Massive replica exchange Monte Carlo algorithm: a tool to access high pressure thermodynamics of hard systems.
We have explored the idea of producing the equilibrium equation of state, i.e. the pressure as a function of packing fraction, βP(φ), of a confined system up to very high pressures to yield the configuration that leads to the maximum packing fraction. For this purpose we have massively implemented the replica exchange Monte Carlo algorithm in graphics processing units (GPUs), in such a way that each GPU core handles a single simulation cell. This yields a very easy scheme to implement parallelization for a very large amount of replicas (thousands), which densely sample configuration space. We have tested this idea with a very well studied system, i.e. discs confined in a circular cavity, for a number of particles N ≤ 125. In all cases, our outcomes for configurations having maximum packing fractions are in perfect agreement with those already reported and conjectured optimal in the literature, for which there is no formal mathematical proof, strongly suggesting that they are indeed optimal configurations. Furthermore, in most cases, we have obtained the same function βP(φ), by compressing loose random configurations and by decompressing copies of the configuration having the largest packing fraction. This reveals numerically that the so obtained maximum packing configurations are the correct answer.


CCL5-CCR5 interaction provides antiapoptotic signals for macrophage survival during viral infection.
Host defense against viruses probably depends on targeted death of infected host cells and then clearance of cellular corpses by macrophages. For this process to be effective, the macrophage must presumably avoid its own virus-induced death. Here we identify one such mechanism. We show that mice lacking the chemokine Ccl5 are immune compromised to the point of delayed viral clearance, excessive airway inflammation and respiratory death after mouse parainfluenza or human influenza virus infection. Virus-inducible levels of Ccl5 are required to prevent apoptosis of virus-infected mouse macrophages in vivo and mouse and human macrophages ex vivo. The protective effect of Ccl5 requires activation of the Ccr5 chemokine receptor and consequent bilateral activation of G(alphai)-PI3K-AKT and G(alphai)-MEK-ERK signaling pathways. The antiapoptotic action of chemokine signaling may therefore allow scavengers to finally stop the host cell-to-cell infectious process.


MSClust: a tool for unsupervised mass spectra extraction of chromatography-mass spectrometry ion-wise aligned data.
Mass peak alignment (ion-wise alignment) has recently become a popular method for unsupervised data analysis in untargeted metabolic profiling. Here we present MSClust-a software tool for analysis GC-MS and LC-MS datasets derived from untargeted profiling. MSClust performs data reduction using unsupervised clustering and extraction of putative metabolite mass spectra from ion-wise chromatographic alignment data. The algorithm is based on the subtractive fuzzy clustering method that allows unsupervised determination of a number of metabolites in a data set and can deal with uncertain memberships of mass peaks in overlapping mass spectra. This approach is based purely on the actual information present in the data and does not require any prior metabolite knowledge. MSClust can be applied for both GC-MS and LC-MS alignment data sets. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11306-011-0368-2) contains supplementary material, which is available to authorized users.


Comparison of the optoacoustic signal generation efficiency of different nanoparticular contrast agents.
Optoacoustic imaging represents a new modality that allows noninvasive in vivo molecular imaging with optical contrast and acoustical resolution. Whereas structural or functional imaging applications such as imaging of vasculature do not require contrast enhancing agents, nanoprobes with defined biochemical binding behavior are needed for molecular imaging tasks. Since the contrast of this modality is based on the local optical absorption coefficient, all particle or molecule types that show significant absorption cross sections in the spectral range of the laser wavelength used for signal generation are suitable contrast agents. Currently, several particle types such as gold nanospheres, nanoshells, nanorods, or polymer particles are used as optoacoustic contrast agents. These particles have specific advantages with respect to their absorption properties, or in terms of biologically relevant features (biodegradability, binding to molecular markers). In the present study, a comparative analysis of the signal generation efficiency of gold nanorods, polymeric particles, and magnetite particles using a 1064 nm Nd:YAG laser for signal generation is described.


A K(ATP) channel gene effect on sleep duration: from genome-wide association studies to function in Drosophila.
Humans sleep approximately a third of their lifetime. The observation that individuals with either long or short sleep duration show associations with metabolic syndrome and psychiatric disorders suggests that the length of sleep is adaptive. Although sleep duration can be influenced by photoperiod (season) and phase of entrainment (chronotype), human familial sleep disorders indicate that there is a strong genetic modulation of sleep. Therefore, we conducted high-density genome-wide association studies for sleep duration in seven European populations (N=4251). We identified an intronic variant (rs11046205; P=3.99 × 10(-8)) in the ABCC9 gene that explains ≈5% of the variation in sleep duration. An influence of season and chronotype on sleep duration was solely observed in the replication sample (N=5949). Meta-analysis of the associations found in a subgroup of the replication sample, chosen for season of entry and chronotype, together with the discovery results showed genome-wide significance. RNA interference knockdown experiments of the conserved ABCC9 homologue in Drosophila neurons renders flies sleepless during the first 3 h of the night. ABCC9 encodes an ATP-sensitive potassium channel subunit (SUR2), serving as a sensor of intracellular energy metabolism.


Associations of self-reported and objectively measured sleep disturbances with depression among primary caregivers of children with disabilities.
The objective of this study was to determine the association between sleep and depression using both self-reported (subjective) and actigraphic (objective) sleep traits. A cross-sectional study was conducted among 175 female primary caregivers of children with disabilities receiving care at a rehabilitation center in Punta Arenas, Chile. The eight-item Patient Health Questionnaire was used to ascertain participants' depression status. The Pittsburgh Sleep Quality Index was used to define subjective, or perceived, sleep quality. Wrist-worn actigraph monitors, worn for seven consecutive nights, were used to characterize objective sleep quality and disturbances. Interviewer-administered questionnaires were used to collect information on sociodemographic and lifestyle factors. Linear regression models were fit using continuous sleep parameters as the dependent variables and depression status as the independent variable. Multivariable models were adjusted for body mass index, marital status, smoking status, education level, and children's disabilities. Using an eight-item Patient Health Questionnaire score ≥10, 26.3% of participants presented with depression. Depressed women were more likely to self-report overall poorer (subjective) sleep compared to non-depressed women; however, differences in sleep were not consistently noted using actigraphic (objective) sleep traits. Among the depressed, both sleep duration and total time in bed were significantly underestimated. In multivariable models, depression was negatively associated with sleep duration using both subjective (β=-0.71, standard error [SE] =0.25; P=0.006) and objective sleep (β=-0.42, SE =0.19; P=0.026). The association between sleep and depression differed comparing subjective and objective methods of assessment. Research strategies allowing for the integration of both perceived and objective measures of sleep traits are encouraged.


"Doing" social inclusion with ELSiTO: empowering learning for social inclusion through occupation.
The European partnership ELSiTO aimed to develop understanding of the nature and processes of social inclusion for persons experiencing mental illness. Partners were from Belgium, Greece and The Netherlands with over 30 members including mental health service users, occupational therapists and other staff. A knowledge-creation learning process was used during four international, experiential, visits and local meetings, which included visiting and describing good practice, telling stories of experiences, reflection and discussion. The partnership developed understandings of the nature and process of social inclusion, including both subjective and objective aspects interrelated with the doing of daily activities in the community. Members' work-related experiences, illustrated through their stories, depict the subjective aspects of social inclusion as they are shaped and framed by the objective conditions within a variety of work opportunities. Experiences in paid work, supported employment and voluntary work may both threaten and enhance mental health. Features of successful (voluntary) work experiences are identified. The importance is revealed of looking critically at current understandings of work and to move beyond a narrow focus on paid work in order to provide a range of work opportunities that will empower the individual's potential and promote inclusive communities.


Aryl hydrocarbon receptor activation and developmental toxicity in zebrafish in response to soil extracts containing unsubstituted and oxygenated PAHs.
Many industrial sites are polluted by complex mixtures of polycyclic aromatic compounds (PACs). Besides polycyclic aromatic hydrocarbons (PAHs), these mixtures often contain significant amounts of more polar PACs including oxygenated PAHs (oxy-PAHs). The effects of oxy-PAHs are, however, poorly known. Here we used zebrafish embryos to examine toxicities and transcriptional changes induced by PAC containing soil extracts from three different industrial sites: a gasworks (GAS), a former wood preservation site (WOOD), and a coke oven (COKE), and to PAH and oxy-PAH containing fractions of these. All extracts induced aryl hydrocarbon receptor (Ahr)-regulated mRNAs, malformations, and mortality. The WOOD extract was most toxic and the GAS extract least toxic. The extracts induced glutathione transferases and heat shock protein 70, suggesting that the toxicity also involved oxidative stress. With all extracts, Ahr2-knock-down reduced the toxicity, indicating a significant Ahr2-dependence on the effects. Ahr2-knock-down was most effective with the PAH fraction of the WOOD extract and with the oxy-PAH fraction of the COKE extract. Our results indicate that oxy-PAH containing mixtures can be as potent Ahr activators and developmental toxicants as PAHs. In addition to Ahr activating potency, the profile of cytochrome P4501 inhibitors may also determine the toxic potency of the extracts.


'Hospice inpatient deep vein thrombosis detection (HIDDen) in advanced non-malignant diseases': a longitudinal pilot study.
To gain preliminary data regarding the prevalence of proximal deep vein thrombosis (DVT) in those with non-malignant conditions admitted to specialist palliative care units (SPCUs). Data were collected as part of a prospective longitudinal observational study in five SPCUs in England, Wales and Northern Ireland (Registration: ISRCTN97567719) to estimate the prevalence of proximal femoral vein DVT in people admitted to SPCUs. The primary outcome for this exploratory substudy was the prevalence of DVT in patients with non-malignant palliative conditions. Consecutive consenting adults underwent bilateral femoral vein ultrasonography within 48 hours of admission. Data were collected on symptoms associated with venous thromboembolism. Patients were ineligible if the estimated prognosis was <5 days. Cross-sectional descriptive analysis was conducted on baseline data and prevalence estimates presented with 95% CIs. 1390 patients were screened, 28 patients had non-malignant disease and all were recruited. The mean age 68·8 (SD 12·0), range 43-86 years; men 61%; survival mean 86 (SD 108.5) range 1-345 days. No patient had a history of venous thromboembolism. Four (14%) were receiving thromboprophylaxis. Of 22 evaluable scans, 8 (36%, 95% CI: 17% to 59%) showed femoral vein DVT. The level of reported relevant symptoms (leg oedema, leg pain, chest pain and breathlessness) was high irrespective of the presence of DVT. Our exploratory data indicate one in three people admitted to an SPCU with non-malignant disease had a femoral vein DVT. Although definitive conclusions cannot be drawn, these data justify a larger prospective survey.


Computational prediction shines light on type III secretion origins.
Type III secretion system is a key bacterial symbiosis and pathogenicity mechanism responsible for a variety of infectious diseases, ranging from food-borne illnesses to the bubonic plague. In many Gram-negative bacteria, the type III secretion system transports effector proteins into host cells, converting resources to bacterial advantage. Here we introduce a computational method that identifies type III effectors by combining homology-based inference with de novo predictions, reaching up to 3-fold higher performance than existing tools. Our work reveals that signals for recognition and transport of effectors are distributed over the entire protein sequence instead of being confined to the N-terminus, as was previously thought. Our scan of hundreds of prokaryotic genomes identified previously unknown effectors, suggesting that type III secretion may have evolved prior to the archaea/bacteria split. Crucially, our method performs well for short sequence fragments, facilitating evaluation of microbial communities and rapid identification of bacterial pathogenicity - no genome assembly required. pEffect and its data sets are available at http://services.bromberglab.org/peffect.


Syndesome Therapeutics for Enhancing Diabetic Wound Healing.
Chronic wounds represent a major healthcare and economic problem worldwide. Advanced wound dressings that incorporate bioactive compounds have great potential for improving outcomes in patients with chronic wounds but significant challenges in designing treatments that are effective in long-standing, nonhealing wounds. Here, an optimized wound healing gel was developed that delivers syndecan-4 proteoliposomes ("syndesomes") with fibroblast growth factor-2 (FGF-2) to enhance diabetic wound healing. In vitro studies demonstrate that syndesomes markedly increase migration of keratinocytes and fibroblasts isolated from both nondiabetic and diabetic donors. In addition, syndesome treatment leads to increased endocytic processing of FGF-2 that includes enhanced recycling of FGF-2 to the cell surface after uptake. The optimized syndesome formulation was incorporated into an alginate wound dressing and tested in a splinted wound model in diabetic, ob/ob mice. It was found that wounds treated with syndesomes and FGF-2 have markedly enhanced wound closure in comparison to wounds treated with only FGF-2. Moreover, syndesomes have an immunomodulatory effect on wound macrophages, leading to a shift toward the M2 macrophage phenotype and alterations in the wound cytokine profile. Together, these studies show that delivery of exogenous syndecan-4 is an effective method for enhancing wound healing in the long-term diabetic diseased state.


Modulation of hippocampal neuronal resilience during aging by the Hsp70/Hsp90 co-chaperone STI1.
Chaperone networks are dysregulated with aging, but whether compromised Hsp70/Hsp90 chaperone function disturbs neuronal resilience is unknown. Stress-inducible phosphoprotein 1 (STI1; STIP1; HOP) is a co-chaperone that simultaneously interacts with Hsp70 and Hsp90, but whose function in vivo remains poorly understood. We combined in-depth analysis of chaperone genes in human datasets, analysis of a neuronal cell line lacking STI1 and of a mouse line with a hypomorphic Stip1 allele to investigate the requirement for STI1 in aging. Our experiments revealed that dysfunctional STI1 activity compromised Hsp70/Hsp90 chaperone network and neuronal resilience. The levels of a set of Hsp90 co-chaperones and client proteins were selectively affected by reduced levels of STI1, suggesting that their stability depends on functional Hsp70/Hsp90 machinery. Analysis of human databases revealed a subset of co-chaperones, including STI1, whose loss of function is incompatible with life in mammals, albeit they are not essential in yeast. Importantly, mice expressing a hypomorphic STI1 allele presented spontaneous age-dependent hippocampal neurodegeneration and reduced hippocampal volume, with consequent spatial memory deficit. We suggest that impaired STI1 function compromises Hsp70/Hsp90 chaperone activity in mammals and can by itself cause age-dependent hippocampal neurodegeneration in mice. Cover Image for this issue: doi: 10.1111/jnc.14749.


Characterization of RNA from Exosomes and Other Extracellular Vesicles Isolated by a Novel Spin Column-Based Method.
Exosomes and other extracellular vesicles (commonly referred to as EVs) have generated a lot of attention for their potential applications in both diagnostics and therapeutics. The contents of these vesicles are the subject of intense research, and the relatively recent discovery of RNA inside EVs has raised interest in the biological function of these RNAs as well as their potential as biomarkers for cancer and other diseases. Traditional ultracentrifugation-based protocols to isolate EVs are labor-intensive and subject to significant variability. Various attempts to develop methods with robust, reproducible performance have not yet been completely successful. Here, we report the development and characterization of a spin column-based method for the isolation of total RNA from EVs in serum and plasma. This method isolates highly pure RNA of equal or higher quantity compared to ultracentrifugation, with high specificity for vesicular over non-vesicular RNA. The spin columns have a capacity to handle up to 4 mL sample volume, enabling detection of low-abundance transcripts in serum and plasma. We conclude that the method is an improvement over traditional methods in providing a faster, more standardized way to achieve reliable high quality RNA preparations from EVs in biofluids such as serum and plasma. The first kit utilizing this new method has recently been made available by Qiagen as "exoRNeasy Serum/Plasma Maxi Kit".


Application of pulsed photoacoustics in water at high pressure.
The application of pulsed photoacoustics to the study of liquids at pressures of up to 350 bars is discussed. The design and development of an in-line sensor for the subsea monitoring of crude oil concentrations in water is reported. Crude oil detection sensitivities at parts per million concentrations were achieved with prototype instrumentation. A comparison of experimental results and a theoretical prediction of the pressure dependence of the pulsed photoacoustic response from water is outlined. The results demonstrate that existing models that describe pulsed photoacoustic generation in liquids are applicable to high-pressure conditions.


Male sex in houseflies is determined by Mdmd, a paralog of the generic splice factor gene CWC22.
Across species, animals have diverse sex determination pathways, each consisting of a hierarchical cascade of genes and its associated regulatory mechanism. Houseflies have a distinctive polymorphic sex determination system in which a dominant male determiner, the M-factor, can reside on any of the chromosomes. We identified a gene, Musca domesticamale determiner (Mdmd), as the M-factor. Mdmd originated from a duplication of the spliceosomal factor gene CWC22 (nucampholin). Targeted Mdmd disruption results in complete sex reversal to fertile females because of a shift from male to female expression of the downstream genes transformer and doublesex The presence of Mdmd on different chromosomes indicates that Mdmd translocated to different genomic sites. Thus, an instructive signal in sex determination can arise by duplication and neofunctionalization of an essential splicing regulator.


Effects of postural drainage, exercise, and cough on mucus clearance in chronic bronchitis.
The effects of postural drainage, exercise, and cough on mucus clearance were compared in 8 patients with chronic bronchitis. A bolus of 99mTc albumin aerosol was inhaled at a high inspiratory flow rate to enhance proximal deposition. Retention of deposited aerosol in the lung, as a function of time, was quantified using a gamma camera and subsequent computer analysis. Coughing greatly accelerated total lung (P less than 0.005) and peripheral (P less than 0.005) mucus clearance. Exercise resulted in much smaller changes than did cough, but significantly increased total lung clearance (P less than 0.005). Postural drainage in which coughing was prohibited did not alter clearance. These results have therapeutic implications and stress the importance of controlling cough when evaluating therapeutic interventions by these techniques.


Concomitant macro and microvascular complications in diabetic nephropathy.
To determine the prevalence of concomitant microvascular and macrovascular complications of diabetic nephropathy we retrospectively reviewed the medical records of all 1,952 type 2 diabetic patients followed-up at Security Forces Hospital, Riyadh, Saudi Arabia from January 1989 to December 2004. There were 626 (32.1%) patients (294 (47%) were males) who developed diabetic nephropathy. Their mean age was 66.9 +/- 11.4 years, mean duration of diabetes was 15.4 +/- 7.5 years, mean age at the onset of nephropathy was 61.5 +/- 12.4 years, and mean duration of nephropathy was 3.9 +/- 3.8 years. Concomitant diabetic complications included cataract (38.2%), acute coronary syndrome (36.1%), peripheral neuropathy (24.9%), myocardial infarction (24.1%), background retinopathy (22.4%), stroke (17.6%), proliferative retinopathy (11.7%), foot infection (7.3%), limb amputation (3.7%) and blindness (3%). Hypertension was documented in 577 (92.2%) patients, dyslipidemia in 266 (42.5%) and mortality from all causes in 86 (13.7%). There were 148 (23.6%) patients with one complication, 81 (12.9%) with two, 83 (13.3%) with three, and 61 (9.7%) with four or more. Deterioration of glomerular filtration rate was observed in 464 (74%) patients and doubling of serum creatinine in 250 (39.9%), while 95 (15.2%) developed end-stage renal disease (ESRD) at the end of study and 79 (12.6%) required dialysis. Complications were significantly more prevalent among males with greater number reaching ESRD level than females (P< 0.05). Relative risks of developing complications were significant after the onset of nephropathy; ACS (1.41), MI (1.49), stroke (1.48), diabetic foot (1.6), amputation (1.58) and death (1.93). We conclude that complications of diabetes are aggressive and progressive including high prevalence of diabetic nephropathy. Careful monitoring and proper institution of management protocols should be implemented to identify diabetic patients at high risk for complications and mitigate progression into ESRD.


A non-linear mixed effect model for innate immune response: In vivo kinetics of endotoxin and its induction of the cytokines tumor necrosis factor alpha and interleukin-6.
Endotoxin, a component of the outer membrane of Gram-negative bacteria, has been extensively studied as a stimulator of the innate immune response. However, the temporal aspects and exposure-response relationship of endotoxin and resulting cytokine induction and tolerance development is less well defined. The aim of this work was to establish an in silico model that simultaneously captures and connects the in vivo time-courses of endotoxin, tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), and associated tolerance development. Data from six studies of porcine endotoxemia in anesthetized piglets (n = 116) were combined and used in the analysis, with purified endotoxin (Escherichia coli O111:B4) being infused intravenously for 1-30 h in rates of 0.063-16.0 μg/kg/h across studies. All data were modelled simultaneously by means of importance sampling in the non-linear mixed effects modelling software NONMEM. The infused endotoxin followed one-compartment disposition and non-linear elimination, and stimulated the production of TNF-α to describe the rapid increase in plasma concentration. Tolerance development, observed as declining TNF-α concentration with continued infusion of endotoxin, was also driven by endotoxin as a concentration-dependent increase in the potency parameter related to TNF-α production (EC50). Production of IL-6 was stimulated by both endotoxin and TNF-α, and four consecutive transit compartments described delayed increase in plasma IL-6. A model which simultaneously account for the time-courses of endotoxin and two immune response markers, the cytokines TNF-α and IL-6, as well as the development of endotoxin tolerance, was successfully established. This model-based approach is unique in its description of the time-courses and their interrelation and may be applied within research on immune response to bacterial endotoxin, or in pre-clinical pharmaceutical research when dealing with study design or translational aspects.


Trends in hospitalization rate, hospital case fatality, and mortality rate of stroke by subtype in Minneapolis-St. Paul, 1980-2002.
The stroke mortality rates have been declining in the USA for decades. Less is known about trends in stroke incidence rates, but some studies indicate they have declined. The stroke case fatality has also been declining. Little information exists on trends in stroke subtypes. We examined trends in mortality, hospitalization rate, and hospital case fatality of stroke by subtype in the Minneapolis-St. Paul area from 1980 to 2002. We estimated hospitalization rates and case fatality for > or =30-year-olds with data from the Minnesota Hospital Association. We estimated mortality rates with counts from the Minnesota Department of Health. Rates were age adjusted to the US 2000 standard by the direct method using census estimates. We tested for significant trends using linear regression. Total stroke mortality and hospital case fatality both declined by almost 50% over the study period, while the rate of stroke hospitalization was relatively stable. Ischemic stroke hospitalization rates increased, while hospital case fatality and mortality rates decreased. Subarachnoid hemorrhage in-hospital case fatality and mortality rates declined, while the hospitalization rate was stable. Intracerebral hemorrhage hospitalization rates increased minimally, hospital case fatality declined, and the mortality rate was stable. These data suggest that declines in total stroke case fatality are contributing most to declining mortality rates in the presence of stable total stroke attack rates.


Protective effects of quercetin from oxidative/nitrosative stress under intermittent hypobaric hypoxia exposure in the rat's heart.
Exposure to high altitude in hypobaric hypoxia (HH) is considered to be a physiological oxidative/nitrosative stress. Quercetin (Que) is an effective antioxidant and free radical scavenger against oxidative/nitrosative stress. The aim of this study was to investigate the cardioprotective effects of Que in animals exposed to intermittent HH (IHH) and therefore exposed to oxidative/nitrosative stress. Wistar albino male rats were exposed to short-term (2 days) or long-term (4 weeks; 5 days/week) IHH in a hypobaric chamber (5,500 m, 8 h/day, 380 mmHg, 12% O2, and 88% N2). Half of the animals received natural antioxidant Que (body weight: 30 mg/kg) daily before each IHH exposure and the remaining rats received vehicle (carboxymethylcellulose solution). Control rats were kept under normobaric normoxia (Nx) and treated in a corresponding manner. One day after the last exposure to IHH, we measured the cardiac hypoxia-induced oxidative/nitrosative stress biomarkers: the malondialdehyde (MDA) level and protein carbonyl (PC) content, the activity of some antioxidant enzymes [superoxide dismutase (SOD) and catalase (CAT)], the nitrite plus nitrate (NOx) production, and the inducible nitric oxide synthase (iNOS) protein expression. Heart tissue MDA and PC levels, NOx level, and iNOS expression of IHH-exposed rats had increased, and SOD and CAT activities had decreased compared with those of the Nx-exposed rats (control groups). MDA, CP, NOx, and iNOS levels had decreased in Que-treated IHH-exposed rats compared with IHH-exposed rats (control groups). However, Que administration increased SOD and CAT activities of the heart tissue in the IHH-exposed rats. HH exposure increases oxidative/nitrosative stress in heart tissue and Que is an effective cardioprotective agent, which further supports the oxidative cardiac dysfunction induced by hypoxia.


Candidalysin Is the Hemolytic Factor of Candida albicans.
Candida albicans produces an important virulence factor, the hypha-associated Ece1-derived secreted peptide toxin candidalysin, which is crucial for the establishment of mucosal and systemic infections. C. albicans has also long been known to be hemolytic, yet the hemolytic factor has not been clearly identified. Here, we show that candidalysin is the hemolytic factor of C. albicans. Its hemolytic activity is modulated by fragments of another Ece1 peptide, P7. Hemolysis by candidalysin can be neutralized by the purinergic receptor antagonist pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS). PPADS also affects candidalysin's ability to intercalate into synthetic membranes. We also describe the neutralization potential of two anti-candidalysin nanobodies, which are promising candidates for future anti-Candida therapy. This work provides evidence that the historically proposed hemolytic factor of C. albicans is in fact candidalysin and sheds more light on the complex roles of this toxin in C. albicans biology and pathogenicity.


Analysis of new Matrilin-1 gene variants in a case-control study related to dental malocclusions in Equus asinus.
Prognathism and brachygnathism are craniofacial deformities that severely affect the health of human and vertebrates, such as donkeys. The multifactorial etiology of this disease makes the genetic analysis a powerful tool for its understanding and prevention of spreading these deformities. This study aims to contribute to the characterization of the genetic basis of prognathism and brachygnathism in donkeys, using the Zamorano-Leonés donkey, an endangered Spanish breed, as a model. Matrilin-1 (MATN1) polymorphisms have been previously described as markers for mandibular prognathism in Korean and Japanese human populations. Genetic variations in MATN1 gene were sought, in order to verify its association in a case-control study, including 30 donkeys presenting brachygnathism, 30 donkeys presenting prognathism and 30 donkeys with normal occlusion phenotypes. One genetic variation (g503G > A) located in an intronic region of MATN1 gene was identified and characterized. Statistically significant differences were detected between the control group and prognathism cases, but no statistical significant results were found between the control group and the brachygnathism cases. These results support evidence for an important role of MATN1 on prognathism in the analyzed population with MATN1 genetic variation - 503G>A - having a protective effect. Further studies should be developed in order to understand the whole role of MATN1 and the mechanisms affected by its genetic variations.


Exploitation of Drought Tolerance-Related Genes for Crop Improvement.
Drought has become a major threat to food security, because it affects crop growth and development. Drought tolerance is an important quantitative trait, which is regulated by hundreds of genes in crop plants. In recent decades, scientists have made considerable progress to uncover the genetic and molecular mechanisms of drought tolerance, especially in model plants. This review summarizes the evaluation criteria for drought tolerance, methods for gene mining, characterization of genes related to drought tolerance, and explores the approaches to enhance crop drought tolerance. Collectively, this review illustrates the application prospect of these genes in improving the drought tolerance breeding of crop plants.


Serious Adverse Effects of Extended-release Niacin/Laropiprant: Results From the Heart Protection Study 2-Treatment of HDL to Reduce the Incidence of Vascular Events (HPS2-THRIVE) Trial.
The Heart Protection Study 2-Treatment of HDL to Reduce the Incidence of Vascular Events (HPS2-THRIVE) trial of patients at high risk of vascular disease found that adding extended-release niacin-laropiprant to intensive statin-based LDL-lowering therapy had no benefit on cardiovascular outcomes. However, the trial also identified previously unrecognized serious adverse effects (including new-onset diabetes, bleeding, and infection). Our objective was to explore the safety profile of niacin-laropiprant and examine whether any patients were at lower (or higher) risk of its adverse effects. HPS2-THRIVE was a randomized, double-blind trial of niacin-laropiprant (2000/40 mg/d) versus placebo among 25,673 patients at high risk of vascular disease. Information on all serious adverse events was collected during a median of 3.9 years of study treatment. Effects of niacin-laropiprant on new-onset diabetes, disturbances of diabetes control, bleeding, infection, and gastrointestinal upset were estimated by (1) time after randomization, (2) severity, (3) baseline characteristics, (4) baseline risk of the adverse event of interest, and (5) risk of major vascular event. The hazard ratio (HR) for new-onset diabetes with niacin/laropiprant was 1.32 (95% CI, 1.16-1.51; P < .001), which corresponded to an absolute excess of 4 people (95% CI, 2-6) developing diabetes per 1000 person-years in the study population as a whole. Among the 8299 participants with diabetes at baseline, the HR for serious disturbances in diabetes control was 1.56 (95% CI, 1.35-1.80), corresponding to an absolute excess of 12 (95% CI, 8-16) per 1000 person-years. The HR was 1.38 (95% CI, 1.17-1.63; P < .001) for serious bleeding, corresponding to an absolute excess of 2 (95% CI, 1-3) per 1000 person-years and 1.22 (95% CI, 1.11-1.34; P < .001) for serious infection, corresponding to an absolute excess of 4 (95% CI, 2-6) per 1000 person-years. The excess risks of these serious adverse events were larger in the first year after starting niacin-laropiprant therapy than in later years (except for the excess of infection, which did not appear to attenuate with time), and the risks of nonfatal and fatal events were similarly increased. The absolute excesses of each of these adverse effects were similar regardless of the baseline risk of the outcome. Practitioners or patients considering the use of niacin (in addition to, or instead of, a statin) despite the lack of evidence of cardiovascular benefits (at least when added to effective statin therapy) should take account of the significant risks of these serious adverse effects when making such decisions. ClinicalTrials.gov identifier: NCT00461630.


Sphingosine-1-phosphate receptor 1 reporter mice reveal receptor activation sites in vivo.
Activation of the GPCR sphingosine-1-phosphate receptor 1 (S1P1) by sphingosine-1-phosphate (S1P) regulates key physiological processes. S1P1 activation also has been implicated in pathologic processes, including autoimmunity and inflammation; however, the in vivo sites of S1P1 activation under normal and disease conditions are unclear. Here, we describe the development of a mouse model that allows in vivo evaluation of S1P1 activation. These mice, known as S1P1 GFP signaling mice, produce a S1P1 fusion protein containing a transcription factor linked by a protease cleavage site at the C terminus as well as a β-arrestin/protease fusion protein. Activated S1P1 recruits the β-arrestin/protease, resulting in the release of the transcription factor, which stimulates the expression of a GFP reporter gene. Under normal conditions, S1P1 was activated in endothelial cells of lymphoid tissues and in cells in the marginal zone of the spleen, while administration of an S1P1 agonist promoted S1P1 activation in endothelial cells and hepatocytes. In S1P1 GFP signaling mice, LPS-mediated systemic inflammation activated S1P1 in endothelial cells and hepatocytes via hematopoietically derived S1P. These data demonstrate that S1P1 GFP signaling mice can be used to evaluate S1P1 activation and S1P1-active compounds in vivo. Furthermore, this strategy could be potentially applied to any GPCR to identify sites of receptor activation during normal physiology and disease.


Ethnicity association of Helicobacter pylori virulence genotype and metronidazole susceptibility.
To characterise the cag pathogenicity island in Helicobacter pylori (H. pylori) isolates by analysing the strains' vacA alleles and metronidazole susceptibilities in light of patient ethnicity and clinical outcome. Ninety-five H. pylori clinical isolates obtained from patients with dyspepsia living in Malaysia were analysed in this study. Six genes in the cagPAI region (cagE, cagM, cagT, cag13, cag10 and cag67) and vacA alleles of the H. pylori isolates were identified by polymerase chain reaction. The isolates' metronidazole susceptibility was also determined using the E-test method, and the resistant gene was characterised by sequencing. More than 90% of the tested isolates had at least one gene in the cagPAI region, and cag67 was predominantly detected in the strains isolated from the Chinese patients, compared with the Malay and Indian patients (P < 0.0001). The majority of the isolates (88%) exhibited partial deletion (rearrangement) in the cagPAI region, with nineteen different patterns observed. Strains with intact or deleted cagPAI regions were detected in 3.2% and 8.4% of isolates, respectively. The prevalence of vacA s1m1 was significantly higher in the Malay and Indian isolates, whereas the isolates from the Chinese patients were predominantly genotyped as vacA s1m2 (P = 0.018). Additionally, the isolates from the Chinese patients were more sensitive to metronidazole than the isolates from the Malay and Indian patients (P = 0.047). Although we attempted to relate the cagPAI genotypes, vacA alleles and metronidazole susceptibilities to disease outcome, no association was observed. The vacA alleles were distributed evenly among the strains with intact, partially deleted or deleted cagPAI regions. Interestingly, the strains exhibiting an intact cagPAI region were sensitive to metronidazole, whereas the strains with a deleted cagPAI were more resistant. Successful colonisation by different H. pylori genotypes is dependent on the host's genetic makeup and may play an important role in the clinical outcome.


Substrate stiffness and oxygen as regulators of stem cell differentiation during skeletal tissue regeneration: a mechanobiological model.
Extrinsic mechanical signals have been implicated as key regulators of mesenchymal stem cell (MSC) differentiation. It has been possible to test different hypotheses for mechano-regulated MSC differentiation by attempting to simulate regenerative events such as bone fracture repair, where repeatable spatial and temporal patterns of tissue differentiation occur. More recently, in vitro studies have identified other environmental cues such as substrate stiffness and oxygen tension as key regulators of MSC differentiation; however it remains unclear if and how such cues determine stem cell fate in vivo. As part of this study, a computational model was developed to test the hypothesis that substrate stiffness and oxygen tension regulate stem cell differentiation during fracture healing. Rather than assuming mechanical signals act directly on stem cells to determine their differentiation pathway, it is postulated that they act indirectly to regulate angiogenesis and hence partially determine the local oxygen environment within a regenerating tissue. Chondrogenesis of MSCs was hypothesized to occur in low oxygen regions, while in well vascularised regions of the regenerating tissue a soft local substrate was hypothesised to facilitate adipogenesis while a stiff substrate facilitated osteogenesis. Predictions from the model were compared to both experimental data and to predictions of a well established computational mechanobiological model where tissue differentiation is assumed to be regulated directly by the local mechanical environment. The model predicted all the major events of fracture repair, including cartilaginous bridging, endosteal and periosteal bony bridging and bone remodelling. It therefore provides support for the hypothesis that substrate stiffness and oxygen play a key role in regulating MSC fate during regenerative events such as fracture healing.


A reversible light-operated nanovalve on mesoporous silica nanoparticles.
Two azobenzene α-cyclodextrin based nanovalves are designed, synthesized and assembled on mesoporous silica nanoparticles. Under aqueous conditions, the cyclodextrin cap is tightly bound to the azobenzene moiety and capable of holding back loaded cargo molecules. Upon irradiation with a near-UV light laser, trans to cis-photoisomerization of azobenzene initiates a dethreading process, which causes the cyclodextrin cap to unbind followed by the release of cargo. The addition of a bulky stopper to the end of the stalk allows this design to be reversible; complete dethreading of cyclodextrin as a result of unbinding with azobenzene is prevented as a consequence of steric interference. As a result, thermal relaxation of cis- to trans-azobenzene allows for the rebinding of cyclodextrin and resealing of the nanopores, a process which entraps the remaining cargo. Two stalks were designed with different lengths and tested with alizarin red S and propidium iodide. No cargo release was observed prior to light irradiation, and the system was capable of multiuse. On/off control was also demonstrated by monitoring the release of cargo when the light stimulus was applied and removed, respectively.


Impact of base analogues within a CpG dinucleotide on the binding of DNA by the methyl-binding domain of MeCP2 and methylation by DNMT1.
The epigenetic control of transcription requires the selective recognition of methylated CpG dinucleotides by methylation-sensitive sequence-specific DNA binding proteins. In order to probe the mechanism of selective interaction of the methyl-binding protein with methylated DNA, we have prepared a series of oligonucleotides containing modified purines and pyrimidines at the recognition site, and we have examined the binding of these oligonucleotides to the methyl-binding domain (MBD) of the methyl-CpG-binding protein 2 (MeCP2). Our results suggest that pyrimidine 5-substituents similar in size to a methyl group facilitate protein binding; however, binding affinity does not correlate with the hydrophobicity of the substituent, and neither the 4-amino group of 5-methylcytosine (mC) nor Watson-Crick base pair geometry is essential for MBD binding. However, 5-substituted uracil analogues in one strand do not direct human DNA methyltransferase 1 (DNMT1) methylation of the opposing strand, as does mC. Important recognition elements do include the guanine O6 and N7 atoms present in the major groove. Unexpectedly, removal of the guanine 2-amino group from the minor groove substantially enhances MBD binding, likely resulting from DNA bending at the substitution site. The enhanced binding of the MBD to oligonucleotides containing several cytosine analogues observed here is better explained by a DNA-protein interface mediated by structured water as opposed to hydrophobic interactions.


Alternative channels for urea in the inner medulla of the rat kidney.
The ascending thin limbs (ATLs) and lower descending thin limbs (DTLs) of Henle's loop in the inner medulla of the rat are highly permeable to urea, and yet no urea transporters have been identified in these sections. We hypothesized that novel, yet-unidentified transporters in these tubule segments could explain the high urea permeability. cDNAs encoding for Na(+)-glucose transporter 1a (SGLT1a), Na(+)-glucose transporter 1 (NaGLT1), urea transporter (UT)-A2c, and UT-A2d were isolated and cloned from the Munich-Wistar rat inner medulla. SGLT1a is a novel NH2-terminal truncated variant of SGLT1. NaGLT1 is a Na(+)-dependent glucose transporter primarily located in the proximal tubules and not previously described in the thin limbs. UT-A2c and UT-A2d are novel variants of UT-A2. UT-A2c is truncated at the COOH terminus, and UT-A2d has one exon skipped. When rats underwent water restriction for 72 h, mRNA levels of SGLT1a increased in ATLs, NaGLT1 levels increased in both ATLs and DTLs, and UT-A2c increased in ATLs. [(14)C]urea uptake assays performed on Xenopus oocytes heterologously expressing these proteins revealed that despite having structural differences from their full-length versions, SGLT1a, UT-A2c, and UT-A2d enhanced urea uptake. NaGLT1 also facilitated urea uptake. Uptakes were Na(+) independent and inhibitable by phloretin and/or phloridzin. Our data indicate that there are several alternative channels for urea in the rat inner medulla that could potentially contribute to the high urea permeabilities in thin limb segments.


Biomarkers of Exposure among Adult Smokeless Tobacco Users in the Population Assessment of Tobacco and Health Study (Wave 1, 2013-2014).
Monitoring population-level toxicant exposures from smokeless tobacco (SLT) use is important for assessing population health risks due to product use. In this study, we assessed tobacco biomarkers of exposure (BOE) among SLT users from the Wave 1 (2013-2014) of the Population Assessment of Tobacco and Health (PATH) Study. Urinary biospecimens were collected from adults ages 18 and older. Biomarkers of nicotine, tobacco-specific nitrosamines (TSNA), polycyclic aromatic hydrocarbons (PAH), volatile organic compounds (VOC), metals, and inorganic arsenic were analyzed and reported among exclusive current established SLT users in comparison with exclusive current established cigarette smokers, dual SLT and cigarette users, and never tobacco users. In general, SLT users (n = 448) have significantly higher concentrations of BOE to nicotine, TSNAs, and PAHs compared with never tobacco users; significant dose-response relationships between frequency of SLT use and biomarker concentrations were also reported among exclusive SLT daily users. Exclusive SLT daily users have higher geometric mean concentrations of total nicotine equivalent-2 (TNE2) and TSNAs than exclusive cigarette daily smokers. In contrast, geometric mean concentrations of PAHs and VOCs were substantially lower among exclusive SLT daily users than exclusive cigarette daily smokers. Our study produced a comprehensive assessment of SLT product use and 52 biomarkers of tobacco exposure. Compared with cigarette smokers, SLT users experience greater concentrations of some tobacco toxicants, including nicotine and TSNAs. Our data add information on the risk assessment of exposure to SLT-related toxicants. High levels of harmful constituents in SLT remain a health concern.


Teaching Third-Year Medical Students to Address Patients' Spiritual Needs in the Surgery/Anesthesiology Clerkship.
Despite many patients wanting physicians to inquire about their religious/spiritual beliefs, most physicians do not make such inquiries. Among physicians who do, surgeons are less likely than family and general practitioners and psychiatrists to do so. To address this gap, we developed a 60-minute curriculum that follows the Kolb cycle of experiential learning for third-year medical students on their surgery/anesthesiology clerkship. The session includes definitions of religion/spirituality, an overview of the literature on spirituality in surgery, a review of the FICA Spiritual History Tool, discussion of the role of the chaplain and the process of initiating a chaplain consult, and three cases regarding the spiritual needs of surgical patients. In total, 165 students participated in 10 sessions over 13 months. Of these, 120 students (73%) provided short-term feedback. Overall, 82% rated the session above average or excellent, and 72% stated the session was very relevant to patient care. To improve the session, students recommended assigning key readings, discussing more cases, role-playing various scenarios, inviting patients to speak, practicing mock interviews, and allowing for more self-reflection and discussion. Long-term feedback was provided by 105 students (64%) and indicated that the spirituality session impacted their attitudes about the role of religion/spirituality in medicine and their behaviors with patients. We have designed a successful session on spirituality for third-year students on their surgery/anesthesiology clerkship. Students reported it to be a positive addition to the curriculum. The session can be modified for other surgical subspecialties and specialties outside of surgery.


How not to get scar(r)ed: pointers to the correct diagnosis in patients with suspected primary cicatricial alopecia.
Primary cicatricial alopecias (PCAs) are a rare, but important, group of disorders that cause irreversible damage to hair follicles resulting in scarring and permanent hair loss. They may also signify an underlying systemic disease. Thus, it is of paramount importance that clinicians who manage patients with hair loss are able to diagnose these disorders accurately. Unfortunately, PCAs are notoriously difficult conditions to diagnose and treat. The aim of this review is to present a rational and pragmatic guide to help clinicians in the professional assessment, investigation and diagnosis of patients with PCA. Illustrating typical clinical and histopathological presentations of key PCA entities we show how dermatoscopy can be profitably used for clinical diagnosis. Further, we advocate the search for loss of follicular ostia as a clinical hallmark of PCA, and suggest pragmatic strategies that allow rapid formulation of a working diagnosis.


Development of Th1 and Th2 populations and the nature of immune responses to hepatitis B virus DNA vaccines can be modulated by codelivery of various cytokine genes.
In this study, we provide direct evidence that the magnitude and nature of the immune response to a DNA vaccine can be differentially regulated by codelivery of various mouse cytokine genes. Mice immunized with a hepatitis B virus (HBV) DNA vaccine and the IL-12 or IFN-gamma gene exhibited a significant enhancement of Th1 cells and increased production of anti-HBV surface IgG2a Ab, as well as a marked inhibition of Th2 cells and decreased production of IgG1 Ab. In contrast, coinjection of the IL-4 gene significantly enhanced the development of specific Th2 cells and increased production of IgG1 Ab, whereas Th1 differentiation and IgG2a production were suppressed. Coinjection of the IL-2 or the granulocyte-macrophage-CSF gene enhanced the development of Th1 cells, while the development of Th2 cells was not affected, and the production of IgG1 and IgG2a Ab were both increased. The CTL activity induced by HBV DNA vaccination was most significantly enhanced by codelivery of the IL-12 or IFN-gamma gene, followed by the IL-2 or granulocyte-macrophage-CSF gene, whereas codelivery of the IL-4 gene suppressed the activity. When challenged with HBV surface Ag (HBsAg)-expressing syngeneic tumors, significant reduction of tumor growth was observed in mice that were coadministered the IL-12 gene but not the IL-4 gene. Taken together, these results demonstrate that application of a cytokine gene in a DNA vaccine formulation can influence the differentiation of Th cells as well as the nature of an immune response and may thus provide a strategy to improve its prophylactic and therapeutic efficacy.


Polymorphisms in MTHFR, MS and CBS genes and premature acute myocardial infarction in a Pakistani population.
High prevalence of premature coronary heart disease in Pakistanis compared to other populations points towards the genetic predisposition of this population to develop this disease. Since no investigations have been carried out in Pakistan to study the relationship of polymorphisms in genes involved in homocysteine cycle, the objective of the present study was to find out if there is any association of methylenetetrahydrofolate reductase (MTHFR) C677T, A1298C; methionine synthase (MS) A2756G; cystathionine-β-synthase (CBS) 844ins68, G919A polymorphisms with premature acute myocardial infarction (AMI) in a population of Pakistani patients with this disease. In a cross-sectional study, DNA samples of 143 AMI patients (age <45 years) and 153 healthy controls were genotyped for the above mentioned polymorphisms using PCR-RFLP methods. Plasma/serum samples of both patients and healthy controls were screened for homocysteine, folate and vitamin B12. One way ANOVA and chi-squared test were used for analysis of data. Mean plasma homocysteine levels in premature AMI patients and healthy controls were found to be 23±17.2 and 23±13.4 μmol/l, respectively which are higher than the upper normal limit of this biomarker (15μmol/l). MTHFR 677 CT genotype in healthy controls and MTHFR 677 TT genotype in AMI patients were found to have significantly increased levels of plasma homocysteine (p value <0.05), while all other polymorphisms did not show any significant difference in mean levels of homocysteine between AMI patients and healthy controls. Moreover, no association was observed between MTHFR C677T, A1298C; MS A2756C; CBS844ins68 polymorphisms and premature AMI in this population. This indicates that common polymorphisms in MTHFR, MS and CBS genes have no role in premature AMI in Pakistani population.


Football Compared with Usual Care in Men with Prostate Cancer (FC Prostate Community Trial): A Pragmatic Multicentre Randomized Controlled Trial.
Physical activity has been shown to mitigate the unwanted psychological and physiological side effects of prostate cancer treatments, but sustainable exercise possibilities are limited. Our objective was to examine whether football in a real-world setting (i.e., local football clubs) was safe and feasible in practice and could improve quality of life, mitigate decline in muscle mass and bone density, and increase fat mass in patients with prostate cancer. In this pragmatic, multicentre, parallel randomized controlled trial, men diagnosed with prostate cancer were recruited from five Danish urological departments. Men (N = 214) diagnosed with prostate cancer were randomly allocated, using random generated lists (block size 4-8) stratified for center and androgen-deprivation therapy status, to either 1 h of football twice weekly in a local football club or to usual care, which was a 15- to 30-min telephone session covering their options for physical activity or free-of-charge rehabilitation delivered as standard in Denmark. Allocation was concealed from the trial investigator performing the randomization, but-given the nature of the intervention-this was not possible for personnel and participants. Assessments were performed at baseline, 12 weeks, and 6 months. The primary outcome was mean change difference in prostate cancer-specific quality of life at 12 weeks. Secondary outcomes were body composition, 12-Item Short Form Health Survey (SF-12) physical and mental health, and safety-reported as fractures, falls, and serious adverse events. Attrition was 1 and 3% at 12 weeks, and 5% and 5% at 6 months for the usual care and football groups, respectively. Prostate cancer-specific quality of life was equal between groups at 12 weeks (mean difference + 1.9 points, 95% confidence interval [CI] -1.0-4.8; P = 0.20) and at 6 months (+ 0.5 points, 95% CI -2.8-3.8; P = 0.76). Fractures were equally distributed, with two fractures in the usual care group and one in the football group. Likewise, body composition outcomes were equal. Mental health improved after 6 months of football (mean difference + 2.7 points, 95% CI 0.8-4.6; P = 0.006). In this trial, community-based football was a feasible exercise strategy for men with prostate cancer. Football did not improve prostate cancer-specific quality of life but did improve mental health; the clinical significance of this is unclear. ClinicalTrials.gov: NCT02430792.


The cyclops mutation blocks specification of the floor plate of the zebrafish central nervous system.
The floor plate is a set of epithelial cells present in the ventral midline of the neural tube in vertebrates that seems to have an important role in the developmental patterning of central nervous system fibre pathways, and arrangements of specific neurons. The floor plate arises from dorsal ectodermal cells closely associated with the mesoderm that forms notochord, and it may depend on interactions from the notochord for its specification. To learn the nature of these interactions we have analysed mutations in zebrafish (Brachydanio rerio). We report here that in wild-type embryos the floor plate develops as a simply organized single cell row, but that its development fails in embryos bearing the newly discovered zygotic lethal 'cyclops' mutation, cyc-1(b16). Mosaic analysis establishes that cyc-1 blocks floor plate development autonomously and reveals the presence of homeogenetic induction between floor plate cells.


Curing hearing loss: Patient expectations, health care practitioners, and basic science.
Millions of patients are debilitated by hearing loss, mainly caused by degeneration of sensory hair cells in the cochlea. The underlying reasons for hair cell loss are highly diverse, ranging from genetic disposition, drug side effects, traumatic noise exposure, to the effects of aging. Whereas modern hearing aids offer some relief of the symptoms of mild hearing loss, the only viable option for patients suffering from profound hearing loss is the cochlear implant. Despite their successes, hearing aids and cochlear implants are not perfect. Particularly frequency discrimination and performance in noisy environments and general efficacy of the devises vary among individual patients. The advent of regenerative medicine, the publicity of stem cells and gene therapy, and recent scientific achievements in inner ear cell regeneration have generated an emerging spirit of optimism among scientists, health care practitioners, and patients. In this review, we place the different points of view of these three groups in perspective with the goal of providing an assessment of patient expectations, health care reality, and potential future treatment options for hearing disorders. (1) Readers will be encouraged to put themselves in the position of a hearing impaired patient or family member of a hearing impaired person. (2) Readers will be able to explain why diagnosis of the underlying pathology of hearing loss is difficult. (3) Readers will be able to list the main directions of current research aimed to cure hearing loss. (4) Readers will be able to understand the different viewpoints of patients and their relatives, health care providers, and scientists with respect to finding novel treatments for hearing loss.


Impact of COVID-19 related social support service closures on people with dementia and unpaid carers: a qualitative study.
Accessing social care and social support services is key to support the well-being of people living with dementia (PLWD) and unpaid carers. COVID-19 has caused sudden closures or radical modifications of these services, and is resulting in prolonged self-isolation. The aim of this study was to explore the effects of COVID-19 related social care and support service changes and closures on the lives of PLWD and unpaid carers. PLWD and unpaid carers were interviewed via telephone in April 2020. Transcripts were analysed using thematic analysis. Demographic characteristics including household Index of Multiple Deprivation score and weekly hours of social support service usage before and since the COVID-19 outbreak were also collected. Paired samples t-tests was used to compare the mean of weekly hours of social support service usage before and since the outbreak. 50 semi-structured interviews were conducted with unpaid carers (n = 42) and PLWD (n = 8). There was a significant reduction in social support service usage since the outbreak. Thematic analysis identified three overarching themes: (1) Loss of control; (2) Uncertainty; (3) Adapting and having to adapt to the new normal. Carers and PLWD were greatly affected by the sudden removal of social support services, and concerned about when services would re-open. Carers were worried about whether the person they cared for would still be able to re-join social support services. PLWD and carers need to receive specific practical and psychological support during the pandemic to support their well-being, which is severely affected by public health restrictions.


Endometrial cancer: reviving progesterone therapy in the molecular age.
Endometrial cancer is the most common gynecologic malignancy and represents a major health concern because overall five-year survival rates have not improved in the last three decades. A great deal of research demonstrates that the endometrium is extremely sensitive to hormones, and a shift in the estrogen:progesterone balance is the major cause for the development of endometrial cancer. Progestin-based therapy has proven effective in a subset of patients, particularly in situations where expression of progesterone receptor (PR) is maintained. However, this approach is not routinely used in the clinic in the U.S. for several reasons. For example, many endometrial tumors have lost PR expression, which limits the clinical application of progestin-based therapy. While the idea that restoring PR expression will resensitize tumors to progestin was proposed over 20 years ago, we only now have the molecular tools to accomplish this goal. Basic science research has revealed several pathways that govern the expression of PR at the DNA, RNA, and protein levels. In this article, we describe one current approach to restore expression of PR at the epigenetic level in endometrial cancer. While still in the preclinical stage, we believe this strategy to re-establish PR expression will result in resensitization of endometrial tumors to progestin therapy.


Osteopontin is a prognostic factor for survival of acute myeloid leukemia patients.
Osteopontin (OPN) is a glycoprotein that is secreted by osteoblasts and hematopoietic cells. OPN suppresses the proliferation of hematopoietic stem cells in vitro and may regulate the hematopoietic stem cell pool. Increased serum OPN concentrations occur in chronic myeloid leukemia, multiple myeloma, and acute myeloid leukemia (AML). In the present study, we analyzed the prognostic impact of OPN in AML by investigating the expression and relevance of OPN in newly diagnosed AML patients from 2 large study groups (the German AML Cooperative Group and the Dutch-Belgian Hematology Oncology Cooperative group). IHC (n = 84), ELISAs of blood/BM sera (n = 41), and microarray data for mRNA levels (n = 261) were performed. Expression of OPN protein was increased in AML patients both in BM blasts (IHC) and in BM serum (ELISA) compared with healthy controls. Patients expressing high levels of OPN within the BM (IHC) experienced shortened overall survival (OS; P = .025). Multivariate analysis identified karyotype, blast clearance (day 16), and the level of OPN expression as independent prognostic factors for OS. This prompted us to analyze microarray data from 261 patients from a third cohort. The analysis confirmed OPN as a prognostic marker. In summary, high OPN mRNA expression indicated decreased event-free survival (P = .0002) and OS (P = .001). The prognostic role of OPN was most prominent in intermediate-risk AML. These data provide evidence that OPN expression is an independent prognostic factor in AML.


Simultaneous inhibition of mTOR-containing complex 1 (mTORC1) and MNK induces apoptosis of cutaneous T-cell lymphoma (CTCL) cells.
mTOR kinase forms the mTORC1 complex by associating with raptor and other proteins and affects a number of key cell functions. mTORC1 activates p70S6kinase 1 (p70S6K1) and inhibits 4E-binding protein 1 (4E-BP1). In turn, p70S6K1 phosphorylates a S6 protein of the 40S ribosomal subunit (S6rp) and 4E-BP1, with the latter negatively regulating eukaryotic initiation factor 4E (eIF-4E). MNK1 and MNK2 kinases phosphorylate and augment activity of eIF4E. Rapamycin and its analogs are highly specific, potent, and relatively non-toxic inhibitors of mTORC1. Although mTORC1 activation is present in many types of malignancies, rapamycin-type inhibitors shows relatively limited clinical efficacy as single agents. Initially usually indolent, CTCL displays a tendency to progress to the aggressive forms with limited response to therapy and poor prognosis. Our previous study (M. Marzec et al. 2008) has demonstrated that CTCL cells display mTORC1 activation and short-term treatment of CTCL-derived cells with rapamycin suppressed their proliferation and had little effect on the cell survival. Cells derived from CTCL were treated with mTORC1 inhibitor rapamycin and MNK inhibitor and evaluated for inhibition of the mTORC1 signaling pathway and cell growth and survival. Whereas the treatment with rapamycin persistently inhibited mTORC1 signaling, it suppressed only partially the cell growth. MNK kinase mediated the eIF4E phosphorylation and inhibition or depletion of MNK markedly suppressed proliferation of the CTCL cells when combined with the rapamycin-mediated inhibition of mTORC1. While MNK inhibition alone mildly suppressed the CTCL cell growth, the combined MNK and mTORC1 inhibition totally abrogated the growth. Similarly, MNK inhibitor alone displayed a minimal pro-apoptotic effect; in combination with rapamycin it triggered profound cell apoptosis. These findings indicate that the combined inhibition of mTORC1 and MNK may prove beneficial in the treatment of CTCL and other malignancies.


The Cost-Effectiveness of Anemia Treatment for Persons with Chronic Kidney Disease.
Although major guidelines uniformly recommend iron supplementation and erythropoietin stimulating agents (ESAs) for managing chronic anemia in persons with chronic kidney disease (CKD), there are differences in the recommended hemoglobin (Hb) treatment target and no guidelines consider the costs or cost-effectiveness of treatment. In this study, we explored the most cost-effective Hb target for anemia treatment in persons with CKD stages 3-4. The CKD Health Policy Model was populated with a synthetic cohort of persons over age 30 with prevalent CKD stages 3-4 (i.e., not on dialysis) and anemia created from the 1999-2010 National Health and Nutrition Examination Survey. Incremental cost-effectiveness ratios (ICERs), computed as incremental cost divided by incremental quality adjusted life years (QALYs), were assessed for Hb targets of 10 g/dl to 13 g/dl at 0.5 g/dl increments. Targeting a Hb of 10 g/dl resulted in an ICER of $32,111 compared with no treatment and targeting a Hb of 10.5 g/dl resulted in an ICER of $32,475 compared with a Hb target of 10 g/dl. QALYs increased to 4.63 for a Hb target of 10 g/dl and to 4.75 for a target of 10.5 g/dl or 11 g/dl. Any treatment target above 11 g/dl increased medical costs and decreased QALYs. In persons over age 30 with CKD stages 3-4, anemia treatment is most cost-effective when targeting a Hb level of 10.5 g/dl. This study provides important information for framing guidelines related to treatment of anemia in persons with CKD.


Prevalence of type 2 diabetes among patients with hypertension under the care of 30 Italian clinics of hypertension: results of the (Iper)tensione and (dia)bete study.
Hypertension is known to be highly prevalent among patients with diabetes and associated with an increased risk of cardiovascular damage. In contrast, relatively few investigations have addressed the prevalence of diabetes among patients with hypertension. The purpose of the present study was to examine the prevalence of type 2 diabetes, the effectiveness of hypertension and diabetes control and the association with other cardiovascular risk factors and previous cardiovascular diseases in a cohort of patients with hypertension referred to 30 hospital outpatient clinics for the treatment of hypertension. Patients were considered as having diabetes if they were already on an antidiabetic treatment either with diet or medications. All other patients had fasting plasma glucose measured on two separate occasions and were classified as having diabetes if both values were at least 140 mg/dl (7.8 mmol/l) and as not having diabetes if both values were less than 110 mg/dl (6.1 mmol/l). In patients with a single determination of at least 110 mg/dl, the final diagnosis of diabetes was established according to the result of an oral glucose tolerance test. A secondary definition of diabetes was also used, that is two fasting plasma glucose values of at least 126 mg/dl (7.0 mmol/l). In all patients, serum total, high-density lipoprotein cholesterol and low-density lipoprotein cholesterol, fasting serum triglycerides, serum creatinine and urinary albumin were also evaluated. Among the 1397 recruited patients, 242 (17.3%) were diagnosed as having diabetes according to the primary definition and 244 (17.5%) according to the secondary definition. In 195 out of the 242 (14%), the diagnosis was already known whereas, in the remaining 47 (3.3%), it was made de novo. In 61.4% of those already having diabetes, plasma glucose was at least 140 mg/dl (7.8 mmol/l), whereas only in 8.4% of them was it less than 110 mg/dl (6.1 mmol/l). Patients with diabetes were older, heavier and with a greater familiar predisposition. Patients with diabetes had higher values of systolic blood pressure than individuals without diabetes (150 +/- 17 vs. 144 +/- 16 mmHg, respectively; P < 0.001), lower high-density lipoprotein cholesterol and higher triglycerides and microalbuminuria. Overall, among patients with hypertension and diabetes, only 3% had blood pressure and HbA1c within the recommended limits. The prevalence of previous cardiovascular disorders was two to three times higher than among individuals without diabetes.


The time-course of the cross-modal semantic modulation of visual picture processing by naturalistic sounds and spoken words.
The time-course of cross-modal semantic interactions between pictures and either naturalistic sounds or spoken words was compared. Participants performed a speeded picture categorization task while hearing a task-irrelevant auditory stimulus presented at various stimulus onset asynchronies (SOAs) with respect to the visual picture. Both naturalistic sounds and spoken words gave rise to cross-modal semantic congruency effects (i.e., facilitation by semantically congruent sounds and inhibition by semantically incongruent sounds, as compared to a baseline noise condition) when the onset of the sound led that of the picture by 240 ms or more. Both naturalistic sounds and spoken words also gave rise to inhibition irrespective of their semantic congruency when presented within 106 ms of the onset of the picture. The peak of this cross-modal inhibitory effect occurred earlier for spoken words than for naturalistic sounds. These results therefore demonstrate that the semantic priming of visual picture categorization by auditory stimuli only occurs when the onset of the sound precedes that of the visual stimulus. The different time-courses observed for naturalistic sounds and spoken words likely reflect the different processing pathways to access the relevant semantic representations.


Identification of centaurin-alpha2: a phosphatidylinositide-binding protein present in fat, heart and skeletal muscle.
We describe here the cloning, expression and characterisation of centaurin-alpha2 from a rat adipocyte cDNA library. The centaurin-alpha2 cDNA contains an open reading frame, which codes for a protein of 376 amino acids with predicted mass of 43.5 kDa. Centaurin-alpha2 shares 51-59% identity with centaurin-alpha1 proteins and has the same domain organisation, consisting of a predicted N-terminal ArfGAP domain followed by two successive pleckstrin homology domains. Despite the sequence similarity, there are a number of notable differences between the previously characterised centaurin-alpha1 proteins and the newly described centaurin-alpha2: (i) in vitro lipid binding experiments with centaurin-alpha2 do not reveal the same selectivity for phosphatidylinositol 3,4,5-trisphosphate over phosphatidylinositol 4,5-bisphosphate that has been shown for centaurin-alpha; (ii) unlike centaurin-alpha1 which is expressed mainly in the brain, centaurin-alpha2 has a broad tissue distribution, being particularly abundant in fat, heart and skeletal muscle; (iii) in contrast to centaurin-alpha1 which is found in both membrane and cytosolic fractions, endogenous centaurin-alpha2 is exclusively present in the dense membrane fractions of cell extracts, suggesting a constitutive membrane association. Insulin stimulation, which stimulates phosphatidylinositol 3,4,5-trisphosphate production, does not alter the subcellular distribution of centaurin-alpha2 between adipocyte membrane fractions. This observation is consistent with the lack of specificity of centaurin-alpha2 for phosphatidylinositol 3,4,5-trisphosphate over phosphatidylinositol 4,5-bisphosphate.


Prefrontal-parietal function: from foraging to foresight.
Comparative neuroanatomy shows that new prefrontal areas emerged during the evolution of anthropoid primates to augment prefrontal, parietal, and temporal areas that had evolved in earlier primates. We recently proposed that the new anthropoid areas reduce foraging errors by generating goals from current contexts and learning to do so rapidly, sometimes based on single events. Among the contexts used to generate these goals, the posterior parietal cortex provides the new prefrontal areas with information about relational metrics such as order, number, duration, length, distance and proportion, which play a crucial role in foraging choices. Here we propose that this specialized network later became adapted to support the human capacity for reasoning and general problem-solving.


Stress-induced antioxidant defense and protein chaperone response in the freeze-tolerant wood frog Rana sylvatica.
Freeze tolerance is an adaptive response utilized by the wood frog Rana sylvatica to endure the sub-zero temperatures of winter. Survival of whole body freezing requires wood frogs to trigger cryoprotective mechanisms to deal with potential injuries associated with conversion of 65-70% of total body water into ice, including multiple consequences of ice formation such as cessation of blood flow and cell dehydration caused by water loss into ice masses. To understand how wood frogs defend against these stressors, we measured the expression of proteins known to be involved in the antioxidant defense and protein chaperone stress responses in brain and heart of wood frogs comparing freezing, anoxia, and dehydration stress. Our results showed that most stress proteins were regulated in a tissue- and stress-specific manner. Notably, protein levels of the cytosolic superoxide dismutase (SOD1) were upregulated by 1.37 ± 0.11-fold in frozen brain, whereas the mitochondrial SOD2 isoform rose by 1.38 ± 0.37-fold in the heart during freezing. Catalase protein levels were upregulated by 3.01 ± 0.47-fold in the brain under anoxia stress, but remained unchanged in the heart. Similar context-specific regulatory patterns were also observed for the heat shock protein (Hsp) molecular chaperones. Hsp27 protein was down-regulated in the brain across the three stress conditions, whereas the mitochondrial Hsp60 was upregulated in anoxic brain by 1.73 ± 0.38-fold and by 2.13 ± 0.57-fold in the frozen heart. Overall, our study provides a snapshot of the regulatory expression of stress proteins in wood frogs under harsh environment conditions and shows that they are controlled in a tissue- and stress-specific manner.


Doctorate in cardiology at FM-USP Heart Institute from 1994 through 2004: defense and publication.
A study evaluated the relationship between the defense of dissertations and their publication within the realm of the entire Medical School of the University of São Paulo (FM-USP). The existence of differences among different areas of knowledge and the time between defense of dissertations and their publication is questioned. To characterize publications related to dissertations defended in graduate courses of the Heart Institute (InCor) of the University of São Paulo Medical School (FM-USP). A retrospective survey was carried out featuring graduate work at FM/USP for the 1994-2004 period. Initially, data were collected on students who defended dissertations during this period, on their advisors, and on the dissertations defended in the course of the period. Then, by crossing these data with data from Medline and Web of Science, publications by these authors and data were located that referred to the respective publications. During that period, 268 dissertations were defended, resulting in 195 publications within a period of up to 10 years after their defense. The median time for publication after defense was one year and nine months, with a median impact factor of 2.1, and a median of 4 citations per paper. There was no statistically significant correlation among any of the data studied. A significant percentage of the dissertations were published, and in the group studied, publication took place within increasingly short times after their defense. This fact may be related to the success of the graduate commissions' policies and to the importance currently lent to debates on this theme, all of which contribute to improvement in the quality of graduate studies.


An association of dominant hand grip strength with some anthropometric variables in Indian collegiate population.
The present study deals with the correlations of dominant hand grip strength and 12 anthropometric variables, namely, height, weight, BMI, upper arm length, forearm length, total arm length, hand breadth, hand length, upper arm circumference, forearm circumference, biceps skinfold and triceps skinfold in randomly selected 303 unrelated, normal, healthy students (151 males and 152 females) aged 18-25 years of Guru Nanak Dev University, Amritsar, Punjab, India. The findings of the present study indicate a strong association of dominant right hand grip strength with all the anthropometric variables, except biceps skinfold in male students and with height (r = 0.200), weight (r = 0.275), BMI (p = 0.217), total arm length (p = 0.218) and upper arm circumference (r = 0.199) in female students. Statistically significant positive correlations were also found between dominant left hand grip strength and height (r = 0.275), weight (p = 0.537), BMI (p = 0.472), hand breadth (p = 0.464), upper arm circumference (r = 0.570), forearm circumference (p = 0.464) and triceps skinfold (p = 0.343) only in male students, but no such association was found between grip strength of left hand dominant female students with any of the twelve anthropometric variables. It may be concluded that hand dominance, especially of the right hand, has some close association with the anthropometric variables related to upper extremities.


Intratumor heterogeneity and precision of microarray-based predictors of breast cancer biology and clinical outcome.
Identifying sources of variation in expression microarray data and the effect of variance in gene expression measurements on complex predictive and diagnostic models is essential when translating microarray-based experimental approaches into clinical assays. The technical reproducibility of microarray platforms is well established. Here, we investigate the additional impact of intratumor heterogeneity, a largely unstudied component of variance, on the performance of several microarray-based assays in breast cancer. Genome-wide expression profiling was performed on 50 core needle biopsies from 18 breast cancer patients using Affymetrix GeneChip Human Genome U133 Plus 2.0 arrays. Global profiles of expression were characterized using unsupervised clustering methods and variance components models. Array-based measures of estrogen receptor (ER) and progesterone receptor (PR) status were compared with immunohistochemistry. The precision of genomic predictors of ER pathway status, recurrence risk, and sensitivity to chemotherapeutics was evaluated by interclass correlation. Global patterns of gene expression demonstrated that intratumor variation was substantially less than the total variation observed across the patient population. Nevertheless, a fraction of genes exhibited significant intratumor heterogeneity in expression. A high degree of reproducibility was observed in single-gene predictors of ER (intraclass correlation coefficient [ICC] = 0.94) and PR expression (ICC = 0.90), and in a multigene predictor of ER pathway activation (ICC = 0.98) with high concordance with immunohistochemistry. Substantial agreement was also observed for multigene signatures of cancer recurrence (ICC = 0.71) and chemotherapeutic sensitivity (ICC = 0.72 and 0.64). Intratumor heterogeneity, although present at the level of individual gene expression, does not preclude precise microarray-based predictions of tumor behavior or clinical outcome in breast cancer patients.


Auxotrophic variants of Pseudomonas aeruginosa are selected from prototrophic wild-type strains in respiratory infections in patients with cystic fibrosis.
Twenty-four nutritionally dependent (auxotrophic) Pseudomonas aeruginosa strains were isolated from 20 cystic fibrosis (CF) patients and tested for their amino acid requirements. Two different methods were necessary to identify the nutritional status of all isolates. Methionine was the most common single amino acid required (9 of 24 isolates), followed by leucine and arginine or ornithine. In total, a requirement for 12 different compounds or combination of compounds was demonstrated. Auxotrophic and prototrophic pairs of isolates from the same patient were compared by macrorestriction analysis of DNA in pulsed-field gel electrophoresis. Thirteen of 18 pairs analyzed presented identical restriction fragment length polymorphism profiles following digestion of DNA with XbaI. Three of the remaining pairs showed percentage similarities of 77, 91, and 98%, and the profiles of two pairs could not be compared because of the excessive degradation of their DNA. These results suggest that auxotrophic and prototrophic P. aeruginosa isolates colonizing the same CF patient constitute an isogenic group and raise the possibility that auxotrophs are selected from the prototrophic population during the course of pulmonary infection in CF patients.


What affects programme engagement for Māori families? A qualitative study of a family-based, multidisciplinary healthy lifestyle programme for children and adolescents.
It is important that intervention programmes are accessible and acceptable for groups most affected by excess weight. This study aimed to understand the barriers to and facilitators of engagement for Māori in a community-based, assessment-and-intervention healthy lifestyle programme (Whānau Pakari). Sixty-four in-depth, home-based interviews were conducted with past service users. Half of these were with families with Māori children and half with non-Māori families. The interviews were thematically analysed with peer debriefing for validity. Māori families experienced barriers due to racism throughout the health system and society, which then affected their ability to engage with the programme. Key barriers included the institutionalised racism evident through substantial structural barriers and socio-economic challenges, the experience of interpersonal racism and its cumulative impact with weight stigma, and internalised racism and beliefs of biological determinism. Responses to these barriers were distrust of health services, followed by renewed engagement or complete disengagement. Participants identified culturally appropriate care as that which was compassionate, respectful, and focused on relationship building. While Whānau Pakari is considered appropriate due to the approach of the delivery team, this is insufficient to retain some Māori families who face increased socio-economic and structural barriers. Past instances of weight stigma and racism have enduring effects when re-engaging with future health services, and inequities are likely to persist until these issues are addressed within the health system and wider society.


NF-kappaB signalling is inhibited by glucocorticoid receptor and STAT6 via distinct mechanisms.
NF-kappaB transcription factors are involved in the cellular response to stress, and are regulated by inhibitor (IkappaB) proteins, which prevent NF-kappaB-mediated transcription by maintaining NF-kappaB in the cytoplasm. Proteins from other pathways are also known to regulate NF-kappaB negatively, notably the glucocorticoid receptor (GR) and IL-4-responsive STAT6. Both pathways were shown to inhibit NF-kappaB-mediated transcription, by expressing either STAT6 or GR and activating the respective pathways. Using fluorescent fusion proteins, we show that GR alters the timing of activated p65 NF-kappaB nuclear occupancy by increasing the export rate of p65 and is independent of whether GR is present as a dimer or monomer. Expression of STAT6 was also shown to alter p65 nuclear occupancy but appeared to affect the import rate and hence the overall maximal level of p65 translocation. Activating STAT6 with IL-4 prior to activating NF-kappaB significantly increased this inhibition. Investigation of IkappaBa showed that activated STAT6 inhibited TNFalpha-mediated IkappaBa phosphorylation and degradation, whereas GR activation did not alter IkappaBalphakinetics. This demonstrates a clear separation of two distinct mechanisms of inhibition by STAT6 and GR upon the NF-kappaB pathway.


Effect of age and sex on the relationship between different socioeconomic indices and self-reported functional health in the EPIC-Norfolk population-based study.
The relationship between different social-economic indices and physical and mental functional health of older people compared with younger people is unclear. To examine the effect of age and sex on the relationship between various social-economic indices and self-reported functional health. A population-based cross-sectional study was conducted in 19,088 participants of European Prospective Investigation into Cancer (EPIC)-Norfolk, UK, ages 40-79 years at baseline. The independent relationships between three different socioeconomic indices; occupational social class, education and residential area deprivation, and functional health measured by anglicized version of 36-item short form questionnaire (UK SF-36), were compared between older (>or=65 years) and younger (<65 years) men and women. Residential area deprivation was significantly associated with poor physical and mental functional health independent of social class and education, and consistent in both age groups in men and women. A low level of education in younger men and being in low social class in younger women were associated with poorer physical functional health compared with their respective older counterparts. Social class had a significantly greater effect in older women compared with younger women. Commonly used socioeconomic indices have differing associations with functional health depending the age and sex of an individual. Residential area deprivation predicts poor functional health in all age and sex groups. This may have implications for health policy.


Psychiatric nurses enhancing consumer and caregiver participation in the state of Victoria: the impact of history and policy.
Australian mental health policy now clearly articulates that consumer and carer (informal caregiver) participation in all aspects of service delivery is an expectation. As the largest professional group, nurses clearly play a key role in translating policy into practice. The aim of this article is to briefly overview the history of mental health service development in Victoria, with specific emphasis on the development of psychiatric nursing. Changing perspectives of consumers of mental health services and their informal carers is discussed. Policy development is described in the context of the development of mental health services. It is argued that an appreciation of the history of punishment and confinement is necessary for providing a climate conducive to consumer and carer participation.


On the nature of consonant/vowel differences in letter position coding: Evidence from developing and adult readers.
In skilled adult readers, transposed-letter effects (jugde-JUDGE) are greater for consonant than for vowel transpositions. These differences are often attributed to phonological rather than orthographic processing. To examine this issue, we employed a scenario in which phonological involvement varies as a function of reading experience: A masked priming lexical decision task with 50-ms primes in adult and developing readers. Indeed, masked phonological priming at this prime duration has been consistently reported in adults, but not in developing readers (Davis, Castles, & Iakovidis, 1998). Thus, if consonant/vowel asymmetries in letter position coding with adults are due to phonological influences, transposed-letter priming should occur for both consonant and vowel transpositions in developing readers. Results with adults (Experiment 1) replicated the usual consonant/vowel asymmetry in transposed-letter priming. In contrast, no signs of an asymmetry were found with developing readers (Experiments 2-3). However, Experiments 1-3 did not directly test the existence of phonological involvement. To study this question, Experiment 4 manipulated the phonological prime-target relationship in developing readers. As expected, we found no signs of masked phonological priming. Thus, the present data favour an interpretation of the consonant/vowel dissociation in letter position coding as due to phonological rather than orthographic processing.


Prediction and Prevention of Sudden Death in Young Patients (<20 years) With Hypertrophic Cardiomyopathy.
Highly reliable identification of adults with hypertrophic cardiomyopathy (HC) at risk for sudden death (SD) has been reported. A significant controversy remains, however, regarding the most reliable risk stratification methodology for children and adolescents with HC. The present study assesses the accuracy of SD prediction and prevention with prophylactic implantable cardioverter-defibrillators (ICDs) in young HC patients. The study group is comprised of 146 HC patients <20 years of age evaluated consecutively over 17 years with prospective risk stratification and ICD decision-making. We relied on ≥1 established individual risk markers considered major within each patient's clinical profile, based on an enhanced American College of Cardiology /American Heart Association (ACC/AHA) guidelines algorithm. Of the 60 largely asymptomatic patients implanted with primary prevention ICDs at age 15 ± 4 years, 9 (15%) experienced device therapy terminating potentially lethal ventricular tachyarrhythmias and restoring sinus rhythm at 19 ± 6 years (range 9 to 29), 5.1 ± 6.0 years after implant; 3 patients had multiple appropriate ICD discharges. The individual risk marker algorithm was associated with 100% sensitivity in predicting SD events (95%CI: 69, 100) and 63% specificity for identifying patients without events (95%CI: 54, 71). Of these patients with device therapy, massive left ventricular hypertrophy (absolute wall thickness ≥30 mm) was the most common predictor, present in 70% of patients either alone or in combination with other risk markers. Each of the 146 study patients have survived to date at 22 ± 5 years, including all 86 without ICD recommendations. In conclusion, an enhanced ACC/AHA risk stratification strategy, based on established individual risk markers, was highly reliable in prospectively predicting SD events in children and adolescents with HC, and preventing arrhythmia-based catastrophes in this susceptible high risk population.


A Comprehensive, Automatically Updated Fungal ITS Sequence Dataset for Reference-Based Chimera Control in Environmental Sequencing Efforts.
The nuclear ribosomal internal transcribed spacer (ITS) region is the most commonly chosen genetic marker for the molecular identification of fungi in environmental sequencing and molecular ecology studies. Several analytical issues complicate such efforts, one of which is the formation of chimeric-artificially joined-DNA sequences during PCR amplification or sequence assembly. Several software tools are currently available for chimera detection, but rely to various degrees on the presence of a chimera-free reference dataset for optimal performance. However, no such dataset is available for use with the fungal ITS region. This study introduces a comprehensive, automatically updated reference dataset for fungal ITS sequences based on the UNITE database for the molecular identification of fungi. This dataset supports chimera detection throughout the fungal kingdom and for full-length ITS sequences as well as partial (ITS1 or ITS2 only) datasets. The performance of the dataset on a large set of artificial chimeras was above 99.5%, and we subsequently used the dataset to remove nearly 1,000 compromised fungal ITS sequences from public circulation. The dataset is available at http://unite.ut.ee/repository.php and is subject to web-based third-party curation.


Interplay of two major repair pathways in the processing of complex double-strand DNA breaks.
Radiation-induced complex double-strand breaks (DSBs) characterised by base lesions, abasic sites or single-strand breaks in close proximity to the break termini, are believed to be a major cause of the biological effects of ionising radiation exposure. It has been hypothesised that complex DSBs pose problems for the repair machinery of the cell. Using a biochemical approach, we have investigated the challenge to two major repair processes: base excision repair and ligation of DSB ends. Double-stranded oligonucleotides were synthesised with 8-oxo-7,8-dihydroguanine (8-oxoG) at defined positions relative to readily ligatable 3'-hydroxy or 5'-phosphate termini. The break termini interfere with removal of 8-oxoG during base excision repair as elucidated from the severely reduced efficiency of 8-oxoG removal by OGG1 with AP endonuclease-1 when in close proximity to break termini. NEIL-1, however, can partially restore processing of complex DSBs in an AP endonuclease-1 independent manner. The influence of 8-oxoG on ligation shows delayed rejoining if 8-oxoG is positioned two to three bases from the 3'-hydroxy or six bases from the 5'-phosphate termini. When two 8-oxoG lesions are positioned across the break junction ligation is severely retarded. This reduced efficiency of repair indicates that complex DSBs are likely to persist longer than simple DSBs in cells, and as a consequence are more significant in contributing to the biological effects of ionising radiation.


Deciding to institutionalize: why do family members cease caregiving at home?
The primary purpose of this secondary analysis was to identify common themes from the statements of caregivers who ultimately decided to institutionalize their relative with Alzheimer or Parkinson disease. Content analysis of transcripts from caregivers (n=11) who institutionalized their relative during their participation in a caregiver intervention study was performed. Two categories identified from the caregivers' stories were anticipating the inevitable and reaching the limit. The results of the descriptive analysis indicated that 3 to 4 months before institutionalization, caregivers discussed knowing that they would not be able to continue caring for their relative. The most frequent reasons for institutionalization were serious health events. The incidental finding that there were more institutionalizations in the Alzheimer disease participant group than in the Parkinson disease group may indicate that caregiving is more difficult for caregivers in Alzheimer disease than in Parkinson disease. This analysis contributes new and important information about the time interval between caregivers' anticipation of the need for alternative care arrangements and the subsequent placement in formal care. Nurses and other healthcare providers should be alert to the fact that when caregivers express anticipation of the need for change in care arrangements, it may be a signal for immediate assessment and referral to appropriate resources for assistance.


Processing of gene expression data generated by quantitative real-time RT-PCR.
Quantitative real-time PCR represents a highly sensitive and powerful technique for the quantitation of nucleic acids. It has a tremendous potential for the high-throughput analysis of gene expression in research and routine diagnostics. However, the major hurdle is not the practical performance of the experiments themselves but rather the efficient evaluation and the mathematical and statistical analysis of the enormous amount of data gained by this technology, as these functions are not included in the software provided by the manufacturers of the detection systems. In this work, we focus on the mathematical evaluation and analysis of the data generated by quantitative real-time PCR, the calculation of the final results, the propagation of experimental variation of the measured values to the final results, and the statistical analysis. We developed a Microsoft Excel-based software application coded in Visual Basic for Applications, called Q-Gene, which addresses these points. Q-Gene manages and expedites the planning, performance, and evaluation of quantitative real-time PCR experiments, as well as the mathematical and statistical analysis, storage, and graphical presentation of the data. The Q-Gene software application is a tool to cope with complex quantitative real-time PCR experiments at a high-throughput scale and considerably expedites and rationalizes the experimental setup, data analysis, and data management while ensuring highest reproducibility.