Combining biological gene expression signatures in predicting outcome in breast cancer: An alternative to supervised classification.
Gene expression profiling has been extensively used to predict outcome in breast cancer patients. We have previously reported on biological hypothesis-driven analysis of gene expression profiling data and we wished to extend this approach through the combinations of various gene signatures to improve the prediction of outcome in breast cancer. We have used gene expression data (25.000 gene probes) from a previously published study of tumours from 295 early stage breast cancer patients from the Netherlands Cancer Institute using updated follow-up. Tumours were assigned to three prognostic groups using the previously reported Wound-response and hypoxia-response signatures, and the outcome in each of these subgroups was evaluated. We have assigned invasive breast carcinomas from 295 stages I and II breast cancer patients to three groups based on gene expression profiles subdivided by the wound-response signature (WS) and hypoxia-response signature (HS). These three groups are (1) quiescent WS/non-hypoxic HS; (2) activated WS/non-hypoxic HS or quiescent WS/hypoxic tumours and (3) activated WS/hypoxic HS. The overall survival at 15 years for patients with tumours in groups 1, 2 and 3 are 79%, 59% and 27%, respectively. In multivariate analysis, this signature is not only independent of clinical and pathological risk factors; it is also the strongest predictor of outcome. Compared to a previously identified 70-gene prognosis profile, obtained with supervised classification, the combination of signatures performs roughly equally well and might have additional value in the ER-negative subgroup. In the subgroup of lymph node positive patients, the combination signature outperforms the 70-gene signature in multivariate analysis. In addition, in multivariate analysis, the WS/HS combination is a stronger predictor of outcome compared to the recently reported invasiveness gene signature combined with the WS. A combination of biological gene expression signatures can be used to identify a powerful and independent predictor for outcome in breast cancer patients.


Pharmacokinetics of cefepime and comparison with those of ceftiofur in horses.
To determine pharmacokinetics of i.v., i.m., and oral administration of cefepime in horses and to compare pharmacokinetics of i.m. administration of cefepime with those of ceftiofur sodium. 6 clinically normal adult horses. Horses received 3 doses of cefepime (11 mg/kg of body weight, PO; 2.2 mg/kg, i.v.; and 2.2 mg/kg, i.m.) and 1 dose of ceftiofur (2.2 mg/kg, i.m.). Two horses also received L-arginine, p.o. and i.v., at doses identical to those contained in the cefepime dihydrochloride-L-arginine preparations previously administered. Blood samples were collected for 24 hours after administration of cefepime or ceftiofur and were assayed for cefepime and ceftiofur concentrations. Pharmacokinetic analysis of disposition data indicated that i.v. administration data were best described by a 2-compartment open model, whereas i.m. administration data were best described by a 1-compartment absorption model. Median elimination half-life and volume of distribution after i.v. administration of cefepime were 125.7 minutes and 225 ml/kg, respectively. After i.m. administration of cefepime, mean maximal plasma concentration of (8.13 microg/ml) was reached at a mean time of 80 minutes. Absorption of cefepime after i.m. administration was complete, with a median bioavailability of 1.11. Intramuscular administration of ceftiofur resulted in similar mean maximal plasma concentration (7.98 microg/ml) and mean time to this concentration (82 minutes). Cefepime was not detected in samples collected after oral administration. Adverse effects consisting principally of gastrointestinal disturbances were observed after oral and i.m. administration of cefepime and after 1 i.m. administration of ceftiofur. Cefepime, administered i.v. or i.m. at a dosage of 2.2 mg/kg, every 8 hours is likely to provide effective antibacterial therapy for cefepime-sensitive organisms in horses. Further studies are needed to evaluate adverse effects on the gastrointestinal tract.


Informed consent in paediatric critical care research--a South African perspective.
Medical care of critically ill and injured infants and children globally should be based on best research evidence to ensure safe, efficacious treatment. In South Africa and other low and middle-income countries, research is needed to optimise care and ensure rational, equitable allocation of scare paediatric critical care resources. Ethical oversight is essential for safe, appropriate research conduct. Informed consent by the parent or legal guardian is usually required for child research participation, but obtaining consent may be challenging in paediatric critical care research. Local regulations may also impede important research if overly restrictive. By narratively synthesising and contextualising the results of a comprehensive literature review, this paper describes ethical principles and regulations; potential barriers to obtaining prospective informed consent; and consent options in the context of paediatric critical care research in South Africa. Voluntary prospective informed consent from a parent or legal guardian is a statutory requirement for child research participation in South Africa. However, parents of critically ill or injured children might be incapable of or unwilling to provide the level of consent required to uphold the ethical principle of autonomy. In emergency care research it may not be practical to obtain consent when urgent action is required. Therapeutic misconceptions and sociocultural and language issues are also barriers to obtaining valid consent. Alternative consent options for paediatric critical care research include a waiver or deferred consent for minimal risk and/or emergency research, whilst prospective informed consent is appropriate for randomised trials of novel therapies or devices. We propose that parents or legal guardians of critically ill or injured children should only be approached to consent for their child's participation in clinical research when it is ethically justifiable and in the best interests of both child participant and parent. Where appropriate, alternatives to prospective informed consent should be considered to ensure that important paediatric critical care research can be undertaken in South Africa, whilst being cognisant of research risk. This document could provide a basis for debate on consent options in paediatric critical care research and contribute to efforts to advocate for South African law reform.


The Association Between Smoking Abstinence and Pain Trajectory Among Veterans Engaged in U.S. Department of Veterans Affairs Mental Health Care.
To prospectively examine associations between smoking and nicotine abstinence and pain trajectory over 12 months among smokers with low, moderate, and severe pain and to assess whether these associations differ over time. A secondary analysis of the "Proactive Outreach for Smokers in VA Mental Health" study, a randomized controlled trial of proactive outreach for veteran smokers engaged in U.S. Department of Veterans Affairs (VA) mental health care. Participants were categorized into "low" (n = 616), "moderate" (n = 479), and "severe" pain (n = 656) groups according to baseline pain score. Associations between self-reported abstinence from smoking and nicotine at 6 and 12 months and pain trajectory, measured via the PEG scale (Pain intensity, Enjoyment of life, General activity) composite score, were assessed through the use of general linear mixed models. Interaction tests assessed whether these associations differed at 6 and 12 months. Analyses were conducted within the overall sample and within the separate pain groups. There were significant interactions in the overall sample and the low and moderate pain groups, such that 7-day point prevalence smoking abstinence was associated with lower pain scores at 6 but not 12 months. In the severe pain group, 7-day abstinence from both smoking and nicotine was associated with lower pain scores across both time points. Six-month prolonged abstinence was not associated with pain scores. In this prospective analysis conducted among veteran smokers engaged in mental health services, 7-day abstinence from smoking and nicotine was associated with significantly lower levels of pain. Education efforts could help better inform smokers on the relationship between smoking and pain.


Validation of two canine STR multiplex-assays following the ISFG recommendations for non-human DNA analysis.
To gain general acceptance forensic DNA testing in animals needs to improve standardization of analysis methods and data interpretation. Recently, the International Society of Forensic Genetics (ISFG) took particular care of this topic by publishing recommendations for forensic non-human DNA analysis following the successful example of human DNA analysis in order to provide a basis for harmonization of the still existing inter-laboratory variability. By following these recommendations we demonstrate the performance of two short tandem repeat (STR) multiplexes for forensic identity testing of canine biological material. Thirteen STRs and two sex-specific markers were selected and validated according to the ISFG guidelines. Population genetic parameters were calculated based on 295 dog samples collected in Austria (124) and Germany (171). A repeat-based nomenclature of the mainly tetrameric STRs and corresponding allelic ladders are presented. All 146 different alleles included in the ladders were sequenced for correct allele calling. Additionally, a canine cell line (DH82-D3167) was evaluated as standard reference material.


Cluster randomized trial in smoking cessation with intensive advice in diabetic patients in primary care. ITADI Study.
It is a priority to achieve smoking cessation in diabetic smokers, given that this is a group of patients with elevated cardiovascular risk. Furthermore, tobacco has a multiplying effect on micro and macro vascular complications. Smoking abstinence rates increase as the intensity of the intervention, length of the intervention and number and diversity of contacts with the healthcare professional during the intervention increases. However, there are few published studies about smoking cessation in diabetics in primary care, a level of healthcare that plays an essential role in these patients. Therefore, the aim of the present study is to evaluate the effectiveness of an intensive smoking cessation intervention in diabetic patients in primary care. Cluster randomized trial, controlled and multicentric. Randomization unit: Primary Care Team. 546 diabetic smokers older than 14 years of age whose disease is controlled by one of the primary care teams in the study. Continuous tobacco abstinence (a person who has not smoked for at least six months and with a CO level of less than 6 ppm measured by a cooximeter) , evolution in the Prochaska and DiClemente's Transtheoretical Model of Change, number of cigarettes/day, length of the visit. Point of assessment: one- year post- inclusion in the study. Brief motivational interview for diabetic smokers at the pre-contemplation and contemplation stage, intensive motivational interview with pharmacotherapy for diabetic smokers in the preparation-action stage and reinforcing intevention in the maintenance stage. A descriptive analysis of all variables will be done, as well as a multilevel logistic regression and a Poisson regression. All analyses will be done with an intention to treatment basis and will be fitted for potential confounding factors and variables of clinical importance. Statistical packages: SPSS15, STATA10 y HLM6. The present study will try to describe the profile of a diabetic smoker who receives the most benefit from an intensive intervention in primary care. The results will be useful for primary care professionals in their usual clinical practice. Clinical Trials.gov Identifier: NCT00954967.


MR imaging of magnetic ink patterns via off-resonance sensitivity.
Printed magnetic ink creates predictable B0 field perturbations based on printed shape and magnetic susceptibility. This can be exploited for contrast in MR imaging techniques that are sensitized to off-resonance. The purpose of this work was to characterize the susceptibility variations of magnetic ink and demonstrate its application for creating MR-visible skin markings. The magnetic susceptibility of the ink was estimated by comparing acquired and simulated B0 field maps of a custom-built phantom. The phantom was also imaged using a 3D gradient echo sequence with a presaturation pulse tuned to different frequencies, which adjusts the range of suppressed frequencies. Healthy volunteers with a magnetic ink pattern pressed to the skin or magnetic ink temporary flexible adhesives applied to the skin were similarly imaged. The volume-average magnetic susceptibility of the ink was estimated to be 131 ± 3 parts per million across a 1-mm isotropic voxel (13,100 parts per million assuming a 10-μm thickness of printed ink). Adjusting the saturation frequency highlights different off-resonant regions created by the ink patterns; for example, if tuned to suppress fat, fat suppression will fail near the ink due to the off-resonance. This causes magnetic ink skin markings placed over a region with underlying subcutaneous fat to be visible on MR images. Patterns printed with magnetic ink can be imaged and identified with MRI. Temporary flexible skin adhesives printed with magnetic ink have the potential to be used as skin markings that are visible both by eye and on MR images.


Identification of estrogenic compounds in wastewater effluent.
In order to identify the dominant contributors to estrogenic activity in environmental waters, a comprehensive fractionation method using silica gel column chromatography, combined with recombinant yeast assay for detecting estrogenic activity and with gas chromatography-mass spectrometry for quantifying endocrine disruptors and natural estrogens, was developed. The method was applied to the municipal sewage treatment plant (STP) secondary effluent discharged to the Tamagawa River in Tokyo, Japan, where endocrine disruption was observed in wild carp. The instrumental analysis demonstrated that averaged concentrations of nonylphenol, bisphenol A, estrone (E1), and 17beta-estradiol (E2) were 564 +/- 127, 27 +/- 19, 33 +/- 11, and 4.6 +/- 3.0 ng/L, respectively. Based on the concentration and relative potency of these compounds, the natural estrogens E1 and E2 represented more than 98% of the total estrogen equivalent concentration (EEQ) in the STP effluent, while the contribution of phenolic compounds to total EEQ was less than 2%. Estrogenic activities associated with the dissolved phase of the effluent samples were detected by a recombinant yeast assay. By using silica gel column chromatography, the dissolved phase was separated into several fractions that were subjected to the bioassay. The polar fractions exhibited estrogenic activity. The greatest estrogenic activity was found in a polar fraction containing E1 and E2 and represented 66 to 88% of the total estrogenic activities estimated from the bioassay data. These results lead to the conclusion that E1 and E2 were the dominant environmental estrogens in the STP effluent, but a significant contribution to estrogenic activities stems from unidentified components in the effluents.


Grape Seed Proanthocyanidin Ameliorates Cardiac Toxicity Induced by Boldenone Undecylenate through Inhibition of NADPH Oxidase and Reduction in the Expression of NOX2 and NOX4.
The effect of anabolic androgenic steroids on the cardiovascular system is poorly understood. Increased production of free radicals is coupled to the pathophysiology of many alterations within the circulatory system. The only function of the enzyme family NADPH oxidases (NOXs) is the generation of reactive oxygen species (ROS). Therefore, this study investigated the beneficial role of grape seed proanthocyanidin extract (GSPE) in ameliorating cardiac toxicity induced by the anabolic steroid Boldenone in male rats through NOX inhibition and reduction in the expression of NOX2 and NOX4. This study was conducted on forty male rats which are divided into four groups (normal control, positive control or GSPE, Boldenone, and posttreatment Boldenone with GSPE). A significant increase in relative body weight, relative heart weight, and hemodynamic parameters, as well as serum concentrations of lactate dehydrogenase, creatine kinase, creatine kinase-muscle brain, myoglobin, cholesterol, low-density lipoprotein cholesterol, risk factor 1/2, K+, and Cl-, in treated rats with Boldenone when compared with control. We also noted a significant increase in the levels of cardiac malondialdehyde, H2O2 generation in heart tissues, mRNA expression of NOX2 and NOX4, and immunoreactivity to proliferating cell nuclear antigen (PCNA). Posttreated rats with Boldenone and GSPE ameliorated cardiac toxicity via inhibition of NOX and a reduction in alteration of the expression of NOX2, NOX4, and PCNA induced by Boldenone. These novel insights into the antioxidative activity of GSPE should serve as a basis for the development of improved chemopreventive or therapeutic strategies for cardiac toxicity.


Determinants of risky sexual behavior among injecting drug users (IDUs) in Georgia.
Injection risk practices and risky sexual behaviors place injection drug users (IDUs) and their sexual partners particularly vulnerable to HIV. The purpose of the study was to describe and understand determinants of high-risk sexual behavior among IDUs in Georgia. A cross-sectional, anonymous survey assessed knowledge, behavior and HIV status in IDUs in five Georgian cities (Tbilisi, Gori, Telavi, Zugdidi, Batumi) in 2009. The study enrolled in total 1,127 (1,112 males, 15 females) IDUs. Results indicate that occasional sexual relationships are common among male IDUs, including married ones. A subsample of 661 male IDUs who reported having occasional and paid sex partners during the last 12 months was analyzed. Multivariate analysis shows that not having a regular partner in the last 12 month (adjusted odds ratio (aOR) 1.57, 95 % CI 1.04 2.37), and using previously used needles/syringes at last injecting (aOR 2.37, 95 % I 1.10-5.11) are independent correlates of inconsistent condom use with occasional and paid sexual partners among IDUs. Buprenorphine injectors have lower odds of inconsistent condom use with occasional and paid sexual partners compared to heroin injectors (aOR 0.47, 95 % CI 0.27-0.80), and IDUs who live in Telavi are twice more likely to engage in such risky sexual behavior than capital city residents (aOR 2.55, 95 % CI 1.46-4.48). More effective programs focused on sexual risk behavior reduction strategies should be designed and implemented.


Filipin-sensitive caveolae-mediated transport in endothelium: reduced transcytosis, scavenger endocytosis, and capillary permeability of select macromolecules.
Caveolae or noncoated plasmalemmal vesicles found in a variety of cells have been implicated in a number of important cellular functions including endocytosis, transcytosis, and potocytosis. Their function in transport across endothelium has been especially controversial, at least in part because there has not been any way to selectively inhibit this putative pathway. We now show that the ability of sterol binding agents such as filipin to disassemble endothelial noncoated but not coated plasmalemmal vesicles selectively inhibits caveolae-mediated intracellular and transcellular transport of select macromolecules in endothelium. Filipin significantly reduces the transcellular transport of insulin and albumin across cultured endothelial cell monolayers. Rat lung microvascular permeability to albumin in situ is significantly decreased after filipin perfusion. Conversely, paracellular transport of the small solute inulin is not inhibited in vitro or in situ. In addition, we show that caveolae mediate the scavenger endocytosis of conformationally modified albumins for delivery to endosomes and lysosomes for degradation. This intracellular transport is inhibited by filipin both in vitro and in situ. Other sterol binding agents including nystatin and digitonin also inhibit this degradative process. Conversely, the endocytosis and degradation of activated alpha 2-macroglobulin, a known ligand of the clathrin-dependent pathway, is not affected. Interestingly, filipin appears to inhibit insulin uptake by endothelium for transcytosis, a caveolae-mediated process, but not endocytosis for degradation, apparently mediated by the clathrin-coated pathway. Such selective inhibition of caveolae not only provides critical evidence for the role of caveolae in the intracellular and transcellular transport of select macromolecules in endothelium but also may be useful for distinguishing transport mediated by coated versus noncoated vesicles.


Retina-specific expression of PDGF-B versus PDGF-A: vascular versus nonvascular proliferative retinopathy.
Platelet-derived growth factor (PDGF) has been implicated in vascular proliferative retinopathies, such as diabetic retinopathy, and in nonvascular retinopathies, such as proliferative vitreoretinopathy. Traction retinal detachment is a central feature of both types of disease. Hemizygous rhodopsin promoter/PDGF-B (rho/PDGF-B) transgenic mice exhibit proliferation of vascular cells, glia, and retinal pigmented epithelial (RPE) cells, resulting in traction retinal detachment. Hemizygous rho/PDGF-A transgenic mice show mild proliferation of glial cells and no traction retinal detachments. This study was undertaken to determine whether higher levels of endogenously produced PDGF-A in the retinas of mice result in retinal detachment. To achieve high-level expression of PDGF-A in the retina, homozygous rho/PDGF-A (rho/PDGF-AA) mice were generated. The phenotype of these mice was compared with that of homozygous rho/PDGF-B (rho/PDGF-BB) mice and double hemizygous rho/PDGF-B-rho/PDGF-A (rho/PDGF-AB) mice. Rho/PDGF-BB and rho/PDGF-AB mice showed a phenotype similar to that previously described in rho/PDGF-B mice. There was extensive proliferation of glial and vascular cells, resulting in fibrovascular membranes that detached the retina. PDGF-AA mice showed extensive proliferation of glial cells and traction retinal detachment. High retinal expression of PDGF-A results in extensive proliferation of glial cells and traction retinal detachment without vascular cell involvement, similar to proliferative vitreoretinopathy in humans. High retinal expression of PDGF-B results in traction retinal detachment from proliferation of both vascular and nonvascular cells, similar to diabetic retinopathy in humans.


Structural and functional neuroprotection in a rat model of Huntington's disease by viral gene transfer of GDNF.
Huntington's disease (HD) is an autosomal dominant disorder caused by an expanded polyglutamine (CAG) tract at the IT15 locus on chromosome 4. These excessive repeats lead to the degeneration of striatal and cortical neurons resulting in a devastating cognitive, psychiatric, and motor disorder for which no treatments are available. Neurotrophic factors support the viability of striatal neurons suggesting that they might prevent the inevitable neural degeneration and its accompanying functional decline associated with HD. The present study investigated whether glial cell line-derived neurotrophic factor (GDNF) delivered by an adeno associated virus could provide structural and functional neuroprotection in a rat model of HD. Lewis rats received bilateral injections of either AAV-GDNF (n = 12) or AAV-green fluorescence protein (AAV-GFP, n = 12) into the striatum followed 2 weeks later by chronic subcutaneous infusions of the mitochondrial toxin, 3-nitropropionic acid (3-NP, 38 mg/kg). All rats underwent 4 weeks of behavioral testing and were then sacrificed. Following 3-NP, the performance by AAV-GFP-treated rats on a raised platform motor task deteriorated while the performance by AAV-GDNF-treated rats was near normal (P < 0.001). AAV-GDNF-treated rats also received better scores on a blinded semi-quantitative neurological scale compared to rats receiving AAV-GFP (P < 0.001). Histological analyses supported our behavioral findings. 3-NP-treated rats receiving AAV-GDNF displayed 70% more NeuN-immunoreactive neurons compared to 3-NP-treated rats receiving AAV-GFP (P = 0.002). Similar findings were seen with dopamine-and-adenosine-3'5'-monophosphate-regulated phosphoprotein (DARPP-32) staining. These data indicate that the viral-mediated gene transfer of GDNF into the striatum provides neuroanatomical and behavioral protection in a rodent model of HD.


Comparative phenotypic and molecular genetic profiling of wild Lactococcus lactis subsp. lactis strains of the L. lactis subsp. lactis and L. lactis subsp. cremoris genotypes, isolated from starter-free cheeses made of raw milk.
Twenty Lactococcus lactis strains with an L. lactis subsp. lactis phenotype isolated from five traditional cheeses made of raw milk with no added starters belonging to the L. lactis subsp. lactis and L. lactis subsp. cremoris genotypes (lactis and cremoris genotypes, respectively; 10 strains each) were subjected to a series of phenotypic and genetic typing methods, with the aims of determining their phylogenetic relationships and suitability as starters. Pulsed-field gel electrophoresis (PFGE) analysis of intact genomes digested with SalI and SmaI proved that all strains were different except for three isolates of the cremoris genotype, which showed identical PFGE profiles. Multilocus sequence typing (MLST) analysis using internal sequences of seven loci (namely, atpA, rpoA, pheS, pepN, bcaT, pepX, and 16S rRNA gene) revealed considerable intergenotype nucleotide polymorphism, although deduced amino acid changes were scarce. Analysis of the MLST data for the present strains and others from other dairy and nondairy sources showed that all of them clustered into the cremoris or lactis genotype group, by using both independent and combined gene sequences. These two groups of strains also showed distinctive carbohydrate fermentation and enzyme activity profiles, with the strains in the cremoris group showing broader profiles. However, the profiles of resistance/susceptibility to 16 antibiotics were very similar, showing no atypical resistance, except for tetracycline resistance in three identical cremoris genotype isolates. The numbers and concentrations of volatile compounds produced in milk by the strains belonging to these two groups were clearly different, with the cremoris genotype strains producing higher concentrations of more branched-chain, derived compounds. Together, the present results support the idea that the lactis and cremoris genotypes of phenotypic Lactococcus lactis subsp. lactis actually represent true subspecies. Some strains of the two subspecies in this study appear to be good starter candidates.


Nomogram to Assess the Survival Benefit of New Salvage Agents for Metastatic Urothelial Carcinoma in the Era of Immunotherapy.
Optimal end points in phase 2 trials evaluating salvage therapy for metastatic urothelial carcinoma are necessary to identify promising drugs, particularly immunotherapeutics, where response and progression-free survival may be unreliable. We developed a nomogram using data from phase 2 trials of historical agents to estimate the 12-month overall survival (OS) for patients to which observed survival of nonrandomized data sets receiving immunotherapies could be compared. Survival and data for major prognostic factors were obtained from phase 2 trials: hemoglobin, performance status, liver metastasis, treatment-free interval, and albumin. A nomogram was developed to estimate 12-month OS. Patients were randomly allotted to discovery:validation data sets in a 2:1 ratio. Calibration plots were constructed in the validation data set and data bootstrapped to assess performance. The nomogram was tested on external nonrandomized cohorts of patients receiving pemetrexed and atezolizumab. Data were available from 340 patients receiving sunitinib, everolimus, docetaxel + vandetanib, docetaxel + placebo, pazopanib, paclitaxel, or docetaxel. Calibration and prognostic ability were acceptable (c index = 0.634; 95% confidence interval [CI], 0.596-0.652). Observed 12-month survival for patients receiving pemetrexed (n = 127, 23.5%; 95% CI, 16.2-31.7) was similar to nomogram-predicted survival (19%; 95% CI, 16.5-21.5; P > .05), while observed results with atezolizumab (n = 403, 39.0%; 95% CI, 34.1-43.9) exceeded predicted results (24.6%; 95% CI, 23.4-25.8; P < .001). This nomogram may be a useful tool to interpret results of nonrandomized phase 2 trials of salvage therapy for metastatic urothelial carcinoma by assessing the OS contributions of drug intervention independent of prognostic variables.


Reconfigurable interconnections using photorefractive holograms.
Using coupled wave theory and the law of refraction, diffraction properties of volume holograms are discussed. Reconfigurable interconnections by either wavelength tuning or spatial division techniques are proposed. Reflection type volume holograms can be used for a large number of reconfigurable interconnections in terms of finite wavelength tunability. Transmission volume holograms encoded in pinhole holograms can be easily reconfigured by spatial light modulator. Experimental demonstrations obtained by using these methods are presented.


Patterns of hepatic and splenic colonization by an attenuated strain of Salmonella typhimurium containing the gene for human interleukin-2: a novel anti-tumor agent.
Currently, there is no effective treatment for unresectable hepatic malignancies. Salmonella sp. are known to naturally track to the liver during active infection. A live biological vector was developed for delivery of Interleukin-2 (IL-2) to the liver for anti-tumor purposes. The avirulent and highly immunogenic c4550 strain of Salmonella typhimurium was used to express the IL-2 protein [renamed c4550(pIL-2)]. We have previously demonstrated that the c4550(pIL-2) produces biologically active IL-2 (up to 46.2 IU/ml) and that a single gavage feeding of 10(7) colony forming units (cfu) of c4550(pIL-2) significantly reduced the number of hepatic metastases when compared to animals fed salmonella lacking the IL-2 gene or non-treated controls. The goal of the current studies was to determine the pattern of splenic and hepatic colonization of Salmonella-IL2. Hepatic and splenic colonization was determined following administration of 10(7) cfu of c4550(pIL-2) and c4550(pYA292) via a single gavage feeding to C57BL/6 mice. Five experiments of antibiotic regimen administration were conducted where splenic and hepatic homogenates were cultured after 14 days of parenteral and/or oral antibiotics. The natural history of hepatic and splenic colonization was also determined for animals without antibiotic treatment. Despite administration of various antibiotic regimens using different routes, eradication of salmonella with and without IL-2 was not achieved. Salmonella, however, was not cultured from hepatic and splenic tissue at 4 months after a single gavage feeding of salmonella with no specific treatment. In conclusion, oral administration of c4550(pIL-2) may represent a novel form of in vivo biotherapy for unresectable hepatic malignancies. Antibiotics do not accelerate eradication of this bacteria and it appears that c4550(pIL-2) follows the natural pathophysiological of salmonella infection in which eradication from the splenic and hepatic tissue occurs over a period of 2-4 months.


Robo signalling controls pancreatic progenitor identity by regulating Tead transcription factors.
A complex interplay of intrinsic factors and extrinsic signalling pathways controls both cell lineage commitment and maintenance of cell identity. Loss of defined cellular states is the cause of many different cancers, including pancreatic cancer. Recent findings suggest a clinical role for the conserved SLIT/ROBO signalling pathway in pancreatic cancer. However, whilst this pathway has been extensively studied in many processes, a role for Slit and Robo genes in pancreas cell identity and plasticity has not been established yet. Here, we identify Slit/Robo signalling as a key regulator of pancreatic progenitor identity. We find that Robo1 and Robo2 are required for preserving pancreatic cell identity shortly after fate induction and, subsequently, for expansion of the pancreatic progenitor pool in the mouse. Furthermore, we show that Robo receptors control the expression of Tead transcription factors as well as its downstream transcriptional activity. Our work identifies an interplay between Slit/Robo pathway and Tead intrinsic regulators, functioning as gatekeeper of pancreatic cell identity.


Expression of Drosophila lamin C is developmentally regulated: analogies with vertebrate A-type lamins.
Vertebrate nuclear lamins form a multigene family with developmentally controlled expression. In contrast, invertebrates have long been thought to contain only a single lamin, which in Drosophila is the well-characterized lamin Dm0. Recently, however, a Drosophila cDNA clone (pG-IF) has been identified that codes for an intermediate filament protein which harbors a nuclear localization signal but lacks a carboxy-terminal CAAX motif. Based on these data the putative protein encoded by pG-IF was tentatively called Drosophila lamin C. To address whether the pG-IF encoded protein is expressed and whether it encodes a cytoplasmic intermediate filament protein or a nuclear lamin we raised antibodies against the recombinant pG-IF protein. The antibodies decorate the nuclear envelope in Drosophila Kc tissue culture cells as well as in salivary and accessory glands demonstrating that pG-IF encodes a nuclear lamin (lamin C). Antibody decoration, in situ hybridization, western and northern blotting studies show that lamin C is acquired late in embryogenesis. In contrast, lamin Dm0 is constitutively expressed. Lamin C is first detected in late stage 12 embryos in oenocytes, hindgut and posterior spiracles and subsequently also in other differentiated tissues. In third instar larvae lamins C and Dm0 are coexpressed in all tissues tested. Thus, Drosophila has two lamins: lamin Dm0, containing a CaaX motif, is expressed throughout, while lamin C, lacking a CaaX motif, is expressed only later in development. Expression of Drosophila lamin C is similar to that of vertebrate lamin A (plus C), which loses its CaaX motif during incorporation into the lamina.


Surgery under extreme conditions in the aftermath of the 2010 Haiti earthquake: the importance of regional anesthesia.
The 12 January 2010 earthquake that struck Port-au-Prince, Haiti caused >200,000 deaths, thousands of injuries requiring immediate surgical interventions, and 1.5 million internally displaced survivors. The earthquake destroyed or disabled most medical facilities in the city, seriously hampering the ability to deliver immediate life- and limb-saving surgical care. A Project Medishare/University of Miami Miller School of Medicine trauma team deployed to Haiti from Miami within 24 hours of the earthquake. The team began work at a pre-existing tent facility in the United Nations (UN) compound based at the airport, where they encountered 225 critically injured patients. However, non-sterile conditions, no means to administer oxygen, the lack of surgical equipment and supplies, and no anesthetics precluded the immediate delivery of general anesthesia. Despite these limitations, resuscitative care was administered, and during the first 72 hours following the event, some amputations were performed with local anesthesia. Because of these austere conditions, an anesthesiologist, experienced and equipped to administer regional block anesthesia, was dispatched three days later to perform anesthesia for limb amputations, debridements, and wound care using single shot block anesthesia until a better equipped tent facility was established. After four weeks, the relief effort evolved into a 250-bed, multi-specialty trauma/intensive care center staffed with >200 medical, nursing, and administrative staff. Within that timeframe, the facility and its staff completed 1,000 surgeries, including spine and pediatric neurological procedures, without major complications. This experience suggests that when local emergency medical resources are completely destroyed or seriously disabled, a surgical team staffed and equipped to provide regional nerve block anesthesia and acute pain management can be dispatched rapidly to serve as a bridge to more advanced field surgical and intensive care, which takes longer to deploy and set up.


Reduced plasma noradrenaline concentrations in simple-obese and diabetic obese patients.
1. Obesity may be associated with a decreased activity of the sympathetic nervous system and also with a deficiency of the response to stimuli activating the sympathetic nervous system. As insulin activates the sympathetic nervous system, the present study was undertaken to measure the plasma noradrenaline concentration in the fasting state and after 75 g of oral glucose in simple-obese patients (n = 13), in non-insulin-dependent (type 2) diabetic patients (n = 37) and in normal control subjects (n = 12). 2. The fasting plasma noradrenaline concentration was similarly reduced in the simple-obese group and in the diabetic obese group compared with the control group (P = 0.005). The glucose-induced increase in plasma noradrenaline concentration was normal in the simple-obese group, but was abolished in the obese diabetic patients (P = 0.008). 3. Step-wise regression analysis indicated that independent effects on the fasting plasma noradrenaline concentration were exerted by age (r = +0.32, P = 0.002), glucose concentration (r = -0.32, P = 0.02) and the degree of obesity (r = -0.37, P = 0.007), but not by plasma insulin concentration. 4. These results show that obese patients have a reduced fasting plasma noradrenaline concentration independently of accompanying diabetes, but that the response of noradrenaline to oral glucose is only abolished in the obese diabetic patients.


[Ill-defined causes of death and unattended deaths, Brazil, 2003].
We studied the distribution of deaths from ill-defined causes that occurred in Brazil during 2003, from which was identified the proportion of unattended deaths. Data were obtained from the Mortality Information System, coordinated by the Ministry of Health. Causes of death included in "Chapter XVIII - Symptoms, signs and abnormal clinical and laboratory findings, not classified elsewhere" of the International Statistical Classification of Diseases and Related Health Problems, tenth revision, were considered ill-defined, among which the category R98 identified "unattended deaths". In Brazil during 2003 the underlying causes of 13.3% of deaths were included in the Chapter of ill-defined causes, and the highest proportions of these deaths occurred in the Northeast and North Regions. Considering the total deaths from ill-defined causes, 53 % correspond to unattended deaths. This proportion increased to over 70% in the states of Maranhão, Piauí, Rio Grande do Norte, Pernambuco, Bahia, Paraíba and Alagoas. Due to the decentralized structure of data collection in the country, we believe that the municipalities bear the major responsibility, followed by the states, for upgrading the quality of mortality statistics.


Individual and collective problem-solving in a foraging context in the leaf-cutting ant Atta colombica.
In this paper we investigate the flexibility of foraging behavior in the leaf-cutting ant Atta colombica, both at the individual and collective levels, following a change in the physical properties of their environment. We studied in laboratory conditions the changes occurring in foraging behavior when a height constraint was placed 1 cm above part of the trail linking the nest to the foraging area. We found that the size and shape of the fragments of foraging material brought back to the nest were significantly modified when the constraint was placed on the trail: independent of their size, forager ants cut smaller and rounder fragments in the presence of a height constraint than in its absence. This size adjustment does not require any direct sensory feedback because it occurred when the ants cut fragments in the foraging area; no further cutting was done when they encountered the constraint. This points to the existence of a template that ants store and use as a reference to adjust their reach while cutting. Remarkably, despite the decrease in the foraging material brought to the nest per capita the colony was still able to improve its foraging performance by doubling the number of transporters. This study illustrates the flexibility of foraging behavior exhibited by an ant colony. It provides a rare example of insects finding an intelligent solution to a problem occurring in a foraging context, at both the individual and collective levels.


Metachronal μ-Cilia for On-Chip Integrated Pumps and Climbing Robots.
Biological cilia often perform metachronal motion, that is, neighboring cilia move out of phase creating a travelling wave, which enables highly efficient fluid pumping and body locomotion. Current methods for creating metachronal artificial cilia suffer from the complex design and sophisticated actuation schemes. This paper demonstrates a simple method to realize metachronal microscopic magnetic artificial cilia (μMAC) through control over the paramagnetic particle distribution within the μMAC based on their tendency to align with an applied magnetic field. Actuated by a 2D rotating uniform magnetic field, the metachronal μMAC enable strong microfluidic pumping and soft robot locomotion. The metachronal μMAC induce twice the pumping efficiency and 3 times the locomotion speed of synchronously moving μMAC. The ciliated soft robots show an unprecedented slope climbing ability (0 to 180°), and they display strong cargo-carrying capacity (>10 times their own weight) in both dry and wet conditions. These findings advance the design of on-chip integrated pumps and versatile soft robots, among others.


Synaptic plasticity in the hippocampus of a APP/PS1 mouse model of Alzheimer's disease is impaired in old but not young mice.
Alzheimer disease (AD) is a neurodegenerative disorder for which there is no cure. We have investigated synaptic plasticity in area CA1 in a novel AD mouse model (APPPS1-21) which expresses the Swedish mutation of APP and the L166P mutation of human PS-1. This model shows initial plaque formation at 2 months in the neocortex and 4 months in the hippocampus and displays beta-amyloid-associated pathologies and learning impairments. We tested long-term potentiation (LTP) and short term potentiation (paired-pulse facilitation, PPF) of synaptic transmission in vivo in area CA1 of the hippocampus. There was no difference in LTP or PPF at 4-5 months of age in APPPS1-21 mice compared to littermate controls. At 6 months of age there was also no difference in LTP but APPPS1-21 mice showed slightly increased PPF (p<0.03). In 8 months old mice, LTP was greatly impaired in APPPS-21 animals (p<0.0001) while PPF was not changed. At 15 months of age, APPPS1-21 mice showed again impaired LTP compared to littermate controls (p<0.005), and PPF was also significantly reduced at 80 ms (p<0.005) and 160 ms (p<0.01) interstimulus interval. Immunohistological analysis showed only modest amyloid deposition in the hippocampus at 4 and 6 months with a robust increase up to 15 months of age. Our results suggest that increased formation and aggregation of beta amyloid with aging is responsible for the impaired LTP with aging in this mouse model, while the transient increase of PPF at 6 months of age is caused by some other mechanism.


Stability of flumazenil with selected drugs in 5% dextrose injection.
The compatibility and stability of flumazenil mixed with aminophylline, dobutamine hydrochloride, dopamine hydrochloride, cimetidine hydrochloride, famotidine, ranitidine hydrochloride, heparin sodium, lidocaine hydrochloride, or procainamide hydrochloride were studied. Flumazenil (1.0 mg) was mixed with each of the other drugs in polyvinyl chloride minibags containing 50 mL of 5% dextrose injection to achieve concentrations commonly used when the drugs are administered by continuous i.v. infusion. All admixtures were analyzed for color change, pH, and particulate matter. Flumazenil concentrations were measured by a stability-indicating high-performance liquid chromatographic assay at baseline and 2, 4, 8, 12, and 24 hours after mixing. No detectable changes in color, pH, or particulate matter were observed. In the presence of each drug except dopamine hydrochloride, at least 95% of initial flumazenil concentration was present 24 hours after mixing. Flumazenil in the presence of dopamine hydrochloride was stable for 12 hours. Flumazenil was visually compatible and chemically stable when admixed with 5% dextrose injection and selected drugs.


Adsorption of reactive dyes onto chitosan/montmorillonite intercalated composite: multi-response optimization, kinetic, isotherm and thermodynamic study.
Chitosan/montmorillonite intercalated composite (CTS/MMT) was used as an effective adsorbent for removal of reactive dyes, i.e. Reactive Black 5 (RB5), Reactive Red 136 (RR136), Reactive Yellow 145 (RY145) and Reactive Blue 222 (RB222). Taguchi method attached grey relational analysis was applied to determine the optimal adsorption conditions, including pH, initial concentration of dye, temperature, adsorbent dosage and contact time, for achieving simultaneous maximization of removal percentage and adsorption capacity. The percentage contribution of each adsorption condition was determined in the analysis of variance and showed that the most effective parameter in removal of RB5, RY145 and RB222 is the dye solution pH, whereas the initial concentration was the determining factor for optimum efficiency for the dye RR136. Under respective optimal condition, the removal percentages and adsorption capacity of four reactive dyes onto CTS/MMT were both found in the following order: RR136 > RY145 > RB5 > RB222. The maximum removal percentages of 78.8 and 49.5%, and the adsorption capacity of 315.20 and 123.75 mg/g were obtained for RR136 and RB222, respectively. The adsorption behaviors showed that the adsorption kinetics and isotherms were in best agreement with Avrami fractionary order model and the Toth isotherm, respectively.


Effects of bisphenol A on incidence and severity of cardiac lesions in the NCTR-Sprague-Dawley rat: A CLARITY-BPA study.
The goal of this study was to determine whether bisphenol A (BPA) had adverse effects indicative of cardiac toxicity. As part of the "Consortium Linking Academic and Regulatory Insights on BPA Toxicity" (CLARITY-BPA), study dams and offspring were exposed by daily gavage to five doses of BPA ranging from 2.5 to 25000μg/kg/day, 0.05 or 0.5μg/kg/day 17α-ethinyl-estradiol (EE) or 0.3% carboxymethylcellulose vehicle. Exposure-related effects were analyzed in isolated hearts by quantitative morphometry and histopathology. No dose-related changes in body weight were detected. Across all exposure groups including vehicle controls, body weight of continuously dosed males was reduced compared to males dosed only until PND21. Heart weight was increased only in females exposed to EE, and consistent alterations in LV wall thickness were not observed. Exposure-related changes in collagen accumulation were minor and limited to highest EE exposure groups with increased collagen accumulation in PND21 males. Decreased collagen was observed in hearts of BPA or EE exposed females at PND90 and PND180. In BPA or EE treated females cardiomyopathy incidence and severity was significantly increased compared to control females at PND21 with myocardial degeneration observed in both males and females at PND21 and PND90.


Severe acute respiratory syndrome-associated coronavirus vaccines formulated with delta inulin adjuvants provide enhanced protection while ameliorating lung eosinophilic immunopathology.
Although the severe acute respiratory syndrome-associated coronavirus (SARS-CoV) epidemic was controlled by nonvaccine measures, coronaviruses remain a major threat to human health. The design of optimal coronavirus vaccines therefore remains a priority. Such vaccines present major challenges: coronavirus immunity often wanes rapidly, individuals needing to be protected include the elderly, and vaccines may exacerbate rather than prevent coronavirus lung immunopathology. To address these issues, we compared in a murine model a range of recombinant spike protein or inactivated whole-virus vaccine candidates alone or adjuvanted with either alum, CpG, or Advax, a new delta inulin-based polysaccharide adjuvant. While all vaccines protected against lethal infection, addition of adjuvant significantly increased serum neutralizing-antibody titers and reduced lung virus titers on day 3 postchallenge. Whereas unadjuvanted or alum-formulated vaccines were associated with significantly increased lung eosinophilic immunopathology on day 6 postchallenge, this was not seen in mice immunized with vaccines formulated with delta inulin adjuvant. Protection against eosinophilic immunopathology by vaccines containing delta inulin adjuvants correlated better with enhanced T-cell gamma interferon (IFN-γ) recall responses rather than reduced interleukin-4 (IL-4) responses, suggesting that immunopathology predominantly reflects an inadequate vaccine-induced Th1 response. This study highlights the critical importance for development of effective and safe coronavirus vaccines of selection of adjuvants based on the ability to induce durable IFN-γ responses. Coronaviruses such as SARS-CoV and Middle East respiratory syndrome-associated coronavirus (MERS-CoV) cause high case fatality rates and remain major human public health threats, creating a need for effective vaccines. While coronavirus antigens that induce protective neutralizing antibodies have been identified, coronavirus vaccines present a unique problem in that immunized individuals when infected by virus can develop lung eosinophilic pathology, a problem that is further exacerbated by the formulation of SARS-CoV vaccines with alum adjuvants. This study shows that formulation of SARS-CoV spike protein or inactivated whole-virus vaccines with novel delta inulin-based polysaccharide adjuvants enhances neutralizing-antibody titers and protection against clinical disease but at the same time also protects against development of lung eosinophilic immunopathology. It also shows that immunity achieved with delta inulin adjuvants is long-lived, thereby overcoming the natural tendency for rapidly waning coronavirus immunity. Thus, delta inulin adjuvants may offer a unique ability to develop safer and more effective coronavirus vaccines.


Prediction of trauma-specific death rates of pedestrians of Fars Province, Iran.
Pedestrians are the most vulnerable group to accidents among road users. Due to the well-known concerns and complications of accidents involving pedestrians, the aim of this study was to identify the rate of such accidents for five-year period. We analyzed all fatalities among pedestrians caused by traffic accidents during years of 2009-2013 in Fars Province in Iran. The study was a cross-sectional study in which logistic regression analysis was used to predict the death rate among pedestrians. Sensitivity analysis using the Monte Carlo method was used to increase the accuracy of the results. Then, we predicted the death rates for the years 2014-2018 predicted and compared the results with the actual data from the previous five-year period (2009-2013). During 2009-2013, 1723 out of 8689 (20.3%) of the people killed in traffic accidents were pedestrians. The death rate for male pedestrians in 2011 was estimated to be 10.86 per 100,000 (with an uncertainty interval of 95% giving a range of 9.85-12.05 per 100,000). Compared to the data for 2006, this represented a decrease of 20% (with a mean decrease of 4% per year). Based on these data, the death date in 2018n was projected to be 8.08 per 100,000 (with an uncertainty interval of 95% giving a range of 7.26-8.87). Similar data and analysis for women indicated that the reduction in the rate of fatalities has been smaller than that for men in recent years, i.e., 2.2% versus 4%. Although great progress has been made in reducing traffic accidents, to date, the death rate is still high among pedestrians. It is essential to continue to find ways to reduce traffic accidents and the pedestrians' deaths associated with them, especially among the elderly, who make up a disproportionate fraction of the deaths.


Design and rationale of FOCUS (PX-171-011): a randomized, open-label, phase 3 study of carfilzomib versus best supportive care regimen in patients with relapsed and refractory multiple myeloma (R/R MM).
Carfilzomib is a next-generation proteasome inhibitor with single-agent activity in patients with relapsed and refractory multiple myeloma (R/R MM). In PX-171-003-A1, a single-arm phase 2 study of carfilzomib monotherapy in heavily pretreated patients, the overall response rate was 23.7%, 37% of patients achieved ≥ minimal response and median overall survival (OS) was 15.6 months. Based on this study, carfilzomib was recently approved by the US Food and Drug Administration for the treatment of R/R MM. Herein we describe the trial design and rationale for a phase 3 randomized study, FOCUS (CarFilzOmib for AdvanCed Refractory MUltiple Myeloma European Study), being conducted to compare OS after treatment with single-agent carfilzomib to best supportive care (BSC) regimen in R/R MM. Patients must have received ≥3 prior regimens, must be responsive to at least 1 line of therapy, and be refractory to their most recent therapy. Eligible patients are randomized 1:1 to receive either carfilzomib (28-day cycles at 20 mg/m(2) IV on Days 1-2 of Cycle 1, escalating to 27 mg/m(2) IV on Days 8, 9, 15, and 16 and continuing at 27 mg/m(2) through Cycle 9 and Days 1, 2, 15, and 16 ≥ Cycle 10) or an active BSC regimen (corticosteroid treatment of prednisolone 30 mg, dexamethasone 6 mg, or equivalent every other day with optional cyclophosphamide 50 mg PO once daily). Patients will continue treatment until disease progression, unacceptable toxicity, or treatment discontinuation and will then enter long-term follow-up for survival. The primary endpoint is OS and secondary endpoints include progression-free survival, overall response rate, and safety. Disease assessments will be determined according to the International Myeloma Working Group Uniform Response Criteria with minimal response per European Blood and Marrow Transplantation Group criteria. This phase 3 trial will provide more rigorous data for carfilzomib, as this is the first carfilzomib study with OS as the primary endpoint and will not be confounded by crossover and will provide more robust secondary response and safety results that will add to the data set from prior phase 2 studies. FOCUS will facilitate regulatory approvals around the world and expand treatment options for patients with R/R MM. EudraCT No. 2009-016840-38; NCT01302392.


Using health provider insights to inform pediatric HIV disclosure: a qualitative study and practice framework from Kenya.
Optimal pediatric HIV disclosure impacts illness and developmental experiences while improving access to timely treatment. However, disclosure rates in high HIV prevalence countries remain low and there are limited data on best practices. We conducted a qualitative study of disclosure practices and interviewed healthcare providers from five pediatric HIV clinics in Kenya. We identified themes central to disclosure practices, rationale for approaches, barriers to implementing disclosure, and creative strategies to overcome challenges. We used these insights to develop a practice-based framework for disclosure that is sensitive to practical challenges. Overall, providers had limited training but extensive experience in disclosure, endorsed individualized disclosure practices, invested substantial time on disclosure despite clinical burden, and noted adverse outcomes associated with unplanned or abrupt disclosure. Providers advocated for an approach to disclosure that is child-centered but respects caregiver fears and values. Caregiver support was provided to enable caregivers to be the person who ultimately disclosed HIV status to children. Unplanned or abrupt disclosure to children was reported to have severe and persistent adverse impact and was a stimulus to accelerate disclosure in scenarios when providers believed children may be suspecting their diagnosis. Based on these expert insights, the framework we developed incorporates concurrent evaluation of child and caregiver readiness, identifies cues to prompt disclosure discussions, includes caregiver education and support, and utilizes a gradual approach of unveiling HIV diagnosis to the child.


Phytoextraction of phosphorus-enriched grassland soils.
High soil P contents in agricultural soils in the Netherlands cause excessive losses of P to surface waters. The reductions in P application rates in the present manure policy are not sufficient to reach surface water quality standards resulting from the European Water Framework Directive in 2015. Accordingly, additional measures are necessary to reduce P loading to surface water. Greenhouse experiments showed that a rapid reduction in soluble P and readily available soil P can be obtained by zero P application. However, field data confirming these findings are scarce. In 2002 a phytoextraction experiment started on four grasslands sites on sand, peat, and clay soils. The phytoextraction (mining) plots receive no P and 300 kg N ha(-1) yr(-1) and the grass is removed by mowing. The experiment showed that zero P application, over a period of 5 yr, led to a strong (30-90%) reduction in P concentrations in soil solution in the upper soil layer (0-0.05 m). The reduction in concentrations declined with depth. Mining also resulted in a decline in P pools in the soil solid phase. The largest decline (10-60%) was found in weakly bound P pools (water extractable P; P(w), and ammonium lactate extractable P; P-AL), whereas reductions in more strongly bound P forms were relatively small. It may be concluded that phytoextraction appears an effective method of reducing soil P concentrations in the uppermost soil layers in a couple of years and prolonged mining may thus be effective in reducing leaching and runoff of P.


Contrasting patterns of cytokinins between years in senescing aspen leaves.
Cytokinins are plant hormones that typically block or delay leaf senescence. We profiled 34 different cytokinins/cytokinin metabolites (including precursors, conjugates and degradation products) in leaves of a free-growing mature aspen (Populus tremula) before and after the initiation of autumnal senescence over three consecutive years. The levels and profiles of individual cytokinin species, or classes/groups, varied greatly between years, despite the fact that the onset of autumn senescence was at the same time each year, and senescence was not associated with depletion of either active or total cytokinin levels. Levels of aromatic cytokinins (topolins) were low and changed little over the autumn period. Diurnal variations and weather-dependent variations in cytokinin content were relatively limited. We also followed the expression patterns of all aspen genes implicated as having roles in cytokinin metabolism or signalling, but neither the pattern of regulation of any group of genes nor the expression of any particular gene supported the notion that decreased cytokinin signalling could explain the onset of senescence. Based on the results from this tree, we therefore suggest that cytokinin depletion is unlikely to explain the onset of autumn leaf senescence in aspen.


Revisiting the putative role of heme as a trigger of inflammation.
Activation of the innate immune system by free heme has been proposed as one of the principal consequences of cell-free hemoglobin (Hb) exposure. Nonetheless, in the absence of infection, heme exposures within a hematoma, during hemolysis, or upon systemic administration of Hb (eg, as a Hb-based oxygen carrier) are typically not accompanied by uncontrolled inflammation, challenging the assumption that heme is a major proinflammatory mediator in vivo. Because of its hydrophobic nature, heme liberated from oxidized hemoglobin is rapidly transferred to alternative protein-binding sites (eg, albumin) or to hydrophobic lipid compartments minimizing protein-free heme under in vivo equilibrium conditions. We demonstrate that the capacity of heme to activate human neutrophil granulocytes strictly depends on the availability of non protein-associated heme. In human endothelial cells as well as in mouse macrophage cell cultures and in mouse models of local and systemic heme exposure, protein-associated heme or Hb do not induce inflammatory gene expression over a broad range of exposure conditions. Only experiments in protein-free culture medium demonstrated a weak capacity of heme-solutions to induce toll-like receptor-(TLR4) dependent TNF-alpha expression in macrophages. Our data suggests that the equilibrium-state of free and protein-associated heme critically determines the proinflammatory capacity of the metallo-porphyrin. Based on these data it appears unlikely that inflammation-promoting equilibrium conditions could ever occur in vivo.


A Laboratory Assessment of 120 Air Pollutant Emissions from Biomass and Fossil Fuel Cookstoves.
Cookstoves emit many pollutants that are harmful to human health and the environment. However, most of the existing scientific literature focuses on fine particulate matter (PM2.5) and carbon monoxide (CO). We present an extensive data set of speciated air pollution emissions from wood, charcoal, kerosene, and liquefied petroleum gas (LPG) cookstoves. One-hundred and twenty gas- and particle-phase constituents-including organic carbon, elemental carbon (EC), ultrafine particles (10-100 nm), inorganic ions, carbohydrates, and volatile/semivolatile organic compounds (e.g., alkanes, alkenes, alkynes, aromatics, carbonyls, and polycyclic aromatic hydrocarbons (PAHs))-were measured in the exhaust from 26 stove/fuel combinations. We find that improved biomass stoves tend to reduce PM2.5 emissions; however, certain design features (e.g., insulation or a fan) tend to increase relative levels of other coemitted pollutants (e.g., EC ultrafine particles, carbonyls, or PAHs, depending on stove type). In contrast, the pressurized kerosene and LPG stoves reduced all pollutants relative to a traditional three-stone fire (≥93% and ≥79%, respectively). Finally, we find that PM2.5 and CO are not strong predictors of coemitted pollutants, which is problematic because these pollutants may not be indicators of other cookstove smoke constituents (such as formaldehyde and acetaldehyde) that may be emitted at concentrations that are harmful to human health.


Hysteroscopic sterilization in women with severe cardiac disease: experience at a tertiary center.
To determine the feasibility and effectiveness of hysteroscopic sterilization as a minimally invasive sterilization method for women with high-risk cardiac disease. In a retrospective cohort study, 18 women with high-risk cardiac conditions that strictly contraindicated pregnancy were compared with a reference cohort of 157 women without cardiac disease. All underwent microinsert hysteroscopic sterilization at Mayo Clinic from January 2003 through February 2007. End points included successful placement, fallopian tube patency determined by hysterosalpingogram 3 months after the procedure, and pregnancy status. Women in the cardiac cohort were younger than those in the reference cohort (median age, 25 vs 39 years; P<.001), had lower parity (median, 0 vs 2; P<.001), and had a higher proportion of patients categorized as American Society of Anesthesiologists' physical status 3 (severe systemic disease) and physical status 4 (systemic disease that is a constant threat to life) (83% vs 6%; P<.001). No significant differences were noted for use of general anesthesia (17% vs 27%; P=.41), successful bilateral device placement (100% vs 95%; P>.99), postoperative pain score (median, 0 for both groups; P=.87), or length of hospitalization (median, 6 vs 6 hours; P=.63). No intraoperative complications occurred. Follow-up hysterosalpingography showed high tubal occlusion rates in both cohorts (100% cardiac; 98% reference; P>.99). No pregnancies occurred during a median follow-up period of 20 months (interquartile range, 8-33 months). For women with cardiac disease and strict contraindications for pregnancy, microinsert hysteroscopic sterilization provided minimally invasive, permanent, and reliable contraception.


Early gamma-oscillations as correlate of localized nociceptive processing in primary sensorimotor cortex.
Recent studies put forward the idea that stimulus-evoked gamma-band oscillations (GBOs; 30-100 Hz) play a specific role in nociception. So far, evidence for the specificity of GBOs for nociception, their possible involvement in nociceptive sensory discriminatory abilities, and knowledge regarding their cortical sources is just starting to grow. To address these questions, we used electroencephalography (EEG) to record brain activity evoked by phasic nociceptive laser stimuli and tactile stimuli applied at different intensities to the right hand and foot of 12 healthy volunteers. The EEG was analyzed in the time domain to extract phase-locked event-related brain potentials (ERPs) and in three regions of interest in the time-frequency domain (delta/theta, 40-Hz gamma, 70-Hz gamma) to extract stimulus-evoked changes in the magnitude of non-phase-locked brain oscillations. Both nociceptive and tactile stimuli, matched with respect to subjective intensity, elicited phase locked ERPs of increasing amplitude with increasing stimulus intensity. In contrast, only nociceptive stimuli elicited a significant enhancement of GBOs (65-85 Hz, 150-230 ms after stimulus onset), whose magnitude encoded stimulus intensity, whereas tactile stimuli led to a GBO decrease. Following nociceptive hand stimulation, the topographical distribution of GBOs was maximal at contralateral electrode C3, whereas maximum activity following foot stimulation was recorded at the midline electrode Cz, compatible with generation of GBOs in the representations of the hand and foot of the primary sensorimotor cortex, respectively. The differential behavior of high-frequency GBOs and low-frequency 40-Hz GBOs is indicating different functional roles and regions in sensory processing.NEW & NOTEWORTHY Gamma-band oscillations show hand-foot somatotopy compatible with generation in primary sensorimotor cortex and are present following nociceptive but not tactile stimulation of the hand and foot in humans.


Novel Clostridium thermocellum type I cohesin-dockerin complexes reveal a single binding mode.
Protein-protein interactions play a pivotal role in a large number of biological processes exemplified by the assembly of the cellulosome. Integration of cellulosomal components occurs through the binding of type I cohesin modules located in a non-catalytic molecular scaffold to type I dockerin modules located at the C terminus of cellulosomal enzymes. The majority of type I dockerins display internal symmetry reflected by the presence of two essentially identical cohesin-binding surfaces. Here we report the crystal structures of two novel Clostridium thermocellum type I cohesin-dockerin complexes (CohOlpC-Doc124A and CohOlpA-Doc918). The data revealed that the two dockerins, Doc918 and Doc124A, are unusual because they lack the structural symmetry required to support a dual binding mode. Thus, in both cases, cohesin recognition is dominated by residues located at positions 11, 12, and 19 of one of the dockerin binding surfaces. The alternative binding mode is not possible (Doc918) or highly limited (Doc124A) because residues that assume the critical interacting positions, when dockerins are reoriented by 180°, make steric clashes with the cohesin. In common with a third dockerin (Doc258) that also presents a single binding mode, Doc124A directs the appended cellulase, Cel124A, to the surface of C. thermocellum and not to cellulosomes because it binds preferentially to type I cohesins located at the cell envelope. Although there are a few exceptions, such as Doc918 described here, these data suggest that there is considerable selective pressure for the evolution of a dual binding mode in type I dockerins that direct enzymes into cellulosomes.


Epidemiological profile of Marfan syndrome in a general population: a national database study.
To explore the current epidemiological profile of Marfan syndrome in a general population. Patients who had received a diagnosis of Marfan syndrome were identified from the Taiwan National Health Insurance database records from January 1, 2000, through December 31, 2012 (average population size, 22,765,535). Cardiovascular events and interventions were identified by using the respective International Classification of Diseases codes. We identified 2329 patients (58% men) with Marfan syndrome. The overall prevalence was 10.2 (95% CI, 9.8-10.7) per 100,000 individuals, with peaks at the age of 15 to 19, 10 to 14, and 20 to 24 years. The minimal birth incidence of 23.3 (95% CI, 21.7-23.3) per 100,000 individuals was estimated in those aged 20 to 29 years. The average annual mortality was 0.23% (69 deaths), mostly owing to cardiac causes (including dissection and sudden death in 40 patients, 58%). Aortic dissection occurred in 226 patients (10%; 61% men) at a mean age of 36.6±10.7 years. The probability of freedom from dissection was 99%, 80%, and 66% at the age of 20, 40, and 50 years, respectively. Of the 69 deaths and 226 dissections during the follow-up period, more than half of the cases occurred before the age of 40 years. Cardiovascular intervention was performed in 360 patients, with early mortality being higher in the emergent operation group (8%) than in the elective group (0%). From this national cohort study, the minimal birth incidence was 23.3 per 100,000 individuals, that is, possibly 1 patient with Marfan syndrome per 4286 people. Despite medical advances, aortic dissection still occurs in about one-tenth of the patients and carries a high mortality risk. Early diagnosis and timely medical interventions are warranted.


Anatolian tree rings and a new chronology for the east Mediterranean Bronze-Iron Ages.
We report an extensive program of high-precision radiocarbon dating to establish the best date for a floating 1599-year Anatolian tree ring chronology that spans the later third millennium B.C. through the earlier first millennium B.C. This chronology is directly associated with a number of key sites and ancient personages. A previously suggested dating is withdrawn and is replaced by a robust new date fix 22 (+4 or -7) years earlier. These new radiocarbon wiggle-matched dates offer a unique independent resource for establishing the precise chronology of the ancient Near East and Aegean and help resolve, among others, a long-standing debate in favor of the so-called Middle Mesopotamian chronology.


A new quinoline based luminescent Zr(iv) metal-organic framework for the ultrasensitive recognition of 4-nitrophenol and Fe(iii) ions.
A Zr(iv) based luminescent metal-organic framework (MOF; 1) was synthesized by a solvothermal method using a mixture of ZrCl4, quinoline-2,6-dicarboxylic acid (H2QDA) ligand and trifluoroacetic acid (modulator) having a 1 : 1 : 10 molar ratio in N,N-dimethylformamide (DMF). For activation, the methanol-exchanged sample was heated under high vacuum at 120 °C for 1 day. Different techniques like X-ray powder diffraction (XRPD), thermogravimetric analysis (TGA), Fourier transform infrared (FT-IR) spectroscopy and energy dispersive X-ray (EDX) analysis were applied to fully characterize 1. The activated form of 1 (called 1') has the formula [Zr6O6(OH)2(CF3COO)2(C11H5NO4)4(H2O)4]. It exhibited quick response and great selectivity for the fluorimetric sensing of 4-nitrophenol (4-NP) in acetonitrile and Fe3+ ions in water. The probe maintained its high selectivity for 4-NP and Fe3+ ions even in the presence of potentially intrusive nitroaromatics and metal ions, respectively. The detection limits for 4-NP and Fe3+ were found to be 1.40 ppt and 0.71 ppb, respectively. These values are the lowest among the existing MOF probes for Fe3+ and 4-NP. The electron and energy transfer processes along with the electrostatic interactions of 4-NP with the MOF can be the possible reasons for the selectivity for 4-NP. The fluorescence resonance energy transfer (FRET) process can be the major reason behind the quenching mechanism for Fe3+ ions. The recyclability test indicates that 1' is a promising probe for the long-term detection of 4-NP and Fe3+ ions.


Dietary fiber intake and risk of breast cancer in postmenopausal women: the National Institutes of Health-AARP Diet and Health Study.
Although dietary fiber has been hypothesized to lower risk of breast cancer by modulating estrogen metabolism, the association between dietary fiber intake and risk of breast cancer by hormone receptor status is unclear. The objective was to examine the relation of dietary fiber intake to breast cancer by hormone receptor status and histologic type among postmenopausal women in the National Institutes of Health-AARP Diet and Health Study (n = 185,598; mean age: 62 y). Dietary intakes were assessed with a food-frequency questionnaire. Incident breast cancer cases were identified through linkage with state cancer registries. Cox proportional hazard models were used to estimate relative risks (RRs) and 2-sided 95% CIs. During an average of 7 y of follow-up, 5461 breast cancer cases were identified, of which 3341 cases had estrogen receptor (ER) and progesterone receptor (PR) status. Dietary fiber intake was inversely associated with breast cancer risk [RR for the highest quintile (Q5) compared with the lowest quintile (Q1): 0.87; 95% CI: 0.77, 0.98; P for trend: 0.02]. The inverse association appeared to be stronger for ER(-)/PR(-) tumors (RR(Q5vsQ1): 0.56; 95% CI: 0.35, 0.90; P for trend: 0.008; 366 cases) than for ER(+)/PR(+) tumors (RR(Q5vsQ1): 0.95; 95% CI: 0.76, 1.20; P for trend: 0.47; 1641 cases). The RR(Q5vsQ1) of lobular tumors was 0.66 (95% CI: 0.44, 0.97; P for trend: 0.04), and the RR(Q5vsQ1) of ductal tumors was 0.90 (95% CI: 0.77, 1.04; P for trend: 0.10). Fiber from grains, fruit, vegetables, and beans was not related to breast cancer. Our findings suggest that dietary fiber can play a role in preventing breast cancer through nonestrogen pathways among postmenopausal women.


Rethinking the Reliability of Eyewitness Memory.
Although certain pockets within the broad field of academic psychology have come to appreciate that eyewitness memory is more reliable than was once believed, the prevailing view, by far, is that eyewitness memory is unreliable-a blanket assessment that increasingly pervades the legal system. On the surface, this verdict seems unavoidable: Research convincingly shows that memory is malleable, and eyewitness misidentifications are known to have played a role in most of the DNA exonerations of the innocent. However, we argue here that, like DNA evidence and other kinds of scientifically validated forensic evidence, eyewitness memory is reliable if it is not contaminated and if proper testing procedures are used. This conclusion applies to eyewitness memory broadly conceived, whether the test involves recognition (from a police lineup) or recall (during a police interview). From this perspective, eyewitness memory has been wrongfully convicted of mistakes that are better construed as having been committed by other actors in the legal system, not by the eyewitnesses themselves. Eyewitnesses typically provide reliable evidence on an initial, uncontaminated memory test, and this is true even for most of the wrongful convictions that were later reversed by DNA evidence.


Necrotizing pancreatitis: a review of multidisciplinary management.
The objective of this review is to summarize the current state of the art of the management of necrotizing pancreatitis, and to clarify some confusing points regarding the terminology and diagnosis of necrotizing pancreatitis, as these points are essential for management decisions and communication between providers and within the literature. Acute pancreatitis varies widely in its clinical presentation. Despite the publication of the Atlanta guidelines, misuse of pancreatitis terminology continues in the literature and in clinical practice, especially regarding the local complications associated with severe acute pancreatitis. Necrotizing pancreatitis is a manifestation of severe acute pancreatitis associated with significant morbidity and mortality. Diagnosis is aided by pancreas-protocol computed tomography or magnetic resonance imaging, ideally 72 h after onset of symptoms to achieve the most accurate characterization of pancreatic necrosis. The extent of necrosis correlates well with the incidence of infected necrosis, organ failure, need for debridement, and morbidity and mortality. Having established the diagnosis of pancreatic necrosis, goals of appropriately aggressive resuscitation should be established and adhered to in a multidisciplinary approach, ideally at a high-volume pancreatic center. The role of antibiotics is determined by the presence of infected necrosis. Early enteral feeds improve outcomes compared with parenteral nutrition. Pancreatic necrosis is associated with a multitude of complications which can lead to long-term morbidity or mortality. Interventional therapy should be guided by available resources and the principle of a minimally invasive approach. When open debridement is necessary, it should be delayed at least 3-6 weeks to allow demarcation of necrotic from viable tissue.


Mandibular rest position: a reliable position influenced by head support and body posture.
This prospective study was designed to establish how the positions of the molars and the condyles are related to incisor position in the mandibular rest position and how their positions are altered by changing head posture. Measurements of the mandibular rest position were taken on 24 men (age range, 23 to 35) with normal Class I occlusion, skeletal patterns, and temporomandibular joint function. The movements of 5 landmarks (lower incisor, and condyles and molars bilaterally) were tracked from maximum intercuspation into 4 independent rest positions (upright supported, upright unsupported, supine supported, and supine unsupported) using an optoelectric (Optotrak; Northern Digital, Waterloo, Ontario, Canada) computer system. The positions were based on minimal electromyographic and verbal instructions to swallow, lick the lips, and say "Mississippi." The results showed significant (P <.01) movements of the incisors, the molars, and the condyles into each of the 4 rest positions. Movements of the molars and the condyles into the supported upright posture and the unsupported upright posture differed slightly but significantly because of greater movement into the supported posture. Patterns of mandibular movement were entirely different between the upright and the supine rest positions; the mandible rotated anteriorly in the supine position and posteriorly in the upright position. We concluded that movement into the mandibular rest position from the intercuspal position is not a simple opening rotation of the mandible, and that the pattern of movement is influenced by head support and body postures.


Serial amniocenteses in the management of twin-twin transfusion syndrome: when is it valuable?
Serial decompressive amniocenteses were performed at 18-23 weeks of gestation in 23 pregnancies referred due to echographic signs typical of twin-twin transfusion syndrome. The procedures were repeated until delivery or permanent normalization of the amniotic fluid volume both in the donor and the recipient twin sac. The overall survival rate was 57%, but in only 39% of the pregnancies did both twins survive without handicaps. The absence of end diastolic flow in the umbilical artery of the donor twin was associated with poor chances of survival for both fetuses; in contrast, the presence of hydrops or ascites in the recipient twin did not worsen the prognosis. A policy of aggressive amniotic fluid decompression may achieve permanent resolution of the fluid in over 50% of the pregnancies complicated by twin-twin transfusion syndrome.


Kinetics of peptide binding to the class II MHC protein I-Ek.
Class II MHC glycoproteins bind short (7-25 amino acid) peptides in an extended type II polyproline-like conformation and present them for immune recognition. Because empty MHC is unstable, measurement of the rate of the second-order reaction between peptide and MHC is challenging. In this report, we use dissociation of a pre-bound peptide to generate the active, peptide-receptive form of the empty class II MHC molecule I-Ek. This allows us to measure directly the rate of reaction between active, empty I-Ek and a set of peptides that vary in structure. We find that all peptides studied, despite having highly variable dissociation rates, bind with similar association rate constants. Thus, the rate-limiting step in peptide binding is minimally sensitive to peptide side-chain structure. An interesting complication to this simple model is that a single peptide can sometimes bind to I-Ek in two kinetically distinguishable conformations, with the stable peptide-MHC complex isomer forming much more slowly than the less-stable one. This demonstrates that an additional free-energy barrier limits the formation of certain specific MHC-peptide complex conformations.


Species differences in the binding of compounds designed to fit a site of known structure in adult human haemoglobin.
1. Oxygen dissociation curves are reported for human haemoglobins A1, FII, FI, A1c and Raleigh (beta1 valine leads to acetylalanine) and for horse haemoglobin in the absence and presence of 2,3-diphosphoglycerate (DPG), or 4,4'-diformyl-2-bibenzyl oxyacetic acid, or the bisulphite addition compound of the latter. 2. These haemoglobins were selected because their amino acid sequences are different at the DPG receptor site of human adult deoxyhaemoglobin. 3. The size of the shifts of the dissociation curves are in the sequence expected from the postulated numbers of interactions made by each compound with each haemoglobin type, based on the assumption of a common receptor site for the three compounds. 4. Multiple linear regression analysis shows that the free energies of interaction of the compounds with the haemoglobins may be predicted, to a first approximation, by summing the number of ionic and covalent bonds predicted for each effector-receptor combination, a reversible covalent bond contributing about twice as much energy (-6.78 kJmol-1) as an ionic interaction (-3.14 kJmol-1).


Unusual in vivo extensive corrosion of a low-silver amalgam restoration involving galvanic coupling: a case report.
The basic mechanism of dental amalgam corrosion has been thoroughly studied during the last 50 years by various experimental techniques, most often carried out in vitro. Electrochemical methods were extensively employed, and it was recognized that a gradual dealloying of the more electroactive components, Zn, Sn, and to a lesser extent Cu, contributed to change the surface composition. It is also well known that, in all circumstances, galvanic coupling threatens the longevity of the restoration. Among the patients examined at the hospital attached to the Department of Dental Surgery of the Paris 5 University, the authors observed, in a few exceptional circumstances of defective restorations, a release of liquid metal droplets in the surrounding tissue. These particles were identified as elemental mercury. This unexpected phenomenon was the result of the use of a low-silver-content alloy, combined with an enhanced corrosion process due to galvanic coupling with a silver alloy crown and brass screw-posts. In the described clinical case, the tooth, which had to be extracted, was axially sliced for observation by scanning electron microscopy coupled with elemental analysis by X-ray fluorescence energy dispersive spectroscopy. The observations showed a dramatic corrosion of the amalgam matrix with the formation of porosity even in the bulk of the material. Superposition of the element mappings proved (1) the electrochemical coupling of the amalgam with the silver-based crown, (2) the decalcification of the dentin, and (3) the formation of tin oxychloride precipitates in the matrix porosities and the interfacial gaps. These experimental results corroborate the corrosion mechanisms described hereupon. They support the current national and international recommendations for the dental amalgam formulation and for a rigorous professional practice.


The Effects of Stress on Cognitive Aging, Physiology and Emotion (ESCAPE) Project.
Despite evidence that psychological stress is an important risk factor for age-related cognitive loss, little research has directly evaluated psychological and physiological mediators of the relationship between stressful experiences and cognitive function. A key objective of the ESCAPE (Effects of Stress on Cognitive Aging, Physiology, and Emotion) project is to evaluate whether engaging in stress-related unconstructive repetitive thought (URT) is a pathway through which stressful experiences negatively affect cognitive health over the short- and long-term. Over the short-term, we hypothesize that engaging in URT will deplete attentional resources and result in worse cognitive performance in daily life. Over the long-term, we expect that the effects of chronic stress, from repeated exposure to stressors and regular engagement in URT, will be apparent in dysregulated hypothalamic-pituitary-adrenal (HPA) axis function and inflammation. Over time, stress-related physiological dysregulation will result in accelerated cognitive decline. This study utilizes a prospective longitudinal measurement-burst design. A systematic probability sample of participants aged 25 to 65 is recruited from residents of the Bronx, NY. Consenting participants complete a baseline assessment and follow-up waves at 9, 18, and 27 months post-baseline. At each wave, participants complete a 14 day measurement burst of brief surveys and cognitive assessments delivered via study smartphones during daily life. Participants provide saliva samples four times each day for five days during the measurement burst and fasting blood samples at the end of each burst from which cortisol and dehydroepiandrosterone sulfate (DHEAS), circulating inflammatory markers, and stimulated inflammatory responses to lipopolysaccharide in whole blood are determined. This study takes a multi-pronged approach to assessing stress (i.e., early adversity, chronic strains, major events, daily hassles), psychological mediators (e.g., URT), biological mechanisms (i.e., HPA function, inflammation) and outcomes across different time-scales (i.e., momentary cognitive performance, cognitive decline across years). The systematic probability sample is locally representative and can be compared with national norms on key markers of health and well-being. The findings will improve our understanding of how environmental, psychological, and physiological stress-related influences accumulate to affect cognitive health and identify potential targets (e.g., URT, inflammation) for prevention and intervention promoting cognitive health.


Enhanced lysis by bispecific oncolytic measles viruses simultaneously using HER2/neu or EpCAM as target receptors.
To target oncolytic measles viruses (MV) to tumors, we exploit the binding specificity of designed ankyrin repeat proteins (DARPins). These DARPin-MVs have high tumor selectivity while maintaining excellent oncolytic potency. Stability, small size, and efficacy of DARPins allowed the generation of MVs simultaneously targeted to tumor marker HER2/neu and cancer stem cell (CSC) marker EpCAM. For optimization, the linker connecting both DARPins was varied in flexibility and length. Flexibility had no impact on fusion helper activity whereas length had. MVs with bispecific MV-H are genetically stable and revealed the desired double-target specificity. In vitro, the cytolytic activity of bispecific MVs was superior or comparable to mono-targeted viruses depending on the target cells. In vivo, therapeutic efficacy of the bispecific viruses was validated in an orthotopic ovarian carcinoma model revealing an effective reduction of tumor mass. Finally, the power of bispecific targeting was demonstrated on cocultures of different tumor cells thereby mimicking tumor heterogeneity in vitro, more closely reflecting real tumors. Here, bispecific excelled monospecific viruses in efficacy. DARPin-based targeting domains thus allow the generation of efficacious oncolytic viruses with double specificity, with the potential to handle intratumoral variation of antigen expression and to simultaneously target CSCs and the bulk tumor mass.


Design and methods for a randomized clinical trial of a diabetes self-management intervention for low-income Latinos: Latinos en Control.
US Latinos have greater prevalence of type 2 diabetes (diabetes), uncontrolled diabetes and diabetes co-morbidities compared to non-Latino Whites. They also have lower literacy levels and are more likely to live in poverty. Interventions are needed to improve diabetes control among low-income Latinos. This randomized clinical trial tested the efficacy of a culturally- and literacy-tailored diabetes self-management intervention (Latinos en Control) on glycemic control among low-income Latinos with diabetes, compared to usual care (control). Participants were recruited from five community health centers (CHCs) in Massachusetts. The theory-based intervention included an intensive phase of 12 weekly sessions and a follow-up maintenance phase of 8 monthly sessions. Assessments occurred at baseline, and at 4 and 12 months. The primary outcome was glycosylated hemoglobin (HbA1c). Secondary outcomes were self-management behaviors, weight, lipids and blood pressure. Additional outcomes included diabetes knowledge, self-efficacy, depression and quality of life. The study was designed for recruitment of 250 participants (estimated 20% dropout rate) to provide 90% power for detecting a 7% or greater change in HbA1c between the intervention and control groups. This is a difference in change of HbA1c of 0.5 to 0.6%. Low-income Latinos bear a great burden of uncontrolled diabetes and are an understudied population. Theory-based interventions that are tailored to the needs of this high-risk population have potential for improving diabetes self-management and reduce health disparities. This article describes the design and methods of a theory driven intervention aimed at addressing this need.


Oral versus intra-vaginal imidazole and triazole anti-fungal treatment of uncomplicated vulvovaginal candidiasis (thrush).
Anti-fungals are available for oral and intra-vaginal treatment of uncomplicated vulvovaginal candidiasis (thrush). The primary objective of this review was to assess the relative effectiveness of oral versus intra-vaginal anti-fungals for the treatment of uncomplicated vulvovaginal candidiasis. The secondary objectives of the review were to assess the cost-effectiveness, safety and patient preference of oral versus intra-vaginal anti-fungals. The following sources were searched: The Cochrane Library (Issue 4, 1999), MEDLINE (January 1985 to May 2000), EMBASE (January 1980 to January 2000) and the Cochrane Collaboration Sexually Transmitted Disease Group Specialised Register of Controlled Trials. The reference lists of retrieved articles were reviewed manually. The manufacturers of anti-fungals available in the UK were contacted. ~bullet~Randomised controlled trials published in any language. ~bullet~Trials had to compare at least one oral anti-fungal with one intra-vaginal anti-fungal. ~bullet~Women (aged 16 years or over) with uncomplicated vulvovaginal candidiasis. ~bullet~The diagnosis of vulvovaginal candidiasis to be made mycologically (i.e. a positive culture and / or microscopy for yeast). ~bullet~Trials were excluded if they solely involved subjects who were HIV positive, immunocompromised, pregnant, breastfeeding or diabetic. ~bullet~The primary outcome measure was clinical cure. Duplicate scrutiny was performed of the titles and abstracts of the electronic search results. Full article formats of all selected abstracts were retrieved and independently assessed by two reviewers. Independent duplicate abstraction was performed by four reviewers. Disagreements regarding trial inclusion or data abstraction were resolved by discussion between the reviewers. Odds ratios were pooled using the random effects model. Chi-squared tests with a p-value of less than 0.1 indicated heterogeneity in the results. Seventeen trials are included in the review, reporting 19 oral versus intra-vaginal anti-fungal comparisons. No statistically significant differences were shown between oral and intra-vaginal anti-fungal treatment for clinical cure at short term (OR 1.00 (95% CI, 0.72 to 1.40)) and long term (OR 1.03 (95% CI, 0.72 to 1.49)) follow-up. No statistically significant differences for mycological cure were observed between oral and intra-vaginal treatment at short term (OR 1.20 (95% CI, 0.87 to 1.65)) or long term follow-up (OR 1.30 (95% CI, 0.99 to 1.71)). Two trials each reported one withdrawal from treatment due to an adverse reaction. Treatment preference data were poorly reported. No differences exist in terms of the relative effectiveness (measured as clinical and mycological cure) of anti-fungals administered by the oral and intra-vaginal routes for the treatment of uncomplicated vaginal candidiasis. No definitive conclusion can be made regarding the relative safety of oral and intra-vaginal anti-fungals for uncomplicated vaginal candidiasis. The oral route of administration is the preferred route for anti-fungals for the treatment of vulvovaginal candidiasis. The decision to prescribe or recommend the purchase of an anti-fungal for oral or intra-vaginal administration should take into consideration: safety, cost and treatment preference. Unless there is a previous history of adverse reaction to one route of administration or contraindications, women who are purchasing their own treatment should be given full information about the characteristics and costs of treatment to make their own decision. If health services are paying the treatment cost, decision-makers should consider whether the higher cost of oral anti-fungal administration is worth the gain in convenience, if this is the patient's preference.


Low-dose gamma irradiation of food protein increases its allergenicity in a chronic oral challenge.
Few chronic food protein models have described the relationship between allergenicity and the molecular structure of food protein after physical processing. The effect of γ-radiation on the structure of food protein was measured by fluorescence, circular dichroism and microcalorimetry. BALB/c mice were intraperitoneally sensitized and then given non-irradiated and irradiated Con-A by daily gavage for 28days. The tendency to form insoluble amorphous aggregates and partially unfolded species was observed after irradiation. The administration of non-irradiated and irradiated samples at low-dose significantly increased weight loss as well as plasma levels of eotaxin in animals repeatedly exposed to Con-A. Significant lymphocytic infiltrate filling completely the stroma of microvilli and tubular glands was observed in the small intestinal of the group given Con-A irradiated at a low dose. This phenotype was not observed in animals treated with Con-A irradiated at a high dose.


Temporal trends in surgical intervention for severe traumatic brain injury caused by extra-axial hemorrhage, 1995 to 2012.
Surgical intervention for severe traumatic brain injury (TBI) caused by extra-axial hemorrhage has declined in recent decades. The effect of this change on patient outcomes is unknown. To determine the change over time in surgical intervention in this population and to assess changes in patient outcomes. In this retrospective cohort study, the Washington State Trauma Registry was queried from 1995 to 2012 for patients with extra-axial hemorrhage and head Abbreviated Injury Scale score of 3 to 5. Data were linked to the state-wide death registry to analyze long-term mortality. The primary outcome was inpatient mortality. Secondary outcomes included 6- and 12-month mortality and modified Functional Independence Measure at discharge. Multivariable analyses were completed for all outcomes. A total of 22974 patients met inclusion criteria. Over the study period, surgical intervention for severe TBI declined from 36% to 7%. There was a decline in case fatality from 22% to 12%. In 2012, the relative risk of inpatient mortality was 23% lower compared with 1995 (adjusted mortality risk ratio, 0.77; 95% confidence interval, 0.63-0.94). Changes in 6- and 12-month adjusted mortality and modified Functional Independence Measure were not statistically significant. The decline in surgical intervention for severe TBI caused by extra-axial hemorrhage in Washington State was ubiquitous across regional, demographic, and injury characteristic strata. There was concurrently a reduction in inpatient mortality in this population. Functional status and long-term mortality, however, have remained the same. Future studies are needed to better identify modifiable risk factors for improvement in functional status and long-term mortality in this population.