Potassium currents responsible for inward and outward rectification in rat neostriatal spiny projection neurons.
Many of the nonlinear membrane properties displayed by neostriatal spiny projection neurons are conferred by their voltage-gated potassium (K+) currents, including an inwardly rectifying current (IKir), fast (IAt), and slowly (IAs)-inactivating A-currents, and a slow, noninactivating current. The relative contribution of these K+ currents to the pronounced inward and outward rectification of the current-voltage (I-V) relationship of spiny neurons was investigated in a neostriatal slice preparation. Manipulation of the equilibrium potential for K+ (EK) showed that the voltage dependence of activation of inward rectification was identical to that of IKir. In addition, application of barium (100 microM), which is known to reduce IKir in a time- and voltage-dependent manner, had equivalent effects on inward rectification. Subsequent application of cesium (3 mM) or tetraethylammonium (TEA, 25 mM) blocked inward rectification in a solely voltage-dependent fashion consistent with the action of these blockers on IKir. Administration of 4-aminopyridine (4-AP, 100 microM) at concentrations that selectively depress IAs, reduced outward rectification of spiny neurons at subthreshold membrane potentials. Higher concentrations of 4-AP (2 mM), which block both IAs and IAt, revealed an early transient overshoot in voltage deflections at potentials near spike threshold, but rectification persisted at the end of the responses. The transient overshoot and the residual rectification were eliminated by TEA (25 mM), a blocker of the slow, noninactivating K+ current. Collectively, these results indicate that all three depolarization-activated K+ currents contribute to outward rectification at different times and membrane potentials defined by their voltage dependence of activation and kinetics of inactivation. The spontaneous activity of neostriatal spiny neurons recorded in intact animals is characterized by sustained and limited shifts in membrane potential from relatively hyperpolarized potentials to depolarized potentials near spike threshold. The present data suggest that the hyperpolarized state is determined principally by IKir and the limits on the depolarized state are defined by IAf, IAs, and the noninactivating current. These outward K+ currents also are hypothesized to govern the spike discharge characteristics once the depolarized state has been reached.


A systematic review of physical activity and quality of life and well-being.
Maintaining or improving quality of life (QoL) and well-being is a universal goal across the lifespan. Being physically active has been suggested as one way to enhance QoL and well-being. In this systematic review, conducted in part for the 2018 U.S. Health and Human Services Physical Activity Guidelines for Americans Scientific Advisory Committee Report, we examined the relationship between physical activity (PA) and QoL and well-being experienced by the general population across the lifespan and by persons with psychiatric and neurologic conditions. Systematic reviews, meta-analyses, and pooled analyses from 2006 to 2018 were used for the evidence base. Strong evidence (predominantly from randomized controlled trials [RCTs]) demonstrated that, for adults aged 18-65 years and older adults (primarily 65 years and older), PA improves QoL and well-being when compared with minimal or no-treatment controls. Moderate evidence indicated that PA improves QoL and well-being in individuals with schizophrenia and Parkinson's disease, and limited evidence indicated that PA improves QoL and well-being for youth and for adults with major clinical depression or bipolar disorder. Insufficient evidence existed for individuals with dementia because of a small number of studies with mixed results. Future high-quality research designs should include RCTs involving longer interventions testing different modes and intensities of PA in diverse populations of healthy people and individuals with cognitive (e.g., dementia) and mental health conditions (e.g., schizophrenia) to precisely characterize the effects of different forms of PA on aspects of QoL and well-being.


Reversible dimerization of acid-denatured ACBP controlled by helix A4.
The peptide segment corresponding to helix A4 in acyl-coenzyme-A-binding protein (ACBP) is an exceptionally stable helix in the denatured state of the protein as well as in its isolated form. Circular dichroism spectroscopy showed an alpha-helix content in the helix A4 peptide (HA4) of 45%, and under denaturing conditions at pH 2.3, helix conformations are still populated in 24% of the ensemble of molecules. The structure of HA4 at atomic resolution was assessed using nuclear magnetic resonance (NMR) spectroscopy. Long-range NOEs between remote residues at opposite peptide ends suggested the formation of an antiparallel homodimer, and the resulting structure was treated as the minimum higher-order structure. The dimerization property of helix A4 is maintained in the full-length protein under denaturing conditions. NMR diffusion studies and concentration-dependent experiments on ACBP at low pH proved the formation of dimers and revealed a cooperative stabilization of helix A4 in this process. This emphasizes its special role in the structure formation in the denatured state of ACBP. No dimers are formed in the presence of guanidine hydrochloride, which underlines the fundamental difference between the nature of these two denatured states.


Ovarian response to stimulation for fertility preservation in women with malignant disease: a systematic review and meta-analysis.
To evaluate the current available data regarding ovarian performance of patients diagnosed with malignant disease undergoing controlled ovarian hyperstimulation (COH) for fertility preservation, before radio/chemotherapy, compared with age-matched, healthy patients undergoing COH for in vitro fertilization/intracytoplasmic sperm injection (IVF-ICSI). Meta-analysis of the data available from a systematic review of the literature. Academic centers of infertility and IVF. Patients with malignant disease, before radio/chemotherapy, undergoing COH for fertility preservation within comparative studies with healthy, age-matched controls. None. Peak estradiol levels on day of human chorionic gonadotropin administration, number of oocytes retrieved, fertilization rate, incidence of low ovarian response, and cycle cancellation. Only seven retrospective, case-controlled studies were found to match our objective. Overall, the results of the meta-analysis indicate that the number of retrieved oocytes rate was statistically significantly lower compared with age-matched healthy IVF patients. The incidence of poor ovarian performance and risk of cycle cancellation as well as the calculated number of two pronuclei zygotes achieved among patients with cancer were comparable with their age-matched controls. Women with malignant disease should expect a lower number of oocytes retrieved after COH for fertility preservation, compared with healthy, age-matched patients. Presently, there is paucity of evidence to assess the effect of a specific malignant disease on ovarian response to COH before IVF for fertility preservation. Multicentric studies should be conducted to resolve these important issues.


East African cassava mosaic-like viruses from Africa to Indian ocean islands: molecular diversity, evolutionary history and geographical dissemination of a bipartite begomovirus.
Cassava (Manihot esculenta) is a major food source for over 200 million sub-Saharan Africans. Unfortunately, its cultivation is severely hampered by cassava mosaic disease (CMD). Caused by a complex of bipartite cassava mosaic geminiviruses (CMG) species (Family: Geminivirideae; Genus: Begomovirus) CMD has been widely described throughout Africa and it is apparent that CMG's are expanding their geographical distribution. Determining where and when CMG movements have occurred could help curtail its spread and reveal the ecological and anthropic factors associated with similar viral invasions. We applied Bayesian phylogeographic inference and recombination analyses to available and newly described CMG sequences to reconstruct a plausible history of CMG diversification and migration between Africa and South West Indian Ocean (SWIO) islands. The isolation and analysis of 114 DNA-A and 41 DNA-B sequences demonstrated the presence of three CMG species circulating in the Comoros and Seychelles archipelagos (East African cassava mosaic virus, EACMV; East African cassava mosaic Kenya virus, EACMKV; and East African cassava mosaic Cameroon virus, EACMCV). Phylogeographic analyses suggest that CMG's presence on these SWIO islands is probably the result of at least four independent introduction events from mainland Africa occurring between 1988 and 2009. Amongst the islands of the Comoros archipelago, two major migration pathways were inferred: One from Grande Comore to Mohéli and the second from Mayotte to Anjouan. While only two recombination events characteristic of SWIO islands isolates were identified, numerous re-assortments events were detected between EACMV and EACMKV, which seem to almost freely interchange their genome components. Rapid and extensive virus spread within the SWIO islands was demonstrated for three CMG complex species. Strong evolutionary or ecological interaction between CMG species may explain both their propensity to exchange components and the absence of recombination with non-CMG begomoviruses. Our results suggest an important role of anthropic factors in CMGs spread as the principal axes of viral migration correspond with major routes of human movement and commercial trade. Finer-scale temporal analyses of CMGs to precisely scale the relative contributions of human and insect transmission to their movement dynamics will require further extensive sampling in the SWIO region.


MeDIP-seq and nCpG analyses illuminate sexually dimorphic methylation of gonadal development genes with high historic methylation in turtle hatchlings with temperature-dependent sex determination.
DNA methylation alters gene expression but not DNA sequence and mediates some cases of phenotypic plasticity. Temperature-dependent sex determination (TSD) epitomizes phenotypic plasticity where environmental temperature drives embryonic sexual fate, as occurs commonly in turtles. Importantly, the temperature-specific transcription of two genes underlying gonadal differentiation is known to be induced by differential methylation in TSD fish, turtle and alligator. Yet, how extensive is the link between DNA methylation and TSD remains unclear. Here we test for broad differences in genome-wide DNA methylation between male and female hatchling gonads of the TSD painted turtle Chrysemys picta using methyl DNA immunoprecipitation sequencing, to identify differentially methylated candidates for future study. We also examine the genome-wide nCpG distribution (which affects DNA methylation) in painted turtles and test for historic methylation in genes regulating vertebrate gonadogenesis. Turtle global methylation was consistent with other vertebrates (57% of the genome, 78% of all CpG dinucleotides). Numerous genes predicted to regulate turtle gonadogenesis exhibited sex-specific methylation and were proximal to methylated repeats. nCpG distribution predicted actual turtle DNA methylation and was bimodal in gene promoters (as other vertebrates) and introns (unlike other vertebrates). Differentially methylated genes, including regulators of sexual development, had lower nCpG content indicative of higher historic methylation. Ours is the first evidence suggesting that sexually dimorphic DNA methylation is pervasive in turtle gonads (perhaps mediated by repeat methylation) and that it targets numerous regulators of gonadal development, consistent with the hypothesis that it may regulate thermosensitive transcription in TSD vertebrates. However, further research during embryogenesis will help test this hypothesis and the alternative that instead, most differential methylation observed in hatchlings is the by-product of sexual differentiation and not its cause.


Synthesis and characterization of UO(2)(2+)-ion imprinted polymer for selective extraction of UO(2)(2+).
Ion-imprinted polymers (IIPs) were prepared for uranyl ion (imprint ion) by formation of binary (salicylaldoxime (SALO) or 4-vinylpyridine (VP)) or ternary (salicylaldoxime and 4-vinylpyridine) complex in 2-methoxy ethanol (porogen) following copolymerization with methacrylic acid (MAA) as a functional monomer and ethylene glycol dimethacrylate (EGDMA) as crosslinking monomer using 2,2'-azobisisobutyronitrile as initiator. Control polymers (CPs) were also prepared under identical experimental conditions without using imprint ion. The above synthesized polymers were characterized by surface area measurement, microanalysis and FT-IR analysis techniques. The imprinted polymer formed with ternary complex of UO(2)(2+)-SALO-VP (1:2:2, IIP3) showed quantitative enrichment of uranyl ion from dilute aqueous solution and hence was chosen for detailed studies. The optimal pH for quantitative enrichment is 3.5-6.5. The adsorbed UO(2)(2+) was completely eluted with 10 mL of 1.0 M HCl. The retention capacity of IIP3 was found to be 0.559 mmol g(-1). Further, the distribution ratio and selectivity coefficients of uranium and other selected inorganic ions were also evaluated. Five replicate determinations of 25 microg L(-1) of uranium(VI) gave a mean absorbance of 0.032 with a relative standard deviation of 2.20%. The detection limit corresponding to three times the standard deviation of the blank was found to be 5 microg L(-1). IIP3 was tested for preconcentration of uranium(VI) from ground, river and sea water samples.


ERCP and endoscopic sphincterotomy (ES): a safe and definitive management of gallstone pancreatitis with the gallbladder left in situ.
UK guidelines recommend that patients with gallstone pancreatitis have cholecystectomy within 2 weeks of their pancreatitis. A proportion of these are elderly with significant comorbidities rendering them high risk for general anaesthesia and surgery. Endoscopic retrograde cholangiopancreatography (ERCP) and endoscopic sphincterotomy (ES) may offer a safe alternative to cholecystectomy as definitive treatment in these patients. A retrospective review of all cases of gallstone pancreatitis presenting between 1999 and 2009 was undertaken. One hundred one patients underwent ERCP and ES as a definitive treatment for gallstone pancreatitis with a median age of 78 years (range, 43-96 years) and a median American Society of Anesthesiologists grade of 2. Three patients died from pancreatitis despite successful ERCP. Eighty-nine patients were successfully treated with an ERCP alone, and 84 patients (94%) had no recurrence of pancreatitis with a mean follow-up of 41 months (±32 months, range 4-118 months). The total patient follow-up was 3,260 months. Twenty-seven patients (33%) died within the follow-up period of unrelated causes, explaining the lower than expected median follow-up. Five patients had a recurrence of pancreatitis during follow-up (6%). ERCP with ES is a safe alternative to laparoscopic cholecystectomy to prevent further attacks of gallstone pancreatitis in high-risk surgical patients and the elderly.


Hydrodynamic Gene Delivery of CC Chemokine Binding Fc Fusion Proteins to Target Acute Vascular Inflammation In Vivo.
Blockade of CC chemokines is an attractive yet under utilized therapeutic strategy. We report the in vivo pharmacokinetics of a broad-spectrum vaccinia virus CC chemokine binding protein (35 K) fused to human IgG1 Fc. We demonstrate that the in vivo efficacy of the protein can be interrogated using hydrodynamic gene delivery of a standard mammalian expression plasmid. High plasma levels of the 35 K-Fc protein are maintained for at least 14 days post gene transfer, with the protein still detectable at 5 weeks. We confirm that the protein has biological activity in acute inflammation, causing a significant reduction in monocyte recruitment during zymosan induced peritonitis. The ability of 35 K-Fc to block more complex pathologies is demonstrated using aortic digests to assess angiotensin II mediated leukocyte recruitment to the aorta. Angiotensin II causes upregulation of mCCL2 in the aorta causing the accumulation of CCR2+ cells. Peak monocyte recruitment to the aorta occurs within 3 days and this process is CC chemokine dependent, being significantly reduced by hydrodynamic delivery of 35 K-Fc.


Lost in translation: "Risks," "needs," and "evidence" in implementing the First Step Act.
In this article, we focus on two highly problematic issues in the manner in which the First Step Act of 2018 is being implemented by the Bureau of Prisons: an uncritical separation of "dynamic risks" and "criminogenic needs"; and a spurious reliance on "evidence-based" interventions to reduce recidivism risk. We argue that if the Act is to live up to its promise of being a game-changing development in efforts to reduce crime while simultaneously shrinking mass incarceration, "needs assessment" must be subject to vastly increased empirical attention, variable and causal risk factors must be identified and validly assessed, and interventions to reduce risk must be rigorously evaluated both for their fidelity of implementation and impact on recidivism. Rather than further proliferating programs that ostensibly reduce risk, we believe that serious consideration should be given to the Bureau of Prisons offering one signature, well-established cognitive-behavioral program that can simultaneously address multiple risk factors for moderate and high-risk prisoners.


Molecular physiology of cation-coupled Cl- cotransport: the SLC12 family.
The electroneutral cation-chloride-coupled cotransporter gene family ( SLC12) was identified initially at the molecular level in fish and then in mammals. This nine-member gene family encompasses two major branches, one including two bumetanide-sensitive Na(+)-K(+)-2Cl(-) cotransporters and the thiazide-sensitive Na(+):Cl(-) cotransporter. Two of the genes in this branch ( SLC12A1 and SLC12A3), exhibit kidney-specific expression and function in renal salt reabsorption, whereas the third gene ( SLC12A2) is expressed ubiquitously and plays a key role in epithelial salt secretion and cell volume regulation. The functional characterization of both alternatively-spliced mammalian Na(+)-K(+)-2Cl(-) cotransporter isoforms and orthologs from distantly related species has generated important structure-function data. The second branch includes four genes ( SLC12A4- 7) encoding electroneutral K(+)-Cl(-) cotransporters. The relative expression level of the neuron-specific SLC12A5 and the Na(+)-K(+)-2Cl(-) cotransporter SLC12A2 appears to determine whether neurons respond to GABA with a depolarizing, excitatory response or with a hyperpolarizing, inhibitory response. The four K(+)-Cl(-) cotransporter genes are co-expressed to varying degrees in most tissues, with further roles in cell volume regulation, transepithelial salt transport, hearing, and function of the peripheral nervous system. The transported substrates of the remaining two SLC12 family members, SLC12A8 and SLC12A9, are as yet unknown. Inactivating mutations in three members of the SLC12 gene family result in Mendelian disease; Bartter syndrome type I in the case of SLC12A1, Gitelman syndrome for SLC12A3, and peripheral neuropathy in the case of SLC12A6. In addition, knockout mice for many members of this family have generated important new information regarding their respective physiological roles.


Our plastic age.
Within the last few decades, plastics have revolutionized our daily lives. Globally we use in excess of 260 million tonnes of plastic per annum, accounting for approximately 8 per cent of world oil production. In this Theme Issue of Philosophical Transactions of the Royal Society, we describe current and future trends in usage, together with the many benefits that plastics bring to society. At the same time, we examine the environmental consequences resulting from the accumulation of waste plastic, the effects of plastic debris on wildlife and concerns for human health that arise from the production, usage and disposal of plastics. Finally, we consider some possible solutions to these problems together with the research and policy priorities necessary for their implementation.


Waterdock 2.0: Water placement prediction for Holo-structures with a pymol plugin.
Water is often found to mediate interactions between a ligand and a protein. It can play a significant role in orientating the ligand within a binding pocket and contribute to the free energy of binding. It would thus be extremely useful to be able to accurately predict the position and orientation of water molecules within a binding pocket. Recently, we developed the WaterDock protocol that was able to predict 97% of the water molecules in a test set. However, this approach generated false positives at a rate of over 20% in most cases and whilst this might be acceptable for some applications, in high throughput scenarios this is not desirable. Here we tackle this problem via the inclusion of knowledge regarding the solvation structure of ligand functional groups. We call this new protocol WaterDock2 and demonstrate that this protocol maintains a similar true positive rate to the original implementation but is capable of reducing the false-positive rate by over 50%. To improve the usability of the method, we have also developed a plugin for the popular graphics program PyMOL. The plugin also contains an implementation of the original WaterDock.


Energy metabolism in human renin-gene transgenic rats: does renin contribute to obesity?
Renin initiates angiotensin II formation and has no other known functions. We observed that transgenic rats (TGR) overexpressing the human renin gene (hREN) developed moderate obesity with increased body fat mass and glucose intolerance compared with nontransgenic Sprague-Dawley (SD) rats. The metabolic changes were not reversed by an angiotensin-converting enzyme inhibitor, a direct renin inhibitor, or by (pro)renin receptor blocker treatment. The obese phenotype in TGR(hREN) originated from higher food intake, which was partly compensated by increases in resting energy expenditure, total thermogenesis (postprandial and exercise activity), and lipid oxidation during the first 8 weeks of life. Once established, the difference in body weight between TGR(hREN) and SD rats remained constant over time. When restricted to the caloric intake of SD, TGR(hREN) developed an even lower body weight than nontransgenic controls. We did not observe significant changes in the cocaine and amphetamine-regulated transcript, pro-opiomelanocortin, both anorexigenic, or neuropeptide Y, orexigenic, mRNA levels in TGR(hREN) versus SD controls. However, the mRNA level of the agouti-related peptide, orexigenic, was significantly reduced in TGR(hREN) versus SD controls at the end of the study, which indicates a compensatory mechanism. We suggest that the human renin transgene initiates a process leading to increased and early appetite, obesity, and metabolic changes not related to angiotensin II. The mechanisms are independent of any currently known renin-related effects.


Habitat-islands in the coastal Atacama Desert: loss of functional redundancy, but not of functional diversity, with decreased precipitation.
Aridity is increasing in many regions of the world, but microclimatic conditions may buffer plant communities from the direct effects of decreased precipitation, creating habitat islands. However, reduced precipitation can also impact these communities indirectly by decreasing the suitability of the surrounding habitat, thus limiting incoming propagules and increasing the chances of population decline and species loss. We test whether decreased precipitation results in loss of species and functional diversity within habitat islands, evaluating in particular whether declines in species diversity and abundance are less likely to result in loss of functional diversity if species/individual loss is stochastic (i.e. independent of species/individual traits) and communities/populations are functionally redundant. Lomas communities are discrete plant communities embedded in the Atacama Desert, maintained by the microclimatic conditions created by fog. We recorded species and functional diversity in six Lomas communities along a 500 km long precipitation gradient in northern Chile. Functional traits were measured in 20 individuals per species, in those species that accounted for approx. 75 % of the abundance at each site. We calculated functional diversity and functional redundancy of the community, and intraspecific functional variation. Decreased precipitation was associated with lower species diversity and lower species abundances. However, no traits or functional strategies increased or decreased consistently with precipitation, suggesting stochastic species/individual loss. Species with stress-tolerant strategies were predominant in all sites. Although species diversity decreased with decreasing precipitation, functional diversity remained unchanged. Lower functional redundancy in the drier sites suggests that mainly functionally redundant species were lost. Likewise, intraspecific functional variation was similar among communities, despite the lower species abundance in drier sites. Decreased precipitation can impact habitat island communities indirectly by decreasing the suitability of the surrounding habitat. Our results support the idea that a stochastic loss of species/individuals from functionally redundant communities and populations does not result in loss of functional diversity.


Fetal exposure to chlordane and permethrin mixtures in relation to inflammatory cytokines and birth outcomes.
We sought to characterize the relationships between cord serum concentrations of chlordane and permethrin pesticides, inflammatory cytokines, gestational age, and size at birth. Umbilical cord serum levels of trans-nonachlor, oxychlordane, cis- and trans-permethrin, piperonyl butoxide, and cytokines (TNF-α, IFN-γ, IL-1β, IL-2, IL-6, IL-8, IL-10, IL-12p70, GMCSF) were quantified in 300 newborns at the Johns Hopkins Hospital in Baltimore, MD (2004-2005). Principal component analyses were used to quantitate chlordane and permethrin mixtures and to identify independent cytokine components. Five cytokine components described 87% of the variance in cord serum cytokine levels; these (and predominant loadings) were as follows: (1) all 9 cytokines; (2) acute phase (IL-1β, IL-6); (3) anti-inflammatory (IL-10); (4) TNF-α; and (5) IL-1β. Of these, the TNF-α component was significantly associated with a 2-day decrease in gestational age. Chlordane was associated with lower levels of the pro-inflammatory IL-1β [β: -0.11 (-0.20, -0.02)]. Permethrin was negatively associated with the anti-inflammatory cytokine IL-10 [β: -0.14 (-0.22, -0.05)]. Neither pesticides nor cytokines were significantly associated with birthweight, length, or head circumference, and pesticides were not associated with gestational age. Our findings suggest that chlordane and permethrin concentrations in cord blood may be associated with levels of inflammatory cytokines in the fetus.


Hemoperitoneum in peripartum: A case-series.
Hemorrhages are the first cause of perinatal deaths in French women. Thirteen percent of these deaths are not linked to obstetrical problems but rather to hemoperitoneum. These incidents are under-diagnosed and as a result, treatment is delayed and fetal and maternal mortality increases. We report three cases of patients, all White female in their last trimester of a non-problematic pregnancy presenting with hemoperitoneum and resulting in different outcomes. The analysis of published materials and of our cases leads us to infer that a diagnosis of hemoperitoneum must be considered in pregnant women when abdominal pain, symptoms of shock and a decrease in hemoglobin are associated. An immediate response and intensive care followed by hemostatic surgery give these patients the best chance to survive.


Development of an amperometric-based glucose biosensor to measure the glucose content of fruit.
An amperometric enzyme-electrode was introduced where glucose oxidase (GOD) was immobilized on chitosan membrane via crosslinking, and then fastened on a platinum working electrode. The immobilized enzyme showed relatively high retention activity. The activity of the immobilized enzyme was influenced by its loading, being suppressed when more than 0.6 mg enzyme was used in the immobilization. The biosensor showing the highest response to glucose utilized 0.21 ml/cm2 thick chitosan membrane. The optimum experimental conditions for the biosensors in analysing glucose dissolved in 0.1 M phosphate buffer (pH 6.0) were found to be 35°C and 0.6 V applied potential. The introduced biosensor reached a steady-state current at 60 s. The apparent Michaelis-Menten constant ([Formula: see text]) of the biosensor was 14.2350 mM, and its detection limit was 0.05 mM at s/n > 3, determined experimentally. The RSD of repeatability and reproducibility of the biosensor were 2.30% and 3.70%, respectively. The biosensor was showed good stability; it retained ~36% of initial activity after two months of investigation. The performance of the biosensors was evaluated by determining the glucose content in fruit homogenates. Their accuracy was compared to that of a commercial glucose assay kit. There was no significance different between two methods, indicating the introduced biosensor is reliable.


A method to preserve sputum for delayed examination.
Examination of sputum cell counts is limited by the need to process samples within hours of expectoration. The validity and repeatability of a method to preserve sputum for delayed processing and examination were investigated in this study. Portions of selected sputum from 39 subjects were dispersed with dithiothreitol (routine method), or were fixed in a dithiothreitol-formaldehyde mixture for 48-72 h before dispersal with trypsin (preservation method). Total and differential cell counts using the two methods were compared; within-method repeatability of the preservation method was also examined. The intraclass correlation coefficient (ICC) for total cell counts and percentage of eosinophils, neutrophils and macrophages in sputum processed by the two methods was 0.89, 0.86, 0.91 and 0.90, respectively. Within-method repeatability (ICC) of the preservation method for the same cellular indices was 0.99, 0.94, 0.97 and 0.97, respectively. The interobserver repeatability for eosinophils, neutrophils and macrophages was 0.96, 0.97 and 0.97 using the preservation method, and 0.96, 0.99 and 0.99 using the routine method, respectively. This method of sputum preservation and dispersal is valid, reliable and convenient, and may be used for delayed processing and examination.


Shape-controlled synthesis of silver nanocrystals by X-ray irradiation for inkjet printing.
A suite of silver (Ag) nanocrystals have been synthesized using a rapid water radiolysis approach via X-ray irradiation. Various shapes including spheroidal, prism, rod, and multifaceted nanoparticles can be produced by varying the initial concentration of polyvinylpyrrolidone (PVP) relative to silver nitrate (AgNO₃). UV-visible spectroscopy and transmission electron microscopy (TEM) coupled with selected area electron diffraction (SAED) have been used to characterize these Ag products. At an optimized reagent ratio, a mixture of high-aspect-ratio rods (tunable to ∼50) and spheroidal particles result. Such a mixture is proven to have highly beneficial melting point and dispersive properties suited to inkjet printing of conductive Ag lines. The resistivity of the printed lines decreases to 77.7 μΩ and 33.1 μΩ after heating to 200 and 350 °C.


Nuclear actin and myosin as control elements in nucleocytoplasmic transport.
Fluorescence redistribution after photobleaching (FRAP) was used to examine the role of actin and myosin in the transport of dextrans through the nuclear pore complex. Anti-actin antibodies added to isolated rat liver nuclei significantly reduced the flux rate of fluorescently labeled 64-kD dextrans. The addition of 3 mM ATP to nuclei, which enhances the flux rate in control nuclei by approximately 250%, had no enhancement effect in the presence of either anti-actin or anti-myosin antibody. Phalloidin (10 microM) and cytochalasin D (1 micrograms/ml) individually inhibited the ATP stimulation of transport. Rabbit serum, anti-fibronectin, and anti-lamins A and C antibodies had no effect on transport. These results suggest a model for nuclear transport in which actin/myosin are involved in an ATP-dependent process that alters the effective transport rate across the nuclear pore complex.


Botulinum toxin to improve facial wound healing: A prospective, blinded, placebo-controlled study.
To test whether botulinum toxin-induced immobillzation of facial lacerations enhances wound healing and results in less noticeable scars. In this blinded, prospective, randomized clinical trial, patients were randomized from February 1, 2002, until January 1, 2004, to botullnum toxin vs placebo injection into the musculature adjacent to the wound within 24 hours after wound closure. Blinded assessment of standardized photographs by experienced facial plastic surgeons using a 10-cm visual analog scale served as the main outcome measure. Thirty-one patients presenting with traumatic forehead lacerations or undergoing elective excisions of forehead masses were included in the study. The overall median visual analog scale score for the botulinum toxin-treated group was 8.9 compared with 7.2 for the placebo group (P=.003), indicating enhanced healing and Improved cosmesis of the experimentally immobilized scars. Botullnum toxin-induced Immobilization of forehead wounds enhances healing and is suggested for use in selected patients to improve the eventual appearance of the scar.


Plasmon-induced optical switching of electrical conductivity in porous anodic aluminum oxide films encapsulated with silver nanoparticle arrays.
We report on plasmon induced optical switching of electrical conductivity in two-dimensional (2D) arrays of silver (Ag) nanoparticles encapsulated inside nanochannels of porous anodic aluminum oxide (AAO) films. The reversible switching of photoconductivity greatly enhanced by an array of closely spaced Ag nanoparticles which are isolated from each other and from the ambient by thin aluminum oxide barrier layers are attributed to the improved electron transport due to the localized surface plasmon resonance and coupling among Ag nanoparticles. The photoconductivity is proportional to the power, and strongly dependent on the wavelength of light illumination. With Ag nanoparticles being isolated from the ambient environments by a thin layer of aluminum oxide barrier layer of controlled thickness in nanometers to tens of nanometers, deterioration of silver nanoparticles caused by environments is minimized. The electrochemically fabricated nanostructured Ag/AAO is inexpensive and promising for applications to integrated plasmonic circuits and sensors.


Molecular evolution of dengue viruses: contributions of phylogenetics to understanding the history and epidemiology of the preeminent arboviral disease.
Dengue viruses (DENV) are the most important arboviral pathogens in tropical and subtropical regions throughout the world, putting at risk of infection nearly a third of the global human population. Evidence from the historical record suggests a long association between these viruses and humans. The transmission of DENV includes a sylvatic, enzootic cycle between nonhuman primates and arboreal mosquitoes of the genus Aedes, and an urban, endemic/epidemic cycle between Aedes aegypti, a mosquito with larval development in peridomestic water containers, and human reservoir hosts. DENV are members of the genus Flavivirus in the Family Flaviviridae and comprise of 4 antigenically distinct serotypes (DENV-1-4). Although they are nearly identical epidemiologically, the 4 DENV serotypes are genetically quite distinct. Utilization of phylogenetic analyses based on partial and/or complete genomic sequences has elucidated the origins, epidemiology (genetic diversity, transmission dynamics and epidemic potential), and the forces that shape DENV molecular evolution (rates of evolution, selection pressures, population sizes, putative recombination and evolutionary constraints) in nature. In this review, we examine how phylogenetics have improved understanding of DENV population dynamics and sizes at various stages of infection and transmission, and how this information may influence pathogenesis and improve our ability to understand and predict DENV emergence.


Probing metal ion binding and conformational properties of the colicin E9 endonuclease by electrospray ionization time-of-flight mass spectrometry.
Nano-electrospray ionization time-of-flight mass spectrometry (ESI-MS) was used to study the conformational consequences of metal ion binding to the colicin E9 endonuclease (E9 DNase) by taking advantage of the unique capability of ESI-MS to allow simultaneous assessment of conformational heterogeneity and metal ion binding. Alterations of charge state distributions on metal ion binding/release were correlated with spectral changes observed in far- and near-UV circular dichroism (CD) and intrinsic tryptophan fluorescence. In addition, hydrogen/deuterium (H/D) exchange experiments were used to probe structural integrity. The present study shows that ESI-MS is sensitive to changes of the thermodynamic stability of E9 DNase as a result of metal ion binding/release in a manner consistent with that deduced from proteolysis and calorimetric experiments. Interestingly, acid-induced release of the metal ion from the E9 DNase causes dramatic conformational instability associated with a loss of fixed tertiary structure, but secondary structure is retained. Furthermore, ESI-MS enabled the direct observation of the noncovalent protein complex of E9 DNase bound to its cognate immunity protein Im9 in the presence and absence of Zn(2+). Gas-phase dissociation experiments of the deuterium-labeled binary and ternary complexes revealed that metal ion binding, not Im9, results in a dramatic exchange protection of E9 DNase in the complex. In addition, our metal ion binding studies and gas-phase dissociation experiments of the ternary E9 DNase-Zn(2+)-Im9 complex have provided further evidence that electrostatic interactions govern the gas phase ion stability.


Island biogeography theory and conservation practice.
The application of island biogeography theory to conservation practice is premature. Theoretically and empirically, a major conclusion of such applications-that refuges should always consist of the largest possible single area-can be incorrect under a variety of biologically feasible conditions. The cost and irreversibility of large-scale conservation programs demand a prudent approach to the application of an insufficiently validated theory.


Reflexology treatment for patients with lower limb amputations and phantom limb pain--an exploratory pilot study.
The objectives of the study were to evaluate the possibility of reflexology being used as a non-invasive form of phantom limb pain relief and of empowering patients to maintain any positive results with self-treatment. Prosthetic Services Centre, Herbert Street, Wolverhampton, West Midlands, England. A same-subject, experimental pilot study, recording the intensity of phantom limb pain in weekly pain diaries over a 30-week period, which was divided into five phases: phase 1 gave a baseline of pain, whilst phase 3 was a resting phase. Phases 2, 4 and 5 provided the reflexology interventions. Ten participants with unilateral lower limb amputations and phantom limb pain were selected from the database at the Prosthetic Centre. REFLEXOLOGY INTERVENTIONS: In phase 2, six weekly reflexology treatments were given, which consisted of: full foot reflexology to the remaining foot and full hand reflexology to the hand of the amputated side of the body. In phase 4, six weekly hand reflexology teaching sessions were carried out; patients copied on their own hands what the therapist did on hers. A hand reflexology booklet gave the sequence of the treatment and was used as a reference. In phase 5, the patients self-treated for 6 weeks at home, using the reference material. Over the 30-week period, there was an improvement in the perception of the presence and the intensity of the phantom limb pain, with a corresponding improvement in the duration of the pain and the affect on the person's lifestyle. The improvement was maintained when the clients self-treated. FOLLOW-UP QUESTIONNAIRE: A follow-up questionnaire was carried out in 2007--12 months after the project had ended--to elicit whether the patients had suffered from phantom pain over the previous 12 months, whether they still had relief from phantom limb pain and whether they still self-treated. The project indicated that reflexology treatment, teaching and self-treatment were effective in eradicating or reducing the intensity and duration of phantom limb pain, in this group of clients. The follow-up questionnaire revealed that there was a maintained improvement in the intensity of phantom limb pain the patients experienced and that the majority still self-treated.


Event coding and motor priming: how attentional modulation may influence binding across action properties.
When we perceive an action it is internally transformed into a motor representation akin to the execution of that same action. Motor priming studies show that action observation facilitates the execution of physically similar actions, but interferes with the performance of dissimilar actions. Some evidence suggests, however, that once a specific motor plan is formed, perceiving an action with partially overlapping features (e.g. a congruent grip-type but and incongruent end-goal) can result in interference. In two experiments we investigate how modulating attention towards observed actions influences the binding that occurs between action features, and therefore the amount of partial-overlap interference to participants' performance. In the first study we directed attention towards a salient action feature (the grip-type). We found that perceiving partially overlapping (i.e. partially congruent) actions slowed participants' responses compared with observation of completely congruent or incongruent actions. In the second experiment attentional resources were taxed through the use of a secondary task. This resulted in the elimination of the partial-overlap interference effect. We discuss results in relation to feature binding and event codes.


Excitation and sensitization of fine articular afferents from cat's knee joint by prostaglandin E2.
1. In cats anaesthetized with alpha-chloralose extracellular recordings were made from fine afferent units belonging to the medial articular nerve of the knee joint. The excitatory and sensitizing effects on articular afferents of prostaglandin E2 (PGE2) applied intra-arterially close to the joint were examined. 2. Bolus injections of PGE2 doses of 0.03-30 micrograms excited about 60% of both the group III (conduction velocity 2.5-20 m/s) and the group IV units (conduction velocity less than 2.5 m/s). The duration and size of the responses were dose dependent consisting in most cases of low-frequency discharges which lasted up to several minutes. Excitation was found among afferents with low and high mechanosensitivity. 3. Among the group III units PGE2 sensitized 64% for their responses to movements and 50% for their responses to bradykinin (applied intra-arterially close to the joint). Sensitization did not depend on the mechanical threshold previous to chemical stimulation. Among the group IV units PGE2 sensitized only 25% for their responses to movements but 75% for their reactions to bradykinin. In group IV fibres a low mechanical threshold predisposed for sensitization to movements and a higher threshold for sensitization to bradykinin. 4. Some units were sensitized and excited, others were either sensitized or excited and some units were not affected by PGE2. We conclude that PGE2 induces in a large proportion of fine articular afferents of normal joints discharges which are similar to those induced by an experimental inflammation. Thus PGE2 may be an inflammatory mediator which has a major role in the generation of the afferent activity developing in the course of an arthritis.


Are there useful physiological or psychological markers for monitoring overload training in elite rowers?
There is a need for markers that would help determine when an athlete's training load is either insufficient or excessive. In this study we examined the relationship between changes in performance and changes in physiological and psychological markers during and following a period of overload training in 10 female and 10 male elite rowers. Change in performance during a 4-wk overload was determined with a weekly 30-min time-trial on a rowing ergometer, whereas an incremental test provided change in lactate-threshold power between the beginning of the study and following a 1-wk taper after the overload. Various psychometric, steroid-hormone, muscle-damage, and inflammatory markers were assayed throughout the overload. Plots of change in performance versus the 4-wk change in each marker were examined for evidence of an inverted-U relationship that would characterize undertraining and excessive training. Linear modeling was also used to estimate the effect of changes in the marker on changes in performance. There was a suggestion of an inverted U only for performance in the incremental test versus some inflammatory markers, due to the relative underperformance of one rower. There were some clear linear relationships between changes in markers and changes in performance, but relationships were inconsistent within classes of markers. For some markers, changes considered to predict excessive training (eg, creatine kinase, several proinflammatory cytokines) had small to large positive linear relationships with performance. In conclusion, some of the markers investigated in this study may be useful for adjusting the training load in individual elite rowers.


Myoblast Phosphoproteomics as a Tool to Investigate Global Signaling Events During Myogenesis.
Protein phosphorylation is a universal covalent chemical modification of amino acids involved in a large number of biological processes including cell signaling, metabolism, proliferation, differentiation, survival/death, ageing, and many more. Regulation of protein phosphorylation is essential in myogenesis and indeed, when the enzymatic activity of protein kinases is distrupted in myoblasts, myogenesis is affected. In this chapter we describe a method to profile the phosphoproteome of myoblasts using mass spectrometry. Phosphate groups are labile and easily lost during the processing of samples for mass spectrometry. Thus, effective methods to enrich for phosphopeptides from protein extracts have been developed. Here, we discuss and present in detail two such methods that we routinely employ. These methods are based on a sample enrichment step performed on titanium dioxide matrices followed by label-free tandem mass spectrometry and semi-quantitation.


In situ detection of individual mRNA molecules and protein complexes or post-translational modifications using padlock probes combined with the in situ proximity ligation assay.
Analysis at the single-cell level is essential for the understanding of cellular responses in heterogeneous cell populations, but it has been difficult to perform because of the strict requirements put on detection methods with regard to selectivity and sensitivity (i.e., owing to the cross-reactivity of probes and limited signal amplification). Here we describe a 1.5-d protocol for enumerating and genotyping mRNA molecules in situ while simultaneously obtaining information on protein interactions or post-translational modifications; this is achieved by combining padlock probes with in situ proximity ligation assays (in situ PLA). In addition, we provide an example of how to design padlock probes and how to optimize staining conditions for fixed cells and tissue sections. Both padlock probes and in situ PLA provide the ability to directly visualize single molecules by standard microscopy in fixed cells or tissue sections, and these methods may thus be valuable for both research and diagnostic purposes.


Harvesting natural populations in a randomly fluctuating environment.
As harvesting effort and yield are increased, animal populations that are being harvested for sustained yield will take longer to recover from environmentally imposed disturbances. One consequence is that the coefficient of variation (the relative variance) of the yield increases as the point of maximum sustained yield (MSY) is approached. When overexploitation has resulted in a population smaller than that for MSY, high effort produces a low average yield with high variance. These observations accord with observed trends in several fish and whaling industries. We expect these effects to be more pronounced for a harvesting strategy based on constant quotas than for one based on constant effort. Although developed in a MSY context, the conclusions also apply if the aim is to maximize the present value of (discounted) net economic revenue.


Acute hyperglycemia and oxidative stress: direct cause and effect?
Oxidative stress is increased in Type 2 diabetes and this appears to underlie the development of diabetic complications. Increased oxidative stress is claimed to be triggered directly by acute (sudden-onset) hyperglycemia, but published data do not clearly support a direct cause and effect relationship. In this article, published evidence of a direct prooxidant effect of acute hyperglycemia is presented and discussed in some detail, and conflicts, controversies, and problems are highlighted. Evidence for glucose variability as a possible important trigger of oxidative stress in diabetes is reviewed, with some speculation as to how the field would be advanced if there were more widespread recognition about the role that wide fluctuations in glucose concentration play in diabetic complications. Possible direct or indirect antioxidative effects of various drugs used in the treatment of diabetic subjects are discussed because these may have influenced current understanding of the link between hyperglycemia and oxidative stress. The aims are to reveal the divergence between the available evidence and the accepted view that acute hyperglycemia is a direct trigger of oxidative stress and to suggest areas of research that will help resolve current controversies in this important and challenging area.


Composite auditory evoked potentials index is not a good indicator of depth of anesthesia in propofol-fentanyl anesthesia: Randomized comparative study.
The composite auditory evoked potentials index (cAAI) was considered a measure of overall balance between noxious stimulation, analgesia, and hypnosis; while bispectral index (BIS) shows only hypnosis, and auditory evoked potentials index (AAI) shows response to stimuli. The present study compared the performance of cAAI, BIS, and AAI in propofol-fentanyl anesthesia. Forty-five patients for abdominal surgery aged 30-65 years with ASA physical status I or II were randomly divided into three groups by an envelope method. Anesthesia was induced with midazolam, propofol, and fentanyl alongwith an epidural block. When hemodynamics were stable during surgery, propofol infusion rate was fixed at 4 mg/kg/h for 10 min, then increased to 6 mg/kg/h and kept it for 10 min. AAI (AEP version 1.4), cAAI (AEP version 1.6), or BIS (A-2000) was monitored in each 15 patients, and the performance of three indices was compared. All three indices decreased significantly before intubation. Only the AAI increased significantly by intubation. During anesthesia except for at propofol 6 mg/kg/h, the cAAI was significantly higher than the AAI. Only the AAI was significantly lower at propofol 6 mg/kg/h than at 4 mg/kg/h. The cAAI had the largest and AAI had the smallest inter-individual variations. The cAAI was higher than the manufacturer's recommended range of general anesthesia. In propofol-fentanyl anesthesia, AAI might be better to discriminate anesthetic depth than cAAI and BIS.


Effects of Helicobacter pylori infection on the link between regenerating gene expression and serum gastrin levels in Mongolian gerbils.
Although regenerating gene (Reg) protein is reported to have a trophic effect on gastric epithelial cells, its involvement in human gastric diseases is not clear. We have recently shown that both gastrin and gastric mucosal inflammation enhance Reg gene expression in the fundic mucosa in rats. This study was designed to clarify whether Reg protein is involved in Helicobacter pylori-induced gastritis and whether Reg gene expression is linked to serum gastrin levels in this condition. Mongolian gerbils were inoculated with an H. pylori strain isolated from a gastric cancer patient. Four weeks later, some of the gerbils with H. pylori infection were eradicated by lansoprazole, amoxicillin, and clarithromycin. The time courses of changes in Reg gene expression, serum gastrin levels, gastric acidity, and histopathologic factors were examined. Four weeks after H. pylori infection, gastritis started spreading to the fundic mucosa, and gastric acidity started reducing. Serum gastrin levels and Reg mRNA expression in the fundus were significantly increased 6 weeks after infection. Reg mRNA expression in the fundus correlated significantly with both serum gastrin levels and the severity of fundic mucosal inflammation. After H. pylori eradication, serum gastrin levels and fundic mucosal inflammation were normalized, and the increase in Reg mRNA expression was abolished. The Reg gene is associated with hypergastrinemia and fundic mucosal inflammation and may be involved in H. pylori-induced gastritis.


Gene-based Therapeutic Tools in the Treatment of Cornea Disease.
As one of the main blinding ocular diseases, corneal blindness resulted from neovascularization that disrupts the angiogenic privilege of corneal avascularity. Following neovascularization, inflammatory cells are infiltrating into cornea to strengthen corneal injury. How to maintain corneal angiogenic privilege to treat corneal disease has been investigated for decades. Local administration of viral and non-viral-mediated anti-angiogenic factors reduces angiogenic protein expression in situ with limited or free of off-target effects upon gene delivery. Recently, Mesenchymal Stem Cells (MSCs) have been studied to treat corneal diseases. Once MSCs are manipulated to express certain genes of interest, they could achieve superior therapeutic efficacy after transplantation. In the text, we first introduce the pathological development of corneal disease in the aspects of neovascularization and inflammation. We summarize how MSCs become an ideal candidate in cell therapy for treating injured cornea, focusing on cell biology, property and features. We provide an updated review of gene-based therapies in animals and preclinical studies in the aspects of controlling target gene expression, safety and efficacy. Gene transfer vectors are potent to induce candidate protein expression. Delivered by vectors, MSCs are equipped with certain characters by expressing a protein of interest, which facilitates better for MSC-mediated therapeutic intervention for the treatment of corneal disease. As the core of this review, we discuss how MSCs could be engineered to be vector system to achieve enhanced therapeutic efficiency after injection.


Pupils' and teachers' experiences of school-based physical education: a qualitative study.
To explore pupils' and teachers' experiences of physical education (PE). A qualitative investigation employing semistructured interviews. Self Determination Theory was used as a guiding theory and Template Analysis was used to analyse the data. A secondary school in the North East of England. 14 pupils (aged 13 and 14, boys and girls) with a range of self-perceived competencies regarding PE and four PE teachers of the pupils (3 male, 1 female). (1) Attitudes and perceptions of PE pupils regarding their experiences of compulsory school PE lessons. (2) PE teachers' experiences of teaching PE. Key results from pupils and teachers suggest pupils enjoy participation in PE when they feel competent, in control and supported by others. Feeling competent depended on (1) the activity within PE and (2) the pupils perceived physical capabilities/aptitude. Feeling in control related to (1) having a choice of activities, (2) being able to set exertion levels and (3) control over clothes worn while taking part. Relationships within pupil groups and between pupils and teachers were perceived as important. Teachers could positively influence their pupils' enjoyment by understanding and supporting their personal goals, as opposed to dictating and controlling what they did and for how long, and by promoting a non-threatening atmosphere between pupils. Rising obesity levels and concerns over the fitness of children and young people has returned the focus of PE to its potential as a vehicle for promoting health. This study suggests schools and PE teachers in particular can positively influence the PE experience of both boys and girls by providing more choice of activities and letting pupils make their own decisions based on their personal needs.


FF483-484 motif of human Polη mediates its interaction with the POLD2 subunit of Polδ and contributes to DNA damage tolerance.
Switching between replicative and translesion synthesis (TLS) DNA polymerases are crucial events for the completion of genomic DNA synthesis when the replication machinery encounters lesions in the DNA template. In eukaryotes, the translesional DNA polymerase η (Polη) plays a central role for accurate bypass of cyclobutane pyrimidine dimers, the predominant DNA lesions induced by ultraviolet irradiation. Polη deficiency is responsible for a variant form of the Xeroderma pigmentosum (XPV) syndrome, characterized by a predisposition to skin cancer. Here, we show that the FF483-484 amino acids in the human Polη (designated F1 motif) are necessary for the interaction of this TLS polymerase with POLD2, the B subunit of the replicative DNA polymerase δ, both in vitro and in vivo. Mutating this motif impairs Polη function in the bypass of both an N-2-acetylaminofluorene adduct and a TT-CPD lesion in cellular extracts. By complementing XPV cells with different forms of Polη, we show that the F1 motif contributes to the progression of DNA synthesis and to the cell survival after UV irradiation. We propose that the integrity of the F1 motif of Polη, necessary for the Polη/POLD2 interaction, is required for the establishment of an efficient TLS complex.


Tissue-specific differential induction of duplicated fatty acid-binding protein genes by the peroxisome proliferator, clofibrate, in zebrafish (Danio rerio).
Force, Lynch and Conery proposed the duplication-degeneration-complementation (DDC) model in which partitioning of ancestral functions (subfunctionalization) and acquisition of novel functions (neofunctionalization) were the two primary mechanisms for the retention of duplicated genes. The DDC model was tested by analyzing the transcriptional induction of the duplicated fatty acid-binding protein (fabp) genes by clofibrate in zebrafish. Clofibrate is a specific ligand of the peroxisome proliferator-activated receptor (PPAR); it activates PPAR which then binds to a peroxisome proliferator response element (PPRE) to induce the transcriptional initiation of genes primarily involved in lipid homeostasis. Zebrafish was chosen as our model organism as it has many duplicated genes owing to a whole genome duplication (WGD) event that occurred ~230-400 million years ago in the teleost fish lineage. We assayed the steady-state levels of fabp mRNA and heterogeneous nuclear RNA (hnRNA) transcripts in liver, intestine, muscle, brain and heart for four sets of duplicated fabp genes, fabp1a/fabp1b.1/fabp1b.2, fabp7a/fabp7b, fabp10a/fabp10b and fabp11a/fabp11b in zebrafish fed different concentrations of clofibrate. Electron microscopy showed an increase in the number of peroxisomes and mitochondria in liver and heart, respectively, in zebrafish fed clofibrate. Clofibrate also increased the steady-state level of acox1 mRNA and hnRNA transcripts in different tissues, a gene with a functional PPRE. These results demonstrate that zebrafish is responsive to clofibrate, unlike some other fishes. The levels of fabp mRNA and hnRNA transcripts for the four sets of duplicated fabp genes was determined by reverse transcription, quantitative polymerase chain reaction (RT-qPCR). The level of hnRNA coded by a gene is an indirect estimate of the rate of transcriptional initiation of that gene. Clofibrate increased the steady-state level of fabp mRNAs and hnRNAs for both the duplicated copies of fabp1a/fabp1b.1, and fabp7a/fabp7b, but in different tissues. Clofibrate also increased the steady-state level of fabp10a and fabp11a mRNAs and hnRNAs in liver, but not for fabp10b and fabp11b. Some duplicated fabp genes have, most likely, retained PPREs, but induction by clofibrate is over-ridden by an, as yet, unknown tissue-specific mechanism(s). Regardless of the tissue-specific mechanism(s), transcriptional control of duplicated zebrafish fabp genes by clofibrate has markedly diverged since the WGD event.


Costimulation and endogenous MHC ligands contribute to T cell recognition.
To initiate an immune response, key receptor-ligand pairs must cluster in "immune synapses" at the T cell-antigen-presenting cell (APC) interface. We visualized the accumulation of a major histocompatibility complex (MHC) class II molecule, I-E(k), at a T cell-B cell interface and found it was dependent on both antigen recognition and costimulation. This suggests that costimulation-driven active transport of T cell surface molecules helps to drive immunological synapse formation. Although only agonist peptide-MHC class II (agonist pMHC class II) complexes can initiate T cell activation, endogenous pMHC class II complexes also appeared to accumulate. To test this directly, we labeled a "null" pMHC class II complex and found that, although it lacked major TCR contact residues, it could be driven into the synapse in a TCR-dependent manner. Thus, low-affinity ligands can contribute to synapse formation and T cell signaling.


Health-promoting substances and heavy metal content in tomatoes grown with different farming techniques.
Organic farming is a production technique that imposes major restrictions on the use of fertilizers, pesticides, feed additives and veterinary drugs and for this reason consumers perceive organic foods to be healthier. The content of health-promoting molecules such as ascorbic acid, beta-carotene, lycopene and salicylic acid are important aspects of the nutritional quality of organic foods. To evaluate health promoting substances and the heavy metal content of tomato berries grown using conventional, integrated pest management (IPM) and organic farming techniques. Moisture was determined by drying, crude protein by the Kjeldhal method, and ashes by incineration at 550 degrees C. Ergosterol, ascorbic acid, beta-carotene, lycopene and salicylic acid were determined by HPLC. The levels of heavy metals were measured by atomic absorption spectroscopy. Compared to crops grown using conventional and IPM methods, organic tomatoes contained more salicylic acid but less vitamin C and lycopene. Organic tomatoes had higher Cd and Pb levels but a lower Cu content. Organic fruits had a slightly higher protein content than conventionally cultivated fruits, but the difference was minimal and consequently the nutritive significance was poor. Farming techniques may have an impact on the quality of tomatoes. Their higher salicylate content supports the notion that organic foodstuffs are more wholesome. However, the lower lycopene and ascorbic acid levels of organic tomatoes are not to be regarded as positive. No residues of pesticides and ergosterol were detected.


A web-based intervention to support increased physical activity among at-risk adults.
Physical inactivity is an important contributor to the development of numerous chronic conditions and alone is an independent risk factor for diabetes, cardiovascular disease, and depression and yet, most Americans consistently fail to achieve the recommended amount of physical activity. As part of Project HealthDesign, we designed and prototyped a personal health record application (PHA) that delivers and supports a highly individualized, behaviorally based lifestyle physical activity intervention for sedentary adults. Through a user centered design approach, we engaged consumers, health care providers, and personal trainers for multiple facilitated group discussions and structured interviews to determine their needs and wants related to an activity PHA. The PHA was developed to include elements of evidence-based approaches which help participants adopt cognitive and behavioral skills such as goal-setting, self-monitoring, accepting social support, cognitive restructuring, contingency management, decisional balance, and relapse prevention. This PHA demonstrated the potential for research-based behavioral interventions to be delivered via a web portal. This finding is important for both consumers and their providers who have the desire to implement physical activity recommendations, but lack the tools to facilitate or undertake such interventions.


Implementation of standard sedation management in paediatric intensive care: effective and feasible?
To study the effects of the introduction of a sedation treatment protocol for children in intensive care, including nurses' compliance. While several sedation guidelines for adults and children have been developed and implemented, there is little evidence on use of sedation protocols in critically ill infants. Pretest-posttest intervention study. Administered sedatives and analgesics over the first seven days of admission were documented for convenience samples, before (n = 27) and after (n = 29) implementation of standard sedation assessments and a sedation protocol. Sedation was assessed with the COMFORT behaviour scale, Nurse Interpretation of Sedation Score and the Visual Analogue Scale for three-month periods, both pretest and posttest. Starting 21 months after the posttest, nurses' compliance with the sedation protocol, as well as administered sedatives and analgesics were evaluated for 12 months. Infants in the posttest period received significantly more midazolam and morphine. The proportion of patients adequately sedated on the grounds of COMFORT scores had increased from 63% pretest to 72% posttest and to 75% in the long run. Adequate sedation as judged from the sedation protocol cutoffs was found in 71% of the assessments. In 45% of assessments indicating undersedation, the infusion rate had been increased on the guidance of the protocol. A survey among staff revealed that most considered the sedation protocol comprehensible and useful. This study showed that regular sedation assessment in critically ill children was feasible and had become standard practice two years after the first posttest. There is insufficient evidence to conclude whether implementation of a sedation treatment protocol indeed improves sedation treatment. This sedation protocol provides decision trees for increasing or weaning of sedatives in both haemodynamically stable and unstable patients. It standardizes sedation management and allows nurses to adapt medication themselves.


Pharmacological interventions for agitation in patients with traumatic brain injury: protocol for a systematic review and meta-analysis.
Traumatic brain injury (TBI) is a worldwide leading cause of mortality and disability. Among TBI complications, agitation is a frequent behavioural problem. Agitation causes potential harm to patients and caregivers, interferes with treatments, leads to unnecessary chemical and physical restraints, increases hospital length of stay, delays rehabilitation, and impedes functional independence. Pharmacological treatments are often considered for agitation management following TBI. Several types of agents have been proposed for the treatment of agitation. However, the benefit and safety of these agents in TBI patients as well as their differential effects and interactions are uncertain. In addition, animal studies and observational studies have suggested impaired cognitive function with the use of certain antipsychotics and benzodiazepines. Hence, a safe and effective treatment for agitation, which does not interfere with neurological recovery, remains to be identified. With the help of Health Sciences librarian, we will design a search strategy in the following databases: PubMed, Ovid MEDLINE®, EMBASE, CINAHL, PsycINFO, Cochrane Library, Google Scholar, Directory of Open Access Journals, LILACS, Web of Science, and Prospero. A grey literature search will be performed using the resources suggested in CADTH's Grey Matters. We will include all randomized controlled, quasi-experimental, and observational studies with control groups. The population of interest is all patients, including children and adults, who have suffered a TBI. We will include studies in which agitation, not further defined, was the presenting symptom or one of the presenting symptoms. We will also include studies where agitation was not the presenting symptom but was measured as an outcome variable and studies assessing the safety of these pharmacological interventions in TBI patients. We will include studies evaluating all pharmacological interventions including beta-adrenergic blockers, typical and atypical antipsychotics, anticonvulsants, dopamine agonists, psychostimulants, antidepressants, alpha-2-adrenergic agonists, hypnotics, and anxiolytics. Although agitation is frequent following TBI and pharmacological agents that are often used, there is no consensus on the most efficacious and safest strategy to treat these complications. There is a need for an updated systematic review to summarize the evidence in order to inform practice and future research. PROSPERO CRD42016033140.


An effective approach to prevent immune rejection of human ESC-derived allografts.
Human embryonic stem cells (hESCs) hold great promise for cell therapy as a source of diverse differentiated cell types. One key bottleneck to realizing such potential is allogenic immune rejection of hESC-derived cells by recipients. Here, we optimized humanized mice (Hu-mice) reconstituted with a functional human immune system that mounts a vigorous rejection of hESCs and their derivatives. We established knockin hESCs that constitutively express CTLA4-Ig and PD-L1 before and after differentiation, denoted CP hESCs. We then demonstrated that allogenic CP hESC-derived teratomas, fibroblasts, and cardiomyocytes are immune protected in Hu-mice, while cells derived from parental hESCs are effectively rejected. Expression of both CTLA4-Ig, which disrupts T cell costimulatory pathways, and PD-L1, which activates T cell inhibitory pathway, is required to confer immune protection, as neither was sufficient on their own. These findings are instrumental for developing a strategy to protect hESC-derived cells from allogenic immune responses without requiring systemic immune suppression.


Human papillomavirus infection and colorectal cancer in the Chinese population: a meta-analysis.
Human papillomavirus (HPV) infection has been reported in colorectal cancer in many studies. We conducted a meta-analysis to assess the association between HPV infection and colorectal cancer/adenomas in the Chinese population. Relevant studies up to January 2018 were searched in PubMed, EMBASE, China National Knowledge Infrastructure and the Wanfang database. We used a random effects model to determine the prevalence of HPV and odds ratios (ORs) with 95% confidence intervals (CIs). The I2 statistic and P-value from the Cochrane Q-test were used to describe the heterogeneity. Ten case-control studies involving 766 colorectal cancer patients and 470 controls were included in the meta-analysis. Among the colorectal cancer patients, the pooled prevalence was 0.45 (95% CI 0.36-0.53). The pooled estimate for OR was 10.78 (95% CI 4.22, 27.53). Among the 193 patients with colorectal adenoma, the pooled prevalence and OR were 0.31 (95% CI 0.24-0.37) and 2.03 (95% CI 0.79, 5.26), respectively. The prevalence of HPV 16 and HPV 18 among HPV-positive cancers ranged from 57.9% to 100% and 0% to 39.7%, respectively. The results indicated that HPV infection, especially HPV 16 and HPV 18, is associated with colorectal cancer in the Chinese population.


Twenty years of bacterial genome sequencing.
Twenty years ago, the publication of the first bacterial genome sequence, from Haemophilus influenzae, shook the world of bacteriology. In this Timeline, we review the first two decades of bacterial genome sequencing, which have been marked by three revolutions: whole-genome shotgun sequencing, high-throughput sequencing and single-molecule long-read sequencing. We summarize the social history of sequencing and its impact on our understanding of the biology, diversity and evolution of bacteria, while also highlighting spin-offs and translational impact in the clinic. We look forward to a 'sequencing singularity', where sequencing becomes the method of choice for as-yet unthinkable applications in bacteriology and beyond.


Loss of TBK1 induces epithelial-mesenchymal transition in the breast cancer cells by ERα downregulation.
Estrogen receptor α (ERα) is the pivotal regulator of proliferation and differentiation in mammary epithelia, where it serves as a crucial prognostic marker and therapeutic target in breast cancer. In this study, we show that the loss of the kinase TANK-binding kinase 1 (TBK1) induces epithelial-mesenchymal transition in ERα-positive breast cancer cells by downregulating ERα expression. TBK1 was overexpressed in ERα-positive breast cancers, where it was associated with distant metastasis-free survival in patients, whereas it was underexpressed in ERα-negative breast cancers. TBK1 silencing decreased expression of epithelial markers and increased expression of mesenchymal markers in ERα-positive breast cancer cells, enhancing tumor growth and lung metastasis in vivo in a manner associated with downregulation of ERα expression. Mechanistically, TBK1 silencing reduced FOXO3A binding to the ERα promoter by inducing the translocation of phosphorylated FOXO3A from the nucleus to the cytoplasm. Thus, our results indicate that the loss of TBK1 expression parallels the loss of ERα expression, in turn helping drive an aggressive breast cancer phenotype.


Systematic review searches must be systematic, comprehensive, and transparent: a critique of Perman et al.
A high quality systematic review search has three core attributes; it is systematic, comprehensive, and transparent. The current over-emphasis on the primacy of systematic reviews over other forms of literature review in health research, however, runs the risk of encouraging publication of reviews whose searches do not meet these three criteria under the guise of being systematic reviews. This correspondence comes in response to Perman S, Turner S, Ramsay AIG, Baim-Lance A, Utley M, Fulop NJ. School-based vaccination programmes: a systematic review of the evidence on organization and delivery in high income countries. 2017; BMC Public Health 17:252, which we assert did not meet these three important quality criteria for systematic reviews, thereby leading to potentially unreliable conclusions. Our aims herein are to emphasize the importance of maintaining a high degree of rigour in the conduct and publication of systematic reviews that may be used by clinicians and policy-makers to guide or alter practice or policy, and to highlight and discuss key evidence omitted in the published review in order to contextualize the findings for readers. By consulting a research librarian, we identified limitations in the search terms, the number and type of databases, and the screening methods used by Perman et al. Using a revised Ovid MEDLINE search strategy, we identified an additional 1016 records in that source alone, and highlighted relevant literature on the organization and delivery of school-based immunization program that was omitted as a result. We argue that a number of the literature gaps noted by Perman et al. may well be addressed by existing literature found through a more systematic and comprehensive search and screening strategy. We commend both the journal and the authors, however, for their transparency in supplying information about the search strategy and providing open access to peer reviewer and editor's comments, which enabled us to understand the reasons for the limitations of that review.


Synthesis of two linear PADRE conjugates bearing a deca- or pentadecasaccharide B epitope as potential synthetic vaccines against Shigella flexneri serotype 2a infection.
The blockwise synthesis of the 2-aminoethyl glycosides of a deca- and a pentadecasaccharide made of two and three repeating units, respectively, of the Shigella flexneri serotype 2a specific polysaccharide is reported. The strategy relies on trifluoromethanesulfonic acid mediated glycosylation of a pentasaccharide building block acting as a glycosyl donor and a potential glycoside acceptor. Both targets were made available in amounts large enough for their subsequent conversion into glycoconjugates. Indeed, efficient elongation of the spacer through an acetylthioacetyl moiety and subsequent conjugation onto a Pan HLA DR-binding epitope (PADRE) T-cell-universal peptide resulted in two fully synthetic neoglycopeptides, which will be evaluated as potential vaccines against S. flexneri serotype 2a infections.


Infectious diseases acquired by international travellers visiting the USA.
Estimates of travel-related illness have focused predominantly on populations from highly developed countries visiting low- or middle-income countries, yet travel to and within high-income countries is very frequent. Despite being a top international tourist destination, few sources describe the spectrum of infectious diseases acquired among travellers to the USA. We performed a descriptive analysis summarizing demographic and travel characteristics, and clinical diagnoses among non-US-resident international travellers seen during or after travel to the USA at a GeoSentinel clinic from 1 January 1997 through 31 December 2016. There were 1222 ill non-US-resident travellers with 1393 diagnoses recorded during the 20-year analysis period. Median age was 40 (range 0-86 years); 52% were female. Patients visited from 63 countries and territories, most commonly Canada (31%), Germany (14%), France (9%) and Japan (7%). Travellers presented with a range of illnesses; skin and soft tissue infections of unspecified aetiology were the most frequently reported during travel (29 diagnoses, 14% of during-travel diagnoses); arthropod bite/sting was the most frequently reported after travel (173 diagnoses, 15% after-travel diagnoses). Lyme disease was the most frequently reported arthropod-borne disease after travel (42, 4%). Nonspecific respiratory, gastrointestinal and systemic infections were also among the most frequently reported diagnoses overall. Low-frequency illnesses (<2% of cases) made up over half of diagnoses during travel and 41% of diagnoses after travel, including 13 cases of coccidioidomycosis and mosquito-borne infections like West Nile, dengue and Zika virus diseases. International travellers to the USA acquired a diverse array of mostly cosmopolitan infectious diseases, including nonspecific respiratory, gastrointestinal, dermatologic and systemic infections comparable to what has been reported among travellers to low- and middle-income countries. Clinicians should consider the specific health risks when preparing visitors to the USA and when evaluating and treating those who become ill.


Haematological studies on pre-menopausal Indian and Caucasian vegetarians compared with Caucasian omnivores.
Full blood counts, serum ferritin, vitamin B12 and folate, erythrocyte folate concentrations and nutrient intakes were estimated in twenty-three Indian vegetarian, twenty-two Caucasian omnivores and eighteen Caucasian vegetarian women aged 25-40 years. Energy and copper intakes were lower in the Indian women than in the Caucasians. Intakes of dietary fibre, vitamin C and folate were greater and the proportion of energy derived from fat was lower in the vegetarians than in the omnivores. Vitamin B12 and protein intakes were lower in both vegetarian groups than in the omnivores. Fe intake was similar in all the groups but haem Fe provided one-quarter of the Fe intake of the omnivores. Haemoglobin concentrations were generally inside the normal range in all groups, but were lower in the Indians as were mean corpuscular volume (MCV) and mean corpuscular haemoglobin (MCH). Higher MCV, MCH and lower erythrocyte (RBC) counts were observed in Caucasian vegetarians compared with the Caucasian omnivores. In both groups of vegetarians, concentrations of serum vitamin B12 and ferritin were markedly lower than in the omnivores. RBC folate concentrations were lower in the Indians than in either of the Caucasian groups when subjects taking supplements were excluded. It is concluded that vegetarians need to ensure they have adequate intakes of Fe and vitamin B12.


Nitric oxide as a mediator for defense responses.
Sequential recognition of invading microbes and rapid induction of plant immune responses comprise at least two recognition systems. Early basal defenses are initiated by pathogen-associated molecular patterns and pattern recognition receptors (PRR) in the plasma membrane. Pathogens produce effectors to suppress defense but plants, in turn, can sense such effectors by dominant plant resistance (R) gene products. Plant PRR and R proteins modulate signaling networks for defense responses that rely on rapid production of reactive nitrogen species (RNS) and reactive oxygen species (ROS). Recent research has shown that nitric oxide (NO) mainly mediates biological function through chemical reactions between locally controlled accumulation of RNS and proteins leading to potential alteration of protein function. Many proteins specifically regulated by NO and participating in signaling during plant defense response have been identified, highlighting the physiological relevance of these modifications in plant immunity. ROS function independently or in cooperation with NO during defense, modulating the RNS signaling functions through the entire process. This review provides an overview of current knowledge about regulatory mechanisms for NO burst and signaling, and crosstalk with ROS in response to pathogen attack.


Garlic and vitamin E provides antioxidant defence in tissues of female rats treated with nicotine.
Nicotine is known to induce oxidative stress in rat tissues and the antioxidant properties of garlic have been reported. This study was designed to determine if the peroxidative damage caused by nicotine administration can be effectively prevented with garlic juice, and vitamin E, a known antioxidant.Four groups of six rats each were divided into: Group I: (control) received 0.2ml of 0.9% normal saline, group II (received nicotine 0.6mg/kg b.w subcutaneously), group III (received nicotine 0.6mg/kg b.w + garlic juice 100mg/kg b.w orally), and group IV (received nicotine 0.6mg/kg b.w + Vitamin E 100mg/kg b.w orally). All animals were treated for 21 days. The pituitary gland, ovary, uterus, heart, liver and kidney of the animals were harvested, weighed and homogenized. Malondialdehyde (MDA), superoxide dismutase (SOD) and reduced glutathione (GSH) were then measured.Concentration of MDA was significantly increased in tissues of nicotine treated rats when compared with the control. In group III and IV, MDA levels were significantly reduced when compared with nicotine group. The activities of SOD and GSH significantly decreased in group II (nicotine only) rat tissues, while it was significantly increased in group III and IV rat tissues. The study showed that garlic juice extract (100mg/kg b.w) and vitamin E (100mg/kg b.w) administration prevented oxidative damage in rat tissues treated with nicotine. The study also showed that vitamin E has a more potent antioxidant activity than garlic juice in preventing nicotine induced oxidative damage in rat.


Pulse Electron Double Resonance Detected Multinuclear NMR Spectra of Distant and Low Sensitivity Nuclei and Its Application to the Structure of Mn(II) Centers in Organisms.
The ability to characterize the structure of metal centers beyond their primary ligands is important to understanding their chemistry. High-magnetic-field pulsed electron double resonance detected NMR (ELDOR-NMR) is shown to be a very sensitive approach to measuring the multinuclear NMR spectra of the nuclei surrounding Mn(II) ions. Resolved spectra of intact organisms with resonances arising from (55)Mn, (31)P, (1)H, (39)K, (35)Cl, (23)Na, and (14)N nuclei surrounding Mn(2+) centers were obtained. Naturally abundant cellular (13)C could be routinely measured as well. The amplitudes of the (14)N and (2)H ELDOR-NMR spectra were found to be linearly dependent on the number of nuclei in the ligand sphere. The evolution of the Mn(II) ELDOR-NMR spectra as a function of excitation time was found to be best described by a saturation phenomenon rather than a coherently driven process. Mn(II) ELDOR-NMR revealed details about not only the immediate ligands to the Mn(II) ions but also more distant nuclei, providing a view of their extended structures. This will be important for understanding the speciation and chemistry of the manganese complexes as well as other metals found in organisms.


Mechanical properties of cortical bone and their relationships with age, gender, composition and microindentation properties in the elderly.
The growing incidence of skeletal fractures poses a significant challenge to ageing societies. Since a major part of physiological loading in the lower limbs is carried by cortical bone, it would be desirable to better understand the structure-mechanical property relationships and scale effects in this tissue. This study aimed at assessing whether microindentation properties combined with chemical and morphological information are usable to predict macroscopic elastic and strength properties in a donor- and site-matched manner. Specimens for quasi-static macroscopic tests in tension, compression, and torsion and microindentation were prepared from a cohort of 19 male and 20 female donors (46 to 99 years). All tests were performed under fully hydrated conditions. The chemical composition of the extra-cellular matrix was investigated with Raman spectroscopy. The results of the micro-mechanical tests were combined with morphological and compositional properties using a power law relationship to predict the macro-mechanical results. Microindentation properties were not gender dependent, remarkably constant over age, and showed an overall small variation with standard deviations of approximately 10 %. Similar results were obtained for chemical tissue composition. Macro-mechanical stiffness and strength were significantly related to porosity for all load cases (p<0.05). In case of macroscopic yield strain and work-to-failure this was only true in torsion and compression, respectively. The correlations of macro-mechanical with micro-mechanical, morphological, and chemical properties showed no significance for cement line density, mineralisation, or variations in the microindentation results and were dominated by porosity with a moderate explanatory power of predominately less than 50 %. The results confirm that age, with minor exceptions gender, and small variations in average mineralisation have negligible effect on the tissue microindentation properties of human lamellar bone in the elderly. Furthermore, our findings suggest that microindentation experiments are suitable to predict macroscopic mechanical properties in the elderly only on average and not on a one to one basis. The presented data may help to form a better understanding of the mechanisms of ageing in bone tissue and of the length scale at which they are active. This may be used for future prediction of fracture risk in the elderly.


Tumor necrosis factor alpha and interleukin 11 secreted by malignant breast epithelial cells inhibit adipocyte differentiation by selectively down-regulating CCAAT/enhancer binding protein alpha and peroxisome proliferator-activated receptor gamma: mechanism of desmoplastic reaction.
The dense layer of fibroblasts that accumulate around malignant breast epithelial cells (i.e., desmoplastic reaction) arises from the breast adipose tissue and provides structural and biochemical support for breast cancer. We report herein a number of epithelial-stromal interactions responsible for desmoplastic reaction in breast cancer using cultured 3T3-L1 murine fibroblasts and human adipose fibroblasts, which can be activated with a mixture of hormones to differentiate to mature adipocytes. Adipocyte differentiation was inhibited by coculturing fibroblasts with various breast cancer cell lines (T47D, MCF-7, SSC202, SSC78, and SSC30) completely or by breast cancer cell conditioned media in a dose-dependent manner; on the other hand, adipocyte differentiation was not inhibited by coculturing with normal human primary mammary epithelial cell conditioned medium. This tumor effect was eliminated using neutralizing antibodies against tumor necrosis factor (TNF)-alpha or interleukin (IL)-11. TNF-alpha and IL-11 levels were 2.5-3 times higher in T47D conditioned medium compared with control medium, and TNF-alpha transcripts were detectable in T47D but not in 3T3-L1 cells in culture, indicating that the malignant epithelial cell is the major site of cytokine production. This was confirmed in vivo in mastectomy specimens, where immunoreactive TNF-alpha and IL-11 were readily detectable in malignant epithelial cells but not in the majority of the surrounding fibroblasts. Adipocyte differentiation is mediated by the expression of a cascade of adipogenic transcription factors, including CCAAT/enhancer binding protein (C/EBP)beta, C/EBPdelta, peroxisome proliferator-activated receptor (PPAR)gamma and C/EBPalpha. C/EBPalpha and PPARgamma are essential for this process. We demonstrated by Northern analysis that exposure of activated 3T3-L1 cells to T47D cell conditioned medium strikingly decreased the levels of PPARgamma and C/EBPalpha transcripts and increased the levels of C/EBPbeta and C/EBPdelta transcripts. In these 3T3-L1 cells, inhibition of differentiation was also confirmed by markedly suppressed levels of aP2 mRNA, which is an adipocyte-specific gene. These in vitro observations were confirmed in sections of human malignant breast tumors, where immunoreactive C/EBPalpha was readily detectable in adipose flbroblasts distant to the tumor but not in intratumoral fibroblasts. Treatment of 3T3-L1 cells with T47D cell conditioned medium or TNF-alpha changed neither the numbers of cells in G0-G1, S, and G2 phases nor the rate of [3H]thymidine incorporation, thus ruling out a proliferative effect of malignant cells on the surrounding fibroblasts. In summary, desmoplastic reaction primarily occurs via the action of cytokines (TNF-alpha and IL-11) secreted by the malignant epithelial cells to inhibit differentiation of adipose fibroblasts to mature adipocytes. This tumor-induced block in adipocyte differentiation is mediated by the selective inhibition of expression of the essential adipogenic transcription factors, i.e., PPARgamma and C/EBPalpha.


Design, synthesis and biological evaluation of novel nitric oxide-donating protoberberine derivatives as antitumor agents.
A novel class of NO-donating protoberberine derivatives were synthesized and initially evaluated for their anti-hepatocellular carcinoma activities. Most of the compounds exhibited more potent activity against HepG2 cells than parent compounds berberine and palmatine. In particular, compound 15a exerted the strongest activity with an IC50 value of 1.36 μM. Moreover, most compounds released moderate levels of NO in vitro, and the antitumor activity of 15a in HepG2 cells was remarkably diminished by an NO scavenger. Interestingly, compound 15a displayed a broad-spectrum antitumor efficacy and possessed good selectivity between tumor cells (HepG2, SMMC-7721, HCT-116, HL-60) and normal liver LO-2 cells. The mechanism studies revealed that 15a blocked the G2 phase of the cell cycle and induced apoptosis of HepG2 cells by mitochondrial depolarization. Furthermore, 15a inhibited tumor growth in H22 liver cancer xenograft mouse model by 62.5% (w/w), which was significantly superior to parent compound palmatine (41.6%, w/w). Overall, the current study may provide a new approach for the discovery of novel antitumor agents.


The Pharmacological Basis of Cannabis Therapy for Epilepsy.
Recently, cannabis has been suggested as a potential alternative therapy for refractory epilepsy, which affects 30% of epilepsy, both adults and children, who do not respond to current medications. There is a large unmet medical need for new antiepileptics that would not interfere with normal function in patients with refractory epilepsy and conditions associated with refractory seizures. The two chief cannabinoids are Δ-9-tetrahyrdrocannabinol, the major psychoactive component of marijuana, and cannabidiol (CBD), the major nonpsychoactive component of marijuana. Claims of clinical efficacy in epilepsy of CBD-predominant cannabis or medical marijuana come mostly from limited studies, surveys, or case reports. However, the mechanisms underlying the antiepileptic efficacy of cannabis remain unclear. This article highlights the pharmacological basis of cannabis therapy, with an emphasis on the endocannabinoid mechanisms underlying the emerging neurotherapeutics of CBD in epilepsy. CBD is anticonvulsant, but it has a low affinity for the cannabinoid receptors CB1 and CB2; therefore the exact mechanism by which it affects seizures remains poorly understood. A rigorous clinical evaluation of pharmaceutical CBD products is needed to establish the safety and efficacy of their use in the treatment of epilepsy. Identification of mechanisms underlying the anticonvulsant efficacy of CBD is also critical for identifying other potential treatment options.


[Pelvic exenterations for gynaecological cancer--a 10 year institutional review].
Pelvic exenteration consists in a radical surgical procedure for patients with advanced cancer but limited to central pelvis. It is used for treatment of primary or recurrent tumours, or, more usually in treatment of recurrent cervical cancer. Retrospective study of medical charts of patients underwent pelvic exenteration for gynaecological cancer between January 1995 and April 2005. Twenty-one pelvic exenterations were performed in this period. The mean age of the patients was 55.7 years. Indication for surgery included 14 (67%) recurrent cervical cancers; 2 uterine cancers (recurrent), 2 vulvar cancer (one recurrent and one primary), 2 vagina carcinoma (one primary and one recurrent) and one patient with synchronous recto and uterine tumours. Exenteration was nine anterior, 3 posterior and 9 totals. The median length of hospitalization was 27 days. Most common postoperative complications were associated with urinary diversion. After surgery 19% of patients develops persistent lesion, 24% develops recurrent lesion with a middle free interval of 12.4 months. After a median follow-up of 29 months, 10 patients (47%) are alive and free of disease. Pelvic exenteration can be offered as a surgical salvage therapy to patients with locally advanced gynaecological cancer.


Development of an intervention to support the implementation of evidence-based strategies for optimising antibiotic prescribing in general practice.
Trials show that antimicrobial stewardship (AMS) strategies, including communication skills training, point-of-care C-reactive protein testing (POC-CRPT) and delayed prescriptions, help optimise antibiotic prescribing and use in primary care. However, the use of these strategies in general practice is limited and inconsistent. We aimed to develop an intervention to enhance uptake and implementation of these strategies in primary care. We drew on the Person-Based Approach to develop an implementation intervention in two stages. (1) Planning and design: We defined the problem in behavioural terms drawing on existing literature and conducting primary qualitative research (nine focus groups) in high-prescribing general practices. We identified 'guiding principles' with intervention objectives and key features and developed logic models representing intended mechanisms of action. (2) Developing the intervention: We created prototype intervention materials and discussed and refined these with input from 13 health professionals and 14 citizens in two sets of design workshops. We further refined the intervention materials following think-aloud interviews with 22 health professionals. Focus groups highlighted uncertainties about how strategies could be used. Health professionals in the workshops suggested having practice champions, brief summaries of each AMS strategy and evidence supporting the AMS strategies, and they and citizens gave examples of helpful communication strategies/phrases. Think-aloud interviews helped clarify and shorten the text and user journey of the intervention materials. The intervention comprised components to support practice-level implementation: antibiotic champions, practice meetings with slides provided, and an 'implementation support' website section, and components to support individual-level uptake: website sections on each AMS strategy (with evidence, instructions, links to electronic resources) and material resources (patient leaflets, POC-CRPT equipment, clinician handouts). We used a systematic, user-focussed process of developing a behavioural intervention, illustrating how it can be used in an implementation context. This resulted in a multicomponent intervention to facilitate practice-wide implementation of evidence-based strategies which now requires implementing and evaluating. Focusing on supporting the uptake and implementation of evidence-based strategies to optimise antibiotic use in general practice is critical to further support appropriate antibiotic use and mitigate antimicrobial resistance.