Restoration of high zinc and lead tailings with municipal biosolids and lime: a field study.
A field study was conducted to test the ability of biosolids (BS) and different types of lime to increase soil pH, neutralize subsoil acidity, and restore a vegetative cover to alluvial mine tailings in Leadville, CO. The tailings had soil pH of 5.2 and total Cd, Pb, and Zn of 75+/-20, 2600+/-1100, and 6700+/-1900 mg kg(-1). Types of lime included agricultural lime (AL), sugar beet lime (SBL), and lime kiln dust (LKD) applied at 224 Mg ha(-1) calcium carbonate equivalent. Plots were established in 2000 and monitored intermittently through 2007. All amendments increased pH in surface and subsurface depths, with LKD, LKD+BS, and SBL+BS being the most effective. Amendments also reduced 0.01 mol L(-1) Ca(NO3)2 extractable Zn and Cd compared to the control. Plant growth was sparse on all treatments with limited yield for three of four harvests. Poor growth may have been related to elevated electrical conductivity (EC). All amendments except LKD alone (5.79 dS m(-1)) increased EC compared to the control treatment (5.28 dS m(-1)). Electrical conductivity was highest in 2002 which had the lowest summer rainfall. In 2005 EC in all treatments except the SBL+BS was similar in the surface soil. Aboveground plant tissue concentrations of Zn and Cd were also elevated. Limited precipitation and high electrical conductivity may be responsible for poor plant growth. Higher rainfall for the last sampling period resulted in significant growth in the LKD+BS, SBL+BS, and LKD alone treatments.


Polyethyleneimine is an effective permeabilizer of gram-negative bacteria.
The effect of the polycation polyethyleneimine (PEI) on the permeability properties of the Gram-negative bacterial outer membrane was investigated using Escherichia coli, Pseudomonas aeruginosa and Salmonella typhimurium as target organisms. At concentrations of less than 20 micrograms ml-1, PEI increased the bacterial uptake of 1-N-phenylnaphthylamine, which is a hydrophobic probe whose quantum yield is greatly increased in a lipid environment, indicating increased hydrophobic permeation of the outer membrane by PEI. The effect of PEI was comparable to that brought about by the well-known permeabilizer EDTA. Permeabilization by PEI was retarded but not completely inhibited by millimolar concentrations of MgCl2. PEI also increased the susceptibility of the test species to the hydrophobic antibiotics clindamycin, erythromycin, fucidin, novobiocin and rifampicin, without being directly bactericidal. PEI sensitized the bacteria to the lytic action of the detergent SDS in assays where the bacteria were pretreated with PEI. In assays where PEI and SDS were simultaneously present, no sensitization was observed, indicating that PEI and SDS were inactivating each other. In addition, a sensitizing effect to the nonionic detergent Triton X-100 was observed for P. aeruginosa. In conclusion, PEI was shown to be a potent permeabilizer of the outer membrane of Gram-negative bacteria.


Grafted metallocalixarenes as single-site surface organometallic catalysts.
Metallocalixarenes were grafted onto silica using a surface organometallic approach and shown to be active and selective catalysts for epoxidation of alkenes using organic hydroperoxides. Calixarene-Ti(IV) precursors were anchored at surface densities from 0.1 to near-monolayer coverages (0.025-0.25 calixarene nm(-2)). Several spectroscopic methods independently detected calixarene-Ti(IV) connectivity before and after epoxidation catalysis. Kinetic analyses of cyclohexene epoxidation confirmed that the active sites were anchored on the silica surface and were significantly more active than their homogeneous analogues. The steric bulk and multidentate binding of the calixarenes led to structural stability and to single-site behavior during epoxidation catalysis. Rate constants were independent of surface density for cyclohexene epoxidation with tert-butyl hydroperoxide (11.1 +/- 0.3 M(-2) s(-1)) or cumene hydroperoxide (25 +/- 2 M(-2) s(-1)). The materials and methods reported here allow the assembly of robust surface organometallic structures in which the active sites behave as isolated species, even near saturation monolayer coverages. In turn, this makes possible the rational design and synthesis of a class of heterogeneous oxide catalysts with atomic-scale precision at the active site.


Analysis of the deubiquitinating enzymes of the yeast Saccharomyces cerevisiae.
Attachment of proteins to ubiquitin is reversed by specialized proteases called deubiquitinating enzymes (Dubs), which are also essential for ubiquitin precursor processing. In the genome of Saccharomyces cerevisiae, 17 potential DUB genes can be discerned. We have now constructed strains deleted for each of these genes. Surprisingly, given the essential nature of the ubiquitin system, none of the mutants is lethal or strongly growth defective under standard conditions, although a number have detectable abnormalities. Including results from this study, 14 of the 17 Dubs have now been shown to have ubiquitin-cleaving activity. The most extensively characterized yeast Dub is Doa4, which is required for both ubiquitin homeostasis and proteasome-dependent proteolysis. To help determine what distinguishes Doa4 functionally from other Dubs, we have cloned a DOA4 ortholog from the yeast Kluyveromyces lactis. The K. lactis protein is 42% identical to Doa4, but unexpectedly the K. lactis gene is slightly closer in nucleotide sequence to UBP5, which cannot substitute for DOA4 even in high dosage. The data suggest that the DOA4 locus underwent a duplication after the divergence of K. lactis and S. cerevisiae. This information will facilitate fine-structure analysis of the Doa4 protein to help delineate its key functional elements.


FourCSeq: analysis of 4C sequencing data.
Circularized Chromosome Conformation Capture (4C) is a powerful technique for studying the spatial interactions of a specific genomic region called the 'viewpoint' with the rest of the genome, both in a single condition or comparing different experimental conditions or cell types. Observed ligation frequencies typically show a strong, regular dependence on genomic distance from the viewpoint, on top of which specific interaction peaks are superimposed. Here, we address the computational task to find these specific peaks and to detect changes between different biological conditions. We model the overall trend of decreasing interaction frequency with genomic distance by fitting a smooth monotonically decreasing function to suitably transformed count data. Based on the fit, z-scores are calculated from the residuals, and high z-scores are interpreted as peaks providing evidence for specific interactions. To compare different conditions, we normalize fragment counts between samples, and call for differential contact frequencies using the statistical method DESEQ2: adapted from RNA-Seq analysis. A full end-to-end analysis pipeline is implemented in the R package FourCSeq available at www.bioconductor.org. firstname.lastname@example.com or email@example.com Supplementary data are available at Bioinformatics online.


Hyponatremic seizure due to huge abdominal cerebrospinal fluid pseudocsyt in a child with ventriculoperitoneal shunt: a case report.
Placement of ventriculoperitoneal(VP) shunt is a worldwide accepted procedure for treatment of hydrocephalus. This procedure have various intra-abdominal complications, of which pseudocyst formation is a rare one. Common presentations of this complication are abdominal mass, abdominal pain, intestinal obstruction, and shunt dysfunction. In this paper, we report a case of 3 year-old boy with cerebrospinal fluid (CSF) pseudocyst of the VP shunt presenting with hyponatremic seizure. To the best of our knowledge, hyponatremic seizure has not been previously reported as a presentation of abdominal CSF pseudocyst in the literature. Our case has also the largest CSF pseudocyst with respect to body surface area of the child in the literature.


France's Évin Law on the control of alcohol advertising: content, effectiveness and limitations.
To assess the effectiveness of the 2015 version of the French Évin Law that was implemented in 1991 with the objective of protecting young people from alcohol advertising. Data were obtained from survey questions measuring exposure and receptivity to alcohol advertisements that were introduced for the first time in the 2015 European School Survey Project on Alcohol and Other Drugs (ESPAD). A representative sample of 6642 10th-12th grade students (mean age 17.3 years) were interviewed in 198 schools in France by a self-administered questionnaire. Information was collected on alcohol advertising exposure in different media (outside billboards, internet, etc.) and receptivity to recent advertisements (attractiveness, incentive to drink, etc.). The majority of students declared that they had been exposed at least once a month to alcohol advertisements in supermarkets (73.2%), in movies (66.1%), magazines and newspapers (59.1%), on billboards in streets (54.5%), and on the internet (54.1%). Concerning the last recalled advertisements, 27.8% remembered the beverage type, 18.2% the brand, 13% felt like having a drink after having seen the advertisement and 19.6% found the advertisement attractive (boys ranked significantly higher than girls for all these indicators; P-value < 0.05). The 2015 version of the French Évin law does not appear to protect young people effectively from exposure to alcohol advertising in France.


Molecular details of the unique mechanism of chloride transport by a cyanobacterial rhodopsin.
Microbial rhodopsins are well known as versatile and ubiquitous light-driven ion transporters and photosensors. While the proton transport mechanism has been studied in great detail, much less is known about various modes of anion transport. Until recently, only two main groups of light-driven anion pumps were known, archaeal halorhodopsins (HRs) and bacterial chloride pumps (known as ClRs or NTQs). Last year, another group of cyanobacterial anion pumps with a very distinct primary structure was reported. Here, we studied the chloride-transporting photocycle of a representative of this new group, Mastigocladopsis repens rhodopsin (MastR), using time-resolved spectroscopy in the infrared and visible ranges and site-directed mutagenesis. We found that, in accordance with its unique amino acid sequence containing many polar residues in the transmembrane region of the protein, its photocycle features a number of unusual molecular events not known for other anion-pumping rhodopsins. It appears that light-driven chloride ion transfers by MastR are coupled with translocation of protons and water molecules as well as perturbation of several polar sidechains. Of particular interest is transient deprotonation of Asp-85, homologous to the cytoplasmic proton donor of light-driven proton pumps (such as Asp-96 of bacteriorhodopsin), which may serve as a regulatory mechanism.


The choice of thujone as drug for diabetes.
The use of thujone, a monoterpene ketone often present in sage (Salvia officinalis L.) or wormwood (Artemisia absinthium L.), for the treatment of diabetes mellitus was recently suggested in a study published in this journal. Evidence was based on the findings obtained in a diabetic rat model. After oral treatment with thujone (5 mg/kg bodyweight (bw)/day for 28 days), the cholesterol and triglyceride levels were significantly adjusted to normal levels when compared to diabetic, untreated rats. While these results sound promising and worthy of further investigation, the well-defined profile of the adverse properties of thujone demands a cautious interpretation of these results. The therapeutic margin of thujone appears to be small, as a dose-related incidence of seizures was noted in 2-year National Toxicology Program studies in rats and mice. The dose level in the diabetic rat study is also considerably higher than a daily intake that is acceptable for humans (0.1 mg/kg bw/day).


Simulation in Urological Training and Education (SIMULATE): Protocol and curriculum development of the first multicentre international randomized controlled trial assessing the transferability of simulation-based surgical training.
To report the study protocol for the first international multicentre randomized controlled trial investigating the effectiveness of simulation-based surgical training and the development process for an evidence-based training curriculum, to be delivered as an educational intervention. This prospective, international, multicentre randomized controlled clinical and educational trial will recruit urology surgical trainees who must not have performed ≥10 of the selected index procedure, ureterorenoscopy (URS). Participants will be randomized to simulation-based training (SBT) or non-simulation-based training (NSBT), the latter of which is the current sole standard of training globally. The primary outcome is the number of procedures required to achieve proficiency, where proficiency is defined as achieving a learning curve plateau of 28 or more on an Objective Structured Assessment of Technical Skills (OSATS) assessment scale, for three consecutive operations, without any complications. All participants will be followed up either until they complete 25 procedures or for 18 months. Development of the URS SBT curriculum took place through a two-round Delphi process. A total of 47 respondents, consisting of trainees (n = 24) with URS experience and urolithiasis specialists (n = 23), participated in round 1 of the Delphi process. Specialists (n = 10) finalized the content of the curriculum in round 2. The developed interventional curriculum consists of initial theoretic knowledge through didactic lectures followed by select tasks and cases on the URO-Mentor (Simbionix, Lod, Israel) VR Simulator, Uro-Scopic Trainer (Limbs & Things, Bristol, UK) and Scope Trainer (Mediskills, Manchester, UK) models for both semi-rigid and flexible URS. Respondents also selected relevant non-technical skills scenarios and cadaveric simulation tasks as additional components, with delivery subject to local availability. SIMULATE is the first multicentre trial investigating the effect and transferability of supplementary SBT on operating performance and patient outcomes. An evidence-based training curriculum is presented, developed with expert and trainee input. Participants will be followed and the primary outcome, number of procedures required to proficiency, will be reported alongside key clinical secondary outcomes, (ISCRTN 12260261).


Vitamin D is a major determinant of bone mineral density at school age.
Vitamin D insufficiency in children may have long-term skeletal consequences as vitamin D affects calcium absorption, bone mineralization and bone mass attainment. This school-based study investigated vitamin D status and its association with vitamin D intake and bone health in 195 Finnish children and adolescents (age range 7-19 years). Clinical characteristics, physical activity and dietary vitamin D intake were evaluated. Blood and urine samples were collected for serum 25-hydroxyvitamin D (25-OHD) and other parameters of calcium homeostasis. Bone mineral density (BMD) and body composition were measured with dual-energy X-ray absorptiometry (DXA). Altogether 71% of the subjects were vitamin D insufficient (25-OHD <50 nmol/L). The median 25-OHD was 41 nmol/L for girls and 45 nmol/L for boys, and the respective median vitamin D intakes 9.1 µg/day and 10 µg/day. In regression analysis, after adjusting for relevant factors, 25-OHD concentration explained 5.6% of the variance in lumbar BMD; 25-OHD and exercise together explained 7.6% of the variance in total hip BMD and 17% of the variance in whole body BMD. S-25-OHD was an independent determinant of lumbar spine and whole body BMD and in magnitude surpassed the effects of physical activity. Vitamin D insufficiency was common even when vitamin D intake exceeded the recommended daily intake. Vitamin D status was a key determinant of BMD. The findings suggest urgent need to increase vitamin D intake to optimize bone health in children.


Does hyperprolactinemia affect hepatic regeneration independent of sex steroids?
Prolactin, administered exogenously, has been shown to be trophic to the liver, causing increases in the liver weight-to-body weight ratio. In ornithine decarboxylase activity, and in thymidine kinase activity. To investigate the effect of endogenous hyperprolactinemia on hepatic regeneration, pituitary isografts were placed beneath the renal capsule in rats 2 weeks before the rats underwent a two-thirds partial hepatectomy. Prolactin levels 2 weeks after the transplant were greater in the animals with the pituitary isografts compared with levels in controls. The increase in the liver weight-to-body weight ratio after hepatectomy was similar in the rats with pituitary transplant and the controls. However, chronic hyperprolactinemia was associated with increased basal levels of ornithine decarboxylase activity and thymidine kinase activity. Both ornithine decarboxylase activity and thymidine kinase activity increased after partial hepatectomy, and the magnitude of the changes was similar for both groups of animals. The levels of estrogen receptor activity before the partial hepatectomy and the reduction in receptor activity that follows partial hepatectomy were similar in the two groups of animals. Moreover, the levels of androgen receptor activity within the liver before partial hepatectomy and the increase in receptor activity after hepatectomy were similar in the two groups of animals. Thus, chronic sustained hyperprolactinemia has no beneficial effect on the hepatic regenerative response, despite induction of both basal ornithine decarboxylase and thymidine kinase activities.


Nilvadipine in mild to moderate Alzheimer disease: A randomised controlled trial.
This study reports the findings of the first large-scale Phase III investigator-driven clinical trial to slow the rate of cognitive decline in Alzheimer disease with a dihydropyridine (DHP) calcium channel blocker, nilvadipine. Nilvadipine, licensed to treat hypertension, reduces amyloid production, increases regional cerebral blood flow, and has demonstrated anti-inflammatory and anti-tau activity in preclinical studies, properties that could have disease-modifying effects for Alzheimer disease. We aimed to determine if nilvadipine was effective in slowing cognitive decline in subjects with mild to moderate Alzheimer disease. NILVAD was an 18-month, randomised, placebo-controlled, double-blind trial that randomised participants between 15 May 2013 and 13 April 2015. The study was conducted at 23 academic centres in nine European countries. Of 577 participants screened, 511 were eligible and were randomised (258 to placebo, 253 to nilvadipine). Participants took a trial treatment capsule once a day after breakfast for 78 weeks. Participants were aged >50 years, meeting National Institute of Neurological and Communicative Disorders and Stroke/Alzheimer's disease Criteria (NINCDS-ADRDA) for diagnosis of probable Alzheimer disease, with a Standardised Mini-Mental State Examination (SMMSE) score of ≥12 and <27. Participants were randomly assigned to 8 mg sustained-release nilvadipine or matched placebo. The a priori defined primary outcome was progression on the Alzheimer's Disease Assessment Scale Cognitive Subscale-12 (ADAS-Cog 12) in the modified intention-to-treat (mITT) population (n = 498), with the Clinical Dementia Rating Scale sum of boxes (CDR-sb) as a gated co-primary outcome, eligible to be promoted to primary end point conditional on a significant effect on the ADAS-Cog 12. The analysis set had a mean age of 73 years and was 62% female. Baseline demographic and Alzheimer disease-specific characteristics were similar between treatment groups, with reported mean of 1.7 years since diagnosis and mean SMMSE of 20.4. The prespecified primary analyses failed to show any treatment benefit for nilvadipine on the co-primary outcome (p = 0.465). Decline from baseline in ADAS-Cog 12 on placebo was 0.79 (95% CI, -0.07-1.64) at 13 weeks, 6.41 (5.33-7.49) at 52 weeks, and 9.63 (8.33-10.93) at 78 weeks and on nilvadipine was 0.88 (0.02-1.74) at 13 weeks, 5.75 (4.66-6.85) at 52 weeks, and 9.41 (8.09-10.73) at 78 weeks. Exploratory analyses of the planned secondary outcomes showed no substantial effects, including on the CDR-sb or the Disability Assessment for Dementia. Nilvadipine appeared to be safe and well tolerated. Mortality was similar between groups (3 on nilvadipine, 4 on placebo); higher counts of adverse events (AEs) on nilvadipine (1,129 versus 1,030), and serious adverse events (SAEs; 146 versus 101), were observed. There were 14 withdrawals because of AEs. Major limitations of this study were that subjects had established dementia and the likelihood that non-Alzheimer subjects were included because of the lack of biomarker confirmation of the presence of brain amyloid. The results do not suggest benefit of nilvadipine as a treatment in a population spanning mild to moderate Alzheimer disease. Clinicaltrials.gov NCT02017340, EudraCT number 2012-002764-27.


Intensive glycemic control is not associated with fractures or falls in the ACCORD randomized trial.
Older adults with type 2 diabetes are at high risk of fractures and falls, but the effect of glycemic control on these outcomes is unknown. To determine the effect of intensive versus standard glycemic control, we assessed fractures and falls as outcomes in the Action to Control Cardiovascular Risk in Diabetes (ACCORD) randomized trial. ACCORD participants were randomized to intensive or standard glycemia strategies, with an achieved median A1C of 6.4 and 7.5%, respectively. In the ACCORD BONE ancillary study, fractures were assessed at 54 of the 77 ACCORD clinical sites that included 7,287 of the 10,251 ACCORD participants. At annual visits, 6,782 participants were asked about falls in the previous year. During an average follow-up of 3.8 (SD 1.3) years, 198 of 3,655 participants in the intensive glycemia and 189 of 3,632 participants in the standard glycemia group experienced at least one nonspine fracture. The average rate of first nonspine fracture was 13.9 and 13.3 per 1,000 person-years in the intensive and standard groups, respectively (hazard ratio 1.04 [95% CI 0.86-1.27]). During an average follow-up of 2.0 years, 1,122 of 3,364 intensive- and 1,133 of 3,418 standard-therapy participants reported at least one fall. The average rate of falls was 60.8 and 55.3 per 100 person-years in the intensive and standard glycemia groups, respectively (1.10 [0.84-1.43]). Compared with standard glycemia, intensive glycemia did not increase or decrease fracture or fall risk in ACCORD.


Genetical toxicogenomics in Drosophila identifies master-modulatory loci that are regulated by developmental exposure to lead.
The genetics of gene expression in recombinant inbred lines (RILs) can be mapped as expression quantitative trait loci (eQTLs). So-called "genetical genomics" studies have identified locally acting eQTLs (cis-eQTLs) for genes that show differences in steady-state RNA levels. These studies have also identified distantly acting master-modulatory trans-eQTLs that regulate tens or hundreds of transcripts (hotspots or transbands). We expand on these studies by performing genetical genomics experiments in two environments in order to identify trans-eQTL that might be regulated by developmental exposure to the neurotoxin lead. Flies from each of 75 RIL were raised from eggs to adults on either control food (made with 250 microM sodium acetate), or lead-treated food (made with 250 microM lead acetate, PbAc). RNA expression analyses of whole adult male flies (5-10 days old) were performed with Affymetrix DrosII whole genome arrays (18,952 probesets). Among the 1389 genes with cis-eQTL, there were 405 genes unique to control flies and 544 genes unique to lead-treated ones (440 genes had the same cis-eQTLs in both samples). There are 2396 genes with trans-eQTL which mapped to 12 major transbands with greater than 95 genes. Permutation analyses of the strain labels but not the expression data suggests that the total number of eQTL and the number of transbands are more important criteria for validation than the size of the transband. Two transbands, one located on the 2nd chromosome and one on the 3rd chromosome, co-regulate 33 lead-induced genes, many of which are involved in neurodevelopmental processes. For these 33 genes, rather than allelic variation at one locus exerting differential effects in two environments, we found that variation at two different loci are required for optimal effects on lead-induced expression.


Effects of amino acid infusion on renal hemodynamics. Role of endothelium-derived relaxing factor.
Ingestion of protein or intravenous infusion of amino acids acutely elevates glomerular filtration rate (GFR) and renal plasma flow (RPF) by unknown mechanisms. Endothelium-derived relaxing factor (EDRF), now known to be nitric oxide derived from metabolism of L-arginine, participates in local regulation of vascular tone. To investigate the hypothesis that EDRF may participate in the renal vasodilatation and increased GFR after amino acid infusion, we characterized the effect of inhibition of EDRF synthesis with NG-monomethyl L-arginine (LNMMA) on basal renal hemodynamics and the response to infusion of a 10% mixed amino acid solution (1 ml/hr i.v.) in the rat. Renal arterial infusion of LNMMA (500 micrograms/kg/min) resulted in a significant increase in mean arterial pressure, decreases in GFR (20%) and RPF (44%), and a significant increase in filtration fraction. Pretreatment with the angiotensin II receptor antagonist Sar-Gly-angiotensin II did not prevent the increase in blood pressure but blunted the decreases in GFR (11%) and RPF (27%) after LNMMA infusion. Amino acid infusion in the untreated, fasted rat resulted in no change in blood pressure but significant increases in GFR and RPF; these effects were completely inhibited by intrarenal LNMMA but not an equihypertensive intravenous infusion of phenylephrine. In summary, EDRF participates in regulation of basal renal hemodynamics. Furthermore, amino acid-induced hyperfiltration and renal vasodilatation are completely prevented by inhibition of EDRF synthesis. We conclude that EDRF may participate in the renal hemodynamic response to amino acid infusion.


Stretching of surface-tethered polymers in pressure-driven flow under confinement.
We study the effect of pressure-driven flow on a single surface-tethered DNA molecule confined between parallel surfaces. The influence of flow and channel parameters as well as the length of the molecules on their extension and orientation is explored. In the experiments the chain conformations are imaged by laser scanning confocal microscopy. We find that the fractional extension of the tethered DNA molecules mainly depends on the wall shear stress, with effects of confinement being very weak. Experiments performed with molecules of different contour length show that the fractional extension is a universal function of the product of the wall shear stress and the contour length, a result that can be obtained from a simple scaling relation. The experimental results are in good agreement with results from coarse-grained molecular dynamics/Lattice-Boltzmann simulations.


Hormone-induced ductal DNA synthesis of human breast tissues maintained in the athymic nude mouse.
Five biopsy specimens of morphologically normal human breast tissues, obtained from the margins of five benign human breast tumors, were processed into slices (4.0 x 4.0 x 0.1 mm) and transplanted s.c. dorsally (eight to ten slices/mouse) into forty-three 6- to 8-week-old female BALB/c athymic nude mice. Each individual human breast tissue specimen was transplanted into seven to ten mice. After 30 days, the mice were divided into four groups and treated for 30 days as follows: (a) controls receiving s.c. cholesterol pellets (38 mg); (b) estrogens that were administered in s.c. pellets containing 2 mg 17 beta-estradiol and 38 mg cholesterol and in drinking water containing 0.5 mg estrone per liter; (c) rat pituitary tumor (RPT), a cell suspension of MtT-W10 RPT that secretes large amounts of prolactin and growth hormone, injected dorsorostrally; and (d) RPT plus estrogens. Three to five human breast tissue grafts were removed from each mouse at the onset of treatment, and the remainder were removed at termination of treatment. DNA synthesis in the ductal epithelium was determined in pre- and posttreatment grafts by [3H]thymidine autoradiography after incubation of grafts for 4 hr in an isotope-enriched medium. The labeling index (LI), mean number of labeled epithelial cells per unit area of epithelial tissue, in pre- and posttreatment grafts was, respectively: (a) control, 7.6 +/- 1.4 and 7.1 +/- 1.4; (b) estrogens, 5.5 +/- 0.6 and 17.9 +/- 2.3; (c) RPT, 6.2 +/- 0.7 and 8.0 +/- 1.5; and (d) RPT plus estrogens, 6.3 +/- 0.8 and 26.6 +/- 2.5. A significant increase in LI was observed after treatment with estrogens (p less than 0.05) or RPT plus estrogen (p less than 0.001). Mean LI after treatment with RPT plus estrogens was significantly greater (p less than 0.02) than after estrogens alone. RPT alone did not significantly alter the LI. Thus, these results provide in vivo evidence that estrogens enhance DNA synthesis of the ductal epithelium of the normal human breast and that a growth factor (or factors) from RPT acts synergistically with estrogens to produce a more pronounced increase in DNA synthesis. RPT growth factors (perhaps prolactin and/or growth hormone) appear to require estrogens for DNA synthesis stimulation in normal human breast ductal epithelium.


Intensive chemotherapy and low-dose radiotherapy for the treatment of advanced-stage Hodgkin's disease in pediatric patients: a Pediatric Oncology Group study.
Sixty-two patients with advanced-stage Hodgkin's disease and a median age of 12 years (range, 3 to 22 years) were treated with four cycles of mechlorethamine, vincristine, procarbazine, and prednisone (MOPP) alternating with four cycles of doxorubicin, vinblastine, bleomycin, and dacarbazine (ABVD) followed by low-dose radiotherapy (RT). We determined the feasibility, immediate safety, and rapidity of response of patients to this regimen, as well as the relationship between prognostic factors and the rate of complete remission (CR), event-free survival (EFS), and overall survival. Therapy was well tolerated, and the major toxicity was hematopoietic. At the end of chemotherapy, 54 of 62 patients (87%) were in CR by clinical restaging, with a biopsy of residual disease where necessary. The actuarial 3-year EFS is 77% (SE, 11%), with a median follow-up of 35 months, and the survival is 91% (SE, 7%). With respect to EFS, female patients and those with stage II or III disease fared statistically better than males and patients with stage IV disease, respectively. Six patients have died: three of progressive Hodgkin's disease, one of secondary acute myelocytic leukemia (AML), one of secondary non-Hodgkin's lymphoma (NHL), and one of overwhelming bacterial sepsis. The Pediatric Oncology Group (POG) is currently engaged in a randomized study of these eight cycles of chemotherapy with and without RT to assess the role of RT in achieving comparable results.


Multi-genome analysis identifies functional and phylogenetic diversity of basidiomycete adenylate-forming reductases.
Among the invaluable benefits of basidiomycete genomics is the dramatically enhanced insight into the potential capacity to biosynthesize natural products. This study focuses on adenylate-forming reductases, which is a group of natural product biosynthesis enzymes that resembles non-ribosomal peptide synthetases, yet serves to modify one substrate, rather than to condense two or more building blocks. Phylogenetically, these reductases fall in four classes. The phylogeny of Heterobasidion annosum (Russulales) and Serpula lacrymans (Boletales) adenylate-forming reductases was investigated. We identified a previously unrecognized phylogenetic branch within class III adenylate-forming reductases. Three representatives were heterologously produced and their substrate preferences determined in vitro: NPS9 and NPS11 of S. lacrymans preferred l-threonine and benzoic acid, respectively, while NPS10 of H. annosum accepted phenylpyruvic acid best. We also investigated two class IV adenylate-forming reductases of Coprinopsis cinerea, which each were active with l-alanine, l-valine, and l-serine as substrates. Our results show that adenylate-forming reductases are functionally more diverse than previously recognized. As none of the natural products known from the species investigated in this study includes the identified substrates of their respective reductases, our findings may help further explore the diversity of these basidiomycete secondary metabolomes.


A taxonomy of needs assessment, elicited from a multiple case study of community nursing education and practice.
The role and expectations of community nurses in carrying out needs assessments changed when an internal quasi-market was introduced to the British health service under the National Health Service (NHS) & Community Care Act 1990. This paper reports on a study commissioned by the English National Board for Nursing, Midwifery and Health Visiting (ENB) to investigate the changing educational needs of community nurses with regard to needs assessment in the context of this legislation. A multiple case study design was utilized and four cases identified, incorporating the geographical variation in England. Recently qualified practitioners (health visitors and district nurses) were observed during a regular shift (n=134 visits), concentrating on their practice of assessing needs, and on liaison and collaboration within teams and across sectors. Participants were interviewed after the observation period (n=33 practitioners), to determine the extent of formality they attached to each assessment, and to elicit information about aspects which may be embedded in everyday practice. Single and multiple case analyses across the four cases used an iterative process of pattern-matching, replication logic and explanation building. The preliminary analysis yielded a descriptive 'taxonomy' which could serve as a basis for classifying the variants of needs assessment and help to clarify the whole phenomenon. When applied further to the data, this revealed the complex interactions between the different ideals (relating to policy, nursing and ascribed worth), the various types (purpose, formality/specificity and complexity) and timing (in relation to client, service and practice issues) within needs assessment.


Salbutamol in cardiogenic shock complicating acute myocardial infarction.
Intravenous salbutamol (13 microgram/min) has been given to 31 patients with cardiogenic shock complicating acute myocardial infarction. Haemodynamic measurements were made in nine of these patients. Salbutamol increased cardiac index by 41 per cent from 1.25 +/- 0.06 l/min per m2 to 1.76 +/- 0.19 l/min per m2 and decreased systemic vascular resistance by 16 per cent from 26.2 +/- 1.9 units to 21.9 +/- 2.1 units. Heart rate rose by 13 per cent from 95 +/- 4.5 beats/min to 106 +/- 6.0 beats/min. Pulmonary artery end-diastolic pressure fell from 20.6 +/- 1.7 mmHg to 16.9+/- 1.9 mmHg. Of the 31 patients, eight survived to leave hospital (27%). Five of the survivors had initial low heart rates and in these patients the clinical improvement was probably attributable to the positive chronotropic action of salbutamol. In the remaining three survivors clinical improvement was probably a result of salbutamol mediated afterload reduction. Salbutamol may be useful in the treatment of cardiogenic shock and pump failure complicating acute myocardial infarction.


Recognition performance for four combinations of FM system and hearing aid microphone signals in adverse listening conditions.
Children with moderate to severe hearing loss routinely use personal frequency modulated (FM) systems in the classroom to improve the signal to noise ratio of teacher-directed speech with notable success. Attention is now being given to the ability of these children to hear other students via the hearing aid (HA) microphone while using an FM system. As a result, a variety of FM system and HA microphone combinations have been recommended for classroom use. To date, there are no studies regarding the efficacy of these FM/HA combinations. The purpose of this study was to evaluate recognition performance using four FM/HA combinations and to characterize that performance for stimuli received primarily through FM system and HA microphone transmission. Recognition performance for FM system and HA microphone signals was evaluated for two symmetrical and two asymmetrical FM/HA combinations using two commercially available FM systems (one conventional and one FM-precedence circuit). Eleven children (ages 9 to 12) with moderate to severe sensorineural hearing loss and eight children (ages 10 to 11) with normal hearing served as subjects. The two symmetrical FM/HA combinations included: 1) binaural FM system and HA microphone input using the conventional FM system, and 2) binaural FM and HA input using the FM-precedence circuit. The conventional FM system was used for the two asymmetrical combinations and included: 1) binaural FM input and monaural HA input, and 2) FM input to one ear and HA input to the other. Stimuli were 33 consonants presented in the form of nonsense syllables. The stimuli were presented through three loudspeakers representing a teacher and two fellow students in a classroom environment. Speech shaped noise was presented through two additional loudspeakers. In general, no statistically significant differences in recognition performance were found between any of the FM/HA combinations. Mean recognition scores for HA microphone transmission (55%) were significantly poorer than those for FM system transmission (75%). As expected, initial consonants were more easily recognized than final consonants via FM system and HA microphone transmission. However, voiceless consonants were more easily recognized than voiced consonants via HA microphone transmission, which was not predicted on the basis of previous research. These results suggest that a certain amount of flexibility is present when choosing an FM/HA combination. However, recognition performance via the HA microphones was consistently poorer than performance via FM transmission. Because relevant material also originates from fellow students (e.g., answering teacher-directed questions), input via the HAs is often as important as information originating from the teacher. The results suggest that attempts to improve performance for signals transmitted through the HA microphones in a classroom setting would benefit children with hearing loss.


Molecular Characteristics of ERCC1-Negative versus ERCC1-Positive Tumors in Resected NSCLC.
Excision repair cross-complementation group 1 (ERCC1) is a protein involved in repair of DNA platinum adducts and stalled DNA replication forks. We and others have previously shown the influence of ERCC1 expression upon survival rates and benefit of cisplatin-based chemotherapy in patients with resected non-small-cell lung cancer (NSCLC). However, little is known about the molecular characteristics of ERCC1-positive and ERCC1-negative tumors. We took advantage of a cohort of 91 patients with resected NSCLC, for which we had matched frozen and paraffin-embedded samples to explore the comparative molecular portraits of ERCC1-positive and ERCC1-negative tumors of NSCLC. We carried out a global molecular analysis including assessment of ERCC1 expression levels by using both immunohistochemistry (IHC) and quantitative reverse transcriptase PCR (qRT-PCR), genomic instability, global gene and miRNA expression, and sequencing of selected key genes involved in lung carcinogenesis. ERCC1 protein and mRNA expression were significantly correlated. However, we observed several cases with clear discrepancies. We noted that ERCC1-negative tumors had a higher rate of genomic abnormalities versus ERCC1-positive tumors. ERCC1-positive tumors seemed to share a common DNA damage response (DDR) phenotype with the overexpression of seven genes linked to DDR. The miRNA expression analysis identified miR-375 as significantly underexpressed in ERCC1-positive tumors. Our data show inconsistencies in ERCC1 expression between IHC and qRT-PCR readouts. Furthermore, ERCC1 status is not linked to specific mutational patterns or frequencies. Finally, ERCC1-negative tumors have a high rate of genomic aberrations that could consequently influence prognosis in patients with resected NSCLC.


Extracorporeal shockwave lithotripsy: first 1000 cases at the London Stone Clinic.
One thousand patients underwent extracorporeal shockwave lithotripsy for renal and ureteric calculi at this clinic. An overall success rate of 91.8% was achieved (stone free or less than 2 mm fragments at three months) and for stones measuring 1 cm 96.3%. Lithotripsy produced extremely low morbidity, and no deaths have occurred at the clinic. Patients who had lithotripsy alone had a mean hospital stay of three days and in most instances were able to perform their full range of activities on discharge. Planned combination of lithotripsy with minimally invasive endourological procedures such as percutaneous nephrolithotomy and ureterorenoscopy has allowed us to extend the range of treatable cases to include large stones. Prophylactic use of Double-J ureteric stents in selected cases has reduced the incidence of obstruction by stone fragments after lithotripsy, thereby decreasing morbidity and hospital stay.


Myeloid PTEN deficiency protects livers from ischemia reperfusion injury by facilitating M2 macrophage differentiation.
Although the role of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) in regulating cell proliferation is well established, its function in immune responses remains to be fully appreciated. In the current study, we analyzed myeloid-specific PTEN function in regulating tissue inflammatory immune response in a murine liver partial warm ischemia model. Myeloid-specific PTEN knockout (KO) resulted in liver protection from ischemia reperfusion injury (IRI) by deviating the local innate immune response against ischemia reperfusion toward the regulatory type: expression of proinflammatory genes was selectively decreased and anti-inflammatory IL-10 was simultaneously increased in ischemia reperfusion livers of PTEN KO mice compared with those of wild-type (WT) mice. PI3K inhibitor and IL-10-neutralizing Abs, but not exogenous LPS, recreated liver IRI in these KO mice. At the cellular level, Kupffer cells and peritoneal macrophages isolated from KO mice expressed higher levels of M2 markers and produced lower TNF-α and higher IL-10 in response to TLR ligands than did their WT counterparts. They had enhanced Stat3- and Stat6-signaling pathway activation, but diminished Stat1-signaling pathway activation, in response to TLR4 stimulation. Inactivation of Kupffer cells by gadolinium chloride enhanced proinflammatory immune activation and increased IRI in livers of myeloid PTEN KO mice. Thus, myeloid PTEN deficiency protects livers from IRI by facilitating M2 macrophage differentiation.


Use of verbal autopsy and social autopsy in humanitarian crises.
Two billion people live in countries affected by conflict, violence and fragility. These are exceptional situations in which mortality shifts dramatically and in which civil registration and vital statistics systems are often weakened or cease to function. Verbal autopsy and social autopsy (VA and SA) are methods used to assign causes of death and understand the contexts in which these occur, in settings where information is otherwise unavailable. This review sought to explore the use of VA and SA in humanitarian crises, with a focus on how these approaches are used to inform policy and programme responses. A rapid scoping review was conducted on the use of VA and SA in humanitarian crises in low and middle-income countries since 1991. Drawing on a maximum variation approach, two settings of application ('application contexts') were selected and investigated via nine semi-structured expert interviews. VA can determine causes of death in crisis-affected populations where no other registration system is in place. Combined with SA and active community involvement, these methods can deliver a holistic view of obstacles to seeking and receiving essential healthcare, yielding context-specific information to inform appropriate responses. The contexts in which VA and SA are used require adaptations to standard tools, and new mobile developments in VA raise specific ethical considerations. Furthermore, collecting and sythesising data in a timely, continuous manner, and ensuring coordination and communication between agencies, is important to realise the potential of these approaches. VA and SA are valuable research methods to foster evidence-informed responses for populations affected by humanitarian crises. When coordinated and communicated effectively, data generated through these methods can help to identify levels, causes and circumstances of deaths among vulnerable groups, and can enable planning and allocating resources effectively, potentially improving health system resilience to future crises.


Imaging performance of annular apertures. 4: Apodization and point spread functions.
Apodizations with decreasing transmissions and with central obstructions transmute the point spread functions in image size and in the distribution of image irradiance. The distributions are characterized by the envelope functions which are exponential functions descending with variable exponents. At high apodization levels and lower central obstruction (<0.1) new extended radial zones in the outer part of the central ring groups are formed. Such transmutations of the image functions are of more than theoretical interest, particularly if the irradiance levels in the outer ring zones are to be compared to background irradiance levels.


Insights from ion mobility-mass spectrometry, infrared spectroscopy, and molecular dynamics simulations on nicotinamide adenine dinucleotide structural dynamics: NAD+vs. NADH.
Nicotinamide adenine dinucleotide (NAD) is found in all living cells where the oxidized (NAD+) and reduced (NADH) forms play important roles in many enzymatic reactions. However, little is known about NAD+ and NADH conformational changes and kinetics as a function of the cell environment. In the present work, an analytical workflow is utilized to study NAD+ and NADH dynamics as a function of the organic content in solution using fluorescence lifetime spectroscopy and in the gas-phase using trapped ion mobility spectrometry coupled to mass spectrometry (TIMS-MS) and infrared multiple photon dissociation (IRMPD) spectroscopy. NAD solution time decay studies showed a two-component distribution, assigned to changes from a "close" to "open" conformation with the increase of the organic content. NAD gas-phase studies using nESI-TIMS-MS displayed two ion mobility bands for NAD+ protonated and sodiated species, while four and two ion mobility bands were observed for NADH protonated and sodiated species, respectively. Changes in the mobility profiles were observed for NADH as a function of the starting solution conditions and the time after desolvation, while NAD+ profiles showed no dependence. IRMPD spectroscopy of NAD+ and NADH protonated species in the 800-1800 and 3200-3700 cm-1 spectral regions showed common and signature bands between the NAD forms. Candidate structures were proposed for NAD+ and NADH kinetically trapped intermediates of the protonated and sodiated species, based on their collision cross sections and IR profiles. Results showed that NAD+ and NADH species exist in open, stack, and closed conformations and that the driving force for conformational dynamics is hydrogen bonding of the N-H-O and O-H-O forms with ribose rings.


Sponge symbiosis is facilitated by adaptive evolution of larval sensory and attachment structures in barnacles.
Symbiotic relations and range of host usage are prominent in coral reefs and crucial to the stability of such systems. In order to explain how symbiotic relations are established and evolve, we used sponge-associated barnacles to ask three questions. (1) Does larval settlement on sponge hosts require novel adaptations facilitating symbiosis? (2) How do larvae settle and start life on their hosts? (3) How has this remarkable symbiotic lifestyle involving many barnacle species evolved? We found that the larvae (cyprids) of sponge-associated barnacles show a remarkably high level of interspecific variation compared with other barnacles. We document that variation in larval attachment devices are specifically related to properties of the surface on which they attach and metamorphose. Mapping of the larval and sponge surface features onto a molecular-based phylogeny showed that sponge symbiosis evolved separately at least three times within barnacles, with the same adaptive features being found in all larvae irrespective of phylogenetic relatedness. Furthermore, the metamorphosis of two species proceeded very differently, with one species remaining superficially on the host and developing a set of white calcareous structures, the other embedding itself into the live host tissue almost immediately after settlement. We argue that such a high degree of evolutionary flexibility of barnacle larvae played an important role in the successful evolution of complex symbiotic relationships in both coral reefs and other marine systems.


Pacing and Performance in Swimming: Differences Between Individual and Relay Events.
Although pacing is considered crucial for success in individual swimming events, there is a lack of research examining pacing in relays. The authors investigated the impact of start lap and pacing strategy on swimming performance and whether these strategies differ between relays and the corresponding individual event. Race data for 716 relay (4 × 200-m freestyle) finals from 14 international competitions between 2010 and 2018 were analyzed retrospectively. Each swimmer's individual 200-m freestyle season's best time for the same year was used for comparison. Races were classified as a fast, average, or slow start lap strategy (lap 1) and as an even, negative, or positive pacing strategy (laps 2-4) to give an overall race strategy, for example, average start lap even pacing. A fast start lap strategy was associated with slower 200-m times (range 0.5-0.9 s, P ≤ .04) irrespective of gender, and positive pacing led to slower 200-m (0.4-0.5 s, P ≤ .03) times in females. A fast start lap strategy led to positive pacing in 71% of swimmers. Half of the swimmers changed pacing strategy, with 13% and 7% more female and male swimmers, respectively, displaying positive pacing in relays compared with individual events. In relays, a fast start lap and positive pacing was utilized more frequently by swimmers positioned on second to fourth relay legs (+13%) compared with lead-off leg swimmers (+3%). To maximize performance, swimmers should be more conservative in the first lap and avoid unnecessary alterations in race strategy in relay events.


Image-based control of skin melanin texture.
We introduce a useful tool for controlling the skin melanin texture of facial photographs. Controlling the skin melanin texture is an important task in the reproduction of posters, TV commercials, movies, and so on. We used component maps of melanin, which were obtained by a previous method [J. Opt. Soc. Am. A 16, 2169 (1999)] as the first processing step. We propose to control the melanin texture continuously and physiologically, based on the analysis of 123 skin textures in our database. The physiological validity for the change of the melanin texture is confirmed by comparing the synthesized image with an ultraviolet image, which can be used to predict the change of melanin texture due to aging. The control processes are implemented on programmable graphics hardware, and real-time processing is achieved for a facial videostream.


An improved specimen loader for cryo-scanning electron microscopy.
Cryo-electron microscopy of cryofixed samples is a well-established and accepted technique for imaging liquid-containing specimens without removing water and other volatile components. There are many steps between cryofixation and cryo-observation in the microscope, during which the sample and sample holder need to be handled. One such major step is the loading of the specimen onto the sample holder and the fixing of the sample holder onto the transfer mechanism. During this handling, the specimen is often exposed (mostly inadvertently) to moisture in the atmosphere, which results in frost deposition. The new specimen loader described here is designed to overcome the traditional tedious handling and to achieve ease in specimen loading. The modifications made are mainly towards allowing movement of the liquid freon cup, eliminating the need for a lock-screw and improving the shape of the stage holder, which makes the mounting of the specimen holder easy, thereby permitting smooth specimen loading without too much handling and with consequent reduced frost deposition.


The myth of translational bioethics.
In recent years, the case has been made for special attention to be paid to a branch of research in the field of bioethics called 'translational bioethics'. In this paper, we start by considering some of the assumptions that those advancing translational approaches to bioethics make about bioethics and compare them to the reality of bioethics as an academic field. We move on to explain how those who make this case, implicitly or explicitly, for translational bioethics go awry because of how they understand the 'gap' between bioethical inquiry and practical settings that requires bridging. We consider three interpretations of this 'translation gap' in bioethics: (i) the gap between theory and practice, (ii) the gap between the force of normative claims and practical contextual realities and (iii) the gap between relevance or applicability to practice and actual application or implementation in practice. In each case, we show how a proper understanding of the nature of the academic field of bioethics undermines how these gaps have been formulated, and how any need for talk of 'translational bioethics' is removed.


Childhood trauma and dissociation in schizophrenia.
This study is concerned with relationships between childhood trauma history, dissociative experiences, and the clinical phenomenology of chronic schizophrenia. Seventy patients with a schizophrenic disorder were evaluated using the Structured Clinical Interview for DSM-IV, Dissociative Experiences Scale, Dissociative Disorders Interview Schedule, Positive and Negative Symptoms Scales, and Childhood Trauma Questionnaire. Childhood trauma scores were correlated with dissociation scale scores and dissociative symptom clusters, but not with core symptoms of the schizophrenic disorder. Cluster analysis identified a subgroup of patients with high dissociation and childhood trauma history. The dissociative subgroup was characterized by higher numbers of general psychiatric comorbidities, secondary features of dissociative identity disorder, Schneiderian symptoms, somatic complaints, and extrasensory perceptions. A significant majority of the dissociative subgroup fit the diagnostic criteria of DSM-IV borderline personality disorder concurrently. Among childhood trauma types, only physical abuse and physical neglect predicted dissociation. A trauma-related dissociative subtype of schizophrenia is supported. Childhood trauma is related to concurrent dissociation among patients with schizophrenic disorder. A duality model based on the interaction of 2 qualitatively distinct psychopathologies and a dimensional approach are proposed as possible explanations for the complex relationship between these 2 psychopathologies and childhood trauma.


Automated geographic atrophy segmentation for SD-OCT images using region-based C-V model via local similarity factor.
Age-related macular degeneration (AMD) is the leading cause of blindness among elderly individuals. Geographic atrophy (GA) is a phenotypic manifestation of the advanced stages of non-exudative AMD. Determination of GA extent in SD-OCT scans allows the quantification of GA-related features, such as radius or area, which could be of important value to monitor AMD progression and possibly identify regions of future GA involvement. The purpose of this work is to develop an automated algorithm to segment GA regions in SD-OCT images. An en face GA fundus image is generated by averaging the axial intensity within an automatically detected sub-volume of the three dimensional SD-OCT data, where an initial coarse GA region is estimated by an iterative threshold segmentation method and an intensity profile set, and subsequently refined by a region-based Chan-Vese model with a local similarity factor. Two image data sets, consisting on 55 SD-OCT scans from twelve eyes in eight patients with GA and 56 SD-OCT scans from 56 eyes in 56 patients with GA, respectively, were utilized to quantitatively evaluate the automated segmentation algorithm. We compared results obtained by the proposed algorithm, manual segmentation by graders, a previously proposed method, and experimental commercial software. When compared to a manually determined gold standard, our algorithm presented a mean overlap ratio (OR) of 81.86% and 70% for the first and second data sets, respectively, while the previously proposed method OR was 72.60% and 65.88% for the first and second data sets, respectively, and the experimental commercial software OR was 62.40% for the second data set.


Blood flow and thrombus formation determine the development of stent neointima.
To establish a relationship between flow, acute thrombus formation, and late intimal formation in implanted arterial stents, canine femoral arteries with normal blood flow were compared to contralateral femoral arteries with restricted blood flow. Thrombocyte activity over the stent segment was evaluated for 3 h after stent placement with nuclear scanning, following administration of In-111 labeled platelets. To evaluate long-term stent patency in relationship to arterial flow, an additional group of dogs were subjected to long-term observation. Matched, symmetrically implanted femoral stents with normal and restricted flow were explanted at 1, 12, and 24 weeks for histological analysis and comparative measurement of neointimal thickness. Angiographic studies were performed before and after nuclear scan in the acute animals and before explant in the chronic animals. Acutely, heparinization prevented subocclusive or occlusive thrombus, regardless of flow. In the absence of heparinization, normal arterial blood flow did not prevent thrombus formation on stents. Chronically, stents with flow restriction had significantly greater neointimal formation in comparison with unrestricted stents. Histological studies suggested that the stent neointima resulted from progressive replacement of stent thrombus.


Heart beats brain: The problem of detecting alpha waves by neuronal current imaging in joint EEG-MRI experiments.
It has been suggested recently that the influence of the neuro-magnetic field should make electrical brain activity directly detectable by MRI. To test this hypothesis, we performed combined EEG-MRI experiments which aim to localize the neuronal current sources of alpha waves (8-12 Hz), one of the most prominent EEG phenomena in humans. A detailed analysis of cross-spectral coherence between simultaneously recorded EEG and MRI time series revealed no sign of alpha waves. Instead the EEG-MRI approach was found to be hampered by artefacts due to cardiac pulsation, which extend into the frequency band of alpha waves. Separate brain displacement mapping experiments confirmed that not only the EEG but also the MRI signal is confounded by harmonics of the cardiac frequency even at 10 Hz and beyond. This well-known ballistocardiogram artefact cannot be avoided or eliminated entirely by available signal processing techniques. Therefore we must conclude that current EEG-MRI methodology based on correlation analysis lacks not only the sensitivity but also the specificity required for the reliable detection of alpha waves.


Capturing Cell-Cell Interactions via SNAP-tag and CLIP-tag Technology.
Juxtacrine or contact-dependent signaling is a major form of cell communication in multicellular organisms. The involved cell-cell and cell-extracellular-matrix (ECM) interactions are crucial for the organization and maintenance of tissue architecture and function. However, because cell-cell contacts are relatively weak, it is difficult to isolate interacting cells in their native state to study, for example, how specific cell types interact with others (e.g., stem cells with niche cells) or where they locate within tissues to execute specific tasks. To achieve this, we propose artificial in situ cell-to-cell linking systems that are based on SNAP-tag and CLIP-tag, engineered mutants of the human O6-alkylguanine-DNA alkyltransferase. Here we demonstrate that SNAP-tag can be utilized to efficiently and covalently tether cells to poly(ethylene glycol) (PEG)-based hydrogel surfaces that have been functionalized with the SNAP-tag substrate benzylguanine (BG). Furthermore, using PEG-based spherical microgels as an artificial cell model, we provide proof-of-principle for inducing clustering that mimics cell-cell pairing.


Anti-tumor necrosis factor antibody therapy fails to prevent lethality after cecal ligation and puncture or endotoxemia.
Cytokines have been studied intensively to delineate their role in the altered pathophysiology observed in septic shock. We studied the role of TNF in the lethality of two well characterized models of septic shock by inhibiting TNF's activity with a specific antibody. In the first model, sepsis was induced by cecal ligation and puncture (CLP), and in the second model sepsis was induced by either an i.p. or i.v. injection of LPS. After CLP, plasma endotoxin was detectable within 4 h and reached a peak at 8 h (136 +/- 109 ng/ml). TNF bioactivity peaked at 12 h (528 +/- 267 pg/ml) at a significantly higher level than sham-operated control mice (64 +/- 31 pg/ml). After i.p. LPS, TNF peaked much more quickly (90 min) compared with CLP and at a significantly higher level (107,900 +/- 25,000 pg/ml). Another cytokine studied in septic shock, IL-6, peaked at 12 h after CLP at 1011 +/- 431 pg/ml, and at 90 min after lethal LPS at 16,300 +/- 3,700 pg/ml. Mice were treated with an anti-TNF antibody that has been shown previously to inhibit in vivo TNF activity. Antibody treatment of mice subjected to CLP significantly reduced TNF bioactivity but did not reduce mortality or pulmonary neutrophilic infiltration. In the i.v. LPS model, anti-TNF antibody treatment concomitant with LPS injection reduced plasma TNF activity from 80,000 +/- 20,000 pg/ml to undetectable levels. However, anti-TNF treatment immediately before either i.v. or i.p. LPS did not reduce mortality. Additionally, when the antibody was administered 4 h before the lethal i.v. LPS, there was no reduction in lethality. These data show that in two separate models of septic shock blockade of TNF biologic activity will not prevent lethality.


Rituals can kill - A fatal case of brucine poisoning.
In some parts of India people follow a religious ritual of drinking an herbal preparation made from the bark of the Alstonia scholaris tree (Blackboard tree) on the day of the new moon in the month of July. This tree could be easily confused with the Strychnos nux vomica tree. Brucine is the predominant alkaloid present in the bark of the Strychnos nux vomica tree. The toxicological property of brucine is similar to strychnine. Brucine is a neurotoxin. A 29-year-old male presented with a history of consumption of an herbal preparation made from the bark of the Strychnos nux vomica tree confusing it for Alstonia scholaris. Soon after, he developed convulsions and later died in hospital on the same day. The aim of this case report is to highlight the fact that people must be cautious when they follow religious rituals.


[Clinical features of pseudoaneurysms complicating pancreatitis: single center experience and review of Korean literature].
Pseudoaneurysm is a life-threatening complication of chronic or acute pancreatitis. This study was undertaken to evaluate the clinical features of pseudoaneurysm complicating pancreatitis. We reviewed the medical records of 7 patients diagnosed as pseudoaneurysms with chronic pancreatitis in Korea University Guro and Anam Hospital from January 1995 to March 2006 and analyzed their demographics, clinical courses and outcomes. All patients were men and mean age was 54.6 years (range, 43-67 years). All the cases occurred in the setting of chronic alcoholic pancreatitis complicated by pseudocyst. Abdominal pain was the unique initial clinical symptom in 5 cases, hematemesis in 1 case, and simultaneous abdominal pain with hematemesis in 1 case. Bleeding into pseudocyst developed in 5 cases, flowing into duodenum through pancreatic duct in 1 case and rupture into the descending colon in 1 case. Mean duration between onset of symptom and diagnosis of pseudoaneurysm was 7.8 days (range, 1-23 days). Six cases were diagnosed by abdominal computed tomography disclosing characteristic finding of focal high density area in the pseudocyst. Pulsed doppler abdominal sonography was performed before computed tomography in 3 cases and results were negative in 2 cases. Transcatheter arterial embolizations were initially performed in 6 cases, and there was no recurrent bleeding except one case of splenic infarction. Distal pancreatectomy was initially performed in 1 case. Pseudoaneurysms complicating chronic pancreatitis shows various clinical features. Transcatheter arterial embolization can be recommended as a primary therapeutic modality.


Paracrine signaling through the epithelial estrogen receptor alpha is required for proliferation and morphogenesis in the mammary gland.
Estradiol is a major regulator of postnatal mammary gland development and thought to exert its effects through estrogen receptor alpha (ERalpha) expressed in the mammary gland stroma and epithelium. Previous studies, however, were confounded by the use of an ERalpha mutant strain that retains some of the protein with transactivation activity. Here, we use an ERalpha-/- mouse strain in which no ERalpha transcript can be detected to analyze mammary gland development in the complete absence of ERalpha signaling. The ERalpha-/- females show no development beyond a rudimentary ductal system. By grafting ERalpha-/- epithelium or stroma in combination with ERalpha WT stroma or epithelium, we show that the primary target for estradiol is the mammary epithelium, whereas a direct response of the mammary stroma is not required for mammary gland development to proceed normally. Mammary glands reconstituted with ERalpha-/- mammary epithelium exposed to pregnancy hormones show increased transcription of milk protein genes, indicating that ERalpha signaling is not an absolute requirement for a transcriptional response to pregnancy hormones. When ERalpha-/- mammary epithelial cells are in close vicinity to ERalpha WT cells, they proliferate and contribute to all aspects of mammary gland development, indicating that estradiol, like progesterone, orchestrates proliferation and morphogenesis by a paracrine mechanism, affecting nearby cells in the mammary epithelium.


A natural mineral supplement provides relief from knee osteoarthritis symptoms: a randomized controlled pilot trial.
This small, pilot study evaluated the impact of treatment with a natural multi-mineral supplement from seaweed (Aquamin) on walking distance, pain and joint mobility in subjects with moderate to severe osteoarthritis of the knee. Subjects (n = 70) with moderate to severe osteoarthritis of the knee were randomized to four double-blinded treatments for 12 weeks: (a) Glucosamine sulfate (1500 mg/d); (b) Aquamin (2400 mg/d); (c) Combined treatment composed of Glucosamine sulfate (1500 mg/d) plus Aquamin (2400 mg/d) and (d) Placebo. Primary outcome measures were WOMAC scores and 6 Minute Walking Distances (6 MWD). Laboratory based blood tests were used as safety measures. Fifty subjects completed the study and analysis of the data showed significant differences between the groups for changes in WOMAC pain scores over time (p = 0.009 ANCOVA); however, these data must be reviewed with caution since significant differences were found between the groups at baseline for WOMAC pain and stiffness scores (p = 0.0039 and p = 0.013, respectively, ANOVA). Only the Aquamin and Glucosamine groups demonstrated significant improvements in symptoms over the course of the study. The combination group (like the placebo group) did not show any significant improvements in OA symptoms in this trial. Within group analysis demonstrated significant improvements over time on treatment for the WOMAC pain, activity, composite and stiffness (Aquamin only) scores as well as the 6 minute walking distances for subjects in the Aquamin and Glucosamine treatment groups. The Aquamin and Glucosamine groups walked 101 feet (+7%) and 56 feet (+3.5%) extra respectively. All treatments were well tolerated and the adverse events profiles were not significantly different between the groups. This small preliminary study suggested that a multi mineral supplement (Aquamin) may reduce the pain and stiffness of osteoarthritis of the knee over 12 weeks of treatment and warrants further study. ClinicalTrials.gov number: NCT00452101.


Self-Healing Dyes-Keeping the Promise?
Self-healing dyes have emerged as a new promising class of fluorescent labels. They consist of two units, a fluorescent dye and a photostabilizer. The latter heals whenever the fluorescent dye is in danger of taking a reaction pathway toward photobleaching. We describe the underlying concepts and summarize the developmental history and state-of-the-art, including latest applications in high-resolution microscopy, live-cell, and single-molecule imaging. We further discuss remaining limitations, which are (i) lower photostabilization of most self-healing dyes when compared to solution additives, (ii) limited mechanistic understanding on the influence of the biochemical environment and molecular oxygen on self-healing, and (iii) the lack of cheap and facile bioconjugation strategies. Finally, we provide ideas on how to further advance self-healing dyes, show new data on redox blinking caused by double-stranded DNA, and highlight forthcoming work on intramolecular photostabilization of fluorescent proteins.


International Union of Pharmacology. LXIII. Retinoid X receptors.
The physiological effects of retinoic acids (RAs) are mediated by members of two families of nuclear receptors, the retinoic acid receptors (RARs) and the retinoid X receptors (RXRs), which are encoded by three distinct human genes, RXRalpha, RXRbeta, and RXRgamma. RARs bind both all-trans- and 9-cis-RA, whereas only the 9-cis-RA stereoisomer binds to RXRs. As RXR/RAR heterodimers, these receptors control the transcription of RA target genes through binding to RA-response elements. This review is focused on the structure, mode of action, ligands, expression, and pharmacology of RXRs. Given their role as common partners to many other members of the nuclear receptor superfamily, these receptors have been the subject of intense scrutiny. Moreover, and despite numerous studies since their initial discovery, RXRs remain enigmatic nuclear receptors, and there is still no consensus regarding their role. Indeed, multiple questions about the actual biological role of RXRs and the existence of an endogenous ligand have still to be answered.


Sensor monitoring to measure and support daily functioning for independently living older people: a systematic review and road map for further development.
To study sensor monitoring (use of a sensor network placed in the home environment to observe individuals' daily functioning (activities of daily living and instrumental activities of daily living)) as a method to measure and support daily functioning for older people living independently at home. Systematic review. Participants' homes. Community-dwelling individuals aged 65 and older. A systematic search in PubMed, Embase, PsycINFO, INSPEC, and The Cochrane Library was performed for articles published between 2000 and October 2012. All study designs, studies that described the use of wireless sensor monitoring to measure or support daily functioning for independently living older people, studies that included community-dwelling individuals aged 65 and older, and studies that focused on daily functioning as a primary outcome measure were included. Seventeen articles met the inclusion criteria. Nine studies used sensor monitoring solely as a method for measuring daily functioning and detecting changes in daily functioning. These studies focused on the technical investigation of the sensor monitoring method used. The other studies investigated clinical applications in daily practice. The sensor data could enable healthcare professionals to detect alert conditions and periods of decline and could enable earlier intervention, although limited evidence of the effect of interventions was found in these studies because of a lack of high methodological quality. Studies on the effectiveness of sensor monitoring to support people in daily functioning remain scarce. A road map for further development is proposed.


Gene synteny comparisons between different vertebrates provide new insights into breakage and fusion events during mammalian karyotype evolution.
Genome comparisons have made possible the reconstruction of the eutherian ancestral karyotype but also have the potential to provide new insights into the evolutionary inter-relationship of the different eutherian orders within the mammalian phylogenetic tree. Such comparisons can additionally reveal (i) the nature of the DNA sequences present within the evolutionary breakpoint regions and (ii) whether or not the evolutionary breakpoints occur randomly across the genome. Gene synteny analysis (E-painting) not only greatly reduces the complexity of comparative genome sequence analysis but also extends its evolutionary reach. E-painting was used to compare the genome sequences of six different mammalian species and chicken. A total of 526 evolutionary breakpoint intervals were identified and these were mapped to a median resolution of 120 kb, the highest level of resolution so far obtained. A marked correlation was noted between evolutionary breakpoint frequency and gene density. This correlation was significant not only at the chromosomal level but also sub-chromosomally when comparing genome intervals of lengths as short as 40 kb. Contrary to previous findings, a comparison of evolutionary breakpoint locations with the chromosomal positions of well mapped common fragile sites and cancer-associated breakpoints failed to reveal any evidence for significant co-location. Primate-specific chromosomal rearrangements were however found to occur preferentially in regions containing segmental duplications and copy number variants. Specific chromosomal regions appear to be prone to recurring rearrangement in different mammalian lineages ('breakpoint reuse') even if the breakpoints themselves are likely to be non-identical. The putative ancestral eutherian genome, reconstructed on the basis of the synteny analysis of 7 vertebrate genome sequences, not only confirmed the results of previous molecular cytogenetic studies but also increased the definition of the inferred structure of ancestral eutherian chromosomes. For the first time in such an analysis, the opossum was included as an outgroup species. This served to confirm our previous model of the ancestral eutherian genome since all ancestral syntenic segment associations were also noted in this marsupial.


Expression of a minimal K+ channel protein in mammalian cells and immunolocalization in guinea pig heart.
The minimal K+ channel protein (minK, also called IsK) is structurally dissimilar to other cloned voltage-gated ion channels. minK is a 15-kD polypeptide with only one potential transmembrane helix. Published data suggest that the current associated with minK expression in Xenopus oocytes may be related to the slow cardiac delayed rectifier K+ current (IKs). However, the fact that minK expression has been limited exclusively to Xenopus oocytes has caused continuing concern about the nature of this protein and its molecular link to known mammalian K+ channels. We report in the present study the first expression of minK activity in transiently transfected mammalian (HEK 293) cells and demonstrate that the characteristics of the expressed minK current are similar to those of IKs recorded from guinea pig heart cells under similar experimental conditions. We also show that an antibody directed against the minK channel protein reacts with a surface antigen on adult guinea pig ventricular myocytes and sinoatrial nodal cells, where IKs is the dominant outward K+ current. The data provide strong evidence that a minK-like protein underlies IKs.


The accessory subunit of Xenopus laevis mitochondrial DNA polymerase gamma increases processivity of the catalytic subunit of human DNA polymerase gamma and is related to class II aminoacyl-tRNA synthetases.
Peptide sequences obtained from the accessory subunit of Xenopus laevis mitochondrial DNA (mtDNA) polymerase gamma (pol gamma) were used to clone the cDNA encoding this protein. Amino-terminal sequencing of the mitochondrial protein indicated the presence of a 44-amino-acid mitochondrial targeting sequence, leaving a predicted mature protein with 419 amino acids and a molecular mass of 47.3 kDa. This protein is associated with the larger, catalytic subunit in preparations of active mtDNA polymerase. The small subunit exhibits homology to its human, mouse, and Drosophila counterparts. Interestingly, significant homology to glycyl-tRNA synthetases from prokaryotic organisms reveals a likely evolutionary relationship. Since attempts to produce an enzymatically active recombinant catalytic subunit of Xenopus DNA pol gamma have not been successful, we tested the effects of adding the small subunit of the Xenopus enzyme to the catalytic subunit of human DNA pol gamma purified from baculovirus-infected insect cells. These experiments provide the first functional evidence that the small subunit of DNA pol gamma stimulates processive DNA synthesis by the human catalytic subunit under physiological salt conditions.


Role of inflammation in diabetic nephropathy.
Diabetic nephropathy (DN) is the leading cause of end stage renal disease (ESRD). Although the pathogenesis of DN is multifactorial, local inflammatory stress may result from both the metabolic and hemodynamic derangements observed in DN. Inflammatory markers such as Interleukin-18 and Tumor Necrosis Factor (TNF)-alpha are increased in the serum of patients with diabetes and DN. This occurs at a very early stage of disease, and correlates with the degree of albuminuria. Recent data suggest that standard pharmacologic interventions for DN, such as angiotensin converting enzyme inhibitors, angiotensin receptor blockers and aldosterone antagonists, may have anti-inflammatory properties that are independent of their hemodynamic effect. Although inflammation is traditionally thought of as a process resulting in macrophage infiltration, current scientific progress has lead to the novel idea that even cells distant from the blood stream, such as podocytes, can produce cytokines and can express molecules that are part of the co-stimulatory pathway. A strong translational research effort is currently aimed at defining the role of such molecules in cells other than lymphocytes and macrophages. Experimental animal models have recently provided evidence that some acute phase markers of inflammation such as intracellular cell adhesion molecule-1 (ICAM-1), TNF-alpha and Monocytes Chemoattractant Protein-1 (MCP-1) may have a causative role in the development of DN. Here, we review the current evidence supporting the role of inflammation in the early phases of clinical and experimental DN. A complete understanding of inflammatory pathways activated in DN may lead to the discovery of earlier and more reliable markers of DN than albuminuria and the identification of novel therapeutic targets.


Selective D-dimer testing for diagnosis of a first suspected episode of deep venous thrombosis: a randomized trial.
D-Dimer testing is sensitive but not specific for diagnosing deep venous thrombosis (DVT). Changing the use of testing and the threshold level for a positive test result on the basis of risk for DVT might improve the tradeoff between sensitivity and specificity and reduce the need for testing. To determine whether using a selective D-dimer testing strategy based on clinical pretest probability (C-PTP) for DVT is safe and reduces diagnostic testing compared with using a single D-dimer threshold for all patients. Randomized, multicenter, controlled trial. Patients were allocated using a central automated system. Ultrasonographers and study adjudicators but not other study personnel were blinded to trial allocation. (ClinicalTrials.gov: NCT00157677) 5 Canadian hospitals. Consecutive symptomatic patients with a first episode of suspected DVT. Selective testing (n = 860), defined as D-dimer testing for outpatients with low or moderate C-PTP (DVT excluded at D-dimer levels <1.0 µg/mL [low C-PTP] or <0.5 µg/mL [moderate C-PTP]) and venous ultrasonography without D-dimer testing for outpatients with high C-PTP and inpatients, or uniform testing (n = 863), defined as D-dimer testing for all participants (DVT excluded at D-dimer levels <0.5 µg/mL). The proportion of patients not diagnosed with DVT during initial testing who had symptomatic venous thromboembolism during 3-month follow-up and the proportion of patients undergoing D-dimer testing and ultrasonography. The incidence of symptomatic venous thromboembolism at 3 months was 0.5% in both study groups (difference, 0.0 percentage point [95% CI, -0.8 to 0.8 percentage points]). Selective testing reduced the proportion of patients who required D-dimer testing by 21.8 percentage points (CI, 19.1 to 24.8 percentage points). It reduced the proportion who required ultrasonography by 7.6 percentage points (CI, 2.9 to 12.2 percentage points) overall and by 21.0 percentage points (CI, 14.2 to 27.6 percentage points) in outpatients with low C-PTP. Results may not be generalizable to all D-dimer assays or patients with previous DVT, study personnel were not blinded, and the trial was stopped prematurely. A selective D-dimer testing strategy seems as safe as and more efficient than having everyone undergo D-dimer testing when diagnosing a first episode of suspected DVT. Heart and Stroke Foundation of Ontario.


Fibronectin-dependent collagen I deposition modulates the cell response to fibronectin.
Communication between cells and the extracellular matrix (ECM) is critical for regulation of cell growth, survival, migration, and differentiation. Remodeling of the ECM can occur under normal physiological conditions, as a result of tissue injury, and in certain pathological conditions. ECM remodeling leads to alterations in ECM composition and organization that can alter many aspects of cell behavior, including cell migration. The cell migratory response varies depending on the type, amount, and organization of ECM molecules present, as well as the integrin and proteoglycan repertoire of the cells. We and others have shown that the deposition of several ECM molecules, including collagen types I and III, depends on the presence and stability of ECM fibronectin. Hence, the effect of fibronectin and fibronectin matrix on cell function may partially depend on its ability to direct the deposition of collagen in the ECM. In this study, we used collagen-binding fibronectin mutants and recombinant peptides that interfere with fibronectin-collagen binding to show that fibronectin-dependent collagen I deposition regulates the cell migratory response to fibronectin. These data show that the ability of fibronectin to organize other proteins in the ECM is an important aspect of fibronectin function and highlight the importance of understanding how interactions between ECM proteins influence cell behavior.


Effect of in utero exposure to diethylstilbestrol on lumbar and femoral bone, articular cartilage, and the intervertebral disc in male and female adult mice progeny with and without swimming exercise.
Developmental exposure to estrogens has been shown to affect the musculoskeletal system. Furthermore, recent studies have shown that environmental exposure to estrogen-like compounds is much higher than originally anticipated. The aim of this study was to determine the effects of diethylstilbestrol (DES), a well-known estrogen agonist, on articular cartilage, intervertebral disc (IVD), and bone phenotype. C57Bl/6 pregnant mice were dosed orally with vehicle (peanut oil) or 0.1, 1.0, and 10 μg/kg/day of DES on gestational days 11 to 14. Male and female pups were allowed to mature without further treatment until 3 months of age, when swim and sedentary groups were formed. After euthanasia, bone mineral density (BMD), bone mineral content (BMC), bone area (BA), and trabecular bone area (TBA) of the lumbar vertebrae and femur were measured by using a PIXImus Bone Densitometer System. Intervertebral disc proteoglycan was measured with the DMMB assay. Histologic analysis of proteoglycan for IVD and articular cartilage was performed with safranin O staining, and degeneration parameters were scored. The lumbar BMC was significantly increased in female swimmers at both the highest and lowest dose of DES, whereas the femoral BMC was increased only at the highest. The males, conversely, showed a decreased BMC at the highest dose of DES for both lumbar and femoral bone. The female swim group had an increased BA at the highest dose of DES, whereas the male counterpart showed a decreased BA for femoral bone. The TBA showed a similar pattern. Proteoglycan analysis of lumbar IVDs showed a decrease at the lowest doses but a significant increase at the highest doses for both males and females. Histologic examination showed morphologic changes of the IVD and articular cartilage for all doses of DES. DES significantly affected the musculoskeletal system of adult mice. Results suggest that environmental estrogen contaminants can have a detrimental effect on the developmental lumbar bone growth and mineralization in mice. Further studies measuring the impact of environmental estrogen mimics, such as bisphenol A, are then warranted.


Technical limitations of dual-energy CT in neuroradiology: 30-month institutional experience and review of literature.
Dual-energy CT (DECT) has been shown to be a useful modality in neuroradiology. To assess failure modes and limitations of DECT in different neuroimaging applications. Dual-source DECT scans were performed in 72 patients over 30 months to differentiate contrast agent staining or extravasation from intracranial hemorrhage (ICH) (n=40); to differentiate calcium from ICH (n=2); for metal-artifact reduction (n=5); and for angiographic assessment (n=25). A three-material decomposition algorithm was used to obtain virtual non-contrast (VNC) and iodine (or calcium) overlay images. Images were analyzed in consensus by two board-certified radiologists to determine the success of the algorithm and to assess confounding factors. Furthermore, a dilution experiment using cylinders containing defined heparinized swine blood, normal saline, and selected iodine concentrations was conducted to assess other possible confounding factors. Dual-energy analysis was successful in 65 (90.2%) patients. However, the algorithm failed when images were affected by beam hardening (n=3, 4.2%), the presence of a fourth material (parenchymal calcification) (n=3, 4.2%), or motion (n=1, 1.4%). In the dilution experiment, a saturation effect was seen at high iodine concentrations (≥37 mg/ml). VNC and iodine overlay images were not reliable above this concentration, and beam-hardening artifacts were noted. DECT material decomposition is usually successful in neuroradiology. However, it can only distinguish up to three preselected materials. A fourth material such as parenchymal calcium may confound the analysis. Artifacts such as beam hardening, metallic streak, or saturation effect can also impair material decomposition.


A Role of Sp1 Binding Motifs in Basal and Large T-Antigen-Induced Promoter Activities of Human Polyomavirus HPyV9 and Its Variant UF-1.
Human polyomavirus 9 (HPyV9) was originally detected in the serum of a renal transplant patient. Seroepidemiological studies showed that ~20-50% of the human population have antibodies against this virus. HPyV9 has not yet been associated with any disease and little is known about the route of infection, transmission, host cell tropism, and genomic variability in circulating strains. Recently, the HPyV9 variant UF-1 with an eight base-pair deletion, a thirteen base-pair insertion and with point mutations, creating three putative Sp1 binding sites in the late promoter was isolated from an AIDS patient. Transient transfection studies with a luciferase reporter plasmid driven by HPyV9 or UF1 promoter demonstrated that UF1 early and late promoters were stronger than HPyV9 promoters in most cell lines, and that the UF1 late promoter was more potently activated by HPyV9 large T-antigen (LTAg). Mutation of two Sp1 motifs strongly reduced trans-activation of the late UF1 promoter by HPyV9 LTAg in HeLa cells. In conclusion, the mutations in the UF1 late promoter seem to strengthen its activity and its response to stimulation by HPyV9 LTAg in certain cells. It remains to be investigated whether these promoter changes have an influence on virus replication and affect the possible pathogenic properties of the virus.


Influence of platelet-vessel wall interactions on leukocyte rolling in vivo.
The influence of platelet-vessel wall interactions on leukocyte rolling was investigated in rabbit mesenteric venules (diameter, 21-40 microns) using intravital videomicroscopy. Puncture of the wall with glass micropipettes (tip, 6-8 microns) evoked the formation of a thrombus in all venules. In most vessels, emboli were produced as well. The rolling of leukocytes (i.e., their movement along the vessel wall at a velocity clearly lower than that of the other blood cells) was quantitated simultaneously in vessel segments upstream and downstream from a thrombus up to 10 minutes after puncture. During embolization the number of rolling leukocytes decreased significantly from the upstream to the downstream vessel segment (median decrease, 45%; p less than or equal to 0.001). It was still decreased by approximately 50% after embolization had stopped, indicating that the decrease in leukocyte rolling was not caused by inclusion of leukocytes in the emboli. In venules without embolization, leukocyte rolling did not change systematically, indicating that fluid dynamic changes induced by the thrombus do not influence leukocyte rolling. Inhibition of prostaglandin formation with aspirin (100 mg/kg) almost completely abolished the influence of the thromboembolic reaction on leukocyte rolling, but blockade of thromboxane A2 receptors with sulotroban (30 mg/kg) had no effect. In conclusion, this is the first report on a functional interaction in vivo, at a site of vessel wall injury, between platelets, vascular cells, and leukocytes. The findings suggest that substances produced by activated platelets and/or damaged vascular cells diminish leukocyte rolling. The identity of these substances is not yet clear, but the present study indicates that prostaglandins other than thromboxane A2 are involved.


Addressing harmful algal blooms (HABs) impacts with ferrate(VI): Simultaneous removal of algal cells and toxins for drinking water treatment.
Although ferrate(VI) has long been recognized as a multi-purpose treatment agent, previous investigations regarding ferrate(VI) for addressing harmful algal blooms (HABs) impacts in drinking water treatment only focused on a single HAB pollutant (e.g. algal cells or algal toxins). Moreover, the performance of ferrate(VI)-driven coagulation was poorly investigated in comparison with ferrate(VI) oxidation, though it has been widely acknowledged as a major ferrate(VI) treatment mechanism. We herein reported ferrate(VI) as an emerging agent for simultaneous and effective removal of algal cells and toxins in a simulated HAB-impacted water. Ferrate(VI)-driven oxidation enabled algal cell inactivation and toxin decomposition. Subsequently, Fe(III) from ferrate(VI) reduction initiated an in-situ coagulation for cell aggregation. Cell viability (initial 4.26 × 104 cells/mL at pH 5.5 and 5.16 × 104 cells/mL at pH 7.5) decreased to 0.0% at ≥ 7 mg/L Fe(VI) at pH 5.5 and 7.5, respectively. Cell density and turbidity were dramatically decreased at pH 5.5 once ferrate(VI) doses were beyond their respective threshold levels, which are defined as minimum effective iron doses (MEIDs). However, the particulate removal at pH 7.5 was poor, likely because the coagulation was principally driven by charge neutralization and a higher pH could not sufficiently lower the particle surface charge. Meanwhile, algal toxins (i.e., microcystins) of 3.98 μg/L could be substantially decomposed at either pH. And the greater degradation achieved at pH 5.5 was due to the higher reactivity of ferrate(VI) at the lower pH. This study represents the first step toward the ferrate(VI) application as a promising approach for addressing multiple HABs impacts for water treatment.


Immunohistochemical panel for distinguishing esophageal adenocarcinoma from squamous cell carcinoma: a combination of p63, cytokeratin 5/6, MUC5AC, and anterior gradient homolog 2 allows optimal subtyping.
Distinguishing adenocarcinoma and squamous cell carcinoma of the esophagus is often based on morphological criteria and can be difficult in small biopsies. We analyzed commonly used immunohistochemical markers (p63, cytokeratin 5/6, cytokeratin 7, CDX2, MUC2, and MUC5AC) and 2 new markers, anterior gradient homolog 2 and SOX2, in esophageal carcinomas to establish the best panel to distinguish these tumors. Tissue microarrays with 69 esophageal adenocarcinomas and 41 whole sections of esophageal squamous cell carcinomas were stained for these markers and semiquantitatively scored. Sensitivities and specificities were calculated for individual markers and select combinations using the morphological diagnosis as a gold standard. All squamous cell carcinomas expressed p63 with 38 of 41 demonstrating reactivity in more than 75% of tumor cells. Cytokeratin 5/6 expression was seen in 40 of 41 squamous cell carcinomas with 39 of 41 demonstrating reactivity in more than 75% of tumor cells. SOX2 expression was present in 35 of 41 of squamous cell carcinomas but also in 24 of 69 of adenocarcinomas, frequently demonstrating extensive reactivity in adenocarcinomas. Anterior gradient homolog 2 was highly sensitive for adenocarcinoma and present in 68 of 69 of cases, but anterior gradient homolog 2 reactivity was also identified in 15 of 41 of squamous cell carcinomas, typically demonstrating focal reactivity in squamous cell carcinoma. MUC5AC expression was seen almost exclusively in adenocarcinomas with only a single squamous cell carcinoma demonstrating focal MUC5AC staining. Overall, the dual expression of both p63 and cytokeratin 5/6 was 99% specific and 98% sensitive for squamous cell carcinoma. In addition, anterior gradient homolog 2 and MUC5AC are useful positive markers of adenocarcinoma in the setting of absent or diminished p63 and cytokeratin 5/6 staining.


Homeostatic imbalance of purine catabolism in first-episode neuroleptic-naïve patients with schizophrenia.
Purine catabolism may be an unappreciated, but important component of the homeostatic response of mitochondria to oxidant stress. Accumulating evidence suggests a pivotal role of oxidative stress in schizophrenia pathology. Using high-pressure liquid chromatography coupled with a coulometric multi-electrode array system, we compared 6 purine metabolites simultaneously in plasma between first-episode neuroleptic-naïve patients with schizophrenia (FENNS, n = 25) and healthy controls (HC, n = 30), as well as between FENNS at baseline (BL) and 4 weeks (4w) after antipsychotic treatment. Significantly higher levels of xanthosine (Xant) and lower levels of guanine (G) were seen in both patient groups compared to HC subjects. Moreover, the ratios of G/guanosine (Gr), uric acid (UA)/Gr, and UA/Xant were significantly lower, whereas the ratio of Xant/G was significantly higher in FENNS-BL than in HC. Such changes remained in FENNS-4w with exception that the ratio of UA/Gr was normalized. All 3 groups had significant correlations between G and UA, and Xan and hypoxanthine (Hx). By contrast, correlations of UA with each of Xan and Hx, and the correlation of Xan with Gr were all quite significant for the HC but not for the FENNS. Finally, correlations of Gr with each of UA and G were significant for both HC and FENNS-BL but not for the FENNS-4w. During purine catabolism, both conversions of Gr to G and of Xant to Xan are reversible. Decreased ratios of product to precursor suggested a shift favorable to Xant production from Xan, resulting in decreased UA levels in the FENNS. Specifically, the reduced UA/Gr ratio was nearly normalized after 4 weeks of antipsychotic treatment. In addition, there are tightly correlated precursor and product relationships within purine pathways; although some of these correlations persist across disease or medication status, others appear to be lost among FENNS. Taken together, these results suggest that the potential for steady formation of antioxidant UA from purine catabolism is altered early in the course of illness.