Recombinant expression of molybdenum reductase fragments of plant nitrate reductase at high levels in Pichia pastoris.
Mo reductase (MoR; formerly cytochrome c reductase) fragments of NADH:NO(3) reductase (NR; EC22.214.171.124) were cytosolically expressed in Pichia pastoris, a methylotrophic yeast, using spinach (Spinacia oleracea) and corn (Zea maize) cDNAs. In fermenter cultures, spinach MoR was expressed at 420 mg L(-1), corn MoR at 32 mg L(-1), and corn MoR plus with putative NR interface domain N terminus (MoR+) at 17 mg L(-1). Constitutively expressed MoR+ was structurally stable while it was degraded when expressed by methanol induction, which suggests methanol growth produces more proteinase. Methanol-induced expression yielded more target protein. All three MoR were purified to homogeneity and their polypeptides were approximately 41 (MoR) and approximately 66 (MoR+) kD. MoR was monomeric and MoR+ dimeric, confirming the predicted role for dimer interface domain of NR. MoR+, although differing in quaternary structure from MoR, has similar kinetic properties for ferricyanide and cytochrome c reductase activities and visible spectra, which were like NR. Redox potentials of MoR and MoR+ were similar for flavin, whereas MoR+ had a more negative potential for heme-iron. Reaction schemes for MoR catalyzed reactions were proposed based on fast-reaction rapid-scan stopped-flow kinetic analysis of MoR. P. pastoris is an excellent system for producing the large amounts of NR fragments needed for detailed biochemical studies.


Statistical shape modeling of the left ventricle: myocardial infarct classification challenge.
Statistical shape modeling is a powerful tool for visualizing and quantifying geometric and functional patterns of the heart. After myocardial infarction (MI), the left ventricle typically remodels in response to physiological challenges. Several methods have been proposed in the literature to describe statistical shape changes. Which method best characterizes left ventricular remodeling after MI is an open research question. A better descriptor of remodeling is expected to provide a more accurate evaluation of disease status in MI patients. We therefore designed a challenge to test shape characterization in MI given a set of three-dimensional left ventricular surface points. The training set comprised 100 MI patients, and 100 asymptomatic volunteers (AV). The challenge was initiated in 2015 at the Statistical Atlases and Computational Models of the Heart workshop, in conjunction with the MICCAI conference. The training set with labels was provided to participants, who were asked to submit the likelihood of MI from a different (validation) set of 200 cases (100 AV and 100 MI). Sensitivity, specificity, accuracy and area under the receiver operating characteristic curve were used as the outcome measures. The goals of this challenge were to (1) establish a common dataset for evaluating statistical shape modeling algorithms in MI, and (2) test whether statistical shape modeling provides additional information characterizing MI patients over standard clinical measures. Eleven groups with a wide variety of classification and feature extraction approaches participated in this challenge. All methods achieved excellent classification results with accuracy ranges from 0.83 to 0.98. The areas under the receiver operating characteristic curves were all above 0.90. Four methods showed significantly higher performance than standard clinical measures. The dataset and software for evaluation are available from the Cardiac Atlas Project website1.


Increased expression of Drosophila tetraspanin, Tsp68C, suppresses the abnormal proliferation of ytr-deficient and Ras/Raf-activated hemocytes.
Tetraspanins are evolutionary conserved transmembrane proteins thought to facilitate cell proliferation, movement or fusion by acting as organizers of different signaling events. Despite their prevalence and conservation, their specific role and functions remain largely elusive, as their redundancy in various organisms has hindered loss of function studies. Here, we take a gain of function approach to study Drosophila tetraspanin Tsp68C and its effect on larval hemocytes. We recently characterized a lethal mutation in ytr, a conserved gene that encodes a nuclear arginine-rich protein of unknown function, which is accompanied by abnormal differentiation and proliferation of the larval hematopoietic tissue in flies. A hemolectin (hml)-Gal4 construct carried by hml-Gal4 transgenic flies was sufficient by itself to abrogate the hematopoietic defects in ytr mutant larvae. This rescue correlated with the overexpression of tsp68C, a tetraspanin gene nested in the hml promoter. The suppression of abnormal proliferation by the hml-Gal4 construct was not restricted to ytr-deficient hemocytes, but was also observed in hemocytes expressing the oncogenic forms of Raf or Ras proteins. However, it had no effect on overproliferation mediated by a constitutively active form of Jak. New hml-Gal4 lines, in which the tsp68C gene was silenced or deleted from the promoter, no longer rescued the hematopoietic defect in ytr mutants nor suppressed the activated Raf-induced overproliferation. Therefore, change in tetraspanin Tsp68C expression has a strong suppressor effect on abnormal proliferation and differentiation of hemocytes in the context of specific lesions, such as overactivation of the Ras/Raf/MAPK pathway.


Seminoma in an adult striped dolphin Stenella coeruleoalba: tomographic, macroscopical, histological and immunohistochemical study.
Testicular neoplasms have been extensively described and characterized in domestic animals, but reports in wildlife species, including marine mammals, are scarce. This case report describes a testicular seminoma in an adult striped dolphin Stenella coeruleoalba stranded along the coasts of the Canary Islands. Post-mortem computerized tomography (CT) showed a prominent enlargement of the cranial pole of the right testicle, displacing the intestinal loops. Necropsy gross findings confirmed the presence of a testicular mass, bulging at the cut surface, with multiple well-delimitated whitish nodular lesions. Histologically, intratubular and diffuse neoplastic germinative cell proliferation was described. Complementary immunohistochemical assessments for vimentin and CD117 antibodies were negative. To the authors' knowledge, this is the first seminoma described in a S. coeruleoalba. We suggest that post-mortem (PM) pre-necropsy CT in dolphins is a useful tool for anatomic-guided pathology in such cases.


Mouse bone marrow-derived mast cells and mast cell lines constitutively produce B cell growth and differentiation activities.
The present report describes a novel function of mast cells that consists of a B cell growth activity. The B cell response occurred without any stimulation or preactivation of mast cells. A small number of mast cells was required, since mast cell/B cell ratios as low as 1/100 to 1/10,000 lead to effective B cell activation. Mast cell-dependent B cell activation resulted, within 48 h of incubation, in blast formation, proliferation, and IgM production. Both low and high density B cells were responsive to mast cells. Supernatants from unstimulated mast cells could also activate B cells, suggesting that a B cell-stimulating activity (MC-BSA) is mediated by a soluble factor(s). The addition of anti-IL-4 or anti-IL-6 mAbs or even proteases to the mast cell-derived supernatants did not alter B cell activation. However, treatment of mast cells with mitomycin C or actinomycin D, or paraformaldehyde fixation totally abrogated MC-BSA. Fractionation of mast cell supernatant by gel filtration chromatography resulted in four peaks, ranging from > 200 to 15 kDa, all of which were biologically active on B cells. Because mast cells are known to continuously release proteoglycans, MC-BSA was subjected to chondroitinase and heparinase treatment, but no significant inhibition of B cell activation was obtained. This direct T cell-independent stimulatory effect of mast cells on B cells could account for a mechanism by which plasma cells are continuously produced in lymphoid organs and particularly in bone marrow.


PE2GO: program evaluation of a physical activity program in elementary schools.
PE2GO is a self-contained physical education (PE) program that provides classroom teachers with the tools they need to lead developmentally appropriate PE lessons. The purpose of this study was to evaluate the PE2GO pilot programs in 6 school districts across the United States. We used paper and pencil surveys at pre intervention (n = 114) and mid intervention (n = 94) and an electronic survey at post intervention (n = 65). In addition an electronic survey was sent to administrators at preintervention (n = 18); focus groups were conducted with teachers at mid intervention for a broader perspective. The study took place September 2004 through May 2005. Results indicate that teachers were satisfied with the PE2GO program and the perceived effects it had on their students. Teachers reported that students increased their time engaged in physical activity (128.7-181.1 minutes per week pre-to-post intervention). Administrator support was important (ie, associated with improvement), but not always present. In conclusion, the PE2GO program holds promise for the concept of providing in-class physical activity opportunities for students.


Distinct muscarinic receptors and signal transduction pathways in gallbladder muscle.
Acetylcholine (ACh) caused a dose-dependent contraction of gallbladder muscle cells in either a normal (1.9 mM) Ca2+, zero-Ca2+ or 4 mM Sr2+ medium, with a maximal contraction about 21 +/- 1% at 10(-6) M. Pirenzepine, methoctramine and p-fluoro-hexahydro-sila-difenidol (the M1, M2 and M3 antagonist, respectively) alone had no inhibitory effect on ACh-induced contraction in normal Ca2+ medium, which was blocked by the combination of methoctramine and p-F-HHSiD. In the 4 mM Sr2+ medium, methoctramine dose dependently inhibited ACh-induced contraction and shifted the ACh dose-response curve to the right. The contraction induced by ACh was further blocked by 10(-4) M propranolol (phosphatidic acid phosphohydrolase inhibitor that prevents the production of diacylglycerol from phospholipase D activation), 10(-5) M H-7 and chelerythrine (the protein kinase C inhibitors) by 64%, 75% and 77%, respectively. In contrast, in the zero-Ca2+ medium, p-fluoro-hexahydro-sila-difenidol dose-dependently inhibited ACh-induced contraction and shifted the ACh dose-response curve to the right. The action of ACh was further blocked by 10(-6) M U-73122 (phospholipase C inhibitor) and 10(-5) M CGS 9343B (calmodulin antagonist) by 95% and 77%, respectively. In conclusion, ACh contracts the gallbladder muscle by stimulating the M2 and M3 muscarinic receptors. The M2 receptors are linked to Ca2+ influx, activation of phospholipase D and protein kinase C-dependent pathway, whereas the M3 receptors are preferentially associated with the activation of phospholipase C, intracellular Ca2+ release and calmodulin-dependent pathway.


Identification of risk factors for radiographic hyperparathyroidism in 422 patients with end-stage renal disease: development of a clinical predictive index.
The hand radiographs of 422 patients with end-stage renal failure were graded for severity of subperiosteal resorption. Two hundred and seventy-three patients (64.7%) had no evidence of resorption; 114 (27.0%) showed resorption, in 32 of whom it was severe (7.6% of the total). Thirty-five patients (8.3%) were assessed as having doubtful evidence of subperiosteal resorption. Age, gender, race, renal diagnosis, duration of renal failure and vitamin D status were assessed as potential risk factors for the development of subperiosteal resorption. Duration of renal failure, female gender, young age, and certain renal diagnostic groups namely obstructive uropathy, unknown diagnosis, presumed glomerulonephritis and tubulointerstitial disease emerged as independent risk factors. Diabetic patients appeared to be least at risk of developing subperiosteal resorption. Patients whose renal failure was of unknown duration showed a degree of risk similar to those whose duration was < 2 years. In order to identify prospectively patients likely to develop subperiosteal resorption by the time they reach renal replacement therapy, the relative risks were used to create a risk index. Use of such an index might allow prophylactic treatment to be given to those particularly at risk. The concept of a risk index requires testing by a prospective study, which is in progress.


Structural mechanisms of HECT-type ubiquitin ligases.
Ubiquitin ligases (E3 enzymes) transfer ubiquitin from ubiquitin-conjugating (E2) enzymes to target proteins. By determining the selection of target proteins, modification sites on those target proteins, and the types of ubiquitin modifications that are formed, E3 enzymes are key specificity factors in ubiquitin signaling. Here, I summarize our knowledge of the structural mechanisms in the HECT E3 subfamily, many members of which play important roles in human disease. I discuss interactions of the conserved HECT domain with E2 enzymes, ubiquitin and target proteins, as well as macromolecular interactions with regulatory functions. While we understand individual steps in the catalytic cycle of HECT E3 enzymes on a structural level, this review also highlights key aspects that have yet to be elucidated. For instance, it remains unclear how diverse target proteins are presented to the catalytic center and how certain HECT E3 enzymes achieve specificity in ubiquitin linkage formation. The structural and functional properties of the N-terminal regions of HECT E3 enzymes that likely act as signaling hubs are also largely unknown. Structural insights into these aspects may open up routes for a therapeutic intervention with specific HECT E3 functions in distinct pathophysiological settings.


SelT, SelW, SelH, and Rdx12: genomics and molecular insights into the functions of selenoproteins of a novel thioredoxin-like family.
Selenium is an essential trace element in many life forms due to its occurrence as a selenocysteine (Sec) residue in selenoproteins. The majority of mammalian selenoproteins, however, have no known function. Herein, we performed extensive sequence similarity searches to define and characterize a new protein family, designated Rdx, that includes mammalian selenoproteins SelW, SelV, SelT and SelH, bacterial SelW-like proteins and cysteine-containing proteins of unknown function in all three domains of life. An additional member of this family is a mammalian cysteine-containing protein, designated Rdx12, and its fish selenoprotein orthologue. Rdx proteins are proposed to possess a thioredoxin-like fold and a conserved CxxC or CxxU (U is Sec) motif, suggesting a redox function. We cloned and characterized three mammalian members of this family, which showed distinct expression patterns in mouse tissues and different localization patterns in cells transfected with the corresponding GFP fusion proteins. By analogy to thioredoxin, Rdx proteins can use catalytic cysteine (or Sec) to form transient mixed disulfides with substrate proteins. We employed this property to identify cellular targets of Rdx proteins using affinity columns containing mutant versions of these proteins. Rdx12 was found to interact with glutathione peroxidase 1, whereas 14-3-3 protein was identified as one of the targets of mammalian SelW, suggesting a mechanism for redox regulation of the 14-3-3 family of proteins.


Reliable blood pressure self-measurement in the obstetric waiting room.
Patients often fail to adhere to clinical recommendations when using current blood pressure self-measurement (BPSM) methods and equipment. As existing BPSM equipment is not able to detect non-adherent behavior, this could result in misdiagnosis and treatment error. To overcome this problem, we suggest introducing an alternative method for achieving reliable BPSM by measuring additional context meta-data for validating patient adherence. To facilitate this, we have developed ValidAid, a context-aware system for determining patient adherence levels during BPSM. The aim of this study was to validate this new reliable BPSM method based on ValidAid in the clinical setting. Specifically, we wanted to evaluate ValidAid's ability to accurately detect and model patient adherence levels during BPSM in the clinic. The validation was done by asking 41 pregnant diabetic patients scheduled for self-measuring their blood pressure (BP) in the waiting room at an obstetrics department's outpatient clinic to perform an additional BPSM using ValidAid. We then compared the automatically measured and classified values from ValidAid with our manual observations. We found that a) the pregnant diabetics did not adhere to given instructions when performing BPSM in the waiting room, and that b) the ValidAid system was able to accurately classify patient adherence to the modeled recommendations. A new method for ensuring reliable BPSM based on the ValidAid system was validated. Results indicate that context-aware technology is useful for accurately modeling important aspects of non-adherent patient behavior. This may be used to identify patients in need of additional training, or to design better aids to actively assist the patients during measurements. ValidAid is also applicable to other self-measurement environments including the home setting and outpatient clinics in remote or underserved areas as it is built using telemedicine technology and thus well-suited for remote monitoring and diagnosis.


Cyclooxygenase isozymes: the biology of prostaglandin synthesis and inhibition.
Nonsteroidal anti-inflammatory drugs (NSAIDs) represent one of the most highly utilized classes of pharmaceutical agents in medicine. All NSAIDs act through inhibiting prostaglandin synthesis, a catalytic activity possessed by two distinct cyclooxygenase (COX) isozymes encoded by separate genes. The discovery of COX-2 launched a new era in NSAID pharmacology, resulting in the synthesis, marketing, and widespread use of COX-2 selective drugs. These pharmaceutical agents have quickly become established as important therapeutic medications with potentially fewer side effects than traditional NSAIDs. Additionally, characterization of the two COX isozymes is allowing the discrimination of the roles each play in physiological processes such as homeostatic maintenance of the gastrointestinal tract, renal function, blood clotting, embryonic implantation, parturition, pain, and fever. Of particular importance has been the investigation of COX-1 and -2 isozymic functions in cancer, dysregulation of inflammation, and Alzheimer's disease. More recently, additional heterogeneity in COX-related proteins has been described, with the finding of variants of COX-1 and COX-2 enzymes. These variants may function in tissue-specific physiological and pathophysiological processes and may represent important new targets for drug therapy.


Interactions between plasma homocysteine and arterial stiffness in chronic kidney disease in community-dwelling individuals: The Maine-Syracuse Study.
Plasma homocysteine (tHcy) is associated with kidney disease. However, few, if any, studies have examined homocysteine in relation to arterial stiffness, with stratification by the presence or absence of early-stage chronic kidney disease (CKD). The aim of this study was to examine prospective associations between tHcy and carotid-femoral pulse wave velocity (PWV) in persons with and without early-stage CKD in a sample of community-living individuals free from end-stage renal disease, dialysis, stroke and dementia. We conducted a prospective study with 498 participants of the Maine-Syracuse Longitudinal Study (mean age 61 years). Levels of tHcy were positively related to PWV measured 4-5 years later for participants with early-stage CKD (estimated glomerular filtration rate <60 ml min(-1) per 1.73 m(2)). Statistical adjustment was made for multiple confounders, including demographic factors, PWV-related variables and cardiovascular risk factors (b=4.27, 95% confidence interval: 0.23-8.31, P=0.04). These associations were not observed in persons free from CKD. Plasma tHcy is an important predictor of arterial stiffness, as indexed by PWV, in community-living individuals with modest CKD.


Synthesis and theoretical studies on rare three-coordinate lead complexes.
A series of beta-diketiminate lead halide complexes has been synthesised LPbCl (2), LPbBr (3) and LPbI (4) (L = {N(2,6-(i)Pr(2)C(6)H(3))C(Me)}2CH]), which includes a rare example of a three-coordinate lead iodide (4). The chloride and bromide complexes, 2 and 3, are relatively stable in both the solid and solution states, only slowly decomposing to elemental lead over the course of a month in solution, the lead iodide 4 appears to be less stable and decomposes after 3 d in the solid state at ambient temperatures. The lead chloride complex 2 was treated with KN(SiMe3)2 to yield an unusual terminal lead amide complex LPbN(SiMe3)2 (5). Unlike three-coordinate beta-diketiminate transition metal-halide complexes, the ligands are present in a pyramidal arrangement around the lead centre, commonly attributed to the presence of a stereochemically active lone pair. We have investigated the influence of this lone pair on the geometry of the metal halide complexes 2-4, as well as the isostructural germanium and tin complexes (6 and 7, respectively) using DFT calculations. The lone pair in the lead complexes is significantly more diffuse than in the tin and germanium analogues and only a small amount of hybridisation between the 6s and 6p orbitals is observed.


Sex differences in the response to resistance exercise training in older people.
Resistance exercise training is known to be effective in increasing muscle mass in older people. Acute measurement of protein metabolism data has indicated that the magnitude of response may differ between sexes. We compared adaptive responses in muscle mass and function to 18 weeks resistance exercise training in a cohort of older (>65 years) men and women. Resistance exercise training improved knee extensor maximal torque, 4 m walk time, time to complete five chair rises, muscle anatomical cross-sectional area (ACSA) and muscle quality with no effect on muscle fat/water ratio or plasma glucose, insulin, triacylglycerol, IL-6, and TNF-α Differences between sexes were observed for knee extensor maximal torque and muscle quality with greater increases observed in men versus women (P < 0.05). Maximal torque increased by 15.8 ± 10.6% in women and 41.7 ± 25.5% in men, whereas muscle quality increased by 8.8 ± 17.5% in women and by 33.7 ± 25.6% in men. In conclusion, this study has demonstrated a difference in the magnitude of adaptation, of some of the outcome measures employed, in response to 18 weeks of resistance exercise training between men and women. The mechanisms underlying this observation remain to be established.


Proteins interacting with Leishmania major PUF1: A proteomic dataset.
Leishmania parasites must deal with stressful environmental conditions (thermal, nutritional and oxidative) along their digenetic life cycles. This requires drastic changes in gene expression, which in this parasite occurs mainly through post-transcriptional mechanisms involving RNA binding proteins (RBPs). PUF proteins, a class of RBPs existing in most eukaryotic organisms, might play too an essential role modulating the fate of mRNAs and regulating gene expression in Leishmania parasites. A proteomic approach to identify putative protein partners (interactome) of the Leishmania major PUF1 protein was performed. The PUF1 interactome was characterized by co-immunoprecipitation using L. major cellular extracts and an anti-LiPUF1 antibody, and a subsequent analysis of the co-immunoprecipitated proteins by mass-spectrometry, identifying 90 LmPUF1 candidate partners. Remarkably, many of the identified proteins are other RBPs and/or putative P bodies and mRNA-exporting machinery components. Raw mass spectrometry data are available via ProteomeXchange with identifier PXD022581.


Kinetic chain exercise in knee rehabilitation.
Rehabilitation is recognised as a critical component in the treatment of the anterior cruciate ligament (ACL) injured athlete, and has been the subject of intense research over the past decade. As a result, sound scientific principles have been applied to this realm of sports medicine, and have improved the outcome of both surgical and nonsurgical treatment. Possibly the most intriguing of these principles is the use of the kinetic chain concept in exercise prescription following ACL reconstruction. The hip, knee, and ankle joints when taken together, comprise the lower extremity kinetic chain. Kinetic chain exercises like the squat recruit all 3 links in unison while exercises such as seated quadriceps extensions isolate one link of the chain. Biomechanical assessment with force diagrams reveals that ACL strain is reduced during kinetic chain exercise by virtue of the axial orientation of the applied load and muscular co-contraction. Additionally, kinetic chain exercise through recruitment of all hip, knee, and ankle extensors in synchrony takes advantage of specificity of training principles. More importantly, however, it is the only way to reproduce the concurrent shift of 'antagonistic' biarticular muscle groups that occurs during simultaneous hip, knee, and ankle extension. Incoordination of the concurrent shift fostered by exercising each muscle group in isolation may ultimately hamper complete recovery. Modifying present day leg press and isokinetic equipment will allow clinicians to make better use of kinetic chain exercise and allow safe isokinetic testing of the ACL reconstructed knee. Reconstruction of the ACL with a strong well placed graft to restore joint kinematics, followed by scientifically sound rehabilitation to improve dynamic control of tibial translation, will improve the outcome after ACL injury.


National and sub-national under-five mortality profiles in Peru: a basis for informed policy decisions.
Information on profiles for under-five causes of death is important to guide choice of child-survival interventions. Global level data have been published, but information at country level is scarce. We aimed at defining national and departmental trends and profiles of under-five mortality in Peru from 1996 through 2000. We used the Ministry of Health registered under-five mortality data. For correction of under-registration, a model life-table that fitted the age distribution of the population and of registered deaths was identified for each year. The mortality rates corresponding to these model life-tables were then assigned to each department in each particular year. Cumulative reduction in under-five mortality rate in the 1996-2000 period was estimated calculating the annual reduction slope for each department. Departmental level mortality profiles were constructed. Differences in mortality profiles and in mortality reduction between coastal, andean and jungle regions were also assessed. At country level, only 4 causes (pneumonia, diarrhoea, neonatal diseases and injuries) accounted for 68% of all deaths in 1996, and for 62% in 2000. There was 32.7% of under-five death reduction from 1996 to 2000. Diarrhoea and pneumonia deaths decreased by 84.5% and 41.8%, respectively, mainly in the andean region, whereas deaths due to neonatal causes and injuries decreased by 37.2% and 21.7%. For 1996-2000 period, the andean, coast and jungle regions accounted for 52.4%, 33.1% and 14.4% of deaths, respectively. These regions represent 41.0%, 46.4% and 12.6% of under-five population. Both diarrhoea and pneumonia constitute 30.6% of under-five deaths in the andean region. As a proportion, neonatal deaths remained stable in the country from 1996 to 2000, accounting for about 30% of under-five deaths, whereas injuries and "other" causes, including congenital anomalies, increased by about 5%. Under-five mortality declined substantially in all departments from 1996 to 2000, which is explained mostly by reduction in diarrhoea and pneumonia deaths, particularly in the andean region. There is the need to emphasize interventions to reduce neonatal deaths and emerging causes of death such as injuries and congenital anomalies.


Concentrating childhood cancer treatment in the Netherlands.
Paediatric tertiary care is highly centralized in the Netherlands. The country is small (16 million inhabitants, overall unemployment rates were approximately 7 % in 2014, while young adult (< 25 years) unemployment rates were 12 %) with the majority of the population living in Amsterdam and Rotterdam and their neighbouring cities/villages. There are 90 hospitals taking care for children in the Netherlands. Specific types of highly specialized care, such as transplantation, are provided in a maximum of three centres (kidney transplants in 3, bone marrow tranplants in 2, liver transplants in only one center), while neonatal intensive care is offered in 10 hospitals. Recently, patients with solid tumours in the thorax and abdomen were concentrated in a single centre with five university centres who provide care during the less intensive part of the treatment (in shared care). Similar changes are planned for congenital surgery, aiming for two such centres in the Netherlands. The general view of the Dutch Paediatric Association underscores the need for centralisation, while high-level care should be guaranteed at those hospitals where no specialized centre is present.


Changing human behavior to prevent disease: the importance of targeting automatic processes.
Much of the global burden of disease is associated with behaviors--overeating, smoking, excessive alcohol consumption, and physical inactivity--that people recognize as health-harming and yet continue to engage in, even when undesired consequences emerge. To date, interventions aimed at changing such behaviors have largely encouraged people to reflect on their behaviors. These approaches are often ineffectual, which is in keeping with the observation that much human behavior is automatic, cued by environmental stimuli, resulting in actions that are largely unaccompanied by conscious reflection. We propose that interventions targeting these automatic bases of behaviors may be more effective. We discuss specific interventions and suggest ways to determine whether and how interventions that target automatic processes can enhance global efforts to prevent disease.


Neuroligin-3 and neuroligin-4X form nanoscopic clusters and regulate growth cone organization and size.
The cell-adhesion proteins neuroligin-3 and neuroligin-4X (NLGN3/4X) have well described roles in synapse formation. NLGN3/4X are also expressed highly during neurodevelopment. However, the role these proteins play during this period is unknown. Here we show that NLGN3/4X localized to the leading edge of growth cones where it promoted neuritogenesis in immature human neurons. Super-resolution microscopy revealed that NLGN3/4X clustering induced growth cone enlargement and influenced actin filament organization. Critically, these morphological effects were not induced by autism spectrum disorder (ASD)-associated NLGN3/4X variants. Finally, actin regulators p21-activated kinase 1 and cofilin were found to be activated by NLGN3/4X and involved in mediating the effects of these adhesion proteins on actin filaments, growth cones and neuritogenesis. These data reveal a novel role for NLGN3 and NLGN4X in the development of neuronal architecture, which may be altered in the presence of ASD-associated variants.


The osteoporosis care gap in Canada.
The presence of a fragility fracture is a major risk factor for osteoporosis, and should be an indicator for osteoporosis diagnosis and therapy. However, the extent to which patients who fracture are assessed and treated for osteoporosis is not clear. We performed a review of the literature to identify the practice patterns in the diagnosis and treatment of osteoporosis in adults over the age of 40 who experience a fragility fracture in Canada. Searches were performed in MEDLINE (1966 to January 2, 2003) and CINAHL (1982 to February 1, 2003) databases. There is evidence of a care gap between the occurrence of a fragility fracture and the diagnosis and treatment of osteoporosis in Canada. The proportion of individuals with a fragility fracture who received an osteoporosis diagnostic test or physician diagnosis ranged from 1.7% to 50%. Therapies such as hormone replacement therapy, bisphosphonates or calcitonin were being prescribed to 5.2% to 37.5% of patients. Calcium and vitamin D supplement intake was variable, and ranged between 2.8% to 61.6% of patients. Many Canadians who experience fragility fracture are not receiving osteoporosis management for the prevention of future fractures.


Use of auscultation and Doppler echocardiography in Boxer puppies to predict development of subaortic or pulmonary stenosis.
Boxers are predisposed to subaortic and pulmonic stenosis (SAS, PS). Screening of puppies may be useful in estimating the risk of their developing a defect that potentially compromises life expectancy or exercise tolerance. Presence of SAS or PS in adult Boxers can be predicted by auscultation and Doppler echocardiography at 9-10 weeks of age. Eighty-five Boxer puppies examined at 9-10 weeks of age and at 12 months of age. Prospective, longitudinal observational study. Auscultation by stethoscope and continuous wave-Doppler echocardiography for peak velocities (V(max)) in the aorta (Ao) and pulmonary artery (PA). Intensity of heart murmurs in puppies correlated with V(max)Ao and V(max)PA in adults. V(max)Ao and V(max)PA in puppies correlated with V(max)Ao and V(max)PA in adults, respectively. From puppy to adult, V(max)Ao increased and V(max)PA remained unchanged. The negative predictive value for absent or only a soft (< or =II/VI) murmur in puppies being associated with V(max)Ao and PA < or = 2.4 m/s as an adult was 90% and < or =3.5 m/s 100%. The negative predictive value of a V(max)< or = 2.4 m/s as a puppy still being < or =2.4 m/s as an adult was 94% for Ao and 96% for PA, and of a V(max)< or = 3.5 m/s, 99% for Ao and 100% for PA. Even though V(max)Ao increases during growth in Boxer puppies, indicating relative narrowing of the aorta, puppies with V(max)Ao < or = 2.4 m/s do not usually progress to clinically have relevant SAS at 12 months of age.


Lipopenia and skin barrier abnormalities in DGAT2-deficient mice.
The synthesis of triglycerides is catalyzed by two known acyl-CoA:diacylglycerol acyltransferase (DGAT) enzymes. Although they catalyze the same biochemical reaction, these enzymes share no sequence homology, and their relative functions are poorly understood. Gene knockout studies in mice have revealed that DGAT1 contributes to triglyceride synthesis in tissues and plays an important role in regulating energy metabolism but is not essential for life. Here we show that DGAT2 plays a fundamental role in mammalian triglyceride synthesis and is required for survival. DGAT2-deficient (Dgat2(-/-)) mice are lipopenic and die soon after birth, apparently from profound reductions in substrates for energy metabolism and from impaired permeability barrier function in the skin. DGAT1 was unable to compensate for the absence of DGAT2, supporting the hypothesis that the two enzymes play fundamentally different roles in mammalian triglyceride metabolism.


Health promotion and empowerment from the perspective of individuals living with head and neck cancer.
The aim of this study was to shed light on health promotion from the perspective of individuals living with head and neck cancer. Eight informants were interviewed and latent content analysis was used. Individuals living with head and neck cancer experienced many problems that had a negative impact on their health. One overarching main theme was demonstrated; the ability to regain control and empower oneself. Three themes emerged: (1) Being enabled by dialogue with one's inner self, including three sub-themes: transformed and improved self-esteem, recognising and embracing existentiality, and increased self-determination. (2) Being enabled by means of contact with a social network, including two sub-themes: emotional support and practical support. (3) Being enabled by means of contact with and a passion for the environment, including two sub-themes: nature, hobbies and activities. Empowerment, the goal of health promotion, was understood as an ongoing process, and the ability to promote health varied and was dependent on internal and external enabling of acting and doing. The interpretation of this ongoing process demonstrates interplay assisted by a dialogue with one's inner self, contact with a social network and a passion for the environment. Altogether, these findings may inspire nurses and other health care professionals to support the individual's empowerment process and pose non-pathology-oriented questions such as "what improves your health?" or "what makes you feel good?"


Perceptions of the seriousness of major public health problems during the COVID-19 pandemic in seven middle-income countries.
Public perception of the seriousness of the COVID-19 pandemic compared to six other major public health problems (alcoholism and drug use, HIV/AIDS, malaria, tuberculosis, lung cancer and respiratory diseases caused by air pollution and smoking, and water-borne diseases like diarrhea) is unclear. We designed a survey to examine this issue using YouGov's internet panels in seven middle-income countries in Africa, Asia, and Latin America in early 2022. Respondents rank ordered the seriousness of the seven health problems using a repeated best-worst question format. Rank-ordered logit models allow comparisons within and across countries and assessment of covariates. In six of the seven countries, respondents perceived other respiratory illnesses to be a more serious problem than COVID-19. Only in Vietnam was COVID-19 ranked above other respiratory illnesses. Alcoholism and drug use was ranked the second most serious problem in the African countries. HIV/AIDS ranked relatively high in all countries. Covariates, particularly a COVID-19 knowledge scale, explained differences within countries; statistics about the pandemic were highly correlated with differences in COVID-19's perceived seriousness. People in the seven middle-income countries perceived COVID-19 to be serious (on par with HIV/AIDS) but not as serious as other respiratory illnesses. In the African countries, respondents perceived alcoholism and drug use as more serious than COVID-19. Our survey-based approach can be used to quickly understand how the threat of a newly emergent disease, like COVID-19, fits into the larger context of public perceptions of the seriousness of health problems.


Fluorescent Proteins Detect Host Structural Rearrangements via Electrostatic Mechanism.
The rational design of genetically encoded fluorescent biosensors, which can detect rearrangements of target proteins via interdomain allostery, is hindered by the absence of mechanistic understanding of the underlying photophysics. Here, we focus on the modulation of fluorescence by mechanical perturbation in a popular biological probe: enhanced Green Fluorescent Protein (eGFP). Using a combination of molecular dynamics (MD) simulations and quantum chemistry, and a set of physically motivated assumptions, we construct a map of fluorescence quantum yield as a function of a 2D electric field imposed by the protein environment on the fluorophore. This map is transferable between Tsien's Class 2 GFP's, and it allows one to estimate the shifts in fluorescence intensity due to mechanical perturbations directly from MD simulations. We use it in combination with steered MD simulations to put forward a hypothesis for the mechanism of a genetically encoded voltage probe (ArcLight) whose mechanism is currently under debate.


A minimal region on the integrin beta4 subunit that is critical to its localization in hemidesmosomes regulates the distribution of HD1/plectin in COS-7 cells.
The integrin alpha6 beta4 is a major component of hemidesmosomes, in which it mediates firm adhesion to laminin 5. Previous studies have shown that the incorporation of alpha6 beta4 into hemidesmosomes requires a 303 amino acid stretch of the cytoplasmic domain of beta4, comprising part of the first fibronectin type III (FNIII) repeat, the second FNIII repeat and the segment that connects the second to the third FNIII repeat (connecting segment). Now, we have further defined sequences within beta4 that are critical for its localization in hemidesmosomes and we demonstrate that these sequences also induce the redistribution of HD1/plectin into junctional complexes containing the integrin alpha6 beta4 in COS-7 cells, transfected with cDNAs encoding alpha6A and beta4. Truncation of the cytoplasmic domain of beta4 after amino acids 1,382 or 1,355 in the connecting segment, by which a potential tyrosine activation motif (TAM) is removed, does not prevent the localization of alpha6 beta4 in hemidesmosomes in the rat bladder carcinoma cell line 804G and neither did it eliminate the ability of alpha6 beta4 to change the subcellular distribution of HD1/plectin in COS-7 cells. In contrast, beta4 subunits in which the entire connecting segment had been deleted or which were truncated after amino acid 1,328, which removes almost the complete segment, had lost both of these functions. Furthermore, when beta4 subunits with either a deletion of the second FNIII repeat or a small deletion in this repeat were co-expressed with alpha6, the integrins were not localized in hemidesmosomes and did not induce the redistribution of HD1/plectin in COS-7 cells. Finally, the fourth FNIII repeat of beta4 could not replace the second in either of these activities. These findings establish that a region in beta4, which encompasses the second FNIII repeat and a stretch of 27 amino acids (1,329-1,355) of the connecting segment, is critical for the localization of alpha6beta4 in hemidesmosomes and that it regulates the distribution of HD1/plectin.


Precursors and prodromata of schizophrenia: findings from the Edinburgh High Risk Study and their literature context.
In schizophrenia research, 'high risk' traditionally referred to studies of the offspring of schizophrenic parents at genetically enhanced risk of illness development. Sixteen major high-risk studies have been undertaken although only six followed through to formal illness so data on prediction remain weak. Recently, 'high risk' has widened to encompass individuals considered 'at risk' by having 'high risk mental states', regardless of family history, in whom initiation of early treatment is postulated to improve outcome. The major familial high-risk studies are reviewed from the perspective of the Edinburgh High Risk Study of Schizophrenia (EHRS), with emphasis on prediction. Familial high-risk studies have established multiple biological markers, the most reproducible of which relate to neuromotor development and cognition, especially aspects of memory/learning. Although most are probably not specific, they support a neurodevelopmental hypothesis. Family and environmental variables point largely to secondary or indirect associations. Pre-illness, non-specific affective symptomatology may be of greater predictive power than most psychotic phenomena. Traditional high-risk designs embody many problems but are able to distinguish non-specific markers from illness predictors, and are ideally suited to exploring the evolution of schizophrenia both clinically and biologically (especially with imaging techniques). The EHRS supports the view that greater specificity may accrue to cognitive domains as precursors of predictive utility.


MAPINS, a Highly Efficient Detection Method That Identifies Insertional Mutations and Complex DNA Rearrangements.
Insertional mutagenesis, in which a piece of exogenous DNA is integrated randomly into the genomic DNA of the recipient cell, is a useful method to generate new mutants with phenotypes of interest. The unicellular green alga Chlamydomonas reinhardtii is an outstanding model for studying many biological processes. We developed a new computational algorithm, MAPINS (mapping insertions), to efficiently identify insertion sites created by the integration of an APHVIII (aminoglycoside 3'-phosphotransferase VIII) cassette that confers paromomycin resistance. Using whole-genome sequencing data, this method eliminates the need for genomic DNA manipulation and retains all the sequencing information provided by paired-end sequencing. We experimentally verified 38 insertion sites out of 41 sites (93%) identified by MAPINS from 20 paromomycin-resistant strains. Using meiotic analysis of 18 of these strains, we identified insertion sites that completely cosegregate with paromomycin resistance. In six of the seven strains with a mutant phenotype, we demonstrated complete cosegregation of the mutant phenotype and the verified insertion site. In addition, we provide direct evidence of complex rearrangements of genomic DNA in five strains, three of which involve the APHVIII insertion site. We suggest that strains obtained by insertional mutagenesis are more complicated than expected from previous analyses in Chlamydomonas To map the locations of some complex insertions, we designed 49 molecular markers based on differences identified via whole-genome sequencing between wild-type strains CC-124 and CC-125. Overall, MAPINS provides a low-cost, efficient method to characterize insertional mutants in Chlamydomonas.


Do medical marijuana laws reduce addictions and deaths related to pain killers?
Recent work finds that medical marijuana laws reduce the daily doses filled for opioid analgesics among Medicare Part-D and Medicaid enrollees, as well as population-wide opioid overdose deaths. We replicate the result for opioid overdose deaths and explore the potential mechanism. The key feature of a medical marijuana law that facilitates a reduction in overdose death rates is a relatively liberal allowance for dispensaries. As states have become more stringent in their regulation of dispensaries, the protective value generally has fallen. These findings suggest that broader access to medical marijuana facilitates substitution of marijuana for powerful and addictive opioids.


Taking Refuge in Spirituality, a Main Strategy of Parents of Children with Cancer: a Qualitative Study
Background: Due to the rise in prevalence of childhood cancer the impact on family caregivers is increasing. Spirituality may be an alleviating factor for improving the quality of parent life. There is a lack of organized spiritual care in health care facilities, so that this aspect needs more emphasis. Thus this study aimed to evaluate the spiritual strategy of parents of children with cancer. Materials: This qualitative study was conducted with a conventional content analysis approach. Fifteen parents of hospitalized children with cancer at the Oncology and Hematology departments of governmental pediatric hospitals nationwide were purposefully selected. Semi-structured in-depth interviews were conducted for data collection and inductive content analysis. Results: Eleven sub-themes were extracted in the data analysis, which resulted in the generation of three themes: "spiritual strategy," "spiritual escape" and "spiritual growth". Conclusion: The spiritual approach of parents of children with cancer had a significant influence on spiritual care and consequently the acceptance of disease and coping with the associated conditions. Spiritual care clearly should be introduced into the health care system of Iran, particular for the parents of children with cancer.


A case for serial examination of sputum inflammatory cells.
In the case reported, serial evaluation of sputum inflammatory cell counts made it possible to identify an unusual series of events in a man with eosinophilic bronchitis. The patient initially presented with a productive cough, which did not respond to treatment with antibiotics or high-dose inhaled corticosteroids. A diagnosis of eosinophilic bronchitis was made after demonstration of intense sputum eosinophilia. When inhaled corticosteroids were stopped, symptoms and sputum eosinophilia became worse and airway hyperresponsiveness developed. Both abnormalities were reversed by a course of prednisone. When the prednisone was stopped the productive cough recurred but on this occasion sputum examination suggested a different disease process and the symptoms resolved after a course of co-trimoxazole. The patient has subsequently remained well on no treatment with little or no sputum eosinophilia.


Harnessing mosquito-Wolbachia symbiosis for vector and disease control.
Mosquito species, members of the genera Aedes, Anopheles and Culex, are the major vectors of human pathogens including protozoa (Plasmodium sp.), filariae and of a variety of viruses (causing dengue, chikungunya, yellow fever, West Nile). There is lack of efficient methods and tools to treat many of the diseases caused by these major human pathogens, since no efficient vaccines or drugs are available; even in malaria where insecticide use and drug therapies have reduced incidence, 219 million cases still occurred in 2010. Therefore efforts are currently focused on the control of vector populations. Insecticides alone are insufficient to control mosquito populations since reduced susceptibility and even resistance is being observed more and more frequently. There is also increased concern about the toxic effects of insecticides on non-target (even beneficial) insect populations, on humans and the environment. During recent years, the role of symbionts in the biology, ecology and evolution of insect species has been well-documented and has led to suggestions that they could potentially be used as tools to control pests and therefore diseases. Wolbachia is perhaps the most renowned insect symbiont, mainly due to its ability to manipulate insect reproduction and to interfere with major human pathogens thus providing new avenues for pest control. We herein present recent achievements in the field of mosquito-Wolbachia symbiosis with an emphasis on Aedes albopictus. We also discuss how Wolbachia symbiosis can be harnessed for vector control as well as the potential to combine the sterile insect technique and Wolbachia-based approaches for the enhancement of population suppression programs.


Quantitative nanoscopy of inhibitory synapses: counting gephyrin molecules and receptor binding sites.
The strength of synaptic transmission is controlled by the number and activity of neurotransmitter receptors. However, little is known about absolute numbers and densities of receptor and scaffold proteins and the stoichiometry of molecular interactions at synapses. Here, we conducted three-dimensional and quantitative nanoscopic imaging based on single-molecule detections to characterize the ultrastructure of inhibitory synapses and to count scaffold proteins and receptor binding sites. We observed a close correspondence between the spatial organization of gephyrin scaffolds and glycine receptors at spinal cord synapses. Endogenous gephyrin was clustered at densities of 5,000-10,000 molecules/μm(2). The stoichiometry between gephyrin molecules and receptor binding sites was approximately 1:1, consistent with a two-dimensional scaffold in which all gephyrin molecules can contribute to receptor binding. The competition of glycine and GABAA receptor complexes for synaptic binding sites highlights the potential of single-molecule imaging to quantify synaptic plasticity on the nanoscopic scale.


Reported Incidence and Survival of Fallopian Tube Carcinomas: A Population-Based Analysis From the North American Association of Central Cancer Registries.
Recognition that serous tubal intraepithelial carcinoma (STIC) may represent the first manifestation of many high-grade cancers that were once considered ovarian primary tumors has led to changes in diagnostic practices that could dramatically increase the reporting of tubal carcinomas in US population-based cancer registries. Further, increased detection of early-stage tubal carcinomas through increased recognition coupled with meticulous pathology processing protocols raises important unanswered questions about the clinical behavior of such lesions, which can only be answered using large data sets. However, rates of tubal carcinomas have not been recently analyzed. Accordingly, we analyzed population-based incidence and survival data for fallopian tube carcinoma in situ (CIS; an imperfect surrogate of STIC), tubal carcinomas, and for comparison, ovarian carcinomas, in the North American Association of Central Cancer Registries (NAACCR) registries. Total counts, standardized incidence rates, and stage-specific survival were computed using 30 NAACCR registries (1999-2012). Temporal incidence rate patterns were analyzed by joinpoint regression with estimates of annual percentage change (APC). All statistical tests were two-sided. Fallopian tube CIS incidence rates were stable from 1999 to 2002, then increased from 2002 to 2012 (APC = 16.2%, 95% confidence interval [CI] = 10.9% to 21.7%, P < .001). Rates of early- and late-stage tubal carcinomas showed similar patterns, whereas high-grade serous ovarian carcinoma rates were relatively stable. Five-year cause-specific survival was 97.9% (95% CI = 93.7% to 99.3%) for tubal CIS and 83.2% (95% CI = 77.3% to 87.7%) for early-stage high-grade serous tubal carcinoma. Reporting of tubal CIS and tubal carcinoma have increased in recent years, likely reflecting changes in pathology processing of specimens and diagnosis. Developing standardized reporting for tubal neoplasms is needed to enable analysis of outcomes for these comparatively uncommon but increasingly recognized tumors.


Physiological Functions of the COPI Complex in Higher Plants.
COPI vesicles are essential to the retrograde transport of proteins in the early secretory pathway. The COPI coatomer complex consists of seven subunits, termed α-, β-, β'-, γ-, δ-, ε-, and ζ-COP, in yeast and mammals. Plant genomes have homologs of these subunits, but the essentiality of their cellular functions has hampered the functional characterization of the subunit genes in plants. Here we have employed virus-induced gene silencing (VIGS) and dexamethasone (DEX)-inducible RNAi of the COPI subunit genes to study the in vivo functions of the COPI coatomer complex in plants. The β'-, γ-, and δ-COP subunits localized to the Golgi as GFP-fusion proteins and interacted with each other in the Golgi. Silencing of β'-, γ-, and δ-COP by VIGS resulted in growth arrest and acute plant death in Nicotiana benthamiana, with the affected leaf cells exhibiting morphological markers of programmed cell death. Depletion of the COPI subunits resulted in disruption of the Golgi structure and accumulation of autolysosome-like structures in earlier stages of gene silencing. In tobacco BY-2 cells, DEX-inducible RNAi of β'-COP caused aberrant cell plate formation during cytokinesis. Collectively, these results suggest that COPI vesicles are essential to plant growth and survival by maintaining the Golgi apparatus and modulating cell plate formation.


Using syndemic theory to understand vulnerability to HIV infection among Black and Latino men in New York City.
HIV is a public health crisis that disproportionately affects Black and Latino men. To understand this crisis, syndemic theory, which takes into account multiple interrelated epidemics, should be used. A syndemic is "two or more afflictions, interacting synergistically, contributing to excess burden of disease in a population." Vulnerability to HIV among Black and Latino men is increased as structural, social, and biological factors interact in the context of social marginalization. In New York City, Black and Latino men experience a syndemic of HIV/AIDS, substance abuse, trauma, incarceration, and poverty; however, current research has yet to fully identify the mechanisms of resilience that may reduce the negative impact of a syndemic or explore the potential adaptive functions of individual-level risk behaviors. To understand HIV risk as part of a syndemic and address HIV prevention in Black and Latino men, we propose the following: (1) the use of complex systems analysis, ethnography, and other mixed-methods approaches to observe changes in relations among social conditions and disease; (2) multidisciplinary and inter-institution collaboration; and (3) involvement of public health practitioners and researchers from diverse and underrepresented backgrounds.


Identification of transcription factors from NF-Y, NAC, and SPL families responding to osmotic stress in multiple tomato varieties.
Identifying osmotic stress-responsive transcription factors (TFs) can facilitate discovery of master regulators mediating salt and/or drought tolerance. To date, few RNA-seq datasets for high resolution time course of salt or drought stress treatments are publicly available for certain crop species. However, such datasets may be available for other crops, and in combination with orthology analysis may be used to infer candidate osmotic stress regulators across distantly related species. Here, we demonstrate the utility of this approach for identification and validation of osmotic stress-responsive transcription factors in tomato. First, we developed physiologically calibrated salt and dehydration-responsive systems for tomato cultivars using real time measurements of transpiration rate and photosynthetic efficiency. Next, we identified differentially expressed TFs in rice using raw RNA-seq datasets for a publicly available salt stress time course. Putative salt stress-responsive TFs in tomato were then inferred based on their orthology with the transcription factors upregulated by salt in rice. Finally, using our osmotic stress system, we experimentally validated stress-responsive expression of predicted tomato candidates representing NUCLEAR FACTOR Y, SQUAMOSA PROMOTER BINDING, and NAC domain TF families. Quantification of transcript copy numbers confirmed that mRNAs encoding all three TFs were strongly upregulated not only by salt but also by drought stress. Induction by both salt and dehydration occurred in a temporal manner across diverse tomato cultivars, suggesting that the identified TFs may play important roles in regulating osmotic stress responses.


Probing the dynamic interface between trimethylamine dehydrogenase (TMADH) and electron transferring flavoprotein (ETF) in the TMADH-2ETF complex: role of the Arg-alpha237 (ETF) and Tyr-442 (TMADH) residue pair.
We have used multiple solution state techniques and crystallographic analysis to investigate the importance of a putative transient interaction formed between Arg-alpha237 in electron transferring flavoprotein (ETF) and Tyr-442 in trimethylamine dehydrogenase (TMADH) in complex assembly, electron transfer, and structural imprinting of ETF by TMADH. We have isolated four mutant forms of ETF altered in the identity of the residue at position 237 (alphaR237A, alphaR237K, alphaR237C, and alphaR237E) and with each form studied electron transfer from TMADH to ETF, investigated the reduction potentials of the bound ETF cofactor, and analyzed complex formation. We show that mutation of Arg-alpha237 substantially destabilizes the semiquinone couple of the bound FAD and impedes electron transfer from TMADH to ETF. Crystallographic structures of the mutant ETF proteins indicate that mutation does not perturb the overall structure of ETF, but leads to disruption of an electrostatic network at an ETF domain boundary that likely affects the dynamic properties of ETF in the crystal and in solution. We show that Arg-alpha237 is required for TMADH to structurally imprint the as-purified semiquinone form of wild-type ETF and that the ability of TMADH to facilitate this structural reorganization is lost following (i) redox cycling of ETF, or simple conversion to the oxidized form, and (ii) mutagenesis of Arg-alpha237. We discuss this result in light of recent apparent conflict in the literature relating to the structural imprinting of wild-type ETF. Our studies support a mechanism of electron transfer by conformational sampling as advanced from our previous analysis of the crystal structure of the TMADH-2ETF complex [Leys, D. , Basran, J. , Sutcliffe, M. J., and Scrutton, N. S. (2003) Nature Struct. Biol. 10, 219-225] and point to a key role for the Tyr-442 (TMADH) and Arg-alpha237 (ETF) residue pair in transiently stabilizing productive electron transfer configurations. Our work also points to the importance of Arg-alpha237 in controlling the thermodynamics of electron transfer, the dynamics of ETF, and the protection of reducing equivalents following disassembly of the TMADH-2ETF complex.


High serum ferritin levels increase the risk of hyperuricemia: a cross-sectional and longitudinal study.
To examine the cross-sectional and longitudinal associations of serum ferritin levels with hyperuricemia. A cross-sectional and subsequently prospective study was performed among the employees of Zhenhai Refining and Chemical Company, Ningbo, China. In a cross-sectional study, the association between serum ferritin levels and the prevalence of hyperuricemia was analyzed. Subjects who were free of hyperuricemia at baseline were followed up annually to explore the prospective association between serum ferritin levels and hyperuricemia incidence. Of the 10,074 subjects enrolled at baseline, 1,731 (17.18%) fulfilled the diagnostic criteria of hyperuricemia. Subjects with hyperuricemia presented significantly higher serum ferritin levels, and the levels were positively correlated with the prevalence of hyperuricemia. During a total of 22,367 person-years of follow-up, 502 subjects developed hyperuricemia. The overall incidence of hyperuricemia for 1,000 person-years of follow-up was 22.4, ranging from 17.6 in subjects with baseline serum ferritin levels in the first quintile to 19.2, 21.7, 23.9, and 30.7 in subjects in quintiles 2, 3, 4, and 5, respectively (p for trend < 0.001). Cox regression analyses showed that serum ferritin levels were positively associated with the risk of incident hyperuricemia. Our cross-sectional and longitudinal results indicate that high serum ferritin levels increase the risk of hyperuricemia.


A New Method for Quick and Easy Hemolymph Collection from Apidae Adults.
Bio-analysis of insects is increasingly dependent on highly sensitive methods that require high quality biological material, such as hemolymph. However, it is difficult to collect fresh and uncontaminated hemolymph from adult bees since they are very active and have the potential to sting, and because hemolymph is rapidly melanized. Here we aimed to develop and test a quick and easy method for sterile and contamination-free hemolymph sampling from adult Apidae. Our novel antennae method for hemolymph sampling (AMHS), entailed the detachment of an antenna, followed by application of delicate pressure to the bee's abdomen. This resulted in the appearance of a drop of hemolymph at the base of the detached antenna, which was then aspirated using an automatic pipetter. Larger insect size corresponded to easier and faster hemolymph sampling, and to a greater sample volume. We obtained 80-100 μL of sterile non-melanized hemolymph in 1 minute from one Bombus terrestris worker, in 6 minutes from 10 Apis mellifera workers, and in 15 minutes from 18 Apis cerana workers (+/-0.5 minutes). Compared to the most popular method of hemolymph collection, in which hemolymph is sampled by puncturing the dorsal sinus of the thorax with a capillary (TCHS), significantly fewer bees were required to collect 80-100 μL hemolymph using our novel AMHS method. Moreover, the time required for hemolymph collection was significantly shorter using the AMHS compared to the TCHS, which protects the acquired hemolymph against melanization, thus providing the highest quality material for biological analysis.


The Orthopaedic Foot and Ankle Outcomes Research (OFAR) network: feasibility of a multicenter network for patient outcomes assessment in foot and ankle.
There is an increasing need for orthopaedic practitioners to measure and collect patient-reported outcomes data. In an effort to better understand outcomes from operative treatment, the American Orthopaedic Foot & Ankle Society (AOFAS) established the Orthopaedic Foot and Ankle Outcomes Research (OFAR) Network, a national consortium of foot and ankle orthopaedic surgeons. We hypothesized that the OFAR Network could successfully collect, aggregate, and report patient-reported outcome (PRO) data using the National Institutes of Health (NIH) Patient Reported Outcomes Measurement Information System (PROMIS). Ten sites enrolled consecutive patients undergoing elective surgery for 1 of 6 foot/ankle disorders. Outcome instruments were collected preoperatively and at 6 months postoperatively using the PROMIS online system: Foot and Ankle Ability Measure (FAAM), Foot Function Index (FFI), and PROMIS physical function (PF) and pain computerized adaptive tests (CAT). During the 3-month period, 328 patients were enrolled; 249 (76%) had completed preoperative patient-reported outcomes data and procedure-specific data. Of these, 140 (56%) also completed 6-month postoperative patient- reported outcomes data. Ankle arthritis and flatfoot demonstrated consistently worse preoperative scores. Five of 6 disorders showed significant improvement at 6 months on PF CAT and FAAM, 4 of 6 showed improvement on pain interference CAT, and no disorders showed improvement on FFI. Ankle arthritis and flatfoot demonstrated the greatest magnitude of change on most patient-reported outcomes scales. We were able to enroll large numbers of patients in a short enrollment period for this preliminary study. Data were easily aggregated and analyzed. Substantial loss of follow-up data indicates a critical area requiring further effort. The AOFAS OFAR Network is undergoing expansion with goals to ultimately facilitate large, prospective multicenter studies and optimize the quality and interpretation of available outcome instruments for the foot and ankle population. Level II, prospective comparative study.


Deletion of prostaglandin E2 synthesizing enzymes in brain endothelial cells attenuates inflammatory fever.
Fever is a hallmark of inflammatory and infectious diseases. The febrile response is triggered by prostaglandin E2 synthesis mediated by induced expression of the enzymes cyclooxygenase-2 (COX-2) and microsomal prostaglandin E synthase 1 (mPGES-1). The cellular source for pyrogenic PGE2 remains a subject of debate; several hypotheses have been forwarded, including immune cells in the periphery and in the brain, as well as the brain endothelium. Here we generated mice with selective deletion of COX-2 and mPGES1 in brain endothelial cells. These mice displayed strongly attenuated febrile responses to peripheral immune challenge. In contrast, inflammation-induced hypoactivity was unaffected, demonstrating the physiological selectivity of the response to the targeted gene deletions. These findings demonstrate that PGE2 synthesis in brain endothelial cells is critical for inflammation-induced fever.


Use of energy reserves in fighting hermit crabs.
When animals engage in fights they face a series of decisions, which are based on the value of the contested resource and either their relative or their absolute fighting ability. Certain correlates of fighting ability or 'resource holding potential' such as body size are fixed but physiological correlates are expected to vary during the encounter. We examine the role of energy reserves in determining fight outcomes and parameters during 'shell fighting' in hermit crabs. During these fights, the two contestants perform very different roles of attacker and defender. We show that the balance of the total energy pool, in the form of glucose and glycogen, determines the ability of defenders to resist eviction from their shells. Low glucose in evicted defenders is not caused by depletion of energy reserves, rather mobilization of glycogen appears to be the result of a strategic decision about whether to resist effectively, based on the perceived fighting ability of the attacker. Attackers, however, always initiate the fight so such a decision for this role appears unlikely. In addition to influencing decisions and ability during fights, physiological correlates of fighting ability can in turn be influenced by strategic decisions.


[Self-rated Caffeine Sensitivity: Implications for Personalized Sleep Medicine?].
The prevalence of the insomnia syndrome and the effects of caffeine on sleep are in part genetically determined. Pharmacogenetic studies in humans demonstrate that functional polymorphisms of the genes encoding adenosine A2A receptors and dopamine transporters contribute to individual differences in impaired sleep quality by caffeine. The A2A receptor and dopamine transporter are preferentially expressed in the striatum. Together, these observations suggest that the striatum plays an important role in sleep-wake regulation. Individual caffeine sensitivity and A2A receptor genotype should be taken into account in the development of possible novel adenosine-based pharmacotherapies of sleep-wake disorders and neurodegenerative disorders such as Parkinson's disease. This may permit the prediction of individual drug effects and improve the reliability of clinical trials.


Arrested demixing opens route to bigels.
Understanding and, ultimately, controlling the properties of amorphous materials is one of the key goals of material science. Among the different amorphous structures, a very important role is played by colloidal gels. It has been only recently understood that colloidal gels are the result of the interplay between phase separation and arrest. When short-ranged attractive colloids are quenched into the phase-separating region, density fluctuations are arrested and this results in ramified amorphous space-spanning structures that are capable of sustaining mechanical stress. We present a mechanism of aggregation through arrested demixing in binary colloidal mixtures, which leads to the formation of a yet unexplored class of materials--bigels. This material is obtained by tuning interspecies interactions. Using a computer model, we investigate the phase behavior and the structural properties of these bigels. We show the topological similarities and the geometrical differences between these binary, interpenetrating, arrested structures and their well-known monodisperse counterparts, colloidal gels. Our findings are supported by confocal microscopy experiments performed on mixtures of DNA-coated colloids. The mechanism of bigel formation is a generalization of arrested phase separation and is therefore universal.


Mutations at the Arabidopsis CHM locus promote rearrangements of the mitochondrial genome.
Nuclear recessive mutations at the chloroplast mutator (CHM) locus of Arabidopsis produce a variegated phenotype that is inherited in a non-Mendelian fashion. Molecular analysis of the cytoplasmic genomes of variegated plants from two independent chm mutant lines, using specific chloroplast and mitochondrial probes, showed that the chm mutations reproducibly induce the appearance of specific new restriction fragments in the mitochondrial genome. The presence of these restriction fragments cosegregated with the variegated phenotype in the progeny of crosses between mutant and wild-type plants. Sequence analysis of one of the new restriction fragments found in the variegated plants suggested that it was the product of a rearrangement event involving regions of the mitochondrial genome. Thus, it appears that the CHM locus may encode a protein involved in the control of specific mitochondrial DNA reorganization events.


Neighborhood Opportunity and Mortality Among Children and Adults in Their Households.
Research has linked neighborhood opportunity to health outcomes in children and adults; however, few studies have examined neighborhood opportunity and mortality risk among children and their caregivers. The objective of this study was to assess associations of neighborhood opportunity and mortality risk in children and their caregivers over 11 years. Participants included 1 025 000 children drawn from the Mortality Disparities in American Communities study, a cohort developed by linking the 2008 American Community Survey to the National Death Index and followed for 11 years. Neighborhood opportunity was measured using the Child Opportunity Index, a measure designed to capture compounding inequities in access to opportunities for health. Using hazard models, we observed inverse associations between Child Opportunity Index quintile and deaths among child and caregivers. Children in very low opportunity neighborhoods at baseline had 1.30 times the risk of dying over follow-up relative to those in very high opportunity neighborhoods (95% confidence interval [CI], 1.15-1.45), and this excess risk attenuated after adjustment for household characteristics (hazard ratio, 1.15; 95% CI, 0.98-1.34). Similarly, children in very low opportunity neighborhoods had 1.57 times the risk of experiencing the death of a caregiver relative to those in very high opportunity neighborhoods (95% CI, 1.50-1.64), which remained after adjustment (hazard ratio, 1.30; 95% CI, 1.23-1.38). Our analyses advance understanding of the adverse consequences of inequitable neighborhood contexts for child well-being and underscore the potential importance of place-based policies for reducing disparities in child and caregiver mortality.


Effects of oxide coating and selected cations on nitrate reduction by iron metal.
Under anoxic conditions, zerovalent iron (Fe(0)) reduces nitrate to ammonium and magnetite (Fe3O4) is produced at near-neutral pH. Nitrate removal was most rapid at low pH (2-4); however, the formation of a black oxide film at pH 5 to 8 temporarily halted or slowed the reaction unless the system was augmented with Fe(2+), Cu(2+), or Al(3+). Bathing the corroding Fe(0) in a Fe(2+) solution greatly enhanced nitrate reduction at near-neutral pH and coincided with the formation of a black precipitate. X-ray diffractometry and scanning electron microscopy confirmed that both the black precipitate and black oxide coating on the iron surface were magnetite. In this system, ferrous iron was determined to be a partial contributor to nitrate removal, but nitrate reduction was not observed in the absence of Fe(0). Nitrate removal was also enhanced by augmenting the Fe(0)-H2O system with Fe(3+), Cu(2+), or Al(3+) but not Ca(2+), Mg(2+), or Zn(2+). Our research indicates that a magnetite coating is not a hindrance to nitrate reduction by Fe(0), provided sufficient aqueous Fe(2+) is present in the system.


Estimation of land surface directional emissivity in mid-infrared channel around 4.0 microm from MODIS data.
This work addressed the estimate of the directional emissivity in the mid-infrared (MIR) channel around 4.0 microm from MODIS data. A series of bidirectional reflectances in MODIS channel 22 (3.97 mum) were retrieved using the method developed by Tang and Li (Int. J. Remote Sens. 29, 4907, 2008) and then were used to estimate the directional emissivity in this channel with the aid of the BRDF model modified by Jiang and Li (Opt. Express 16, 19310, 2008). To validate the estimated directional emissivity, a cross-comparison of MODIS derived emissivities in channel 22 using the proposed method were performed with those provided by the MODIS land surface temperature/emissivity product MYD11B1 data. The results show that the proposed method for estimating the directional emissivity in MIR channel gives results comparable to those of MYD11B1 product with a Mean Error of -0.007 and a Root Mean Square Error of 0.024.


Vaccinia as a vector for tumor-directed gene therapy: biodistribution of a thymidine kinase-deleted mutant.
Tumor-directed gene therapy, such as "suicide gene" therapy, requires high levels of gene expression in a high percentage of tumor cells in vivo to be effective. Current vector strategies have been ineffective in achieving these goals. This report introduces the attenuated (thymidine kinase (TK)-negative) replication-competent vaccinia virus (VV) as a potential vector for tumor-directed gene therapy by studying the biodistribution of VV in animal tumor models. A TK-deleted recombinant VV (Western Reserve strain) expressing luciferase on a synthetic promoter was constructed. Luciferase activity was measured in vitro after transduction of a variety of human and murine tumor cell lines and in vivo after intraperitoneal (i.p.) delivery in C57BL/6 mice with 7-day i.p. tumors (10(6) MC-38 cells). Three other in vivo tumor models were examined for tumor-specific gene expression after intravenous delivery of VV (human melanoma in nude mice, adenocarcinoma liver metastasis in immunocompetent mice, and subcutaneous sarcoma in the rat). In addition, a replication-incompetent vaccinia (1 microg of psoralen and ultraviolet light, 365 nm, 4 minutes) was tested in vitro and in vivo and compared with active virus. Luciferase activity in i.p. tumors at 4 days after i.p. injection of VV was >7000-fold higher than lung, >3000-fold higher than liver, and >250-fold higher than ovary. In addition, intravenous injection of VV resulted in markedly higher tumor luciferase activity compared with any other organ in every model tested (up to 188,000-fold higher than liver and 77,000-fold higher than lung). Inactivation of the virus resulted in negligible gene expression in vivo. In summary, VV has a high transduction efficiency in tumor cells with high levels of gene expression. The results suggest a selective in vivo replication of TK-deleted VV in tumor cells. Replication competent, TK-deleted VV appears to be an ideal vector for testing the in vivo delivery of toxic genes to tumor cells.


Prenatal ultrasound scanning and the risk of schizophrenia and other psychoses.
Prenatal ultrasound exposure has been associated with increased prevalence of left-hand or mixed-hand preference, and has been suggested to affect the normal lateralization of the fetal brain. Atypical lateralization is more common in patients with schizophrenia. We evaluated possible associations of prenatal ultrasound with schizophrenia and other psychoses. We identified a cohort of individuals born in Sweden 1973-1978. During this period, one Swedish hospital (Malmö University Hospital) performed prenatal ultrasound on a routine basis, and all individuals born at that hospital were considered exposed to ultrasound. Children born at hospitals where ultrasound was not used routinely or selectively were considered unexposed. We used Poisson regression analysis to estimate the effect of ultrasound exposure on the incidence of schizophrenia and other psychoses. In all, 370,945 individuals were included in the study, of whom 13,212 were exposed to ultrasound. The exposed group demonstrated a tendency toward a higher risk of schizophrenia (among men, crude incidence rate ratio = 1.58 [95% confidence interval = 0.99-2.51]; among women, 1.26 [0.62-2.55]). However, men and women born in several of the 7 tertiary level hospitals without ultrasound scanning also had higher risks of schizophrenia compared with those born in other hospitals. For other psychoses there were no differences between groups. No clear associations between prenatal ultrasound exposure and schizophrenia or other psychoses were found. Other factors related to place of birth might have influenced the results.


Maternal magnesium deficiency in mice leads to maternal metabolic dysfunction and altered lipid metabolism with fetal growth restriction.
Inadequate magnesium (Mg) intake is a widespread problem, with over 50% of women of reproductive age consuming less than the Recommended Dietary Allowance (RDA). Because pregnancy increases the requirement for Mg and the beneficial effects of magnesium sulfate for preeclampsia/eclampsia and fetal neuroprotection are well described, we examined the outcomes of Mg deficiency during pregnancy. Briefly, pregnant Swiss Webster mice were fed either control or Mg-deficient diets starting on gestational day (GD) 6 through euthanasia on GD17. Mg-deficient dams had significantly reduced weight gain and higher plasma adipokines, in the absence of inflammation. Livers of Mg-deficient dams had significantly higher saturated fatty acids (SFAs) and monounsaturated fatty acids (MUFAs) and lower polyunsaturated fatty acids (PUFAs), including docosahexaenoic acid (DHA) (P < 0.0001) and arachidonic acid (AA) (P < 0.0001). Mechanistically, Mg deficiency was accompanied by enhanced desaturase and elongase mRNA expression in maternal livers along with higher circulating insulin and glucose concentrations (P < 0.05) and increased mRNA expression of Srebf1 and Chrebp, regulators of fatty acid synthesis (P < 0.05). Fetal pups exposed to Mg deficiency were growth-restricted and exhibited reduced survival. Mg-deficient fetal livers showed lower MUFAs and higher PUFAs, with lower desaturase and elongase mRNA expression than controls. In addition, DHA concentrations were lower in Mg-deficient fetal brains (P < 0.05). These results indicate that Mg deficiency during pregnancy influences both maternal and fetal fatty acid metabolism, fetal growth and fetal survival, and support better understanding maternal Mg status before and during pregnancy.


Cell fate specification and differentiation in the adult mammalian intestine.
Intestinal stem cells at the bottom of crypts fuel the rapid renewal of the different cell types that constitute a multitasking tissue. The intestinal epithelium facilitates selective uptake of nutrients while acting as a barrier for hostile luminal contents. Recent discoveries have revealed that the lineage plasticity of committed cells - combined with redundant sources of niche signals - enables the epithelium to efficiently repair tissue damage. New approaches such as single-cell transcriptomics and the use of organoid models have led to the identification of the signals that guide fate specification of stem cell progeny into the six intestinal cell lineages. These cell types display context-dependent functionality and can adapt to different requirements over their lifetime, as dictated by their microenvironment. These new insights into stem cell regulation and fate specification could aid the development of therapies that exploit the regenerative capacity and functionality of the gut.


The microRNA156-SQUAMOSA PROMOTER BINDING PROTEIN-LIKE3 module regulates ambient temperature-responsive flowering via FLOWERING LOCUS T in Arabidopsis.
The flowering time of plants is affected by modest changes in ambient temperature. However, little is known about the regulation of ambient temperature-responsive flowering by small RNAs. In this study, we show that the microRNA156 (miR156)-SQUAMOSA PROMOTER BINDING PROTEIN-LIKE3 (SPL3) module directly regulates FLOWERING LOCUS T (FT) expression in the leaf to control ambient temperature-responsive flowering. Overexpression of miR156 led to more delayed flowering at a lower ambient temperature (16°C), which was associated with down-regulation of FT and FRUITFULL expression. Among miR156 target genes, SPL3 mRNA levels were mainly reduced, probably because miR156-mediated cleavage of SPL3 mRNA was higher at 16°C. Overexpression of miR156-resistant SPL3 [SPL3(-)] caused early flowering, regardless of the ambient temperature, which was associated with up-regulation of FT and FRUITFULL expression. Reduction of miR156 activity by target mimicry led to a phenotype similar to that of SUC2::rSPL3 plants. FT up-regulation was observed after dexamethasone treatment in GVG-rSPL3 plants. Misexpression and artificial microRNA-mediated suppression of FT in the leaf dramatically altered the ambient temperature-responsive flowering of plants overexpressing miR156 and SPL3(-). Chromatin immunoprecipitation assay showed that the SPL3 protein directly binds to GTAC motifs within the FT promoter. Lesions in TERMINAL FLOWER1, SHORT VEGETATIVE PHASE, and EARLY FLOWERING3 did not alter the expression of miR156 and SPL3. Taken together, our data suggest that the interaction between the miR156-SPL3 module and FT is part of the regulatory mechanism controlling flowering time in response to ambient temperature.


Terahertz spectroscopy and computational investigation of the flufenamic acid/nicotinamide cocrystal.
Terahertz spectroscopy probes the low-frequency vibrations that are sensitive to both the intermolecular and intramolecular interactions of molecules in the solid state. Thus, terahertz spectroscopy can be a useful tool in the investigation of crystalline pharmaceutical compounds, where slight changes in the packing arrangement can modify the overall effectiveness of a drug formulation. This is especially true for cases of polymorphic systems, hydrates/solvates, and cocrystals. In this work, the cocrystal of flufenamic acid with nicotinamide was investigated using terahertz spectroscopy and solid-state density functional theory. The solid-state simulations enable understanding of the low-frequency vibrations seen in the terahertz spectra, while also providing insight into the energetics involved in the formation of the cocrystal. The comparison of the cocrystal to the pure forms of the molecular components reveals that the cocrystal has better overall binding energy, driven by increased intermolecular hydrogen bond strength and greater London dispersion forces and that the trifluoromethyl torsional potential is significantly different between the studied solids.


microRNAs with AAGUGC seed motif constitute an integral part of an oncogenic signaling network.
microRNA (miRNA) dysregulation is a common feature of cancer cells, but the complex roles of miRNAs in cancer are not fully elucidated. Here, we used functional genomics to identify oncogenic miRNAs in non-small cell lung cancer and evaluate their impact on response to epidermal growth factor (EGFR)-targeting therapy. Our data demonstrate that miRNAs with an AAGUGC motif in their seed sequence increase both cancer cell proliferation and sensitivity to EGFR inhibitors. Global transcriptomics, proteomics and target prediction resulted in the identification of several tumor suppressors involved in the G1/S transition as AAGUGC-miRNA targets. The clinical implications of our findings were evaluated by analysis of AAGUGC-miRNA expression in multiple cancer types, supporting the link between this miRNA seed family, their tumor suppressor targets and cancer cell proliferation. In conclusion, we propose the AAGUGC seed motif as an oncomotif and that oncomotif-miRNAs promote cancer cell proliferation. These findings have potential therapeutic implications, especially in selecting patients for EGFR-targeting therapy.


Atlas-based diffusion tensor imaging correlates of executive function.
Impairment in executive function (EF) is commonly found in Alzheimer's disease (AD) and mild cognitive impairment (MCI). Atlas-based diffusion tensor imaging (DTI) methods may be useful in relating regional integrity to EF measures in MCI and AD. Sixty-six participants (25 normal controls, 22 MCI, and 19 AD) received DTI scans and clinical evaluation. DTI scans were applied to a pre-segmented atlas and fractional anisotropy (FA) and mean diffusivity (MD) were calculated. ANOVA was used to assess group differences in frontal, parietal, and cerebellar regions. For regions differing between groups (p < 0.01), linear regression examined the relationship between EF scores and regional FA and MD. Anisotropy and diffusivity in frontal and parietal lobe white matter structures were associated with EF scores in MCI and only frontal lobe structures in AD. EF was more strongly associated with FA than MD. The relationship between EF and anisotropy and diffusivity was strongest in MCI. These results suggest that regional white matter integrity is compromised in MCI and AD and that FA may be a better correlate of EF than MD.


Delayed hypersensitivity in man: effects of systemic anticoagulation.
Skin test reactivity, lymphocyte transformation, and mononuclear cell tissue factor generation were evaluated both before and during systemic anticoagulation in 24 volunteers. Anticoagulation with warfarin decreased skin test induration and tissue factor generation, but lymphocyte trnasformation remained unchanged. An intact coagulation mechanism, including tissue factor generation, appears to be important for the development of skin test induration in humans.


Is it necessary to re-evaluate diagnostic criteria for Wilson disease in children?
The differential diagnosis of Wilson Disease (WD) is challenging, especially in children, because liver copper levels may also increase in other chronic liver diseases with bile stasis. The aim of this study is to determine urine and liver copper cut-off values to differentiate WD from other chronic liver diseases (non-WD, NWD) in children. Seventy-six patients participated in the study, 35 with WD and 41 with NWD. The two groups were divided into two subgroups according to the presence of cholestasis. At the time of diagnosis, age, sex, biochemical test results, serum ceruloplasmin, baseline 24-h urinary copper levels, liver biopsy histological findings, liver copper levels, and Child-Pugh scores were obtained from medical records. Copper content in liver tissue and copper levels in urine were measured by atomic absorption spectrometry. Cut-off values for differentiation of WD from NWD were determined by receiver operating characteristic (ROC) analysis. A liver copper cut-off value of 98 µg/g indicated WD with 91% sensitivity and 65.4% specificity (area under the curve =0.838, 95% CI: 0.749-0.927). A 24-h urinary copper cut-off value of 67.5 µg/24h indicated WD with 85% sensitivity and 71% specificity (area under the curve =0.843, 95% CI: 0.752-0.934). In this study of pediatric chronic liver disease patients, copper cut-off values for distinguishing WD differed substantially from those used for diagnosis. A larger scale study is warranted to re-evaluate liver copper and 24-h urinary copper cut-offs for children with suspected WD.


Incidence, morbidity and mortality of patients with achalasia in England: findings from a study of nationwide hospital and primary care data.
Achalasia is an uncommon condition characterised by failed lower oesophageal sphincter relaxation. Data regarding its incidence, prevalence, disease associations and long-term outcomes are very limited. Hospital Episode Statistics (HES) include demographic and diagnostic data for all English hospital attendances. The Health Improvement Network (THIN) includes the primary care records of 4.5 million UK subjects, representative of national demographics. Both were searched for incident cases between 2006 and 2016 and THIN for prevalent cases. Subjects with achalasia in THIN were compared with age, sex, deprivation tand smoking status matched controls for important comorbidities and mortality. There were 10 509 and 711 new achalasia diagnoses identified in HES and THIN, respectively. The mean incidence per 100 000 people in HES was 1.99 (95% CI 1.87 to 2.11) and 1.53 (1.42 to 1.64) per 100 000 person-years in THIN. The prevalence in THIN was 27.1 (25.4 to 28.9) per 100 000 population. Incidence rate ratios (IRRs) were significantly higher in subjects with achalasia (n=2369) compared with controls (n=3865) for: oesophageal cancer (IRR 5.22 (95% CI: 1.88 to 14.45), p<0.001), aspiration pneumonia (13.38 (1.66 to 107.79), p=0.015), lower respiratory tract infection (1.33 (1.05 to 1.70), p=0.02) and mortality (1.33 (1.17 to 1.51), p<0.001). The median time from achalasia diagnosis to oesophageal cancer diagnosis was 15.5 (IQR 20.4) years. The incidence of achalasia is 1.99 per 100 000 population in secondary care data and 1.53 per 100 000 person-years in primary care data. Subjects with achalasia have an increased incidence of oesophageal cancer, aspiration pneumonia, lower respiratory tract infections and higher mortality. Clinicians treating patients with achalasia should be made aware of these associated morbidities and its increased mortality.


Quantitative in vitro drug potency and drug susceptibility evaluation of Leishmania ssp. from patients unresponsive to pentavalent antimony therapy.
Quantitative in vitro drug sensitivity of 32 Leishmania isolates (16 from patients failing pentavalent antimony [SbV] therapy) was determined using a radiorespirometric microtechnique (RAM). Of 30 isolates with histories, 22 (73%) RAM tests agreed with patient history; the remaining 8 (27%) did not. There was no difference in RAM drug sensitivity: clinical correlation between 15 isolates tested blindly and 15 with known clinical history (4 did not agree with clinical history in both). Test sensitivity appeared to be limited only by the sensitivity of the Leishmania to SbV and could be detected at 2 micrograms/ml Sb (about 10% of serum drug level). An isolate from a patient with untreated self-healing cutaneous disease was drug resistant. Using RAM, parasite drug sensitivity can be quantified apart from patient physiologic and immunologic variables intrinsic to clinical data. Potency differed a maximum of 100% (weight% Sb:weight% Sb) among drug lots and also between Glucantime and Pentostam. Potency changes between drug lots were not explainable based on Sb content or test-to-test variability. This microtest offers a rapid method for evaluating the drug sensitivity of patient isolates and for determining of the activity of pentavalent antimonials and other candidate anti-leishmanials prior to the initiation of therapy.


Revealing Interaction of Organic Adsorbates with Semiconductor Surfaces Using Chemically Enhanced Raman.
Surface enhanced Raman spectroscopy (SERS) is frequently associated with "chemical enhancement" (CE), which is an effect of the chemical coupling between reporting molecules and surfaces. While SERS technique is mainly attributed to the studies of metallic surfaces, chemical coupling must be present on semiconductor surfaces as well. Here, we examine binding of trans-1,2-two(4-pyridyl) ethylene (BPE) to various crystallographic facets of PbSe semiconductor. The calculated off-resonant Raman spectra vary significantly on different crystallographic facets of PbSe, correlating with the electronic structure of each type of semiconductor surface. We distinguish situations when the charge transfer is present and when it is not, which raises the question about what exactly should be called the "chemical enhancement". We attempt to clarify this situation by introducing the concept of the "charge-transfer" and "charge-transfer-less" chemical enhancement. We also demonstrate a transition between these two regimes, which exhibits a nonlinear behavior of the vibrational coupling and a significantly stronger contribution to the Raman intensity.


Drug effects analysis on cells using a high throughput microfluidic chip.
Usually cell-based assay is performed using titer plates. Because of the large library of chemical compounds, robust and rapid methods are required to find, refine and test a potential drug candidate in an efficient manner. In this article, the drug effects analysis on human breast cancer cells with a droplet microfluidic chip is reported. Each droplet serves as a nanoliter-volume titer plate and contains a human breast cancer cell MDA-MB-231, Cytochalasin D drug solution and cell viability indicator such as Calcein AM, which emits cytoplasmic green fluorescence. The drug effects on each cell are monitored in real time using a fluorescence microscope and by analyzing the fluorescence image of each cell. Clear change of the cell shape and size has been observed after the drug treatment, which is similar to that of conventional petri dish technique, suggesting this approach is a potential viable technical platform for drug effect analysis and for high throughput drug screen and discovery.


An "off-on" phosphorescent aptasensor for the detection of thrombin based on PRET.
Thrombin plays an important role in the blood coagulation cascade and it stimulates the process of platelet aggregation. Herein, we developed a highly efficient and sensitive phosphorescent aptasensor system for the quantitative analysis of thrombin. The phosphorescence of 3-mercaptopropionic acid capped Mn-doped ZnS quantum dots (MPA-Mn:ZnS QDs) was gradually quenched with the addition of thrombin binding aptamers-BHQ2 (TBA-BHQ2) based on phosphorescence resonance energy transfer (PRET). With the addition of the target analyte thrombin into the system, TBA-BHQ2 could change its spatial structure from a random coil to an antiparallel G-quadruplex which resulted from the combination of thrombin and TBA-BHQ2, leading to the phosphorescence recovery. Finally, the concentration of thrombin could be accurately determined by means of measuring the phosphorescence intensity change value (ΔP). The limit of detection (LOD) was obtained as low as 15.26 pM with wide linear ranges both from 60 to 2000 pM and from 2 to 900 nM. The proposed strategy was also successfully applied for thrombin detection in human serum samples and plasma samples with satisfactory recoveries from 96% to 99% and 95% to 104%, respectively. The long lifetime of phosphorescent QDs possessed a suitable time delay to eliminate autofluorescence and scattered light interference from biological matrices effectively. Thus, the signal to noise ratio of the phosphorescent aptasensor was improved visibly for the analysis of target analytes.