Females report higher postoperative pain scores than males after ankle surgery.
The majority of patients experience moderate-to-intense pain following ankle surgery. Early, adequate treatment of postoperative pain is desirable for optimal pain relief, which in turn may facilitate optimal pulmonary function, normal respiration pattern, rehabilitation and prevention of a chronic pain condition. In this retrospective study, we aimed to identify possible predictors of moderate-to-intense postoperative pain while in the Post Anaesthesia Care Unit (PACU) in patients operated for ankle fractures. Social demographics and clinical characteristics from admission throughout the stay in the PACU were collected from the hospital patient record system in retrospect. Pain was assessed using a Visual Analogue Scale (VAS) or a verbal Numeric Rating Scale (vNRS). A VAS/vNRS score 4-6 was classified as moderate and 7-10 as intense pain. Other factors which were investigated were time from ankle fracture to surgery, anaesthetic procedure, pre-, per- and postoperative medical treatment, radiological classification, complexity of fracture, operative technique, and time using tourniquet procedure. Data from 336 patients who underwent surgery to repair an ankle fracture between January 2009 and December 2010 were analysed. None of the following variables had a statistically significant effect on pain; age, weight, smoking, timeframe from fracture to operation, type of anaesthesia, opioids given peroperatively, complexity of the fracture, operation technique or tourniquet inflation procedure. Female sex predicted moderate-to-intense postoperative pain in the PACU with odds ratio 2.31 (95% confidence interval 1.39-3.86), P=0.001. As far as we know, this is the first study to show a sex difference in reporting pain in the first hours after surgery for ankle fracture. Female patients operated for ankle fracture report higher pain-intensity-score than male patients while in the PACU. Our findings suggest that treatment strategies to prevent high peaks of pain should particularly target women operated for an ankle fracture.


Direct and indirect climate change effects on carbon dioxide fluxes in a thawing boreal forest-wetland landscape.
In the sporadic permafrost zone of northwestern Canada, boreal forest carbon dioxide (CO2 ) fluxes will be altered directly by climate change through changing meteorological forcing and indirectly through changes in landscape functioning associated with thaw-induced collapse-scar bog ('wetland') expansion. However, their combined effect on landscape-scale net ecosystem CO2 exchange (NEELAND ), resulting from changing gross primary productivity (GPP) and ecosystem respiration (ER), remains unknown. Here, we quantify indirect land cover change impacts on NEELAND and direct climate change impacts on modeled temperature- and light-limited NEELAND of a boreal forest-wetland landscape. Using nested eddy covariance flux towers, we find both GPP and ER to be larger at the landscape compared to the wetland level. However, annual NEELAND (-20 g C m-2 ) and wetland NEE (-24 g C m-2 ) were similar, suggesting negligible wetland expansion effects on NEELAND . In contrast, we find non-negligible direct climate change impacts when modeling NEELAND using projected air temperature and incoming shortwave radiation. At the end of the 21st century, modeled GPP mainly increases in spring and fall due to reduced temperature limitation, but becomes more frequently light-limited in fall. In a warmer climate, ER increases year-round in the absence of moisture stress resulting in net CO2 uptake increases in the shoulder seasons and decreases during the summer. Annually, landscape net CO2 uptake is projected to decline by 25 ± 14 g C m-2 for a moderate and 103 ± 38 g C m-2 for a high warming scenario, potentially reversing recently observed positive net CO2 uptake trends across the boreal biome. Thus, even without moisture stress, net CO2 uptake of boreal forest-wetland landscapes may decline, and ultimately, these landscapes may turn into net CO2 sources under continued anthropogenic CO2 emissions. We conclude that NEELAND changes are more likely to be driven by direct climate change rather than by indirect land cover change impacts.


Comparing alcohol affordability in 65 cities worldwide.
To develop a measure of alcohol affordability (AA) and compare the AA of 65 cities worldwide. In this paper, AA is defined as the proportion of median daily income needed to buy a certain quantity of certain alcoholic beverages. The income data and the price of alcoholic beverages were drawn from the Union Bank of Switzerland survey and the Economist Intelligence Unit respectively. A large majority of cities (87.7%, n = 57) had a high level of AA. The top 20 ranking was occupied by European and American cities with Tokyo in the Western Pacific region being the exception. All cities belonging to high-income countries had high levels of AA. However, two cities with low-level AA came from low-middle-income countries instead of low-income countries. The findings have shown that alcohol consumption is highly affordable in many cities. If price policy is being considered as policy instrument of alcohol control, it is in urgent need of price adjustments. More specifically, the new emerging economies play a significant role in the world alcohol control movement because of their bright economic performance with huge population size. Further studies on AA, especially periodical monitoring and its impacts on alcohol consumption and alcohol related health problems, should be conducted so as to facilitate the formulation and evaluation price measure of alcohol control.


The relationship between body temperature, heart rate and respiratory rate in children.
To describe and quantify the effect that increasing body temperature has on heart rate and respiratory rate in children attending a paediatric emergency department (ED). Data on pulse, temperature, respiratory rate and age were collected from attendances to two children's ED in the UK between 2003 and 2006. Triage observations as documented at the time were collated and analysed. Data on a total of 63 857 attendances were examined, 31 851 with complete data. Data on children not sent home from the ED were removed, to exclude any patient with haemodynamic shock. The remaining dataset of 21 033 patients with data for heart rate and 14 487 with data for respiratory rate were studied. The state of agitation of the patient was not considered in this study. Body temperature is an independent determinant of heart rate, causing an increase of approximately 10 beats per minute per degree centigrade. Body temperature is also an independent determinant of respiratory rate. This quantification may help in the assessment of the hot and unwell child, to determine whether any tachycardia or tachypnoea is caused solely by fever, or whether there may be an element of concurrent shock.


When a Child Unexpectedly Draws a Violent Scene.
Carter is a 12-year-old boy who has been seeing a developmental-behavioral pediatrician since the age of 7 years for problems with behavioral regulation. Around that time, he began to receive special education services after an educational assessment of autism. He has average intellectual abilities, with below-average semantic-pragmatic speech (e.g., conversations are one-sided). His medical diagnoses included attention-deficit hyperactivity disorder (ADHD), combined presentation, and generalized anxiety disorder. He has never met the DSM criteria for autistic spectrum disorder (ASD) because although he has atypical sensory behaviors (e.g., preoccupied with sniffing objects), he has otherwise lacked restricted, repetitive behaviors. Other medical problems include obesity.His functional impairments associated with impulsivity, inattention, and anxiety improved with combined pharmacotherapy (a long-acting stimulant and a selective serotonin reuptake inhibitor [SSRI], on which he remains) and cognitive-behavioral therapy (CBT). After starting sixth grade, his Individualized Educational Plan (IEP) was modified to address his social impairments, with a self-contained classroom without windows. Soon thereafter, he began to talk about "hating myself" and developed mild-to-moderate depression, which improved after several weeks of a higher dose of SSRI and more frequent visits with his therapist.Several weeks after starting seventh grade, the teacher sent an email to Carter's parents, which they forwarded to his developmental-behavioral pediatrician: "Carter drew a picture of himself shooting and stabbing a student he was mad at today (). He was very upset when I told him I was going to tell you. We haven't processed it through yet but I think a conversation at home about appropriate drawings and using other ways to calm down would help this not happen again."


Can Ketones Help Rescue Brain Fuel Supply in Later Life? Implications for Cognitive Health during Aging and the Treatment of Alzheimer's Disease.
We propose that brain energy deficit is an important pre-symptomatic feature of Alzheimer's disease (AD) that requires closer attention in the development of AD therapeutics. Our rationale is fourfold: (i) Glucose uptake is lower in the frontal cortex of people >65 years-old despite cognitive scores that are normal for age. (ii) The regional deficit in brain glucose uptake is present in adults <40 years-old who have genetic or lifestyle risk factors for AD but in whom cognitive decline has not yet started. Examples include young adult carriers of presenilin-1 or apolipoprotein E4, and young adults with mild insulin resistance or with a maternal family history of AD. (iii) Regional brain glucose uptake is impaired in AD and mild cognitive impairment (MCI), but brain uptake of ketones (beta-hydroxybutyrate and acetoacetate), remains the same in AD and MCI as in cognitively healthy age-matched controls. These observations point to a brain fuel deficit which appears to be specific to glucose, precedes cognitive decline associated with AD, and becomes more severe as MCI progresses toward AD. Since glucose is the brain's main fuel, we suggest that gradual brain glucose exhaustion is contributing significantly to the onset or progression of AD. (iv) Interventions that raise ketone availability to the brain improve cognitive outcomes in both MCI and AD as well as in acute experimental hypoglycemia. Ketones are the brain's main alternative fuel to glucose and brain ketone uptake is still normal in MCI and in early AD, which would help explain why ketogenic interventions improve some cognitive outcomes in MCI and AD. We suggest that the brain energy deficit needs to be overcome in order to successfully develop more effective therapeutics for AD. At present, oral ketogenic supplements are the most promising means of achieving this goal.


Phosphate starvation decouples cell differentiation from DNA replication control in the dimorphic bacterium Caulobacter crescentus.
Upon nutrient depletion, bacteria stop proliferating and undergo physiological and morphological changes to ensure their survival. Yet, how these processes are coordinated in response to distinct starvation conditions is poorly understood. Here we compare the cellular responses of Caulobacter crescentus to carbon (C), nitrogen (N) and phosphorus (P) starvation conditions. We find that DNA replication initiation and abundance of the replication initiator DnaA are, under all three starvation conditions, regulated by a common mechanism involving the inhibition of DnaA translation. By contrast, cell differentiation from a motile swarmer cell to a sessile stalked cell is regulated differently under the three starvation conditions. During C and N starvation, production of the signaling molecules (p)ppGpp is required to arrest cell development in the motile swarmer stage. By contrast, our data suggest that low (p)ppGpp levels under P starvation allow P-starved swarmer cells to differentiate into sessile stalked cells. Further, we show that limited DnaA availability, and consequently absence of DNA replication initiation, is the main reason that prevents P-starved stalked cells from completing the cell cycle. Together, our findings demonstrate that C. crescentus decouples cell differentiation from DNA replication initiation under certain starvation conditions, two otherwise intimately coupled processes. We hypothesize that arresting the developmental program either as motile swarmer cells or as sessile stalked cells improves the chances of survival of C. crescentus during the different starvation conditions.


Identification of a Van der Woude syndrome mutation in the cleft palate 1 mutant mouse.
Mutations in Interferon Regulatory Factor 6 (IRF6) have been identified in two human allelic syndromes with cleft lip and/or palate: Van der Woude (VWS) and Popliteal Pterygium syndromes (PPS). Furthermore, common IRF6 haplotypes and single nucleotide polymorphisms (SNP) alleles are strongly associated with nonsyndromic clefting defects in multiple ethnic populations. Mutations in the mouse often provide good models for the study of human diseases and developmental processes. We identified the cleft palate 1 (clft1) mouse mutant in a forward genetic screen for phenotypes modeling human congenital disease. In the clft1 mutant, we have identified a novel missense point mutation in the mouse Irf6 gene, which confers an amino acid alteration that has been found in a VWS family. Phenotypic comparison of clft1 mutants to previously reported Irf6 mutant alleles demonstrates the Irf6(clft1) allele is a hypomorphic allele. The cleft palate seen in these mutants appears to be due to abnormal adhesion between the palate and tongue. The Irf6(clft1) allele provides the first mouse model for the study of an etiologic IRF6 missense mutation observed in a human VWS family.


Patterns of food safety knowledge among Australians: a latent class approach.
This study aimed to examine food safety knowledge and its associations among Australians. An Internet-based nationwide survey of 2,022 consumers was conducted in 2011. Quota sampling was used to ensure that the age, gender, educational background, and state of residence of the respondents were representative of the Australian population. A list of 10 food safety knowledge items was administered along with questions about the respondents' food attitudes, demographics, school education, and diet practices. Overall, the results showed that safety knowledge was relatively poor. Latent class analysis identified two groups of respondents with different levels of food safety knowledge. Poor knowledge was negatively associated with age, the female gender, university education, experience of home economics or health education at school, the use of salt reduction diets, and general interest in food issues. The study's limitations and implications are discussed.


General practitioners and completeness of cancer registry.
The aim of the study was to evaluate the role of the general practitioner as a source of information for a cancer registry. The study involved a random sample of respondents to a letter inviting participation which was sent to all general practitioners in a specific area. Participating doctors were visited to maximise cooperation. Data collection consisted of setting up a retrospective (prevalence) registry of cancer patients diagnosed over a 20 month period, and a prospective (incidence) register over a subsequent 12 month period. The general practitioner cancer file was then linked to the total cancer registry data base to estimate missed cases. The study took place in the catchment area of the Comprehensive Cancer Centre Middle-Netherlands (IKMN). Of 448 general practitioners in the IKMN region, 152 were willing to participate and of these 52 were randomly selected to take part (11% of all general practitioners in the region). A total of 1637 tumours were identified from the general practitioners, of which 252 (15.4%) were not included in the cancer registry. Of these, only 22 (1.3%) were not included in the registry because they had tumours diagnosed clinically in outpatients and therefore had been omitted from the usual sources of information available to the cancer registry (pathology reports, hospital discharge letters). The missed cases were mostly older patients with digestive tract tumours. On cost-benefit grounds it was not considered feasible to initiate an active cancer registration system among general practitioners, provided that notification of pathological examinations to the registry is complete. Limited under-registration will occur when death certificates cannot be used as an additional source of information.


Expression of 60-kD heat shock protein increases during carcinogenesis in the uterine exocervix.
The aim of the present study was to determine the presence and expression of the 60-kD heat shock protein (HSP60) in the dysplasia-carcinoma sequence in the uterine exocervix and to evaluate its diagnostic and prognostic significance. We performed Western blot and immunohistochemical analyses on biopsies from 40 cases, consisting of 10 normal exocervical biopsies, 10 low-grade squamous intraepithelial lesions (L-SIL), 10 high-grade squamous intraepithelial lesions (H-SIL) and 10 cancerous exocervices (G2 grade). The immunohistochemical results were quantified by computer-assisted image analysis. Western blot analysis showed that HSP60 was undetectable in normal tissues and that there was a gradual increase of protein expression from L-SIL to carcinoma. Immunostaining for HSP60 was negative in normal tissue and positive in basal and parabasal layers of L-SIL epithelium; H-SIL were markedly stained in all layers of epithelium, and carcinomas showed an even stronger positivity. The increasing expression correlated with the malignancy grade. Finally, koilocytes were mostly negative in L-SIL and positive in H-SIL. The increasing degree of expression of HSP60 from L-SIL to carcinoma and the different intraepithelial distribution between L-SIL and H-SIL could be used as a new diagnostic tool. Moreover, HSP60 could have a role in cervical carcinogenesis.


Linking Human Destruction of Nature to COVID-19 Increases Support for Wildlife Conservation Policies.
This paper investigates if narratives varying the cause of the COVID-19 pandemic affects pro-wildlife conservation outcomes. In a pre-registered online experiment (N = 1081), we randomly allocated subjects to either a control group or to one of three narrative treatment groups, each presenting a different likely cause of the COVID-19 outbreak: an animal cause; an animal and human cause (AHC); and an animal, human or lab cause. We found that the AHC narrative elicited significantly greater pro-conservation policy support, especially for bans in the commercial trade of wildlife, when compared to the control group. Possible mechanisms driving this effect are that AHC narratives were less familiar, elicited higher mental and emotional engagement, and induced feelings that firms and governments are responsible for mitigating wildlife extinction.


Lover, mother or worker: women's multiple roles and the HIV/AIDS and reproductive health agenda in Tanzania.
International and national campaigns to prevent HIV/AIDS and efforts to promote reproductive health remain separate in terms of conceptualisation and implementation. Local negotiations around reproductive health issues similarly seem to lack explicit attention to HIV/AIDS. This paper argues that even in reproductive health clinics a gap exists between the extent of knowledge of HIV/AIDS and AIDS talk. There also appears to be a mismatch between collective knowledge of the behavioural and biomedical context of HIV/AIDS and the socio-economic context of AIDS as a lived experience. Using an ethnographic account, I explore how one woman's lived experience and her knowledge of AIDS can teach us to take HIV/AIDS into account when theorising, promoting or providing services for improving African women's reproductive health. The background for this ethnography comes from data collected during 25 months of fieldwork at 10 maternal and child health/family planning (MCH/FP) clinic sites in the Morogoro, Ruvuma and Kilimanjaro regions of Tanzania. Rehema's story shows that AIDS, like other diseases, is significantly linked to host-susceptibility and economic vulnerability. Separate and competing vertical programmes on AIDS and MCH/FP, as commonly encountered throughout Africa, cannot meet the needs of women in countries like Tanzania. Yet, we still hear most often of abstinence, anti-retrovirals and condom use as the primary focus of HIV/AIDS prevention and intervention in Africa.


Combined reconstruction of fluorescent and optical parameters using time-resolved data.
We present an algorithm for simultaneous reconstruction of optical parameters, quantum yield, and lifetime in turbid media with embedded fluorescent inclusions. This algorithm is designed in the Fourier domain as an iterative solution of a system of differential equations of the Helmholtz type and does not involve full ill-conditioned matrix computations. The approach is based on allowing the unknown optical parameters, quantum yield, and lifetime to depend on the Fourier spectral parameter. The algorithm was applied to a time-gated experimental data set acquired by imaging a highly scattering cylindrical phantom concealing small fluorescent tubes. Relatively accurate reconstruction demonstrates the potential of the method.


Gastrostomy in patients with amyotrophic lateral sclerosis (ProGas): a prospective cohort study.
Gastrostomy feeding is commonly used to support patients with amyotrophic lateral sclerosis who develop severe dysphagia. Although recommended by both the American Academy of Neurology and the European Federation of Neurological Societies, currently little evidence indicates the optimum method and timing for gastrostomy insertion. We aimed to compare gastrostomy insertion approaches in terms of safety and clinical outcomes. In this large, longitudinal, prospective cohort study (ProGas), we enrolled patients with a diagnosis of definite, probable, laboratory supported, or possible amyotrophic lateral sclerosis who had agreed with their treating clinicians to undergo gastrostomy at 24 motor neuron disease care centres or clinics in the UK. The primary outcome was 30-day mortality after gastrostomy. This study was registered on the UK Clinical Research Network database, identification number 9923. Between Nov 2, 2010, and Jan 31, 2014, 345 patients were recruited of whom 330 had gastrostomy. 163 (49%) patients underwent percutaneous endoscopic gastrostomy, 121 (37%) underwent radiologically inserted gastrostomy, 43 (13%) underwent per-oral image-guided gastrostomy, and three (1%) underwent surgical gastrostomy. 12 patients (4%, 95% CI 2·1-6·2) died within the first 30 days after gastrostomy: five (3%) of 163 after percutaneous endoscopic gastrostomy, four (3%) of 121 after radiologically inserted gastrostomy, and three (7%) of 43 after per-oral image-guided gastrostomy (p=0·46). Including repeat attempts in 14 patients, 21 (6%) of 344 gastrostomy procedures could not be completed: 11 (6%) of 171 percutaneous endoscopic gastrostomies, seven (6%) of 121 radiologically inserted gastrostomies, and three (6%) of 45 per-oral image-guided gastrostomies (p=0·947). The three methods of gastrostomy seemed to be as safe as each other in relation to survival and procedural complications. In the absence of data from randomised trials, our findings could inform clinicians and patients in reaching decisions about gastrostomy and will stimulate further research into the nutritional management in patients with amyotrophic lateral sclerosis. Motor Neurone Disease Association of Great Britain and Northern Ireland (MNDA) and the Sheffield Institute for Translational Neuroscience (SITraN).


Introgression through rare hybridization: A genetic study of a hybrid zone between red and sika deer (genus Cervus) in Argyll, Scotland.
In this article we describe the structure of a hybrid zone in Argyll, Scotland, between native red deer (Cervus elaphus) and introduced Japanese sika deer (Cervus nippon), on the basis of a genetic analysis using 11 microsatellite markers and mitochondrial DNA. In contrast to the findings of a previous study of the same population, we conclude that the deer fall into two distinct genetic classes, corresponding to either a sika-like or red-like phenotype. Introgression is rare at any one locus, but where the taxa overlap up to 40% of deer carry apparently introgressed alleles. While most putative hybrids are heterozygous at only one locus, there are rare multiple heterozygotes, reflecting significant linkage disequilibrium within both sika- and red-like populations. The rate of backcrossing into the sika population is estimated as H = 0.002 per generation and into red, H = 0.001 per generation. On the basis of historical evidence that red deer entered Kintyre only recently, a diffusion model evaluated by maximum likelihood shows that sika have increased at approximately 9.2% yr-1 from low frequency and disperse at a rate of approximately 3.7 km yr-1. Introgression into the red-like population is greater in the south, while introgression into sika varies little along the transect. For both sika- and red-like populations, the degree of introgression is 30-40% of that predicted from the rates of current hybridization inferred from linkage disequilibria; however, in neither case is this statistically significant evidence for selection against introgression.


Design and analysis of an optoelectronic inclinometer for simultaneous measurement of inclinations along two orthogonal directions.
Conventional inclination measurement systems such as precision vials and capacitance measuring systems can measure inclination in only one direction at a time. We present what is believed to be a new optoelectronic system that can measure inclination angles along two orthogonal directions simultaneously by using a simple pendulum, two mirrors, a 2D position-sensing detector (PSD), and a laser diode. The light ray from the laser is projected onto a mirror that is fixed to a pendulum whose relative angle modifies in response to inclination changes of the inclinometer's housing. The light ray reflected by the mirror is sensed by the PSD, after which the signal can be interpreted by a PSD signal processor, recorded, or output to a computer. This study uses skew-ray tracing methodology to obtain implicit nonlinear system equations to model the relations of the relative inclination angles of the various components, PSD position, and world frame. A first-order Taylor series expansion is used to obtain a linear form of the system equations. To validate the proposed methodology, an actual prototype system is built and experimental results show that the performance of this system is excellent.


Squalus clarkae sp. nov., a new dogfish shark from the Northwest Atlantic and Gulf of Mexico, with comments on the Squalus mitsukurii species complex.
Sharks of the genus Squalus have slow reproductive rates coupled with low genetic diversity, as is typical of deep-water sharks, making this group slow to rebound from depletion due to overfishing. The number of species within Squalus has been expanding recently due to increased attention on taxonomic revision, and a growing research focus on little-known deep-water sharks in general. Here we use genetics and morphology to describe a new species of dogfish shark, Squalus clarkae sp. nov. from the Gulf of Mexico (GoM) which replaces Squalus mitsukurii in this region, and place it in the context of congeners from the Atlantic and elsewhere. Previously, S. clarkae sp. nov. was considered a part of the Squalus mitsukurii species complex, a group of closely related but distinct species. We sequenced the mitochondrial cytochrome oxidase I and the NADH Dehydrogenase II gene of S. mitsukurii from the type location in Japan, S. clarkae sp. nov. from the GoM, as well as three closely related species (S. cubensis, S. blainville, and S. megalops) and S. cf. mitsukurii from Brazil. Squalus clarkae sp. nov. is genetically distinct from other species with significant statistical support (>98.6% bootstrap support/posterior probability), and 2.8% divergent from S. mitsukurii in the type location of Japan. Morphological estimates also revealed differences between S. clarkae sp. nov., S. mitsukurii, and other Atlantic Squalus species, with S. clarkae sp. nov. exhibiting a longer body, smaller interorbital space, shorter caudal fin, and a differently-proportioned first dorsal fin. In general, dogfish sharks in the Atlantic and GoM are characterized by similar but distinct morphology, significant genetic variation, and small species ranges.


Synergistic inhibition of Haemonchus contortus exsheathment by flavonoid monomers and condensed tannins.
This study investigated the separate and combined anthelmintic (AH) effects of different phenolic compounds, including condensed tannins and flavonoids, all of which are known to occur in willow leaves, a potentially valuable dry season feed. A range of contrasting model tannins, which span the whole range of willow tannins, were isolated from tilia flowers, goat willow leaves, black currant leaves and red currant leaves. All together, the tested compounds represented the major tannin types (procyanidins and prodelphinidins) and flavonoid types (flavonols, flavones and flavanones). The larval exsheathment inhibition assay (LEIA) was used to assess their in vitro effects on Haemonchus contortus third stage larvae. Arbutin, vanillic acid, and taxifolin proved to be ineffective whereas naringenin, quercetin and luteolin were highly effective at 250 μM concentrations. Procyanidin (PC) tannins tended to be less active than prodelphinidin tannins (PD). Experiments with combinations of tannins and quercetin or luteolin revealed for the first time the existence of synergistic AH effects between tannins and flavonoid monomers. They also provided evidence that synergistic effects appear to occur at slightly lower concentrations of PC than PD. This suggests that the AH activity of condensed tannins can be significantly enhanced by the addition of quercetin or luteolin. This information may prove useful for plant breeding or selection and for designing optimal feed mixtures.


Cloning and characterization of 5E6(Ly-49C), a receptor molecule expressed on a subset of murine natural killer cells.
5E6 is a cell surface molecule expressed on a subpopulation of murine natural killer (NK) cells that are involved in the specific rejection of H-2d or H-2f (hemopoietic histocompatibility determinant 2) bone marrow cell grafts. Here, we isolated and cloned the gene encoding 5E6 and determined the nucleotide sequence of the cDNA. 5E6 is nearly identical to Ly-49C; the deduced amino acid sequence reveals a polypeptide of 266 amino acids with a molecular weight of 31,284 that contains multiple cysteine residues to explain its disulfide-linked homodimer structure and five potential N-linked glycosylation sites. 5E6 is a type II integral membrane protein with an extracellular carbohydrate recognition domain characteristic of C-type (Ca(2+)-dependent) animal lectins. Chromosomal mapping indicates that 5E6 is located within the NK gene complex on chromosome 6. The sequence of 5E6 mRNA and the degree of glycosylation of 5E6 protein are under genetic control. Immunoprecipitation before removal of N-linked sugars reveals different size molecules. There are several nucleotide differences among BALB/c, B6, and NZB mRNAs; however, none of them would be expected to affect N-glycosylation. Of particular interest are two findings: (a) BALB/c, B6, and (BALB/c x B6)F1 5E6 reduced molecules are approximately 65, 54, and 54 kD, and (b) the cDNA sequence of (BALB/c x B6)F1 is identical to B6. Thus, there appears to be allelic exclusion of 5E6 expression that may be related to the ability of F1 hybrid mice to reject parental H-2d bone marrow cell grafts.


Finite element model predictions of intracranial hemorrhage from non-impact, rapid head rotations in the piglet.
Clinicians are charged with the significant task of distinguishing between accidental and inflicted head trauma. Oftentimes this distinction is straightforward, but many times probabilities of injuries from accidental scenarios are unknown making the differential diagnosis difficult. For example, it is unknown whether intracranial hemorrhage (IH) can occur at a location other than a focal contact site following a low height fall. To create a foundation for predicting regional IH in infants, we sought to identify the biomechanical response and injury threshold best able to predict IH in 3-5 day old piglets. First, finite element (FE) model simulations of in situ animal studies were performed to ascertain the optimal representation of the pia-arachnoid complex, cerebrospinal fluid and cortical vasculature (PCC) for predicting brain strain and brain/skull displacement. Second, rapid head rotations resulting in various degrees of IH were simulated (n=24) to determine the biomechanical predictor and injury threshold most closely correlated with IH. FE models representing the PCC with either spring connectors or solid elements between the brain and skull resulted in peak brain strain and brain/skull displacement similar to measured values in situ. However, when predicting IH, the spring connector representation of the PCC had the best predictive capability for IH with a sensitivity of 80% and a specificity of 85% when ≥ 1% of all spring connectors had at least a peak strain of 0.31 mm/mm. These findings and reported methodology will be used in the development of a human infant FE model to simulate real-world falls and identify injury thresholds for predicting IH in infants.


Myocardial perfusion imaging with 13N-ammonia PET is a strong predictor for outcome.
The aim of the present study was to assess the long-term predictive value of myocardial perfusion imaging (MPI) with (13)N-ammonia positron emission tomography (PET) in patients with suspected myocardial perfusion abnormality. At present, outcome data on the predictive value of MPI in (13)N-ammonia PET exist only for rather small patient populations. Cardiac perfusion was assessed in 943 consecutive patients using (13)N-ammonia PET, and follow-up was obtained in 698 (74%). 77 patients who underwent early revascularization (<60 days) were excluded and 621 patients were assigned to normal versus abnormal perfusion for outcome analysis. Hard events (cardiac death and non-fatal myocardial infarction) and major adverse cardiac events (MACE; hard events, hospitalization for cardiac reasons and late revascularization) were investigated using the Kaplan-Meier method. Independent predictors for various cardiac events were identified using Cox proportional hazard regression analysis. During follow-up (5.7 ± 2.5 years), 275 patients had at least 1 cardiac event, including 102 cardiac deaths and 33 non-fatal myocardial infarction. Abnormal perfusion (n=469) was associated with a higher incidence of MACE (P<0.001) and hard events (P<0.001) throughout the 10-year follow-up period. Cardiac perfusion findings in (13)N-ammonia PET are strong predictors of long-term outcome.


Novel therapeutic agents targeting the glucocorticoid receptor for inflammation and cancer.
Glucocorticoids, through their interaction with the ubiquitous glucocorticoid receptor (GR), have extensive and disparate effects on different cells and tissues. They have long been used in the treatment of asthma, arthritis and autoimmune diseases based on their anti-inflammatory and immunosuppressive effects. For these reasons, as well as for their ability to induce massive apoptosis in hematological malignancies, they are also commonly used as cotreatment in cancers. Despite their wide usage, chronic glucocorticoid therapy has deleterious side effects, including weight gain, osteoporosis and diabetes mellitus, and has been shown to diminish the tumor toxicity of chemotherapy, preventing the full potential of glucocorticoid treatment from being realized. Technological advances have contributed to a better understanding of the mechanism of glucocorticoid action, prompting the development of tailored therapeutics targeting the desired outcomes of GR signaling. This review discusses recent advances in the development of novel therapeutic agents for inflammation and cancer through targeting the GR.


Applying clinical guidelines in general practice: a qualitative study of potential complications.
Clinical guidelines for single diseases often pose problems in general practice work with multimorbid patients. However, little research focuses on how general practice is affected by the demand to follow multiple guidelines. This study explored Norwegian general practitioners' (GPs') experiences with and reflections upon the consequences for general practice of applying multiple guidelines. Qualitative focus group study carried out in Mid-Norway. The study involved a purposeful sample of 25 Norwegian GPs from four pre-existing groups. Interviews were audio-recorded, transcribed and analyzed using systematic text condensation, i.e. applying a phenomenological approach. The GPs' responses clustered around two major topics: 1) Complications for the GPs of applying multiple guidelines; and, 2) Complications for their patients when GPs apply multiple guidelines. For the GPs, applying multiple guidelines created a highly problematic situation as they felt obliged to implement guidelines that were not suited to their patients: too often, the map and the terrain did not match. They also experienced greater insecurity regarding their own practice which, they admitted, resulted in an increased tendency to practice 'defensive medicine'. For their patients, the GPs experienced that applying multiple guidelines increased the risk of polypharmacy, excessive non-pharmacological recommendations, a tendency toward medicalization and, for some, a reduction in quality of life. The GPs experienced negative consequences when obliged to apply a variety of single disease guidelines to multimorbid patients, including increased risk of polypharmacy and overtreatment. We believe patient-centered care and the GPs' courage to non-comply when necessary may aid in reducing these risks. Health care authorities and guideline developers need to be aware of the potential negative effects of applying a single disease focus in general practice, where multimorbidity is highly prevalent.


Combined endoscopic third ventriculostomy and choroid plexus cauterization as primary treatment of hydrocephalus for infants with myelomeningocele: long-term results of a prospective intent-to-treat study in 115 East African infants.
Shunt dependence is more dangerous for children in less developed countries. Combining endoscopic third ventriculostomy (ETV) with choroid plexus cauterization (CPC) was previously shown to treat hydrocephalus more effectively than ETV alone in infants < 1 year of age. The goal of this prospective study was to evaluate the effectiveness of ETV-CPC as primary treatment of hydrocephalus in infants with myelomeningocele. One hundred fifteen consecutive East African infants with myelomeningocele requiring treatment for hydrocephalus were intended for primary management using ETV-CPC. Patient information was prospectively entered into a database. Outcomes were evaluated by life table analysis. Potential predictors for treatment failure were evaluated using multivariate logistic regression. Ninety-three patients had a completed ETV-CPC with > 1 month of follow-up. The ETV-CPC procedure was successful in 71 patients (76%), with a mean and median follow-up of 19.0 months. Treatment failures occurred before 6 months in 86% of the patients, and none occurred after 10 months. The operative mortality rate was 1.1%, and there were no infections. Life table analysis suggested that 72% of the patients would be successfully treated using a single ETV-CPC and 78% would remain shunt-independent with reopening of a closed ETV stoma. Multivariate logistic regression showed scarring of the cistern (p = 0.021) or choroid plexus (p = 0.026) as predictors of failure, but age at the time of surgery was not a significant predictor. Using ETV-CPC appears to successfully provide a more durable primary treatment of hydrocephalus for infants with spina bifida than does shunt placement. These results support ETV-CPC as the better treatment option for these children in developing countries.


In vitro measurement of platelet glycoprotein IIb/IIIa receptor blockade by abciximab: interindividual variation and increased platelet secretion.
Inhibition of soluble fibrinogen binding to activated platelets represents the target of pharmacologic approach with antagonists of the glycoprotein IIb/IIIa (GPIIb/IIIa) complex. In this study we assessed the effects of abciximab, a recombinant chimeric Fab fraction of the antibody against GPIIb/IIIa, on several markers of platelet activation. The platelet surface expression of GPIIb/IIIa was measured by a flow cytometry technique using a two-color assay. GPIIb/IIIa was detected by FITC-conjugated antibodies in whole blood, either unstimulated or exposed to platelet stimuli. The following antibodies were used: CD41, which recognizes the IIb/IIIa complex both in activated and non-activated conformers, and PAC-1, which is directed toward the activated conformer of GPIIb/IIIa. In addition, the same blood sample was incubated with CD62 antibody to measure P-selectin, as a marker of a-granule degranulation. The effect of abciximab was also assessed by experiments carried out on shear stress-induced platelet aggregation, a test that appears to be a predictor of platelet hemostatic function. Abciximab inhibited CD41 binding to glycoprotein IIb (GPIIb) in a concentration-dependent manner and also inhibited the binding of PAC-1 to active GPIIb/IIIa. In contrast, membrane-associated P-selectin was significantly increased by the drug, which suggests that blockade of GPIIb/IIIa receptors results in an increased platelet degranulation in response to agonists. Shear stress-induced platelet aggregation was inhibited by abciximab, with a more pronounced effect on blood filtration, which represents an index of platelet aggregate formation. Our results indicate that GPIIb/IIIa blockade by abciximab is accompanied by an increase of a-granule secretion, suggesting that different mechanisms regulate these aspects of platelet activation. The described flow cytometry technique, that allows the simultaneous in vitro detection of several platelet markers, is a suitable method for assessing the effects of agents which interfere with platelet function.


The promise, problems and pitfalls of mass drug administration for malaria elimination: a qualitative study with scientists and policymakers.
The emergence of artemisinin resistance in the Greater Mekong Subregion (GMS) has prompted urgent containment measures. One possible approach is mass drug administration (MDA). This article explores attitudes towards and perceptions of MDA for malaria elimination among policymakers and leading malariologists. Thirty-two semistructured interviews (SSI) were conducted with policymakers (n=17) and principal investigators (n=15) selected based on their involvement in malaria prevention, control and elimination in the GMS. Interviews were audio recorded and transcribed for qualitative content (thematic) analysis using NVivo (QSR International, Doncaster, Victoria, Australia). Researchers and policymakers described reluctance and consequently delays to pilot MDA for malaria elimination. Most policymakers and some researchers reported concerns around the evidence base, citing a lack of data on its effectiveness and appropriate target populations. There were also worries about promoting resistance. Other issues included a previous lack of support from the World Health Organization, past MDAs, the remoteness of target populations and challenges explaining the rationale for MDA. The complex rationale for MDA for malaria elimination, mistaking pilot studies for implementation, past experiences with MDA, difficulties in selecting appropriate sites and the WHO's lack of clear backing undermined the support for MDA for malaria elimination.


Mapping of determinants of the host range for canine cells in the genome of canine parvovirus using canine parvovirus/mink enteritis virus chimeric viruses.
Feline panleukopenia virus (FPLV), mink enteritis virus (MEV) and canine parvovirus (CPV) are more than 98% similar in DNA and predicted amino acid sequences, but they show different host-cell specificities; CPV is able to replicate in canine cells in culture, whereas FPLV and MEV cannot or replicate only to a low titre. To map the genomic region responsible for the host range of CPV in vitro, CPV/MEV chimeric viruses were generated by transfecting infectious CPV/MEV chimeric plasmids into a cultured feline kidney cell line, and their host cell ranges were analysed. The 60 to 91 map units (m.u.) region of the CPV genome, which contains a part of the capsid protein (VP) gene encoding from amino acid 91 (in the VP2 sequence) to the carboxy terminus of VP protein, was required to impart the ability to replicate in canine cells to MEV, although the chimeric virus containing the 60 to 91 m.u. region of the CPV genome in the MEV background did not replicate in canine cells as efficiently as did CPV derived from the infectious plasmid of CPV. Not only the VP gene, but also a part of the NS gene of CPV were considered to participate in the full expression of the ability to replicate in canine cells. Within the 60 to 91 m.u. region, five of nine amino acid changes between MEV-Abashiri and CPV-Y1 were thought to be phylogenetically CPV-common; however, a recombinant virus containing all five amino acid changes of CPV in the MEV background replicated minimally in canine cells.


De novo assembly, characterization and functional annotation of pineapple fruit transcriptome through massively parallel sequencing.
Pineapple (Ananas comosus var. comosus), is an important tropical non-climacteric fruit with high commercial potential. Understanding the mechanism and processes underlying fruit ripening would enable scientists to enhance the improvement of quality traits such as, flavor, texture, appearance and fruit sweetness. Although, the pineapple is an important fruit, there is insufficient transcriptomic or genomic information that is available in public databases. Application of high throughput transcriptome sequencing to profile the pineapple fruit transcripts is therefore needed. To facilitate this, we have performed transcriptome sequencing of ripe yellow pineapple fruit flesh using Illumina technology. About 4.7 millions Illumina paired-end reads were generated and assembled using the Velvet de novo assembler. The assembly produced 28,728 unique transcripts with a mean length of approximately 200 bp. Sequence similarity search against non-redundant NCBI database identified a total of 16,932 unique transcripts (58.93%) with significant hits. Out of these, 15,507 unique transcripts were assigned to gene ontology terms. Functional annotation against Kyoto Encyclopedia of Genes and Genomes pathway database identified 13,598 unique transcripts (47.33%) which were mapped to 126 pathways. The assembly revealed many transcripts that were previously unknown. The unique transcripts derived from this work have rapidly increased of the number of the pineapple fruit mRNA transcripts as it is now available in public databases. This information can be further utilized in gene expression, genomics and other functional genomics studies in pineapple.


Landscape-Scale Controls on Aboveground Forest Carbon Stocks on the Osa Peninsula, Costa Rica.
Tropical forests store large amounts of carbon in tree biomass, although the environmental controls on forest carbon stocks remain poorly resolved. Emerging airborne remote sensing techniques offer a powerful approach to understand how aboveground carbon density (ACD) varies across tropical landscapes. In this study, we evaluate the accuracy of the Carnegie Airborne Observatory (CAO) Light Detection and Ranging (LiDAR) system to detect top-of-canopy tree height (TCH) and ACD across the Osa Peninsula, Costa Rica. LiDAR and field-estimated TCH and ACD were highly correlated across a wide range of forest ages and types. Top-of-canopy height (TCH) reached 67 m, and ACD surpassed 225 Mg C ha-1, indicating both that airborne CAO LiDAR-based estimates of ACD are accurate in tall, high-biomass forests and that the Osa Peninsula harbors some of the most carbon-rich forests in the Neotropics. We also examined the relative influence of lithologic, topoedaphic and climatic factors on regional patterns in ACD, which are known to influence ACD by regulating forest productivity and turnover. Analyses revealed a spatially nested set of factors controlling ACD patterns, with geologic variation explaining up to 16% of the mapped ACD variation at the regional scale, while local variation in topographic slope explained an additional 18%. Lithologic and topoedaphic factors also explained more ACD variation at 30-m than at 100-m spatial resolution, suggesting that environmental filtering depends on the spatial scale of terrain variation. Our result indicate that patterns in ACD are partially controlled by spatial variation in geologic history and geomorphic processes underpinning topographic diversity across landscapes. ACD also exhibited spatial autocorrelation, which may reflect biological processes that influence ACD, such as the assembly of species or phenotypes across the landscape, but additional research is needed to resolve how abiotic and biotic factors contribute to ACD variation across high biomass, high diversity tropical landscapes.


Technical note: protein conjugate-based immunoassay of whole milk progesterone.
The objectives were to develop a novel protein conjugate-based ELISA test for whole milk progesterone with a dynamic range capable of fully profiling estrous cycles in the dairy cow and to study effects of whole milk medium on antibody binding to progesterone-protein conjugates. A series of progesterone-4-ovalbumin conjugates with different length intermediate linkers were applied as coating antigens in an ELISA format to determine antibody-binding performance in whole milk. Use of an 18-atom linker gave higher binding than use of a 4- or 11-atom linker at certain conjugate concentrations, but no further increase was observed with increasing linker length. An ELISA constructed with the 18-atom linker conjugate gave a detection limit of 0.089 ng/mL progesterone and correlated well to an established radioimmunoassay procedure (r = 0.94). The assay has the distinct advantages of a wide linear range (0.1 to 100 ng/mL), allowing full profiling of bovine estrous cycles, use of whole milk directly without extraction or prior dilution, and employing more easily purified protein conjugates as coating antigens compared with commercial progesterone-enzyme conjugate for milk ELISA assays.


Does Central Vision Loss Impair Visual Search Performance of Adults More than Children?
In general, young adults with normal vision show the best visual search performance when compared with children and older adults. Through our study, we show that this trend is not observed in individuals with vision impairment. An interaction effect of vision impairment with visual development and aging is observed. Performance in many visual tasks typically shows improvement with age until young adulthood and then declines with aging. Using a visual search task, this study investigated whether a similar age effect on performance is present in people with central vision loss. A total of 98 participants, 37 with normal sight (NS) and 61 with visual impairment (VI) searched for targets in 150 real-world digital images. Search performance was quantified by an integrated measure combining speed and accuracy. Participant ages ranged from 5 to 74 years, visual acuity from -0.14 (20/14.5) to 1.16 logMAR (20/290), and log contrast sensitivity (CS) from 0.48 to 2.0. Data analysis was performed with participants divided into three age groups: children (aged <14 years, n = 25), young adults (aged 14 to 45 years, n = 47), and older adults (aged >45 years, n = 26). Regression (r = 0.7) revealed CS (P < .001) and age (P = .003) were significant predictors of search performance. Performance of VI participants was normalized to the age-matched average performance of the NS group. In the VI group, it was found that children's normalized performance (52%) was better than both young (39%, P = .05) and older (40%, P = .048) adults. Unlike NS participants, young adults in the VI group may not have search ability superior to children with VI, despite having the same level of visual functions (quantified by visual acuity and CS). This could be because of vision impairment limiting the developmental acquisition of the age dividend for peak performance. Older adults in the VI group had the worst performance, indicating an interaction of aging.


Postgraduate studies (1978-1985) at the Medical Faculty of the University of Tuzla, Tuzla, Bosnia and Herzegovina.
The Postgraduate studies (PS) at the Medical Faculty (MF) of the University of Tuzla (UT) were founded with the aim of training future staff of the MF in scientific research work. The course lasted four semesters. Up to 1986, classes were attended by five generations or 141 postgraduate students, and 57 of them received their Master's degree (MSc's). Classes were held every week on Fridays and Saturdays. One part of the classes was taught at the MF in Szeged (Hungary). Besides teachers from the UT, classes were also taught by teachers from other universities from the former Yugoslavia and abroad. The most important textbooks were: Cell and Molecular Biology by de Robertisa, Mathematics and statistics for use in pharmacy, biology, and chemistry by Saunders & Fleming, and Kako se pišu saopštenja o medicinskim istraživanjima (How to write reports of medical research) by Rajko Igić. Searching the index base Pub Med at the end of 2014, by the surnames and initials of the names of the 57 masters, we found that they had published 14 articles before completing their MSc's and 821 articles after completing their Master of Science. Later, 35 masters received PhDs and were appointed assistant professors, and later they were also appointed to higher ranks. Looking at the results of the PS, MF of the UT in the above mentioned period, it can be said that the PS was the place where the formation began of the future scientific and teaching staff of the MF in Tuzla.


Utilization rates and perceptions of (VCT) services in Kisii Central District, Kenya.
Voluntary counseling and testing (VCT) services have been set up in most Districts in Kenya due to the rising surge of HIV/AIDS. However, the use of these services among married persons has not been fully explored. In Kissi, the issue of VCT is pressing as the rate of HIV prevalence is close to 3%. In 2006, about 20 000 clients came for VCT services in Kenya yet only 165 of these were married persons. In the Keumbu sub-district hospital, of the more than 1000 clients that came for VCT services, approximately 29% were married persons. This paper therefore aims at determining the utilization of VCT services by married persons in the study area. The qualitative data was obtained principally through two focus group discussions (FGDs) in which the respondents were asked to comment on their use of VCT services while the quantitative data was obtained from interviews with 245 respondents. The qualitative data was analyzed through verbatim transcription while for the quantitative data; the responses were coded and populated into SPSS from which the frequencies and percentages were calculated. The results show that actual use of the VCT services is low (28.1%) but slightly higher among female respondents than males. The low usage may be attributed to (a) fear of results, (b) death anxiety, (c) lack of confidentiality and lastly, (d) fear of stigmatization. Female respondents were found to have a greater awareness of VCT and thus its potential use.


Does rising crime lead to increasing distress? Longitudinal analysis of a natural experiment with dynamic objective neighbourhood measures.
Identifying 'neighbourhood effects' to support widespread beliefs that where we live matters for our health remains a major challenge due to the reliance upon observational data. In this study we reassess the issue of local crime rates and psychological distress by applying unobserved ('fixed') effects models to a sample of participants who remain in the same neighbourhoods throughout the study. Baseline data was extracted from the 45 and Up Study between 2006 and 2008 and followed up as part of the Social Economic and Environmental Factors (SEEF) Study between 2009 and 2010. Kessler 10 scores were recorded for 25,545 men and 29,299 women reported valid outcomes. Annual crime rates per 1000 (including non-domestic violence, malicious damage, break and enter, and stealing, theft and robbery) from 2006 to 2010 inclusive were linked to the person-level data. Change in exposure to crime among participants in this study, therefore, occurs as a result of a change in the local crime rate, rather than a process of neighbourhood selection. Gender stratified unobserved effects logistic regression adjusting for sources of time-varying confounding (age, income, employment, couple status and physical functioning) indicated that an increase in the risk of experiencing psychological distress was generally associated with an increase in the level of neighbourhood crime. Effect sizes were particularly high for women, especially for an increase in malicious damage (Odds Ratio Tertile 3 vs Tertile 1 2.40, 95% Confidence Interval 1.88, 3.05), which may indicate that damage to local built environment is an important pathway linking neighbourhood crime with psychological distress. No statistically significant association was detected for an increase in non-domestic violence, although the effect was in the hypothesised direction. In summary, the application of unobserved effects models to analyse data that takes into account the temporally dynamic characteristics of where people live warrants further investigation.


Complex regulation of Hsf1-Skn7 activities by the catalytic subunits of PKA in Saccharomyces cerevisiae: experimental and computational evidences.
The cAMP-dependent protein kinase regulatory network (PKA-RN) regulates metabolism, memory, learning, development, and response to stress. Previous models of this network considered the catalytic subunits (CS) as a single entity, overlooking their functional individualities. Furthermore, PKA-RN dynamics are often measured through cAMP levels in nutrient-depleted cells shortly after being fed with glucose, dismissing downstream physiological processes. Here we show that temperature stress, along with deletion of PKA-RN genes, significantly affected HSE-dependent gene expression and the dynamics of the PKA-RN in cells growing in exponential phase. Our genetic analysis revealed complex regulatory interactions between the CS that influenced the inhibition of Hsf1/Skn7 transcription factors. Accordingly, we found new roles in growth control and stress response for Hsf1/Skn7 when PKA activity was low (cdc25Δ cells). Experimental results were used to propose an interaction scheme for the PKA-RN and to build an extension of a classic synchronous discrete modeling framework. Our computational model reproduced the experimental data and predicted complex interactions between the CS and the existence of a repressor of Hsf1/Skn7 that is activated by the CS. Additional genetic analysis identified Ssa1 and Ssa2 chaperones as such repressors. Further modeling of the new data foresaw a third repressor of Hsf1/Skn7, active only in the absence of Tpk2. By averaging the network state over all its attractors, a good quantitative agreement between computational and experimental results was obtained, as the averages reflected more accurately the population measurements. The assumption of PKA being one molecular entity has hindered the study of a wide range of behaviors. Additionally, the dynamics of HSE-dependent gene expression cannot be simulated accurately by considering the activity of single PKA-RN components (i.e., cAMP, individual CS, Bcy1, etc.). We show that the differential roles of the CS are essential to understand the dynamics of the PKA-RN and its targets. Our systems level approach, which combined experimental results with theoretical modeling, unveils the relevance of the interaction scheme for the CS and offers quantitative predictions for several scenarios (WT vs. mutants in PKA-RN genes and growth at optimal temperature vs. heat shock).


Flow-based image abstraction.
We present a non-photorealistic rendering technique that automatically delivers a stylized abstraction of a photograph. Our approach is based on shape/color filtering guided by a vector field that describes the flow of salient features in the image. This flow-based filtering significantly improves the abstraction performance in terms of feature enhancement and stylization. Our method is simple, fast, and easy to implement. Experimental results demonstrate the effectiveness of our method in producing stylistic and feature-enhancing illustrations from photographs.


Association Between Time to Treatment With Endovascular Reperfusion Therapy and Outcomes in Patients With Acute Ischemic Stroke Treated in Clinical Practice.
Randomized clinical trials suggest benefit of endovascular-reperfusion therapy for large vessel occlusion in acute ischemic stroke (AIS) is time dependent, but the extent to which it influences outcome and generalizability to routine clinical practice remains uncertain. To characterize the association of speed of treatment with outcome among patients with AIS undergoing endovascular-reperfusion therapy. Retrospective cohort study using data prospectively collected from January 2015 to December 2016 in the Get With The Guidelines-Stroke nationwide US quality registry, with final follow-up through April 15, 2017. Participants were 6756 patients with anterior circulation large vessel occlusion AIS treated with endovascular-reperfusion therapy with onset-to-puncture time of 8 hours or less. Onset (last-known well time) to arterial puncture, and hospital arrival to arterial puncture (door-to-puncture time). Substantial reperfusion (modified Thrombolysis in Cerebral Infarction score 2b-3), ambulatory status, global disability (modified Rankin Scale [mRS]) and destination at discharge, symptomatic intracranial hemorrhage (sICH), and in-hospital mortality/hospice discharge. Among 6756 patients, the mean (SD) age was 69.5 (14.8) years, 51.2% (3460/6756) were women, and median pretreatment score on the National Institutes of Health Stroke Scale was 17 (IQR, 12-22). Median onset-to-puncture time was 230 minutes (IQR, 170-305) and median door-to-puncture time was 87 minutes (IQR, 62-116), with substantial reperfusion in 85.9% (5433/6324) of patients. Adverse events were sICH in 6.7% (449/6693) of patients and in-hospital mortality/hospice discharge in 19.6% (1326/6756) of patients. At discharge, 36.9% (2132/5783) ambulated independently and 23.0% (1225/5334) had functional independence (mRS 0-2). In onset-to-puncture adjusted analysis, time-outcome relationships were nonlinear with steeper slopes between 30 to 270 minutes than 271 to 480 minutes. In the 30- to 270-minute time frame, faster onset to puncture in 15-minute increments was associated with higher likelihood of achieving independent ambulation at discharge (absolute increase, 1.14% [95% CI, 0.75%-1.53%]), lower in-hospital mortality/hospice discharge (absolute decrease, -0.77% [95% CI, -1.07% to -0.47%]), and lower risk of sICH (absolute decrease, -0.22% [95% CI, -0.40% to -0.03%]). Faster door-to-puncture times were similarly associated with improved outcomes, including in the 30- to 120-minute window, higher likelihood of achieving discharge to home (absolute increase, 2.13% [95% CI, 0.81%-3.44%]) and lower in-hospital mortality/hospice discharge (absolute decrease, -1.48% [95% CI, -2.60% to -0.36%]) for each 15-minute increment. Among patients with AIS due to large vessel occlusion treated in routine clinical practice, shorter time to endovascular-reperfusion therapy was significantly associated with better outcomes. These findings support efforts to reduce time to hospital and endovascular treatment in patients with stroke.


Water quality, compliance, and health outcomes among utilities implementing Water Safety Plans in France and Spain.
Water Safety Plans (WSPs), recommended by the World Health Organization since 2004, seek to proactively identify potential risks to drinking water supplies and implement preventive barriers that improve safety. To evaluate the outcomes of WSP application in large drinking water systems in France and Spain, we undertook analysis of water quality and compliance indicators between 2003 and 2015, in conjunction with an observational retrospective cohort study of acute gastroenteritis incidence, before and after WSPs were implemented at five locations. Measured water quality indicators included bacteria (E. coli, fecal streptococci, total coliform, heterotrophic plate count), disinfectants (residual free and total chlorine), disinfection by-products (trihalomethanes, bromate), aluminum, pH, turbidity, and total organic carbon, comprising about 240K manual samples and 1.2M automated sensor readings. We used multiple, Poisson, or Tobit regression models to evaluate water quality before and after the WSP intervention. The compliance assessment analyzed exceedances of regulated, recommended, or operational water quality thresholds using chi-squared or Fisher's exact tests. Poisson regression was used to examine acute gastroenteritis incidence rates in WSP-affected drinking water service areas relative to a comparison area. Implementation of a WSP generally resulted in unchanged or improved water quality, while compliance improved at most locations. Evidence for reduced acute gastroenteritis incidence following WSP implementation was found at only one of the three locations examined. Outcomes of WSPs should be expected to vary across large water utilities in developed nations, as the intervention itself is adapted to the needs of each location. The approach may translate to diverse water quality, compliance, and health outcomes.


Human auditory cortical processing of changes in interaural correlation.
Sensitivity to the similarity of the acoustic waveforms at the two ears, and specifically to changes in similarity, is crucial to auditory scene analysis and extraction of objects from background. Here, we use the high temporal resolution of magnetoencephalography to investigate the dynamics of cortical processing of changes in interaural correlation, a measure of interaural similarity, and compare them with behavior. Stimuli are interaurally correlated or uncorrelated wideband noise, immediately followed by the same noise with intermediate degrees of interaural correlation. Behaviorally, listeners' sensitivity to changes in interaural correlation is asymmetrical. Listeners are faster and better at detecting transitions from correlated noise than transitions from uncorrelated noise. The cortical response to the change in correlation is characterized by an activation sequence starting from approximately 50 ms after change. The strength of this response parallels behavioral performance: auditory cortical mechanisms are much less sensitive to transitions from uncorrelated noise than from correlated noise. In each case, sensitivity increases with interaural correlation difference. Brain responses to transitions from uncorrelated noise lag those from correlated noise by approximately 80 ms, which may be the neural correlate of the observed behavioral response time differences. Importantly, we demonstrate differences in location and time course of neural processing: transitions from correlated noise are processed by a distinct neural population, and with greater speed, than transitions from uncorrelated noise.


High intra-specific variation in avian body condition responses to climate limits generalisation across species.
It is generally assumed that populations of a species will have similar responses to climate change, and thereby that a single value of sensitivity will reflect species-specific responses. However, this assumption is rarely systematically tested. High intraspecific variation will have consequences for identifying species- or population-level traits that can predict differences in sensitivity, which in turn can affect the reliability of projections of future climate change impacts. We investigate avian body condition responses to changes in six climatic variables and how consistent and generalisable these responses are both across and within species, using 21 years of data from 46 common passerines across 80 Dutch sites. We show that body condition decreases with warmer spring/early summer temperatures and increases with higher humidity, but other climate variables do not show consistent trends across species. In the future, body condition is projected to decrease by 2050, mainly driven by temperature effects. Strikingly, populations of the same species generally responded just as differently as populations of different species implying that a single species signal is not meaningful. Consequently, species-level traits did not explain interspecific differences in sensitivities, rather population-level traits were more important. The absence of a clear species signal in body condition responses implies that generalisation and identifying species for conservation prioritisation is problematic, which sharply contrasts conclusions of previous studies on the climate sensitivity of phenology.


Adaptation of Plasmodium falciparum to its transmission environment.
Success in eliminating malaria will depend on whether parasite evolution outpaces control efforts. Here, we show that Plasmodium falciparum parasites (the deadliest of the species causing human malaria) found in low-transmission-intensity areas have evolved to invest more in transmission to new hosts (reproduction) and less in within-host replication (growth) than parasites found in high-transmission areas. At the cellular level, this adaptation manifests as increased production of reproductive forms (gametocytes) early in the infection at the expense of processes associated with multiplication inside red blood cells, especially membrane transport and protein trafficking. At the molecular level, this manifests as changes in the expression levels of genes encoding epigenetic and translational machinery. Specifically, expression levels of the gene encoding AP2-G-the transcription factor that initiates reproduction-increase as transmission intensity decreases. This is accompanied by downregulation and upregulation of genes encoding HDAC1 and HDA1-two histone deacetylases that epigenetically regulate the parasite's replicative and reproductive life-stage programmes, respectively. Parasites in reproductive mode show increased reliance on the prokaryotic translation machinery found inside the plastid-derived organelles. Thus, our dissection of the parasite's adaptive regulatory architecture has identified new potential molecular targets for malaria control.


Stereotactic radiosurgery with versus without prior Onyx embolization for brain arteriovenous malformations.
Investigations of the combined effects of neoadjuvant Onyx embolization and stereotactic radiosurgery (SRS) on brain arteriovenous malformations (AVMs) have not accounted for initial angioarchitectural features prior to neuroendovascular intervention. The aim of this retrospective, multicenter matched cohort study is to compare the outcomes of SRS with versus without upfront Onyx embolization for AVMs using de novo characteristics of the preembolized nidus. The International Radiosurgery Research Foundation AVM databases from 1987 to 2018 were retrospectively reviewed. Patients were categorized based on AVM treatment approach into Onyx embolization (OE) and SRS (OE+SRS) or SRS alone (SRS-only) cohorts and then propensity score matched in a 1:1 ratio. The primary outcome was AVM obliteration. Secondary outcomes were post-SRS hemorrhage, all-cause mortality, radiological and symptomatic radiation-induced changes (RICs), and cyst formation. Comparisons were analyzed using crude rates and cumulative probabilities adjusted for competing risk of death. The matched OE+SRS and SRS-only cohorts each comprised 53 patients. Crude rates (37.7% vs 47.2% for the OE+SRS vs SRS-only cohorts, respectively; OR 0.679, p = 0.327) and cumulative probabilities at 3, 4, 5, and 6 years (33.7%, 44.1%, 57.5%, and 65.7% for the OE+SRS cohort vs 34.8%, 45.5%, 59.0%, and 67.1% for the SRS-only cohort, respectively; subhazard ratio 0.961, p = 0.896) of AVM obliteration were similar between the matched cohorts. The secondary outcomes of the matched cohorts were also similar. Asymptomatic and symptomatic embolization-related complication rates in the matched OE+SRS cohort were 18.9% and 9.4%, respectively. Pre-SRS AVM embolization with Onyx does not appear to negatively influence outcomes after SRS. These analyses, based on de novo nidal characteristics, thereby refute previous studies that found detrimental effects of Onyx embolization on SRS-induced AVM obliteration. However, given the risks incurred by nidal embolization using Onyx, this neoadjuvant intervention should be used judiciously in multimodal treatment strategies involving SRS for appropriately selected large-volume or angioarchitecturally high-risk AVMs.


Effects of somatostatin on vascular endothelial growth factor (VEGF) secretion from non-functioning pituitary tumoral cells incubated in vitro.
The aim of the study was to examine the effect of somatostatin (SST) on vascular endothelial growth factor (VEGF) secretion from clinically non-functioning pituitary tumors incubated in vitro. Eight pituitary tumors surgically removed were investigated. All of the tumors were diagnosed before surgery as non-functioning. Seven of them were diagnosed after surgery as pituitary adenomas and expressed either gonadotropins or their subunits as detected by immunohistochemistry. Two tumors additionally expressed prolactin and growth hormone. One tumor was immunonegative for pituitary hormones and was diagnosed as haemangiopericytoma. All tumors but one were investigated immunohistochemically to detect the somatostatin receptors and expressed at least 3 of 5 subtypes of somatostatin receptors. The cells isolated from the examined tumors were exposed in vitro to native SST-14. The concentration of VEGF in the culture media was performed by means of ELISA method. It was found that the exposure on SST-14 resulted in the divergent changes (increase in 4 cases and decrease in 3 cases) in VEGF concentrations in the medium. It seems that the inhibition of VEGF secretion is related to the expression of somatostatin receptor subtypes sst1 and sst2. The response of VEGF secretion from pituitary tumoral cells to SST seems to depend on the spectrum of expressed somatostatin receptor subtypes. However, this presumption needs further studies on the larger material.


Hyaluronan-CD44 interactions mediate contractility and migration in periodontal ligament cells.
The role of hyaluronan (HA) in periodontal healing has been speculated via its interaction with the CD44 receptor. While HA-CD44 interactions have previously been implicated in numerous cell types; effect and mechanism of exogenous HA on periodontal ligament (PDL) cells is less clear. Herein, we examine the effect of exogenous HA on contractility and migration in human and murine PDL cells using arrays of microposts and time-lapse microscopy. Our findings observed HA-treated human PDL cells as more contractile and less migratory than untreated cells. Moreover, the effect of HA on contractility and focal adhesion area was abrogated when PDL cells were treated with Y27632, an inhibitor of rho-dependent kinase, but not when these cells were treated with ML-7, an inhibitor of myosin light chain kinase. Our results provide insight into the mechanobiology of PDL cells, which may contribute towards the development of therapeutic strategies for periodontal healing and tissue regeneration.


Genome-scale analysis of regulatory protein acetylation enzymes from photosynthetic eukaryotes.
Reversible protein acetylation occurring on Lys-Ne has emerged as a key regulatory post-translational modification in eukaryotes. It is mediated by two groups of enzymes: lysine acetyltransferases (KATs) and lysine deacetylases (KDACs) that catalyze the addition and removal of acetyl groups from target proteins. Estimates indicate that protein acetylation is second to protein phosphorylation in abundance, with thousands of acetylated sites now identified in different subcellular compartments. Considering the important regulatory role of protein phosphorylation, elucidating the diversity of KATs and KDACs across photosynthetic eukaryotes is essential in furthering our understanding of the impact of reversible protein acetylation on plant cell processes. We report a genome-scale analysis of lysine acetyltransferase (KAT)- and lysine deacetylase (KDAC)-families from 53 photosynthetic eukaryotes. KAT and KDAC orthologs were identified in sequenced genomes ranging from glaucophytes and algae to land plants and then analyzed for evolutionary relationships. Based on consensus molecular phylogenetic and subcellular localization data we found new sub-classes of enzymes in established KAT- and KDAC-families. Specifically, we identified a non-photosynthetic origin of the HD-tuin family KDACs, a new monocot-specific Class I HDA-family sub-class, and a phylogenetically distinct Class II algal/heterokont sub-class which maintains an ankyrin domain not conserved in land plant Class II KDACs. Protein structure analysis showed that HDA- and SRT-KDACs exist as bare catalytic subunits with highly conserved median protein length, while all KATs maintained auxiliary domains, with CBP- and TAFII250-KATs displaying protein domain gain and loss over the course of photosynthetic eukaryote evolution in addition to variable protein length. Lastly, promoter element enrichment analyses across species revealed conserved cis-regulatory sequences that support KAT and KDAC involvement in the regulation of plant development, cold/drought stress response, as well as cellular processes such as the circadian clock. Our results reveal new evolutionary, structural, and biological insights into the KAT- and KDAC-families of photosynthetic eukaryotes, including evolutionary parallels to protein kinases and protein phosphatases. Further, we provide a comprehensive annotation framework through our extensive phylogenetic analysis, from which future research investigating aspects of protein acetylation in plants can use to position new findings in a broader context.


Neurodegenerative diseases in children.
The detection of neurodegenerative and neurometabolic diseases in children relies on a high index of suspicion as most will present as common paediatric problems such as recurrent vomiting, feeding problem, failure to thrive, sepsis, or developmental delay. Alternatively, children may present with an acute encephalopathy or with a chronic progressive encephalopathy. Clinical clues suggestive of neurometabolic disorders include encephalopathic features such as microcephaly, macrocephaly, developmental regression, developmental arrest, change in sensorium, seizures, hypotonia, hypertonia, abnormal eye signs; also extrapyramidal or cerebellar signs and systemic features like abnormal respiration, hepatosplenomegaly, abnormal hair, liver dysfunction, renal tubular dysfunction, cardiomyopathy, and feeding difficulties or growth problems. Initial screening include tests for acidosis, ketosis, hyperlacticemia, and hyperammonemia. Further investigations should amino acid chromatography, assays of organic acids, specific enzyme assay of white cell or fibroblast culture, and histopatholgy of cell and tissue biopsy (white blood cell, skin, muscle, conjunctiva, bone marrow, liver, rectum, or brain). The correct diagnosis holds implications for targeted therapeutic intervention, genetic counselling, and possibly, prenatal diagnosis.


Crystal structure of thermostable DNA photolyase: pyrimidine-dimer recognition mechanism.
DNA photolyase is a pyrimidine-dimer repair enzyme that uses visible light. Photolyase generally contains two chromophore cofactors. One is a catalytic cofactor directly contributing to the repair of a pyrimidine-dimer. The other is a light-harvesting cofactor, which absorbs visible light and transfers energy to the catalytic cofactor. Photolyases are classified according to their second cofactor into either a folate- or deazaflavin-type. The native structures of both types of photolyases have already been determined, but the mechanism of substrate recognition remains largely unclear because of the lack of structural information regarding the photolyase-substrate complex. Photolyase from Thermus thermophilus, the first thermostable class I photolyase found, is favorable for function analysis, but even the type of the second cofactor has not been identified. Here, we report the crystal structures of T. thermophilus photolyase in both forms of the native enzyme and the complex along with a part of its substrate, thymine. A structural comparison with other photolyases suggests that T. thermophilus photolyase has structural features allowing for thermostability and that its light-harvesting cofactor binding site bears a close resemblance to a deazaflavin-type photolyase. One thymine base is found at the hole, a putative substrate-binding site near the catalytic cofactor in the complex form. This structural data for the photolyase-thymine complex allow us to propose a detailed model for the pyrimidine-dimer recognition mechanism.


Gestalt neurons and emergent properties in visual perception: A novel concept for the transformation from local to global processing.
Gestalten in visual perception are defined by emergent properties of the whole, which cannot be predicted from the sum of its parts; rather, they arise by virtue of inherent principles, the Laws of Seeing. This review attempts to assign neurophysiological correlates to select emergent properties in motion and contour perception and proposes parallels to the processing of local versus global attributes by classical versus contextual receptive fields. The aim is to identify Gestalt neurons in the visual system to account for the Laws of Seeing in causal terms and to explain "Why do things look as they do" (Koffka, 1935, p. 76).


Narrow linewidth operation of buried-heterostructure photonic crystal nanolaser.
We investigate the spectral linewidth of a monolithic photonic crystal nanocavity laser. The nanocavity laser is based on a buried heterostructure cavity in which an ultra-small InGaAsP active region is embedded in an InP photonic crystal. Although it was difficult to achieve narrow linewidth operation in previously reported photonic crystal nanocavity lasers, we have successfully demonstrated a linewidth of 143.5 MHz, which is far narrower than the cold cavity linewidth and the narrowest value yet reported for nanolasers and photonic crystal lasers. The narrow linewidth is accompanied by a low power consumption and an ultrasmall footprint, thus making this particular laser especially suitable for use as an integrated multi-purpose sensor.


Circles of Support and personalization: Exploring the economic case.
Circles of Support aim to enable people with learning disabilities (and others) to live full lives as part of their communities. As part of a wider study of the economic case for community capacity building conducted from 2012 to 2014, we conducted a mixed methods study of five Circles in North West England. Members of these Circles were supporting adults with moderate to profound learning disabilities and provided accounts of success in enabling the core member to live more independent lives with improved social care outcomes within cost envelopes that appeared to be less than more traditional types of support. The Circles also reported success in harnessing community resources to promote social inclusion and improve well-being. This very small-scale study can only offer tentative evidence but does appear to justify more rigorous research into the potential of Circles to secure cost-effective means of providing support to people with learning disabilities than the alternative, which in most cases would have been a long-term residential care placement.


The substantiation of the elastic-viscoplastic model of the human spine for modeling the correction process of kyphoscoliotic deformation.
The relevance of the problem is caused by an increase in the number of spine-related diseases among children, including scoliosis. Currently, there are no methodologies for the treatment of scoliosis, which ensure an unambiguous positive result. The purpose of the article is to justify the spinal model as an elastic viscoplastic body for further mathematical modeling of the process of spine correction and search for its optimal conditions. The leading approach to the study of this problem is the development of techniques for the surgical treatment of deformities of the vertebral column with the aid of an external fixation device for the spine, providing for a rigid connection of the elements of the apparatus with each other and with the spine. The rigid connection between the elements of the external fixation device increases the degree of static indeterminacy of the design, which leads to the occurrence of additional dangerous stresses in the details of the apparatus and in the vertebrae. The control actions in such devices do not provide an adequate result for the process of correction of the vertebral column. The main result is the substantiation of the spine model as an elastic viscoplastic body. This will allow more detailed consideration of the medical and biological features of the spine and the physical and mechanical properties of human bone and soft tissues. The proposed model will allow developing an adaptive design of the device, taking into account specific features of the organism and more effectively managing the correction process. The materials of the article can be useful for scientists, doctors and specialists in conducting scientific research on the problem of spine deformation correction and the development of appropriate technical means.


Rhythmic neural activity is comodulated with short-term gait modifications during first-time use of a dummy prosthesis: a pilot study.
After transfemoral amputation, many hours of practice are needed to re-learn walking with a prosthesis. The long adaptation process that consolidates a novel gait pattern seems to depend on cerebellar function for reinforcement of specific gait modifications, but the precise, step-by-step gait modifications (e.g., foot placement) most likely rely on top-down commands from the brainstem and cerebral cortex. The aim of this study was to identify, in able-bodied individuals, the specific modulations of cortical rhythms that accompany short-term gait modifications during first-time use of a dummy prosthesis. Fourteen naïve participants walked on a treadmill without (one block, 4 min) and with a dummy prosthesis (three blocks, 3 × 4 min), while ground reaction forces and 32-channel EEG were recorded. Gait cycle duration, stance phase duration, step width, maximal ground reaction force and, ground reaction force trace over time were measured to identify gait modifications. Independent component analysis of EEG data isolated brain-related activity from distinct anatomical sources. The source-level data were segmented into gait cycles and analyzed in the time-frequency domain to reveal relative enhancement or suppression of intrinsic cortical oscillations. Differences between walking conditions were evaluated with one-way ANOVA and post-hoc testing (α = 0.05). Immediate modifications occurred in the gait parameters when participants were introduced to the dummy prosthesis. Except for gait cycle duration, these modifications remained throughout the duration of the experimental session. Power modulations of the theta, mu, beta, and gamma rhythms, of sources presumably from the fronto-central and the parietal cortices, were found across the experimental session. Significant power modulations of the theta, beta, and gamma rhythms within the gait cycle were predominately found around the heel strike of both feet and the swing phase of the right (prosthetic) leg. The modulations of cortical activity could be related to whole-body coordination, including the swing phase and placing of the prosthesis, and the bodyweight transfer between legs and arms. Reduced power modulation of the gamma rhythm within the experimental session may indicate initial motor memories being formed. Better understanding of the sensorimotor processes behind gait modifications may inform the development of neurofeedback strategies to assist gait rehabilitation.


Methods for evaluating endothelial function: a position statement from the European Society of Cardiology Working Group on Peripheral Circulation.
The endothelium holds a pivotal role in cardiovascular health and disease. Assessment of its function was until recently limited to experimental designs due to its location. The advent of novel techniques has facilitated testing on a more detailed basis, with focus on distinct pathways. This review presents available in-vivo and ex-vivo methods for evaluating endothelial function with special focus on more recent ones. The diagnostic modalities covered include assessment of epicardial and microvascular coronary endothelial function, local vasodilation by venous occlusion plethysmography and flow-mediated dilatation, arterial pulse wave analysis and pulse amplitude tonometry, microvascular blood flow by laser Doppler flowmetry, biochemical markers and bioassays, measurement of endothelial-derived microparticles and progenitor cells, and glycocalyx measurements. Insights and practical information on the theoretical basis, methodological aspects, and clinical application in various disease states are discussed. The ability of these methods to detect endothelial dysfunction before overt cardiovascular disease manifests make them attractive clinical tools for prevention and rehabilitation.


LeishGEdit: A Method for Rapid Gene Knockout and Tagging Using CRISPR-Cas9.
Postgenomic analyses of Leishmania biology benefit from rapid and precise methods for gene manipulation. Traditional methods of gene knockout or tagging by homologous recombination have limitations: they tend to be slow and require successive transfection and selection rounds to knock out multiple alleles of a gene. Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 systems overcome these limitations. We describe here in detail a simple, rapid, and scalable method for CRISPR-Cas9-mediated gene knockout and tagging in Leishmania. This method details how to use simple PCR to generate (1) templates for single guide RNA (sgRNA) transcription in cells expressing Cas9 and T7 RNA polymerase and (2) drug-selectable editing cassettes, using a modular set of plasmids as templates. pT plasmids allow for amplification of drug resistance genes for knockouts and pPLOT plasmids provide a choice of different tags to generate N- or C-terminally tagged proteins. We describe how to use an online platform ( LeishGEdit.net ) for automated primer design and how to perform PCRs and transfections in small batches or on 96-well plates for large-scale knockout or tagging screens. This method allows generation of knockout mutants or tagged cell lines within 1 week.


Learning from mistakes. Factors that influence how students and residents learn from medical errors.
Trainees are exposed to medical errors throughout medical school and residency. Little is known about what facilitates and limits learning from these experiences. To identify major factors and areas of tension in trainees' learning from medical errors. Structured telephone interviews with 59 trainees (medical students and residents) from 1 academic medical center. Five authors reviewed transcripts of audiotaped interviews using content analysis. Trainees were aware that medical errors occur from early in medical school. Many had an intense emotional response to the idea of committing errors in patient care. Students and residents noted variation and conflict in institutional recommendations and individual actions. Many expressed role confusion regarding whether and how to initiate discussion after errors occurred. Some noted the conflict between reporting errors to seniors who were responsible for their evaluation. Learners requested more open discussion of actual errors and faculty disclosure. No students or residents felt that they learned better from near misses than from actual errors, and many believed that they learned the most when harm was caused. Trainees are aware of medical errors, but remaining tensions may limit learning. Institutions can immediately address variability in faculty response and local culture by disseminating clear, accessible algorithms to guide behavior when errors occur. Educators should develop longitudinal curricula that integrate actual cases and faculty disclosure. Future multi-institutional work should focus on identified themes such as teaching and learning in emotionally charged situations, learning from errors and near misses and balance between individual and systems responsibility.


Development of the escape response in teleost fishes: do ontogenetic changes enable improved performance?
Teleost fishes typically first encounter the environment as free-swimming embryos or larvae. Larvae are morphologically distinct from adults, and major anatomical structures are unformed. Thus, larvae undergo a series of dramatic morphological changes until they reach adult morphology (but are reproductively immature) and are considered juveniles. Free-swimming embryos and larvae are able to perform a C-start, an effective escape response that is used evade predators. However, escape response performance improves during early development: as young fish grow, they swim faster (length-specific maximum velocity increases) and perform the escape more rapidly (time to complete the behavior decreases). These improvements cease when fish become juveniles, although absolute swimming velocity (m s(-1)) continues to increase. We use studies of escape behavior and ontogeny in California halibut (Paralichthys californicus), rainbow trout (Oncorhynchus mykiss), and razorback suckers (Xyrauchen texanus) to test the hypothesis that specific morphological changes improve escape performance. We suggest that formation of the caudal fin improves energy transfer to the water and therefore increases thrust production and swimming velocity. In addition, changes to the axial skeleton during the larval period produce increased axial stiffness, which in turn allows the production of a more rapid and effective escape response. Because escape performance improves as adult morphology develops, fish that enter the environment in an advanced stage of development (i.e., those with direct development) should have a greater ability to evade predators than do fish that enter the environment at an early stage of development (i.e., those with indirect development).


Expression of the p16INK4A/Cdkn2a gene is prevalently downregulated in human pheochromocytoma tumor specimens.
A number of hereditary syndromes have been found to be associated with pheochromocytoma development, but there is a paucity of data regarding secondary molecular events, such as downregulation of the p16INK4A/Cdkn2a gene (hereafter p16), contributing to pheochromocytoma tumorigenesis. Using tissue microarray and immunohistochemistry, we evaluated the expression of p16 in 31 pheochromocytoma tumor specimens. Our results showed that the p16 gene was expressed at low level or even not expressed in all but one specimens [30/31 (96.8%)], indicative of the prevalence of p16 downregulation in pheochromocytomas. In contrast, high expression of pl6 was observed in the majority of control "normal" specimens [5/7 (71.6%)]. To further investigate the molecular mechanisms underlying pl6 downregulation in pheochromocytomas, we used quantitative real-time PCR, methylation-specific PCR, and direct DNA sequencing to analyze these specimens for potential genetic alterations of the p16 gene. Deletions and aberrant CpG methylation of pl6 were identified in 9 (29.0%) and 11 (35.5%) specimens, respectively, while one specimen harbored a point mutation, Ala --> Pro at residue 20 of P16, and this mutation led to an eightfold decrease in the CDK4-inhibitory activity of P16. The overall frequency of pl6 genetic alterations is 67.7%. Taken together, our results demonstrate that reduced expression of pl6 is a common event in human pheochromocytomas, and the primary cause for such downregulation is inactivating genetic abnormalities in the p16 gene.


Density-functional theory for vacancies in hard-sphere crystals
The equilibrium vacancy concentration in solids can be computed from density-functional theory (DFT) if allowance is made for density profiles with less than one particle per lattice site. For the fundamental-measure theory (FMT), this approach predicts reasonably small vacancy concentrations in hard sphere crystals, in contrast to earlier DFTs. Using an asymptotic analysis of the FMT functional, it is shown that the number of vacancies depends exponentially on the distance to the close packing density, as expected from heuristic arguments. The prefactor of the exponential is calculated for three recently suggested variants of the theory, using density profiles obtained from a quasifree minimization. Extrapolation of the asymptotic behavior to the melting density yields good agreement with other estimates and computer simulation results.