Autoregulation of tubulin synthesis in hepatocytes and fibroblasts.
Microtubule polymer levels in mouse 3T6 fibroblasts and primary cultures of rat hepatocytes can be manipulated by treatment of cells with long term, low doses of colcemid. Such treatment produces a rather uniform population of cells with microtubules of reduced lengths. Using this system, we demonstrate (a) that the rate of tubulin synthesis is sensitive to small changes (10%) in microtubule polymer mass and (b) that the percent of inhibition of synthesis is proportional to the level of soluble tubulin. Experiments with hepatocytes indicate that not only synthesis but the stability of tubulin protein was also regulated to maintain a specific level of tubulin. Treatment of hepatocytes with colcemid or other microtubule-depolymerizing drugs reduced the half-life of tubulin from 50 to 2 h, whereas taxol, which stabilizes microtubules, increased the half-life. To assess the consequences of altering microtubule polymer mass, we have analyzed the effect of controlled depolymerization of microtubules in rat hepatocytes on the processing of endocytosed ligands and found it sensitive to small changes in microtubule polymer levels.


A distinctive avian assemblage (Aves: Passeriformes) in Western Darién, Panama is uncovered through a disease surveillance program.
Basic knowledge about the distribution of flora and fauna is lacking for most tropical areas. Improving our knowledge of the tropical biota will help address contemporary global problems, including emerging tropical diseases. Less appreciated is the role that applied studies can have in improving our understanding of basic biological patterns and processes in the tropics. Here, I describe a novel avifauna assemblage uncovered in Western Darién province in the Republic of Panama that was uncovered during a vector-borne disease surveillance program. I compared the passerine bird species composition at 16 sites using records from recent ornithological expeditions sponsored by the Smithsonian Tropical Research Institute in Central and Eastern Panama. Based on the results of a Mantel test, geographic distance did not correlate with pairwise distinctiveness of sites. instead, based on an index of distinctiveness modified from the Chao-Jaccard index, most sites were more or less similarly distinctive, with one site, Aruza Abajo, significantly more distinctive than the rest. I found that the distinctiveness of this site was due not only to the presence of several rare and range-restricted taxa, but also to the absence of taxa that are common elsewhere. This finding provides more evidence of high species composition turnover (beta-diversity) in the Panamanian biota, which appears to be driven by a combination of soil and climate differences over narrow distances.


Fusion of 3D ultrasound images of the fetal femur improves boundary definition and volume measurement.
To combine multiple 3D volumes of the same fetal femur into one composite image data set using image registration and wavelet-based fusion. Fused and single data sets were compared in terms of image quality and femur volume (FV) measurement repeatability. In healthy pregnant volunteers, six volumes of the same femur were acquired and fused into a composite data set. Image quality scores were given to the fused and single data sets by an independent assessor in a blinded fashion; repeatability of FV measurement was assessed using coefficients of variation (CV), intraclass correlation coefficients (ICC) and Bland-Altman plots. Fusion was successful in 24 out of 25 cases. Median image quality score was 7/10 in fused data sets, compared to 6/10 in single data sets (p = 0.096). Repeatability of FV measurement was better in fused data sets (intraobserver CV 4.6% and ICC 0.987; interobserver CV 4.9%, ICC 0.985) compared to single ones (intraobserver CV 5.8%, ICC 0.977; interobserver CV 10.0%, ICC 0.931). The measured FV was significantly higher in fused data sets (mean FV 1.7 vs. 1.3 ml, p < 0.001). Image registration and wavelet-based fusion can improve image quality and FV repeatability; it also results in an increased FV measurement.


Inter-rater reliability of categorical versus continuous scoring of fish vitality: Does it affect the utility of the reflex action mortality predictor (RAMP) approach?
Scoring reflex responsiveness and injury of aquatic organisms has gained popularity as predictors of discard survival. Given this method relies upon the individual interpretation of scoring criteria, an evaluation of its robustness is done here to test whether protocol-instructed, multiple raters with diverse backgrounds (research scientist, technician, and student) are able to produce similar or the same reflex and injury score for one of the same flatfish (European plaice, Pleuronectes platessa) after experiencing commercial fishing stressors. Inter-rater reliability for three raters was assessed by using a 3-point categorical scale ('absent', 'weak', 'strong') and a tagged visual analogue continuous scale (tVAS, a 10 cm bar split in three labelled sections: 0 for 'absent', 'weak', 'moderate', and 'strong') for six reflex responses, and a 4-point scale for four injury types. Plaice (n = 304) were sampled from 17 research beam-trawl deployments during four trips. Fleiss kappa (categorical scores) and intra-class correlation coefficients (ICC, continuous scores) indicated variable inter-rater agreement by reflex type (ranging between 0.55 and 0.88, and 67% and 91% for Fleiss kappa and ICC, respectively), with least agreement among raters on extent of injury (Fleiss kappa between 0.08 and 0.27). Despite differences among raters, which did not significantly influence the relationship between impairment and predicted survival, combining categorical reflex and injury scores always produced a close relationship of such vitality indices and observed delayed mortality. The use of the continuous scale did not improve fit of these models compared with using the reflex impairment index based on categorical scores. Given these findings, we recommend using a 3-point categorical over a continuous scale. We also determined that training rather than experience of raters minimised inter-rater differences. Our results suggest that cost-efficient reflex impairment and injury scoring may be considered a robust technique to evaluate lethal stress and damage of this flatfish species on-board commercial beam-trawl vessels.


Exogenous tetrahydrobiopterin causes endothelium-dependent contractions in isolated canine basilar artery.
Tetrahydrobiopterin is an essential cofactor in the biosynthesis of nitric oxide, but in the presence of O2, autooxidation of tetrahydrobiopterin leads to the production of superoxide anions and the subsequent chemical inactivation of nitric oxide. As a result, the present experiments were designed to determine the effect of exogenous tetrahydrobiopterin on isolated canine basilar arteries. Rings with and without endothelium were suspended for isometric force recording in modified Krebs-Ringer bicarbonate solution bubbled with 94% O2-6% CO2 (37 degrees C; pH 7.4). A radioimmunoassay technique was used to measure guanosine 3',5' cyclic monophosphate production (cGMP). Tetrahydrobiopterin (10(-7) to 10(-4) M), but not dihydrobiopterin or biopterin, caused endothelium-dependent contractions. Superoxide dismutase (150 U/ml) abolished tetrahydrobiopterin-induced contractions, but a H2O2 scavenger, catalase (1,200 U/ml), and hydroxyl radical scavengers deferoxamine (10(-4) M) and dimethylsulfoxide (10(-4) to 10(-3) M) did not significantly affect the contractions. A cyclooxygenase inhibitor, indomethacin (10(-5) M), significantly reduced the contractile effect of tetrahydrobiopterin. With the prostaglandin H2/ thromboxane A2 receptor antagonist, SQ-29548 (10(-6) M) present, contractions reversed to relaxations. In rings with endothelium, tetrahydrobiopterin (10(-4) M) significantly decreased the levels of cGMP. These studies suggest that autooxidation of exogenous tetrahydrobiopterin induces endothelium-dependent contractions by 1) chemical inactivation of nitric oxide by superoxide anions and 2) activation of arachidonic acid metabolism via the cyclooxygenase pathway with subsequent release of endoperoxide and/or thromboxane A2 from endothelial cells.


Interactions between glutathione S-transferase P1, tumor necrosis factor, and traffic-related air pollution for development of childhood allergic disease.
Air pollutants may induce airway inflammation and sensitization due to generation of reactive oxygen species. The genetic background to these mechanisms could be important effect modifiers. Our goal was to assess interactions between exposure to air pollution and single nucleotide polymorphisms (SNPs) in the beta2-adrenergic receptor (ADRB2), glutathione S-transferase P1 (GSTP1), and tumor necrosis factor (TNF) genes for development of childhood allergic disease. In a birth cohort originally of 4,089 children, we assessed air pollution from local traffic using nitrogen oxides (traffic NO(x)) as an indicator based on emission databases and dispersion modeling and estimated individual exposure through geocoding of home addresses. We measured peak expiratory flow rates and specific IgE for inhalant and food allergens at 4 years of age, and selected children with asthma symptoms up to 4 years of age (n = 542) and controls (n = 542) for genotyping. Interaction effects on allergic sensitization were indicated between several GSTP1 SNPs and traffic NO(x) exposure during the first year of life (p(nominal) < 0.001-0.06). Children with Ile105Val/Val105Val genotypes were at increased risk of sensitization to any allergen when exposed to elevated levels of traffic NO(x) (for a difference between the 5th and 95th percentile of exposure: odds ratio = 2.4; 95% confidence interval, 1.0-5.3). In children with TNF-308 GA/AA genotypes, the GSTP1-NO(x) interaction effect was even more pronounced. We observed no conclusive interaction effects for ADRB2. The effect of air pollution from traffic on childhood allergy appears to be modified by GSTP1 and TNF variants, supporting a role of genes controlling the antioxidative system and inflammatory response in allergy.


Endozepine stupor. Recurring stupor linked to endozepine-4 accumulation.
Recurring stupor can be caused by repeated metabolic, toxic or structural brain disturbances. Recently, cases of recurring stupor, with fast EEG activity were shown to display increased endogenous benzodiazepine-like activity during the episodes of stupor. Patients with recurring stupor underwent extensive metabolic and toxicologic screening, EEG and brain imaging. Endozepines and exogenously administered benzodiazepines were assayed in plasma and CSF by means of mass spectrometry. Flumazenil, a benzodiazepine antagonist was administered and the behavioural and EEG responses monitored. Treatment with oral flumazenil was attempted in selected cases. Twenty patients were found with recurring stupor. Episodes had begun between ages 18 and 67 years, and in nine patients, had disappeared spontaneously after 4-6 years with symptoms. Stupor lasted hours or days. Onset of the episodes and frequency were unpredictable. Patients were normal between attacks. Stupor was characterized by initial drowsiness, staggering and behavioural changes, followed by deep sleep and spontaneous recovery with post-ictal amnesia. Biochemical screening and brain imaging were always normal. Ictal EEG showed fast background activity, and flumazenil transiently awoke the patients and normalized the EEG. In the nine cases examined, endozepine-4 levels were increased during the stupor. Oral flumazenil reduced the frequency of the attacks in three of these nine patients. Recurring episodes of stupor may be due to increased endozepine-4. We propose the term 'endozepine stupor' for such episodes. Endozepine-4 is an endogenous ligand for the benzodiazepine recognition site at the GABAA receptor, with unknown molecular structure.


The Association Between Cyberbullying, School Bullying, and Suicidality Among Adolescents.
Bullying and suicidality are serious worldwide problems with negative effects on the young population and therefore international comparisons in this field are of paramount importance. To analyze the prevalence of bullying and cyberbullying and their association with suicidal behavior among school-aged children in Israel, Lithuania, and Luxembourg. In total, 3,814 15-year-olds from schools in Israel, Lithuania, and Luxembourg were surveyed in the Health Behavior in School-Aged Children (HBSC) cross-national survey in 2013/2014 using standardized anonymous questionnaires. Data analysis employed logistic regression and structural equation modeling (SEM). In all, 6.5% of the adolescents reported being cyberbullied, 15.6% reported being bullied at school. In the previous 12 months, 38.6% reported experiencing emotions that stopped them from doing their usual activities, 17.8% considered attempting suicide, 12.0% made a suicide plan, and 9.5% attempted suicide. Victims of cyberbullying and school bullying had a significantly higher risk of suicidal ideations, plans, and attempts. The SEM analysis confirmed a significant overall effect of bullying on adolescent suicidality. The strongest effect was seen among Israeli students. The prevalence estimates were obtained by self-report. The prevalence of adolescent cyberbullying, school bullying, and suicidal behavior is relatively high in Israel, Lithuania, and Luxembourg. Cyberbullying is a strong predictor of adolescent suicidality.


The effect of copper(II), iron(II) sulphate, and vitamin C combinations on the weak antimicrobial activity of (+)-catechin against Staphylococcus aureus and other microbes.
Few attempts have been made to improve the activity of plant compounds with low antimicrobial efficacy. (+)-Catechin, a weak antimicrobial tea flavanol, was combined with putative adjuncts and tested against different species of bacteria. Copper(II) sulphate enhanced (+)-catechin activity against Pseudomonas aeruginosa but not Staphylococcus aureus, Proteus mirabilis or Escherichia coli. Attempts to raise the activity of (+)-catechin against two unresponsive species, S. aureus and E. coli, with iron(II) sulphate, iron(III) chloride, and vitamin C, showed that iron(II) enhanced (+)-catechin against S. aureus, but not E. coli; neither iron(III) nor combined iron(II) and copper(II), enhanced (+)-catechin activity against either species. Vitamin C enhanced copper(II) containing combinations against both species in the absence of iron(II). Catalase or EDTA added to active samples removed viability effects suggesting that active mixtures had produced H(2)O(2)via the action of added metal(II) ions. H(2)O(2) generation by (+)-catechin plus copper(II) mixtures and copper(II) alone could account for the principal effect of bacterial growth inhibition following 30 minute exposures as well as the antimicrobial effect of (+)-catechin-iron(II) against S. aureus. These novel findings about a weak antimicrobial flavanol contrast with previous knowledge of more active flavanols with transition metal combinations. Weak antimicrobial compounds like (+)-catechin within enhancement mixtures may therefore be used as efficacious agents. (+)-Catechin may provide a means of lowering copper(II) or iron(II) contents in certain crop protection and other products.


Perceived exercise benefits and barriers of non-exercising female university students in the United Kingdom.
Many individuals do not engage in sufficient physical activity due to low perceived benefits and high perceived barriers to exercise. Given the increasing incidence of obesity and obesity related health disorders, this topic requires further exploration. We used the Exercise Benefits/Barriers Scale to assess perceived benefit and barrier intensities to exercise in 200 non-exercising female university students (mean age 19.3 years, SD = 1.06) in the UK. Although our participants were selected because they self reported themselves to be non-exercising, however they reported significantly higher perceived benefits from exercise than perceived barriers to exercise [t(199) = 6.18, p < 0.001], and their perceived benefit/barrier ratio was 1.33. The greatest perceived benefit from exercise was physical performance followed by the benefits of psychological outlook, preventive health, life enhancement, and then social interaction. Physical performance was rated significantly higher than all other benefits. Psychological outlook and preventive health were not rated significantly different, although both were significantly higher than life enhancement and social interaction. Life enhancement was also rated significantly higher than social interaction. The greatest perceived barrier to exercise was physical exertion, which was rated significantly higher than time expenditure, exercise milieu, and family discouragement barriers. Implications from this investigation for the design of physical activity programmes include the importance, for females, of a perception of high benefit/barrier ratio that could be conducive to participation in exercise. Applied interventions need to assist female students to 'disengage' from or overcome any perceived 'unpleasantness' of physical exertion during physical activity (decrease their perceived barriers), and to further highlight the multiple health and other benefits of regular exercising (increase their perceived benefits).


BMP4 promotes prostate tumor growth in bone through osteogenesis.
Induction of new bone formation is frequently seen in the bone lesions from prostate cancer. However, whether osteogenesis is necessary for prostate tumor growth in bone is unknown. Recently, 2 xenografts, MDA-PCa-118b and MDA-PCa-133, were generated from prostate cancer bone metastases. When implanted subcutaneously in severe combined immunodeficient (SCID) mice, MDA-PCa-118b induced strong ectopic bone formation while MDA-PCa-133 did not. To identify the factors that are involved in bone formation, we compared the expression of secreted factors (secretome) from MDA-PCa-118b and MDA-PCa-133 by cytokine array. We found that the osteogenic MDA-PCa-118b xenograft expressed higher levels of bone morphogenetic protein BMP4 and several cytokines including interleukin-8, growth-related protein (GRO), and CCL2. We showed that BMP4 secreted from MDA-PCa-118b contributed to about a third of the osteogenic differentiation seen in MDA-PCa-118b tumors. The conditioned media from MDA-PCa-118b induced a higher level of osteoblast differentiation, which was significantly reduced by treatment with BMP4 neutralizing antibody or the small molecule BMP receptor 1 inhibitor LDN-193189. BMP4 did not elicit an autocrine effect on MDA-PCa-118b, which expressed low to undetectable levels of BMP receptors. Treatment of SCID mice bearing MDA-PCa-118b tumors with LDN-193189 significantly reduced tumor growth. Thus, these studies support a role of BMP4-mediated osteogenesis in the progression of prostate cancer in bone.


Pheromone-Mediated Mate Location and Discrimination by Two Syntopic Sibling Species of Dendroctonus Bark Beetles in Chiapas, Mexico.
Where their geographic and host ranges overlap, sibling species of tree-killing bark beetles may simultaneously attack and reproduce on the same hosts. However, sustainability of these potentially mutually beneficial associations demands effective prezygotic reproductive isolation mechanisms between the interacting species. The pine bark beetle, Dendroctonus frontalis Zimmermann, is syntopic in the Central American region with a recently described sibling species, Dendroctonus mesoamericanus Armendáriz-Toledano and Sullivan, but mechanisms for their reproductive isolation are uncertain. We investigated whether semiochemicals mediate species discrimination by mate-seeking males of both species. In olfactometer bioassays, walking males of both species strongly preferred odors from gallery entrances of conspecific females. Coupled gas chromatography-electroantennographic detection and gas chromatography-mass spectrometry isolated 16 olfactory stimulants for males in these odors, but only two, ipsdienol and endo-brevicomin (both from D. mesoamericanus females), differed in quantity in female-associated odors between the species. In olfactometer bioassays, with 10, 1, or 0.1 female entrance equivalents of synthetic semiochemicals, the combination of ipsdienol and endo-brevicomin inhibited responses of male D. frontalis and enhanced responses of male D. mesoamericanus to two compounds associated with female entrances of both species (the pheromone component frontalin and host odor α-pinene). We conclude that ipsdienol and endo-brevicomin, pheromone components produced by females of just one of the two species (D. mesoamericanus), mediate interspecific mate discrimination by males of both species and provide an apparently symmetrical reproductive isolation mechanism.


In Vitro Anti-Cholinesterase and Antioxidant Activity of Extracts of Moringa oleifera Plants from Rivers State, Niger Delta, Nigeria.
This study evaluated Moringa oleifera extracts from two locations in Niger Delta for in vitro anti-cholinesterase and antioxidant activities. Methanolic, aqueous and ethanolic extracts of Moringa oleifera were evaluated for inhibition of acetylcholinesterase (AChE) activity, antioxidant properties, and total phenolic and flavonoid contents using standard procedures. M. oleifera extracts possessed significant and concentration dependent AChE inhibitory activity for methanolic, aqueous, and ethanolic extracts. For the most potent extracts, the percentage AChE inhibition/IC50 (µg/mL) values were Moringa oleifera root methanolic extracts (MORME): ~80%/0.00845; Moringa oleifera root ethanolic extract 1 (MOREE1): ~90%/0.0563; Moringa oleifera root ethanolic extract 2 (MOREE2): ~70%/0.00175; and Moringa oleifera bark ethanolic extract (MOBEE): ~70%/0.0173. The descending order of AChE inhibitory potency of plant parts were: root > bark > leaf > flowers > seed. All M. oleifera methanolic extracts at a concentration of 1000 µg/mL displayed significant (p < 0.05⁻0.001) DPPH radical scavenging activity, with values of ~20⁻50% of that of ascorbic acid. The total phenolic content and total flavonoid content (TPC/TFC) of MORME, Moringa Oju bark methanolic extract (MOBME), MOREE1, MOREE2 and Moringa leaf ethanolic leaf extract (MLEE) were (287/254), (212/113), (223/185), (203/343) and (201/102) mg gallic acid equivalents/g and quercetin equivalents/g, respectively. There was an inverse correlation between plant extract AChE inhibition and total phenolic (p < 0.0001) and total flavonoid contents (p < 0.0012). In summary, this study revealed 5 of 19 extracts of M. oleifera that have potent in vitro anti-cholinesterase and antioxidant activities.


Guideline for the diagnosis and treatment of celiac disease in children: recommendations of the North American Society for Pediatric Gastroenterology, Hepatology and Nutrition.
Celiac disease is an immune-mediated enteropathy caused by a permanent sensitivity to gluten in genetically susceptible individuals. It occurs in children and adolescents with gastrointestinal symptoms, dermatitis herpetiformis, dental enamel defects, osteoporosis, short stature, delayed puberty and persistent iron deficiency anemia and in asymptomatic individuals with type 1 diabetes, Down syndrome, Turner syndrome, Williams syndrome, selective immunoglobulin (Ig)A deficiency and first degree relatives of individuals with celiac disease. The Celiac Disease Guideline Committee of the North American Society for Pediatric Gastroenterology, Hepatology and Nutrition has formulated a clinical practice guideline for the diagnosis and treatment of pediatric celiac disease based on an integration of a systematic review of the medical literature combined with expert opinion. The Committee examined the indications for testing, the value of serological tests, human leukocyte antigen (HLA) typing and histopathology and the treatment and monitoring of children with celiac disease. It is recommended that children and adolescents with symptoms of celiac disease or an increased risk for celiac disease have a blood test for antibody to tissue transglutaminase (TTG), that those with an elevated TTG be referred to a pediatric gastroenterologist for an intestinal biopsy and that those with the characteristics of celiac disease on intestinal histopathology be treated with a strict gluten-free diet. This document represents the official recommendations of the North American Society for Pediatric Gastroenterology, Hepatology and Nutrition on the diagnosis and treatment of celiac disease in children and adolescents.


Effects of disc degeneration on neurophysiological responses during dorsoventral mechanical excitation of the ovine lumbar spine.
Mechanisms of spinal manipulation and mobilization include the elicitation of neuromuscular responses, but it is not clear how these responses are affected or altered by disc degeneration. We studied the neurophysiological responses of the normal and degenerated ovine spine subjected to mechanical excitation (varying force amplitude and duration) consistent with spinal manipulative therapy (SMT). Needle electromyographic (EMG) multifidus muscle activation adjacent to the L3 and L4 spinous processes and compound action potentials (CAPs) of the L4 nerve roots were measured during the application of dorsoventral mechanical excitation forces designed to mimic SMT force-time profiles used routinely in clinical practice. The magnitude and percentage of positive EMG responses increased with increasing SMT force magnitude, but not SMT pulse duration, whereas CAP responses were greatest for shorter duration pulses. Disc degeneration was associated with a reduction (20-25%) in positive EMG responses, and a concomitant increase (4.5-10.2%) in CAP responses.


Transcriptional analysis of left-sided colitis, pancolitis, and ulcerative colitis-associated dysplasia.
It is unknown why patients with extensive ulcerative colitis (UC) have a higher risk of colorectal cancer compared with patients with left-sided UC. This study characterizes the inflammatory processes in left-sided UC, pancolitis, and UC-associated dysplasia at the transcriptional level to identify potential biomarkers and transcripts of importance for the carcinogenic behavior of chronic inflammation. The Affymetrix GeneChip Human Genome U133 Plus 2.0 was applied on colonic biopsies from UC patients with left-sided UC, pancolitis, dysplasia, and controls. Reverse transcription polymerase chain reaction and immunohistochemistry were performed for validating selected transcripts in the initial cohort and in 2 independent cohorts of patients with UC. Microarray data were analyzed by principal component analysis, and reverse transcription polymerase chain reaction and immunohistochemistry data by the Wilcoxon's rank-sum test. The principal component analysis results revealed separate clusters for left-sided UC, pancolitis, dysplasia, and controls. Close clustering of dysplastic and pancolitic samples indicated similarities in gene expression. Indeed, 101 and 656 parallel upregulated and downregulated transcripts, respectively, were identified in specimens from dysplasia and pancolitis. Validation of selected transcripts hereof identified insulin receptor alpha (INSRA) and MAP kinase interacting serine/threonine kinase 2 (MKNK2) with an enhanced expression in dysplasia compared with left-sided UC and controls, whereas laminin γ2 (LAMC2) was found with a lower expression in dysplasia compared with the remaining 3 groups. This study demonstrates pancolitis and left-sided UC as distinct inflammatory processes at the transcriptional level, and identifies INSRA, MKNK2, and LAMC2 as potential critical transcripts in the inflammation-driven preneoplastic process of UC.


Taxol metabolism and disposition in cancer patients.
The objective of this study was to determine the metabolic fate and disposition of taxol in cancer patients. Five patients received 225 or 250 mg/m2 of taxol together with 100 microCi of [3H]taxol as a 3-hr infusion, followed by cisplatin and 5-fluorouracil. Urine, feces, and blood samples were collected for 120 hr and analyzed for total radioactivity, taxol, and metabolites by reversed-phase HPLC and tandem MS. Total urinary excretion was 14.3 +/- 1.4% (SE) of the dose, with unchanged taxol and an unknown polar metabolite as the main excretion products. Total fecal excretion was 71.1 +/- 8.2%, with 6 alpha-hydroxytaxol being the largest component by far. Unchanged taxol and four other metabolites could also be identified from fecal extracts. The plasma area under the curve for unchanged taxol was 20.5 +/- 2.3 microM.hr and that for total taxol metabolites was 14.2 +/- 4.5 microM.hr. The half-life of total metabolites (5.6 +/- 0.4 hr), however, greatly exceeded that of unchanged taxol (2.9 +/- 0.3 hr). Thus, at 5-hr posttaxol infusion, the plasma concentrations of the five metabolites together exceeded the taxol concentration by 2.4-fold. The findings from this study should be of importance as a guide to further therapeutic evaluation of this drug.


Linezolid for the treatment of methicillin-resistant Staphylococcus aureus infections in children.
Infections caused by methicillin-resistant Staphylococcus aureus (MRSA) are becoming increasingly prevalent. Linezolid is effective and well-tolerated in the treatment of adults with MRSA infections. To evaluate the clinical efficacy and safety of iv/oral linezolid in children with MRSA infections. Data were obtained from two independent clinical trials. In an outpatient trial children (5 to 17 years of age) with uncomplicated skin and skin structure infections (SSSIs) were treated with linezolid or cefadroxil. In an inpatient trial hospitalized children (0 to 11 years of age) with pneumonia, bacteremia or complicated SSSI caused by resistant Gram-positive pathogens were administered iv linezolid with the option to switch to oral suspension (patients >90 days of age) or iv vancomycin. A subset of patients with MRSA infections from the two clinical trials is analyzed herein. In the outpatient trial children with skin infections caused by MRSA were treated with linezolid (15 patients) and cefadroxil (10 patients). In the microbiologically evaluable population, the clinical cure rate was 92.3% in the linezolid group and 85.7% in the cefadroxil group (P = 0.64). The pathogen eradication rate for MRSA was 92.3 and 85.7% in the linezolid and cefadroxil groups, respectively (P = 0.64). There were very few adverse events or drug-related adverse events and no serious adverse events in the outpatient trial. In the inpatient trial 20 children treated with linezolid and 14 treated with vancomycin had infections caused by MRSA. In the microbiologically evaluable population, the clinical cure rate was 94.1% in the linezolid group and 90.0% in the vancomycin group (P = 0.69). Pathogen eradication rates were 88.2 and 90.0% for the linezolid and vancomycin groups, respectively (P = 0.89). Susceptibility patterns of the MRSA isolates showed distinct patterns between the outpatient and inpatient trials. In the inpatient trial fewer patients in the linezolid group had drug-related adverse events than did those in the vancomycin group (20% vs. 43%; P = 0.15). Intravenous/oral linezolid is effective and well-tolerated in children with MRSA infections.


Remodeling of the notochord during development of vertebral fusions in Atlantic salmon (Salmo salar).
Histological characterization of spinal fusions in Atlantic salmon (Salmo salar) has demonstrated shape alterations of vertebral body endplates, a reduced intervertebral space, and replacement of intervertebral cells by ectopic bone. However, the significance of the notochord during the fusion process has not been addressed. We have therefore investigated structural and cellular events in the notochord during the development of vertebral fusions. In order to induce vertebral fusions, Atlantic salmon were exposed to elevated temperatures from fertilization until they attained a size of 15g. Based on results from radiography, intermediate and terminal stages of the fusion process were investigated by immunohistochemistry and real-time quantitative polymerase chain reaction. Examination of structural extracellular matrix proteins such as Perlecan, Aggrecan, Elastin, and Laminin revealed reduced activity and reorganization at early stages in the pathology. Staining for elastic fibers visualized a thinner elastic membrane surrounding the notochord of developing fusions, and immunohistochemistry for Perlecan showed that the notochordal sheath was stretched during fusion. These findings in the outer notochord correlated with the loss of Aggrecan- and Substance-P-positive signals and the further loss of vacuoles from the chordocytes in the central notochord. At more progressed stages of fusion, chordocytes condensed, and the expression of Aggrecan and Substance P reappeared. The hyperdense regions seem to be of importance for the formation of notochordal tissue into bone. Thus, the remodeling of notochord integrity by reduced elasticity, structural alterations, and cellular changes is probably involved in the development of vertebral fusions.


Amino acid-mediated regulation of spontaneous synaptic activity patterns in the rat basolateral amygdala.
1. Spontaneous postsynaptic currents (PSCs) were examined in the basolateral amygdala using whole cell patch-clamp recordings in coronal slices (400 microns) from young rats (postnatal day 6-25). In most cells, Cs+ was used in the electrode to block putative voltage-activated K(+)-currents. Both inward and outward spontaneous PSCs were examined. 2. The alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate receptor antagonist, 6,7-nitroquinoxaline-2,3-dione (DNQX) blocked all inward PSCs, which reversed near 0 mV. They therefore were considered to be glutamate-mediated excitatory postsynaptic currents (EPSCs). Averaged EPSCs had a rapid 10-90% rise time (1.0 +/- 0.04 ms; mean +/- SD) and monoexponential decay (tau = 3.6 +/- 0.18 ms) at potentials negative to about -50 mV. Above this potential, a second, slower time constant (tau 1 = 41 +/- 4.5 ms at -30 mV), accounting for 10-30% of the total EPSC amplitude was resolved in 8 of 10 cells examined. The slower decay time constant was sensitive to the N-methyl-D-aspartate (NMDA)-receptor antagonist, DL-2-amino-5-phosphonovaleric acid (AP5) and therefore probably was due to activation of NMDA receptors. 3. The gamma-aminobutyric acid-A (GABAA) antagonist, bicuculline, blocked all outward PSCs, which reversed near -70 mV. They therefore were considered to be GABA-mediated inhibitory postsynaptic currents (IPSCs). Averaged IPSCs displayed rapid 10-90% rise times (1.0 +/- 0.03 ms) and monoexponential decay time constants (tau = 5.16 +/- 0.14 ms). 4. Tetrodotoxin (TTX) reduced the frequency of synaptic activity and eliminated the largest PSCs, thus reducing slightly the mean EPSC and IPSC amplitude. Most cells received bursts of spontaneous IPSCs and/or EPSCs (30-68 Hz lasting 0.5-6 s), which were also TTX sensitive. The TTX data suggest that the somata of the cells responsible for the largest PSCs and the PSC bursts were contained within the slice. 5. In addition to blocking EPSCs, DNQX blocked the bursts of IPSCs, but not all individual IPSCs. DNQX had similar effects as TTX on the bursts and frequency of the IPSCs. 6. Bicuculline enhanced spontaneous EPSC frequency (231 +/- 90%). Much of this increase was due to an increase in the bursts of EPSCs. 7. Neurons in the basolateral amygdala therefore appear to receive both excitatory (glutamatergic) and inhibitory (GABAergic) synaptic input from local neurons. The activity of the neurons responsible for these inputs are themselves largely regulated by glutamatergic and GABAergic inputs. The relevance of this local circuitry to seizures and epilepsy is discussed briefly.


Yeast DNA polymerase zeta is an efficient extender of primer ends opposite from 7,8-dihydro-8-Oxoguanine and O6-methylguanine.
Genetic studies in Saccharomyces cerevisiae have indicated the requirement of DNA polymerase (Pol) zeta for mutagenesis induced by UV light and by other DNA damaging agents. However, on its own, Pol zeta is highly inefficient at replicating through DNA lesions; rather, it promotes their mutagenic bypass by extending from the nucleotide inserted opposite the lesion by another DNA polymerase. So far, such a role for Pol zeta has been established for cyclobutane pyrimidine dimers, (6-4) dipyrimidine photoproducts, and abasic sites. Here, we examine whether Pol zeta can replicate through the 7,8-dihydro-8-oxoguanine (8-oxoG) and O(6)-methylguanine (m6G) lesions. We chose these two lesions for this study because the replicative polymerase, Pol delta, can replicate through them, albeit weakly. We found that Pol zeta is very inefficient at inserting nucleotides opposite both these lesions, but it can efficiently extend from the nucleotides inserted opposite them by Pol delta. Also, the most efficient bypass of 8-oxoG and m6G lesions occurs when Pol delta is combined with Pol zeta, indicating a role for Polzeta in extending from the nucleotides inserted opposite these lesions by Pol delta. Thus, Pol zeta is a highly specialized polymerase that can proficiently extend from the primer ends opposite DNA lesions, irrespective of their degree of geometric distortion. Pol zeta, however, is unusually sensitive to geometric distortion of the templating residue, as it is highly inefficient at incorporating nucleotides even opposite the moderately distorting 8-oxoG and m6G lesions.


Bidirectional transendothelial migration of monocytes across hepatic sinusoidal endothelium shapes monocyte differentiation and regulates the balance between immunity and tolerance in liver.
Monocytes are versatile cells that can fulfill proinflammatory and anti-inflammatory functions when recruited to the liver. Recruited monocytes differentiate into tissue macrophages and dendritic cells, which sample antigens and migrate to lymph nodes to elicit T-cell responses. The signals that determine monocyte differentiation and the role of hepatic sinusoidal endothelial cells (HSECs) in this process are poorly understood. HSECs are known to modulate T-cell activation, which led us to investigate whether transendothelial migration of monocytes across HSECs influences their phenotype and function. Subsets of blood-derived monocytes were allowed to transmigrate across human HSECs into a collagen matrix. Most migrated cells remained in the subendothelial matrix, but ~10% underwent spontaneous basal to apical transendothelial migration. The maturation, cytokine secretion, and T-cell stimulatory capacity of reverse transmigrating (RT) and subendothelial (SE) monocytes were compared. SE monocytes were mainly CD16(-) , whereas 75%-80% of RT monocytes were CD16(+) . SE monocytes derived from the CD14(++) CD16(-) subset and exhibited high phagocytic activity, whereas RT monocytes originated from CD14(++) CD16(+) and CD14(+) CD16(++) monocytes, displayed an immature dendritic cell-like phenotype (CD11c(pos) HLA-DR(pos) CD80lo CD86lo ), and expressed higher levels of chemokine (C-C motif) receptor 8. Consistent with a dendritic cell phenotype, RT monocytes secreted inflammatory cytokines and induced antigen-specific CD4(+) T-cell activation. In contrast, SE monocytes suppressed T-cell proliferation and activation and exhibited endotoxin tolerance. Transcriptome analysis underscored the functional differences between SE and RT monocytes. Migration across HSECs shapes the subsequent fate of monocytes, giving rise to anergic macrophage-like cells in tissue and the release of immunocompetent pre-dendritic cells into the circulation.


Synaptic protein synthesis associated with memory is regulated by the RISC pathway in Drosophila.
Long-lasting forms of memory require protein synthesis, but how the pattern of synthesis is related to the storage of a memory has not been determined. Here we show that neural activity directs the mRNA of the Drosophila Ca(2+), Calcium/Calmodulin-dependent Kinase II (CaMKII), to postsynaptic sites, where it is rapidly translated. These features of CaMKII synthesis are recapitulated during the induction of a long-term memory and produce patterns of local protein synthesis specific to the memory. We show that mRNA transport and synaptic protein synthesis are regulated by components of the RISC pathway, including the SDE3 helicase Armitage, which is specifically required for long-lasting memory. Armitage is localized to synapses and lost in a memory-specific pattern that is inversely related to the pattern of synaptic protein synthesis. Therefore, we propose that degradative control of the RISC pathway underlies the pattern of synaptic protein synthesis associated with a stable memory.


Clinical risk assessment of organ manifestations in systemic sclerosis: a report from the EULAR Scleroderma Trials And Research group database.
Systemic sclerosis (SSc) is a multisystem autoimmune disease, which is classified into a diffuse cutaneous (dcSSc) and a limited cutaneous (lcSSc) subset according to the skin involvement. In order to better understand the vascular, immunological and fibrotic processes of SSc and to guide its treatment, the EULAR Scleroderma Trials And Research (EUSTAR) group was formed in June 2004. EUSTAR collects prospectively the Minimal Essential Data Set (MEDS) on all sequential patients fulfilling the American College of Rheumatology diagnostic criteria in participating centres. We aimed to characterise demographic, clinical and laboratory characteristics of disease presentation in SSc and analysed EUSTAR baseline visits. In April 2006, a total of 3656 patients (1349 with dcSSc and 2101 with lcSSc) were enrolled in 102 centres and 30 countries. 1330 individuals had autoantibodies against Scl70 and 1106 against anticentromere antibodies. 87% of patients were women. On multivariate analysis, scleroderma subsets (dcSSc vs lcSSc), antibody status and age at onset of Raynaud's phenomenon, but not gender, were found to be independently associated with the prevalence of organ manifestations. Autoantibody status in this analysis was more closely associated with clinical manifestations than were SSc subsets. dcSSc and lcSSc subsets are associated with particular organ manifestations, but in this analysis the clinical distinction seemed to be superseded by an antibody-based classification in predicting some scleroderma complications. The EUSTAR MEDS database facilitates the analysis of clinical patterns in SSc, and contributes to the standardised assessment and monitoring of SSc internationally.


Aromatherapy positively affects mood, EEG patterns of alertness and math computations.
EEG activity, alertness, and mood were assessed in 40 adults given 3 minutes of aromatherapy using two aromas, lavender (considered a relaxing odor) or rosemary (considered a stimulating odor). Participants were also given simple math computations before and after the therapy. The lavender group showed increased beta power, suggesting increased drowsiness, they had less depressed mood (POMS) and reported feeling more relaxed and performed the math computations faster and more accurately following aromatherapy. The rosemary group, on the other hand, showed decreased frontal alpha and beta power, suggesting increased alertness. They also had lower state anxiety scores, reported feeling more relaxed and alert and they were only faster, not more accurate, at completing the math computations after the aromatherapy session.


An empirical comparison of methods used to estimate carcinogenic potency in long-term animal bioassays: lifetable vs summary incidence data.
Two methods for estimating carcinogenic potency from animal carcinogenesis bioassays (TD50-defined in the paper) are compared, one based on lifetable data and one based on summary incidence data. The lifetable analysis adjusts for the differential effects of toxicity among dose groups and for differences in the time pattern of tumor incidence, while summary incidence analysis does not. However, summary data are all that are usually available in the published results of animal cancer tests. Using NCI bioassay results which provide full lifetable data, we compare lifetable and summary estimates of potency and their statistical significance as well as the estimated shape of the dose-response curve. There is substantial agreement between these methods of analysis in terms of potency estimation, although lifetable estimates are usually more potent. But, there are some notable differences in the estimated shape of the dose-response curve, suggesting that both target site selection and method of analysis play an important role in risk estimation.


Prevalence and significance of coagulase-negative staphylococci isolated from blood cultures in a tertiary hospital.
Blood cultures (BC) are the most important tool in the diagnosis of bloodstream infections. However, false positive results are associated with increased laboratory costs and inappropriate antibiotic use. In order to determine the prevalence and location of blood cultures contaminated with coagulase-negative staphylococci (CNS), we performed a retrospective analysis of all blood cultures performed at St. Vincent's Hospital, Sydney during a 6-month period. From a total of 4234 patients with BC collected, CNS was isolated from 109 patients (2.6%). 94% of all CNS isolates (101/109) were contaminants. In the emergency department (ED), CNS isolates were significantly more likely to be contaminants (62/63, p<0.02) compared with the rest of the hospital, representing a 3.3% patient BC contamination rate. Treatment for a contaminant with vancomycin was significantly more likely to occur in ward patients (14/28, p<0.01) compared to the rest of the hospital. Duration of therapy did not differ across the hospital. Strategies to reduce the numbers of contaminants should be directed at medical staff in ED. Inappropriate vancomycin therapy could be curtailed by greater clinical microbiology liaison and vancomycin stewardship.


Older people living at home: associations between falls and health complaints in men and women.
To investigate the associations between self-reported falls and health complaints, among persons aged 75 years and older living at home, and to investigate gender differences in the associations. There are several studies concerned with risk factors for falling, and others related to health complaints, but not many with associations between falls and health complaints. There are some inconsistent data of incidence and gender-related differences in falling. Case-control community-based study. In total, 1243 persons living in two municipal districts in Sweden answered a questionnaire. Odds ratios (OR) and regression models with 95% confidence interval (CI) were used to determine the associations between self-reported falls and different health complaints. The adjusted (multivariate) linear regression showed that urinary incontinence, self-rated health and tiredness were significantly associated with falls for both men and women living at home. The gender-related differences in falling were associated with the variables such as self-rated health for men and tiredness and pain in the hands, elbows, legs or knees for women. An association is evident between falls and urinary incontinence, poor self-rated health and tiredness for older persons living at home. Gender differences in falls show an association with poor self-rated health, tiredness and pain in the hands, elbows, legs or knees. As older people are expected to live in their own homes as long as possible, more knowledge is required about what determines the risk of falling. Nurses in community care are recommended to use assessment tools that include urinary incontinence in order to detect the risk of falling.


Familial evaluation for diagnosis of arrhythmogenic right ventricular dysplasia.
Most sudden cardiac deaths in young athletes are caused by previously undetected inherited cardiac diseases. Here, we report a case of a young male athlete in whom a presumptive diagnosis of hypertrophic cardiomyopathy (HCM) was made following a near sudden cardiac death. Although his imaging studies initially suggested HCM, a detailed clinical and genetic evaluation of the patient and his asymptomatic father led to the diagnosis of arrhythmogenic right ventricular cardiomyopathy/dysplasia (ARVD) in both. DNA sequencing revealed that each individual was heterozygous for two rare variants in the PKP2 and DSC2 genes, both of which were previously shown to be associated with ARVD and to encode desmosomal proteins, i.e. the previously reported splicing variant c2489 + 1A > G in the PKP2 gene and the novel p.I109M variant in the DSC2 gene. Imaging and electrophysiologic studies further supported a diagnosis of ARVD in the father. This case highlights the importance of detailed clinical evaluation and genetic testing of family members when dealing with sudden cardiac death or unexplained cardiomyopathies in the young.


Technical note: detection and adjustment of abnormal test-day yields.
A method to detect and to adjust or exclude abnormally low or high milk, fat, and protein yields on test-day (TD) was developed. Predicted TD yield is calculated based on preceding and subsequent (if available) TD yields. Observed TD yields that are < 60% or > 150% of predicted TD yield are defined as abnormal. Most abnormal yields are adjusted to this floor or ceiling, but some are excluded. Yields of < 4.5 kg that are identified as from a cow that was sick or that are less than half the mean of adjacent tests are excluded as are yields of > 59 kg above predicted yield. Lactation yields are calculated from the restricted TD yields. When this procedure was applied to 2002 data, 1.8% of milk, 2.4% of fat, and 1.6% of protein yields on TD were below the acceptance range and 0.1% of milk and protein and 0.8% of fat were above. Predicted TD yield was calculated as preceding TD yield plus preceding test interval multiplied by daily yield change (slope) based on days in milk (DIM), DIM2, previous normal TD yield, and interaction between DIM and previous TD yield. To accommodate changes in slope at peak yield, separate coefficients were estimated for < 50 and > or = 50 DIM. Herd mean was used when only one TD was recorded for a cow (or when two were recorded and the second was designated as abnormal based on the first) and to determine an acceptable range for component percentages. Predicted TD yield for first TD was based on subsequent rather than previous normal TD. To test the adjustments, lactation records with one abnormal TD yield or more were matched with subsequent lactation records. Correlation between consecutive lactations increased from 0.692 to 0.693 for milk (561,063 lactation pairs), from 0.653 to 0.660 for fat (951,387 lactation pairs), and from 0.686 to 0.694 for protein (488,653 lactation pairs). Outlier adjustment improved the correlation between consecutive lactation yields and is applied routinely to TD records of cows for calvings since 1997.


Allelic loss of p53 gene is associated with genesis and maintenance, but not invasion, of mouse carcinoma in situ of the bladder.
Carcinoma in situ (CIS) of the bladder has recently been proposed to be a heterogeneous group of diseases with varied histogenesis and biological behavior. In this study, we describe the sequential steps of CIS development and progression in a transgenic mouse model expressing low levels of the SV40 large T antigen. We found that CIS in transgenic mice arose from urothelial dysplasia, that CIS could persist for an extended period of time without invasion, and that the majority of CIS eventually evolved into high-grade, superficial, papillary tumors before a small fraction of them advanced to invasion/metastasis. A genome-wide search of chromosomal imbalances by comparative genomic hybridization revealed that 9 of 11 (82%) of CIS had losses on chromosome 11. Southern blotting demonstrated the allelic loss of the p53 gene, which resides on mouse chromosome 11, in four comparative genomic hybridization-tested tumors and 10 of 11 (91%) additional CIS examined. Consistent with the reduced p53 gene dosage because of the allelic loss and the functional inactivation of p53 protein of the remaining allele by SV40T antigen, there was a dramatic decrease in CIS of Mdm-2, a major p53 target. In contrast, the level of p21, another p53 target, was largely unaltered, suggesting that p21 expression can be regulated by p53-independent mechanisms. These results delineate the early stages of bladder tumorigenesis and suggest that the loss of a p53-bearing chromosome is an early event in bladder tumorigenesis and is crucial for the genesis and the maintenance, but not the progression, of bladder CIS. On the basis of our current and previous transgenic studies, we have proposed an integrated pathway progression model of bladder cancer.


The EULAR Outcome Measures Library: development and an example from a systematic review for systemic lupus erythematous instruments.
Patient reported outcomes (PROs) are relevant in rheumatology. Variable accessibility and validity of commonly used PROs are obstacles to homogeneity in evidence synthesis. The objective of this project was to provide a comprehensive library of "validated PROs". A launch meeting with rheumatologists, PROs methodological experts, and patients, was held to define the library's aims and scope, and basic requirements. To feed the library we performed systematic reviews on selected diseases and domains. Relevant information on PROs was collected using standardised data collection forms based on the COSMIN checklist. The EULAR Outcomes Measures Library (OML), whose aims are to provide and to advise on PROs on a user-friendly manner albeit based on scientific grounds, has been launched and made accessible to all. PROs currently included cover any domain and, are generic or specifically target to the following diseases: rheumatoid arthritis, osteoarthritis, spondyloarthritis, low back pain, systemic lupus erythematosus, gout, osteoporosis, juvenile idiopathic arthritis, and fibromyalgia. Up to 236 instruments (106 generic and 130 specific) have been identified, evaluated, and included. The systematic review for SLE, which yielded 10 specific instruments, is presented here as an example. The OML website includes, for each PRO, information on the construct being measured and the extent of validation, recommendations for use, and available versions; it also contains a glossary on common validation terms. The OML is an in progress library led by rheumatologists, related professionals and patients, that will help to better understand and apply PROs in rheumatic and musculoskeletal diseases.


When health policy and empirical evidence collide: the case of cigarette package warning labels and economic consumer surplus.
In its graphic warning label regulations on cigarette packages, the Food and Drug Administration severely discounts the benefits of reduced smoking because of the lost "pleasure" smokers experience when they stop smoking; this is quantified as lost "consumer surplus." Consumer surplus is grounded in rational choice theory. However, empirical evidence from psychological cognitive science and behavioral economics demonstrates that the assumptions of rational choice are inconsistent with complex multidimensional decisions, particularly smoking. Rational choice does not account for the roles of emotions, misperceptions, optimistic bias, regret, and cognitive inefficiency that are germane to smoking, particularly because most smokers begin smoking in their youth. Continued application of a consumer surplus discount will undermine sensible policies to reduce tobacco use and other policies to promote public health.


Structural analysis of histone deacetylase 8 mutants associated with Cornelia de Lange Syndrome spectrum disorders.
Cornelia de Lange Syndrome (CdLS) and associated spectrum disorders are characterized by one or more congenital anomalies including distinctive facial features, upper limb abnormalities, intellectual disability, and other symptoms. The molecular genetic basis of CdLS is linked to defects in cohesin, a protein complex that functions in sister chromatid cohesion, chromatin organization, and transcriptional regulation. Histone deacetylase 8 (HDAC8) plays an important role in cohesin function by catalyzing the deacetylation of SMC3, which is required for efficient recycling of the cohesin complex. Missense mutations in HDAC8 have been identified in children diagnosed with CdLS spectrum disorders, and here we outline structure-function relationships for four of these mutations. Specifically, we report the 1.50 Å-resolution structure of the I45T HDAC8-suberoylanilide hydroxamic acid complex, the 1.84 Å-resolution structure of E66D/Y306F HDAC8 complexed with a peptide assay substrate, and the 2.40 Å-resolution structure of G320R HDAC8 complexed with the inhibitor M344. Additionally, we present a computationally generated model of D176G HDAC8. These structures illuminate new structure-function relationships for HDAC8 and highlight the importance of long-range interactions in the protein scaffold that can influence catalytic function.


Distinct motifs in the chemokine receptor CCR7 regulate signal transduction, receptor trafficking and chemotaxis.
The chemokine receptor CCR7, together with its ligands CCL19 and CCL21, is responsible for the correct homing and trafficking of dendritic cells and lymphocytes to secondary lymphoid tissues. Moreover, cancer cells can utilize CCR7 to metastasize to draining lymph nodes. However, information on CCR7 signaling leading to cell migration or receptor trafficking is sparse. Using novel CCR7 deletion mutants with successive truncations of the intracellular C-terminus and a mutant with impaired G-protein coupling, we identified distinct motifs responsible for various aspects of CCR7 signal transduction. Deleting a Ser/Thr motif at the tip of the intracellular tail of CCR7 resulted in an impaired chemokine-mediated activation of Erk1/2 kinases. Interestingly, deleting an additional adjacent motif restored the ability of CCL19-mediated Erk1/2 phosphorylation, suggesting the presence of a regulatory motif. Both the Ser/Thr and the regulatory motif are dispensable for signaling events leading to cell migration and receptor trafficking. A CCR7 mutant lacking virtually the complete C-terminus readily bound CCL19 and was internalized, but was unable to activate the G protein and to transmit signals required for cell migration, mobilization of [Ca2+](i) and Erk1/2 activation. Finally, G-protein coupling was critical for [Ca2+](i) mobilization, Erk1/2 phosphorylation and chemotaxis, but not for CCR7 trafficking.


Decompression of the superficial peroneal nerve: clinical outcomes and anatomical study.
OBJECTIVE The authors of this study sought to determine the outcomes of patients undergoing superficial peroneal nerve (SPN) release to treat lower-extremity pain and describe consistent anatomical landmarks to direct surgical planning. METHODS This retrospective cohort study examined 54 patients with pain in the SPN distribution who were treated with decompression between 2011 and 2014. Patients rated pain and the effect of pain on quality of life (QOL) on the visual analog scale (VAS) from 0 to 10. Scores were then converted to percentages. Linear regression analysis was performed to assess the impact of the preoperative effect of pain on QOL, age, body mass index (BMI), and preoperative duration of pain on the postoperative effect of pain on QOL. Measurements were made intraoperatively in 13 patients to determine the landmarks for identifying the SPN. RESULTS A higher BMI was a negative predictor for improvement in the effect of pain on QOL. A decrease in pain compared with the initial level of pain suggested a nonlinear relationship between these variables. A minority of patients (7 of 16) with a preoperative pain VAS score ≤ 60 reported less pain after surgery. A large majority (30 of 36 patients) of those with a preoperative pain VAS score > 60 reported improvement. Intraoperative measurements demonstrated that the SPN was consistently found to be 5 ± 1.1, 5 ± 1.1, and 6 ± 1.2 cm lateral to the tibia at 10, 15, and 20 cm proximal to the lateral malleolus, respectively. CONCLUSIONS A majority of patients with a preoperative pain VAS score > 60 showed a decrease in postoperative pain. A higher BMI was associated with less improvement in the effect of pain on QOL. This information can be useful when counseling patients on treatment options. Based on the intraoperative data, the authors found that the SPN can be located at reliable points in reference to the tibia and lateral malleolus.


Cultured mast cells from patients with asthma and controls respond with similar sensitivity to recombinant Der p2-induced, IgE-mediated activation.
The function of cultured mast cells may depend on genetic or environmental influence on the stem cell donor. This study investigates whether asthma or atopy in the donor influenced the growth and sensitivity of mast cells cultured from patients with asthma and healthy controls under identical conditions. Mast cells were cultured from peripheral blood from twelve patients with an objectively confirmed asthma diagnosis and eight healthy subjects. During the last 2 weeks of culture, mast cells were incubated with IL-4 and 80 kU/l recombinant human IgE containing two clones (7% + 7%) specific for mite allergen Der p2. The sensitivity of IgE-mediated activation of mast cells was investigated as FcεRI-mediated upregulation of CD63. Ten subjects were atopic, defined as a positive skin prick test (>3 mm) to at least one of ten common allergens. After activation with recombinant Der p2, the maximum CD63 median fluorescence intensity was 20 456 ± 1640 (SE) for patients with asthma and 22,275 ± 1971 (SE) for controls (ns). The fraction of CD63 positive cells was 54.4% in patients with asthma and 48.4% in controls (ns). The allergen concentration inducing 50% of the maximal CD63 response was similar in patients with asthma [-0.4795 log ng/ml ± 0.092 (SE)] and controls (-0.6351 log ng/ml ± 0.083, ns) and in atopic and non-atopic subjects. When cultured, sensitized and activated under identical conditions, mast cells from allergic asthmatics and healthy controls respond similar. Activation of cultured mast cells appears to depend on culture conditions (IL-4, IgE) rather than on donor status as atopy and asthma.


A private ultraviolet channel in visual communication.
Although private communication is considered an important diversifying force in evolution, there is little direct behavioural evidence to support this notion. Here, we show that ultraviolet (UV) signalling in northern swordtails (Xiphophorus) affords a channel for communication that is not accessible to their major predator, Astyanax mexicanus, the Mexican tetra. Laboratory and field behavioural experiments with swordtails (X. nigrensis) and predators (A. mexicanus) demonstrate that male UV ornamentation significantly increases their attractiveness to females but not to this predator, which is less sensitive to UV. UV reflectance among swordtail species correlates positively with tetra densities across habitats, and visual contrast estimates suggest that UV signals are highly conspicuous to swordtails in their natural environment. Cross-species comparisons also support the hypothesis that natural selection drives the use of UV communication. We compared two species, one with high (X. nigrensis) and one with low (X. malinche) Mexican tetra densities. Xiphophorus nigrensis males reflect significantly more UV than X. malinche, exhibit significant UV sexual dimorphism, and UV is a salient component of the sexual communication system. In X. malinche, however, males reflect minimally in the UV, there is no UV sexual dimorphism, and UV does not play a part in its communication system.


Hyperdense middle cerebral arteries identified on CT as a false sign of vascular occlusion.
To evaluate the middle cerebral artery (MCA) on CT, including its relationship to cerebral infarction. Thirteen patients with either a unilateral or bilateral hyperdense M1 segment of the MCA were evaluated. History of hypertension, diabetes, and hematocrit were obtained and compared with a control group of patients without a hyperdense MCA. None of the patients had a unilateral hyperdense MCA ipsilateral to a clinically identifiable stroke. Patients with a hyperdense MCA had a statistically higher hematocrit and also a higher prevalence of hypertension and diabetes mellitus than patients without a hyperdense MCA. The higher hematocrit may have increased the density of the blood, while both diabetes and hypertension are associated with calcification within blood vessel walls. A hyperdense MCA is not a reliable indicator of occlusion of this vessel or subsequent infarction.


Molecular insights into an ancient form of Paget's disease of bone.
Paget's disease of bone (PDB) is a chronic skeletal disorder that can affect one or several bones in individuals older than 55 y of age. PDB-like changes have been reported in archaeological remains as old as Roman, although accurate diagnosis and natural history of the disease is lacking. Six skeletons from a collection of 130 excavated at Norton Priory in the North West of England, which dates to medieval times, show atypical and extensive pathological changes resembling contemporary PDB affecting as many as 75% of individual skeletons. Disease prevalence in the remaining collection is high, at least 16% of adults, with age at death estimations as low as 35 y. Despite these atypical features, paleoproteomic analysis identified sequestosome 1 (SQSTM1) or p62, a protein central to the pathological milieu of PDB, as one of the few noncollagenous human sequences preserved in skeletal samples. Targeted proteomic analysis detected >60% of the ancient p62 primary sequence, with Western blotting indicating p62 abnormalities, including in dentition. Direct sequencing of ancient DNA excluded contemporary PDB-associated SQSTM1 mutations. Our observations indicate that the ancient p62 protein is likely modified within its C-terminal ubiquitin-associated domain. Ancient miRNAs were remarkably preserved in an osteosarcoma from a skeleton with extensive disease, with miR-16 expression consistent with that reported in contemporary PDB-associated bone tumors. Our work displays the use of proteomics to inform diagnosis of ancient diseases such as atypical PDB, which has unusual features presumably potentiated by yet-unidentified environmental or genetic factors.


Venlafaxine extended release in posttraumatic stress disorder: a sertraline- and placebo-controlled study.
This 12-week, double-blind, multicenter trial evaluated the efficacy of venlafaxine extended release (ER), sertraline, and placebo in adult outpatients (N = 538) with a primary diagnosis of posttraumatic stress disorder (PTSD), as defined in the Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition, symptoms for 6 months or more and 17-item Clinician-administered PTSD Scale (CAPS-SX17) score of 60 or more. Patients were randomly assigned to receive placebo or flexible doses of venlafaxine ER (37.5-300 mg/d) or sertraline (25-200 mg/d) for 12 weeks or less. The primary outcome was the baseline-to-end point change in total CAPS-SX17 score (last observation carried forward). Secondary measures included CAPS-SX17 symptom cluster scores for reexperiencing/intrusion, avoidance/numbing, and hyperarousal; frequency of remission (CAPS-SX17 < or =20); and changes in Davidson Trauma Scale total score and symptom cluster scores for avoidance/numbing, hyperarousal, and reexperiencing/intrusion. Mean changes in CAPS-SX17 scores were -41.8, -39.4, and -33.9 for venlafaxine ER (P < 0.05 vs. placebo), sertraline, and placebo, respectively. Mean changes for venlafaxine ER, sertraline, and placebo in CAPS-SX17 cluster scores were -13.0, -11.7, and -11.0 for reexperiencing; -17.1, -16.8, and -13.7 (P < 0.05 both active treatments vs. placebo) for avoidance/numbing; and -11.8, -10.9, and -9.2 (P < 0.05 venlafaxine vs. placebo) for hyperarousal. Week 12 remission rates were venlafaxine ER 30.2% (P < 0.05 vs. placebo), sertraline 24.3%, and placebo 19.6%. The venlafaxine ER group had significantly better Davidson Trauma Scale total and cluster scores than placebo. Mean maximum daily doses were 225-mg venlafaxine ER and 151-mg sertraline. Both treatments were generally well tolerated. Study results suggest that venlafaxine ER is effective and well tolerated in the short-term treatment of PTSD.


Checkpoint Proteins in Pediatric Brain and Extracranial Solid Tumors: Opportunities for Immunotherapy.
Pediatric brain and extracranial solid tumors are a diverse group of malignancies that represent almost half of all pediatric cancers. Standard therapy includes various combinations of surgery, cytotoxic chemotherapy, and radiation, which can be very harmful to a developing child, and survivors carry a substantial burden of long-term morbidities. Although these therapies have improved survival rates for children with solid tumors, outcomes still remain extremely poor for subsets of patients. Recently, immunosuppressive checkpoint molecules that negatively regulate immune cell function have been described. When found on malignant cells or in the tumor microenvironment, they contribute to immune evasion and tumor escape. Agents designed to inhibit these proteins have demonstrated significant efficacy in human adult solid tumor studies. However, there is limited research focusing on immune checkpoint molecules and inhibitors in pediatric solid tumors. In this review, we examine the current knowledge on immune checkpoint proteins with an emphasis on cytotoxic T lymphocyte antigen-4 (CTLA-4); programmed cell death protein-1 (PD-1) and programmed death-ligand 1 (PD-L1); OX-2 membrane glycoprotein (CD200); and indoleamine 2,3-dioxygenase (IDO). We review T-cell signaling, the mechanisms of action of these checkpoint molecules, pediatric preclinical studies on checkpoint proteins and checkpoint blockade, pediatric checkpoint inhibitor clinical trials conducted to date, and future immunotherapy opportunities for childhood cancers. Clin Cancer Res; 23(2); 342-50. ©2016 AACR.


Structure of MurNAc 6-phosphate hydrolase (MurQ) from Haemophilus influenzae with a bound inhibitor.
The breakdown and recycling of peptidoglycan, an essential polymeric cell structure, occur in a number of bacterial species. A key enzyme in the recycling pathway of one of the components of the peptidoglycan layer, N-acetylmuramic acid (MurNAc), is MurNAc 6-phosphate hydrolase (MurQ). This enzyme catalyzes the cofactor-independent cleavage of a relatively nonlabile ether bond and presents an interesting target for mechanistic studies. Open chain product and substrate analogues were synthesized and tested as competitive inhibitors (K(is) values of 1.1 ± 0.3 and 0.23 ± 0.02 mM, respectively) of the MurNAc 6P hydrolase from Escherichia coli (MurQ-EC). To identify the roles of active site residues that are important for catalysis, the substrate analogue was cocrystallized with the MurNAc 6P hydrolase from Haemophilus influenzae (MurQ-HI) that was amenable to crystallographic studies. The cocrystal structure of MurQ-HI with the substrate analogue showed that Glu89 was located in the proximity of both the C2 atom and the oxygen at the C3 position of the bound inhibitor and that no other potential acid/base residue that could act as an active site acid/base was located in the vicinity. The conserved residues Glu120 and Lys239 were found within hydrogen bonding distance of the C5 hydroxyl group and C6 phosphate group, suggesting that they play a role in substrate binding and ring opening. Combining these results with previous biochemical data, we propose a one-base mechanism of action in which Glu89 functions to both deprotonate at the C2 position and assist in the departure of the lactyl ether at the C3 position. This same residue would serve to deprotonate the incoming water and reprotonate the enolate in the second half of the catalytic cycle.


Mycotoxin Testing Paradigm: Challenges and Opportunities for the Future.
Mycotoxins are one of the great global challenges to agri-food and feed safety. Industry requires fast, reliable, and economical testing methods for the most important regulated mycotoxins to manage this problem. Climate change and changes in agricultural practice are complicating this situation, triggering the movement of some mycotoxins into new regions, which are unprepared for their management. Modern LC-tandem MS (LC-MS/MS) instruments have addressed this analytical challenge, but such instruments are expensive and require highly qualified personnel and dedicated facilities. As a result of these limitations, traditional LC-MS/MS is not amenable for use on farms or at small to midsized processing facilities, such as a grain elevator. To address the need for on-site rapid testing, the mycotoxin community has focused on antibody-based and spectrophotometric approaches. The development of innovative technologies such as miniaturized MS would allow for the acquisition of more information on mixtures of toxins present in a sample at costs comparable to those of the existing rapid methods such as ELISA. The capital costs are higher, but it would reduce per-sample testing costs and time requirements and provide better value for money while maintaining the accuracy and selectivity achieved in a laboratory setting. In this article, we review the available techniques and contrast them in the context of three main criteria: method performance, speed of analysis, and cost. We define the integration of these three parameters as the "mycotoxin testing paradigm."


Fractalkine Receptor Deficiency Is Associated with Early Protection but Late Worsening of Outcome following Brain Trauma in Mice.
An impaired ability to regulate microglia activation by fractalkine (CX3CL1) leads to microglia chronic sub-activation. How this condition affects outcome after acute brain injury is still debated, with studies showing contrasting results depending on the timing and the brain pathology. Here, we investigated the early and delayed consequences of fractalkine receptor (CX3CR1) deletion on neurological outcome and on the phenotypical features of the myeloid cells present in the lesions of mice with traumatic brain injury (TBI). Wild type (WT) and CX3CR1(-/-) C57Bl/6 mice were subjected to sham or controlled cortical impact brain injury. Outcome was assessed at 4 days and 5 weeks after TBI by neuroscore, neuronal count, and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. Compared with WT mice, CX3CR1(-/-) TBI mice showed a significant reduction of sensorimotor deficits and lower cellular damage in the injured cortex 4 days post-TBI. Conversely, at 5 weeks, they showed a worsening of sensorimotor deficits and pericontusional cell death. Microglia (M) and macrophage (μ) activation and polarization were assessed by quantitative immunohistochemistry for CD11b, CD68, Ym1, and inducible nitric oxide synthase (iNOS)-markers of M/μ activation, phagocytosis, M2, and M1 phenotypes, respectively. Morphological analysis revealed a decreased area and perimeter of CD11b(+) cells in CX3CR1(-/-) mice at 4 days post-TBI, whereas, at 5 weeks, both parameters were significantly higher, compared with WT mice. At 4 days, CX3CR1(-/-) mice showed significantly decreased CD68 and iNOS immunoreactivity, while at 5 weeks post-injury, they showed a selective increase of iNOS. Gene expression on CD11b(+) sorted cells revealed an increase of interleukin 10 and insulin-like growth factor 1 (IGF1) at 1 day and a decrease of IGF1 4 days and 5 weeks post-TBI in CX3CR1(-/-), compared with WT mice. These data show an early protection followed by a chronic exacerbation of TBI outcome in the absence of CX3CR1. Thus, longitudinal effects of myeloid cell manipulation at different stages of pathology should be investigated to understand how and when their modulation may offer therapeutic chances.


Harnessing preclinical mouse models to inform human clinical cancer trials.
The urgent need for better cancer treatments has stimulated interest in employing small-animal models to evaluate potential drug therapies. Robust mouse models of many human cancers have been generated using sophisticated technologies for engineering germ-line mutations. As we enter into an age of targeted therapeutics, these strains provide novel platforms for validating new anticancer drugs, assessing therapeutic index, identifying surrogate markers of tumor progression, and defining epigenetic and environmental influences on tumorigenesis.


Altered prostaglandin synthesis by glomeruli from rats with unilateral ureteral ligation.
There is evidence that prostaglandins modulate renal hemodynamics in unilateral ureteral obstruction. We examined prostaglandin synthesis by glomeruli from rats with 24- and 72-h unilateral ureteral ligation. During prelabeling of the lipid pools with [14C]arachidonate glomeruli from the ligated kidney incorporated less 14C label than those from contralateral or normal control kidneys. However, the incorporation of [14C]arachidonate into phospholipids of glomeruli was proportionately higher in the ligated kidney. Incubation of the prelabeled glomeruli with either angiotensin II or bradykinin significantly increased release of labeled prostaglandins and turnover of [14C]arachidonate on the ligated side. Radioimmunoassay determinations showed that after 24-h ligation, glomeruli from both sides produced comparable amounts of PGF2 alpha, 6-keto-PGF1 alpha, TXB2, and PGE2. After 72 h, glomeruli from the ligated kidney produced more PGF2 alpha, 6-keto-PGF1 alpha, and TXB2 than the contralateral glomeruli. In contrast, PGE2 synthesis was higher in contralateral glomeruli. Neither angiotensin II nor bradykinin had a measurable effect on radioimmunoassayable prostaglandins. We conclude that unilateral ureteral ligation is associated with specific alterations of glomerular prostaglandin synthesis and phospholipid metabolism that are intrinsic to the glomerulus.


Effects of systematic partial volume errors on the estimation of gray matter cerebral blood flow with arterial spin labeling MRI.
Partial volume (PV) correction is an important step in arterial spin labeling (ASL) MRI that is used to separate perfusion from structural effects when computing the mean gray matter (GM) perfusion. There are three main methods for performing this correction: (1) GM-threshold, which includes only voxels with GM volume above a preset threshold; (2) GM-weighted, which uses voxel-wise GM contribution combined with thresholding; and (3) PVC, which applies a spatial linear regression algorithm to estimate the flow contribution of each tissue at a given voxel. In all cases, GM volume is obtained using PV maps extracted from the segmentation of the T1-weighted (T1w) image. As such, PV maps contain errors due to the difference in readout type and spatial resolution between ASL and T1w images. Here, we estimated these errors and evaluated their effect on the performance of each PV correction method in computing GM cerebral blood flow (CBF). Twenty-two volunteers underwent scanning using 2D echo planar imaging (EPI) and 3D spiral ASL. For each PV correction method, GM CBF was computed using PV maps simulated to contain estimated errors due to spatial resolution mismatch and geometric distortions which are caused by the mismatch in readout between ASL and T1w images. Results were analyzed to assess the effect of each error on the estimation of GM CBF from ASL data. Geometric distortion had the largest effect on the 2D EPI data, whereas the 3D spiral was most affected by the resolution mismatch. The PVC method outperformed the GM-threshold even in the presence of combined errors from resolution mismatch and geometric distortions. The quantitative advantage of PVC was 16% without and 10% with the combined errors for both 2D and 3D ASL. Consistent with theoretical expectations, for error-free PV maps, the PVC method extracted the true GM CBF. In contrast, GM-weighted overestimated GM CBF by 5%, while GM-threshold underestimated it by 16%. The presence of PV map errors decreased the calculated GM CBF for all methods. The quality of PV maps presents no argument for the preferential use of the GM-threshold method over PVC in the clinical application of ASL.


OPENER Is a Nuclear Envelope and Mitochondria Localized Protein Required for Cell Cycle Progression in Arabidopsis.
Currently one-third of the proteins encoded by the Arabidopsis (Arabidopsis thaliana) genome are of unknown function. Some of these unknown proteins are likely to be involved in uncharacterized vital biological processes. Evolutionarily conserved single copy genes in flowering plants have been shown to be enriched in essential housekeeping functions. This together with publicly available gene expression data allows for a focused search for uncharacterized essential genes. Here we identify an essential single copy gene called OPENER (OPNR) in Arabidopsis. We show that OPNR is predominantly expressed in actively dividing cells and performs essential functions in seed development and root meristem maintenance. Cell cycle tracking using 5-ethynyl-2'-deoxyuridine staining and fluorescent cell cycle markers together with the increased size of nucleolus and nucleus in opnr mutants indicate that OPNR is required for cell cycle progression through the S or G2 phases. Intriguingly, OPNR localizes to the nuclear envelope and mitochondria. Furthermore, the nuclear envelope localization of OPNR is dependent on its interaction with nuclear inner membrane Sad1/UNC-84 (SUN) domain proteins SUN1 and SUN2. Taken together our results open a line of investigation into an evolutionarily conserved essential cellular process occurring in both the nuclear envelopes and mitochondria of dividing cells.


Role of NMDA, non-NMDA, and GABA receptors in signal propagation in the amygdala formation.
Although the synaptic physiology of the amygdala has been studied with single neuron recordings, the properties of the networks between the various nuclei have resisted characterization because of the limitations of field recording in a neuronally diffuse structure. We addressed this issue in the rat amygdala complex in vitro by using a photodiode array coupled with a voltage-sensitive dye. Low-intensity single pulse stimulation of the lateral amygdala nucleus produced a complex multi-phasic potential. This signal propagated to the basolateral nucleus and the amygdalostriatal transition zone but not to the central nucleus. The local potential, which depended on both synaptic responses and activation of voltage-dependent ion channels, was reduced in amplitude by the non-N-methyl-D-aspartate (non-NMDA) glutamate receptor antagonist 6,7-dinitroquinoxaline (DNQX) and reduced to a lesser extent by the NMDA glutamate receptor antagonist D-2-amino-5-phosphonovaleric acid (D-APV). We next characterized the less complex signals that propagated to more distal regions with or without the addition of the GABA receptor antagonist bicuculline (BIC). BIC alone greatly increased the signal propagation and permitted activation of previously silent areas within the amygdala. DNQX blocked signal propagation to amygdala regions outside of La, even in the presence of BIC, whereas D-APV had minimal effects on these distal signals. These data represent several novel findings: the characterization of the multi-component potential near the site of stimulation, the gating of signal propagation within the amygdala by GABAergic inhibition, the critical role of non-NMDA receptor-mediated depolarization in signal propagation, and the lack of a role for NMDA receptors in maintaining propagation.


Hysteroscopic removal of intrauterine devices in pregnancy.
To introduce and demonstrate an approach to the hysteroscopic removal of retained intrauterine devices (IUDs) in pregnancy. Pregnancy risks associated with retained IUDs are also discussed, specifically spontaneous abortion and preterm labor. A step-by-step explanation of the procedure with surgical video footage. Academic medical center. We present four patients undergoing hysteroscopic removal of IUDs in pregnancy. All patients have imaging documenting an IUD in utero, desire pregnancy continuation, and undergo a physical examination where the IUD strings are not retrievable from the cervical os. Before the procedure, informed consent is obtained, imaging is reviewed, viability is confirmed, and preoperative antibiotics are administered. Hysteroscopy is then performed with the use of a stepwise approach: 1) vaginoscopic hysteroscopy; 2) IUD localization with or without ultrasound guidance; and 3) IUD removal with the use of a hysteroscopic grasper. Technical tips include using a small-caliber hysteroscope and infusion of small volumes of isotonic distension media. Uncomplicated removal of the IUD with postoperative confirmation of viability. Hysteroscopic IUD removal was successfully performed in all four cases presented. After the procedure, all four patients delivered live births at term. The surgical approach presented in this video allows for the successful removal of IUDs in early pregnancy. This technique has a low complication rate and is associated with a >90% ongoing pregnancy rate. Patients with retained IUDs in pregnancy should be offered IUD removal in pregnancy, and referred to physicians experienced in advanced hysteroscopy for consideration of this procedure.


The morphological and molecular changes of brain cells exposed to direct current electric field stimulation.
The application of low-intensity direct current electric fields has been experimentally used in the clinic to treat a number of brain disorders, predominantly using transcranial direct current stimulation approaches. However, the cellular and molecular changes induced by such treatment remain largely unknown. Here, we tested various intensities of direct current electric fields (0, 25, 50, and 100V/m) in a well-controlled in vitro environment in order to investigate the responses of neurons, microglia, and astrocytes to this type of stimulation. This included morphological assessments of the cells, viability, as well as shape and fiber outgrowth relative to the orientation of the direct current electric field. We also undertook enzyme-linked immunosorbent assays and western immunoblotting to identify which molecular pathways were affected by direct current electric fields. In response to direct current electric field, neurons developed an elongated cell body shape with neurite outgrowth that was associated with a significant increase in growth associated protein-43. Fetal midbrain dopaminergic explants grown in a collagen gel matrix also showed a reorientation of their neurites towards the cathode. BV2 microglial cells adopted distinct morphological changes with an increase in cyclooxygenase-2 expression, but these were dependent on whether they had already been activated with lipopolysaccharide. Finally, astrocytes displayed elongated cell bodies with cellular filopodia that were oriented perpendicularly to the direct current electric field. We show that cells of the central nervous system can respond to direct current electric fields both in terms of their morphological shape and molecular expression of certain proteins, and this in turn can help us to begin understand the mechanisms underlying the clinical benefits of direct current electric field.


Brain activity from stimuli that are not perceived: Visual mismatch negativity during binocular rivalry suppression.
Predictive coding explains visual perception as the result of an interaction between bottom-up sensory input and top-down generative models at each level of the visual hierarchy. Evidence for this comes from the visual mismatch negativity (vMMN): a more negative ERP for rare, unpredictable visual stimuli-deviants, than for frequent, predictable visual stimuli-standards. Here, we show that the vMMN does not require conscious experience. We measured the vMMN from monocular luminance-decrement deviants that were either perceived or not during binocular rivalry dominance or suppression, respectively. We found that both sorts of deviants elicited the vMMN at about 250 ms after stimulus onset, with perceived deviants eliciting a bigger vMMN than not-perceived deviants. These results show that vMMN occurs in the absence of consciousness, and that consciousness enhances the processing underlying vMMN. We conclude that generative models of visual perception are tested, even when sensory input for those models is not perceived.


cAMP-GEFII is a direct target of cAMP in regulated exocytosis.
Although cAMP is well known to regulate exocytosis in many secretory cells, its direct target in the exocytotic machinery is not known. Here we show that cAMP-GEFII, a cAMP sensor, binds to Rim (Rab3-interacting molecule, Rab3 being a small G protein) and to a new isoform, Rim2, both of which are putative regulators of fusion of vesicles to the plasma membrane. We also show that cAMP-GEFII, through its interaction with Rim2, mediates cAMP-induced, Ca2+-dependent secretion that is not blocked by an inhibitor of cAMP-dependent protein kinase (PKA). Accordingly, cAMP-GEFII is a direct target of cAMP in regulated exocytosis and is responsible for cAMP-dependent, PKA-independent exocytosis.


Applying Latent Class Analysis to Risk Stratification for Perioperative Mortality in Patients Undergoing Intraabdominal General Surgery.
Perioperative risk stratification is often performed using individual risk factors without consideration of the syndemic of these risk factors. We used latent class analysis (LCA) to identify the classes of comorbidities and risk factors associated with perioperative mortality in patients presenting for intraabdominal general surgery. The 2005 to 2010 American College of Surgeons National Surgical Quality Improvement Program was used to obtain a cohort of patients undergoing intraabdominal general surgery. Risk factors and comorbidities were entered into LCA models to identify the latent classes, and individuals were assigned to a class based on the highest posterior probability of class membership. Relative risk regression was used to determine the associations between the latent classes and 30-day mortality, with adjustments for procedure. A 9-class model was fit using LCA on 466,177 observations. After combining classes with similar adjusted mortality risks, 5 risk classes were obtained. Compared with the class with average mortality risk (class 4), the risk ratios (95% confidence interval) ranged from 0.020 (0.014-0.027) in the lowest risk class (class 1) to 6.75 (6.46-7.02) in the highest risk class. After adjusting for procedure and ASA physical status, the latent classes remained significantly associated with 30-day mortality. The addition of the risk class variable to a model containing ASA physical status and surgical procedure demonstrated a significant increase in the area under the receiver operator characteristic curve (0.892 vs 0.915; P < 0.0001). Latent classes of risk factors and comorbidities in patients undergoing intraabdominal surgery are predictive of 30-day mortality independent of the ASA physical status and improve risk prediction with the ASA physical status.


Science review: searching for gene candidates in acute lung injury.
Acute lung injury (ALI) is a complex and devastating illness, often occurring within the setting of sepsis, and carries an annual mortality rate of 30-50%. Although the genetic basis of ALI has not been fully established, an increasing body of evidence suggests that genetic predisposition contributes to disease susceptibility and severity. Significant difficulty exists, however, in defining the exact nature of these genetic factors, including large phenotypic variance, incomplete penetrance, complex gene-environment interactions, and strong potential for locus heterogeneity. We utilized the candidate gene approach and an ortholog gene database to provide relevant gene ontologies and insights into the genetic basis of ALI. We employed a Medline search of selected basic and clinical studies in the English literature and studies sponsored by the HopGene National Institutes of Health sponsored Program in Genomic Applications. Extensive gene expression profiling studies in animal models of ALI (rat, murine, canine), as well as in humans, were performed to identify potential candidate genes http://www.hopkins-genomics.org/. We identified a number of candidate genes for ALI, with blood coagulation and inflammation gene ontologies being the most highly represented. The candidate gene approach coupled with extensive gene profiling and novel bioinformatics approaches is a valuable way to identify genes that are involved in ALI.


CTLA4 methylation predicts response to anti-PD-1 and anti-CTLA-4 immunotherapy in melanoma patients.
Recent years have witnessed the groundbreaking success of immune checkpoint blockage (ICB) in metastasized malignant melanoma. However, biomarkers predicting the response to ICB are still urgently needed. In the present study, we investigated CTLA4 promoter methylation (mCTLA4) in 470 malignant melanoma patients from The Cancer Genome Atlas (non-ICB cohort) and in 50 individuals with metastasized malignant melanomas under PD-1/CTLA-4-targeted immunotherapy (ICB cohort). mCTLA4 levels were quantified using the Infinium HumanMethylation450 BeadChip (non-ICB cohort) and methylation-specific quantitative real-time PCR in DNA formalin-fixed and paraffin-embedded tissues (ICB cohort). Methylation levels were associated with molecular and clinicopathological variables and analyzed with respect to response (irRECIST) and overall survival. CTLA-4 mRNA and mCTLA4 showed a significant inverse correlation (non-ICB cohort: Spearman's ρ = -0.416, P < 0.001). In ICB-treated melanoma patients, low mCTLA4 was further strongly correlated with response to therapy (P = 0.009, ANOVA) and overall survival (hazard ratio = 2.06 [95% CI: 1.29-3.29], P = 0.003). Our data strongly support the assumption that mCTLA4 predicts response to both anti-PD-1 and anti-CTLA-4 targeted ICB in melanoma and provides paramount information for the selection of patients likely to respond to ICB.


Comparative demography of an epiphytic lichen: support for general life history patterns and solutions to common problems in demographic parameter estimation.
Lichens are major components in many terrestrial ecosystems, yet their population ecology is at best only poorly understood. Few studies have fully quantified the life history or demographic patterns of any lichen, with particularly little attention to epiphytic species. We conducted a 6-year demographic study of Vulpicida pinastri, an epiphytic foliose lichen, in south-central Alaska. After testing multiple size-structured functions to describe patterns in each V. pinastri demographic rate, we used the resulting estimates to construct a stochastic demographic model for the species. This model development led us to propose solutions to two general problems in construction of demographic models for many taxa: how to simply but accurately characterize highly skewed growth rates, and how to estimate recruitment rates that are exceptionally difficult to directly observe. Our results show that V. pinastri has rapid and variable growth and, for small individuals, low and variable survival, but that these traits are coupled with considerable longevity (e.g., >50 years mean future life span for a 4-cm(2) thallus) and little deviation of the stochastic population growth rate from the deterministic expectation. Comparisons of the demographic patterns we found with those of other lichen studies suggest that their relatively simple architecture may allow clearer generalities about growth patterns for lichens than for other taxa, and that the expected pattern of faster growth rates for epiphytic species is substantiated.


Guidelines for auditing pediatric blood transfusion practices.
Although transfusion of blood products is an essential and potentially life-saving measure, not all blood transfusions are beneficial to patients. The associated risks, particularly transfusion-transmitted viral illnesses, such as hepatitis and acquired immunodeficiency syndrome, require that careful consideration be given before a decision is made to transfuse any blood product. Many institutions have established a local committee to monitor transfusion practices and audit such practices regularly. To assist in this task, the Pediatric Hemotherapy Committee of the American Association of Blood Banks has developed guidelines for the conduct of pediatric blood transfusion audits. These guidelines, summarized herein, cover transfusion of red blood cells, platelets, white blood cells, fresh-frozen plasma, albumin, and clotting concentrates. The use of cytomegalovirus low-risk and irradiated blood products is also discussed. Throughout the report, special attention is given to the transfusion needs of newborn infants.


Single-cell transcriptomic analysis of Alzheimer's disease.
Alzheimer's disease is a pervasive neurodegenerative disorder, the molecular complexity of which remains poorly understood. Here, we analysed 80,660 single-nucleus transcriptomes from the prefrontal cortex of 48 individuals with varying degrees of Alzheimer's disease pathology. Across six major brain cell types, we identified transcriptionally distinct subpopulations, including those associated with pathology and characterized by regulators of myelination, inflammation, and neuron survival. The strongest disease-associated changes appeared early in pathological progression and were highly cell-type specific, whereas genes upregulated at late stages were common across cell types and primarily involved in the global stress response. Notably, we found that female cells were overrepresented in disease-associated subpopulations, and that transcriptional responses were substantially different between sexes in several cell types, including oligodendrocytes. Overall, myelination-related processes were recurrently perturbed in multiple cell types, suggesting that myelination has a key role in Alzheimer's disease pathophysiology. Our single-cell transcriptomic resource provides a blueprint for interrogating the molecular and cellular basis of Alzheimer's disease.