Myocardial infarction related atrial fibrillation: role of endogenous adenosine.
Exogenous administration of adenosine induces atrial fibrillation in up to 7.0% of patients. Animal studies affirm endogenous adenosine released in response to tissue hypoxia may play a mechanistic role in arrhythmias associated with myocardial ischaemia or hypoxia. Therefore, atrial fibrillation occurring early after the acute phase of myocardial infarction involving atrial tissue may be secondary to an excessive accumulation of adenosine that leads to a shortening of atrial refractory period. Early in the course of acute inferior myocardial infarction, two patients (males aged 45 and 68) suffered new onset sustained atrial fibrillation that was abrupt in onset and complicated their clinical management. They were administered 250 mg theophylline as a slow intravenous injection at a rate of 100 mg/min or until conversion to normal sinus rhythm occurred. Both patients converted to normal sinus rhythm within five minutes of the administration of theophylline. In up to 52 hours of continuous ECG monitoring after the theophylline administration the atrial fibrillation did not recur. Neither patient experienced any adverse outcome from theophylline administration. These observations are the first reported in humans or laboratory animals to suggest that atrial fibrillation, presumably due to elevated interstitial atrial concentration of adenosine caused by myocardial ischaemia, can be terminated with an adenosine receptor antagonist. However, the hypothesis that excessive accumulation of endogenous adenosine in atrial tissue may induce atrial fibrillation is well substantiated by other investigators. Thus, A1 adenosine receptor antagonists may prove to be valuable in the management of ischaemia related atrial fibrillation.


MicroRNA-21 induces resistance to 5-fluorouracil by down-regulating human DNA MutS homolog 2 (hMSH2).
The overexpression of microRNA-21 (miR-21) is linked to a number of human tumors including colorectal cancer, where it appears to regulate the expression of tumor suppressor genes including p21, phosphatase and tensin homolog, TGFβ receptor II, and B-cell leukemia/lymphoma 2 -associated X protein. Here we demonstrate that miR-21 targets and down-regulates the core mismatch repair (MMR) recognition protein complex, human mutS homolog 2 (hMSH2) and 6 (hMSH6). Colorectal tumors that express a high level of miR-21 display reduced hMSH2 protein expression. Cells that overproduce miR-21 exhibit significantly reduced 5-fluorouracil (5-FU)-induced G2/M damage arrest and apoptosis that is characteristic of defects in the core MMR component. Moreover, xenograft studies demonstrate that miR-21 overexpression dramatically reduces the therapeutic efficacy of 5-FU. These studies suggest that the down-regulation of the MMR mutator gene associated with miR-21 overexpression may be an important clinical indicator of therapeutic efficacy in colorectal cancer.


Performance of the applied biosystems ViroSeq human immunodeficiency virus type 1 (HIV-1) genotyping system for sequence-based analysis of HIV-1 in pediatric plasma samples.
The ViroSeq HIV-1 Genotyping System is a commercially available, integrated sequence-based system for analysis of human immunodeficiency virus type 1 (HIV-1) drug resistance. We evaluated the performance of this system by analyzing HIV-1 in pediatric plasma samples. Plasma samples from children 4 months to 17 years of age were obtained from a clinical trial protocol (PACTG 377). Children in PACTG 377 were randomized to four treatment arms, including different combinations of antiretroviral drugs. HIV-1 genotyping was performed using samples collected prior to antiretroviral therapy (baseline) and at the time of virologic failure. Performance of the genotyping system was compared in three university laboratories. A total of 196 samples were analyzed, including 135 baseline and 61 failure samples. Plasma volumes ranged from 0.05 to 0.5 ml, and viral loads ranged from 1,084 to 3,484,991 copies/ml. PCR products suitable for sequencing were obtained for 192 of the 196 samples. Complete sequences for protease and reverse transcriptase were obtained for all of these 192 samples. For 180 samples, data were obtained from both DNA strands for the entire region analyzed. There was no evidence of sample cross-contamination based on phylogenetic analysis of HIV-1 sequences. Performance of the genotyping system was similar in three laboratories. This genotyping system performs well for analysis of HIV-1 in pediatric plasma samples, including those with low volume and low viral load. The availability of this system should facilitate studies of HIV-1 drug resistance.


Data from complete mtDNA sequencing of Tunisian centenarians: testing haplogroup association and the "golden mean" to longevity.
Since the mitochondrial theory of ageing was proposed, mitochondrial DNA (mtDNA) diversity has been largely studied in old people, however complete genomes are still rare, being limited to Japanese and UK/US samples. In this work, we evaluated possible longevity associated polymorphisms/haplogroups in an African population, from Tunisia, by performing complete mtDNA sequencing. This population has a mixed Eurasian/sub-Saharan mtDNA gene pool, which could potentially facilitate the evaluation of association for sub-Saharan lineages. Sub-Saharan haplogroups were shown to be significantly less represented in centenarians (9.5%) than in controls (54.5%), but it is not possible to rule out an influence of population structure, which is high in these populations. No recurrent polymorphism were more frequent in centenarians than in controls, and although the Tunisian centenarians presented less synonymous and replacement polymorphisms than controls, this difference was not statistically significant. So far, it does not seem that centenarians have significantly less mildly deleterious substitutions, not only in Tunisia but also in Japanese and UK/US samples, as tested here, not favouring a "golden mean" to longevity.


Hurricane debbie modification experiments, august 1969.
Maximum winds in Hurricane Debbie, August 1969, decreased after modification experiments by Project Stormfury. Clouds surrounding the center of Debbie were seeded with silver iodide particles five times at approximately 2-hour intervals on both 18 and 20 August. Before the first seeding on 18 August, the maximum speed of winds at 3600 meters was 182 kilometers per hour, but, 5 hours after the fifth seeding, these winds decreased to 126 kilometers per hour. On 20 August, the corresponding change was from 183 to 156 kilometers per hour. Analyses of the data suggest that the storm was modified.


Indirect effects of species interactions on habitat provisioning.
Species that shelter in a biogenic habitat can influence their refugia and, in turn, play an essential role in shaping local patterns of biodiversity. Here we explore a positive feedback loop between the provisioning rate of habitat-forming branching corals and their associated fishes and show how interactions between two groups of fish--the planktivorous damselfish and predatory hawkfish--altered the feedback. A field experiment confirmed that skeletal growth of branching coral (genus Pocillopora) increased substantially with increasing numbers (biomass) of resident fishes, likely because they greatly increased the interstitial concentrations of nutrients. Because there is a positive relationship between colony size and number (biomass) of associated fishes (primarily damselfishes in the Family Pomacentridae), a structure-function feedback loop exists in which increasing numbers of damselfish enhance coral growth and larger corals host greater abundances (and species richness) of fish. However, interactions between damselfishes and arc-eye hawkfish, Paracirrhites arcatus, a largely solitary resident, can disrupt this positive feedback loop. Field surveys revealed a marked pattern of fish occupancy related to coral size: Pocillopora colonies of sufficient size to host fish (>40 cm circumference) had either groups of damselfish or an arc-eye hawkfish; only larger colonies (>75 cm) were occupied by both the damselfish and hawkfish. Subsequent short- and long-term experiments revealed that on intermediate-sized Pocillopora colonies, arc-eye hawkfish prevented the establishment of damselfish by suppressing their recruitment. The demographic consequences to the host coral were substantial; in a 1-year-long experiment, intermediate-size Pocillopora occupied by hawkfish grew at half the rate of corals that hosted groups of damselfish. These findings indicate that: (1) species which occupy a biogenic habitat can enhance the provisioning rate of their habitat; (2) such positive feedbacks between community structure and ecosystem function can be disrupted by a strong interactor; (3) even substantial consequences on ecosystem processes that arise can be difficult to discern.


Lack of Benefit and Potential Harm of Induction Therapy in Simultaneous Liver-Kidney Transplants.
The number of simultaneous liver-kidney transplantations (SLKTs) and use of induction therapy for SLKT have increased recently, without much published evidence, especially in the context of maintenance immunosuppression containing tacrolimus (TAC) and mycophenolic acid (MPA). We queried the Organ Procurement and Transplant Network registry for SLKT recipients maintained on TAC/MPA at discharge in the United States for 2002-2016. The cohort was divided into 3 groups on the basis of induction type: rabbit antithymocyte globulin (r-ATG; n = 831), interleukin 2 receptor antagonist (IL2RA; n = 1558), and no induction (n = 2333). Primary outcomes were posttransplant all-cause mortality and acute rejection rates in kidney and liver allografts at 12 months. Survival rates were analyzed by the Kaplan-Meier method. A propensity score analysis was used to control potential selection bias. Multivariate inverse probability weighted Cox proportional hazard and logistic regression models were used to estimate the hazard ratios (HRs) and odds ratios. Among SLKT recipients, survival estimates at 3 years were lower for recipients receiving r-ATG (P = 0.05). Compared with no induction, the multivariate analyses showed an increased mortality risk with r-ATG (HR, 1.29; 95% confidence interval [CI], 1.10-1.52; P = 0.002) and no difference in acute liver or kidney rejection rates at 12 months across all induction categories. No difference in outcomes was noted with IL2RA induction over the no induction category. In conclusion, there appears to be no survival benefit nor reduction in rejection rates for SLKT recipients who receive induction therapy, and r-ATG appears to increase mortality risk compared with no induction.


Opportunities and challenges for using the zebrafish to study neuronal connectivity as an endpoint of developmental neurotoxicity.
Chemical exposures have been implicated as environmental risk factors that interact with genetic susceptibilities to influence individual risk for complex neurodevelopmental disorders, including autism spectrum disorder, schizophrenia, attention deficit hyperactivity disorder and intellectual disabilities. Altered patterns of neuronal connectivity represent a convergent mechanism of pathogenesis for these and other neurodevelopmental disorders, and growing evidence suggests that chemicals can interfere with specific signaling pathways that regulate the development of neuronal connections. There is, therefore, a growing interest in developing screening platforms to identify chemicals that alter neuronal connectivity. Cell-cell, cell-matrix interactions and systemic influences are known to be important in defining neuronal connectivity in the developing brain, thus, a systems-based model offers significant advantages over cell-based models for screening chemicals for effects on neuronal connectivity. The embryonic zebrafish represents a vertebrate model amenable to higher throughput chemical screening that has proven useful in characterizing conserved mechanisms of neurodevelopment. Moreover, the zebrafish is readily amenable to gene editing to integrate genetic susceptibilities. Although use of the zebrafish model in toxicity testing has increased in recent years, the diverse tools available for imaging structural differences in the developing zebrafish brain have not been widely applied to studies of the influence of gene by environment interactions on neuronal connectivity in the developing zebrafish brain. Here, we discuss tools available for imaging of neuronal connectivity in the developing zebrafish, review what has been published in this regard, and suggest a path forward for applying this information to developmental neurotoxicity testing.


EphA2 Expression Is a Key Driver of Migration and Invasion and a Poor Prognostic Marker in Colorectal Cancer.
EphA2, a member of the Eph receptor tyrosine kinases family, is an important regulator of tumor initiation, neovascularization, and metastasis in a wide range of epithelial and mesenchymal cancers; however, its role in colorectal cancer recurrence and progression is unclear. EphA2 expression was determined by immunohistochemistry in stage II/III colorectal tumors (N = 338), and findings correlated with clinical outcome. The correlation between EphA2 expression and stem cell markers CD44 and Lgr5 was examined. The role of EphA2 in migration/invasion was assessed using a panel of KRAS wild-type (WT) and mutant (MT) parental and invasive colorectal cancer cell line models. Colorectal tumors displayed significantly higher expression levels of EphA2 compared with matched normal tissue, which positively correlated with high CD44 and Lgr5 expression levels. Moreover, high EphA2 mRNA and protein expression were found to be associated with poor overall survival in stage II/III colorectal cancer tissues, in both univariate and multivariate analyses. Preclinically, we found that EphA2 was highly expressed in KRASMT colorectal cancer cells and that EphA2 levels are regulated by the KRAS-driven MAPK and RalGDS-RalA pathways. Moreover, EphA2 levels were elevated in several invasive daughter cell lines, and downregulation of EphA2 using RNAi or recombinant EFNA1 suppressed migration and invasion of KRASMT colorectal cancer cells. These data show that EpHA2 is a poor prognostic marker in stage II/III colorectal cancer, which may be due to its ability to promote cell migration and invasion, providing support for the further investigation of EphA2 as a novel prognostic biomarker and therapeutic target.


Mean annual precipitation predicts primary production resistance and resilience to extreme drought.
Extreme drought is increasing in frequency and intensity in many regions globally, with uncertain consequences for the resistance and resilience of ecosystem functions, including primary production. Primary production resistance, the capacity to withstand change during extreme drought, and resilience, the degree to which production recovers, vary among and within ecosystem types, obscuring generalized patterns of ecological stability. Theory and many observations suggest forest production is more resistant but less resilient than grassland production to extreme drought; however, studies of production sensitivity to precipitation variability indicate that the processes controlling resistance and resilience may be influenced more by mean annual precipitation (MAP) than ecosystem type. Here, we conducted a global meta-analysis to investigate primary production resistance and resilience to extreme drought in 64 forests and grasslands across a broad MAP gradient. We found resistance to extreme drought was predicted by MAP; however, grasslands (positive) and forests (negative) exhibited opposing resilience relationships with MAP. Our findings indicate that common plant physiological mechanisms may determine grassland and forest resistance to extreme drought, whereas differences among plant residents in turnover time, plant architecture, and drought adaptive strategies likely underlie divergent resilience patterns. The low resistance and resilience of dry grasslands suggests that these ecosystems are the most vulnerable to extreme drought - a vulnerability that is expected to compound as extreme drought frequency increases in the future.


Imaging studies of bacterial biofilms on cochlear implants-Bioactive glass (BAG) inhibits mature biofilm.
The capability of Pseudomonas aeruginosa and Staphylococcus aureus to form biofilm on varying CI component materials differs in the presence and absence of bioactive glass (BAG). The application of BAG induces significant changes in biofilm morphology which can be visualized via scanning electron microscopy (SEM). Bacterial biofilm formation on medical devices, such as cochlear implants (CI), can lead to chronic infections. Interestingly, BAG of type S53P4 seems to be a promising tool for use in the reduction of biofilm development. Primarily, four bacterial species known to cause implant-related infections, P.aeruginosa (ATCC9027), S. aureus (ATCC6538), Staphylococcus epidermidis (ATCC12228) and Streptococcus pyogenes (ATCC19615) were analyzed regarding their capacity to form biofilm on CI components manufactured from three kinds of material: silicone, platinum and titanium. Subsequently, P. aeruginosa and S. aureus biofilms were visualized using scanning electron microscopy, comparing BAG-treated biofilm with non-treated biofilm. The four bacterial species presented biofilm-forming capabilities in a species and surface dependent manner. Metal CI components allowed for the greatest proliferation of biofilm. S. aureus and P. aeruginosa showed the highest rate of biofilm formation on polystyrene surfaces. For both species, SEM revealed altered biofilm morphology after treatment of S53P4 BAG. This study indicates that bacterial biofilm formation and structure on CI components is dependent on the surface composition, altering between metal and silicone surfaces. After application of BAG, changes in biofilm morphology on CI components were observed. These data highlight the impact of BAG on bacterial biofilm morphology.


Atypical mycobacterial infections: a clinical study of 92 patients.
Between July 1970 and December 1979, 92 patients with atypical mycobacterial infections of the lung were hospitalized 110 times at Blue Ridge Hospital (Charlottesville, Va). These patients comprised less than 3% of all patients hospitalized for active mycobacterial disease. Sixteen had Mycobacterium kansasii or group I disease, three had group II disease (two M scrofulaceum, one M szulgai), 70 had group III disease (68 M avium-intracellulare, two M xenopi), and three had M fortuitum or group IV disease. M kansasii infections comprised 23% of the total during the first five years, but only 6% during the second half of the decade. Clinical and roentgenographic findings were similar to those in patients with tuberculosis. As anticipated, most of the M kansasii organisms were sensitive to antimycobacterial drugs, and these patients generally responded well to chemotherapy. In contrast, most of the group III organisms, including one of the M xenopi, exhibited resistance to several drugs. Despite the high incidence of resistance, 59% of the patients with group III infections who were treated for at least three months in the hospital had sputum cultures converted to negative.


Cannabidiol, a Major Non-Psychotropic Cannabis Constituent Enhances Fracture Healing and Stimulates Lysyl Hydroxylase Activity in Osteoblasts.
Cannabinoid ligands regulate bone mass, but skeletal effects of cannabis (marijuana and hashish) have not been reported. Bone fractures are highly prevalent, involving prolonged immobilization and discomfort. Here we report that the major non-psychoactive cannabis constituent, cannabidiol (CBD), enhances the biomechanical properties of healing rat mid-femoral fractures. The maximal load and work-to-failure, but not the stiffness, of femurs from rats given a mixture of CBD and Δ(9) -tetrahydrocannabinol (THC) for 8 weeks were markedly increased by CBD. This effect is not shared by THC (the psychoactive component of cannabis), but THC potentiates the CBD stimulated work-to-failure at 6 weeks postfracture followed by attenuation of the CBD effect at 8 weeks. Using micro-computed tomography (μCT), the fracture callus size was transiently reduced by either CBD or THC 4 weeks after fracture but reached control level after 6 and 8 weeks. The callus material density was unaffected by CBD and/or THC. By contrast, CBD stimulated mRNA expression of Plod1 in primary osteoblast cultures, encoding an enzyme that catalyzes lysine hydroxylation, which is in turn involved in collagen crosslinking and stabilization. Using Fourier transform infrared (FTIR) spectroscopy we confirmed the increase in collagen crosslink ratio by CBD, which is likely to contribute to the improved biomechanical properties of the fracture callus. Taken together, these data show that CBD leads to improvement in fracture healing and demonstrate the critical mechanical role of collagen crosslinking enzymes.


Therapeutic Potential of Pterostilbene and Resveratrol on Biomechanic, Biochemical, and Histological Parameters in Streptozotocin-Induced Diabetic Rats.
The aim of this study was to investigate the effects of pterostilbene (PTS) (trans-3,5-dimethoxy-4'-hydroxystilbene) and resveratrol (RSV) (trans-3,5,4' trihydroxystilbene) applied at different doses for the treatment of streptozotocin- (STZ-) induced diabetic rats. At the end of the 5-week experimental period, the right gastrocnemius muscles of the rats were examined biomechanically, while the left ones were examined histologically. In addition, blood glucose, serum insulin, and malondialdehyde (MDA) levels were analyzed in blood samples taken from the rats. The skeletal muscle isometric contraction forces, which showed a decrease with diabetes, were observed to increase with antioxidant applications. Blood glucose, serum insulin, and MDA levels in diabetic rats approached normal levels after applying PTS. When the electron microscopic images of the rat skeletal muscle were examined, those in the combination treatment group were observed to show a better enhancement in the skeletal muscle morphological structure compared to the other diabetic and treatment groups. According to the findings, we suggest that these antioxidant treatments might have good therapeutic nutraceutical potential for some muscle diseases that coexist with diabetes. These treatments should be comprehensively investigated in the future.


Prostate-specific antigen, sexual behavior, and sexually transmitted infections in US men 40-59 years old, 2001-2004: a cross-sectional study.
Sexually transmitted infections (STIs) are hypothesized to play a role in the development of prostate cancer, perhaps due to inflammation-induced oncogenesis. We assessed in a nationally representative population of middle-aged men whether sexual behavior indicators for an increased risk of genital infection were associated with serum prostate-specific antigen (PSA) concentration, a marker of prostatic disease and inflammation. The percentage of men between the ages of 40 and 59 with a PSA > or = 4.0 ng/ml was 2.6% (95% confidence interval [CI], 1.8% - 3.8%). The percentage of men between the ages of 40 and 59 self-reporting a past diagnosis of genital warts or genital herpes, or a recent diagnosis of gonorrhea or chlamydia is estimated to be 7.3% (95% CI, 6.2% - 8.6%). Men self-reporting that they had had sex without using a condom in the past month had a lower PSA concentration and higher %fPSA than those who did not. There were no associations between any of the other sexual activity or laboratory measures and PSA or %fPSA. In this nationally representative sample of middle-aged American men, we did not find consistent evidence for an association between sexual behavior or a history of STIs and PSA levels. Therefore, sexual factors are unlikely to lead to falsely elevated PSA tests in this population. We cannot rule out the role of these factors in causing false positive PSA tests in subgroups of the population that have a higher prevalence of high-risk sexual behavior, and more protracted or recent exposures to these agents.


Examining reliability of WHOBARS: a tool to measure the quality of administration of WHO surgical safety checklist using generalisability theory with surgical teams from three New Zealand hospitals.
To extend reliability of WHO Behaviourally Anchored Rating Scale (WHOBARS) to measure the quality of WHO Surgical Safety Checklist administration using generalisability theory. In this context, extending reliability refers to establishing generalisability of the tool scores across populations of teams and raters by accounting for the relevant sources of measurement errors. Cross-sectional random effect measurement design assessing surgical teams by the five items on the three Checklist phases, and at three sites by two trained raters simultaneously. The data were collected in three tertiary hospitals in Auckland, New Zealand in 2016 and included 60 teams observed in 60 different cases with an equal number of teams (n=20) per site. All elective and acute cases (adults and children) involving surgery under general anaesthesia during normal working hours were eligible. The study included 243 surgical staff members, 138 (50.12%) women. Absolute generalisability coefficient that accounts for variance due to items, phases, sites and raters for the WHOBARS measure of the quality of WHO Surgical Safety Checklist administration. The WHOBARS in its present form has demonstrated good generalisability of scores across teams and raters (G absolute=0.83). The largest source of measurement error was the interaction between the surgical team and the rater, accounting for 16.7% (95% CI 16.4 to 16.9) of the total variance in the data. Removing any items from the WHOBARS led to a decrease in the overall reliability of the instrument. Assessing checklist administration quality is important for promoting improvement in its use, and WHOBARS offers a reliable approach for doing this.


Kinematic analysis of a multi-segment foot model for research and clinical applications: a repeatability analysis.
An unbiased understanding of foot kinematics has been difficult to achieve due to the complexity of foot structure and motion. We have developed a protocol for evaluation of foot kinematics during barefoot walking based on a multi-segment foot model. Stereophotogrammetry was used to measure retroreflective markers on three segments of the foot plus the tibia. Repeatability was evaluated between-trial, between-day and between-tester using two subjects and two testers. Subtle patterns and ranges of motion between segments of the foot were consistently detected. We found that repeatability between different days or different testers is primarily subject to variability of marker placement more than inter-tester variability or skin movement. Differences between inter-segment angle curves primarily represent a shift in the absolute value of joint angles from one set of trials to another. In the hallux, variability was greater than desired due to vibration of the marker array used. The method permits objective foot measurement in gait analysis using skin-mounted markers. Quantitative and objective characterisation of the kinematics of the foot during activity is an important area of clinical and research evaluation. With this work we hope to have provided a firm basis for a common protocol for in vivo foot study.


All-age whole mount in situ hybridization to reveal larval and juvenile expression patterns in zebrafish.
The zebrafish Danio rerio is a valuable and common model for scientists in the fields of genetics and developmental biology. Since zebrafish are also amenable to genetic manipulation, modelling of human diseases or behavioral experiments have moved into the focus of zebrafish research. Consequently, gene expression data beyond embryonic and larval stages become more important, yet there is a dramatic knowledge gap of gene expression beyond day four of development. Like in other model organisms, the visualization of spatial and temporal gene expression by whole mount in situ hybridization (ISH) becomes increasingly difficult when zebrafish embryos develop further and hence the growing tissues become dense and less permeable. Here we introduce a modified method for whole mount ISH, which overcomes these penetration and detection problem. The method is an all in one solution that enables the detection and visualization of gene expression patterns up to the late larval stage in a 3D manner without the need for tissue sectioning and offers a valuable extension for whole mount ISH by immunohistochemistry in the zebrafish field.


A High-Fat Diet Modifies Brain Neurotransmitter Profile and Hippocampal Proteome and Morphology in an IUGR Pig Model.
Intrauterine Growth Restriction (IUGR) hinders the correct growth of the fetus during pregnancy due to the lack of oxygen or nutrients. The developing fetus gives priority to brain development ("brain sparing"), but the risk exists of neurological and cognitive deficits at short or long term. On the other hand, diets rich in fat exert pernicious effects on brain function. Using a pig model of spontaneous IUGR, we have studied the effect on the adult of a long-term high-fat diet (HFD) on the neurotransmitter profile in several brain areas, and the morphology and the proteome of the hippocampus. Our hypothesis was that animals affected by IUGR (born with low birth weight) would present a different susceptibility to an HFD when they become adults, compared with normal birth-weight animals. Our results indicate that HFD affected the serotoninergic pathway, but it did not provoke relevant changes in the morphology of the hippocampus. Finally, the proteomic analysis revealed that, in some instances, NBW and LBW individuals respond to HFD in different ways. In particular, NBW animals presented changes in oxidative phosphorylation and the extracellular matrix, whereas LBW animals presented differences in RNA splicing, anterograde and retrograde transport and the mTOR pathway.


Evaluation of fluorophores to label SNAP-tag fused proteins for multicolor single-molecule tracking microscopy in live cells.
Single-molecule tracking has become a widely used technique for studying protein dynamics and their organization in the complex environment of the cell. In particular, the spatiotemporal distribution of membrane receptors is an active field of study due to its putative role in the regulation of signal transduction. The SNAP-tag is an intrinsically monovalent and highly specific genetic tag for attaching a fluorescent label to a protein of interest. Little information is currently available on the choice of optimal fluorescent dyes for single-molecule microscopy utilizing the SNAP-tag labeling system. We surveyed 6 green and 16 red excitable dyes for their suitability in single-molecule microscopy of SNAP-tag fusion proteins in live cells. We determined the nonspecific binding levels and photostability of these dye conjugates when bound to a SNAP-tag fused membrane protein in live cells. We found that only a limited subset of the dyes tested is suitable for single-molecule tracking microscopy. The results show that a careful choice of the dye to conjugate to the SNAP-substrate to label SNAP-tag fusion proteins is very important, as many dyes suffer from either rapid photobleaching or high nonspecific staining. These characteristics appear to be unpredictable, which motivated the need to perform the systematic survey presented here. We have developed a protocol for evaluating the best dyes, and for the conditions that we evaluated, we find that Dy 549 and CF 640 are the best choices tested for single-molecule tracking. Using an optimal dye pair, we also demonstrate the possibility of dual-color single-molecule imaging of SNAP-tag fusion proteins. This survey provides an overview of the photophysical and imaging properties of a range of SNAP-tag fluorescent substrates, enabling the selection of optimal dyes and conditions for single-molecule imaging of SNAP-tagged fusion proteins in eukaryotic cell lines.


The genome of the intracellular bacterium of the coastal bivalve, Solemya velum: a blueprint for thriving in and out of symbiosis.
Symbioses between chemoautotrophic bacteria and marine invertebrates are rare examples of living systems that are virtually independent of photosynthetic primary production. These associations have evolved multiple times in marine habitats, such as deep-sea hydrothermal vents and reducing sediments, characterized by steep gradients of oxygen and reduced chemicals. Due to difficulties associated with maintaining these symbioses in the laboratory and culturing the symbiotic bacteria, studies of chemosynthetic symbioses rely heavily on culture independent methods. The symbiosis between the coastal bivalve, Solemya velum, and its intracellular symbiont is a model for chemosynthetic symbioses given its accessibility in intertidal environments and the ability to maintain it under laboratory conditions. To better understand this symbiosis, the genome of the S. velum endosymbiont was sequenced. Relative to the genomes of obligate symbiotic bacteria, which commonly undergo erosion and reduction, the S. velum symbiont genome was large (2.7 Mb), GC-rich (51%), and contained a large number (78) of mobile genetic elements. Comparative genomics identified sets of genes specific to the chemosynthetic lifestyle and necessary to sustain the symbiosis. In addition, a number of inferred metabolic pathways and cellular processes, including heterotrophy, branched electron transport, and motility, suggested that besides the ability to function as an endosymbiont, the bacterium may have the capacity to live outside the host. The physiological dexterity indicated by the genome substantially improves our understanding of the genetic and metabolic capabilities of the S. velum symbiont and the breadth of niches the partners may inhabit during their lifecycle.


Intramolecular group transfer is a characteristic of neurotoxic esterase and is independent of the tissue source of the enzyme. A comparison of the aging behaviour of di-isopropyl phosphorofluoridate-labelled proteins in brain, spinal cord, liver, kidney and spleen from hen and in human placenta.
Neurotoxic esterase activity was measured in homogenates of human placenta and hen brain, spinal cord, liver, kidney and spleen. The activity in liver comprised less than 20% of the Paraoxon-resistant esterases, but in the other tissues neurotoxic esterase accounted for over 50%. The same tissues were labelled with [3H]di-isopropyl phosphorofluoridate, and any isopropyl group transferred on to protein during 'aging' of the labelled enzymes (alkali-volatilizable tritium) was measured. No Paraoxon-sensitive labelled sites were found to age in this way in any tissue. In brain, the Paraoxon-resistant alkali-volatilizable-tritium-labelled sites correlated with the number of neurotoxic esterase labelled sites, indicating that 'aging' and isopropyl group transfer were 100% efficient. The site receiving the transferred isopropyl group was characterized by analysing the distribution of radiolabelled proteins on gel-filtration chromatography in the presence of SDS. In particulate preparations from each tissue, the protein-bound alkali-volatilizable tritium (transferred isopropyl group) was attached to a polypeptide of Mr 178 000. This same polypeptide also bore the isopropyl-phosphoryl group of neurotoxic esterase, indicating that aging of neurotoxic esterase is an intramolecular group transfer. The apparent turnover number for the enzyme (average 1.6 X 10(5) min-1) was approximately the same in each hen tissue, confirming that closely similar enzymes were present in brain, spinal cord, liver and spleen. The apparent turnover for the human enzyme was 1.8-fold higher than that for the hen enzyme. The concentration of the neurotoxic esterase phosphorylated subunit in brain, spinal cord, spleen, placenta and liver was 14.6, 3.8, 7.4, 3.3 and 3.8 pmol/g of tissue. The evidence indicated that neurotoxic esterase is present in each tissue except kidney, and that isopropyl group transfer on 'aging' occurs on this enzyme only. This process is an intramolecular transfer of the group within the same polypeptide.


Family intervention approach to loss of clinical effect during long-term antidepressant treatment: a pilot study.
The return of depressive symptoms during maintenance antidepressant treatment is a common phenomenon, but has attracted very limited research attention. The aims of this investigation were to explore the feasibility of a family intervention approach to loss of clinical effect during long-term antidepressant therapy and to compare this approach with dose increase. Twenty outpatients with recurrent major depressive disorder (diagnosed using Research Diagnostic Criteria, i.e., patients were at their third or greater episode of major depressive disorder, with the immediately preceding episode being no more than 2.5 years before the onset of the episode which led to antidepressant treatment) who lived with a partner and relapsed while taking antidepressant drugs were randomly assigned to (1) family intervention approach according to the McMaster Model and maintenance of the anti-depressant drug at the same dosage or (2) dose increase and clinical management. A 1-year follow-up was performed. The study was conducted from January 2002 to December 2004. Seven of 10 patients responded to an increased dosage; all but 1 relapsed again on that dosage during follow-up. Seven of 10 patients responded to family intervention, but only 1 relapsed during follow-up. The difference in relapse was significant (p < .05). The data suggest that application of a family intervention approach is feasible when there is a loss of clinical effect during long-term antidepressant treatment, and this approach may carry long-term benefits. The results need to be confirmed by large-scale controlled studies but should alert the physician to explore the psychosocial correlates of loss of clinical effect.


Cannabis; adverse effects from an oromucosal spray.
An oromucosal spray has been developed from the major components of marijuana (cannabis), including tetrahydrocannabinol (THC) and cannabidiol (CBD), in alcohol with a peppermint flavouring, designed to be administered as a spray under the tongue or on the buccal mucosa to relieve pain in multiple sclerosis. Although the available evidence indicates its efficacy in this respect, some patients develop oral burning sensation, stinging or white lesions, probably burns. To investigate the oral side-effects of oromucosal cannabis spray in multiple sclerosis (MS) patients. A small open observational study. A series of nine patients with MS who had been using a marijuana oromucosal spray for at least four weeks, were asked to attend for oral examination. Patients were asked whether they had ever experienced symptoms (dryness; bad taste; stinging) associated with use of the spray. A standard oral examination was carried out using a dental light, and the presence of any mucosal lesions recorded. Where mucosal lesions were present, patients were advised to discontinue the spray and re-attend after four weeks for re-examination. For ethical reasons, biopsies were not undertaken at the first visit. Of nine patients invited to participate, eight attended. All admitted to a stinging sensation on using the oromucosal cannabis spray, and four had visible oral mucosal white lesions in the floor of the mouth. Although the white lesions observed were almost certainly burns, resolving or improving on discontinuation of use of the medication, the high alcohol concentration of the oromucosal cannabis spray raises concern in relation to chronic oral use.


Hypoxia-inducible factor plays a gut-injurious role in intestinal ischemia reperfusion injury.
Gut injury and loss of normal intestinal barrier function are key elements in the paradigm of gut-origin systemic inflammatory response syndrome, acute lung injury, and multiple organ dysfunction syndrome (MODS). As hypoxia-inducible factor (HIF-1) is a critical determinant of the physiological and pathophysiological response to hypoxia and ischemia, we asked whether HIF-1 plays a proximal role in the induction of gut injury and subsequent lung injury. Using partially HIF-1α-deficient mice in an isolated superior mesenteric artery occlusion (SMAO) intestinal ischemia reperfusion (I/R) injury model (45 min SMAO followed by 3 h of reperfusion), we showed a direct relationship between HIF-1 activation and intestinal I/R injury. Specifically, partial HIF-1α deficiency attenuated SMAO-induced increases in intestinal permeability, lipid peroxidation, mucosal caspase-3 activity, and IL-1β mRNA levels. Furthermore, partial HIF-1α deficiency prevented the induction of ileal mucosal inducible nitric oxide synthase (iNOS) protein levels after SMAO and iNOS deficiency ameliorated SMAO-induced villus injury. Resistance to SMAO-induced gut injury was also associated with resistance to lung injury, as reflected by decreased levels of myeloperoxidase, IL-6 and IL-10 in the lungs of HIF-1α(+/-) mice. In contrast, a short duration of SMAO (15 min) followed by 3 h of reperfusion neither induced mucosal HIF-1α protein levels nor caused significant gut and lung injury in wild-type or HIF-1α(+/-) mice. This study indicates that intestinal HIF-1 activation is a proximal regulator of I/R-induced gut mucosal injury and gut-induced lung injury. However, the duration and severity of the gut I/R insult dictate whether HIF-1 plays a gut-protective or deleterious role.


Comparison of outcomes for African Americans, Hispanics, and Non-Hispanic Whites in the CATIE study.
Medication outcome literature in schizophrenia across racial-ethnic groups is sparse, with inconsistent findings. The Clinical Antipsychotic Trials of Intervention Effectiveness (CATIE) study provided an opportunity for exploratory analyses of racial-ethnic outcomes. The study objective was to examine race-ethnicity outcomes for CATIE's main outcome (study discontinuation) and secondary outcomes. CATIE participants included whites (non-Hispanic) (N=722), African Americans (N=506), and Hispanics (N=170). Survival analyses and mixed-effects regression modeling were conducted, with adjustment for baseline sociodemographic differences and baseline scores of the secondary outcomes. Racial-ethnic groups had unique patterns of outcomes. Hispanics were much more likely to discontinue for lack of efficacy from perphenazine (64% versus 42% non-Hispanic whites and 24% African Americans) and ziprasidone (71% versus 40% non-Hispanic whites and 24% African Americans); Hispanics' quality of life also declined on these medications. Non-Hispanic whites were more likely to discontinue for lack of efficacy in general (averaging olanzapine, quetiapine, and risperidone discontinuation rates). African Americans were less likely to continue after the first phase (32% continuing versus 40% for non-Hispanic whites and 41% Hispanics). Discontinuations were driven by research burden, personal issues, and unspecified loss to follow-up. Non-Hispanic whites had higher depression scores during the follow-up period. African Americans had fewer side effects. CATIE results did not show disparities favoring non-Hispanic whites. CATIE may have provided state-of-the-art treatment and thus reduced disparate treatments observed in community clinics. African Americans discontinued even after consideration of socioeconomic differences. Why perphenazine and ziprasidone may be less effective with Hispanics should be explored.


Decursin attenuates kainic acid-induced seizures in mice.
Epilepsy is a neurological disorder with recurrent unprovoked seizures as the main symptom. Of the coumarin derivatives in Angelica gigas, decursin, a major coumarin component, was reported to exhibit significant protective activity against glutamate-induced neurotoxicity when added to primary cultures of rat cortical cells. This study served to investigate the effects of decursin on a kainic acid (KA)-induced status epilepticus model. Thirty minutes after intraperitoneal injections of decursin (20 mg/kg) in male 7-week-old C57BL/6 mice, the animals were treated with KA (30 mg/kg, intraperitoneally) and then examined for behavioral seizure score, electroencephalogram, seizure-related expressed protein levels, neuronal cell loss, neurodegeneration, and astrogliosis. KA injections significantly enhanced neurodegenerative conditions but treatment with decursin 30 min before KA injection reduced the detrimental effects of KA in mice. The decursin-treated KA-injected group showed significantly decreased behavioral seizure activity and remarkably attenuated intense and high-frequency seizure discharges in the parietal cortex for 2 h compared with the group treated only with KA. Furthermore, in-vivo results indicated that decursin strongly inhibits selective neuronal death, astrogliosis, and oxidative stress induced by KA administration. Therefore decursin is able to attenuate KA-induced seizures and could have potential as an antiepileptic drug.


Antiemetic use among pregnant women in the United States: the escalating use of ondansetron.
To examine ondansetron use in pregnancy in the context of other antiemetic use among a large insured United States population of women delivering live births. We assessed ondansetron and other antiemetic use among pregnant women delivering live births between 2001 and 2015 in 15 data partners contributing data to the Mini-Sentinel Distributed Database. We identified live birth pregnancies using a validated algorithm, and all forms of ondansetron and other available antiemetics were identified using National Drug Codes or procedure codes. We assessed the prevalence of antiemetic use by trimester, calendar year, and formulation. In over 2.3 million pregnancies, the prevalence of ondansetron, promethazine, metoclopramide, or doxylamine/pyridoxine use anytime in pregnancy was 15.2, 10.3, 4.0, and 0.4%, respectively. Ondansetron use increased from <1% of pregnancies in 2001 to 22.2% in 2014, with much of the increase attributable to oral ondansetron beginning in 2006. Promethazine and metoclopramide use increased modestly between 2001 (13.8%, 3.2%) and 2006 (16.0%, 6.0%) but decreased annually through 2014 (8.0%, 3.2%). Doxylamine/pyridoxine, approved for management of nausea and vomiting in pregnancy in 2013, was used in 1.8% of pregnancies in 2014. For all antiemetics, use was highest in the first trimester. We observed a marked increase in ondansetron use by study year, prescribed to nearly one-quarter of insured pregnant women in 2014, occurring in conjunction with decreased use of promethazine and metoclopramide. Given the widespread use of ondansetron in pregnancy, data establishing product efficacy and methodologically rigorous evaluation of post-marketing safety are needed. Published 2017. This article is a U.S. Government work and is in the public domain in the USA.


Penguin colony attendance under artificial lights for ecotourism.
Wildlife watching is an emerging ecotourism activity around the world. In Australia and New Zealand, night viewing of little penguins attracts hundreds of thousands of visitors per year. As penguins start coming ashore after sunset, artificial lighting is essential to allow visitors to view them in the dark. This alteration of the nightscape warrants investigation for any potential effects of artificial lighting on penguin behavior. We experimentally tested how penguins respond to different light wavelengths (colors) and intensities to examine effects on the colony attendance behavior at two sites on Phillip Island, Australia. At one site, nocturnal artificial illumination has been used for penguin viewing for decades, whereas at the other site, the only light is from the natural night sky. Light intensity did not affect colony attendance behaviors of penguins at the artificially lit site, probably due to penguin habituation to lights. At the not previously lit site, penguins preferred lit paths over dark paths to reach their nests. Thus, artificial light might enhance penguin vision at night and consequently it might reduce predation risk and energetic costs of locomotion through obstacle and path detection. Although penguins are faithful to their path, they can be drawn to artificial lights at small spatial scale, so light pollution could attract penguins to undesirable lit areas. When artificial lighting is required, we recommend keeping lighting as dim and time-restricted as possible to mitigate any negative effects on the behavior of penguins and their natural habitat.


Mislocalization of AQP4 precedes chronic seizures in the kainate model of temporal lobe epilepsy.
It has been suggested that loss of the astrocytic water channel aquaporin-4 (AQP4) from perivascular endfeet in sclerotic hippocampi contributes to increased seizure propensity in human mesial temporal lobe epilepsy (MTLE). Whether this loss occurs prior to or as a consequence of epilepsy development remains to be resolved. In the present study, we investigated whether the expression and distribution of AQP4 was altered prior to (i.e., in the latent phase) or after the onset of chronic epileptic seizures (i.e., in the chronic phase) in the kainate (KA) model of MTLE. Immunogold electron microscopic analysis revealed that AQP4 density in adluminal endfoot membranes was reduced in KA treated rats already in the latent phase, while the AQP4 density in the abluminal endfoot membrane was stable or slightly increased. The decrease in adluminal AQP4 immunogold labeling was accompanied by a reduction in the density of AQP4's anchoring protein alpha-syntrophin. The latent and chronic phases were associated with an upregulation of the M1 isoform of AQP4, as judged by semi-quantitative Western blot analysis. Taken together, the findings in this model suggest that a mislocalization of AQP4--reflecting a loss of astrocyte polarization--is an integral part of the epileptogenic process.


Tissue and cellular distribution of the extended family of protein kinase C isoenzymes.
Polyclonal isoenzyme-specific antisera were developed against four calcium-independent protein kinase C (PKC) isoenzymes (delta, epsilon, epsilon', and zeta) as well as the calcium-dependent isoforms (alpha, beta I, beta II, and gamma). These antisera showed high specificities, high titers, and high binding affinities (3-370 nM) for the peptide antigens to which they were raised. Each antiserum detected a species of the predicted molecular weight by Western blot that could be blocked with the immunizing peptide. PKC was sequentially purified from rat brain, and the calcium-dependent forms were finally resolved by hydroxyapatite chromatography. Peak I reacted exclusively with antisera to PKC gamma, peak II with PKC beta I and -beta II, and peak III with PKC alpha. These same fractions, however, were devoid of immunoreactivity for the calcium-independent isoenzymes. The PKC isoenzymes demonstrated a distinctive tissue distribution when evaluated by Western blot and immunocytochemistry. PCK delta was present in brain, heart, spleen, lung, liver, ovary, pancreas, and adrenal tissues. PKC epsilon was present in brain, kidney, and pancreas, whereas PKC epsilon' was present predominantly in brain. PKC zeta was present in most tissues, particularly the lung, brain, and liver. Both PKC delta and PKC zeta showed some heterogeneity of size among the different tissues. PKC alpha was present in all organs and tissues examined. PKC beta I and -beta II were present in greatest amount in brain and spleen. Although the brain contained the most PKC gamma immunoreactivity, some immunostaining was also seen in adrenal tissue. These studies provide the first evidence of selective organ and tissue distributions of the calcium-independent PKC isoenzymes.


Hypergraphs and centrality measures identifying key features in gene expression data.
Multidisciplinary approaches can significantly advance our understanding of complex systems. For instance, gene co-expression networks align prior knowledge of biological systems with studies in graph theory, emphasising pairwise gene to gene interactions. In this paper, we extend these ideas, promoting hypergraphs as an investigative tool for studying multi-way interactions in gene expression data. Additional freedoms are achieved by representing individual genes with hyperedges, and simultaneously testing each gene against many features/vertices. Further gene/hyperedge interactions can be captured and explored using the line graph representations, a technique that reduces the complexity of dense hypergraphs. Such an approach provides access to graph centrality measures, which identifies salient features within a data set. For instance dominant or hub-like hyperedges, leading to key knowledge on gene expression. The validity of this approach is established through the study of gene expression data for the plant species Senecio lautus and results will be interpreted within this biological setting.


Thermal infrared near-field spectroscopy.
Despite the seminal contributions of Kirchhoff and Planck describing far-field thermal emission, fundamentally distinct spectral characteristics of the electromagnetic thermal near-field have been predicted. However, due to their evanescent nature their direct experimental characterization has remained elusive. Combining scattering scanning near-field optical microscopy with Fourier-transform spectroscopy using a heated atomic force microscope tip as both a local thermal source and scattering probe, we spectroscopically characterize the thermal near-field in the mid-infrared. We observe the spectrally distinct and orders of magnitude enhanced resonant spectral near-field energy density associated with vibrational, phonon, and phonon-polariton modes. We describe this behavior and the associated distinct on- and off-resonance nanoscale field localization with model calculations of the near-field electromagnetic local density of states. Our results provide a basis for intrinsic and extrinsic resonant manipulation of optical forces, control of nanoscale radiative heat transfer with optical antennas, and use of this new technique of thermal infrared near-field spectroscopy for broadband chemical nanospectroscopy.


Aqueous-phase synthesis of highly luminescent CdTe/ZnTe core/shell quantum dots optimized for targeted bioimaging.
Semiconductor quantum dots (QDs) have traditionally been synthesized in organic phase and transferred to aqueous solution by functionalizing their surface with silica, polymers, short-chain thiol ligand, or phospholipid micelles. However, these complex steps result in i) a reduction of the quantum yield (QY) of QDs, ii) partial degrdation of the QDs, and iii) a drastic increase in the hydrodynamic size of QDs, which may hinder their biomedical applications. In this work, the fabrication and applications of cysteine-capped CdTe/ZnTe QDs, which are directly synthesized in aqueous media, as optical probes for specific targeting of pancreatic and esophageal cancer cells in vitro are reported, as well as their capability for in vivo imaging. The CdTe/ZnTe QDs are synthesized in a one-pot method and capped with amino acid cysteine, which contains both carboxyl and amine functional groups on their surfaces for bioconjugation. The fabricated QDs have an ultrasmall hydrodynamic diameter (3-5 nm), possess high QY (52%), and are non-toxic to cells at experimental dosages. Confocal imaging is used to demonstrate a receptor-mediated uptake of antibody-conjugated QDs into pancreatic cancer cells in vitro. In vitro cytotoxicity studies (MTS-assay) show that the IC(50) value of these QDs is approximately 160 microg mL(-1), demonstrating low toxicity. In addition, the QDs are used for small-animal imaging where the in vivo biocompatiblity of these QDs and their clearance following systemic injection is studied.


Prevalence and risk factors for peri-implant diseases in Japanese adult dental patients.
We investigated the prevalences and risk factors for peri-implant diseases in Japanese adult dental patients attending a follow-up visit at dental hospitals or clinics as part of their maintenance program. This cross-sectional multicenter study enrolled patients with dental implants who attended regular check-ups as part of a periodontal maintenance program during the period from October 2012 through September 2013. Patients with implants with at least 3 years of loading time were included in the study. The condition of peri-implant tissue was examined and classified into the following categories: healthy, peri-implant mucositis, and peri-implantitis. Patients were also evaluated for implant risk factors. A total of 267 patients (110 men, 157 women; mean age: 62.5 ± 10.7 years) were analyzed. The prevalence of patient-based peri-implant mucositis was 33.3% (n = 89), and the prevalence of peri-implantitis was 9.7% (n = 26). Poor oral hygiene and a history of periodontitis were strong risk factors for peri-implant disease. The present prevalences were lower than those previously reported. The quality of periodontal therapy before and after implant installation and patient compliance and motivation, as indicated by plaque control level, appear to be important in maintaining peri-implant tissue health.


Anticancer Potential of Nutraceutical Formulations in MNU-induced Mammary Cancer in Sprague Dawley Rats.
Nutraceuticals help in combating some of the major health problems of the century including cancer, and 'nutraceutical formulations' have led to the new era of medicine and health. To develop different nutraceutical formulations and to assess the anticancer potential of nutraceutical formulations in N-methyl-N-nitrosourea (MNU)-induced mammary cancer in Sprague Dawley rats. Different nutraceutical formulations were prepared using fine powders of amla, apple, garlic, onion, papaya, turmeric, and wheat grass with and without cow urine distillate. Total phenolic content, acute oral toxicity, and microbial load of nutraceutical formulations were assessed. The anticancer potential of nutraceutical formulations was evaluated against MNU-induced mammary cancer in female Sprague Dawley rats. Improvement in total phenolic content was significant (P < 0.001) after self-fortification process. Toxicity studies showed that the nutraceutical formulations were safe to use in animals. Microbial load was within the limits. Significant longer tumor-free days (P < 0.01), lower tumor incidence (P < 0.01), lower tumor multiplicity (P < 0.05) and tumor burden (P < 0.01) were observed for nutraceutical formulation-treated groups. Combination of whole food-based nutraceuticals acted synergistically in the prevention of mammary cancer. Further, the process of fortification is novel and enhanced the anticancer potential of nutraceutical formulations. Nutraceuticals help in combating some of the major health problems of the century including cancer, and 'nutraceutical formulations' have led to the new era of medicine and health. In this study, different nutraceutical formulations using fine powders of amla, apple, garlic, onion, papaya, turmeric, and wheat grass with and without cow urine distillate. Total phenolic content, acute oral toxicity, and microbial load of nutraceutical formulations were assessed. The anticancer potential of nutraceutical formulations was evaluated against MNU-induced mammary cancer in female Sprague Dawley rats. Improvement in total phenolic content was observed after self-fortification process. Toxicity studies showed that the nutraceutical formulations were safe to use in animals. Microbial load was within the limits. Longer tumor-free days, lower tumor incidence, lower tumor multiplicity and tumor burden were observed for nutraceutical formulation-treated groups. This suggests that combination of whole food-based nutraceuticals acted synergistically in the prevention of mammary cancer. Further, the process of fortification enhanced the anticancer potential of nutraceutical formulations. Abbreviations used: HMNU: N-methyl-N-nitrosourea, CAM: Complementary and Alternative Medicine, NF: Nutraceutical Formulation, SFNF: Self-Fortitfied Nutraceutical Formulation, NFCUD: Nutraceutical Formulation fortified with Cow Urine Disstillate, SFNFCUD: Self-Fortified Nutraceutical Formulation fortified with Cow Urine Disstillate, CPCSEA: Committee for the Purpose of Control and Supervision of Experiments on Animals, OECD: Organisation for Economic Co-operation and Development, TPC: Total Phenolic Content, ANOVA: Analysis of Variance, GAE: Gallic Acid Equivalent, cfu/g: Colony forming unit per g.


Prognostic role of PET scanning before and after reduced-intensity allogeneic stem cell transplantation for lymphoma.
Allogeneic stem cell transplantation (SCT) is an established therapy for patients with relapsed lymphoma, but the role of positron emission tomography (PET) scanning preallogeneic and postallogeneic SCT is uncertain. We investigated whether pretransplantation PET status predicted outcome after allogeneic SCT and whether PET surveillance after transplantation provided additional information compared with computed tomography (CT) scanning. Eighty consecutive patients with lymphoma who received a reduced-intensity allogeneic SCT were entered onto a prospective trial. PET and CT scans were performed before transplantation and up to 36 months after transplantation. Forty-two patients were PET-positive before transplantation. Pretransplantation PET status had no significant impact on either relapse rate or overall survival. Thirty-four relapses were observed, of which 17 were PET-positive with a normal CT scan at relapse. Donor lymphocyte infusion (DLI) was administered in 26 episodes of relapse and was guided by PET alone in 14 patients. These findings suggest that, in contrast to autologous SCT, pretransplantation PET status is not predictive of relapse and survival after allogeneic SCT for lymphoma. Posttransplantation surveillance by PET detected relapse before CT in half of episodes, often allowing earlier administration of DLI in patients with recurrent lymphoma, and permitted withholding of potentially harmful DLI in those with PET-negative masses on CT scans.


Health literacy in geriatric patients: An integrative review of the literature.
The concept of health literacy is a recent addition to healthcare literature, appearing in the early to mid 1990s. Health literacy refers to individuals' ability to understand their healthcare issues and effectively care for themselves in the healthcare system. Individuals' health literacy abilities vary greatly and can be viewed on a continuum ranging from adequate to marginal to inadequate. Repeated research has demonstrated that older individuals, the geriatric patient population, are at risk of inadequate or marginal health literacy. This risk impacts health outcomes and cost for caring for the older people in our society who are the consumers of a large quantity of healthcare services. The demographic data from the research show that multiple factors influence an individual's health literacy. Besides age, individuals with less education, lower income, "blue collar" jobs, and poor health status (mental and physical) can be at risk of marginal or inadequate health literacy. Providing care to low health literacy patients is complicated by commercially prepared healthcare materials and presentations that are often written well above the average national literacy level. Studies in this review examine various approaches to healthcare education and provide viable options for educating those with low health literacy. This review stresses the importance of healthcare professionals identifying older individuals with marginal or inadequate health literacy and being able to provide education in an effective manner to this segment of the population.


The multivariate physical activity signature associated with metabolic health in children.
Physical activity is a cornerstone for promoting good metabolic health in children, but it is heavily debated which intensities (including sedentary time) are most influential. A fundamental limitation to current evidence for this relationship is the reliance on analytic approaches that cannot handle collinear variables. The aim of the present study was to determine the physical activity signature related to metabolic health in children, by investigating the association pattern for the whole spectrum of physical activity intensities using multivariate pattern analysis. We used a sample of 841 children (age 10.2 ± 0.3 years; BMI 18.0 ± 3.0; 50% boys) from the Active Smarter Kids study, who provided valid data on accelerometry (ActiGraph GT3X+) and several indices of metabolic health (aerobic fitness, abdominal fatness, insulin sensitivity, lipid metabolism, blood pressure) that were used to create a composite metabolic health score. We created 16 physical activity variables covering the whole intensity spectrum (from 0-100 to ≥ 8000 counts per minute) and used multivariate pattern analysis to analyze the data. Physical activity intensities in the vigorous range (5000-7000 counts per minute) were most strongly associated with metabolic health. Moderate intensity physical activity was weakly related to health, and sedentary time and light physical activity were not related to health. This study is the first to determine the multivariate physical activity signature related to metabolic health in children across the whole intensity spectrum. This novel approach shows that vigorous physical activity is strongest related to metabolic health. We recommend future studies adapt a multivariate analytic approach to further develop the field of physical activity epidemiology. The study was registered in Clinicaltrials.gov (www.clinicaltrials.gov) 7th of April 2014 with identification number NCT02132494 .


ERP Evidence for Ultra-Fast Semantic Processing in the Picture-Word Interference Paradigm.
We used the event-related potential (ERP) approach combined with a subtraction technique to explore the timecourse of activation of semantic and phonological representations in the picture-word interference paradigm. Subjects were exposed to to-be-named pictures superimposed on to-be-ignored semantically related, phonologically related, or unrelated words, and distinct ERP waveforms were generated time-locked to these different classes of stimuli. Difference ERP waveforms were generated in the semantic condition and in the phonological condition by subtracting ERP activity associated with unrelated picture-word stimuli from ERP activity associated with related picture-word stimuli. We measured both latency and amplitude of these difference ERP waveforms in a pre-articulatory time-window. The behavioral results showed standard interference effects in the semantic condition, and facilitatory effects in the phonological condition. The ERP results indicated a bimodal distribution of semantic effects, characterized by the extremely rapid onset (at about 100 ms) of a primary component followed by a later, distinct, component. Phonological effects in ERPs were characterized by components with later onsets and distinct scalp topography of ERP sources relative to semantic ERP components. Regression analyses revealed a covariation between semantic and phonological behavioral effect sizes and ERP component amplitudes, and no covariation between the behavioral effects and ERP component latency. The early effect of semantic distractors is thought to reflect very fast access to semantic representations from picture stimuli modulating on-going orthographic processing of distractor words.


Behavior of spinal neurons deprived of supraspinal input.
This Review discusses the spinal neuronal changes that occur after a complete spinal cord injury (SCI) in humans. Early after an SCI, neither locomotor nor spinal reflex activity can be evoked. Once spinal shock has resolved, locomotor activity and an early spinal reflex component reappear in response to appropriate peripheral afferent input. In the subsequent 4-8 months, clinical signs of spasticity appear, largely as a result of non-neuronal (for example, muscular) changes, whereas locomotor and spinal reflex activity undergo little change. At 9-12 months, the electromyographic amplitude in the leg muscles during assisted locomotion declines, accompanied by a decrease in the amplitude of the early spinal reflex component and an increase in the amplitude of a late spinal reflex component. This exhaustion of locomotor activity also occurs in nonambulatory patients with incomplete SCI. Neuronal dysfunction is fully established 1 year after the injury without further alterations in subsequent years. In chronic SCI, the absence of input from supraspinal sources has been suggested to lead to degradation of neuronal function below the level of the lesion or, alternatively, a predominance of inhibitory signaling to the locomotor pattern generator. Appropriate training and/or provision of afferent input to spinal neurons might help to prevent neuronal dysfunction in chronic SCI.


Long-term effect of antiepileptic drug switch on serum lipids and C-reactive protein.
Prior studies have shown that switching patients from inducing antiepileptic drugs (AEDs) to lamotrigine, levetiracetam, or topiramate reduces serum lipids and C-reactive protein (CRP). These studies were all of short duration, and some drugs, such as zonisamide, have not been investigated. We recruited 41 patients taking phenytoin or carbamazepine who were being switched to zonisamide, lamotrigine, or levetiracetam. We measured serum lipids and CRP before the switch, >6weeks after, and >6months after. An untreated control group (n=14) underwent similar measurement. We combined these data with those of our previous investigation (n=34 patients and 16 controls) of a very similar design. There were no differences in outcome measures between the two inducing AEDs nor among the three noninducing AEDs. Total cholesterol (TC), atherogenic lipids, and CRP were higher under inducer treatment than in controls. All measures were elevated under inducer treatment relative to noninducer treatment, including TC (24mg/dL higher, 95% CI: 17.5-29.9, p<0.001) and CRP (72% higher, 95% CI: 41%-111%, p<0.001). The difference between drug treatments was clinically meaningful for atherogenic lipids (16%, 95% CI: 11%-20%, p<0.001) but small for high-density lipoprotein cholesterol (5%, 95% CI: 1%-9%, p<0.05). All measures were stable between 6weeks and 6months after drug switch. We demonstrate that switching from inducing to noninducing AEDs produces an enduring reduction in serum lipids and CRP. These results provide further evidence that inducing AEDs may be associated with elevated vascular disease risk. These are the first vascular risk marker data in patients taking zonisamide, which shows a profile similar to that of other noninducing AEDs.


Automated Survival Prediction in Metastatic Cancer Patients Using High-Dimensional Electronic Medical Record Data.
Oncologists use patients' life expectancy to guide decisions and may benefit from a tool that accurately predicts prognosis. Existing prognostic models generally use only a few predictor variables. We used an electronic medical record dataset to train a prognostic model for patients with metastatic cancer. The model was trained and tested using 12 588 patients treated for metastatic cancer in the Stanford Health Care system from 2008 to 2017. Data sources included provider note text, labs, vital signs, procedures, medication orders, and diagnosis codes. Patients were divided randomly into a training set used to fit the model coefficients and a test set used to evaluate model performance (80%/20% split). A regularized Cox model with 4126 predictor variables was used. A landmarking approach was used due to the multiple observations per patient, with t0 set to the time of metastatic cancer diagnosis. Performance was also evaluated using 399 palliative radiation courses in test set patients. The C-index for overall survival was 0.786 in the test set (averaged across landmark times). For palliative radiation courses, the C-index was 0.745 (95% confidence interval [CI] = 0.715 to 0.775) compared with 0.635 (95% CI = 0.601 to 0.669) for a published model using performance status, primary tumor site, and treated site (two-sided P < .001). Our model's predictions were well-calibrated. The model showed high predictive performance, which will need to be validated using external data. Because it is fully automated, the model can be used to examine providers' practice patterns and could be deployed in a decision support tool to help improve quality of care.


Evaluating protein incorporation and release in electrospun composite scaffolds for bone tissue engineering applications.
Electrospun polymer/ceramic composites have gained interest for use as scaffolds for bone tissue engineering applications. In this study, we investigated methods to incorporate Platelet Derived Growth Factor-BB (PDGF-BB) in electrospun polycaprolactone (PCL) or PCL prepared with polyethylene oxide (PEO), where both contained varying levels (up to 30 wt %) of ceramic composed of biphasic calcium phosphates, hydroxyapatite (HA)/β-tricalcium phosphate (TCP). Using a model protein, lysozyme, we compared two methods of protein incorporation, adsorption and emulsion electrospinning. Adsorption of lysozyme on scaffolds with ceramic resulted in minimal release of lysozyme over time. Using emulsion electrospinning, lysozyme released from scaffolds containing a high concentration of ceramic where the majority of the release occurred at later time points. We investigated the effect of reducing the electrostatic interaction between the protein and the ceramic on protein release with the addition of the cationic surfactant, cetyl trimethylammonium bromide (CTAB). In vitro release studies demonstrated that electrospun scaffolds prepared with CTAB released more lysozyme or PDGF-BB compared with scaffolds without the cationic surfactant. Human mesenchymal stem cells (MSCs) on composite scaffolds containing PDGF-BB incorporated through emulsion electrospinning expressed higher levels of osteogenic markers compared to scaffolds without PDGF-BB, indicating that the bioactivity of the growth factor was maintained. This study revealed methods for incorporating growth factors in polymer/ceramic scaffolds to promote osteoinduction and thereby facilitate bone regeneration.


Local scale prediction of Plasmodium falciparum malaria transmission in an endemic region using temperature and rainfall.
To support malaria control strategies, prior knowledge of disease risk is necessary. Developing a model to explain the transmission of malaria, in endemic and epidemic regions, is of high priority in developing health system interventions. We develop, fit and validate a non-spatial dynamic model driven by meteorological conditions that can capture seasonal malaria transmission dynamics at the village level in a malaria holoendemic area of north-western Burkina Faso. A total of 676 children aged 6-59 months took part in this study. Trained interviewers visited children at home weekly from December 2003 to November 2004 for Plasmodium falciparum malaria infection detection. Anopheles daily biting rate, mortality rate and growth rate were evaluated. Digital meteorological stations measured ambient temperature, humidity and rainfall in each site. The overall P. falciparum malaria infection incidence was 1.1 episodes per person year. There was strong seasonal variation in P. falciparum malaria infection incidence with a peak observed in August and September, corresponding to the rainy season and a high number of mosquitoes. The model estimates of monthly mosquito abundance and the incidence of malaria infection correlated well with observed values. The fit was sensitive to daily mosquito survival and daily human parasite clearance. The model has demonstrated potential for local scale seasonal prediction of P. falciparum malaria infection. It could therefore be used to understand malaria transmission dynamics using meteorological parameters as the driving force and to help district health managers in identifying high-risk periods for more focused interventions.


Genome-wide association of polycystic ovary syndrome implicates alterations in gonadotropin secretion in European ancestry populations.
Polycystic ovary syndrome (PCOS) is a common, highly heritable complex disorder of unknown aetiology characterized by hyperandrogenism, chronic anovulation and defects in glucose homeostasis. Increased luteinizing hormone relative to follicle-stimulating hormone secretion, insulin resistance and developmental exposure to androgens are hypothesized to play a causal role in PCOS. Here we map common genetic susceptibility loci in European ancestry women for the National Institutes of Health PCOS phenotype, which confers the highest risk for metabolic morbidities, as well as reproductive hormone levels. Three loci reach genome-wide significance in the case-control meta-analysis, two novel loci mapping to chr 8p23.1 [Corrected] and chr 11p14.1, and a chr 9q22.32 locus previously found in Chinese PCOS. The same chr 11p14.1 SNP, rs11031006, in the region of the follicle-stimulating hormone B polypeptide (FSHB) gene strongly associates with PCOS diagnosis and luteinizing hormone levels. These findings implicate neuroendocrine changes in disease pathogenesis.


Developing primary care review criteria from evidence-based guidelines: coronary heart disease as a model.
National Health Service (NHS) initiatives such as Clinical Governance, National Service Frameworks and the National Institute of Clinical Excellence (NICE) clinical guidelines programme create demand for tools to enable performance review by healthcare professionals. Ideally such tools should enable clinical teams to assess quality of care and highlight areas of good practice or where improvement is needed. They should also be able to be used to demonstrate progress towards goals and promote quality, while not unnecessarily increasing demand on limited resources or weakening professional control. To formulate and evaluate a method for developing, from clinical guidelines, evidence-based review criteria that are proritised, useful and relevant to general practices assessing quality of care for the primary care management of coronary heart disease (CHD). A two-stage study comprising, first, a review of available evidence-based guidelines for CHD and, second, the definition and prioritization of associated review criteria from the most highly rated guidelines. Primary healthcare teams in England. Using structured methods, evidence-based clinical guidelines for CHD were identified and appraised to ensure their suitability as the basis for developing review criteria. Recommendations common to a number of guidelines were priortszid by a panel of general practitioners to develop review criteria suitable for use in primary care. A standardised method has been developed for constructing evidence-based review criteria from clinical guidelines. A limited, prioritized set of review criteria was developed for the primary care management of CHD. This was distributed around the NHS through the Royal College of General Practitioners for use by primary care teams across the United Kingdom. Developing useful, evidence-based review criteria is not a straightforward process, partly because of a lack of consistency and clarity in guidelines currently available. A method was developed which accommodated these limitations and which can be applied to the development and evaluation of review criteria from guidelines for other conditions.


Clinical assessment is a neglected component of outbreak preparedness: evidence from refugee camps in Greece.
Refugees may have an increased vulnerability to infectious diseases, and the consequences of an outbreak are more severe in a refugee camp. When an outbreak is suspected, access to clinical information is critical for investigators to verify that an outbreak is occurring, to determine the cause and to select interventions to control it. Experience from previous outbreaks suggests that the accuracy and completeness of this information is poor. This study is the first to assess the adequacy of clinical characterisation of acute medical illnesses in refugee camps. The objective is to direct improvements in outbreak identification and management in this vulnerable setting. We collected prospective data in 13 refugee camps in Greece. We passively observed consultations where patients presented with syndromes that might warrant inclusion into an existing syndromic surveillance system and then undertook a structured assessment of routine clinical data collection to examine the extent to which key clinical parameters required for an outbreak response were ascertained and then documented. A total of 528 patient consultations were included. The most common presenting condition was an acute respiratory illness. Clinicians often made a comprehensive clinical assessment, especially for common syndromes of respiratory and gastrointestinal conditions, but documented their findings less frequently. For fewer than 5% of patients were a full set of vital signs ascertained and so the severity of patient illnesses was largely unknown. In only 11% of consultations was it verified that a patient who met the case criteria for syndromic surveillance reporting based on an independent assessment was reported into the system. Opportunities exist to strengthen clinical data capture and recording in refugee camps, which will produce a better calibrated and directed public health response. Information of significant utility for outbreak response is collected at the clinical interface and we recommend improving how this information is recorded and linked into surveillance systems.


Effects of one year of supplementation with zinc and other micronutrients on cellular immunity in the elderly.
The objective of this study was to determine the effects of a year of Zn supplementation on Zn concentrations in circulating cells and on cellular immune functions in the elderly. Subjects, aged 60-89, were given a placebo, 15 mg Zn, or 100 mg Zn daily for 12 months. All subjects also received a multivitamin/mineral supplement that contained no additional Zn. Blood samples were drawn and immune functions assessed prior to and at 3, 6, 12, and 16 months after beginning Zn supplementation. Subject diets were also assessed at each visit. Dietary folate, pyridoxine, alpha-tocopherol, copper, zinc, and magnesium were consistently below recommended intakes. Although plasma Zn increased significantly in the 100 mg Zn treatment group, concentrations of Zn in erythrocytes, mononuclear cells, polymorphonuclear leukocytes, and platelets were not significantly increased by zinc supplementation. Natural killer cell activity was transiently enhanced by the 100 mg/day dose of Zn. There was a progressive improvement in delayed dermal hypersensitivity (DDH) and in lymphocyte proliferative responses to two mitogens; this may have been due to one or more components of the multivitamin/mineral supplement administered to all study subjects. The enhancement of DDH was significantly greater in the placebo group than in either zinc treatment group. Thus, zinc had a beneficial effect on one measure of cellular immune function while simultaneously having an adverse effect on another measure of cellular immunity.


Effect of Mexiletine on Muscle Stiffness in Patients With Nondystrophic Myotonia Evaluated Using Aggregated N-of-1 Trials.
In rare diseases it is difficult to achieve high-quality evidence of treatment efficacy because of small cohorts and clinical heterogeneity. With emerging treatments for rare diseases, innovative trial designs are needed. To investigate the effectiveness of mexiletine in nondystrophic myotonia using an aggregated N-of-1 trials design and compare results between this innovative design and a previously conducted RCT. A series of aggregated, double-blind, randomized, placebo-controlled N-of-1-trials, performed in a single academic referral center. Thirty Dutch adult patients with genetically confirmed nondystrophic myotonia (38 patients screened) were enrolled between February 2014 and June 2015. Follow-up was completed in September 2016. Mexiletine (600 mg daily) vs placebo during multiple treatment periods of 4 weeks. Reduction in daily-reported muscle stiffness on a scale of 1 to 9, with higher scores indicating more impairment. A Bayesian hierarchical model aggregated individual N-of-1 trial data to determine the posterior probability of reaching a clinically meaningful effect of a greater than 0.75-point difference. Among 30 enrolled patients (mean age, 43.4 [SD, 15.24] years; 22% men; 19 CLCN1 and 11 SCN4A genotype), 27 completed the study and 3 dropped out (1 because of a serious adverse event). In 24 of the 27 completers, a clinically meaningful treatment effect was found. In the Bayesian hierarchical model, mexiletine resulted in a 100% posterior probability of reaching a clinically meaningful reduction in self-reported muscle stiffness for the nondystrophic myotonia group overall and the CLCN1 genotype subgroup and 93% posterior probability for the SCN4A genotype subgroup. In the total nondystrophic myotonia group, the median muscle stiffness score was 6.08 (interquartile range, 4.71-6.80) at baseline and was 2.50 (95% credible interval [CrI], 1.77-3.24) during the mexiletine period and 5.56 (95% CrI, 4.73-6.39) during the placebo period; difference in symptom score reduction, 3.06 (95% CrI, 1.96-4.15; n = 27) favoring mexiletine. The most common adverse event was gastrointestinal discomfort (21 mexiletine [70%], 1 placebo [3%]). One serious adverse event occurred (1 mexiletine [3%]; allergic skin reaction). Using frequentist reanalysis, mexiletine compared with placebo resulted in a mean reduction in daily-reported muscle stiffness of 3.12 (95% CI, 2.46-3.78), consistent with the previous RCT treatment effect of 2.69 (95% CI, 2.12-3.26). In a series of N-of-1 trials of mexiletine vs placebo in patients with nondystrophic myotonia, there was a reduction in mean daily-reported muscle stiffness that was consistent with the treatment effect in a previous randomized clinical trial. These findings support the efficacy of mexiletine for treatment of nondystrophic myotonia as well as the feasibility of N-of-1 trials for assessing interventions in some chronic rare diseases. ClinicalTrials.gov Identifier: NCT02045667.


The neurobiology of uncertainty: implications for statistical learning.
The capacity for assessing the degree of uncertainty in the environment relies on estimating statistics of temporally unfolding inputs. This, in turn, allows calibration of predictive and bottom-up processing, and signalling changes in temporally unfolding environmental features. In the last decade, several studies have examined how the brain codes for and responds to input uncertainty. Initial neurobiological experiments implicated frontoparietal and hippocampal systems, based largely on paradigms that manipulated distributional features of visual stimuli. However, later work in the auditory domain pointed to different systems, whose activation profiles have interesting implications for computational and neurobiological models of statistical learning (SL). This review begins by briefly recapping the historical development of ideas pertaining to the sensitivity to uncertainty in temporally unfolding inputs. It then discusses several issues at the interface of studies of uncertainty and SL. Following, it presents several current treatments of the neurobiology of uncertainty and reviews recent findings that point to principles that serve as important constraints on future neurobiological theories of uncertainty, and relatedly, SL. This review suggests it may be useful to establish closer links between neurobiological research on uncertainty and SL, considering particularly mechanisms sensitive to local and global structure in inputs, the degree of input uncertainty, the complexity of the system generating the input, learning mechanisms that operate on different temporal scales and the use of learnt information for online prediction.This article is part of the themed issue 'New frontiers for statistical learning in the cognitive sciences'.


Results of the multicenter natural interferon-alpha treatment for chronic hepatitis C: correlation between total dose, administration periods and efficacy of therapy. Keio Natural IFN-alpha study group.
The efficacy of different doses and administration periods of interferon (IFN)-alpha treatment for patients with chronic hepatitis C was retrospectively evaluated by using the return of serum alanine aminotransferase (ALT) to normal levels 12 months after therapy as an index. Two hundred forty-one patients with chronic hepatitis C received 6 to 9 mega units of IFN-alpha once, twice, or three times per week for a different number of weeks. ALT levels generally returned to normal in 77 of the 241 patients (32.0%). The efficacy of IFN-alpha therapy with a total dose of 350 mega units or more was significantly superior to that with 350 mega units or less (30 to 45%, p < 0.05). However, the efficacy was the same if a total dose was more than 350 mega units, and more than 700 mega units did not improve the response rate. Administration periods of more than 6 months and less than 1 year appeared to be the most effective in this study. To achieve a complete response, administration three times a week was better than once a week. The higher the dose administered within a certain period, the higher was the efficacy of the IFN-alpha treatment. However, the factors affecting response must be further studied, because there are patients who respond to therapy with a total of only about 200 mega units, and patients who do not respond even to 800 mega units given for more than 1 year.


Probing key elements of teixobactin-lipid II interactions in membranes.
Teixobactin (Txb) is a recently discovered antibiotic against Gram-positive bacteria that induces no detectable resistance. The bactericidal mechanism is believed to be the inhibition of cell wall biosynthesis by Txb binding to lipid II and lipid III. Txb binding specificity likely arises from targeting of the shared lipid component, the pyrophosphate moiety. Despite synthesis and functional assessment of numerous chemical analogs of Txb, and consequent identification of the Txb pharmacophore, the detailed structural information of Txb-substrate binding is still lacking. Here, we use molecular modeling and microsecond-scale molecular dynamics simulations to capture the formation of Txb-lipid II complexes at a membrane surface. Two dominant binding conformations were observed, both showing characteristic lipid II phosphate binding by the Txb backbone amides near the C-terminal cyclodepsipeptide (d-Thr8-Ile11) ring. Additionally, binding by Txb also involved the side chain hydroxyl group of Ser7, as well as a secondary phosphate binding provided by the side chain of l-allo-enduracididine. Interestingly, those conformations differ by swapping two groups of hydrogen bond donors that coordinate the two phosphate moieties of lipid II, resulting in opposite orientations of lipid II binding. In addition, residues d-allo-Ile5 and Ile6 serve as the membrane anchors in both Txb conformations, regardless of the detailed phosphate binding interactions near the cyclodepsipeptide ring. The role of hydrophobic residues in Txb activity is primarily for its membrane insertion, and subsidiarily to provide non-polar interactions with the lipid II tail. Based on the Txb-lipid II interactions captured in their complexes, as well as their partitioning depths into the membrane, we propose that the bactericidal mechanism of Txb is to arrest cell wall synthesis by selectively inhibiting the transglycosylation of peptidoglycan, while possibly leaving the transpeptidation step unaffected. The observed "pyrophosphate caging" mechanism of lipid II inhibition appears to be similar to some lantibiotics, but different from that of vancomycin or bacitracin.


IRF5 guides monocytes toward an inflammatory CD11c+ macrophage phenotype and promotes intestinal inflammation.
Mononuclear phagocytes (MNPs) are vital for maintaining intestinal homeostasis but, in response to acute microbial stimulation, can also trigger immunopathology, accelerating recruitment of Ly6Chi monocytes to the gut. The regulators that control monocyte tissue adaptation in the gut remain poorly understood. Interferon regulatory factor 5 (IRF5) is a transcription factor previously shown to play a key role in maintaining the inflammatory phenotype of macrophages. Here, we investigate the impact of IRF5 on the MNP system and physiology of the gut at homeostasis and during inflammation. We demonstrate that IRF5 deficiency has a limited impact on colon physiology at steady state but ameliorates immunopathology during Helicobacter hepaticus-induced colitis. Inhibition of IRF5 activity in MNPs phenocopies global IRF5 deficiency. Using a combination of bone marrow chimera and single-cell RNA-sequencing approaches, we examined the intrinsic role of IRF5 in controlling colonic MNP development. We demonstrate that IRF5 promotes differentiation of Ly6Chi monocytes into CD11c+ macrophages and controls the production of antimicrobial and inflammatory mediators by these cells. Thus, we identify IRF5 as a key transcriptional regulator of the colonic MNP system during intestinal inflammation.


Kidney transplantation from organ donors following cardiopulmonary death using extracorporeal membrane oxygenation support.
Extracorporeal membrane oxygenation (ECMO) has the ability to provide normal organ perfusion and oxygenation in the absence of cardiac function and thus has the potential to improve viability of subsequently transplanted kidneys. In addition, ECMO support allows the donation following cardiopulmonary death (DCD) process to occur in a controlled manner that is acceptable to staff unfamiliar with DCD. In 1999 our center implemented a controlled DCD program that incorporates post-mortem ECMO support of the organs. Arterial and venous cannulae are placed following consent to donate, but prior to withdrawal of support. ECMO circulation is initiated immediately following declaration of death. Following a brief period where the donor family is allowed to grieve, the donor is moved to the operating room where organ recovery occurs. We reviewed the results of 20 kidney transplants from 13 ECMO supported donors that occurred between October 2000 and August 2003. One renal allograft was lost due to surgical complications, all 19 remaining grafts functioned. An 11% (2/19) delayed graft function rate was observed. Kidneys donated from "controlled" ECMO supported CPD donors are a viable source of organs for renal transplantation. This recovery method warrants further investigation.


Telling the truth about antibiotics: benefits, harms and moral duty in prescribing for children in primary care.
Antimicrobial resistance represents a growing threat to global health, yet antibiotics are frequently prescribed in primary care for acute childhood illness, where there is evidence of very limited clinical effectiveness. Moral philosophy supports the need for doctors to consider wider society, including future patients, when treating present individuals, and it is clearly wrong to waste antibiotics in situations where they are largely clinically ineffective at the expense of future generations. Doctors should feel confident in applying principles of antibiotic stewardship when treating children in primary care, but they must explain these to parents. Provision of accurate, accessible information about the benefits and harms of antibiotics is key to an ethical approach to antimicrobial stewardship and to supporting shared decision making. Openness and honesty about drivers for antibiotic requests and prescribing may further allow parents to have their concerns heard and help clinicians to develop with them an understanding of shared goals.


SNRPN methylation patterns in germ cell tumors as a reflection of primordial germ cell development.
Studies examining altered imprinted gene expression in cancer compare the observed expression pattern to the normal expression pattern for a given tissue of origin, usually the somatic expression pattern for the imprinted gene. Germ cell tumors (GCTs), however, require a developmental stage-dependent comparison. To explore using methylation as an indicator of germ cell development, we determined the pattern of methylation at the 5' untranslated region of SNRPN in 89 GCTs from both children and adults. Fifty-one of 84 tumors (60.7%) (12/30 (40%) of cultured pediatric GCTs, 23/36 (63.9%) of frozen adult GCTs, and 16/23 (69.5%) of frozen pediatric GCTs, with five samples having results from both cultured and uncultured material) demonstrated a nonsomatic methylation pattern after dual digestion with XbaI, NotI, and Southern blot analysis. In contrast, only 2 of 18 (11%) control samples (16 non-GCTs and 2 normal ovaries) exhibited a nonsomatic pattern. In both cases, the result was shown to be due to copy number differences between maternal and paternal homologs, unlike the GCTs in which there was no evidence of an uneven homolog number. A comparison of the data for only the gonadal GCTs and the control data showed a highly significant difference in the proportion of tumors with methylation alterations at this locus (P = 0.0000539). Since there is no published evidence of the involvement of SNRPN methylation changes in the development of malignancy, the data suggest that the methylation pattern of SNRPN in GCTs reflects that of the primordial germ cell giving rise to the tumor.