Protective Action of Anandamide and Its COX-2 Metabolite against l-Homocysteine-Induced NLRP3 Inflammasome Activation and Injury in Podocytes.
Recent studies have demonstrated that l-homocysteine (Hcys)-induced podocyte injury leading to glomerular damage or sclerosis is attributable to the activation of the nucleotide-binding oligomerization domain-like receptor containing pyrin domain 3 (NLRP3) inflammasome. Given the demonstrated anti-inflammatory effects of endocannabinoids, the present study was designed to test whether anandamide (AEA) or its metabolites diminish NLRP3 inflammasome activation and prevent podocyte injury and associated glomerular damage during hyperhomocysteinemia (hHcys). AEA (100 μM) inhibited Hcys-induced NLRP3 inflammasome activation in cultured podocytes, as indicated by elevated caspase-1 activity and interleukin-1β levels, and attenuated podocyte dysfunction, as shown by reduced vascular endothelial growth factor production. These effects of AEA were inhibited by the cyclooxygenase-2 (COX-2) inhibitor celecoxib (CEL). In mice in vivo, AEA treatment attenuated glomerular NLRP3 inflammasome activation induced by hHcys accompanying a folate-free diet, on the basis of inhibition of hHcys-induced colocalization of NLRP3 molecules and increased interleukin-1β levels in glomeruli. Correspondingly, AEA prevented hHcys-induced proteinuria, albuminuria, and glomerular damage observed microscopically. Hcys- and AEA-induced effects were absent in NLRP3-knockout mice. These beneficial effects of AEA against hHcys-induced NLRP3 inflammasome activation and glomerular injury were not observed in mice cotreated with CEL. We further demonstrated that prostaglandin E2-ethanolamide (PGE2-EA), a COX-2 product of AEA, at 10 μM had a similar inhibitory effect to that of 100 μM AEA on Hcys-induced NLRP3 inflammasome formation and activation in cultured podocytes. From these results, we conclude that AEA has anti-inflammatory properties, protecting podocytes from Hcys-induced injury by inhibition of NLRP3 inflammasome activation through its COX-2 metabolite, PGE2-EA.


Endogenous Leu332Gln mutation in p53 disrupts the tetramerization ability in a canine mammary gland tumor cell line.
Mutations in the p53 gene are associated with more than half of all human cancers. These mutations often cause a disruption of the tumor-suppressor function of p53 and induce genomic instabilities. Wild‑type p53 requires tetramerization to function as an initiator of cell cycle arrest and apoptosis. Although alterations in p53 tetramerization caused by mutation have been well studied, there are few cell lines containing an endogenous mutation in the tetramerization domain of p53. Here, we report the discovery of a canine mammary gland tumor cell line CTB‑m2, which contains the Leu332Gln (L332Q) mutation corresponding to Leu344 in the tetramerization domain of human p53. Although CTB‑m2 cells are genetically heterozygous for the Leu332Gln mutation, the mutant mRNA was almost exclusively expressed. CTB‑m2 cells showed enhanced cell proliferation compared to wild‑type p53-expressing CTB‑m cells of the same lineage. A p53 tetramerization reporter assay showed that the ability of the p53 mutant to form tetramers was significantly lower than that of wild‑type p53. An immunoblot analysis of cross-linked p53 oligomerized forms demonstrated that the L332Q mutant lacked the ability to form tetramers but retained the ability to form dimers. These data suggest that the p53 mutant cell line CTB‑m2 could be a useful tool for analyzing the precise tetramerization mechanisms of p53 and verifying the effects of therapeutic agents against tumors expressing p53 mutants that lack the ability to tetramerize.


Deaf people's help-seeking following trauma: Experiences with and recommendations for the Massachusetts behavioral health care system.
Deaf trauma survivors are one of the more underserved populations in behavioral health care and experience significant obstacles to seeking help. Repeated encounters with these barriers fuel negative perceptions and avoidance of behavioral health treatment. The current study sought to explore Deaf trauma survivors' help-seeking experiences and elicit their recommendations for improving Deaf behavioral health services in Massachusetts. We conducted semistructured American Sign Language interviews with 16 trauma-exposed Deaf individuals that included questions from the Life Events Checklist and the PTSD Symptom Scale Interview and questions about Deaf individuals' help-seeking behaviors. Qualitative responses regarding help-seeking experiences were analyzed using a grounded theory approach. In the aftermath of trauma, our participants emphasized a desire to work with a signing provider who is highly knowledgeable about Deaf culture, history, and experience and to interact with clinic staff who possess basic sign language skills and training in Deaf awareness. Most stressed the need for providers to better outreach into the Deaf community-to provide education about trauma, to describe available treatment resources, and to prove one's qualifications. Participants also provided suggestions for how behavioral health clinics can better protect Deaf survivors' confidentiality in a small-community context. Deaf-friendly trauma treatment should incorporate the components of trauma-informed care but also carefully consider key criteria expressed by our participants: direct signed communication, understanding of Deaf history and experience, stringent practices to protect confidentiality, provider visibility in the community, and reliance on peer support and Deaf role models in treatment interventions. (PsycINFO Database Record


Type 1 angiotensin receptors on macrophages ameliorate IL-1 receptor-mediated kidney fibrosis.
In a wide array of kidney diseases, type 1 angiotensin (AT1) receptors are present on the immune cells that infiltrate the renal interstitium. Here, we examined the actions of AT1 receptors on macrophages in progressive renal fibrosis and found that macrophage-specific AT1 receptor deficiency exacerbates kidney fibrosis induced by unilateral ureteral obstruction (UUO). Macrophages isolated from obstructed kidneys of mice lacking AT1 receptors solely on macrophages had heightened expression of proinflammatory M1 cytokines, including IL-1. Evaluation of isolated AT1 receptor-deficient macrophages confirmed the propensity of these cells to produce exaggerated levels of M1 cytokines, which led to more severe renal epithelial cell damage via IL-1 receptor activation in coculture compared with WT macrophages. A murine kidney crosstransplantation concomitant with UUO model revealed that augmentation of renal fibrosis instigated by AT1 receptor-deficient macrophages is mediated by IL-1 receptor stimulation in the kidney. This study indicates that a key role of AT1 receptors on macrophages is to protect the kidney from fibrosis by limiting activation of IL-1 receptors in the kidney.


Capturing intrusive re-experiencing in trauma survivors' daily lives using ecological momentary assessment.
Intrusive memories are common following traumatic events and among the hallmark symptoms of posttraumatic stress disorder (PTSD). Most studies assess summarized accounts of intrusions retrospectively. We used an ecological momentary approach and index intrusive memories in trauma survivors with and without PTSD using electronic diaries. Forty-six trauma survivors completed daily diaries for 7 consecutive days recording a total of 294 intrusions. Participants with PTSD experienced only marginally more intrusions than those without PTSD, but experienced them with more "here and now quality," and responded with more fear, helplessness, anger, and shame than those without PTSD. Most frequent intrusion triggers were stimuli that were perceptually similar to stimuli from the trauma. Individuals with PTSD experienced diary-prompted voluntary trauma memories with the same sense of nowness and vividness as involuntary intrusive trauma memories. The findings contribute to a better understanding of everyday experiences of intrusive reexperiencing in trauma survivors with PTSD and offer clinical treatment implications.


The expected time to cross extended fitness plateaus.
For a population to acquire a complex adaptation requiring multiple individually neutral mutations, it must cross a plateau in the fitness landscape. We consider plateaus involving three mutations, and show that large populations can cross them rapidly via lineages that acquire multiple mutations while remaining at low frequency, much faster than the ∝μ3 rate for simultaneous triple mutations. Plateau-crossing is fastest for very large populations. At intermediate population sizes, recombination can greatly accelerate adaptation by combining independent mutant lineages to form triple-mutants. For more frequent recombination, such that the population is kept near linkage equilibrium, we extend our analysis to find simple expressions for the expected time to cross plateaus of arbitrary width.


Diffusion-based microfluidic PCR for "one-pot" analysis of cells.
Genetic analysis starting with cell samples often requires multi-step processing including cell lysis, DNA isolation/purification, and polymerase chain reaction (PCR) based assays. When conducted on a microfluidic platform, the compatibility among various steps often demands a complicated procedure and a complex device structure. Here we present a microfluidic device that permits a "one-pot" strategy for multi-step PCR analysis starting from cells. Taking advantage of the diffusivity difference, we replace the smaller molecules in the reaction chamber by diffusion while retaining DNA molecules inside. This simple scheme effectively removes reagents from the previous step to avoid interference and thus permits multi-step processing in the same reaction chamber. Our approach shows high efficiency for PCR and potential for a wide range of genetic analysis including assays based on single cells.


Pyrosequencing reveals the influence of organic and conventional farming systems on bacterial communities.
It has been debated how different farming systems influence the composition of soil bacterial communities, which are crucial for maintaining soil health. In this research, we applied high-throughput pyrosequencing of V1 to V3 regions of bacterial 16S rRNA genes to gain further insight into how organic and conventional farming systems and crop rotation influence bulk soil bacterial communities. A 2×2 factorial experiment consisted of two agriculture management systems (organic versus conventional) and two crop rotations (flax-oat-fababean-wheat versus flax-alfalfa-alfalfa-wheat) was conducted at the Glenlea Long-Term Crop Rotation and Management Station, which is Canada's oldest organic-conventional management study field. Results revealed that there is a significant difference in the composition of bacterial genera between organic and conventional management systems but crop rotation was not a discriminator factor. Organic farming was associated with higher relative abundance of Proteobacteria, while Actinobacteria and Chloroflexi were more abundant in conventional farming. The dominant genera including Blastococcus, Microlunatus, Pseudonocardia, Solirubrobacter, Brevundimonas, Pseudomonas, and Stenotrophomonas exhibited significant variation between the organic and conventional farming systems. The relative abundance of bacterial communities at the phylum and class level was correlated to soil pH rather than other edaphic properties. In addition, it was found that Proteobacteria and Actinobacteria were more sensitive to pH variation.


Warfarin pharmacogenetics: a single VKORC1 polymorphism is predictive of dose across 3 racial groups.
Warfarin-dosing algorithms incorporating CYP2C9 and VKORC1 -1639G>A improve dose prediction compared with algorithms based solely on clinical and demographic factors. However, these algorithms better capture dose variability among whites than Asians or blacks. Herein, we evaluate whether other VKORC1 polymorphisms and haplotypes explain additional variation in warfarin dose beyond that explained by VKORC1 -1639G>A among Asians (n = 1103), blacks (n = 670), and whites (n = 3113). Participants were recruited from 11 countries as part of the International Warfarin Pharmacogenetics Consortium effort. Evaluation of the effects of individual VKORC1 single nucleotide polymorphisms (SNPs) and haplotypes on warfarin dose used both univariate and multi variable linear regression. VKORC1 -1639G>A and 1173C>T individually explained the greatest variance in dose in all 3 racial groups. Incorporation of additional VKORC1 SNPs or haplotypes did not further improve dose prediction. VKORC1 explained greater variability in dose among whites than blacks and Asians. Differences in the percentage of variance in dose explained by VKORC1 across race were largely accounted for by the frequency of the -1639A (or 1173T) allele. Thus, clinicians should recognize that, although at a population level, the contribution of VKORC1 toward dose requirements is higher in whites than in nonwhites; genotype predicts similar dose requirements across racial groups.


Attachment to roots and virulence of a chvB mutant of Agrobacterium tumefaciens are temperature sensitive.
Agrobacterium tumefaciens chvB mutants are unable to produce beta-1,2 glucan. They are nonattaching and avirulent and show reduced motility at room temperature. At lower temperatures (16 degrees C), chvB mutants became virulent on Bryophyllum daigremontiana and Lycopersicon esculentum and were able to attach to L. esculentum, Arabidopsis thaliana, Daucus carota, and Tagetes erecta roots. The mutant bacteria also recovered wild-type motility at lower temperatures. Two other nonattaching mutants of A. tumefaciens, AttR and AtrA, were unaffected by the lowered temperature, remaining nonattaching and avirulent.


Higher fidelity of RNA-dependent DNA mispair extension by M184V drug-resistant than wild-type reverse transcriptase of human immunodeficiency virus type 1.
Reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1) has low fidelity compared with RTs of other retroviruses and cellular DNA polymerases. We and others have previously found that the fidelity of DNA-dependent DNA polymerization (DDDP) of M184V-mutated HIV-1 RT is significantly higher than that of wild-type RT. Viruses containing the M184V substitution are highly resistant to (-)-2'-dideoxy-3'-thiacytidine (3TC) in vitro and in patients treated with 3TC monotherapy. It was of interest to determine the fidelity of RNA-dependent DNA polymerization (RDDP) of M184V RT compared with wild-type because this step occurs first in reverse transcription; errors made during this step may be copied in subsequent polymerization steps. Using an in vitro mispaired primer extension assay, M184V-mutated RT exhibited 3-49-fold decreased frequency of mispair extension compared with wild-type RT. Fidelity differences between M184V and wild-type RT were most marked in extension of A:G (49-fold) and A:C (16-fold) mispairs, with only a marginal (3-fold) decrease in the extension of A:A mispairs. RT containing a methionine to isoleucine (M184I) mutation showed only slight increases in RDDP fidelity compared with wild-type, ranging from 1.5- to 6-fold increases. Of the three RTs tested, wild-type RT was the most error-prone, with mispair extension frequencies ranging from 6.674 x 10(-1) to 7.454 x10(-2).


Bringing High-Rate, CO2-Based Microbial Electrosynthesis Closer to Practical Implementation through Improved Electrode Design and Operating Conditions.
The enhancement of microbial electrosynthesis (MES) of acetate from CO2 to performance levels that could potentially support practical implementations of the technology must go through the optimization of key design and operating conditions. We report that higher proton availability drastically increases the acetate production rate, with pH 5.2 found to be optimal, which will likely suppress methanogenic activity without inhibitor addition. Applied cathode potential as low as -1.1 V versus SHE still achieved 99% of electron recovery in the form of acetate at a current density of around -200 A m(-2). These current densities are leading to an exceptional acetate production rate of up to 1330 g m(-2) day(-1) at pH 6.7. Using highly open macroporous reticulated vitreous carbon electrodes with macropore sizes of about 0.6 mm in diameter was found to be optimal for achieving a good balance between total surface area available for biofilm formation and effective mass transfer between the bulk liquid and the electrode and biofilm surface. Furthermore, we also successfully demonstrated the use of a synthetic biogas mixture as carbon dioxide source, yielding similarly high MES performance as pure CO2. This would allow this process to be used effectively for both biogas quality improvement and conversion of the available CO2 to acetate.


Mind the gap: Opportunities for improving end-of-life care for patients with advanced heart failure.
Patients with advanced heart failure (HF) experience progressive symptoms, decreased quality of life, and more frequent hospitalizations as they approach the end of life (EOL). Understanding patient perspectives and preferences regarding EOL issues is necessary to identify key opportunities for improving care. To identify, from the patient's perspective, the major opportunities for improving EOL care for patients hospitalized because of advanced HF. A cross-sectional survey of patient perspectives regarding EOL care was administered via interview of 106 hospitalized patients who had advanced HF in five tertiary care centres across Canada. The study compared which aspects of EOL care patients rated as 'extremely important' and their level of satisfaction with these aspects of EOL care to identify key opportunities for improvement of care. The greatest opportunities for improvement in EOL care were reducing the emotional and physical burden on family, having an adequate plan of care following discharge, effective symptom relief and opportunities for honest communication. The three most important issues ranked by patients were avoidance of life support if there was no hope for a meaningful recovery, communication of information by the doctor and avoidance of burden for the family. Advanced care planning that seamlessly bridges hospital and home must be standard care for patients who have advanced HF. Components must include coordination of care, caregiver support, comprehensive symptom management, and effective communication regarding HF and EOL issues.


Morphological and Phylogenetic Characterization of New Gephyrocapsa Isolates Suggests Introgressive Hybridization in the Emiliania/Gephyrocapsa Complex (Haptophyta).
The coccolithophore genus Gephyrocapsa contains a cosmopolitan assemblage of pelagic species, including the bloom-forming Gephyrocapsa oceanica, and is closely related to the emblematic coccolithophore Emiliania huxleyi within the Noëlaerhabdaceae. These two species have been extensively studied and are well represented in culture collections, whereas cultures of other species of this family are lacking. We report on three new strains of Gephyrocapsa isolated into culture from samples from the Chilean coastal upwelling zone using a novel flow cytometric single-cell sorting technique. The strains were characterized by morphological analysis using scanning electron microscopy and phylogenetic analysis of 6 genes (nuclear 18S and 28S rDNA, plastidial 16S and tufA, and mitochondrial cox1 and cox3 genes). Morphometric features of the coccoliths indicate that these isolates are distinct from G. oceanica and best correspond to G. muellerae. Surprisingly, both plastidial and mitochondrial gene phylogenies placed these strains within the E. huxleyi clade and well separated from G. oceanica isolates, making Emiliania appear polyphyletic. The only nuclear sequence difference, 1bp in the 28S rDNA region, also grouped E. huxleyi with the new Gephyrocapsa isolates and apart from G. oceanica. Specifically, the G. muellerae morphotype strains clustered with the mitochondrial β clade of E. huxleyi, which, like G. muellerae, has been associated with cold (temperate and sub-polar) waters. Among putative evolutionary scenarios that could explain these results we discuss the possibility that E. huxleyi is not a valid taxonomic unit, or, alternatively the possibility of past hybridization and introgression between each E. huxleyi clade and older Gephyrocapsa clades. In either case, the results support the transfer of Emiliania to Gephyrocapsa. These results have important implications for relating morphological species concepts to ecological and evolutionary units of diversity.


Stepping down from inhaled corticosteroids with leukotriene inhibitors in asthma: a systematic review and meta-analysis.
The risks of using leukotriene receptor antagonists (LTRA) as part of a strategy for stepping down inhaled corticosteroid (ICS) are not well known. To estimate the risk of asthma exacerbation in individuals with stable asthma who start LTRA when stopping ICS or reducing ICS dose. We identified articles from a systematic review of English and non-English articles by using a number of data bases. We included randomized controlled trials with a stable asthma run-in period of 4 weeks or more and a follow-up period of at least 3 months. We included studies of individuals with stable asthma who stopped ICS and substituted LTRA (versus continuing ICS) and who reduced ICS while starting LTRA (versus placebo). The search strategy identified 1132 potential articles, of which 52 were reviewed at the full-text level, and four met criteria for inclusion. The single article that met the inclusion criteria for substitution of LTRA for ICS as a step-down strategy found a statistically increased risk of treatment failure of 30.3% for substituting LTRA compared with 20.2% for continuing ICS. The three articles that met the inclusion criteria for comparing LTRA versus placebo in patients with stable asthma who reduce ICS found a modestly decreased risk ratio that favored LTRA of 0.57 (95% confidence interval, 0.36-0.90; I(2) = 0%) in studies that only included individuals >15 years old. Only one study addressed the risk of substitution of LTRA for ICS in stable asthma, which limited any strong conclusions about this step-down strategy.


Perspectives on Comets, Comet-like Asteroids, and Their Predisposition to Provide an Environment That Is Friendly to Life.
In recent years, studies have shown that there are many similarities between comets and asteroids. In some cases, it cannot even be determined to which of these groups an object belongs. This is especially true for objects found beyond the main asteroid belt. Because of the lack of comet fragments, more progress has been made concerning the chemical composition of asteroids. In particular, the SMASSII classification establishes a link between the reflecting spectra and chemical composition of asteroids and meteorites. To find clues for the chemical structure of comets, the parameters of all known asteroids of the SMASSII classification were compared to those of comet groups like the Encke-type comets, the Jupiter-family comets, and the Halley-type comets, as well as comet-like objects like the damocloids and the centaurs. Fifty-six SMASSII objects similar to comets were found and are categorized as comet-like asteroids in this work. Aside from the chemistry, it is assumed that the available energy on these celestial bodies plays an important role concerning habitability. For the determination of the available energy, the effective temperature was calculated. Additionally, the size of these objects was considered in order to evaluate the possibility of a liquid water core, which provides an environment that is more likely to support processes necessary to create the building blocks of life. Further study of such objects could be notable for the period of the Late Heavy Bombardment and could therefore provide important implications for our understanding of the inner workings of the prebiotic evolution within the Solar System since the beginning.


Association of schizophrenia and autoimmune diseases: linkage of Danish national registers.
Individuals with schizophrenia and their relatives tend to have either higher or lower than expected prevalences of autoimmune disorders, especially rheumatoid arthritis, celiac disease, autoimmune thyroid diseases, and type 1 diabetes. The purpose of the study was to estimate the association of schizophrenia with these disorders as well as a range of other autoimmune diseases in a single large epidemiologic study. The Danish Psychiatric Register, the National Patient Register, and a register with socioeconomic information were linked to form a data file that included all 7,704 persons in Denmark diagnosed with schizophrenia from 1981 to 1998 and their parents along with a sample of matched comparison subjects and their parents. The data linkage required that the autoimmune disease occur before the diagnosis of schizophrenia. A history of any autoimmune disease was associated with a 45% increase in risk for schizophrenia. Nine autoimmune disorders had higher prevalence rates among patients with schizophrenia than among comparison subjects (crude incidence rate ratios ranging from 1.9 to 12.5), and 12 autoimmune diseases had higher prevalence rates among parents of schizophrenia patients than among parents of comparison subjects (adjusted incidence rate ratios ranging from 1.3 to 3.8). Thyrotoxicosis, celiac disease, acquired hemolytic anemia, interstitial cystitis, and Sjögren's syndrome had higher prevalence rates among patients with schizophrenia than among comparison subjects and also among family members of schizophrenia patients than among family members of comparison subjects. Schizophrenia is associated with a larger range of autoimmune diseases than heretofore suspected. Future research on comorbidity has the potential to advance understanding of pathogenesis of both psychiatric and autoimmune disorders.


Genetic analysis of Pseudomonas aeruginosa adherence: distinct genetic loci control attachment to epithelial cells and mucins.
Infection of mucosal tissues by the opportunistic pathogen Pseudomonas aeruginosa is initiated by attachment of the bacterium to host tissues. To gain a better understanding of this interaction, we used two methods to isolate mutants of P. aeruginosa with altered adherence to cultured A549 cells and to mucins. First, from a population of nonpiliated mutants of P. aeruginosa mutagenized with transposon Tn5G, we have isolated variants that are defective in binding to both A549 cells and respiratory mucins. Using a cloned transposon plus flanking DNA from one such mutant as a DNA probe, we have isolated plasmids from a cosmid bank, which, upon reintroduction to the original mutants, restored adhesion to both A549 cells and mucin. The second strategy to identify genes involved in adhesion used mutagenesis of P. aeruginosa N1G, an rpoN mutant which is unable to bind to either A549 cells or mucin, with transposon Tn5 containing an outward-directed promoter. From this bank of mutagenized P. aeruginosa N1G, two classes of adhesion variants were isolated; one class attached to A549 cells and to mucin, and the other class restored binding of the rpoN mutant to mucin but not to A549 cells. These findings suggest that P. aeruginosa can express at least two adhesins distinct from pili, one recognizing receptors shared by epithelial cells and mucins and the other recognizing mucins alone.


Analysis of endovascular treatment of ruptured microaneurysms compared with ruptured larger aneurysms.
To retrospectively analyze the complications and outcome of the endovascular treatment of ruptured microaneurysms compared with the treatment of ruptured larger aneurysms. 40 ruptured cerebral microaneurysms treated by endovascular techniques were selected retrospectively and compared with 207 larger ruptured cerebral aneurysms treated by endovascular techniques during the same time period. Medical charts and imaging studies were reviewed to analyze baseline clinical and epidemiologic characteristics, procedural complications, and clinical outcomes RESULTS: Cerebral microaneurysms had a higher incidence of intraoperative technical ruptures (13.5% vs 2.9%, p<0.005). The number of thromboembolic complications was not increased. Patient prognosis was similar for the two groups (mean modified Rankin Scale score 1.81 vs 2.09, p>0.1). Coiling of cerebral microaneurysms has a reasonable safety profile with good clinical outcomes, similar to coiling of larger aneurysms. In our experience, the systematic use of remodeling balloons, operator experience, and the ability to manage complications are the reasons for the satisfactory results.


Revisiting the interaction of heme with hemopexin.
In hemolytic disorders, erythrocyte lysis results in massive release of hemoglobin and, subsequently, toxic heme. Hemopexin is the major protective factor against heme toxicity in human blood and currently considered for therapeutic use. It has been widely accepted that hemopexin binds heme with extraordinarily high affinity of <1 pM in a 1:1 ratio. However, several lines of evidence point to a higher stoichiometry and lower affinity than determined 50 years ago. Here, we re-analyzed these data. SPR and UV/Vis spectroscopy were used to monitor the interaction of heme with the human protein. The heme-binding sites of hemopexin were characterized using hemopexin-derived peptide models and competitive displacement assays. We obtained a KD value of 0.32 ± 0.04 nM and the ratio for the interaction was determined to be 1:1 at low heme concentrations and at least 2:1 (heme:hemopexin) at high concentrations. We were able to identify two yet unknown potential heme-binding sites on hemopexin. Furthermore, molecular modelling with a newly created homology model of human hemopexin suggested a possible recruiting mechanism by which heme could consecutively bind several histidine residues on its way into the binding pocket. Our findings have direct implications for the potential administration of hemopexin in hemolytic disorders.


A Biomathematical Model of Pneumococcal Lung Infection and Antibiotic Treatment in Mice.
Pneumonia is considered to be one of the leading causes of death worldwide. The outcome depends on both, proper antibiotic treatment and the effectivity of the immune response of the host. However, due to the complexity of the immunologic cascade initiated during infection, the latter cannot be predicted easily. We construct a biomathematical model of the murine immune response during infection with pneumococcus aiming at predicting the outcome of antibiotic treatment. The model consists of a number of non-linear ordinary differential equations describing dynamics of pneumococcal population, the inflammatory cytokine IL-6, neutrophils and macrophages fighting the infection and destruction of alveolar tissue due to pneumococcus. Equations were derived by translating known biological mechanisms and assuming certain response kinetics. Antibiotic therapy is modelled by a transient depletion of bacteria. Unknown model parameters were determined by fitting the predictions of the model to data sets derived from mice experiments of pneumococcal lung infection with and without antibiotic treatment. Time series of pneumococcal population, debris, neutrophils, activated epithelial cells, macrophages, monocytes and IL-6 serum concentrations were available for this purpose. The antibiotics Ampicillin and Moxifloxacin were considered. Parameter fittings resulted in a good agreement of model and data for all experimental scenarios. Identifiability of parameters is also estimated. The model can be used to predict the performance of alternative schedules of antibiotic treatment. We conclude that we established a biomathematical model of pneumococcal lung infection in mice allowing predictions regarding the outcome of different schedules of antibiotic treatment. We aim at translating the model to the human situation in the near future.


Change in disgust reactions following cognitive-behavioral therapy for childhood anxiety disorders.
Disgust, in addition to fear, is a prominent emotional state associated with avoidance of distressing stimuli. While most of the research in disgust has been conducted in relation to Obsessive-Compulsive Disorder (OCD), recent data has also implicated disgust in the etiology of anxiety disorders in general. Studies have shown that decreases in disgust are key to symptom reduction in individuals with OCD. However, there has been little empirical work exploring whether these interventions are efficacious for childhood anxiety disorders that present with prominent disgust components. This study examined how disgust propensity in children with anxiety disorders responds to cognitive-behavioral therapy (CBT) with an emphasis on exposure. Forty-one children, ages 7 to 17, with anxiety disorders were evaluated for disgust propensity and were treated with intensive, weekly, CBT. It was found that disgust levels decreased following treatment across all anxiety disorder diagnoses, where children with primary OCD exhibited significantly greater reductions. Clinical implications and suggestions for further research of the treatment of disgust in relation to childhood anxiety disorders are discussed.


An extensive interaction interface between thrombin and factor V is required for factor V activation.
The interaction interface between human thrombin and human factor V (FV), necessary for complex formation and cleavage to generate factor Va, was investigated using a site-directed mutagenesis strategy. Fifty-three recombinant thrombins, with a total of 78 solvent-exposed basic and polar residues substituted with alanine, were used in a two-stage clotting assay with human FV. Seventeen mutants with less than 50% of wild-type (WT) thrombin FV activation were identified and mapped to anion-binding exosite I (ABE-I), anion-binding exosite II (ABE-II), the Leu(45)-Asn(57) insertion loop, and the Na(+) binding loop of thrombin. Three ABE-I mutants (R68A, R70A, and Y71A) and the ABE-II mutant R98A had less than 30% of WT activity. The thrombin Na(+) binding loop mutants, E229A and R233A, and the Leu(45)-Asn(57) insertion loop mutant, W50A, had a major effect on FV activation with 5, 15, and 29% of WT activity, respectively. The K52A mutant, which maps to the S' specificity pocket, had 29% of WT activity. SDS-polyacrylamide gel electrophoresis analysis of cleavage reactions using the thrombin ABE mutants R68A, Y71A, and R98A, the Na(+) binding loop mutant E229A, and the Leu(45)-Asn(57) insertion loop mutant W50A showed a requirement for both ABEs and the Na(+)-bound form of thrombin for efficient cleavage at the FV residue Arg(709). Several basic residues in both ABEs have moderate decreases in FV activation (40-60% of WT activity), indicating a role for the positive electrostatic fields generated by both ABEs in enhancing complex formation with complementary negative electrostatic fields generated by FV. The data show that thrombin activation of FV requires an extensive interaction interface with thrombin. Both ABE-I and ABE-II and the S' subsite are required for optimal cleavage, and the Na(+)-bound form of thrombin is important for its procoagulant activity.


On the classification capability of sign-constrained perceptrons.
The perceptron (also referred to as McCulloch-Pitts neuron, or linear threshold gate) is commonly used as a simplified model for the discrimination and learning capability of a biological neuron. Criteria that tell us when a perceptron can implement (or learn to implement) all possible dichotomies over a given set of input patterns are well known, but only for the idealized case, where one assumes that the sign of a synaptic weight can be switched during learning. We present in this letter an analysis of the classification capability of the biologically more realistic model of a sign-constrained perceptron, where the signs of synaptic weights remain fixed during learning (which is the case for most types of biological synapses). In particular, the VC-dimension of sign-constrained perceptrons is determined, and a necessary and sufficient criterion is provided that tells us when all 2(m) dichotomies over a given set of m patterns can be learned by a sign-constrained perceptron. We also show that uniformity of L(1) norms of input patterns is a sufficient condition for full representation power in the case where all weights are required to be nonnegative. Finally, we exhibit cases where the sign constraint of a perceptron drastically reduces its classification capability. Our theoretical analysis is complemented by computer simulations, which demonstrate in particular that sparse input patterns improve the classification capability of sign-constrained perceptrons.


Reactions of Nicotiana species to inoculation with monopartite and bipartite begomoviruses.
Some Nicotiana species are widely used as experimental hosts for plant viruses. Nicotiana species differ in ploidy levels, chromosome numbers and have diverse geographical origins. Thus, these species are useful model systems to investigate virus-host interactions, co-evolution of pathogens and hosts and the effects of ploidy level on virus resistance/susceptibility. Here we have studied the responses of seven Nicotiana species to inoculation with Cotton leaf curl Multan virus (CLCuMV), a monopartite begomovirus, and Tomato leaf curl New Delhi virus (ToLCNDV), a bipartite begomovirus, both from the Indian subcontinent. All Nicotiana species supported the replication of both begomoviruses in inoculated leaves. However, only three Nicotiana species, namely N. benthamiana, N. tabacum and N. sylvestris showed symptoms when inoculated with ToLCNDV, while N. benthamiana was the only species that developed leaf curl symptoms when inoculated with CLCuMV. CLCuMV accumulated to detectable levels in N. tabacum, but plants remained asymptomatic. A previously identified mutation of RNA dependent RNA polymerase 1 was shown to be present only in N. benthamiana. The finding is in line with earlier results showing that the susceptibility of this species to a diverse range of plant viruses correlates with a defective RNA silencing-mediated host defense. The results presented show that individual Nicotiana species respond differently to inoculation with begomoviruses. The inability of begomoviruses to systemically infect several Nicotiana species is likely due to inhibition of virus movement, rather than replication, and thus provides a novel model to study virus-host interactions in resistant/susceptible hosts.


Use of an automated blood culture system (BD BACTEC™) for diagnosis of prosthetic joint infections: easy and fast.
For the diagnosis of prosthetic joint infection (PJI) automated BACTEC™ blood culture bottle methods have comparable sensitivity, specificity and a shorter time to positivity than traditional cooked meat enrichment broth methods. We evaluate the culture incubation period required to maximise sensitivity and specificity of microbiological diagnosis, and the ability of BACTEC™ to detect slow growing Propionibacteria spp. Multiple periprosthetic tissue samples taken by a standardised method from 332 patients undergoing prosthetic joint revision arthroplasty were cultured for 14 days, using a BD BACTEC™ instrumented blood culture system, in a prospective study from 1st January to 31st August 2012. The "gold standard" definition for PJI was the presence of at least one histological criterion, the presence of a sinus tract or purulence around the device. Cases where > =2 samples yielded indistinguishable isolates were considered culture-positive. 1000 BACTEC™ bottle cultures which were negative after 14 days incubation were sub-cultured for Propionibacteria spp. 79 patients fulfilled the definition for PJI, and 66 of these were culture-positive. All but 1 of these 66 culture-positive cases of PJI were detected within 3 days of incubation. Only one additional (clinically-insignificant) Propionibacterium spp. was identified on terminal subculture of 1000 bottles. Prolonged microbiological culture for 2 weeks is unnecessary when using BACTEC™ culture methods. The majority of clinically significant organisms grow within 3 days, and Propionibacteria spp. are identified without the need for terminal subculture. These findings should facilitate earlier decisions on final antimicrobial prescribing.


Coated photonic bandgap fibres for low-index sensing applications: cutoff analysis.
We investigate theoretically the performance of photonic crystal fibres with coated holes as refractive index sensors. We show that coating the holes with a high-index material allows to extend the extreme sensitivities analyte-waveguide based geometries offer to the case of low-index analytes, including water-based solutions. As the sensitivity of these sensors is intricately linked to the sensitivity of the cutoff of a single inclusion to the analyte refractive index, our approach relies on the derivation of cutoff equations for coated inclusions. This is performed analytically without approximations, in the fully vectorial case, for modes of all orders. Our analytic approach allows us to rapidly cover the parameter space, and to quickly identify promising geometries. The best results are obtained when considering fluorinated polymer fibres, for which the index of the background material is not too different to that of water, and with thin high-index coatings. Using these results, we propose a sensor based on a directional coupler geometry that would lead to a sensitivity of 2.2 x 10(4) nm/RIU for water based solutions with achievable smallest detectable refractive index changes below 10(-6).


Volitional control of food transport and bolus formation during feeding.
When healthy individuals eat solid food, chewed food is usually transported to the oropharynx where it accumulates before swallowing (stage II transport). We tested the hypothesis that this transport process can be altered by volition. Eight healthy young subjects ate 8 g pieces of cookie with barium while movements were recorded with videofluorography. There were two trials for each subject, each with different instructions: 1) without command: to eat the cookie in his/her usual manner; 2) with command: to chew the cookie, give a signal when ready to swallow, and then swallow on command of the investigator. We measured the number of chewing cycles, the duration of each stage in the feeding sequence, and the position of the leading edge of the barium at time of command and at swallow onset. Sequence duration was longer with than without command (P=0.02), primarily because of an increase in the number of chewing cycles (P=0.02). The leading edge was typically higher in the foodway at the time of swallow onset with than without command (P=0.06). Under the command condition, stage II transport was delayed, and transport to the valleculae was inhibited. Volition alters swallow initiation in both the timing and location of the food bolus relative to the airway.


Collectrin is involved in the development of salt-sensitive hypertension by facilitating the membrane trafficking of apical membrane proteins via interaction with soluble N-ethylmaleiamide-sensitive factor attachment protein receptor complex.
Collectrin, a homologue of angiotensin converting enzyme 2, is expressed in pancreatic beta cells and renal proximal tubular and collecting duct cells under the control of hepatocyte nuclear factors-1alpha and -1beta. Because collectrin interacts with the soluble N-ethylmaleiamide-sensitive factor attachment protein receptor (SNARE) complexes, we investigated whether collectrin is involved in sodium handling in hypertension by vesicle trafficking of apical membrane proteins. Collectrin physically interacts with the SNARE complex: snapin, synaptosomal-associated protein 23 kDa, syntaxin-4, and vesicle-associated membrane protein-2 in mIMCD-3 cells. siRNA knockdown of collectrin resulted in a reduction in membrane-associated aquaporin-2, alpha-epithelial Na+ channel, and H+-ATPase. Collectrin and SNARE proteins were abundantly expressed in collecting ducts of Wistar-Kyoto rats. Wistar-Kyoto rats and spontaneously hypertensive rats 7 weeks of age were subjected to normal-salt (1% NaCl) and high-salt (8% NaCl) chow for 10 weeks. High-salt chow prominently elevated blood pressure, oral intake, and urinary excretion of NaCl and water in both groups. Although urinary excretion of aldosterone was significantly suppressed in both groups, collectrin expression was upregulated and associated with the maintenance of aquaporin-2, alpha-epithelial Na+ channel, and H+-ATPase in membrane fractions. Collectrin promoter activities and mRNA and protein expressions were upregulated and ubiquitinated collectrin was reduced by high NaCl (175 to 225 mmol/L) and not altered by 1 micromol/L aldosterone in mIMCD-3 cells. Upregulation of collectrin by high NaCl independent of aldosterone functionally links to the trafficking of apical membrane proteins via the SNARE complex, and collectrin may be responsible for the sodium retention in salt-sensitive hypertension.


Activation of caspase-9 on the apoptosome as studied by methyl-TROSY NMR.
Mitochondrial apoptotic signaling cascades lead to the formation of the apoptosome, a 1.1-MDa heptameric protein scaffold that recruits and activates the caspase-9 protease. Once activated, caspase-9 cleaves and activates downstream effector caspases, triggering the onset of cell death through caspase-mediated proteolysis of cellular proteins. Failure to activate caspase-9 enables the evasion of programmed cell death, which occurs in various forms of cancer. Despite the critical apoptotic function of caspase-9, the structural mechanism by which it is activated on the apoptosome has remained elusive. Here, we used a combination of methyl-transverse relaxation-optimized NMR spectroscopy, protein engineering, and biochemical assays to study the activation of caspase-9 bound to the apoptosome. In the absence of peptide substrate, we observed that both caspase-9 and its isolated protease domain (PD) only very weakly dimerize with dissociation constants in the millimolar range. Methyl-NMR spectra of isotope-labeled caspase-9, within the 1.3-MDa native apoptosome complex or an engineered 480-kDa apoptosome mimic, reveal that the caspase-9 PD remains monomeric after recruitment to the scaffold. Binding to the apoptosome, therefore, organizes caspase-9 PDs so that they can rapidly and extensively dimerize only when substrate is present, providing an important layer in the regulation of caspase-9 activation. Our work highlights the unique role of NMR spectroscopy to structurally characterize protein domains that are flexibly tethered to large scaffolds, even in cases where the molecular targets are in excess of 1 MDa, as in the present example.


Bacterial gene transfer by natural genetic transformation in the environment.
Natural genetic transformation is the active uptake of free DNA by bacterial cells and the heritable incorporation of its genetic information. Since the famous discovery of transformation in Streptococcus pneumoniae by Griffith in 1928 and the demonstration of DNA as the transforming principle by Avery and coworkers in 1944, cellular processes involved in transformation have been studied extensively by in vitro experimentation with a few transformable species. Only more recently has it been considered that transformation may be a powerful mechanism of horizontal gene transfer in natural bacterial populations. In this review the current understanding of the biology of transformation is summarized to provide the platform on which aspects of bacterial transformation in water, soil, and sediments and the habitat of pathogens are discussed. Direct and indirect evidence for gene transfer routes by transformation within species and between different species will be presented, along with data suggesting that plasmids as well as chromosomal DNA are subject to genetic exchange via transformation. Experiments exploring the prerequisites for transformation in the environment, including the production and persistence of free DNA and factors important for the uptake of DNA by cells, will be compiled, as well as possible natural barriers to transformation. The efficiency of gene transfer by transformation in bacterial habitats is possibly genetically adjusted to submaximal levels. The fact that natural transformation has been detected among bacteria from all trophic and taxonomic groups including archaebacteria suggests that transformability evolved early in phylogeny. Probable functions of DNA uptake other than gene acquisition will be discussed. The body of information presently available suggests that transformation has a great impact on bacterial population dynamics as well as on bacterial evolution and speciation.


Small nuclear ribonucleoprotein remodeling during catalytic activation of the spliceosome.
Major structural changes occur in the spliceosome during its activation just before catalyzing the splicing of pre-messenger RNAs (pre-mRNAs). Whereas changes in small nuclear RNA (snRNA) conformation are well documented, little is known about remodeling of small nuclear ribonucleoprotein (snRNP) structures during spliceosome activation. Here, human 45S activated spliceosomes and a previously unknown 35S U5 snRNP were isolated by immunoaffinity selection and were characterized by mass spectrometry. Comparison of their protein components with those of other snRNP and spliceosomal complexes revealed a major change in protein composition during spliceosome activation. Our data also suggest that the U5 snRNP is dramatically remodeled at this stage, with the Prp19 complex and other factors tightly associating, possibly in exchange for other U5 proteins, and suggest that after catalysis the remodeled U5 is eventually released from the postsplicing complex as a 35S snRNP particle.


Exploring the epididymis: a personal perspective on careers in science.
Science is a profession of inquiry. We ask ourselves what is it we see and why our observations happen the way they do. Answering those two question puts us in the company of those early explorers, who from Europe found the New World, and from Asia reached west to encounter Europe. Vasco Núñez de Balboa of Spain was such an explorer. He was the first European to see or "discover" the Pacific Ocean. One can imagine his amazement, his excitement when he first saw from a mountain top that vast ocean previously unknown to his culture. A career in science sends each of us seeking our own "Balboa Moments," those observations or results that surprise or even amaze us, those discoveries that open our eyes to new views of nature and medicine. Scientists aim to do what those early explorers did: discover what has previously been unknown, see what has previously been unseen, and reveal what has previously been hidden. Science requires the scientist to discover the facts from among many fictions and to separate the important facts from the trivial so that knowledge can be properly developed. It is only with knowledge that old dogmas can be challenged and corrected. Careers in science produce specific sets of knowledge. When pooled with other knowledge sets they eventually contribute to wisdom and it is wisdom, we hope, that will improve the human condition.


The association of tumor volume with mortality following radical prostatectomy.
Data regarding the prognostic significance of tumor volume (TV) in prostate cancer are conflicting. Herein, we evaluated the association of TV with prostate cancer mortality following radical prostatectomy (RP), and assessed the additive prognostic value of TV to an established predictive model. We identified 13,687 patients who underwent RP without preoperative therapy between 1987 and 2009. TV was estimated using the prolate ellipsoid formula. Survival was estimated using the Kaplan-Meier method and compared with the log-rank test. Cox proportional hazard regression models were used to evaluate the association of TV with mortality. The ability of TV to enhance the performance of an established prognostic model (Mayo Clinic GPSM (Gleason, PSA, seminal vesicle and margin status) score) was assessed using the c-index. Median TV was 1.57 cm(3) (interquartile range (IQR) 0.48-4.19). Increasing TV was associated with significantly higher risks of seminal vesicle invasion (hazard ratio (HR) 1.58; P<0.0001), positive surgical margins (HR 1.28; P<0.0001) and lymph node involvement (HR 1.26; P<0.0001). Median postoperative follow-up was 9.4 years (IQR 5.0-14.5). Patient grouping into quartiles according to TV resulted in a significant stratification of outcome, as the 15-year cancer-specific survival by TV quartile was 99%, 98%, 95% and 88%, respectively (P<0.0001). Moreover, on multivariate analysis, greater TV remained associated with significantly increased risks of systemic progression (HR 1.27; P<0.0001), death from prostate cancer (HR 1.29; P<0.0001) and all-cause mortality (HR 1.05; P<0.0001). Meanwhile, addition of TV to the GPSM score increased the c-index for the model's prediction of prostate cancer mortality from 0.803 to 0.822. TV is associated with survival following RP, and enhances, although modestly, the performance of an established prediction model. As such, TV warrants continued assessment in risk stratification tools.


Why high-error-rate random mutagenesis libraries are enriched in functional and improved proteins.
The fraction of proteins that retain wild-type function after mutation has long been observed to decline exponentially as the average number of mutations per gene increases. Recently, several groups have used error-prone polymerase chain reactions (PCR) to generate libraries with 15 to 30 mutations per gene, on average, and have reported that orders of magnitude more proteins retain function than would be expected from the low-mutation-rate trend. Proteins with improved or novel function were isolated disproportionately from these high-error-rate libraries, leading to claims that high mutation rates unlock regions of sequence space that are enriched in positively coupled mutations. Here, we show experimentally that error-prone PCR produces a broader non-Poisson distribution of mutations consistent with a detailed model of PCR. As error rates increase, this distribution leads directly to the observed excesses in functional clones. We then show that while very low mutation rates result in many functional sequences, only a small number are unique. By contrast, very high mutation rates produce mostly unique sequences, but few retain function. Thus an optimal mutation rate exists that balances uniqueness and retention of function. Overall, high-error-rate mutagenesis libraries are enriched in improved sequences because they contain more unique, functional clones. Our findings demonstrate how optimal error-prone PCR mutation rates may be calculated, and indicate that "optimal" rates depend on both the protein and the mutagenesis protocol.


Structure of mycobacterial 3'-to-5' RNA:DNA helicase Lhr bound to a ssDNA tracking strand highlights distinctive features of a novel family of bacterial helicases.
Mycobacterial Lhr is a DNA damage-inducible superfamily 2 helicase that uses adenosine triphosphate (ATP) hydrolysis to drive unidirectional 3'-to-5' translocation along single-stranded DNA (ssDNA) and to unwind RNA:DNA duplexes en route. ATPase, translocase and helicase activities are encompassed within the N-terminal 856-amino acid segment. The crystal structure of Lhr-(1-856) in complex with AMPPNP•Mg2+ and ssDNA defines a new helicase family. The enzyme comprises two N-terminal RecA-like modules, a winged helix (WH) domain and a unique C-terminal domain. The 3' ssDNA end binds in a crescent-shaped groove at the interface between the first RecA domain and the WH domain and tracks 5' into a groove between the second RecA and C domains. A kissing interaction between the second RecA and C domains forms an aperture that demarcates a putative junction between the loading strand tail and the duplex, with the first duplex nucleoside bookended by stacking on Trp597. Intercalation of Ile528 between nucleosides of the loading strand creates another bookend. Coupling of ATP hydrolysis to RNA:DNA unwinding is dependent on Trp597 and Ile528, and on Thr145 and Arg279 that contact phosphates of the loading strand. The structural and functional data suggest a ratchet mechanism of translocation and unwinding coupled to ATP-driven domain movements.


Bovine papillomavirus type 2 (BPV-2) E5 oncoprotein binds to the subunit D of the V₁-ATPase proton pump in naturally occurring urothelial tumors of the urinary bladder of cattle.
Active infection by bovine papillomavirus type 2 (BPV-2) was documented for fifteen urinary bladder tumors in cattle. Two were diagnosed as papillary urothelial neoplasm of low malignant potential (PUNLMP), nine as papillary and four as invasive urothelial cancers. In all cancer samples, PCR analysis revealed a BPV-2-specific 503 bp DNA fragment. E5 protein, the major oncoprotein of the virus, was shown both by immunoprecipitation and immunohistochemical analysis. E5 was found to bind to the activated (phosphorylated) form of the platelet derived growth factor β receptor. PDGFβR immunoprecipitation from bladder tumor samples and from normal bladder tissue used as control revealed a protein band which was present in the pull-down from bladder cancer samples only. The protein was identified with mass spectrometry as "V₁-ATPase subunit D", a component of the central stalk of the V₁-ATPase vacuolar pump. The subunit D was confirmed in this complex by coimmunoprecipitation investigations and it was found to colocalize with the receptor. The subunit D was also shown to be overexpressed by Western blot, RT-PCR and immunofluorescence analyses. Immunoprecipitation and immunofluorescence also revealed that E5 oncoprotein was bound to the subunit D. For the first time, a tri-component complex composed of E5/PDGFβR/subunit D has been documented in vivo. Previous in vitro studies have shown that the BPV-2 E5 oncoprotein binds to the proteolipid c ring of the V₀-ATPase sector. We suggest that the E5/PDGFβR/subunit D complex may perturb proteostasis, organelle and cytosol homeostasis, which can result in altered protein degradation and in autophagic responses.


Differential lectin labelling of circulating hemocytes from Biomphalaria glabrata and Biomphalaria tenagophila resistant or susceptible to Schistosoma mansoni infection.
Lectins/carbohydrate binding can be involved in the Schistosoma mansoni recognition and activation of the Biomphalaria hemocytes. Therefore, expression of lectin ligands on Biomphalaria hemocytes would be associated with snail resistance against S. mansoni infection. To test this hypothesis, circulating hemocytes were isolated from B. glabrata BH (snail strain highy susceptible to S. mansoni), B. tenagophila Cabo Frio (moderate susceptibility), and B. tenagophila Taim (completely resistant strains), labelled with FITC conjugated lectins (ConA, PNA, SBA, and WGA) and analyzed under fluorescence microscopy. The results demonstrated that although lectin-labelled hemocytes were detected in hemolymph of all snail species tested, circulating hemocytes from both strains of B. tenagophila showed a larger number of lectin-labelled cells than B. glabrata. Moreover, most of circulating hemocytes of B. tenagophila were intensively labelled by lectins PNA-FITC and WGA-FITC, while in B. glabrata small hemocytes were labeled mainly by ConA. Upon S. mansoni infection, lectin-labelled hemocytes almost disappeared from the hemolymph of Taim and accumulated in B. glabrata BH. The role of lectins/carbohydrate binding in resistance of B. tengophila infection to S. mansoni is still not fully understood, but the data suggest that there may be a correlation to its presence with susceptibility or resistance to the parasite.


Automated stimulus-response mapping of high-electrode-count neural implants.
Over the past decade, research in the field of functional electrical stimulation (FES) has led to a new generation of high-electrode-count (HEC) devices that offer increasingly selective access to neural populations. Incorporation of these devices into research and clinical applications, however, has been hampered by the lack of hardware and software platforms capable of taking full advantage of them. In this paper, we present the first generation of a closed-loop FES platform built specifically for HEC neural interface devices. The platform was designed to support a wide range of stimulus-response mapping and feedback-based control routines. It includes a central control module, a 1100-channel stimulator, an array of biometric devices, and a 160-channel data recording module. To demonstrate the unique capabilities of this platform, two automated software routines for mapping stimulus-response properties of implanted HEC devices were implemented and tested. The first routine determines stimulation levels that produce perithreshold muscle activity, and the second generates recruitment curves (as measured by peak impulse response). Both routines were tested on 100-electrode Utah Slanted Electrode Arrays (USEAs) implanted in cat hindlimb nerves using joint torque or emg as muscle output metric. Mean time to map perithreshold stimulus level was 16.4 s for electrodes that evoked responses (n = 3200), and 3.6 s for electrodes that did not evoke responses (n = 1800). Mean time to locate recruitment curve asymptote for an electrode (n = 155) was 9.6 s , and each point in the recruitment curve required 0.87 s. These results demonstrate the utility of our FES platform by showing that it can be used to completely automate a typically time- and effort-intensive procedure associated with using HEC devices.


Predictors of treatment outcome in an effectiveness trial of cognitive behavioral therapy for children with anxiety disorders.
A substantial number of children with anxiety disorders do not improve following cognitive behavioral therapy (CBT). Recent effectiveness studies have found poorer outcome for CBT programs than what is typically found in efficacy studies. The present study examined predictors of treatment outcome among 181 children (aged 8-15 years), with separation anxiety, social phobia, or generalized anxiety disorder, who participated in a randomized, controlled effectiveness trial of a 10-session CBT program in community clinics. Potential predictors included baseline demographic, child, and parent factors. Outcomes were as follows: a) remission from all inclusion anxiety disorders; b) remission from the primary anxiety disorder; and c) child- and parent-rated reduction of anxiety symptoms at post-treatment and at 1-year follow-up. The most consistent findings across outcome measures and informants were that child-rated anxiety symptoms, functional impairment, a primary diagnosis of social phobia or separation anxiety disorder, and parent internalizing symptoms predicted poorer outcome at post-treatment. Child-rated anxiety symptoms, lower family social class, lower pretreatment child motivation, and parent internalizing symptoms predicted poorer outcome at 1-year follow-up. These results suggest that anxious children with more severe problems, and children of parents with elevated internalizing symptom levels, may be in need of modified, additional, or alternative interventions to achieve a positive treatment outcome.


EuroSCORE as predictor of in-hospital mortality after percutaneous coronary intervention.
To date, no common risk stratification system is available to predict the risk of surgical or percutaneous myocardial revascularisation in patients with coronary artery disease (CAD). Thus, we sought to assess the European System for Cardiac Operative Risk Evaluation (EuroSCORE) validity to predict in-hospital mortality after percutaneous coronary intervention (PCI). EuroSCORE was prospectively and systematically assessed in 1173 consecutive patients undergoing PCI in a high-volume single centre between April 2005 and October 2006. The receiver-operating characteristics (ROC) curve was used to describe performance and accuracy of the EuroSCORE risk model for the prediction of in-hospital mortality after PCI. The EuroSCORE model demonstrated an overall relation between EuroSCORE rank and the incidence of in-hospital mortality, showing consistency in predicting patient risk across many subgroups and levels of global risk. At multivariable logistic regression analysis the EuroSCORE value was an independent in-hospital mortality predictor (p = 0.002) together with left main disease (p = 0.005), procedural urgency (p = 0.001), ACC/AHA C type lesion (p = 0.02) and PCI failure (p = 0.01). The area under the ROC curve for the EuroSCORE system was 0.91 (95% CI 0.86 to 0.97), indicating a good ability of the model to discriminate patients at risk of dying during the index hospitalisation. The EuroSCORE risk model, already extensively validated for the prediction of early mortality following open-heart surgery, can also be efficiently utilised in the setting of PCI. The introduction of the EuroSCORE assessment in patients with documented CAD may help to improve the revascularisation strategy decision-making process.


Surgical Outcomes for Drop Body Syndrome in Adult Spinal Deformity.
A multicenter retrospective case series of patients treated surgically for adult spinal deformity (ASD). The aim of this study was to compare clinical outcomes between propensity score matched ASD patients with or without drop body syndrome (DBS). DBS is an extreme primary sagittal-plane deformity often seen in Asian countries. Although the importance of sagittal alignment is widely recognized, surgical outcomes for deformities purely in the sagittal plane are poorly understood. This study included 243 consecutive patients (age 66 ± 17 years; range 22-78) who were treated surgically for ASD and were followed at least 2 years (mean follow-up 3.7 ± 2.3 years). DBS was defined as a primary lumbar kyphosis with PI-LL >40°, Cobb angle <30°, and multifidus cross-sectional area <300 mm. DBS patients were matched with non-DBS patients by propensity scores for age, gender, lowest instrumented vertebra (LIV) level, and number of levels fused. Demographics, radiographic findings, and clinical outcomes were compared between DBS and non-DBS patients. Of 243 patients with ASD, 34 had DBS (14%); 28 of these were propensity-matched with ASD patients without DBS. Baseline bone mineral density (BMD), body mass index (BMI), and frailty were similar in DBS and non-DBS patients. Baseline sagittal alignment was worse in DBS than in non-DBS patients [C7SVA 14 ± 5 vs. 8 ± 5 cm; pelvic incidence (PI) - lumbar lordosis (LL) 60 ± 14 vs. 36 ± 20°], and scoliosis research society (SRS)22 scores were also worse for DBS patients (2.5 ± 0.6 vs. 2.9 ± 0.8). Although DBS patients had more complications (20 DBS vs. 16 non-DBS), the clinical outcomes were similarly improved in both groups after surgery. At the 2-year follow-up, the spinopelvic malalignment was worse in DBS than non-DBS patients (PI-LL 17 ± 16° vs. 8 ± 13°, P < 0.05). DBS affected 14% of 234 ASD patients. Although DBS patients had inferior baseline SRS22 scores than non-DBS patients, ASD surgery resulted in similar clinical improvement in both groups. Future studies should examine the influence of lifestyle and genetics on clinical outcomes after surgery for DBS. 3.


Glutamine-Induced Secretion of Intestinal Secretory Immunoglobulin A: A Mechanistic Perspective.
Secretory immunoglobulin A (SIgA) is one important line of defense in the intestinal mucosal surface to protect the intestinal epithelium from enteric toxins and pathogenic microorganisms. Multiple factors, such as intestinal microbiota, intestinal cytokines, and nutrients are highly involved in production of SIgA in the intestine. Recently, glutamine has been shown to affect intestinal SIgA production; however, the underlying mechanism by which glutamine stimulates secretion of intestinal SIgA is unknown. Here, we review current knowledge regarding glutamine in intestinal immunity and show that glutamine-enhanced secretion of SIgA in the intestine may involve intestinal microbiota, intestinal antigen sampling and presentation, induction pathways for SIgA production by plasma cells (both T-dependent and T-independent pathway), and even transport of SIgA. Altogether, the glutamine-intestinal SIgA axis has broad therapeutic implications for intestinal SIgA-associated diseases, such as celiac disease, allergies, and inflammatory bowel disease.


Analysis of changes in trends in the consumption rates of benzodiazepines and benzodiazepine-related drugs.
To analyse trends in the rates of consumption of benzodiazepine (BZD) anxiolytics, BZD hypnotics and non-BZD hypnotics and the association with contextual factors. Descriptive time series study. Units of analysis were monthly dose per inhabitant per day (DID) and dose per medical card per day(DCD) of benzodiazepine(BZD anxiolytics(BZD-A), BZD hypnotics(BZD-H) and non-BZD hypnotics(Non-BZD-H) between January 2006-December 2015. We analysed 6 primary healthcare districts(PHD) and used defined daily doses (DDDs) to calculate the monthly DIDs(overall and by ATC group). Trends and monthly percentage change (MPC) were analysed through joinpoint regression. The annual DID increased by 26% overall, the trend was different across ATC groups. Consumption in BZD-A and BZD-H increased (27.1%,61.9%), consumption in Non-BZD-H decreased by 35%. There was high variability in DCD across the PHD, with an overall increase of 10.2%(5.7%-22.9%). By ATC, DCD increased by 10.4% in BZD-A(4.2%-22.2%) and by 44.2% in BZD-H(33.2%-76.5%). The overall DCD in the Non-BZD-H decreased by 42.1%(19.7%-50.8%). We found an initial upward trend in consumption of BZD-A until April/2008(monthly percentage change -MPC- +0.5%), followed by a slightly slower increase (+0.1%). No changes in trend were detected in BZD-H. In Non-BZD-H, we observed an upward trend until February/2013(+0.1%), followed by a sharp decrease until August/2013(-6.3%), and finally a slight decrease(-0.3%). BZD consumption has increased in the last decade, with variability across areas. The changes in trends do not coincide with the financial crisis, introduction of prescriptions by active ingredient, electronic prescriptions or copayment. The only decrease in the Non-BZD-H may be linked to an intervention.


Effects of anthocyanidin derivative (HK-008) on relaxation in rat perfused mesenterial bed.
Anthocyanins, which are responsible for a variety of bright colors (including red, blue, and purple) in fruits, vegetables, and flowers, are consumed as dietary polyphenols. Anthocyanin-containing fruits are thought to decrease coronary heart disease and are used in anti-diabetic preparations. Diabetes is associated with a variety of cardiovascular complications that may be mediated by endothelial dysfunction, and so this study was designed mainly to characterize the influence of a synthesized anthocyanidin derivative (HK-008) over acetylcholine (ACh)-induced relaxation in mesenteric arterial beds isolated from rats. In a glucose-tolerance test in intact rats, HK-008 (30 mg/kg) reduced the glucose level as effectively as the same dose of glibenclamide. The aortic relaxation induced by pinacidil (an ATP-sensitive potassium channel opener) was greatly inhibited by glibenclamide (10 microM), and also significantly inhibited by HK-008 (10 microM). Interestingly, the ACh-induced relaxation in the perfused, preconstricted mesenteric arterial bed was significantly enhanced by HK-008 (10 microM), and this enhancement was significantly attenuated by indomethacin (10 microM). The ACh-induced mesenteric relaxation was impaired by an increase in oxidative stress, viz. superoxide-generating treatment [xanthine oxidase (XO; 0.1 U/ml) plus hypoxanthine (HX; 10 microM)]. However, this impairment was strongly suppressed by HK-008 (10 microM). These results suggest that HK-008 increases endothelium-induced relaxation by suppressing oxidative stress or modulating prostanoids signaling. This compound may therefore be useful against certain cardiovascular disorders.


Development and optimization of a sublingual tablet formulation for physostigmine salicylate.
This study is aimed to design and optimize a sublingual tablet formulation of physostigmine salicylate, an effective drug in Alzheimer's disease and nerve gas poisoning, by means of the D-optimal experimental design methodology. Polyvinyl pyrrolidone, lactose, starch 1500 and sodium starch glycolate were used in the formulations as independent variables. Tablets were prepared by the direct compression method and evaluated for their physical properties (tablet hardness, disintegration time and friability), which were regarded as responses in a D-optimal design. Due to the significance of the special cubic model for data fitted, compared to other models, it was used to examine the obtained results. Response surface plots were plotted to study the tablet properties and the optimized overlay plot was generated based on the results and targets considered for the responses. After verification of the optimum checkpoint formulations, an optimized formulation was chosen due to its desirable physical properties and closely observed and predicted values. Drug assay, content uniformity of the dosage unit, drug dissolution and accelerated stability studies were done on the optimum formulation as further experiments. All the obtained results complied with the requirements of a sublingual tablet formulation.


What can we learn from Plausible Values?
In this paper, we show that the marginal distribution of plausible values is a consistent estimator of the true latent variable distribution, and, furthermore, that convergence is monotone in an embedding in which the number of items tends to infinity. We use this result to clarify some of the misconceptions that exist about plausible values, and also show how they can be used in the analyses of educational surveys.


BMP promotes motility and represses growth of smooth muscle cells by activation of tandem Wnt pathways.
We present a novel cell-signaling paradigm in which bone morphogenetic protein 2 (BMP-2) consecutively and interdependently activates the wingless (Wnt)-β-catenin (βC) and Wnt-planar cell polarity (PCP) signaling pathways to facilitate vascular smooth muscle motility while simultaneously suppressing growth. We show that BMP-2, in a phospho-Akt-dependent manner, induces βC transcriptional activity to produce fibronectin, which then activates integrin-linked kinase 1 (ILK-1) via α4-integrins. ILK-1 then induces the Wnt-PCP pathway by binding a proline-rich motif in disheveled (Dvl) and consequently activating RhoA-Rac1-mediated motility. Transfection of a Dvl mutant that binds βC without activating RhoA-Rac1 not only prevents BMP-2-mediated vascular smooth muscle cell motility but promotes proliferation in association with persistent βC activity. Interfering with the Dvl-dependent Wnt-PCP activation in a murine stented aortic graft injury model promotes extensive neointima formation, as shown by optical coherence tomography and histopathology. We speculate that, in response to injury, factors that subvert BMP-2-mediated tandem activation of Wnt-βC and Wnt-PCP pathways contribute to obliterative vascular disease in both the systemic and pulmonary circulations.


Compensatory activation in fronto-parietal cortices among HIV-infected persons during a monetary decision-making task.
HIV infection can cause direct and indirect damage to the brain and is consistently associated with neurocognitive disorders, including impairments in decision-making capacities. The tendency to devalue rewards that are delayed (temporal discounting) is relevant to a range of health risk behaviors. Making choices about delayed rewards engages the executive control network of the brain, which has been found to be affected by HIV. In this case-control study of 18 HIV-positive and 17 HIV-negative adults, we examined the effects of HIV on brain activation during a temporal discounting task. Functional MRI (fMRI) data were collected while participants made choices between smaller, sooner rewards and larger, delayed rewards. Choices were individualized based on participants' unique discount functions, so each participant experienced hard (similarly valued), easy (disparately valued), and control choices. fMRI data were analyzed using a mixed-effects model to identify group-related differences associated with choice difficulty. While there was no difference between groups in behavioral performance, the HIV-positive group demonstrated significantly larger increases in activation within left parietal regions and bilateral prefrontal regions during easy trials and within the right prefrontal cortex and anterior cingulate during hard trials. Increasing activation within the prefrontal regions was associated with lower nadir CD4 cell count and risk-taking propensity. These results support the hypothesis that HIV infection can alter brain functioning in regions that support decision making, providing further evidence for HIV-associated compensatory activation within fronto-parietal cortices. A history of immunosuppression may contribute to these brain changes. Hum Brain Mapp 37:2455-2467, 2016. © 2016 Wiley Periodicals, Inc.


Induction, adaptation and recovery of lysosomal integrity in green-lipped mussel Perna viridis.
Biomarkers are generally applied to detect pollution in environmental monitoring. Such biological responses should accurately reflect the stress over time in a quantitative manner. As such, the initial and maximum responses induced by stress, as well as adaptation and recovery of these biomarkers, need to be fully understood or else erroneous false-negative or false-positive may be arrived. However, most of the biomarker studies only provided information on initially induced responses under different concentrations of toxicants, while biological adaptation and recovery were poorly known. In this study, the time required for induction, adaptation and recovery of lysosomal integrity in green-lipped mussel Perna viridis upon exposure to benzo[a]pyrene was investigated over a period of 62 days. Maximum induction occurred on day 6 when lysosomal integrity was significantly reduced by 51%, and no further change or adaptation was detected thereafter. When mussels were depurated in clean seawater after 18 days of exposure to benzo[a]pyrene, a gradual recovery was observed, with lysosomal integrity returning to its background level and showing a complete recovery after 20 days of depuration. Lysosomal integrity was significantly correlated with the body burden concentrations of benzo[a]pyrene and condition index of the mussels. The relatively fast induction (6 days) and recovery (20 days) without apparent adaptation suggested that lysosomal integrity in P. viridis can serve as a good biomarker in biomonitoring, as its response is not likely to generate both false-negative and false-positive results.


SH2-containing phosphotyrosine phosphatase as a target of protein-tyrosine kinases.
A mouse phosphotyrosine phosphatase containing two Src homology 2 (SH2) domains, Syp, was identified. Syp bound to autophosphorylated epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) receptors through its SH2 domains and was rapidly phosphorylated on tyrosine in PDGF- and EGF-stimulated cells. Furthermore, Syp was constitutively phosphorylated on tyrosine in cells transformed by v-src. This mammalian phosphatase is most closely related, especially in its SH2 domains, to the corkscrew (csw) gene product of Drosophila, which is required for signal transduction downstream of the Torso receptor tyrosine kinase. The Syp gene is widely expressed throughout embryonic mouse development and in adult tissues. Thus, Syp may function in mammalian embryonic development and as a common target of both receptor and nonreceptor tyrosine kinases.


Short-term homeostatic regulation of blood/interstitial fluid Ca2+ concentration by the scales of anadromous sea trout Salmo trutta L. during smoltification and migration.
The elasmoid scales of anadromous sea trout Salmo trutta L. represent a significant internal reservoir of Ca2+ . Although more is known about long-term remodelling of scales in response to calciotropic challenges encountered during smoltification and migration, very little is known about the contribution made by scales to the short-term, minute-to-minute regulation of Ca2+ homeostasis in the extracellular fluid (ECF) during these phases of the life cycle. This gap in the knowledge is partly due to the technical challenges involved in measuring small Ca2+ fluxes around the scales of live fish in real time. Here, this study describes exfoliating, mounting and culturing scales and their resident cells from parr, smolt and adult sea trout from a freshwater environment, as well as from adult sea trout caught in sea or brackish water. All the scales were then examined using an extracellular, non-invasive, surface-scanning Ca2+ -sensitive microelectrode. The authors quantified the Ca2+ fluxes, in the absence of any systemic or local regulators, into and out of scales on both the episquamal and hyposquamal sides under different extracellular calcemic challenges set to mimic a variety of ECF-Ca2+ concentrations. Scales from the life-cycle stages as well as from adult fish taken from sea, brackish or fresh water all showed a consistent efflux or influx of Ca2+ under hypo- or hypercalcemic conditions, respectively. What were considered to be isocalcemic conditions resulted in minimal flux of Ca2+ in either direction, or in the case of adult scales, a consistent but small influx. Indeed, adult scales appeared to display the largest flux densities in either direction. These new data extend the current understanding of the role played by fish scales in the short-term, minute-to-minute homeostatic regulation of ECF-Ca2+ concentration, and are similar to those recently reported from zebrafish Danio rerio scales. This suggests that this short-term regulatory response might be a common feature of teleost scales.


Periodontal bacteria and hypertension: the oral infections and vascular disease epidemiology study (INVEST).
Chronic infections, including periodontal infections, may predispose to cardiovascular disease. We investigated the relationship between periodontal microbiota and hypertension. Six hundred and fifty-three dentate men and women with no history of stroke or myocardial infarction were enrolled in INVEST. We collected 4533 subgingival plaque samples (average of seven samples per participant). These were quantitatively assessed for 11 periodontal bacteria using DNA-DNA checkerboard hybridization. Cardiovascular risk factor measurements were obtained. Blood pressure and hypertension (SBP > or =140 mmHg, DBP > or =90 mmHg or taking antihypertensive medication, or self-reported history) were each regressed on the level of bacteria: considered causative of periodontal disease (etiologic bacterial burden); associated with periodontal disease (putative bacterial burden); and associated with periodontal health (health-associated bacterial burden). All analyses were adjusted for age, race/ethnicity, sex, education, BMI, smoking, diabetes, low-density lipoprotein and high-density lipoprotein cholesterol. Etiologic bacterial burden was positively associated with both blood pressure and prevalent hypertension. Comparing the highest and lowest tertiles of etiologic bacterial burden, SBP was 9 mmHg higher, DBP was 5 mmHg higher (P for linear trend was less than 0.001 in each case), and the odds ratio for prevalent hypertension was 3.05 (95% confidence interval 1.60-5.82) after multivariable adjustment. Our data provide evidence of a direct relationship between the levels of subgingival periodontal bacteria and both SBP and DBP as well as hypertension prevalence.


Harnessing the Angiogenic Potential of Stem Cell-Derived Exosomes for Vascular Regeneration.
Mesenchymal stem cells (MSCs) are known to display important regenerative properties through the secretion of proangiogenic factors. Recent evidence pointed at the key role played by exosomes released from MSCs in this paracrine mechanism. Exosomes are key mediators of intercellular communication and contain a cargo that includes a modifiable content of microRNA (miRNA), mRNA, and proteins. Since the biogenesis of the MSCs-derived exosomes is regulated by the cross talk between MSCs and their niche, the content of the exosomes and consequently their biological function are dependent on the cell of origin and the physiologic or pathologic status of their microenvironment. Recent preclinical studies revealed that MSCs-derived exosomes have a critical implication in the angiogenic process since the use of exosomes-depleted conditioned medium impaired the MSCs angiogenesis response. In this review, we discuss the current knowledge related to the angiogenic potential of MSCs-exosomes and methods to enhance their biological activities for improved vascular regeneration. The current gain of insight in exosomes studies highlights the power of combining cell based therapies and their secreted products in therapeutic angiogenesis.


Quantitative Laser Diffraction for Quantification of Protein Aggregates: Comparison With Resonant Mass Measurement, Nanoparticle Tracking Analysis, Flow Imaging, and Light Obscuration.
In the past, analysis of micron-sized (>1.0 μm) aggregates of therapeutic proteins has been limited to light obscuration (LO), and appropriate quantitative methods of evaluating protein aggregates need to be developed. Recently, novel methods with enhanced reliability and sensitivity, such as nanoparticle tracking analysis (NTA), resonant mass measurement (RMM), and flow imaging (FI), have emerged. We have found that quantitative laser diffraction (qLD) is also effective for quantitative evaluation of protein aggregates over a wide size range. However, the different detection principles of the methods potentially lead to inconsistencies in results. This study aimed to compare particle size distributions and concentrations of protein aggregates using the orthogonal methods. Protein aggregates were generated by stirring an immunoglobulin solution. Serial dilutions of the aggregates stock were analyzed by RMM, FI, and qLD to obtain the particle size distribution and concentration using each method. In addition, size distribution of a protein aggregates solution was compared by RMM, NTA, FI, LO, and qLD. Both particle size distribution and concentration were in good agreement between RMM and qLD (0.3-2 μm) and between FI and qLD (2-20 μm). Thus, we concluded that qLD enables covering of the overlapping particle size range between RMM and FI.


Engagement with protective behaviours in the UK during the COVID-19 pandemic: a series of cross-sectional surveys (the COVID-19 rapid survey of adherence to interventions and responses [CORSAIR] study).
Behaviour is key to suppressing the COVID-19 pandemic. Maintaining behaviour change can be difficult. We investigated engagement with hand cleaning, reducing the number of outings, and wearing a face covering over the course of the pandemic. We used a series of 64 cross-sectional surveys between 10 February 2020 and 20 January 2022 (n ≈ 2000 per wave). Surveys investigated uptake of hand cleaning behaviours, out of home activity (England only, n ≈ 1700 per wave) and wearing a face covering (England only, restricted to those who reported going out shopping in the last week, n ≈ 1400 per wave). Reported hand cleaning has been high throughout the pandemic period (85 to 90% of participants consistently reporting washing their hands thoroughly and regularly with soap and water frequently or very frequently). Out of home activity has mirrored the easing and re-introduction of restrictive measures. Total number of outings were higher in the second national lockdown than in the first and third lockdowns. Wearing a face covering increased steadily between April to August 2020, plateauing until the end of measurement in May 2021, with approximately 80% of those who had been out shopping in the previous week reporting wearing a face covering frequently or very frequently. Engagement with protective behaviours increased at the start of the pandemic and has remained high since. The greatest variations in behaviour reflected changes to Government rules. Despite the duration of restrictions, people have continued to adopt personal protective behaviours that were intended to prevent the spread of COVID-19.


Alteration of ultraviolet-induced mutagenesis in yeast through molecular modulation of the REV3 and REV7 gene expression.
DNA damage can lead to mutations during replication. The damage-induced mutagenesis pathway is an important mechanism that fixes DNA lesions into mutations. DNA polymerase zeta (Pol zeta), formed by Rev3 and Rev7 protein complex, and Rev1 are components of the damage-induced mutagenesis pathway. Since mutagenesis is an important factor during the initiation and progression of human cancer, we postulate that this mutagenesis pathway may provide an inhibiting target for cancer prevention and therapy. In this study, we tested if UV-induced mutagenesis can be altered by molecular modulation of Rev3 enzyme levels using the yeast Saccharomyces cerevisiae as a eukaryotic model system. Reducing the REV3 expression in yeast cells through molecular techniques was employed to mimic Pol zeta inhibition. Lower levels of Pol zeta significantly decreased UV-induced mutation frequency, thus achieving inhibition of mutagenesis. In contrast, elevating the Pol zeta level by enhanced expression of both REV3 and REV7 genes led to a approximately 3-fold increase in UV-induced mutagenesis as determined by the arg4-17 mutation reversion assays. In vivo, UV lesion bypass by Pol zeta requires the Rev1 protein. Even overexpression of Pol zeta could not alleviate the defective UV mutagenesis in the rev1 mutant cells. These observations provide evidence that the mutagenesis pathway could be used as a target for inhibiting damage-induced mutations.


Modulating perceptual complexity and load reveals degradation of the visual working memory network in ageing.
Previous neuroimaging studies have reported a posterior to anterior shift of activation in ageing (PASA). Here, we explore the nature of this shift by modulating load (1,2 or 3 items) and perceptual complexity in two variants of a visual working memory task (VWM): a 'simple' color and a 'complex' shape change detection task. Functional near-infrared spectroscopy (fNIRS) was used to record changes in activation in younger (N=24) and older adults (N=24). Older adults exhibited PASA by showing lesser activation in the posterior cortex and greater activation in the anterior cortex when compared to younger adults. Further, they showed reduced accuracy at loads 2 and 3 for the simple task and across all loads for the complex task. Activation in the posterior and anterior cortices was modulated differently for younger and older adults. In older adults, increasing load in the simple task was accompanied by decreasing activation in the posterior cortex and lack of modulation in the anterior cortex, suggesting the inability to encode and/or maintain representations without much aid from higher-order centres. In the complex task, older adults recruited verbal working memory areas in the posterior cortex, suggesting that they used adaptive strategies such as labelling the shape stimuli. This was accompanied by reduced activation in the anterior cortex reflecting the inability to exert top-down modulation to typical VWM areas in the posterior cortex to improve behavioral performance.


Mercury in the seafood and human exposure in coastal area of Guangdong province, South China.
Consumption of fish and aquatic products is considered to be the main pathway of human exposure to methylmercury (MeHg). To assess human health risk through seafood consumption in a coastal area of Guangdong Province, South China, a total of 518 seafood samples (including fish, shrimp, crabs, and mollusks) were collected from 11 coastal cities for total mercury (THg) analysis. The THg concentrations varied significantly among different seafood groups and ranged from 0.11 to 317 ng/g (with a mean of 37.2 ng/g). The THg concentrations were relatively low compared with fish from Europe and North America, and no samples exceeded the national limit recommended by the Standardization Administration of China (0.5 ng/g wet wt). The ecological functional groups and cultured styles have significant effects on THg concentrations in the fish tissue. The median of probable daily intakes (PDIs) of MeHg via seafood consumption ranged from 42.6 to 71.4 ng/g for different age groups, with fish contributing the major portion (84%). The results indicated potential health risks associated with seafood consumption for the general population in the coastal area of Guangdong Province, South China.


Early Life Exposure in Mexico to ENvironmental Toxicants (ELEMENT) Project.
The Early Life Exposure in Mexico to ENvironmental Toxicants (ELEMENT) Project is a mother-child pregnancy and birth cohort originally initiated in the mid-1990s to explore: (1) whether enhanced mobilisation of lead from maternal bone stores during pregnancy poses a risk to fetal and subsequent offspring neurodevelopment; and (2) whether maternal calcium supplementation during pregnancy and lactation can suppress bone lead mobilisation and mitigate the adverse effects of lead exposure on offspring health and development. Through utilisation of carefully archived biospecimens to measure other prenatal exposures, banking of DNA and rigorous measurement of a diverse array of outcomes, ELEMENT has since evolved into a major resource for research on early life exposures and developmental outcomes. n=1643 mother-child pairs sequentially recruited (between 1994 and 2003) during pregnancy or at delivery from maternity hospitals in Mexico City, Mexico. Maternal bone (eg, patella, tibia) is an endogenous source for fetal lead exposure due to mobilisation of stored lead into circulation during pregnancy and lactation, leading to increased risk of miscarriage, low birth weight and smaller head circumference, and transfer of lead into breastmilk. Daily supplementation with 1200 mg of elemental calcium during pregnancy and lactation reduces lead resorption from maternal bone and thereby, levels of circulating lead. Beyond perinatal outcomes, early life exposure to lead is associated with neurocognitive deficits, behavioural disorders, higher blood pressure and lower weight in offspring during childhood. Some of these relationships were modified by dietary factors; genetic polymorphisms specific for iron, folate and lipid metabolism; and timing of exposure. Research has also expanded to include findings published on other toxicants such as those associated with personal care products and plastics (eg, phthalates, bisphenol A), other metals (eg, mercury, manganese, cadmium), pesticides (organophosphates) and fluoride; other biomarkers (eg, toxicant levels in plasma, hair and teeth); other outcomes (eg, sexual maturation, metabolic syndrome, dental caries); and identification of novel mechanisms via epigenetic and metabolomics profiling. As the ELEMENT mothers and children age, we plan to (1) continue studying the long-term consequences of toxicant exposure during the perinatal period on adolescent and young adult outcomes as well as outcomes related to the original ELEMENT mothers, such as their metabolic and bone health during perimenopause; and (2) follow the third generation of participants (children of the children) to study intergenerational effects of in utero exposures. NCT00558623.


Cost effectiveness of a computer-delivered intervention to improve HIV medication adherence.
High levels of adherence to medications for HIV infection are essential for optimal clinical outcomes and to reduce viral transmission, but many patients do not achieve required levels. Clinician-delivered interventions can improve patients' adherence, but usually require substantial effort by trained individuals and may not be widely available. Computer-delivered interventions can address this problem by reducing required staff time for delivery and by making the interventions widely available via the Internet. We previously developed a computer-delivered intervention designed to improve patients' level of health literacy as a strategy to improve their HIV medication adherence. The intervention was shown to increase patients' adherence, but it was not clear that the benefits resulting from the increase in adherence could justify the costs of developing and deploying the intervention. The purpose of this study was to evaluate the relation of development and deployment costs to the effectiveness of the intervention. Costs of intervention development were drawn from accounting reports for the grant under which its development was supported, adjusted for costs primarily resulting from the project's research purpose. Effectiveness of the intervention was drawn from results of the parent study. The relation of the intervention's effects to changes in health status, expressed as utilities, was also evaluated in order to assess the net cost of the intervention in terms of quality adjusted life years (QALYs). Sensitivity analyses evaluated ranges of possible intervention effectiveness and durations of its effects, and costs were evaluated over several deployment scenarios. The intervention's cost effectiveness depends largely on the number of persons using it and the duration of its effectiveness. Even with modest effects for a small number of patients the intervention was associated with net cost savings in some scenarios and for durations greater than three months and longer it was usually associated with a favorable cost per QALY. For intermediate and larger assumed effects and longer durations of intervention effectiveness, the intervention was associated with net cost savings. Computer-delivered adherence interventions may be a cost-effective strategy to improve adherence in persons treated for HIV. Clinicaltrials.gov identifier NCT01304186.