Endotoxin-neutralizing properties of the 25 kD N-terminal fragment and a newly isolated 30 kD C-terminal fragment of the 55-60 kD bactericidal/permeability-increasing protein of human neutrophils.
The bactericidal/permeability-increasing protein (BPI) of polymorphonuclear leukocytes (PMN) is a potent cytotoxin, specific for Gram-negative bacteria, that also inhibits endotoxin activity by neutralizing isolated bacterial lipopolysaccharides (LPS). We have previously shown that an isolated 25 kD N-terminal fragment of human BPI carries all the antibacterial activities of the parent 55-60 kD molecule. In this study we have compared the LPS-neutralizing activities of human holo-BPI, the N-terminal fragment and a 30 kD C-terminal fragment that we have now isolated. We show that the N-terminal fragment also has LPS-neutralizing activity as detected by inhibition (up to 95%) of (a) activation by LPS of procoagulant proteases in Limulus amebocyte lysates, (b) LPS "priming" of PMN, and (c) LPS-mediated production of tumor necrosis factor in whole human blood. Holo-BPI and the 25 kD fragment have similar neutralizing potency (in nanomolar range) in all assays toward "smooth" LPS from Escherichia coli O111:B4 and O55:B5 (possessing long chain polysaccharide or O-antigen), and "deep rough" LPS from Salmonella minnesota Re595 mutant (possessing no O-antigen). The C-terminal fragment of BPI is devoid of antibacterial activity when tested against BPI-sensitive E. coli J5, but does have endotoxin-neutralizing activity. This activity is weak relative to holo-BPI and the 25 kD N-terminal fragment in the Limulus and PMN-priming assay, but is comparable for inhibition of TNF production in whole blood. We conclude that the principal determinants for LPS recognition and neutralization, like those for antibacterial action, reside in the N-terminal half of the BPI molecule, but that sites within the C-terminal half can also contribute to BPI-LPS interaction once LPS is detached from the bacterial envelope.


Evidence valued and used by health promotion practitioners.
The use of evidence has become a foundational part of health promotion practice. Although there is a general consensus that adopting an evidence-based approach is necessary for practice, disagreement remains about what types of evidence practitioners should use to guide their work. An empirical understanding of how practitioners conceptualize and use evidence has been lacking in the literature. In this article, we explore (i) practitioners' purposes for using evidence, (ii) types of evidence they valued, and (iii) qualities that made evidence useful for practice. 58 semi-structured interviews and 250 h of participant and non-participant observation were conducted with 54 health promotion practitioners working across New South Wales, Australia. Interviews were recorded and transcribed, and field notes were written during the observations; these were analysed using Grounded Theory methods. Practitioners used evidence for practical and strategic purposes, and valued four different types of evidence according to their relevance and usefulness for these purposes. Practitioners' ideal evidence was generated within their practice settings, and met both substantive and procedural evaluation criteria. We argue that due to the complex nature of their work, practitioners rely on a diverse range of evidence and require organizational structures that will support them in doing so.


Survival of Chlamydia pneumoniae-infected Mono Mac 6 cells is dependent on NF-kappaB binding activity.
The respiratory tract pathogen Chlamydia pneumoniae has been associated with atherosclerosis. Monocytes are supposed to serve as a vehicle for systemic dissemination of intracellular C. pneumoniae from the lung to the artery vessel wall. We were therefore interested in pathogen-induced cellular events associated with NF-kappaB, a crucial transcription factor for both inflammatory cytokines and antiapoptotic molecules. In this study we demonstrate by electrophoretic mobility shift assay that C. pneumoniae infection of the human monocytic cell line Mono Mac 6 induces activation of NF-kappaB over 48 h, with a maximum level at 1 h postinfection. As shown by supershift assay, the activated NF-kappaB complex consists of the subunits RelA (p65) and NF-kappaB1 (p50). Apoptotic host cells were not detected during the early stages of the infection when maximal activation of NF-kappaB was detected. Pretreatment of Mono Mac 6 with the antioxidant and NF-kappaB inhibitor PDTC (pyrrolidine dithiocarbamate) induced activation of caspase-3 and led to apoptotic cell death. The C. pneumoniae-induced activation of the NF-kappaB complex was reduced by PDTC, which in parallel resulted in an increased apoptosis, as quantified by annexin V labeling and terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling reaction. In the complete absence of activated NF-kappaB, when Mono Mac 6 cells were pretreated with the more potent NF-kappaB inhibitors MG-132 and parthenolide a C. pneumoniae-mediated rescue of cells from induced apoptosis could not be achieved. Our results indicate that activation of NF-kappaB in C. pneumoniae-infected Mono Mac 6 cells is associated with protection of Mono Mac 6 cells against apoptosis and might thereby contribute to systemic spread of the pathogen.


Patient compliance in rheumatoid arthritis, polymyalgia rheumatica, and gout.
(1) To explore patient compliance with prescribed drug regimens in the setting of usual care for outpatients with rheumatoid arthritis (RA), gout, and polymyalgia rheumatica (PMR) by utilizing electronic medication event monitors (MEMS(R)) to register openings of the medication package. (2) To examine the influence of disease, frequency of intake of the drug, and class of drug on compliance. (3) To explore the influence of demographic factors, quality of life measures, coping, health status, and functional ability as potential predictors of patient compliance. A total of 127 consenting consecutive patients were enrolled: 81 patients with RA, 33 taking nonsteroidal antiinflammatory drugs (13 diclofenac TID and 20 naproxen BID) and 48 taking disease modifying antirheumatic drugs [25 sulfasalazine (SSZ) BID and 23 methotrexate (MTX) once weekly]; 17 patients with PMR starting with prednisolone QD; and 29 patients with gout starting with colchicine (12, QD) or starting with uric acid lowering agents (17, QD). All patients received first prescriptions and were instructed to take the medication as prescribed. Followup was 6 months (gout 12 mo). All patients were aware of the monitoring capability of the package. At baseline a series of questionnaires was completed. We summarized the dosing histories as "taking compliance" (percentage of total prescribed doses taken), "correct dosing" (percentage of doses taken as prescribed), and "timing compliance" (percentage of doses taken within +/- 25% of prescribed interdose intervals). A total of 26,685 days (> 73 patient-years) were monitored. Compliance expressed as "taking compliance," mean (95% CI), "correct dosing," mean (95% CI), and "timing compliance," mean (95% CI) are: naproxen: 82% (75-90), 68% (57-80), 48% (34-61); diclofenac: 77% (61-93), 67% (47-87), 39% (21-57); MTX: 107% (98-117), 81% (75-87), 83% (76-90); SSZ: 72% (60-84), 55% (44-67), 25% (18-33); prednisolone: 96% (89-102), 88% (83-92), 82% (74-89); colchicine: 65% (48-81), 44% (26-62), 32% (18-46); and uric acid lowering agents: 84% (76-92), 74% (63-85), 65% (52-79). Missed doses occurred more frequently than taking of extra doses: in RA, on 10% of all monitored days there was no evidence of dosing, while on 3% of all monitored days extra doses were taken. In PMR and gout these data are 10% and 4%, and 15% and 7%, respectively. We observed a decline of compliance over time in all study medication groups. Multiple regression analyses showed that the class of medication (symptom modifying or disease controlling), the dosing frequency, the patient's sex, coping pattern (avoidance, passive reaction pattern, and expression of emotions), and the overall health (total Nottingham Health Profile score) together explained 67% of the variance in taking compliance (adjusted R2) (p = 0.002). Studying patient compliance with prescribed drug regimens utilizing electronic medication event monitors in RA, gout, and PMR showed that large differences exist in compliance between the various medication groups. Compliance declines over time. A regression model shows that it is possible to relate differences in patient compliance to a number of medication and patient related factors.


Wastewater treatment plant effluent as a source of microplastics: review of the fate, chemical interactions and potential risks to aquatic organisms.
Wastewater treatment plant (WWTP) effluent has been identified as a potential source of microplastics in the aquatic environment. Microplastics have recently been detected in wastewater effluent in Western Europe, Russia and the USA. As there are only a handful of studies on microplastics in wastewater, it is difficult to accurately determine the contribution of wastewater effluent as a source of microplastics. However, even the small amounts of microplastics detected in wastewater effluent may be a remarkable source given the large volumes of wastewater treatment effluent discharged to the aquatic environment annually. Further, there is strong evidence that microplastics can interact with wastewater-associated contaminants, which has the potential to transport chemicals to aquatic organisms after exposure to contaminated microplastics. In this review we apply lessons learned from the literature on microplastics in the aquatic environment and knowledge on current wastewater treatment technologies, with the aim of identifying the research gaps in terms of (i) the fate of microplastics in WWTPs, (ii) the potential interaction of wastewater-based microplastics with trace organic contaminants and metals, and (iii) the risk for aquatic organisms.


Effect of domestic cooking on carotenoids, tocopherols, fatty acids, phenolics, and antioxidant activities of lentils (Lens culinaris).
The phytochemicals and antioxidant activity in lipophilic and hydrophilic (extractable and bound) fractions of lentils before and after domestic cooking were investigated. The hydrophilic fractions in lentils contributed much more to the antioxidant activity than the lipophilic fraction. The phenolic content of lentils was mainly composed of extractable compounds. Significant changes (P < 0.05) in carotenoid, tocopherol, total phenolic, and condensed tannin contents of both extractable and bound phenolics fractions, as well as in antioxidant activities, were found in lentils before and after cooking. More specifically, cooking was found to favor the release of carotenoids and tocopherols and flavonols (kaempferol glycosides), but led to losses of flavanols (monomeric and condensed tannin). Whereas reduced flavanols and other phenolic compounds may have negatively affected the antioxidant activity, other components, especially the lipophilic antioxidants, were increased. The present study suggests that incorporation of cooked lentils into the diet will not cause significant loss to the phytochemical antioxidants and thus will retain the potential health benefits.


Light-induced gauge fields for ultracold atoms.
Gauge fields are central in our modern understanding of physics at all scales. At the highest energy scales known, the microscopic universe is governed by particles interacting with each other through the exchange of gauge bosons. At the largest length scales, our Universe is ruled by gravity, whose gauge structure suggests the existence of a particle-the graviton-that mediates the gravitational force. At the mesoscopic scale, solid-state systems are subjected to gauge fields of different nature: materials can be immersed in external electromagnetic fields, but they can also feature emerging gauge fields in their low-energy description. In this review, we focus on another kind of gauge field: those engineered in systems of ultracold neutral atoms. In these setups, atoms are suitably coupled to laser fields that generate effective gauge potentials in their description. Neutral atoms 'feeling' laser-induced gauge potentials can potentially mimic the behavior of an electron gas subjected to a magnetic field, but also, the interaction of elementary particles with non-Abelian gauge fields. Here, we review different realized and proposed techniques for creating gauge potentials-both Abelian and non-Abelian-in atomic systems and discuss their implication in the context of quantum simulation. While most of these setups concern the realization of background and classical gauge potentials, we conclude with more exotic proposals where these synthetic fields might be made dynamical, in view of simulating interacting gauge theories with cold atoms.


Digital junk: food and beverage marketing on Facebook.
We assessed the amount, reach, and nature of energy-dense, nutrient-poor (EDNP) food and beverage marketing on Facebook. We conducted a content analysis of the marketing techniques used by the 27 most popular food and beverage brand Facebook pages in Australia. We coded content across 19 marketing categories; data were collected from the day each page launched (mean = 3.65 years of activity per page). We analyzed 13 international pages and 14 Australian-based brand pages; 4 brands (Subway, Coca-Cola, Slurpee, Maltesers) had both national and international pages. Pages widely used marketing features unique to social media that increase consumer interaction and engagement. Common techniques were competitions based on user-generated content, interactive games, and apps. Four pages included apps that allowed followers to place an order directly through Facebook. Adolescent and young adult Facebook users appeared most receptive to engaging with this content. By using the interactive and social aspects of Facebook to market products, EDNP food brands capitalize on users' social networks and magnify the reach and personal relevance of their marketing messages.


Can Fiber Application Affect the Fracture Strength of Endodontically Treated Teeth Restored with a Low Viscosity Bulk-Fill Composite?
The aim of this study is to evaluate the effects of different fiber insertion techniques and thermomechanical aging on the fracture resistance of endodontically treated mandibular premolar teeth restored using bulk-fill composites. Eighty human mandibular premolar teeth were randomly divided into eight groups: Group IN, Group BF, Group PRF1, Group PRF2, Group IN-TMA, Group BF-TMA, Group PRF1-TMA ,and Group PRF2-TMA. Group IN (intact) and Group IN-TMA (intact but subjected to thermomechanical aging) served as control groups. In the other six groups, endodontic treatment was performed and standardized mesio-occluso-distal (MOD) cavities were prepared. In BF, PRF1, and PRF2, the cavities were restored with bulk-fill composite only, bulk-fill/Ribbond, and bulk-fill/additional Ribbond, respectively. In BF-TMA, PRF1-TMA, and PRF2-TMA, the teeth were subjected to thermomechanical aging after the restorations. All of the teeth were fractured on the universal testing machine. Fracture surfaces were analyzed with a stereomicroscope. Control groups showed significantly higher fracture strengths than tested groups (P<0.05). No statistically significant difference was observed among the tested groups (P>0.05). Most of the favorable fractures were seen in PRF1, PRF2, and PRF2-TMA. Most of the unfavorable fractures were seen in BF-TMA. Although fiber insertion with different techniques did not increase the fracture strength of teeth restored with bulk-fill composites, it increased the favorable fracture modes. Thermomechanical aging did not change the fracture strength of the groups.


Flexible Fiberoptic Bronchoscopy Through the Laryngeal Mask Airway in a Small Premature Infant.
Flexible bronchoscopy (FB) can be used safely for wider indications in children. Ultra-thin bronchoscopes are used for premature or newborn infants and are of limited diagnostic value. Bronchoscopes with a suction channel, may lead to problems when the nasal passage is narrow, particularly in patients under 2.5 kg. In addition, it may cause bronchospasm and hypoxia in small infants during the procedure because of an almost complete obstruction of the airway. A laryngeal mask airway (LMA) may prevent both bronchospasm and hypoxia because it does not need a nasal route. In addition, the LMA allows positive pressure ventilation during the procedure. We performed FB with a 3.7 mm bronchoscope through the LMA in a 75-day-old and 1910 g premature baby with atelectasis. This is the first and successful FB experience in such a small premature infant reported in the literature using a 3.7 mm bronchoscope through the LMA.


POR hits the road: import and assembly of a plastid protein.
The biosynthesis of chlorophyll is a strictly light-dependent multistep process in higher plants. The light-dependent step is catalysed by NADPH:protochlorophyllide oxidoreductase (POR, EC.126.96.36.199), which reduces protochlorophyllide (Pchlide) to chlorophyllide (Chlide). POR is nucleus-encoded and post-translationally imported into plastids. It has been proposed that the import of a POR protein isozyme (PORA) is totally dependent on Pchlide and uses a novel import pathway. This proposal is based on findings that PORA import only occurs in the presence of Pchlide and that the presence of overexpressed precursor of Rubisco small subunit (pSS), a protein which is known to use the general import pathway, does not outcompete PORA import. Another study demonstrated that POR precursor protein (pPOR) can be cross-linked to one of the components in the translocation machinery, Toc75, in the absence of Pchlide, and that its import can be outcompeted by the addition of the pSS. This indicates that pSS and pPOR may use the same translocation mechanism. Thus, POR does not necessarily need Pchlide for import--which is in contrast to earlier observations--and the exact POR import mechanism remains unresolved. Once in the stroma, the POR transit peptide is cleaved off and the mature POR protein is associated to the plastid inner membranes. Formation of the correct membrane-associated, thermolysin-protected assembly is strictly dependent of NADPH. As a final step, the formation of the NADPH-Pchlide-POR complex occurs. When POR accumulates in the membranes of proplastids, an attraction of monogalactosyl diacylglycerol (MGDG) can occur, leading to the formation of prolamellar bodies (PLBs) and the development of etioplasts in darkness.


Bayesian Calibration of Computational Knee Models to Estimate Subject-Specific Ligament Properties, Tibiofemoral Kinematics, and Anterior Cruciate Ligament Force With Uncertainty Quantification.
High-grade knee laxity is associated with early anterior cruciate ligament (ACL) graft failure, poor function, and compromised clinical outcome. Yet, the specific ligaments and ligament properties driving knee laxity remain poorly understood. We described a Bayesian calibration methodology for predicting unknown ligament properties in a computational knee model. Then, we applied the method to estimate unknown ligament properties with uncertainty bounds using tibiofemoral kinematics and ACL force measurements from two cadaver knees that spanned a range of laxities; these knees were tested using a robotic manipulator. The unknown ligament properties were from the Bayesian set of plausible ligament properties, as specified by their posterior distribution. Finally, we developed a calibrated predictor of tibiofemoral kinematics and ACL force with their own uncertainty bounds. The calibrated predictor was developed by first collecting the posterior draws of the kinematics and ACL force that are induced by the posterior draws of the ligament properties and model parameters. Bayesian calibration identified unique ligament slack lengths for the two knee models and produced ACL force and kinematic predictions that were closer to the corresponding in vitro measurement than those from a standard optimization technique. This Bayesian framework quantifies uncertainty in both ligament properties and model outputs; an important step towards developing subject-specific computational models to improve treatment for ACL injury.


Automated screening of microtubule growth dynamics identifies MARK2 as a regulator of leading edge microtubules downstream of Rac1 in migrating cells.
Polarized microtubule (MT) growth in the leading edge is critical to directed cell migration, and is mediated by Rac1 GTPase. To find downstream targets of Rac1 that affect MT assembly dynamics, we performed an RNAi screen of 23 MT binding and regulatory factors and identified RNAi treatments that suppressed changes in MT dynamics induced by constitutively activated Rac1. By analyzing fluorescent EB3 dynamics with automated tracking, we found that RNAi treatments targeting p150(glued), APC2, spastin, EB1, Op18, or MARK2 blocked Rac1-mediated MT growth in lamellipodia. MARK2 was the only protein whose RNAi targeting additionally suppressed Rac1 effects on MT orientation in lamellipodia, and thus became the focus of further study. We show that GFP-MARK2 rescued effects of MARK2 depletion on MT growth lifetime and orientation, and GFP-MARK2 localized in lamellipodia in a Rac1-activity-dependent manner. In a wound-edge motility assay, MARK2-depleted cells failed to polarize their centrosomes or exhibit oriented MT growth in the leading edge, and displayed defects in directional cell migration. Thus, automated image analysis of MT assembly dynamics identified MARK2 as a target regulated downstream of Rac1 that promotes oriented MT growth in the leading edge to mediate directed cell migration.


Differential recognition of Staphylococcus aureus quorum-sensing signals depends on both extracellular loops 1 and 2 of the transmembrane sensor AgrC.
Virulence in Staphylococcus aureus is regulated via agr-dependent quorum sensing in which an autoinducing peptide (AIP) activates AgrC, a histidine protein kinase. AIPs are usually thiolactones containing seven to nine amino acid residues in which the thiol of the central cysteine is linked to the alpha-carboxyl of the C-terminal amino acid residue. The staphylococcal agr locus has diverged such that the AIPs of the four different S. aureus agr groups self-activate but cross-inhibit. Consequently, although the agr system is conserved among the staphylococci, it has undergone significant evolutionary divergence whereby to retain functionality, any changes in the AIP-encoding gene (agrD) that modifies AIP structure must be accompanied by corresponding changes in the AgrC receptor. Since AIP-1 and AIP-4 only differ by a single amino acid, we compared the transmembrane topology of AgrC1 and AgrC4 to identify amino acid residues involved in AIP recognition. As only two of the three predicted extracellular loops exhibited amino acid differences, site-specific mutagenesis was used to exchange the key AgrC1 and AgrC4 amino acid residues in each loop either singly or in combination. A novel lux-based agrP3 reporter gene fusion was constructed to evaluate the response of the mutated AgrC receptors. The data obtained revealed that while differential recognition of AIP-1 and AIP-4 depends primarily on three amino acid residues in loop 2, loop 1 is essential for receptor activation by the cognate AIP. Furthermore, a single mutation in the AgrC1 loop 2 resulted in conversion of (Ala5)AIP-1 from a potent antagonist to an activator, essentially resulting in the forced evolution of a new AIP group. Taken together, our data indicate that loop 2 constitutes the predicted hydrophobic pocket that binds the AIP thiolactone ring while the exocyclic amino acid tail interacts with loop 1 to facilitate receptor activation.


Testosterone-induced relaxation of coronary arteries: activation of BKCa channels via the cGMP-dependent protein kinase.
Androgens are reported to have both beneficial and detrimental effects on human cardiovascular health. The aim of this study was to characterize nongenomic signaling mechanisms in coronary artery smooth muscle (CASM) and define the ionic basis of testosterone (TES) action. TES-induced relaxation of endothelium-denuded porcine coronary arteries was nearly abolished by 20 nM iberiotoxin, a highly specific inhibitor of large-conductance, calcium-activated potassium (BK(Ca)) channels. Molecular patch-clamp studies confirmed that nanomolar concentrations of TES stimulated BK(Ca) channel activity by ∼100-fold and that inhibition of nitric oxide synthase (NOS) activity by N(G)-monomethyl-L-arginine nearly abolished this effect. Inhibition of nitric oxide (NO) synthesis or guanylyl cyclase activity also attenuated TES-induced coronary artery relaxation but did not alter relaxation due to 8-bromo-cGMP. Furthermore, we detected TES-stimulated NO production in porcine coronary arteries and in human CASM cells via stimulation of the type 1 neuronal NOS isoform. Inhibition of the cGMP-dependent protein kinase (PKG) attenuated TES-stimulated BK(Ca) channel activity, and direct assay determined that TES increased activity of PKG in a concentration-dependent fashion. Last, the stimulatory effect of TES on BK(Ca) channel activity was mimicked by addition of purified PKG to the cytoplasmic surface of a cell-free membrane patch from CASM myocytes (∼100-fold increase). These findings indicate that TES-induced relaxation of endothelium-denuded coronary arteries is mediated, at least in part, by enhanced NO production, leading to cGMP synthesis and PKG activation, which, in turn, opens BK(Ca) channels. These findings provide a molecular mechanism that could help explain why androgens have been reported to relax coronary arteries and relieve angina pectoris.


Quantifying fear effects on prey demography in nature.
In recent years, it has been argued that the effect of predator fear exacts a greater demographic toll on prey populations than the direct killing of prey. However, efforts to quantify the effects of fear have primarily relied on experiments that replace predators with predator cues. Interpretation of these experiments must consider two important caveats: (1) the magnitude of experimenter-induced predator cues may not be realistically comparable to those of the prey's natural sensory environment, and (2) given functional predators are removed from the treatments, the fear effect is measured in the absence of any consumptive effects, a situation which never occurs in nature. We contend that demographic consequences of fear in natural populations may have been overestimated because the intensity of predator cues applied by experimenters in the majority of studies has been unnaturally high, in some instances rarely occurring in nature without consumption. Furthermore, the removal of consumption from the treatments creates the potential situation that individual prey in poor condition (those most likely to contribute strongly to the observed fear effects via starvation or reduced reproductive output) may have been consumed by predators in nature prior to the expression of fear effects, thus confounding consumptive and fear effects. Here, we describe an alternative treatment design that does not utilize predator cues, and in so doing, better quantifies the demographic effect of fear on wild populations. This treatment substitutes the traditional cue experiment where consumptive effects are eliminated and fear is simulated with a design where fear is removed and consumptive effects are simulated through the experimental removal of prey. Comparison to a natural population would give a more robust estimate of the effect of fear in the presence of consumption on the demographic variable of interest. This approach represents a critical advance in quantifying the mechanistic pathways through which predation structures ecological communities. Discussing the merits of both treatments will motivate researchers to go beyond simply describing the existence of fear effects and focus on testing their true magnitude in wild populations and natural communities.


Exercise Training in Group 2 Pulmonary Hypertension: Which Intensity and What Modality.
Pulmonary hypertension (PH) due to left-sided heart disease (LSHD) is a common and disconcerting occurrence. For example, both heart failure (HF) with preserved and reduced ejection fraction (HFpEF and HFrEF) often lead to PH as a consequence of a chronic elevation in left atrial filling pressure. A wealth of literature demonstrates the value of exercise training (ET) in patients with LSHD, which is particularly robust in patients with HFrEF and growing in patients with HFpEF. While the effects of ET have not been specifically explored in the LSHD-PH phenotype (i.e., composite pathophysiologic characteristics of patients in this advanced disease state), the overall body of evidence supports clinical application in this subgroup. Moderate intensity aerobic ET significantly improves peak oxygen consumption, quality of life and prognosis in patients with HF. Resistance ET significantly improves muscle strength and endurance in patients with HF, which further enhance functional capacity. When warranted, inspiratory muscle training and neuromuscular electrical stimulation are becoming recognized as important components of a comprehensive rehabilitation program. This review will provide a detailed account of ET programing considerations in patients with LSHD with a particular focus on those concomitantly diagnosed with PH.


Cannabinoids in the Treatment of Epilepsy: Hard Evidence at Last?
The interest in cannabis-based products for the treatment of refractory epilepsy has skyrocketed in recent years. Marijuana and other cannabis products with high content in Δ(9) - tetrahydrocannabinol (THC), utilized primarily for recreational purposes, are generally unsuitable for this indication, primarily because THC is associated with many undesired effects. Compared with THC, cannabidiol (CBD) shows a better defined anticonvulsant profile in animal models and is largely devoid of adverse psychoactive effects and abuse liability. Over the years, this has led to an increasing use of CBD-enriched extracts in seizure disorders, particularly in children. Although improvement in seizure control and other benefits on sleep and behavior have been often reported, interpretation of the data is made difficult by the uncontrolled nature of these observations. Evidence concerning the potential anti-seizure efficacy of cannabinoids reached a turning point in the last 12 months, with the completion of three high-quality placebo-controlled adjunctive-therapy trials of a purified CBD product in patients with Dravet syndrome and Lennox-Gastaut syndrome. In these studies, CBD was found to be superior to placebo in reducing the frequency of convulsive (tonic-clonic, tonic, clonic, and atonic) seizures in patients with Dravet syndrome, and the frequency of drop seizures in patients with Lennox-Gastaut syndrome. For the first time, there is now class 1 evidence that adjunctive use of CBD improves seizure control in patients with specific epilepsy syndromes. Based on currently available information, however, it is unclear whether the improved seizure control described in these trials was related to a direct action of CBD, or was mediated by drug interactions with concomitant medications, particularly a marked increased in plasma levels of N-desmethylclobazam, the active metabolite of clobazam. Clarification of the relative contribution of CBD to improved seizure outcome requires re-assessment of trial data for the subgroup of patients not comedicated with clobazam, or the conduction of further studies controlling for the confounding effect of this interaction.


Postprandial lipid responses of butter blend containing fish oil in a single-meal study in humans.
The postprandial effects of a butter product containing fish oil were investigated in a single-meal, randomized crossover study with a commercial butter product as the control. Twelve healthy males consumed two test meals with (13)C-labelled cholesterol (45 mg) and either an interesterified butter blend with fish oil (352 mg n-3 long-chain PUFA (LCPUFA)) or the commercial butter blend. Blood samples were collected after the meals and in the fasting condition on the test day and the following morning, and were analysed for cholesterol absorption, plasma lipid profile and fatty acid composition. No significant difference in the postprandial plasma fatty acid composition was observed between the groups, neither difference in cholesterol absorption, plasma cholesterol or the cholesterol contents of plasma lipoproteins. The incorporation of fish oil in the butter resulted in a significant lower concentration of triacylglycerols in the plasma 2 h after the meal in comparison with the commercial butter blend (p = 0.02); there was, however, no significant difference 24 h after the meal. In conclusion, fish oil-enriched butter blend provides a source to increase the intake of n-3 LCPUFA in the population, but has no acute effect on cholesterol absorption and plasma cholesterol concentration in human.


Impact of the Spanish smoking law on exposure to second-hand smoke and respiratory health in hospitality workers: a cohort study.
A smoke-free law came into effect in Spain on 1st January 2006, affecting all enclosed workplaces except hospitality venues, whose proprietors can choose among totally a smoke-free policy, a partial restriction with designated smoking areas, or no restriction on smoking on the premises. We aimed to evaluate the impact of the law among hospitality workers by assessing second-hand smoke (SHS) exposure and the frequency of respiratory symptoms before and one year after the ban. We formed a baseline cohort of 431 hospitality workers in Spain and 45 workers in Portugal and Andorra. Of them, 318 (66.8%) were successfully followed up 12 months after the ban, and 137 nonsmokers were included in this analysis. We obtained self-reported exposure to SHS and the presence of respiratory symptoms, and collected saliva samples for cotinine measurement. Salivary cotinine decreased by 55.6% after the ban among nonsmoker workers in venues where smoking was totally prohibited (from median of 1.6 ng/ml before to 0.5 ng/ml, p<0.01). Cotinine concentration decreased by 27.6% (p = 0.068) among workers in venues with designated smoking areas, and by 10.7% (p = 0.475) among workers in venues where smoking was allowed. In Portugal and Andorra, no differences between cotinine concentration were found before (1.2 ng/ml) and after the ban (1.2 ng/ml). In Spain, reported respiratory symptom declined significantly (by 71.9%; p<0.05) among workers in venues that became smoke-free. After adjustment for potential confounders, salivary cotinine and respiratory symptoms decreased significantly among workers in Spanish hospitality venues where smoking was totally banned. Among nonsmoker hospitality workers in bars and restaurants where smoking was allowed, exposure to SHS after the ban remained similar to pre-law levels. The partial restrictions on smoking in Spanish hospitality venues do not sufficiently protect hospitality workers against SHS or its consequences for respiratory health.


Validity and reliability of a multiple-group measurement scale for interprofessional collaboration.
Many measurement scales for interprofessional collaboration are developed for one health professional group, typically nurses. Evaluating interprofessional collaborative relationships can benefit from employing a measurement scale suitable for multiple health provider groups, including physicians and other health professionals. To this end, the paper begins development of a new interprofessional collaboration measurement scale designed for use with nurses, physicians, and other professionals practicing in contemporary acute care settings. The paper investigates validity and reliability of data from nurses evaluating interprofessional collaboration of physicians and shows initial results for other rater/target combinations. Items from a published scale originally designed for nurses were adapted to a round robin proxy report format appropriate for multiple health provider groups. Registered nurses, physicians, and allied health professionals practicing in inpatient wards/services of 15 community and academic hospitals in Toronto, Canada completed the adapted scale. Exploratory and confirmatory factor analysis of responses to the adapted scale examined dimensionality, construct and concurrent validity, and reliability of nurses' response data. Correlations between the adapted scale, the nurse-physician relations subscale of the Nursing Work Index, and the Attitudes Toward Health Care Teams Scale were calculated. Differences of mean scores on the Nursing Work Index and the interprofessional collaboration scale were compared between hospitals. Exploratory factor analysis revealed 3 factors in the adapted interprofessional collaboration scale - labeled Communication, Accommodation, and Isolation - which were subsequently corroborated by confirmatory factor analysis. Nurses' scale responses about physician collaboration had convergent, discriminant, and concurrent validity, and acceptable reliability. The new scale is suitable for use with nurses assessing physicians. The scale may yield valid and reliable data from physicians and others, but measurement equivalence and other properties of the scale should be investigated before it is used with multiple health professional groups.


Sprouty2 expression controls endothelial monolayer integrity and quiescence.
Vascular integrity is fundamental to the formation of mature blood vessels and depends on a functional, quiescent endothelial monolayer. However, how endothelial cells enter and maintain quiescence in the presence of angiogenic factors is still poorly understood. Here we identify the fibroblast growth factor (FGF) antagonist Sprouty2 (Spry2) as a key player in mediating endothelial quiescence and barrier integrity in mouse aortic endothelial cells (MAECs): Spry2 knockout MAECs show spindle-like shapes and are incapable of forming a functional, impermeable endothelial monolayer in the presence of FGF2. Whereas dense wild type cells exhibit contact inhibition and stop to proliferate, Spry2 knockout MAECs remain responsive to FGF2 and continue to proliferate even at high cell densities. Importantly, the anti-proliferative effect of Spry2 is absent in sparsely plated cells. This cell density-dependent Spry2 function correlates with highly increased Spry2 expression in confluent wild type MAECs. Spry2 protein expression is barely detectable in single cells but steadily increases in cells growing to high cell densities, with hypoxia being one contributing factor. At confluence, Spry2 expression correlates with intact cell-cell contacts, whereas disruption of cell-cell contacts by EGTA, TNFα and thrombin decreases Spry2 protein expression. In confluent cells, high Spry2 levels correlate with decreased extracellular signal-regulated kinase 1/2 (Erk1/2) phosphorylation. In contrast, dense Spry2 knockout MAECs exhibit enhanced signaling by Erk1/2. Moreover, inhibiting Erk1/2 activity in Spry2 knockout cells restores wild type cobblestone monolayer morphology. This study thus reveals a novel Spry2 function, which mediates endothelial contact inhibition and barrier integrity.


Evaluation of the medicinal use of clay minerals as antibacterial agents.
Natural clays have been used to heal skin infections since the earliest recorded history. Recently our attention was drawn to a clinical use of French green clay (rich in Fe-smectite) for healing Buruli ulcer, a necrotizing fasciitis ('flesh-eating' infection) caused by Mycobacterium ulcerans. These clays and others like them are interesting as they may reveal an antibacterial mechanism that could provide an inexpensive treatment for this and other skin infections, especially in global areas with limited hospitals and medical resources.Microbiological testing of two French green clays, and other clays used traditionally for healing, identified three samples that were effective at killing a broad-spectrum of human pathogens. A clear distinction must be made between 'healing clays' and those we have identified as antibacterial clays. The highly adsorptive properties of many clays may contribute to healing a variety of ailments, although they are not antibacterial. The antibacterial process displayed by the three identified clays is unknown. Therefore, we have investigated the mineralogical and chemical compositions of the antibacterial clays for comparison with non-antibacterial clays in an attempt to elucidate differences that may lead to identification of the antibacterial mechanism(s).The two French green clays used to treat Buruli ulcer, while similar in mineralogy, crystal size, and major element chemistry, have opposite effects on the bacterial populations tested. One clay deposit promoted bacterial growth whereas another killed the bacteria. The reasons for the difference in antibacterial properties thus far show that the bactericidal mechanism is not physical (e.g., an attraction between clay and bacteria), but by a chemical transfer or reaction. The chemical variables are still under investigation.Cation exchange experiments showed that the antibacterial component of the clay can be removed, implicating exchangeable cations in the antibacterial process. Furthermore, aqueous leachates of the antibacterial clays effectively kill the bacteria. Progressively heating the clay leads first to dehydration (200 degrees C), then dehydroxylation (550 degrees C or more), and finally to destruction of the clay mineral structure by (~900 degrees C). By identifying the elements lost after each heating step, and testing the bactericidal effect of the heated product, we eliminated many toxins from consideration (e.g., microbes, organic compounds, volatile elements) and identified several redox-sensitive refractory metals that are common among antibacterial clays. We conclude that the pH and oxidation state buffered by the clay mineral surfaces is key to controlling the solution chemistry and redox related reactions occurring at the bacterial cell wall.


Impact of HIV-1 Subtypes on AIDS Progression in a Brazilian Cohort.
Viral and host factors are known to play a role in the different patterns of AIDS progression. The cocirculation of HIV-1 subtypes B, F1, BBR, and BF1; the occasional detection of HIV-1 subtype D; and an increasing prevalence of subtype C and other recombinant forms have been described in Rio de Janeiro, Brazil. The aim of this study was to evaluate the potential association of HIV-1 subtypes circulating among HIV-1+ individuals in Rio de Janeiro with AIDS disease progression. For this purpose, 246 HIV-1 individuals under clinical and laboratory follow-up from 1986 to 2011 were classified according to their progression to AIDS in typical progressors (n = 133), rapid progressors (n = 95), and long-term nonprogressors (n = 18). The env-gp120 region was amplified and sequenced. Neighbor-joining phylogenetic inferences were performed in Mega 6 and bootscan analysis was performed in Simplot 3.5.1. The Kaplan-Meier method and Cox modeling were performed to determine the time until an AIDS-defining event based on the HIV-1 subtypes/variants. Similar AIDS progression rates were observed among individuals infected with HIV-1 subtype B and variant BBR. However, a direct association between more rapid AIDS progression and HIV-1 subtypes, D and BF1, was confirmed in the multivariate analysis, corroborating previous results. Our findings contribute to the investigation of the possible influence of HIV-1 subtypes in AIDS outcome.


Effects of chronic lithium, amitriptyline and mianserin on glucoregulation, corticosterone and energy balance in the rat.
Major negative side-effects reported for mood-stabilizing and antidepressant drugs in humans are excess weight gain and carbohydrate craving. The aim of the present study was to establish whether the rat could usefully be employed in investigation of these phenomena. Three experiments investigated the effects of chronic lithium (40 mg/kg LiCl), amitriptyline (2.5 mg/kg), mianserin (2.5 mg/kg) and saline administration (15-20 days, one subcutaneous injection/day) on body weight, food intake and fluid intake. Water and food cubes were provided in all experiments. Additionally available, as separate fluid sources, in Experiment 2 were 24% sucrose and 0.6% saccharin and in Experiment 3, 0.6% saccharin. Blood was collected for plasma glucose and insulin determinations 20-24 hours after the final injections. Lithium administration resulted in a marked increase in weight gain but only if both sucrose and saccharin were available (Experiment 2). Saccharin intake was increased with lithium treatment as was total caloric intake with sucrose available. Amitriptyline induced a sweetness craving; however, weight gain was somewhat depressed with just cubes available (Experiment 1) and only normalised by the additional availability of sucrose and saccharin (Experiment 2). With amitriptyline, total caloric intake was never different from controls. Weight gain was slightly suppressed and caloric intake slightly elevated by mianserin but importantly the two effects combined for a decrease in metabolic efficiency which was particularly exaggerated under the condition of carbohydrate availability (Experiment 2). Lithium and amitriptyline both produced hyperinsulinemia with normoglycemia whether or not the rate of weight gain was changed and whether or not intake was increased. Corticosterone levels were elevated by all drug treatments in Experiment 1.(ABSTRACT TRUNCATED AT 250 WORDS)


Association between beliefs about medicines and self-medication with analgesics among patients with dental pain.
Self-medication with analgesics in dental pain management is a common practice as most of these medicines are available over-the-counter (OTC). The study aims to examine the relationship between beliefs about medicines and self-medication with analgesics in dental pain management in Malaysia. This cross-sectional study was conducted among conveniently sampled patients attending dental clinics, located in Kuala Lumpur, Malaysia to assess association between self-medication with analgesics and patient's beliefs about medicines via Beliefs about Medicines Questionnaire. Participants were evaluated for their self-medication practices via 4 items. Further assessment was done via Quantitative Analgesic Questionnaire (QAQ) regarding the analgesics taken. Statistical analyses were performed using SPSS version 24, with 0.05 as level of significance. The prevalence of self-medication with analgesics was 29.4%, with 95.6% of the participants took analgesics when necessary. Participants practising self-medication for dental pain reported more positive beliefs in General-Necessity (13.04 vs. 9.98, p = 0.001) than those not practising self-medication. However, these participants had weaker beliefs in General-Harm (12.00 vs. 10.29, p = 0.006) and General-Overuse (11.38 vs. 10.31, p = 0.032) than those not practising self-medication. Participants beliefs in General-Harm (r = -0.243; p = 0.003) and General-Overuse (r = -0.203; p = 0.012) were negatively correlated with total QAQ point. The study found that individuals who practised self-medication had stronger beliefs about the benefits of medicines and weaker beliefs in viewing medicines as harmful and overused. Findings can guide public education to improve the safety aspects of self-medication with analgesics in dental practice.


Mothers' experiences of a telephone based breastfeeding support intervention after discharge from neonatal intensive care units: a mixed-method study.
After discharge from a neonatal intensive care unit (NICU), many mothers of preterm infants (gestational age < 37 weeks) experience a lack of support for breastfeeding. An intervention study was designed to evaluate the effects of proactive (a daily telephone call initiated by a member of a breastfeeding support team) and/or reactive (mothers could call the breastfeeding support team) telephone based breastfeeding support for mothers after discharge from the NICU. The mothers in the intervention group had access to both proactive and reactive support; the mothers in the control group only had access to reactive support. The aim of this study was to explore the mothers' experiences of the proactive and reactive telephone support. This study was a qualitatively driven, mixed-method evaluation using three data sources: questionnaires with qualitative open-ended questions, visual analogue scales and telephone interviews. In total, 365 mothers contributed data for this study. The qualitative data were analysed with an inductive thematic network analysis, while the quantitative data were analysed with Student's t-test and the chi-square test. Proactive support contributed to greater satisfaction and involvement in breastfeeding support. The mothers who received proactive support reported that they felt strengthened, supported and secure, as a result of the continuous care provided by staff who were knowledgeable and experienced (i.e., in breastfeeding and preterm infants), which resulted in the global theme 'Empowered by proactive support'. The mothers who received reactive support experienced contradictory feelings; some felt secure because they had the opportunity to call for support, whereas others found it difficult to decide when and if they should use the service, which resulted in the global theme; 'Duality of reactive support'. There were positive aspects of both proactive (i.e., greater satisfaction and feelings of empowerment) and reactive support (i.e., the opportunity to call for support); however, the provision of reactive support alone may be inadequate for those with the greatest need for support as they are the least likely to access it. NCT01806480 on 5 March 2013.


Prevention of infections associated with combat-related extremity injuries.
During combat operations, extremities continue to be the most common sites of injury with associated high rates of infectious complications. Overall, ∼ 15% of patients with extremity injuries develop osteomyelitis, and ∼ 17% of those infections relapse or recur. The bacteria infecting these wounds have included multidrug-resistant bacteria such as Acinetobacter baumannii, Pseudomonas aeruginosa, extended-spectrum β-lactamase-producing Klebsiella species and Escherichia coli, and methicillin-resistant Staphylococcus aureus. The goals of extremity injury care are to prevent infection, promote fracture healing, and restore function. In this review, we use a systematic assessment of military and civilian extremity trauma data to provide evidence-based recommendations for the varying management strategies to care for combat-related extremity injuries to decrease infection rates. We emphasize postinjury antimicrobial therapy, debridement and irrigation, and surgical wound management including addressing ongoing areas of controversy and needed research. In addition, we address adjuvants that are increasingly being examined, including local antimicrobial therapy, flap closure, oxygen therapy, negative pressure wound therapy, and wound effluent characterization. This evidence-based medicine review was produced to support the Guidelines for the Prevention of Infections Associated With Combat-Related Injuries: 2011 Update contained in this supplement of Journal of Trauma.


Combined C-H functionalization/Cope rearrangement with vinyl ethers as a surrogate for the vinylogous Mukaiyama aldol reaction.
Vinyl ethers selectively undergo the combined C-H functionalization/Cope rearrangement reaction via an s-cis/boat transition state. With chiral dirhodium catalysts, products are generated in a highly diastereoselective and enantioselective fashion. This reaction can be considered as a surrogate to the traditional vinylogous Mukaiyama aldol reaction. Effective kinetic resolution has been achieved, leading to the recovery of a cyclic vinyl ether with axial chirality of high enantiomeric purity.


Impact of the introduction of emergency ultrasound to one large UK emergency department: the REBUS study.
Firstly, to detail the experiences of one UK training region in establishing an emergency ultrasound (EU) training programme and secondly, to report the initial 30 months of EU scanning experience. Prospective study of all documented emergency department (ED) ultrasound scans. Results were extracted from written paper reports and/or electronically saved images. Details of scan date, time, type of scan, grade of operator, supervision status (whether supervised by a level 1 competent scanner) and whether the scan was clinical (performed or supervised by a level 1 operator) or training, were recorded. EU scans were reviewed for quality (internal quality assurance) and for diagnostic accuracy (external quality assurance). Between 14 January 2009 and 4 July 2011, 626 scans were performed by 41 operators. 263 (42%) scans were completed outside of normal working hours (09:00 to 17:00). There were 251 abdominal aorta and inferior vena cava scans (40% of all scans) and 198 focused assessment with sonography in trauma scans (32%). The number of scans performed by each operator varied widely. 87 scans (14%) were supervised but the majority (459; 73%) were not. 484 (77%) scans were for training purposes, 124 (20%) were clinical scans and the majority (401; 63%) were performed by either speciality registrars (ST4-6) or specialist registrars (SpR). When the three commonest types of scans performed were analysed, eight false positives and 11 false negatives were identified. Only seven of these were deemed of poor quality and none led to poor patient outcome. Since the acquisition of our ED ultrasound machine and the development of a quality assured training programme, on average 20 scans per month have been performed in the ED, with no known adverse patient outcomes.


Simulation in the medical undergraduate curriculum to promote interprofessional collaboration for acute care: a systematic review.
This literature review summarises interprofessional, simulation-based interventions in the context of preparing undergraduate and prelicensure healthcare students for the management of acutely unstable patients. There was a particular focus on the impact of such interventions on medical students. The review sought to identify the range of described interprofessional education (IPE) learning outcomes, types of learners, methods used to evaluate intervention effectiveness and study conclusions. We systematically compiled this information and generated review findings through narrative summary. A total of 18 articles fulfilled the review criteria. The diversity of IPE interventions described suggests a developing field where the opportunities provided by simulation are still being explored. With significant heterogeneity among the studies, comparison between them was unfeasible, but each study provided a unique narrative on the complex interplay between intervention, curriculum, learning activities, learners and facilitators. Together, the narratives provided in these studies reflect positively on undergraduate simulation-based interventions to promote interprofessional collaboration in acute care settings, and provide the basis for recommendations for future IPE design and delivery, and areas requiring further research.


Regulation of oxytocin receptor responsiveness by G protein-coupled receptor kinase 6 in human myometrial smooth muscle.
Oxytocin plays an important role in the progression, timing, and modulation of uterine contraction during labor and is widely used as an uterotonic agent. We investigated the mechanisms regulating oxytocin receptor (OTR) signaling in human primary myometrial smooth muscle cells and the ULTR cell-line. Oxytocin produced concentration-dependent increases in both total [(3)H]inositol phosphate accumulation and intracellular Ca(2+) concentration ([Ca(2+)](i)); however, responses were greater and more reproducible in the ULTR cell line. Assessment of phospholipase C activity in single cells revealed that the OTR desensitizes rapidly (within 5 min) in the presence of oxytocin (100 nm). To characterize OTR desensitization further, cells were stimulated with a maximally effective concentration of oxytocin (100 nm, 30 sec) followed by a variable washout period and a second identical application of oxytocin. This brief exposure to oxytocin caused a marked decrease (>70%) in OTR responsiveness to rechallenge and was fully reversed by increasing the time period between agonist challenges. To assess involvement of G protein-coupled receptor kinases (GRKs) in OTR desensitization, cells were transfected with small interfering RNAs to cause specific > or =75% knockdown of GRKs 2, 3, 5, or 6. In both primary myometrial and ULTR cells, knockdown of GRK6 largely prevented oxytocin-induced OTR desensitization; in contrast, selective depletion of GRKs 2, 3, or 5 was without effect. These data indicate that GRK6 recruitment is a cardinal effector of OTR responsiveness and provide mechanistic insight into the likely in vivo regulation of OTR signaling in uterine smooth muscle.


Communicating curriculum reform to students: advice in hindsight...
In view of the changing health care needs of communities, curriculum reform of traditional curricula is inevitable. In order to allay the apprehension that may accompany such change, curriculum development and implementation should be an inclusive process, with both staff and students being well informed of the planned reform. In 2001, the Nelson R. Mandela School of Medicine implemented Year 1 of a problem-based learning curriculum. During the design phase, students and staff were invited to take part in the development and were kept abreast of developments through meetings and newsletters. A survey of Years 1-5 students of the last intake into the traditional curriculum was undertaken a few months prior to the implementation of the new programme. Students were generally well informed about the impending change, having heard about it from fellow students and staff. The more senior the students, the less the perceived impact of the reform. Although most of what students had heard was correct, some, however, had misconceptions that were generally extreme views (e.g. all self-directed learning; no Anatomy) about the new programme. Others expressed valid concerns (e.g. underpreparedness of students from disadvantaged schools; overcrowding in hospitals). Advice offered to institutions considering curriculum reform include using various methods to inform internal and external affected parties, ensuring that the student representative body and staff is well informed, reiterating the need for the change, confirming that the new programme meets recognised standards and that the students most affected are reassured about their future studies.


Pharmacogenetics of morphine: Potential implications in sickle cell disease.
Morphine is frequently used to treat painful episodes associated with sickle cell disease (SCD) but may fail to provide adequate analgesia in many patients. This concise review focuses on unique disease related changes in physiologic variables associated with SCD that impacts pharmacokinetics and pharmacodynamics of morphine and may contribute to the variability in analgesia. Emerging evidence suggests that the allelic variants in the genes involving the opioid (UGT2B7, OPRM1, and ABCB1 genes) and nonopioid system (COMT gene) can alter the efficacy of morphine.


In hot pursuit: fluctuating mating system and sexual selection in sand lizards.
A changing climate is expected to have profound effects on many aspects of ectotherm biology. We report on a decade-long study of free-ranging sand lizards (Lacerta agilis), exposed to an increasing mean mating season temperature and with known operational sex ratios. We assessed year-to-year variation in sexual selection on body size and postcopulatory sperm competition and cryptic female choice. Higher temperature was not linked to strength of sexual selection on body mass, but operational sex ratio (more males) did increase the strength of sexual selection on body size. Elevated temperature increased mating rate and number of sires per clutch with positive effects on offspring fitness. In years when the "quality" of a female's partners was more variable (in standard errors of a male sexual ornament), clutches showed less multiple paternity. This agrees with prior laboratory trials in which females exercised stronger cryptic female choice when male quality varied more. An increased number of sires contributing to within-clutch paternity decreased the risk of having malformed offspring. Ultimately, such variation may contribute to highly dynamic and shifting selection mosaics in the wild, with potential implications for the evolutionary ecology of mating systems and population responses to rapidly changing environmental conditions.


The redox imbalanced mutants of Arabidopsis differentiate signaling pathways for redox regulation of chloroplast antioxidant enzymes.
A network of enzymatic and nonenzymatic antioxidants protects chloroplasts from photooxidative damage. With all enzymatic components being nuclear encoded, the control of the antioxidant capacity depends on chloroplast-to-nucleus redox signaling. Using an Arabidopsis (Arabidopsis thaliana) reporter gene line expressing luciferase under control of the redox-sensitive 2-cysteine peroxiredoxin A (2CPA) promoter, six mutants with low 2CPA promoter activity were isolated, of which five mutants show limitations in redox-box regulation of the 2CPA promoter. In addition to 2CPA, the transcript levels for other chloroplast antioxidant enzymes were decreased, although a higher oxidation status of the ascorbate pool, a higher reduction state of the plastoquinone pool, and an increased oxidation status of the 2-Cys peroxiredoxin pool demonstrated photooxidative stress conditions. Greening of the mutants, chloroplast ultrastructure, steady-state photosynthesis, and the responses to the stress hormone abscisic acid were wild type like. In the rosette state, the mutants were more sensitive to low CO2 and to hydrogen peroxide. Comparison of gene expression patterns and stress sensitivity characterizes the mutants as redox imbalanced in the regulation of nuclear-encoded chloroplast antioxidant enzymes and differentiates redox signaling cascades.


B7-H5 costimulates human T cells via CD28H.
The B7/CD28 family has profound modulatory effects in immune responses and constitutes an important target for the development of novel therapeutic drugs against human diseases. Here we describe a new CD28 homologue (CD28H) that has unique functions in the regulation of the human immune response and is absent in mice. CD28H is constitutively expressed on all naive T cells. Repetitive antigenic exposure, however, induces a complete loss of CD28H on many T cells, and CD28H negative T cells have a phenotype of terminal differentiation and senescence. After extensive screening in a receptor array, a B7-like molecule, B7 homologue 5 (B7-H5), was identified as a specific ligand for CD28H. B7-H5 is constitutively found in macrophages and could be induced on dendritic cells. The B7-H5/CD28H interaction selectively costimulates human T-cell growth and cytokine production via an AKT-dependent signalling cascade. Our study identifies a novel costimulatory pathway regulating human T-cell responses.


1995 Volvo Award in clinical sciences. Determinants of lumbar disc degeneration. A study relating lifetime exposures and magnetic resonance imaging findings in identical twins.
Retrospective cohort. To investigate the effects of lifetime exposure to commonly suspected risk factors on disc degeneration using magnetic resonance imaging, and to estimate the effects of these suspected risk factors relative to age and familial aggregation, reflecting genetic and shared environmental influences. Structural and biochemical changes associated with disc degeneration are suspected as the underlying conditions of many back-related symptoms. Little is known about the determinants of disc degeneration. Based on lifetime discordance in suspected environmental risk factors for disc degeneration, 115 male identical twin pairs were selected. An in-depth interview was conducted of occupational and leisure time physical loading, driving, and smoking. Disc degeneration was evaluated using observational and digital magnetic resonance imaging assessment methods. Heavier lifetime occupational and leisure physical loading was associated with greater disc degeneration in the upper lumbar levels (P = 0.055 - 0.001), whereas sedentary work was associated with lesser degeneration (P = 0.006). These univariate associations did not reach statistical significance in the lower lumbar region. In multivariate analyses of the upper lumbar levels, the mean job code explained 7% of the variability in observational disc degeneration scores; the addition of age explained 16%, and familial aggregation improved the model such that 77% of the variability was explained. In the lower lumbar levels, leisure time physical loading entered the multivariate model, explaining 2% of the variability. Adding age explained 9%, and familial aggregation raised the variability in disc degeneration scores explained to 43%. The present study findings suggest that disc degeneration may be explained primarily by genetic influences and by unidentified factors, which may include complex, unpredictable interactions. The particular environmental factors studied, which have been among those most widely suspected of accelerating disc degeneration, had very modest effects.


BU10038 as a safe opioid analgesic with fewer side-effects after systemic and intrathecal administration in primates.
The marked increase in mis-use of prescription opioids has greatly affected our society. One potential solution is to develop improved analgesics which have agonist action at both mu opioid peptide (MOP) and nociceptin/orphanin FQ peptide (NOP) receptors. BU10038 is a recently identified bifunctional MOP/NOP partial agonist. The aim of this study was to determine the functional profile of systemic or spinal delivery of BU10038 in primates after acute and chronic administration. A series of behavioural and physiological assays have been established specifically to reflect the therapeutic (analgesia) and side-effects (abuse potential, respiratory depression, itch, physical dependence, and tolerance) of opioid analgesics in rhesus monkeys. After systemic administration, BU10038 (0.001-0.01 mg kg-1) dose-dependently produced long-lasting antinociceptive and antihypersensitive effects. Unlike the MOP agonist oxycodone, BU10038 lacked reinforcing effects (i.e. little or no abuse liability), and BU10038 did not compromise the physiological functions of primates including respiration, cardiovascular activities, and body temperature at antinociceptive doses and a 10-30-fold higher dose (0.01-0.1 mg kg-1). After intrathecal administration, BU10038 (3 μg) exerted morphine-comparable antinociception and antihypersensitivity without itch scratching responses. Unlike morphine, BU10038 did not cause the development of physical dependence and tolerance after repeated and chronic administration. These in vivo findings demonstrate the translational potential of bifunctional MOP/NOP receptor agonists such as BU10038 as a safe, non-addictive analgesic with fewer side-effects in primates. This study strongly supports that bifunctional MOP/NOP agonists may provide improved analgesics and an alternative solution for the ongoing prescription opioid crisis.


Genetic interactions in yeast between Ypt GTPases and Arf guanine nucleotide exchangers.
Two families of GTPases, Arfs and Ypt/rabs, are key regulators of vesicular transport. While Arf proteins are implicated in vesicle budding from the donor compartment, Ypt/rab proteins are involved in the targeting of vesicles to the acceptor compartment. Recently, we have shown a role for Ypt31/32p in exit from the yeast trans-Golgi, suggesting a possible function for Ypt/rab proteins in vesicle budding as well. Here we report the identification of a new member of the Sec7-domain family, SYT1, as a high-copy suppressor of a ypt31/32 mutation. Several proteins that belong to the Sec7-domain family, including the yeast Gea1p, have recently been shown to stimulate nucleotide exchange by Arf GTPases. Nucleotide exchange by Arf GTPases, the switch from the GDP- to the GTP-bound form, is thought to be crucial for their function. Sec7p itself has an important role in the yeast secretory pathway. However, its mechanism of action is not yet understood. We show that all members of the Sec7-domain family exhibit distinct genetic interactions with the YPT genes. Biochemical assays demonstrate that, although the homology between the members of the Sec7-domain family is relatively low (20-35%) and limited to a small domain, they all can act as guanine nucleotide exchange factors (GEFs) for Arf proteins, but not for Ypt GTPases. The Sec7-domain of Sec7p is sufficient for this activity. Interestingly, the Sec7 domain activity is inhibited by brefeldin A (BFA), a fungal metabolite that inhibits some of the Arf-GEFs, indicating that this domain is a target for BFA. These results demonstrate that the ability to act as Arf-GEFs is a general property of all Sec7-domain proteins in yeast. The genetic interactions observed between Arf GEFs and Ypt GTPases suggest the existence of a Ypt-Arf GTPase cascade in the secretory pathway.


Production of asexual and sexual offspring in the triploid sexual planarian Dugesia ryukyuensis.
Certain freshwater planarians reproduce asexually as well as sexually, and their chromosomal ploidies include polyploidy, aneuploidy and mixoploidy. Previously, we successfully performed an experiment in which a clonal population produced by asexual reproduction of the Dugesia ryukyuensis (OH strain) switched to the sexual mode of reproduction. Worms of this strain are triploid with a pericentric inversion on Chromosome 4. The worms were switched to sexual reproduction after being fed with sexually mature Bdellocephala brunnea, which is a sexually reproducing species. The resulting sexualized OH strain produced cocoons filled with several eggs. Two putative factors, Mendelian factor(s) and chromosomal control(s), have been proposed as determining the reproductive mode. The present study demonstrated that inbreeding of the resultant sexualized worms produced the following four types of offspring through sexual reproduction: diploid asexual worms, triploid asexual worms, diploid sexual worms and triploid sexual worms. The chromosomal mutation on Chromosome 4 was inherited by these offspring independent of their reproductive mode. These results provide two important pieces of information: (i) the putative genetic factor was not necessarily inherited in a Mendelian fashion; and (ii) the reproductive mode is not regulated by chromosomal changes such as polyploidy or chromosomal mutations. This suggests that asexuality in D. ryukyuensis is regulated by an unknown factor(s) other than a Mendelian factor or a chromosomal control.


Mitochondrial depletion in CD4+ and CD19+ peripheral lymphocytes in early stage Alzheimer's disease.
Alzheimer's disease (AD) may be associated with mitochondrial defects. The aim of the present study was to investigate changes in mitochondrial abundance in peripheral lymphocytes of early and late stage AD patients. We analysed levels of mitochondrial DNA (mtDNA) and mean fluorescence intensity (MFI) of the mitochondria-specific antibody 113-1 in CD4+, CD8+, CD19+ and CD56+ peripheral lymphocytes of early and late stage AD by quantitative real-time PCR and flow cytometry, respectively. In early stage AD, the levels of mtDNA were significantly decreased in CD4+, CD19+ and CD56+ peripheral lymphocytes while the MFI of 113-1 staining was significantly decreased in CD4+ and CD19+ cells. Thus, CD4+ and CD19+ peripheral lymphocytes of early stage AD patients exhibit mitochondrial depletion, as seen both at the level of DNA and protein.


Mechanisms for size-dependent protein segregation at immune synapses assessed with molecular rulers.
Immunological synapses are specialized intercellular contacts formed by several types of immune cells in contact with target cells or antigen-presenting cells. A late-stage immune synapse is commonly a bulls-eye pattern of immune cell receptor-ligand pairs surrounded by integrin complexes. Based on crystal structures, the intermembrane distance would be ∼15 nm for many immune cell receptor-ligand pairs, but ∼40 nm for integrin-ligand pairs. Close proximity of these two classes of intermembrane bonds would require significant membrane bending and such proteins can segregate according to their size, which may be key for receptor triggering. However, tools available to evaluate the intermembrane organization of the synapse are limited. Here, we present what we believe to be a novel approach to test the importance of size in the intercellular organization of proteins, using live-cell microscopy of a size-series of fluorescently-labeled molecules and quantum dots to act as molecular rulers. Small particles readily colocalized at the synapse with MHC class I bound to its cognate natural killer cell receptor, whereas particles larger than 15 nm were increasingly segregated from this interaction. Combined with modeling of the partitioning of the particles by scaled-particle adsorption theory, these molecular rulers show how membrane-bending elasticity can drive size-dependent exclusion of proteins within immune synapses.


[Actinomyces with anti-candida activity isolated from leaf-cutting ants Atta cephalotes (Formicidae: Myrmicinae: Attini)].
To isolate, select and identify actinomyces associated to leaf-cutting ants Atta cephalotes (Linnaeus, 1758), that present a greater anti-Candida activity. Cross-sectional study made with ants collected at a location in Huánuco, Peru, from which strands of actinomyces were isolated and later evaluated by in vitro testing in order to determine its antagonistic capacity against species of Candida. The actinomyces with greater antagonism were selected and cultured by agitation, then the reliable extracellular metabolites were obtained with organic solvents, and finally the crude extracts were evaluated to determine quantitatively the minimum inhibiting concentration (MIC). Thirty (30) actinomyces were isolated, of which 47% exhibited antagonistic activity against Candida albicans (C. albicans) ATCC 7516, 43% to Candida parapsilosis ATCC 7307, 37% to Candida tropicalis ATCC 7206 and C. albicans ATCC 10231, and 30% to C. albicans ATCC 98028. Organic extracts of the HAA-16 and HAA-17 strands exhibited noticeable anti-Candida activity, being the ethyl acetate extract of the HAA-17 strand the one with the highest performance thanks to a wider activity spectrum of MIC 3.25 mg/mL against C. albicans ATCC 7516 and Candida parapsilosis ATCC 7307. The selected actinomyces were identified by means of molecular techniques as members of the Streptomyces genus. Actinomyces associated to Atta cephalotes are excellent producers of bioactive compounds, being able to inhibit the growth of pathogenic mold of the Candida genus and with potential for application in the development of new natural products for the biomedical field.


Complete genome sequence of Rickettsia typhi and comparison with sequences of other rickettsiae.
Rickettsia typhi, the causative agent of murine typhus, is an obligate intracellular bacterium with a life cycle involving both vertebrate and invertebrate hosts. Here we present the complete genome sequence of R. typhi (1,111,496 bp) and compare it to the two published rickettsial genome sequences: R. prowazekii and R. conorii. We identified 877 genes in R. typhi encoding 3 rRNAs, 33 tRNAs, 3 noncoding RNAs, and 838 proteins, 3 of which are frameshifts. In addition, we discovered more than 40 pseudogenes, including the entire cytochrome c oxidase system. The three rickettsial genomes share 775 genes: 23 are found only in R. prowazekii and R. typhi, 15 are found only in R. conorii and R. typhi, and 24 are unique to R. typhi. Although most of the genes are colinear, there is a 35-kb inversion in gene order, which is close to the replication terminus, in R. typhi, compared to R. prowazekii and R. conorii. In addition, we found a 124-kb R. typhi-specific inversion, starting 19 kb from the origin of replication, compared to R. prowazekii and R. conorii. Inversions in this region are also seen in the unpublished genome sequences of R. sibirica and R. rickettsii, indicating that this region is a hot spot for rearrangements. Genome comparisons also revealed a 12-kb insertion in the R. prowazekii genome, relative to R. typhi and R. conorii, which appears to have occurred after the typhus (R. prowazekii and R. typhi) and spotted fever (R. conorii) groups diverged. The three-way comparison allowed further in silico analysis of the SpoT split genes, leading us to propose that the stringent response system is still functional in these rickettsiae.


Panax ginseng C.A Meyer root extract for moderate chronic obstructive pulmonary disease (COPD): study protocol for a randomised controlled trial.
Chronic obstructive pulmonary disease (COPD) impairs quality of life and leads to premature mortality. COPD sufferers experience progressive deterioration of lung function and decreased ability to undertake day-to-day activities. Ginseng has been used for thousands of years in Chinese medicine for respiratory symptoms. Several controlled clinical trials using ginseng for COPD have shown promising clinical effect, however these studies were generally small and with some potential bias, prompting the need for rigorously designed studies. The objective of this study is to evaluate the therapeutic value and safety profile of a standardised root extract of Panax ginseng C.A Meyer (ginseng) for symptomatic relief, with a focus on quality of life (QoL) improvements in individuals with moderate (Stage II) COPD FEV1/FVC < 0.7 and FEV₁ 50%-80% predicted. This paper describes the design of a randomised, multi-centre, double-blind, placebo controlled, two-armed parallel clinical trial. Two trial sites in Melbourne Australia will proportionately randomise a total of 168 participants to receive either ginseng capsule (100 mg) or matching placebo twice daily for 24 weeks. The primary outcomes will be based on three validated QoL questionnaires, St Georges Respiratory Questionnaire (SGRQ), Short Form Health Survey (SF-36) and the COPD Assessment Test (CAT). Secondary outcomes are based on lung function testing, relief medication usage and exacerbation frequency and severity. Safety endpoints include blood tests and adverse event reporting. Intention-to-treat will be applied to all data analyses. Findings from this study may lead to new therapeutic development for chronic respiratory diseases, particularly COPD. This protocol may also guide other investigators to develop quality herbal medicine clinical trials in the future. Australia and New Zealand Clinical Trials Register (ANZCTR): ACTRN12610000768099.


Do cardiac troponins provide prognostic insight in hemodialysis patients?
The diagnosis of myocardial necrosis in patients with chronic renal failure is often difficult because biochemical markers of cardiac damage such as creatine kinase MB (CKMB) and cardiac troponin T (cTnT) may be spuriously elevated. Recent small studies also report unexplained elevations in cardiac troponin I (cTnI) in chronic renal failure patients undergoing hemodialysis. The relative incidence of elevated cardiac troponins in this population and their relationship to clinical events remain unknown. To determine the incidence and prognostic significance of asymptomatic elevations of cTnT and cTnI in patients undergoing hemodialysis for chronic renal failure. Prospective cohort study. University tertiary care teaching hospital. One hundred thirteen patients over 21 years of age undergoing onsite hemodialysis were enrolled between December 1997 and February 1998. All-cause and cardiovascular mortality, hospitalization for acute myocardial infarction, unstable angina or congestive heart failure, new onset sustained arrhythmia or need for unscheduled emergency hemodialysis due to volume overload at 30 days and six months. The incidence of abnormal results for cTnT, cTnI and CKMB were 42%, 15% and 4%, respectively. Independent predictors of mortality at six months were median age greater than 63 years (odds ratio 14.3, 95% CI 1.5 to 130.3, P=0.019) and positive cTnT (odds ratio 13.6, 95% CI 2.5 to 73.2, P=0.002). Diabetics were more likely to have positive cTnI and cTnT results than nondiabetics (P<0.001 and P=0.023, respectively). cTnT is commonly elevated in patients with chronic renal failure even in the absence of acute coronary syndromes. cTnT may be an important independent prognostic marker in patients on hemodialysis for chronic renal failure. While less common, elevations of cTnI are more frequent than CKMB elevations. The basis of these cardiac troponin elevations is unclear. These findings may represent, in part, a subclinical myocardial injury, an inflammatory response to chronic renal failure or a chronically volume overloaded state.


Immunological Molecular Responses of Human Retinal Pigment Epithelial Cells to Infection With Toxoplasma gondii.
Ocular toxoplasmosis is the commonest clinical manifestation of infection with obligate intracellular parasite, Toxoplasma gondii. Active ocular toxoplasmosis is characterized by replication of T. gondii tachyzoites in the retina, with reactive inflammation. The multifunctional retinal pigment epithelium is a key target cell population for T. gondii. Since the global gene expression profile is germane to understanding molecular involvements of retinal pigment epithelial cells in ocular toxoplasmosis, we performed RNA-Sequencing (RNA-Seq) of human cells following infection with T. gondii tachyzoites. Primary cell isolates from eyes of cadaveric donors (n = 3), and the ARPE-19 human retinal pigment epithelial cell line, were infected for 24 h with GT-1 strain T. gondii tachyzoites (multiplicity of infection = 5) or incubated uninfected as control. Total and small RNA were extracted from cells and sequenced on the Illumina NextSeq 500 platform; results were aligned to the human hg19 reference sequence. Multidimensional scaling showed good separation between transcriptomes of infected and uninfected primary cell isolates, which were compared in edgeR software. This differential expression analysis revealed a sizeable response in the total RNA transcriptome-with significantly differentially expressed genes totaling 7,234 (28.9% of assigned transcripts)-but very limited changes in the small RNA transcriptome-totaling 30 (0.35% of assigned transcripts) and including 8 microRNA. Gene ontology and pathway enrichment analyses of differentially expressed total RNA in CAMERA software, identified a strong immunologic transcriptomic signature. We conducted RT-qPCR for 26 immune response-related protein-coding and long non-coding transcripts in epithelial cell isolates from different cadaveric donors (n = 3), extracted by a different isolation protocol but similarly infected with T. gondii, to confirm immunological activity of infected cells. For microRNA, increases in miR-146b and miR-212 were detected by RT-qPCR in 2 and 3 of these independent cell isolates. Biological network analysis in the InnateDB platform, including 735 annotated differentially expressed genes plus 2,046 first-order interactors, identified 10 contextural hubs and 5 subnetworks in the transcriptomic immune response of cells to T. gondii. Our observations provide a solid base for future studies of molecular and cellular interactions between T. gondii and the human retinal pigment epithelium to illuminate mechanisms of ocular toxoplasmosis.


Nail bed INJury Assessment Pilot (NINJA-P) study: should the nail plate be replaced or discarded after nail bed repair in children? Study protocol for a pilot randomised controlled trial.
Nail bed injuries account for the majority of paediatric hand trauma cases. Despite their frequency, controversy remains regarding their treatment. The accepted teaching is to remove the fingernail, repair the underlying nail bed with fine sutures and replace the nail under the nail fold. A recent study by Miranda et al. (Plast Reconst Surg. 129(2):394e-396e, 2012) suggests that replacing the nail is associated with increased complications, in particular post-operative infection. Nail bed INJury Assessment Pilot (NINJA-P) is an external pilot study for a large pragmatic, multicentre, randomised, controlled study (NINJA) to assess whether the nail should be replaced or discarded after nail bed repair in children under the age of 16. NINJA-P is a randomised pilot study. The participants are patients below 16 years of age who require surgical repair of the nail bed. Eligible patients will be randomised to receive one of two possible interventions. Group 1 will have the nail replaced after nail bed repair, and group 2 will have the nail discarded. The clinical outcome measures include the presence of post-operative complications at 2 weeks and 30 days, the cosmetic appearance of the nail at 4 months and the level of pain experienced by the child at their first dressings change at 2 weeks. In order to inform the design of the main NINJA trial, the following feasibility data will also be recorded: the number of potentially eligible children and the proportion which agree to take part in the study, the proportion of children who received the allocated treatment and reasons for any non-compliance and the proportion of participants with a valid response at each follow-up point. Neither the patient, family members nor treating physicians will be blinded. A replaced nail can take several weeks to fall off once a new nail has grown out. The cosmetic appearance of the nail at 4 months will be assessed by a blinded assessor. The NINJA-P pilot study will inform the design and execution of the NINJA trial, which will assess whether the nail should be replaced or discarded after nail bed repair in children under 16. It will provide randomised comparative evidence for the treatment of this common injury. First participant randomised: 21/04/2015, UKCRN Portfolio ID: 18516, ISRCTN16571591.


[Community-acquired pneumonia in hospitalized adult patients. Clinical presentation and prognostic factors].
Community-acquired pneumonia (CAP) is a serious health problem in Chile. To study prognostic factors on admission and outcome of CAP, in immune competent adult patients, hospitalized in the Catholic University Clinical Hospital. All adult patients admitted with a CAP in a period of 2 years were prospectively studied. Patients with immunodeficiency, solid tumors or receiving oral adrenal steroids were excluded from the study. In the study period, 463 patients (69 +/- 19 years, 55% male) were evaluated. Ninety four percent were treated with 2nd or 3rd generation cephalosporins. Mean hospital length of stay was 10 days. Mortality during hospital stay was 8% and in the ensuing 30 days, it was 12%. Bacterial etiology was established in 25% of cases. The most frequent pathogens isolated were Streptococcus pneumoniae (10.2%), Haemophilus influenzae (3.7%), Staphylococcus aureus (2.8%) and Gram negative bacilli (5.2%). Admission prognostic factors associated with hospital mortality were an age over 65 years, presence of comorbidity, chronic neurological and hepatic disease, suspicion of aspiration, duration of symptoms for less than 3 days, presence of dyspnea and altered mental status, absence of cough, fever and chills; low blood pressure, tachypnea, metabolic acidosis, hypoxemia, high blood urea nitrogen, hypernatremia, hyperkalemia, hyperphosphatemia, hypoalbuminemia, multilobar radiographic pulmonary infiltrates, bacteremia, high risk categories of the Fine Index (IV and V), and admission to Intermediate Care Unit or ICU. The features of community acquired pneumonia of these patients are similar to those reported abroad.


Erectile dysfunction in fit and healthy young men: psychological or pathological?
Epidemiological studies consistently show that prevalence of erectile dysfunction (ED) increases with ageing. Nonetheless, complaints of ED even in younger men are becoming more and more frequent. Healthcare professionals working in Sexual Medicine but even those operating in different clinical contexts might be adequately prepared to answer this increasing requirement. ED in younger men is likely to be overlooked and dismissed without performing any medical assessment, even the most basic ones, such as collection of medical history and physical exam. This is due to the widespread assumption that ED in younger individuals is a self-limiting condition, which does not deserve any clinical evaluation or therapy and can be managed only with patient reassurance. However, evidence shows that, in younger subjects, organic, psychological and relational conditions can contribute to the pathogenesis of ED and all these conditions might be evaluated and treated, whenever necessary. Among the organic conditions contributing to the onset of ED, metabolic and cardiovascular (CV) risk factors are surprisingly of particular relevance in this age group. In fact, in younger men with ED, even more than in older ones, recognizing CV risk factors or conditions suggestive of cardio-metabolic derangements can help identifying men who, although at low absolute risk due to young age, carry a high relative risk for development of CV events. In this view, the assessment of a possible organic component of ED even in younger individuals acquires a pivotal importance, because it offers the unique opportunity to unearth the presence of CV risk factors, thus allowing effective and high quality preventive interventions.


Behavior of CD34+ cells isolated from patients with polycythemia vera in NOD/SCID mice.
We investigated if polycythemia vera (PV) peripheral blood (PB) CD34+ cells contain cells capable of engrafting nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice and if the JAK2V617F mutational burden of these cells alters their behavior in NOD/SCID mice. CD34+ cells isolated from patients with PV, idiopathic myelofibrosis (IM), or granulocyte colony-stimulating factor-mobilized normal donors were transplanted into sublethally irradiated NOD/SCID mice. Cells engrafted into the NOD/SCID mice were analyzed flow cytometrically using lineage-specific antibodies. Genomic DNA was extracted from granulocytes, CD34+ cells, and sorted human CD45(+) cells purified from the bone marrow cells of these mice to examine their JAK2V617F mutational burdens. Multilineage human cell engraftment was observed in mice transplanted with CD34+ cells from mobilized normal volunteers, IM patients and PV patients with high JAK2V617F burden, but not in mice receiving grafts from PV patients with low JAK2V617F burden. The differentiation program of engrafting PV CD34+ cells with high JAK2V617F burden was remarkably different than that of IM CD34+ cells. The JAK2V617F allele frequency in the human CD45+ cells isolated from the mice receiving CD34+ cells was lower than that observed in the CD34+ cell grafts, indicating the persistence of a JAK2V617F negative compartment of stem cells. We conclude that PB CD34+ cells from PV patients with high JAK2V617F burden and patients with IM contain NOD/SCID repopulating cells, and that differentiation program of IM and PV CD34+ cells are dramatically different.


The ribosome biogenesis factor Nol11 is required for optimal rDNA transcription and craniofacial development in Xenopus.
The production of ribosomes is ubiquitous and fundamental to life. As such, it is surprising that defects in ribosome biogenesis underlie a growing number of symptomatically distinct inherited disorders, collectively called ribosomopathies. We previously determined that the nucleolar protein, NOL11, is essential for optimal pre-rRNA transcription and processing in human tissue culture cells. However, the role of NOL11 in the development of a multicellular organism remains unknown. Here, we reveal a critical function for NOL11 in vertebrate ribosome biogenesis and craniofacial development. Nol11 is strongly expressed in the developing cranial neural crest (CNC) of both amphibians and mammals, and knockdown of Xenopus nol11 results in impaired pre-rRNA transcription and processing, increased apoptosis, and abnormal development of the craniofacial cartilages. Inhibition of p53 rescues this skeletal phenotype, but not the underlying ribosome biogenesis defect, demonstrating an evolutionarily conserved control mechanism through which ribosome-impaired craniofacial cells are removed. Excessive activation of this mechanism impairs craniofacial development. Together, our findings reveal a novel requirement for Nol11 in craniofacial development, present the first frog model of a ribosomopathy, and provide further insight into the clinically important relationship between specific ribosome biogenesis proteins and craniofacial cell survival.


Automated SDS Depletion for Mass Spectrometry of Intact Membrane Proteins though Transmembrane Electrophoresis.
Membrane proteins are underrepresented in proteome analysis platforms because of their hydrophobic character, contributing to decreased solubility. Sodium dodecyl sulfate is a favored denaturant in proteomic workflows, facilitating cell lysis and protein dissolution; however, SDS impedes MS detection and therefore must be removed prior to analysis. Although strategies exist for SDS removal, they provide low recovery, purity, or reproducibility. Here we present a simple automated device, termed transmembrane electrophoresis (TME), incorporating the principles of membrane filtration, but with an applied electric current to ensure near-complete (99.9%) removal of the surfactant, including protein-bound SDS. Intact proteins are recovered in solution phase in high yield (90-100%) within 1 h of operation. The strategy is applied to protein standards and proteome mixtures, including an enriched membrane fraction from E. coli, resulting in quality MS spectra free of SDS adducts. The TME platform is applicable to both bottom-up MS/MS as well as LC-ESI-MS analysis of intact proteins. SDS-depleted fractions reveal a similar number of protein identifications (285) compared wit a non-SDS control (280), being highly correlated in terms of protein spectral counts. This fully automated approach to SDS removal presents a viable tool for proteome sample processing ahead of MS analysis. Data are available via ProteomeXchange, identifier PXD003941.


The effects of dopamine and adrenaline infusions on acid-base balance and systemic haemodynamics in severe infection.
Adrenaline is used increasingly in the management of septic shock, but its efficacy and safety are uncertain. In an open, randomised, crossover study we compared the effects of stepped doses of adrenaline 0.1 to 0.5 microgram/kg per min and dopamine 2.5 to 10 micrograms/kg per min on the haemodynamic and acid-base status of 23 patients critically ill with severe sepsis (n = 10) or severe malaria (n = 13). All patients completed the dopamine study whereas in 16 (84%) patients the adrenaline infusion had to be terminated before reaching, or during, the maximum dose because of lactic acidosis (p < 0.0002). Adrenaline was associated with a mean (95% CI) increase in plasma lactate of 3.2 (2.6 to 3.8) mmol/L, and mean falls in arterial pH of 0.052 (0.035-0.068) pH units and base excess of 3.8 (2.8-4.7) mmol/L. The geometric mean (95% CI) lactate increment per unit adrenaline dose was 8.2 (5.8-10.5) mmol/L per microgram/kg per min. In contrast dopamine was associated with a fall in lactate of 1.0 (0.4-1.5) mmol/L, a rise in base excess of 1.4 (0.7 to 2.0) mmol/L (p < 0.0001 in each case), and no effect on arterial pH. Both drugs induced significant increases in cardiac index and oxygen delivery with smaller increases in oxygen consumption and falls in systemic vascular resistance which were similar in severe malaria and severe sepsis (p > 0.1 in each case) [corrected]. Infusion of inotropic doses of adrenaline in severe infections causes lactic acidosis.


Structure of Kluyveromyces lactis subtelomeres: duplications and gene content.
We have constructed a map of the duplicated regions of Kluyveromyces lactis subtelomeres. Seven out of 12 subtelomeres contain an almost identical 9 kb long segment starting from the end. This segment is bordered by a long terminal repeat element. Two of the subtelomeres share sequence similarity that extends over a total of 20 kb. The other subtelomeres also contain duplicated regions of 1-6 kb. Nonduplicated regions contain unique genes and genes from paralog families. All duplicated segments are in the same orientation with respect to the telomere, probably as a result of genetic exchange. We map the only two copies of retrotransposons in the genome, in subtelomeres. Low-complexity gene sequences that encode threonine- and serine-rich peptides are associated with the subtelomeres of K. lactis, as in Saccharomyces cerevisiae. The ubiquity of these sequences in hemiascomycete genomes, and the propensity they have to encode proteins with extracellular localization, make these genes ideal candidates for fast evolving 'contingency' genes involved in the adaptation of a species to its environment.