotic conditions. Furthermore, no additional salt needs to be added for the cationic vesicle caps to induce the loading of the vesicles into the nanotubes due to the presence of counterions.


How useful are anti-neural IgM antibodies in the diagnosis of chronic immune-mediated neuropathies?
Antibodies against several neural antigens have been associated with different chronic immune-mediated neuropathies but their practical clinical relevance remains unclear. To determine the possible diagnostic usefulness of these antibodies we reviewed the clinical correlate of IgM antibodies to the myelin-associated glycoprotein (MAG), sulfatide, the gangliosides GM1, GM2, GD1a and GD1b in 539 consecutive patients examined for neuropathy or related diseases in our Neuropathy Clinics and tested for these antibodies in our laboratory since 1985. 302 patients (56%) had an established diagnosis of definite or possible chronic immune-mediated neuropathy while 237 had a neuropathy of non-immune-mediated origin or of unknown aetiology or a closely related disease. Antibodies to one or more antigen were more frequent (chi(2)=63.32; p<0.00001) in patients with chronic immune-mediated neuropathy (37.7%) than with other neuropathy or related diseases (7.2%) and their presence was associated in 87% of the patients with an immune-mediated neuropathy, incrementing by 31% the probability of having this form. Testing for MAG permitted to identify 24.8% of patients with an immune-mediated neuropathy, GM1 an additional 9.9%, while GM2, GD1b, GD1a and sulfatide altogether an additional 3% of the patients. Concerning clinical correlations, all 75 patients with anti-MAG IgM had neuropathy and IgM monoclonal gammopathy (PN+IgM) with a positive predictive value for this neuropathy of 100%. A similarly high predictive value for neuropathy (91.4%) was observed among 269 patients with IgM monoclonal gammopathy including 103 patients without neuropathy. Anti-sulfatide IgM, though rare, were also significantly and constantly associated with PN+IgM and permitted to identify few patients not bearing anti-MAG IgM. Anti-GM1 IgM were significantly associated with multifocal motor neuropathy (MMN) (29.2%) but where also found in a few patients with other immune or non-immune neuropathies or related diseases with a positive predictive value for MMN of 25.5%. Anti-GM2 IgM were also significantly associated with MMN and increased the sensitivity (36.2%) for MMN obtained with anti-GM1 IgM only, without affecting its specificity and positive predictive value. Anti-GD1a, GD1b, though not significantly more frequent in patients with immune-mediated neuropathy, were associated in 80 to 100% of patients with these neuropathies. In conclusion anti-neural IgM antibodies may help in identifying patients with a chronic immune-mediated neuropathy, even if only anti-MAG and anti-sulfatide IgM appear to be strictly associated with a definite clinical syndrome.


A single bout of resistance exercise improves postprandial lipid metabolism in overweight/obese men with prediabetes.
Prediabetes is associated with postprandial hypertriacylglycerolaemia. Resistance exercise acutely lowers postprandial plasma triacylglycerol (TG); however, the changes in lipid metabolism that mediate this reduction are poorly understood. The aim of this study was to identify the constitutive metabolic mechanisms underlying the changes in postprandial lipid metabolism after resistance exercise in obese men with prediabetes. We evaluated the effect of a single bout of whole-body resistance exercise (seven exercises, three sets, 10-12 repetitions at 80% of one-repetition maximum) on postprandial lipid metabolism in ten middle-aged (50 ± 9 years), overweight/obese (BMI: 33 ± 3 kg/m2), sedentary men with prediabetes (HbA1c >38 but <48 mmol/mol [>5.7% but <6.5%]), or fasting plasma glucose >5.6 mmol/l but <7.0 mmol/l or 2 h OGTT glucose >7.8 mmol/l but <11.1 mmol/l). We used a randomised, crossover design with a triple-tracer mixed meal test (ingested [(13C4)3]tripalmitin, i.v. [U-13C16]palmitate and [2H5]glycerol) to evaluate chylomicron-TG and total triacylglycerol-rich lipoprotein (TRL)-TG kinetics. We used adipose tissue and skeletal muscle biopsies to evaluate the expression of genes regulating lipolysis and lipid oxidation, skeletal muscle respirometry to evaluate oxidative capacity, and indirect calorimetry to assess whole-body lipid oxidation. The single bout of resistance exercise reduced the lipaemic response to a mixed meal in obese men with prediabetes without changing chylomicron-TG or TRL-TG fractional clearance rates. However, resistance exercise reduced endogenous and meal-derived fatty acid incorporation into chylomicron-TG and TRL-TG. Resistance exercise also increased whole-body lipid oxidation, skeletal muscle mitochondrial respiration, oxidative gene expression in skeletal muscle, and the expression of key lipolysis genes in adipose tissue. A single bout of resistance exercise improves postprandial lipid metabolism in obese men with prediabetes, which may mitigate the risk for cardiovascular disease and type 2 diabetes.


Whole-genome resequencing of Hanwoo (Korean cattle) and insight into regions of homozygosity.
Hanwoo (Korean cattle), which originated from natural crossbreeding between taurine and zebu cattle, migrated to the Korean peninsula through North China. Hanwoo were raised as draft animals until the 1970s without the introduction of foreign germplasm. Since 1979, Hanwoo has been bred as beef cattle. Genetic variation was analyzed by whole-genome deep resequencing of a Hanwoo bull. The Hanwoo genome was compared to that of two other breeds, Black Angus and Holstein, and genes within regions of homozygosity were investigated to elucidate the genetic and genomic characteristics of Hanwoo. The Hanwoo bull genome was sequenced to 45.6-fold coverage using the ABI SOLiD system. In total, 4.7 million single-nucleotide polymorphisms and 0.4 million small indels were identified by comparison with the Btau4.0 reference assembly. Of the total number of SNPs and indels, 58% and 87%, respectively, were novel. The overall genotype concordance between the SNPs and BovineSNP50 BeadChip data was 96.4%. Of 1.6 million genetic differences in Hanwoo, approximately 25,000 non-synonymous SNPs, splice-site variants, and coding indels (NS/SS/Is) were detected in 8,360 genes. Among 1,045 genes containing reliable specific NS/SS/Is in Hanwoo, 109 genes contained more than one novel damaging NS/SS/I. Of the genes containing NS/SS/Is, 610 genes were assigned as trait-associated genes. Moreover, 16, 78, and 51 regions of homozygosity (ROHs) were detected in Hanwoo, Black Angus, and Holstein, respectively. 'Regulation of actin filament length' was revealed as a significant gene ontology term and 25 trait-associated genes for meat quality and disease resistance were found in 753 genes that resided in the ROHs of Hanwoo. In Hanwoo, 43 genes were located in common ROHs between whole-genome resequencing and SNP chips in BTA2, 10, and 13 coincided with quantitative trait loci for meat fat traits. In addition, the common ROHs in BTA2 and 16 were in agreement between Hanwoo and Black Angus. We identified 4.7 million SNPs and 0.4 million small indels by whole-genome resequencing of a Hanwoo bull. Approximately 25,000 non-synonymous SNPs, splice-site variants, and coding indels (NS/SS/Is) were detected in 8,360 genes. Additionally, we found 25 trait-associated genes for meat quality and disease resistance among 753 genes that resided in the ROHs of Hanwoo. These findings will provide useful genomic information for identifying genes or casual mutations associated with economically important traits in cattle.


How should we establish the clinical case numbers required to achieve proficiency in flexible endoscopy?
Recommended procedure numbers for upper endoscopy (UE) and colonoscopy (C) are 35 and 50 for surgical residents, and 130 and 140 for gastroenterology fellows, respectively. The purpose of this study was to challenge the methods used to determine proficiency in flexible endoscopy. Global assessment of gastrointestinal endoscopic skills (GAGES) was used to evaluate 139 procedures. Scores for UE were compared using self-reported case numbers and grouped according to requirements for each discipline. C scores were compared using the requirements to define novice and experienced endoscopists. Procedure volumes were plotted against GAGES scores. Three groups were compared for UE based on case volumes: fewer than 35 cases (group 1), 35 to 130 cases (group 2), and more than 130 cases (group 3). There was no difference between group 2 (17.8 +/- 1.8) and group 3 (19.1 +/- 1.1), but both scored higher than group 1 (14.4 +/- 3.7; P < .05). For C, the scores were 11.8 +/- 3.8 (novices) and 18.8 +/- 1.34 (experienced; P < .001) at a 50-case minimum and 12.4 +/- 4.2 and 18.8 +/- 1.3 (P < .001) for a 140-case proficiency cut-off level, respectively. The curve of procedures versus GAGES plateaued at 50 (UE) and 75 (C). The surgical and gastroenterology case recommendations may not represent the experience needed to achieve proficiency. GAGES scores could help define proficiency in basic endoscopy.


Swelling-induced and controlled curving in layered gel beams.
We describe swelling-driven curving in originally straight and non-homogeneous beams. We present and verify a structural model of swollen beams, based on a new point of view adopted to describe swelling-induced deformation processes in bilayered gel beams, that is based on the split of the swelling-induced deformation of the beam at equilibrium into two components, both depending on the elastic properties of the gel. The method allows us to: (i) determine beam stretching and curving, once assigned the characteristics of the solvent bath and of the non-homogeneous beam, and (ii) estimate the characteristics of non-homogeneous flat gel beams in such a way as to obtain, under free-swelling conditions, three-dimensional shapes. The study was pursued by means of analytical, semi-analytical and numerical tools; excellent agreement of the outcomes of the different techniques was found, thus confirming the strength of the method.


Validation of an atrial fibrillation risk algorithm in whites and African Americans.
We sought to validate a recently published risk algorithm for incident atrial fibrillation (AF) in independent cohorts and other racial groups. We evaluated the performance of a Framingham Heart Study (FHS)-derived risk algorithm modified for 5-year incidence of AF in the FHS (n = 4764 participants) and 2 geographically and racially diverse cohorts in the age range 45 to 95 years: AGES (the Age, Gene/Environment Susceptibility-Reykjavik Study) (n = 4238) and CHS (the Cardiovascular Health Study) (n = 5410, of whom 874 [16.2%] were African Americans). The risk algorithm included age, sex, body mass index, systolic blood pressure, electrocardiographic PR interval, hypertension treatment, and heart failure. We found 1359 incident AF events in 100 074 person-years of follow-up. Unadjusted 5-year event rates differed by cohort (AGES, 12.8 cases/1000 person-years; CHS whites, 22.7 cases/1000 person-years; and FHS, 4.5 cases/1000 person-years) and by race (CHS African Americans, 18.4 cases/1000 person-years). The strongest risk factors in all samples were age and heart failure. The relative risks for incident AF associated with risk factors were comparable across cohorts and race groups. After recalibration for baseline incidence and risk factor distribution, the Framingham algorithm, reported in C statistic, performed reasonably well in all samples: AGES, 0.67 (95% confidence interval [CI], 0.64-0.71); CHS whites, 0.68 (95% CI, 0.66-0.70); and CHS African Americans, 0.66 (95% CI, 0.61-0.71). Risk factors combined in the algorithm explained between 47.0% (AGES) and 63.6% (FHS) of the population-attributable risk. Risk of incident AF in community-dwelling whites and African Americans can be assessed reliably by routinely available and potentially modifiable clinical variables. Seven risk factors accounted for up to 64% of risk.