A Low-Power MEMS IDE Capacitor with Integrated Microhotplate: Application as Methanol Sensor using a Metal-Organic Framework Coating as Affinity Layer.
Capacitors made of interdigitated electrodes (IDEs) as a transducer platform for the sensing of volatile organic compounds (VOCs) have advantages due to their lower power operation and fabrication using standard micro-fabrication techniques. Integrating a micro-electromechanical system (MEMS), such as a microhotplate with IDE capacitor, further allows study of the temperature- dependent sensing response of VOCs. In this paper, the design, fabrication, and characterization of a low-power MEMS microhotplate with IDE capacitor to study the temperature-dependent sensing response to methanol using Zeolitic imidazolate framework (ZIF-8), a class of metal-organic framework (MOF), is presented. A Titanium nitride (TiN) microhotplate with aluminum IDEs suspended on a silicon nitride membrane is fabricated and characterized. The power consumption of the ZIF-8 MOF-coated device at an operating temperature of 50 ∘ C is 4.5 mW and at 200 ∘ C it is 26 mW. A calibration methodology for the effects of temperature of the isolation layer between the microhotplate electrodes and the capacitor IDEs is developed. The device coated with ZIF-8 MOF shows a response to methanol in the concentration range of 500 ppm to 7000 ppm. The detection limit of the sensor for methanol vapor at 20 ∘ C is 100 ppm. In situ study of sensing properties of ZIF-8 MOF to methanol in the temperature range from 20 ∘ C to 50 ∘ C using the integrated microhotplate and IDE capacitor is presented. The kinetics of temperature-dependent adsorption and desorption of methanol by ZIF-8 MOF are fitted with double-exponential models. With the increase in temperature from 20 ∘ C to 50 ∘ C, the response time for sensing of methanol vapor concentration of 5000 ppm decreases by 28%, whereas the recovery time decreases by 70%.


Functionally associated targets in mantle cell lymphoma as defined by DNA microarrays and RNA interference.
Mantle cell lymphoma (MCL) is a non-Hodgkin lymphoma with poor prognosis. Its hallmark is the translocation t(11:14)q (13;32), leading to overexpression of cyclin D1, a positive regulator of the cell cycle. As cyclin D1 up-regulation is not sufficient for inducing malignant transformation, we combined DNA microarray and RNA interference (RNAi) approaches to identify novel deregulated genes involved in the progression of MCL. DNA microarray analysis identified 46 genes specifically up-regulated in MCL compared with normal B cells; 20 of these were chosen for further studies based on their cellular functions, such as growth and proliferation. The Granta 519 cell line was selected as an MCL in vitro model, to set up the RNAi protocol. To confirm the functionality of overexpression of the 20 disease-associated genes, they were knocked down using small interfering RNAs (siRNAs). In particular, knockdown of 3 genes, encoding the hepatoma-derived growth factor related protein 3 (HDGFRP3), the frizzled homolog 2 (FZD2), and the dual specificity phosphatase 5 (DUSP5), induced proliferative arrest in Granta 519 MCL cells. These genes emerged as functionally associated in MCL, in relation to growth and survival, and interfering with their function would increase insight into lymphoma growth regulation, potentially leading to novel clinical intervention modalities.


4-Trifluoromethyl-substituted coumarins with large Stokes shifts: synthesis, bioconjugates, and their use in super-resolution fluorescence microscopy.
Bright and photostable fluorescent dyes with large Stokes shifts are widely used as sensors, molecular probes, and light-emitting markers in chemistry, life sciences, and optical microscopy. In this study, new 7-dialkylamino-4-trifluoromethylcoumarins have been designed for use in bioconjugation reactions and optical microscopy. Their synthesis was based on the Stille reaction of 3-chloro-4-trifluoromethylcoumarins and available (hetero)aryl- or (hetero)arylethenyltin derivatives. Alternatively, the acylation of 2-trifluoroacetyl-5-dialkylaminophenols with available (hetero)aryl- or (hetero)arylethenylacetic acids followed by intramolecular condensation afforded coumarins with 3-(hetero)aryl or 3-[2-(hetero)aryl]ethenyl groups. Hydrophilic properties were provided by the introduction of a sulfonic acid residue or by phosphorylation of a primary hydroxy group attached at C-4 of the 2,2,4-trimethyl-1,2-dihydroquinoline fragment fused to the coumarin fluorophore. For use in immunolabeling procedures, the dyes were decorated with an (activated) carboxy group. The positions of the absorption and emission maxima vary in the ranges 413-480 and 527-668 nm, respectively. The phosphorylated dye, 9,CH=CH-2-py,H, with the 1-(3-carboxypropyl)-4-hydroxymethyl-2,2-dimethyl-1,2-dihydroquinoline fragment fused to the coumarin fluorophore bearing the 3-[2-(2-pyridyl)ethenyl] residue (absorption and emission maxima at 472 and 623 nm, respectively) was used in super-resolution light microscopy with stimulated emission depletion and provided an optical resolution better than 70 nm with a low background signal. As a result of their large Stokes shifts, good fluorescence quantum yields, and adequate photostabilities, phosphorylated coumarins enable two-color imaging (using several excitation sources and a single depletion laser) to be combined with subdiffractional optical resolution.


Multimodal imaging of neural progenitor cell fate in rodents.
For clinical application of stem cell-based therapies, noninvasive detection of applied stem cells is of high importance. We report on the feasibility of detecting implanted neural progenitor cells (NPCs) noninvasively and follow their fate and functional status by sequential multimodal molecular imaging and reporter gene technology. We investigated C17.2 cells stably expressing herpes simplex virus type 1-thymidine kinase (HSV-1-tk) and green fluorescent protein (gfp) (C17.2-tkIRESgfp = C17.2-TIG) or HSV-1-tk, gfp, and firefly luciferase (luc) (C17.2-lucIREStkgfp = C17.2-LITG) and determined the detection sensitivity of positron emission tomography (PET) and bioluminescence imaging (BLI) for these cells in culture and in vivo in subcutaneous and intracranial glioma models. In addition, PET and BLI were used to further investigate and follow the fate of implanted C17.2-LITG cells in an intracranial glioma model. We show that both imaging modalities are sensitive in detecting reporter gene expressing NPCs; however, PET, by the use of 9-[4-[(18)F]fluoro-3-hydroxymethyl)butyl]guanine ([(18)F]FHBG), detects NPCs only at sites of disrupted blood-brain barrier. Furthermore, both imaging modalities can be used to detect stem cell fate and migration and indicate excessive proliferation and aberrant migration. In conclusion, multimodal imaging can be used for longitudinal noninvasive monitoring of grafted NPCs in rodents.


Immunosuppressive treatment for immune thrombocytopenia which masked Graves' disease.
A 71-year-old female patient followed primary immune thrombocytopenia (ITP) was admitted to endocrinology unit with excessive sweating. We started methimazole for Graves' disease. Without any additional immunosuppressive treatment, at week 12 of methimazole therapy, thyroid stimulating hormone (TSH) levels returned to normal, and platelet counts rose to tolerable levels. When her hospital records were analyzed, they revealed that a year ago, when she had been diagnosed with ITP, her TSH values had been suppressed. After immunosuppressive therapy, her platelet values were maintained at normal levels, and during her control visits, her TSH levels were measured twice and were within normal limits. We think that immunosuppressive therapy for ITP without considering thyroid function tests may result in a transient euthyroid state, which potentially masks Graves' disease accompanying immunosuppressive therapy and associated recurrent ITP attacks.


Potential serum biomarkers from a metabolomics study of autism.
Early detection and diagnosis are very important for autism. Current diagnosis of autism relies mainly on some observational questionnaires and interview tools that may involve a great variability. We performed a metabolomics analysis of serum to identify potential biomarkers for the early diagnosis and clinical evaluation of autism. We analyzed a discovery cohort of patients with autism and participants without autism in the Chinese Han population using ultra-performance liquid chromatography quadrupole time-of-flight tandem mass spectrometry (UPLC/Q-TOF MS/MS) to detect metabolic changes in serum associated with autism. The potential metabolite candidates for biomarkers were individually validated in an additional independent cohort of cases and controls. We built a multiple logistic regression model to evaluate the validated biomarkers. We included 73 patients and 63 controls in the discovery cohort and 100 cases and 100 controls in the validation cohort. Metabolomic analysis of serum in the discovery stage identified 17 metabolites, 11 of which were validated in an independent cohort. A multiple logistic regression model built on the 11 validated metabolites fit well in both cohorts. The model consistently showed that autism was associated with 2 particular metabolites: sphingosine 1-phosphate and docosahexaenoic acid. While autism is diagnosed predominantly in boys, we were unable to perform the analysis by sex owing to difficulty recruiting enough female patients. Other limitations include the need to perform test-retest assessment within the same individual and the relatively small sample size. Two metabolites have potential as biomarkers for the clinical diagnosis and evaluation of autism.


A computer controlled step-scanning vacuum ultraviolet spectrometer for beam foil spectroscopy.
A McPherson 225 vacuum uv spectrometer has been interfaced with an IBM 1130 computer. The electronic interface, consisting mainly of integrated circuits, translates computer commands for a dc synchronous stepping motor which controls the grating position. Intensity information is recorded in digital form on a magnetic disk and may be plotted on a Calcomp plotter. A strip chart recorder is operated simultaneously. Reproducibility of the grating position as determined with a mercury source is within 0.03 A on the average.


Misfolding-Associated Exposure of Natively Buried Residues in Mutant SOD1 Facilitates Binding to TRAF6.
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease primarily impacting motor neurons. Mutations in superoxide dismutase 1 (SOD1) are the second most common cause of familial ALS. Several of these mutations lead to misfolding or toxic gain of function in the SOD1 protein. Recently, we reported that misfolded SOD1 interacts with TNF receptor-associated factor 6 (TRAF6) in the SOD1G93A rat model of ALS. Further, we showed in cultured cells that several mutant SOD1 proteins, but not wildtype SOD1 protein, interact with TRAF6 via the MATH domain. Here, we sought to uncover the structural details of this interaction through molecular dynamics (MD) simulations of a dimeric model system, coarse grained using the AWSEM force field. We used direct MD simulations to identify buried residues, and predict binding poses by clustering frames from the trajectories. Metadynamics simulations were also used to deduce preferred binding regions on the protein surfaces from the potential of the mean force in orientation space. Well-folded SOD1 was found to bind TRAF6 via co-option of its native homodimer interface. However, if loops IV and VII of SOD1 were disordered, as typically occurs in the absence of stabilizing Zn2+ ion binding, these disordered loops now participated in novel interactions with TRAF6. On TRAF6, multiple interaction hot-spots were distributed around the equatorial region of the MATH domain beta barrel. Expression of TRAF6 variants with mutations in this region in cultured cells demonstrated that TRAF6T475 facilitates interaction with different SOD1 mutants. These findings contribute to our understanding of the disease mechanism and uncover potential targets for the development of therapeutics.


Effects of omega-3 fatty acid supplementation on lipid levels in endstage renal disease patients.
Our purpose was to determine if over-the-counter fish oil improves the cardiovascular-disease risk profile of endstage renal disease patients. This study used a double-blind, permuted-block, randomized, placebo-controlled design. The experimental intervention consisted of fish-oil concentrate supplementation, whereas corn-oil capsules were used as a control. Compliance follow-ups were performed 3 times per week. Patients of Central Texas Nephrology Associates clinics were eligible for this study. Exclusion criteria comprised a life-expectancy of less than 6 months, pregnancy, a history of hemodialysis or medication noncompliance, or age below 18 years. The final sample size was 87 patients. The attrition rate was 9%. Participants in the experimental group consumed six 1-g soft-gel capsules of fish-oil concentrate each day for 6 months. The control group consumed corn-oil capsules, following the same protocol. Venous blood samples were acquired before and after the intervention. We assessed a number of serum lipid indicators. There were significant supplement/time interactions in low-density lipoprotein cholesterol (LDL) levels (P = .0001) and LDL particle number (P = .0001). Repeated-measures analysis of variance revealed significant time trends in high-density lipoprotein cholesterol (P = .012) and LDL (P = .001). High-density lipoprotein cholesterol levels significantly decreased in the control group, and increased in the fish-oil group, at 6 months, and LDL levels increased significantly in both groups. The analysis indicates mixed results with respect to cardiovascular disease risk. Further research is needed to assess the benefits of an over-the-counter fish-oil supplement in the renal population.


Identification of approved drugs that inhibit the binding of amyloid β oligomers to ephrin type-B receptor 2.
Ephrin type-B receptor 2 (EphB2) is a member of the receptor tyrosine kinase family and plays an important role in learning and memory functions. In patients with Alzheimer's disease (AD) and in mouse models of AD, a reduction in the hippocampal EphB2 level is observed. It was recently reported that normalization of the EphB2 level in the dentate gyrus rescues memory function in a mouse model of AD, suggesting that drugs that restore EphB2 levels may be beneficial in the treatment of AD. Amyloid β (Aβ) oligomers, which are believed to be key molecules involved in the pathogenesis of AD, induce EphB2 degradation through their direct binding to EphB2. Thus, compounds that inhibit the binding of Aβ oligomers to EphB2 may be beneficial. Here, we screened for such compounds from drugs already approved for clinical use in humans. Utilizing a cell-free screening assay, we determined that dihydroergotamine mesilate, bromocriptine mesilate, cepharanthine, and levonorgestrel inhibited the binding of Aβ oligomers to EphB2 but not to cellular prion protein, another endogenous receptor for Aβ oligomers. Additionally, these four compounds did not affect the binding between EphB2 and ephrinB2, an endogenous ligand for EphB2, suggesting that the compounds selectively inhibited the binding of Aβ oligomers to EphB2. This is the first identification of compounds that selectively inhibit the binding of Aβ oligomers to EphB2. These results suggest that these four compounds may be safe and effective drugs for treatment of AD.


Cannabis shenanigans: advocating for the restoration of an effective treatment of pain following spinal cord injury.
Cannabis is an effective treatment for pain following spinal cord injury that should be available to patients and researchers. The major argument against the rescheduling of cannabis is that the published research is not convincing. This argument is disingenuous at best, given that the evidence has been presented and rejected at many points during the political dialog. Moreover, the original decision to criminalize cannabis did not utilize scientific or medical data. There is tension between the needs of a society to protect the vulnerable by restricting the rights of others to live well and with less pain. It is clear that this 70-year war on cannabis has had little effect in controlling the supply of cannabis. Prohibition can never succeed; "it is a tyranny from which every independent mind revolts." People living with chronic pain should not have to risk addiction, social stigma, restrictions on employment and even criminal prosecution in order to deal with their pain. It is time to end the shenanigans and have an open, transparent discussion of the true benefits of this much-beleaguered medicine.


Methionine adenosyltransferase 2A/2B and methylation: gene sequence variation and functional genomics.
Methionine adenosyltransferase (MAT) catalyzes the synthesis of S-adenosylmethionine, the major biological methyl donor. MAT1A and MAT2A encode two distinct MAT isoforms in mammals. MAT2A is expressed in nonhepatic tissues, whereas MAT1A is expressed in the liver. A third gene, MAT2B, encodes a MAT2A regulatory protein. We resequenced MAT2A and MAT2B exons, splice junctions, and flanking regions using 288 DNA samples from three ethnic groups and also imputed additional single nucleotide polymorphisms (SNPs) across both genes using data from the 1000 Genomes Project. For MAT2A, resequencing identified 74 polymorphisms, including two nonsynonymous (ns) SNPs. Functional genomic studies of wild type and the two MAT2A variant allozymes (Val11 and Val205) showed that the Val11 allozyme had approximately 40% decreases in levels of enzyme activity and immunoreactive protein after COS-1 cell transfection. For MAT2B, 44 polymorphisms, 2 nonsynonymous, were identified during resequencing. Neither of the two MAT2B nsSNPs displayed alterations in levels of protein. Imputation using 1000 Genomes Project data resulted in 1730 additional MAT2A and 1997 MAT2B polymorphisms within ± 200 kilobases of each gene, respectively. Coexpression of MAT2A and MAT2B in COS-1 cells resulted in significantly increased MAT enzyme activity that correlated with increased MAT2A and MAT2B immunoreactive protein, apparently as a result of decreased degradation. Finally, studies of mRNA expression in lymphoblastoid cells showed that 7 SNPs in MAT2A and 16 SNPs in MAT2B were significantly associated with mRNA expression with p < 0.01. These observations provide a foundation for future mechanistic and clinical translational pharmacogenomic studies of MAT2A/2B.


Involvement of decreased glutamate receptor subunit GluR2 expression in lead-induced neuronal cell death.
Lead is known to induce neurotoxicity, particularly in young children, and GluR2, an AMPA-type glutamate receptor subunit, plays an important role in neuronal cell survival. Therefore, we hypothesized that altered GluR2 expression plays a role in lead-induced neuronal cell death. To test this idea, we investigated the effect of exposure to 5 and 20 µM lead for 1-9 days on the viability and GluR2 expression of primary-cultured rat cortical neurons. The number of trypan-blue stained cells was increased by exposure to 5 µM lead for 9 days or 20 µM lead for 7-9 days, and LDH release was increased after exposure to 20 µM lead for 9 days. GluR2 expression was reduced by exposure to 5-100 µM lead, but not 0.1-1 µM lead, for 9 days. Immunocytochemistry also confirmed that GluR2 expression was decreased in the presence of lead. Application of 50 ng/ml brain-derived neurotrophic factor (BDNF) led to a recovery of lead-induced neuronal cell death, accompanied with increased GluR2 expression. Our results suggest that long-term exposure to lead induces neuronal cell death, in association with a decrease of GluR2 expression.


MLL gene fusions in human leukaemias: in vivo modelling to recapitulate these primary tumourigenic events.
Recurrent reciprocal chromosomal translocations are frequently found in leukaemias and sarcomas as initiating events in these cancers. Mouse models of chromosomal translocations are not only important for the elucidation of the mechanism of these factors underlying the disease but are also important pre-clinical models for assessing new drug combinations, developing new rational therapeutic strategies based on new drugs and testing novel macromolecular drugs. We describe three technologies for creating chromosomal translocation mimics in mice, applied specifically to understand how the MLL-fusions contribute to leukaemia. An important finding of this work is that the lineage of the tumours can be controlled by the MLL-protein fusion. The translocation mimic methods can be applied to any human reciprocal chromosomal translocation.


Development of the International Classification of Functioning, Disability and Health as a brief head and neck cancer patient questionnaire.
WHO has adopted the International Classification of Functioning, Disability and Health (ICF) to assess functioning and disability. A Brief ICF Core Set for head and neck cancer comprises 19 items. This study developed a patient self-completed questionnaire from the items of the brief core set (BCSQ-H&N), compared the BCSQ-H&N questionnaire with the University of Washington v.4 (UW-QOLv4) and compared the BCSQ-H&N results with a clinician-rated evaluation. UW-QOL v4 and BCSQ-H&N were sent to 751 disease-free head and neck cancer patients in April 2008. 376 patients responded to the questionnaire and 25 were interviewed. The percentage reporting significant problems in BCSQ-H&N items ranged between 11% and 43%. The type of problem varied with tumour site. Patients with smaller tumours and patients without radiotherapy reported better outcomes. The BCSQ-H&N correlated well with appropriate items in the UW-QOLv4 especially for functional outcome. There were systematic differences between observer-rated scores and patient self-completed questionnaire responses. Patients suggested additional items for inclusion, namely taste, jaw opening, articulation function, structure of shoulder region, loss of function at the free flap donor site, and intimate relationships. Further validation is required but BCSQ-H&N shows promise as an outcome measure for global use.


Pain management by the family physician: the family practice pain education project.
Pain is a common complaint of patients who visit a family physician, and its appropriate management is a medical mandate. The fundamental principles for pain management are: placing the patient at the center of care; adequately assessing and quantifying pain; treating pain adequately; maximizing function; accounting for culture and gender differences; identifying red and yellow flags early; understanding and differentiating tolerance, dependence and addiction; minimizing side effects; and being familiar with and using CAM therapies when good evidence of efficacy exists. The pharmacologic management of pain requires thorough knowledge of nonsteroidal anti-inflammatory drugs, cyclo-oxygenase-2-specific inhibitors, and opioids. A table of equianalgesic dosages is useful because patients may need to move from one opioid to another. Accompanying this article are papers discussing 5 common pain disorders seen by family physicians, including: neck pain, low back pain, joint pain, pelvic pain, and cancer/end of life pain. The family physician who learns these principles of pain management and the algorithms for these common pain disorders can serve patients well.


Physical Fitness Tests and Type 2 Diabetes Among Japanese: A Longitudinal Study From the Niigata Wellness Study.
Simple physical fitness test can be a useful potential predictor of type 2 diabetes (T2DM). We examined the association between performances on simple physical fitness tests and the incidence of T2DM. This longitudinal study was conducted in 21,802 nondiabetic Japanese (6,649 women) aged 20 to 92 years, who underwent all physical fitness tests at baseline (April 2001 to March 2002). From April 2001 to March 2008, physical fitness tests, including grip strength, vertical jump, single-leg balance with eyes closed, forward bending, whole-body reaction time, and supine legs-up, were performed every year. Participants had physical fitness tests at least two times during the period. T2DM was also annually determined based on fasting blood glucose, glycated hemoglobin, and self-reported diabetes during the period. Discrete-time logit models were used to examine the influence of the serial level of each physical fitness test on the incidence of T2DM. During the entire study period, 972 participants developed diabetes. Lower relative grip strength (grip strength/body weight) and single-leg balance performance were associated with a higher incidence of T2DM. For relative grip strength, as compared with the fourth quartile group, the odds ratios for other groups ranged from 1.16 to 1.56 (P for trend < 0.001). For single-leg balance, the odds ratios ranged from 1.03 to 1.49 (P for trend < 0.001). The performance of a simple single-leg balance test as well as that of a grip strength test were negatively associated with the risk of T2DM among Japanese.


The relationship of p53 immunostaining to survival in carcinoma of the lung.
In this study 125 primary lung tumours have been immunostained with a panel of 5 anti-p53 antibodies (PAb240, PAb421, PAb1801, CM-1 and C19). These antibodies recognise different epitopes over the full extent of the p53 gene. It is generally believed that immunolabelling identifies only mutant p53 proteins due to the short half life of the wild type protein. The aims of this study were to confirm earlier studies of p53 positivity in human lung tumours and to establish whether or not this bore any relationship to survival. Immunostaining was demonstrated within the nuclei of affected cells in 54% of the 125 lung tumours (59% of 78 squamous cell carcinomas, 52% of 42 adenocarcinomas and 20% of five small cell carcinomas). This confirms previous smaller studies of p53 protein expression in human lung tumours. Survival curves have been drawn for all of the cases considered together and for squamous and adenocarcinomas separately. No differences in survival between p53 positive and negative cases were seen for any group of tumours. This indicates that although p53 may be of considerable importance in the initiation of malignancy it is probably of little significance once a tumour has developed.


Binding and Action of Amino Acid Analogs of Chloramphenicol upon the Bacterial Ribosome.
Antibiotic chloramphenicol (CHL) binds with a moderate affinity at the peptidyl transferase center of the bacterial ribosome and inhibits peptide bond formation. As an approach for modifying and potentially improving properties of this inhibitor, we explored ribosome binding and inhibitory activity of a number of amino acid analogs of CHL. The L-histidyl analog binds to the ribosome with the affinity exceeding that of CHL by 10 fold. Several of the newly synthesized analogs were able to inhibit protein synthesis and exhibited the mode of action that was distinct from the action of CHL. However, the inhibitory properties of the semi-synthetic CHL analogs did not correlate with their affinity and in general, the amino acid analogs of CHL were less active inhibitors of translation in comparison with the original antibiotic. The X-ray crystal structures of the Thermus thermophilus 70S ribosome in complex with three semi-synthetic analogs showed that CHL derivatives bind at the peptidyl transferase center, where the aminoacyl moiety of the tested compounds established idiosyncratic interactions with rRNA. Although still fairly inefficient inhibitors of translation, the synthesized compounds represent promising chemical scaffolds that target the peptidyl transferase center of the ribosome and potentially are suitable for further exploration.


Experimental demonstration of nonbilocal quantum correlations.
Quantum mechanics admits correlations that cannot be explained by local realistic models. The most studied models are the standard local hidden variable models, which satisfy the well-known Bell inequalities. To date, most works have focused on bipartite entangled systems. We consider correlations between three parties connected via two independent entangled states. We investigate the new type of so-called "bilocal" models, which correspondingly involve two independent hidden variables. These models describe scenarios that naturally arise in quantum networks, where several independent entanglement sources are used. Using photonic qubits, we build such a linear three-node quantum network and demonstrate nonbilocal correlations by violating a Bell-like inequality tailored for bilocal models. Furthermore, we show that the demonstration of nonbilocality is more noise-tolerant than that of standard Bell nonlocality in our three-party quantum network.


Changes in procoagulant and fibrinolytic gene expression during bleomycin-induced lung injury in the mouse.
Bleomycin-induced lung injury is an established murine model of human pulmonary fibrosis. Although procoagulant molecules (e.g., tissue factor [TF]) and fibrinolytic components (e.g., urokinase [u-PA] and type 1 plasminogen activator inhibitor [PAI-1]) have been detected in alveolar fluid from injured lungs, the origin of these molecules remains unknown. We therefore examined the expression of procoagulant and fibrinolytic components in relation to the distribution of parenchymal fibrin in bleomycin-injured lungs. Extravascular fibrin localized to the alveolar and extracellular matrix in injured lung tissue. Injured lung tissue extracts contained elevated levels of PAI-1 activity and decreased levels of u-PA activity. Whole lung PAI-1 and TF mRNAs were dramatically induced by lung injury. In situ hybridization of injured lungs revealed that PAI-1, u-PA, and TF mRNAs were induced within the fibrin-rich fibroproliferative lesions, primarily in fibroblast-like and macrophagelike cells, respectively, while TF mRNA was also induced in perilesional alveolar cells. Taken together, these observations suggest that the induction of PAI-1 and TF gene expression plays and important role in the formation and persistence of extracellular fibrin in bleomycin injured murine lungs.


Day-to-day inconsistency in parent knowledge: links with youth health and parents' stress.
Considerable evidence documents the linkages between higher levels of parental knowledge about youth activities and positive youth outcomes. This study investigated how day-to-day inconsistency in parental knowledge of youth activities was linked to youth behavioral, psychological, and physical health and parents' stress. Participants were employees in the Information Technology Division of a Fortune 500 company and their children (N = 129, mean age of youth = 13.39 years, 55% female). Data were collected from parents and youth via separate workplace and in-home surveys as well as telephone diary surveys on eight consecutive evenings. We assessed day-to-day inconsistency in parental knowledge across these eight calls. Parents differed in their knowledge from day to day almost as much as their average knowledge scores differed from those of other parents. Controlling for mean levels of knowledge, youth whose parents exhibited more knowledge inconsistency reported more physical health symptoms (e.g., colds and flu). Knowledge inconsistency was also associated with more risky behavior for girls but greater psychological well-being for older adolescents. Parents who reported more stressors also had higher knowledge inconsistency. Assessing only average levels of parental knowledge does not fully capture how this parenting dimension is associated with youth health. Consistent knowledge may promote youth physical health and less risky behavior for girls. Yet knowledge inconsistency also may reflect normative increases in autonomy as it was positively associated with psychological well-being for older adolescents. Given the linkages between parental stress and knowledge inconsistency, parent interventions should include stress management components.


Object-guided spatial attention in touch: holding the same object with both hands delays attentional selection.
Previous research has shown that attention to a specific location on a uniform visual object spreads throughout the entire object. Here we demonstrate that, similar to the visual system, spatial attention in touch can be object guided. We measured event-related brain potentials to tactile stimuli arising from objects held by observers' hands, when the hands were placed either near each other or far apart, holding two separate objects, or when they were far apart but holding a common object. Observers covertly oriented their attention to the left, to the right, or to both hands, following bilaterally presented tactile cues indicating likely tactile target location(s). Attentional modulations for tactile stimuli at attended compared to unattended locations were present in the time range of early somatosensory components only when the hands were far apart, but not when they were near. This was found to reflect enhanced somatosensory processing at attended locations rather than suppressed processing at unattended locations. Crucially, holding a common object with both hands delayed attentional selection, similar to when the hands were near. This shows that the proprioceptive distance effect on tactile attentional selection arises when distant event locations can be treated as separate and unconnected sources of tactile stimulation, but not when they form part of the same object. These findings suggest that, similar to visual attention, both space- and object-based attentional mechanisms can operate when we select between tactile events on our body surface.


Cell-Free Prototyping of AND-Logic Gates Based on Heterogeneous RNA Activators.
RNA-based devices controlling gene expression bear great promise for synthetic biology, as they offer many advantages such as short response times and light metabolic burden compared to protein-circuits. However, little work has been done regarding their integration to multilevel regulated circuits. In this work, we combined a variety of small transcriptional activator RNAs (STARs) and toehold switches to build highly effective AND-gates. To characterize the components and their dynamic range, we used an Escherichia coli (E. coli) cell-free transcription-translation (TX-TL) system dispensed via nanoliter droplets. We analyzed a prototype gate in vitro as well as in silico, employing parametrized ordinary differential equations (ODEs), for which parameters were inferred via parallel tempering, a Markov chain Monte Carlo (MCMC) method. On the basis of this analysis, we created nine additional AND-gates and tested them in vitro. The functionality of the gates was found to be highly dependent on the concentration of the activating RNA for either the STAR or the toehold switch. All gates were successfully implemented in vivo, offering a dynamic range comparable to the level of protein circuits. This study shows the potential of a rapid prototyping approach for RNA circuit design, using cell-free systems in combination with a model prediction.


The formation of intracellular glyceraldehyde-derived advanced glycation end-products and cytotoxicity.
Nonalcoholic steatohepatitis (NASH) is a feature of metabolic syndrome. Advanced glycation end-products (AGEs) are formed by the Maillard reaction, which contributes to aging and to certain pathological complications of diabetes. A recent study has suggested that glyceraldehyde-derived AGEs (Glycer-AGEs) are elevated in the sera of patients with NASH. Furthermore, immunohistochemistry of Glycer-AGEs showed intense staining in the livers of patients with NASH. The present study aimed to examine the effect of intracellular Glycer-AGEs on hepatocellular carcinoma (Hep3B) cells. Cell viability was determined by the WST-1 assay. The slot blot and Western blot were used to detect intracellular Glycer-AGEs, and their localization was analyzed by confocal microscopy. Real-time reverse transcription-polymerase chain reaction was used to quantify the mRNA for the acute phase reactant C-reactive protein (CRP). Glyceraldehyde (GA), which is the precursor of Glycer-AGEs, induced a concentration- and time-dependent increase in cell death, which was associated with an increase in intracellular Glycer-AGEs formation. Aminoguanidine (AG), which prevents AGEs formation, inhibited the formation of intracellular Glycer-AGEs and prevented cell death. Among the intracellular Glycer-AGEs that were formed, heat shock cognate 70 (Hsc70) was identified as a GA-modified protein, and its modification reduced the activity of Hsc70. Furthermore, intracellular Glycer-AGEs increased the CRP mRNA concentration. These results suggest that intracellular Glycer-AGEs play important roles in promoting inflammation and hepatocellular death.


Relative Modification of Prompt ψ(2S) and J/ψ Yields from pp to PbPb Collisions at sqrt[s_{NN}]=5.02 TeV.
The relative modification of the prompt ψ(2S) and J/ψ yields from pp to PbPb collisions, at the center-of-mass energy of 5.02 TeV per nucleon pair, is presented. The analysis is based on pp and PbPb data samples collected by the CMS experiment at the LHC in 2015, corresponding to integrated luminosities of 28.0 pb^{-1} and 464 μb^{-1}, respectively. The double ratio of measured yields of prompt charmonia reconstructed through their decays into muon pairs, (N_{ψ(2S)}/N_{J/ψ})_{PbPb}/(N_{ψ(2S)}/N_{J/ψ})_{pp}, is determined as a function of PbPb collision centrality and charmonium transverse momentum p_{T}, in two kinematic intervals: |y|<1.6 covering 6.5<p_{T}<30 GeV/c and 1.6<|y|<2.4 covering 3<p_{T}<30 GeV/c. The centrality-integrated double ratios are 0.36±0.08(stat)±0.05(syst) in the first interval and 0.24±0.22(stat)±0.09(syst) in the second. The double ratio is lower than unity in all the measured bins, suggesting that the ψ(2S) yield is more suppressed than the J/ψ yield in the explored phase space.


Treatment of unfavourable results of flexor tendon surgery: Ruptured repairs, tethered repairs and pulley incompetence.
As primary repair of divided flexor tendons becomes more common, secondary tendon surgery becomes largely that of the complications of primary repair, namely ruptured and adherent repairs. These occur with an incidence of each in most reported series world-wide of around 5%, with these problems having changed little in the last two decades, despite strengthening our suture repairs. Where the primary referral service is less well-developed, and as a more occasional occurrence where primary treatment is the routine, the surgeon faces different problems. Patients arrive at a hand unit variable, but longer, times after the primary insult, having had no, or bad, previous treatment. Sometimes the situation is the same, viz. an extended finger with no active flexion, but now no longer amenable to primary repair. Frequently, it is much more complex as a result of injuries to the other tissues of the digit and, also, as a result of the unaided healing process within the digit in the presence of an inactive flexor system. We present our experience in dealing with ruptured repairs, tethered repairs and pulley incompetence.


penMesh--Monte Carlo radiation transport simulation in a triangle mesh geometry.
We have developed a general-purpose Monte Carlo simulation code, called penMesh, that combines the accuracy of the radiation transport physics subroutines from PENELOPE and the flexibility of a geometry based on triangle meshes. While the geometric models implemented in most general-purpose codes--such as PENELOPE's quadric geometry--impose some limitations in the shape of the objects that can be simulated, triangle meshes can be used to describe any free-form (arbitrary) object. Triangle meshes are extensively used in computer-aided design and computer graphics. We took advantage of the sophisticated tools already developed in these fields, such as an octree structure and an efficient ray-triangle intersection algorithm, to significantly accelerate the triangle mesh ray-tracing. A detailed description of the new simulation code and its ray-tracing algorithm is provided in this paper. Furthermore, we show how it can be readily used in medical imaging applications thanks to the detailed anatomical phantoms already available. In particular, we present a whole body radiography simulation using a triangulated version of the anthropomorphic NCAT phantom. An example simulation of scatter fraction measurements using a standardized abdomen and lumbar spine phantom, and a benchmark of the triangle mesh and quadric geometries in the ray-tracing of a mathematical breast model, are also presented to show some of the capabilities of penMesh.


Cucurbitacin D induces cell cycle arrest and apoptosis by inhibiting STAT3 and NF-κB signaling in doxorubicin-resistant human breast carcinoma (MCF7/ADR) cells.
Breast cancer is the most common cancer for women and is a major cause of mortality in women. Doxorubicin is a generally used chemotherapy drug for breast cancer. However, multidrug resistance of breast cancer interferes with the chemotherapy. We examined whether cucurbitacin D affects doxorubicin resistance of MCF7/ADR breast cancer cells. Cell viability was measured by MTT assay. Levels of p-STAT3, p-NF-κB, IκB, and caspases were measured by Western blot analysis. Nuclear staining of Stat3 and NF-κB was measured by immunocytochemistry. STAT3 and NF-κB transcriptional activity was detected by STAT3 and NF-κB luciferase reporter gene assays. Analysis of cell cycle arrest was performed by flow cytometry. Induction of apoptosis by cucurbitacin D was measured by Annexin V-FITC/propidium iodide assay. More than 90% of MCF7/ADR cells lived upon treatment with doxorubicin for 24 h. However, upon treatment with cucurbitacin D, cell death was more than 60%. Co-administration of cucurbitacin D and doxorubicin induced apoptosis, and G2/M cell cycle arrest, and inhibited upregulated Stat3 by doxorubicin on MCF7/ADR cells. Additionally, cucurbitacin D led to an increase in the IκBα level in the cytosol and a decrease in the p-NF-κB level in the nucleus. Finally, cucurbitacin D inhibited translocation of Stat3 and NF-κB and decreased transcriptional activity in the nucleus. Cucurbitacin D decreases cell proliferation and induces apoptosis by inhibiting Stat3 and NF-κB signaling in doxorubicin-resistant breast cancer cells. Cucurbitacin D could be used as a useful compound to treat adriamycin-resistant patients.


Emerging biosensor platforms for the assessment of water-borne pathogens.
Pathogens are key contaminants in water that are responsible for the generation of various water-borne diseases, and include viruses, fungi, bacteria, and protozoan parasites. The pathogenic effects of these species in water depend on their shape, size, composition, and structure. The resulting water-borne diseases are a serious threat to the environment, including to humans and animals, and are directly responsible for environmental deterioration and pollution. The potential presence of these pathogens requires sensitive, powerful, efficient, and ideally real-time monitoring methods for their reproducible quantification. Conventional methods for pathogen detection mainly rely on time-consuming enrichment steps followed by biochemical identification strategies, which require assay times ranging from 24 h to up to a week. However, in recent years, significant efforts have been made towards the development of biosensing technologies enabling rapid and close-to-real-time detection of water-borne pathogens. This review summarizes recent developments in biosensors and sensing systems based on a variety of transducer technologies for water-quality monitoring, with specific focus on rapid pathogen detection.


Tea tree oil attenuates experimental contact dermatitis.
Herbs and minerals have been used in clinical dermatology for hundreds of years and herbal ingredients are becoming increasingly popular with the public in treatment of various dermatological conditions characterised by inflammation and pruritus. The aim of this study was to compare the efficacy of traditional topical therapeutic agents with a moderate potency topical glucocorticoid on experimental contact dermatitis and contact urticaria. The effects of ichthammol 10% pet, zinc oxide 20% pet, camphor 20% pet, levomenthol 10% pet, tea tree oil 20 or 50% and clobetason butyrate 0.05% ointment were studied in the following experimental models: elicitation of allergic contact dermatitis to nickel, irritant contact dermatitis to benzalkonium chloride, and in immediate reactions to histamine and benzoic acid (non-immunological contact utricaria) respectively. Delayed reactions were evaluated using a clinical scoring system and immediate reactions were estimated by planimetry. Histamine-induced pruritus was evaluated using VAS. Tea tree oil reduced allergic contact dermatitis by 40.5% (p = 0.003), zinc oxide by 17.4% (p = 0.04) and clobetason butyrate by 23.5% (p = 0.01). Zinc oxide reduced histamine induced flare by 18.5% (p = 0.01), ichthammol by 19.2% (p = 0.02) and clobetason butyrate by 44.1% (p = 0.02). Irritant contact dermatitis and non-immunological contact urticaria were not influenced by the pre-treatments. Pruritus induced by histamine also remained unchanged. In conclusion, tea tree oil seems to be a more effective anti-eczematic agent than zinc oxide and clobetasone butyrate, while clobetasone butyrate is superior to both ichthammol and zinc oxide in topical treatment of urticarial reactions.


B-cell maturation in chimaeric mice deficient for the heat stable antigen (HSA/mouse CD24).
The murine differentiation marker heat stable antigen (HSA) is a GPI-anchored surface glycoprotein showing strong expression on immature B- and T-lymphocytes and gradually reduced expression during maturation. Although HSA has been suggested to be involved in adhesion and/or signalling, its function has not been clearly demonstrated so far. In order to elucidate the function of HSA, we analysed chimaeric mice that were generated by targeted disruption of both HSA alleles in ES cells. These mice contain normal numbers of peripheral B-cells and normal serum IgM and IgG titres of ES cell-derived allotype, demonstrating that HSA expression on B-cells is not an absolute requirement for their maturation. However, a reduction in immature B-cells in the bone marrow and an altered degree of bone marrow and blood chimaerism suggest that HSA expression influences the maturation of B-cells.


Moderate altitude but not additional endurance training increases markers of oxidative stress in exhaled breath condensate.
Oxidative stress occurs at altitude, and physical exertion might enhance this stress. In the present study, we investigated the combined effects of exercise and moderate altitude on redox balance in ten endurance exercising biathletes, and five sedentary volunteers during a 6-week-stay at 2,800 m. As a marker for oxidative stress, hydrogen peroxide (H(2)O(2)) was analyzed by the biosensor measuring system Ecocheck, and 8-iso prostaglandin F2alpha (8-iso PGF2alpha) was determined by enzyme immunoassay in exhaled breath condensate (EBC). To determine the whole blood antioxidative capacity, we measured reduced glutathione (GSH) enzymatically using Ellman's reagent. Exercising athletes and sedentary volunteers showed increased levels of oxidative markers at moderate altitude, contrary to our expectations; there was no difference between both groups. Therefore, all subjects' data were pooled to examine the oxidative stress response exclusively due to altitude exposure. H(2)O(2) levels increased at altitude and remained elevated for 3 days after returning to sea level (p < or = 0.05). On the other hand, 8-iso PGF2alpha levels showed a tendency to increase at altitude, but declined immediately after returning to sea level (p < or = 0.001). Hypoxic exposure during the first day at altitude resulted in elevated GSH levels (p < or = 0.05), that decreased during prolonged sojourn at altitude (p < or = 0.001). In conclusion, a stay at moderate altitude for up to 6 weeks increases markers of oxidative stress in EBC independent of additional endurance training. Notably, this oxidative stress is still detectable 3 days upon return to sea level.


Growth of epitaxial Pt1-xPbx alloys by surface limited redox replacement and study of their adsorption properties.
The surface limited redox replacement (SLRR) method has been used to design two-dimensional Pt-Pb nanoalloys with controlled thickness, composition, and structure. The electrochemical behavior of these alloys has been systematically studied as a function of alloy composition. A single-cell, two-step SLRR protocol based on the galvanic replacement of underpotentially deposited monolayers of Pb with Pt was used to grow epitaxial Pt1-xPbx (x < 0.1) alloys of up to 10 ML thickness on Au substrates. It is shown that by varying the terminating potential of the galvanic replacement step, the Pb atomic content can be controlled in the films. Electrochemical analysis of the alloys showed that the adsorption of both H and CO exhibits similar, and systematic, decreases with small increases in the Pb content. These measurements, commonly used in electrocatalysis for the determination of active surface areas of Pt, suggested area values much lower than those expected based on the net Pt composition in the alloy as measured by XPS. These results show that Pb has a strong screening effect on the adsorption of both H and CO. Moreover, changes in alloy composition result in a negative shift in the potential of the peaks of CO oxidation that scales with the increase of Pb content. The results suggest electronic and bifunctional effects of incorporated Pb on the electrochemical behavior of Pt. The study illustrates the potential of the SLRR methodology, which could be employed in the design of 2-dimensional bimetallic Pt nanoalloys for fundamental studies of electrocatalytic behavior in fuel cell reactions dependent on the nature of alloying metal and its composition.


Comparison of access-hole filling materials for screw retained implant prostheses: 12-month in vivo study.
Screw retained implant prostheses seem to be an efficient restorative method to prevent peri-implantitis caused by cement excess around the abutment. The drawback of the screw-retained prostheses is the difficulty to realize an efficient access-hole filling functionally and aesthetically. Up to now, few in vitro and in vivo studies were reported in the literature. The aim of this study was to evaluate clinical performances of two direct filling materials through a period of 12 months. To pursue a previous in vitro evaluation, this in vivo 12 months prospective study followed up and compared the access-hole filling integrity of a modified 4-META (4-methacryloxyethyl trimellitate anhydride)/MMA-TBB (methyl methacrylate-tri-n-butyl borane) - based resin (M4M) and a photo-polymerizing nano-hybrid composite resin (CR). Twenty-eight access-holes were filled with both materials respectively, then impressions and intra-oral photographs were taken at T = 0, T = 1 M (month), 3, 6, and 12 M. The access-hole surface measurement and the margin analysis (depth and angle) were carried out. The VAS (visual analogue scale) value on marginal discoloration and integrity at the baseline T = 0 and T = 12 M was recorded. The mean values of the surface areas changes from T = 1 to T = 12 M were 83.3 ± 11.5% for group CR and 77.1 ± 13.1% for group M4M, respectively. (Mann-Whitney test p < 0.05, p = 0.046). The mean marginal depth at T = 12 M for group CR were 141.2 ± 125.5 μm and 132.1 ± 107.8 μm for the group M4M, respectively. (Mann-Whitney test p > 0.05, p = 0.58). The mean values of the angle formed at the margin (T = 12 M) were for group CR 39.5 ± 19.4° and 28.2 ± 17.2° for group M4M, respectively (Mann-Whitney test p < 0.0001). The photographical analysis by VAS values showed no significant difference between CR and M4M groups (Mann-Whitney test p > 0.05, p = 0.848). Based on intra- and extra-oral evaluations with the limitation, both CR and M4M combined with a ceramic primer are indicated as promising materials to fill the access-hole. Further long-term investigation is necessary to confirm this finding.


Nano-sized paramagnetic and fluorescent fluorinated carbon fiber with high NIR absorbance for cancer chemo-photothermal therapy.
Nano-sized fluorinated carbon fiber possesses unique charge distribution and special chemical bonds and holds great promise in biomedicine. However, its synthesis remains a big challenge on account of the chemical inertness and strong hydrophobicity of C-F bonds. Herein, an up-bottom method to prepare nano-sized and water-soluble fluorinated carbon fiber oxide (FCO) with tunable lengths and adjustable fluorine levels was first developed. It was found that the effective control over structure and composition endowed the FCO with high photoluminescence (PL) and near-infrared (NIR) light absorbance, which was further employed to monitor the drug loading by turn-off PL and treat cancer by photothermal therapy (PTT). We also reported the first experimental example of paramagnetic FCO, whose novel paramagnetism was generated by point defects from fluorine invasion. Moreover, the FCO was noncovalently functionalized with polyvinylpyrrolidone, loaded with doxorubicin of high capacity (1.15 mg mg-1) and exhibited acid-triggered and photothermally enhanced drug release behavior. The application of FCO in cancer treatment achieved much better therapeutic effects than single chemotherapy or PTT. This work established FCO as a novel nano-carrier with high PL, NIR absorbance, paramagnetism, and drug loading capacity, and demonstrated its first application in cancer chemo-photothermal therapy.


Plurigon: three dimensional visualization and classification of high-dimensionality data.
High-dimensionality data is rapidly becoming the norm for biomedical sciences and many other analytical disciplines. Not only is the collection and processing time for such data becoming problematic, but it has become increasingly difficult to form a comprehensive appreciation of high-dimensionality data. Though data analysis methods for coping with multivariate data are well-documented in technical fields such as computer science, little effort is currently being expended to condense data vectors that exist beyond the realm of physical space into an easily interpretable and aesthetic form. To address this important need, we have developed Plurigon, a data visualization and classification tool for the integration of high-dimensionality visualization algorithms with a user-friendly, interactive graphical interface. Unlike existing data visualization methods, which are focused on an ensemble of data points, Plurigon places a strong emphasis upon the visualization of a single data point and its determining characteristics. Multivariate data vectors are represented in the form of a deformed sphere with a distinct topology of hills, valleys, plateaus, peaks, and crevices. The gestalt structure of the resultant Plurigon object generates an easily-appreciable model. User interaction with the Plurigon is extensive; zoom, rotation, axial and vector display, feature extraction, and anaglyph stereoscopy are currently supported. With Plurigon and its ability to analyze high-complexity data, we hope to see a unification of biomedical and computational sciences as well as practical applications in a wide array of scientific disciplines. Increased accessibility to the analysis of high-dimensionality data may increase the number of new discoveries and breakthroughs, ranging from drug screening to disease diagnosis to medical literature mining.


Host genetic control of mosquito-borne Flavivirus infections.
Flaviviruses are arthropod-borne viruses, several of which represent emerging or re-emerging pathogens responsible for widespread infections with consequences ranging from asymptomatic seroconversion to severe clinical diseases and congenital developmental deficits. This variability is due to multiple factors including host genetic determinants, the role of which has been investigated in mouse models and human genetic studies. In this review, we provide an overview of the host genes and variants which modify susceptibility or resistance to major mosquito-borne flaviviruses infections in mice and humans.


Opportunities to Strengthen Fish Supply Chain Policy to Improve External Food Environments for Nutrition in the Solomon Islands.
Malnutrition and food insecurity have significant social and economic impacts in small island developing states, such as the Solomon Islands. Enhancing the domestic supply of fish, the main source of local protein, can contribute to improved nutrition and food security. This research aimed to improve understanding of the policy interface between the fisheries and health sectors and identify opportunities to strengthen fish supply chain policy to improve domestic (particularly urban) access to fish in the Solomon Islands. The study design drew on theories of policy learning and policy change and analysed policies using a consumption-oriented supply chain approach. Interviews were conducted with 12 key informants in the Solomon Islands, and 15 policy documents were analysed. Analysis of policy documents and interview data indicated that there were strengths as well as opportunities in the existing policy context. In particular, community-based fisheries management approaches and explicit recognition of the links between fisheries and nutrition were key strengths. Challenges included gaps in implementation, variations in capacities across government actors and communities, and limited attention to domestic monitoring and enforcement. Improving the effectiveness of resource management efforts may result in sustainable outcomes for both livelihoods and health, which will accomplish priorities at the national and sub-national levels and support the achievement of the Solomon Islands' commitments to the Sustainable Development Goals.


Prognostic Significance of Elevated Cardiac Troponin in Acute Gastrointestinal Bleeding.
Acute gastrointestinal bleeding (AGIB) is responsible for over 140,000 hospitalizations annually. Cardiovascular-related deaths account for 30% of the patients surviving the initial episode of AGIB. The purpose of this study was to identify the impact of elevated troponin on short-term mortality and length of stay (LOS) of these patients. From July 2013 to July 2016, 290 patients admitted with a diagnosis of AGIB and who had cardiac troponin I measured within 24 h of presentation were retrospectively reviewed. Clinical variables including 30-day mortality, 30-day readmission and LOS were then compared between the groups of troponin elevation and no troponin elevation. The overall 30-day mortality among patients with AGIB was 6.5% (19/290). Cardiac troponin was elevated in 10% of patients (29/290). Among patients with normal troponin, 5% (13/261) died within 30 days. In patients with troponin elevation, 21% died in the same period (6/29, P = 0.001). The LOS was also higher in patients with troponin elevation (6 vs. 5 days, P = 0.02). There was no difference in 30-day readmission among the two groups. Past history of coronary artery disease, congestive heart failure, hypertension, aspirin use and elevated creatinine was more common in patients with troponin elevation. On multivariate analysis, troponin elevation on presentation is associated with increased mortality (odds: 5.50, CI: 1.73 - 17.47, P = 0.004). In patients admitted to the inpatient service with AGIB, elevated troponin I on presentation is associated with high short-term mortality and longer hospital stay.


Asthma and allergic symptoms in relation to house dust endotoxin: Phase Two of the International Study on Asthma and Allergies in Childhood (ISAAC II).
Several studies have consistently reported inverse associations between exposure to endotoxin in house dust and atopy. With regard to the association between house dust endotoxin and asthma, the results are inconsistent. To study the association between house dust endotoxin levels and respiratory symptoms and atopy in populations from largely different countries. Data were collected within the International Study on Asthma and Allergies in Childhood Phase Two, a multi-centre cross-sectional study of 840 children aged 9-12 years from six centres in the five countries of Albania, Italy, New Zealand, Sweden and the United Kingdom. Living room floor dust was collected and analysed for endotoxin. Health end-points and demographics were assessed by standardized questionnaires. Atopy was assessed by measurements of allergen-specific IgE against a panel of inhalant allergens. Associations between house dust endotoxin and health outcomes were analysed by logistic regression. Odds ratios (ORs) were presented for an overall interquartile range increase in exposure. Many associations between house dust endotoxin in living room floor dust and health outcomes varied between countries. Combined across countries, endotoxin levels were inversely associated with asthma ever [adjusted OR (95% confidence interval (CI)) 0.53 (0.29-0.96) for endotoxin levels per m(2) of living room floor] and current wheeze [adjusted OR (95% CI) 0.77 (0.64-0.93) for endotoxin levels per gram of living room floor dust]. There were inverse associations between endotoxin concentrations and atopy, which were statistically significant in unadjusted analyses, but not after adjustment for gender, parental allergies, cat and house dust mite allergens. No associations were found with dust quantity and between endotoxin exposure and hayfever. These findings suggest an inverse association between endotoxin levels in living room floor dust and asthma in children.


Differential T helper cell responses to human papillomavirus type 16 E7 related to viral clearance or persistence in patients with cervical neoplasia: a longitudinal study.
T-cell-mediated immune responses against oncogenic human papillomaviruses (HPVs) are believed to play a role in the prevention of cervical carcinogenesis. The in vitro production of interleukin 2 by CD4+ T helper (Th) cells in response to overlapping 20-mer peptides covering the HPV-16 E7 oncoprotein sequence was determined in 72 women with cytological evidence of premalignant cervical intraepithelial neoplasia (CIN) who participated in a nonintervention follow-up (FU) study. In addition, 15 HPV-16 + cervical carcinoma patients were tested. Positive Th cell reactivity was restricted to patients infected by HPV-16 and related types and showed a strong association with viral persistence and disease progression, as evidenced by the high frequency of positive responders among women with persistent HPV-16 infections who ended FU with high-grade CIN III lesions [14 of 15 (93%)]. Women with cervical carcinoma showed responses at a significantly reduced rate [7 of 15 (47%); P = 0.014]. Over the FU period (10-34 months), the level of E7-induced interleukin 2 production from the lymphocytes of CIN patients who had cleared HPV-16 infection showed an inverse correlation with time relative to the last positive HPV DNA test, with 8 of 13 of these patients showing positive responses after clearance. By contrast, among women with persistent HPV-16 infections and developing CIN III lesions (n = 8), there was a rise in Th cell activity over the course of FU. The majority of women responded to an immunogenic region in the carboxyl terminus of the E7 protein (amino acids 67-98). The observed HPV-16 E7-specific Th cell responses may develop as a consequence of increased antigen availability resulting either from clearance or from progression of cervical lesions.


Synthesis, characterization and reactivity of an imidazolin-2-iminato aluminium dihydride.
The reaction of bis(2,6-diisopropylphenyl)imidazolin-2-imine (LH, 1) with Me3N·AlH3 furnishes {μ-LAlH2}2 (2). The marked tendency of 2 to release its hydride substituents is ascribed to the strong electron-donor character of the imidazolin-2-iminato ligand. This is supported by its reactivity study and DFT calculations. In fact, compound 2 was further converted with Me3SiOTf, Me2S·BH3, Me2S·BBr3, and BX3 (with X = Cl, Br, and I) into {μ-LAl(H)OTf}2 (3), {μ-LAl(BH4)2}2 (4), and {μ-LAlX2}2 (5, X = Br; 6, X = Cl; 7, X = I), respectively. For all new aluminium complexes the formulation as dimers was evidenced by high resolution mass spectrometry, as well as single-crystal X-ray diffraction analysis. A prominent structural motif of these compounds is the square-planar four-membered Al2N2 ring with two bridging bulky imidazolin-2-imino moieties.


Impact of ion binding on poly-L-lysine (un)folding energy landscape and kinetics.
We utilize T-jump UV resonance Raman spectroscopy (UVRR) to study the impact of ion binding on the equilibrium energy landscape and on (un)folding kinetics of poly-L-lysine (PLL). We observe that the relaxation rates of the folded conformations (including π-helix (bulge), pure α-helix, and turns) of PLL are slower than those of short alanine-based peptides. The PLL pure α-helix folding time is similar to that of short alanine-based peptides. We for the first time have directly observed that turn conformations are α-helix and π-helix (bulge) unfolding intermediates. ClO(4)(-) binding to the Lys side chain -NH(3)(+) groups and the peptide backbone slows the α-helix unfolding rate compared to that in pure water, but little impacts the folding rate, resulting in an increased α-helix stability. ClO(4)(-) binding significantly increases the PLL unfolding activation barrier but little impacts the folding barrier. Thus, the PLL folding coordinate(s) differs from the unfolding coordinate(s). The-π helix (bulge) unfolding and folding coordinates do not directly go through the α-helix energy well. Our results clearly demonstrate that PLL (un)folding is not a two-state process.


Mucopolysaccharidosis type IIID: 12 new patients and 15 novel mutations.
Mucopolysaccharidosis III D (Sanfilippo disease type D, MPS IIID) is a rare autosomal recessive lysosomal storage disorder previously described in only 20 patients. MPS IIID is caused by a deficiency of N-acetylglucosamine-6-sulphate sulphatase (GNS), one of the enzymes required for the degradation of heparan sulphate. So far only seven mutations in the GNS gene have been reported. The clinical phenotype of 12 new MPS IIID patients from 10 families was studied. Mutation analysis of GNS was performed in 16 patients (14 index cases). Clinical signs and symptoms of the MPS IIID patients appeared to be similar to previously described patients with MPS III. Early development was normal with onset of behavioral problems around the age of 4 years, followed by developmental stagnation, deterioration of verbal communication and subsequent deterioration of motor functions. Sequence analysis of the coding regions of the gene encoding GNS (GNS) resulted in the identification of 15 novel mutations: 3 missense mutations, 1 nonsense mutation, 4 splice site mutations, 3 frame shift mutations, 3 large deletions and 1 in-frame small deletion. They include the first missense mutations and a relatively high proportion of large rearrangements, which warrants the inclusion of quantitative techniques in routine mutation screening of the GNS gene.


Improvements in Pyrosequencing technology by employing Sequenase polymerase.
Pyrosequencing is a DNA sequencing technique based on the bioluminometric detection of inorganic pyrophosphate, which is released when nucleotides are incorporated into a target DNA. Since the technique is based on an enzymatic cascade, the choice of enzymes is a critical factor for efficient performance of the sequencing reaction. In this study we have analyzed the performance of an alternative DNA polymerase, Sequenase, on the sequencing performance of the Pyrosequencing technology. Compared to the Klenow fragment of DNA polymerase I, Sequenase could read through homopolymeric regions with more than five T bases. In addition, Sequenase reduces remarkably interference from primer-dimers and loop structures that give rise to false sequence signals. By using Sequenase, synchronized extensions and longer reads can be obtained on challenging templates, thereby opening new avenues for applications of Pyrosequencing technology.


Creating a culture of inquiry in family medicine.
Strengthening the contribution of reflective practice and new knowledge generation to the learning relationships forged during graduate and undergraduate medical training offers a possibility to create a climate more conducive to the recruitment and retention of family physicians. The Culture of Inquiry (CI) fellowship, an immersive, experientially based training program, combines didactic instruction, workshops, and mentoring to develop the capacity of family medicine's teachers to imagine, implement, and disseminate clinically relevant research and stimulate collaborations with those whom they train. This article outlines the CI fellowship program, summarizes its outcomes, and offers insights about programmatic features contributing to its success. The Department of Family Medicine and Community Health at Case Western Reserve University selected CI fellows from interested local family physicians who train residents and medical students. Over 10 months, with 10% effort expected from fellows, the CI fellowship exposed each fellow to the entire research process and provided technical and logistical support for the design and completion of two research projects. Quantitative and qualitative program evaluation were used to assess outcomes. Scholarly productivity of fellows exceeded expectations. Collaborations with students and residents produced a ripple effect that amplified the fellowship's impact by strengthening those relationships crucial to the creation of a culture of inquiry among family medicine's teachers, learners, and practitioners. The CI fellowship represents a highly replicable program to connect committed and interested clinicians to research mentors with the goal of increasing scholarship and creating a growing culture of inquiry in family medicine.


[Prokaryotic expression systems].
For overproduction of recombinant proteins both eukaryotic and prokaryotic expression systems are used. Choosing the right system depends, among other things, on the growth rate and culture of host cells, level of the target gene expression and posttranslational processing of the synthesized protein. Regardless of the type of expression system, its basic elements are the vector and the expression host. The most widely used system for protein overproduction, both on a laboratory and industrial scale, is the prokaryotic system. This system is based primarily on the bacteria E. coli, although increasingly often Bacillus species are used. The prokaryotic system allows one to obtain large quantities of recombinant proteins in a short time. A simple and inexpensive bacterial cell culture and well-known mechanisms of transcription and translation facilitate the use of these microorganisms. The simplicity of genetic modifications and the availability of many bacterial mutants are additional advantages of the prokaryotic system. In this article we characterize the structural elements of prokaryotic expression vectors. Also strategies for preparation of the target protein gene that increase productivity, facilitate detection and purification of recombinant protein and provide its activity are discussed. Bacterial strains often used as host cells in expression systems as well as the potential location of heterologous proteins are characterized. Knowledge of the basic elements of the prokaryotic expression system allows for production of biologically active proteins in a short time and in satisfactory quantities.


Quantification and kinetics of viral RNA transcripts produced in Orthohantavirus infected cells.
Rodent borne viruses of the Orthohantavirus genus cause hemorrhagic fever with renal syndrome among people in Eurasia, and hantavirus cardiopulmonary syndrome in the Americas. At present, there are no specific treatments or efficient vaccines against these diseases. Improved understanding of viral transcription and replication may instigate targeted treatment of Orthohantavirus infections. For this purpose, we investigated the kinetics and levels of viral RNA transcription during an ongoing infection in-vitro. Vero E6 cells were infected with Puumala Orthohantavirus (strain Kazan) before cells and supernatants were collected at different time points post infection for the detection of viral RNAs. A plasmid containing primer binding sites of the three Orthohantavirus segments small (S), medium (M) and large (L) was constructed and standard curves were generated to calculate the copy numbers of the individual transcripts in the collected samples. Our results indicated a rapid increase in the copy number of viral RNAs after 9 h post infection. At peak days, 2-6 days after infection, the S- and M-segment transcripts became thousand and hundred-fold more abundant than the copy number of the L-segment RNA, respectively. The presence of viral RNA in the cell culture media was detected at later time-points. We have developed a method to follow RNA transcription in-vitro after synchronous infection of Vero cells. The obtained results may contribute to the understanding of the viral replication, and may have implications in the development of antiviral drugs targeting transcription or replication of negative stranded RNA viruses.


Synthesis of 3-alkyl enol mimics inhibitors of type II dehydroquinase: factors influencing their inhibition potency.
Several 3-alkylaryl mimics of the enol intermediate in the reaction catalyzed by type II dehydroquinase were synthesized to investigate the effect on the inhibition potency of replacing the oxygen atom in the side chain by a carbon atom. The length and the rigidity of the spacer was also studied. The inhibitory properties of the reported compounds against type II dehydroquinase from Mycobacterium tuberculosis and Helicobacter pylori are also reported. The binding modes of these analogs in the active site of both enzymes were studied by molecular docking using GOLD 5.0 and dynamic simulations studies.


Advances in CRISPR-Cas9 genome engineering: lessons learned from RNA interference.
The discovery that the machinery of the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 bacterial immune system can be re-purposed to easily create deletions, insertions and replacements in the mammalian genome has revolutionized the field of genome engineering and re-invigorated the field of gene therapy. Many parallels have been drawn between the newly discovered CRISPR-Cas9 system and the RNA interference (RNAi) pathway in terms of their utility for understanding and interrogating gene function in mammalian cells. Given this similarity, the CRISPR-Cas9 field stands to benefit immensely from lessons learned during the development of RNAi technology. We examine how the history of RNAi can inform today's challenges in CRISPR-Cas9 genome engineering such as efficiency, specificity, high-throughput screening and delivery for in vivo and therapeutic applications.


Management of Metastatic Hormone-Sensitive Prostate Cancer (mHSPC): an Evolving Treatment Paradigm.
Combination systemic therapy is now standard of care for all men with metastatic, hormone-sensitive prostate cancer (mHSPC). Patients with mHSPC should be treated with standard androgen deprivation therapy (ADT) and abiraterone acetate with prednisone or docetaxel (chemohormoanl therapy) unless there are contraindications to combination therapy. Based on the Chemohormonal Therapy Versus Androgen Ablation Randomized Trial for Extensive Disease in Prostate Cancer (CHAARTED) study subgroup analysis, chemohormonal therapy may be most beneficial in men with high-volume disease burden, as men with low-volume metastatic disease do not appear to experience a survival benefit with chemohormonal therapy, while abiraterone in combination with ADT appears to be beneficial across both disease volume subgroups. Decisions regarding whether to use chemohormonal therapy or abiraterone and ADT for men with mHSPC should integrate consideration of volume of disease burden, quality of life effects, duration of therapy, and patient preferences for treatment as there is no formally powered prospective head-to-head comparison of these options demonstrating superiority of one approach over the other. Treatment of the primary tumor with radiation should be considered in men with de novo low-volume metastatic disease as radiation is associated with prolonged survival and a tolerable toxicity profile. Men with de novo high-volume metastatic disease do not appear to have improved survival with radiation of the primary tumor. Numerous clinical trials are ongoing to evaluate treatment approaches that may benefit men with mHSPC. Radical prostatectomy in men with mHSPC in combination with optimal systemic therapy is currently being assessed in a clinical trial, but should not be considered outside of a clinical trial. Metastasis-directed therapy with radiotherapy directed at metastatic lesions is still investigational, but can be considered in clinical trials in men with oligometastatic disease. Multiple studies are enrolling worldwide for men with mHSPC, and these should be considered for all interested patients.


Potential biological fate of ingested nanoemulsions: influence of particle characteristics.
Edible nanoemulsions have great potential for utilization in the food and beverage industries to encapsulate, protect, and deliver lipophilic functional components claimed to have health benefits ("nutraceuticals"), such as carotenoids, flavonoids, phytosterols, polyunsaturated lipids, and oil-soluble vitamins. Nanoemulsions have a number of possible advantages over conventional emulsions for these applications, including high optical clarity, high stability to particle aggregation and gravitational separation, and increased bioavailability of lipophilic substances. Nevertheless, there are concerns about the potential risks associated with ingestion of nanoemulsions due to their ability to alter the behavior of bioactive components within the gastrointestinal tract. At present, there is still a relatively poor understanding of the biological fate of nanoemulsions in the human GI tract, which is holding back the rational design and application of nanoemulsion-based delivery systems for lipophilic bioactive components. This article provides a brief review of the current status of the formation, properties, and potential biological fate of food-grade nanoemulsions. In particular, it focuses on the influence of particle characteristics, such as size and interfacial properties, on the digestion and absorption of lipid nanoparticles.


Past and current gene flow in the selfing, wind-dispersed species Mycelis muralis in western Europe.
The distribution of genetic diversity in Mycelis muralis, or wall lettuce, was investigated at a European scale using 12 microsatellite markers to infer historical and contemporary forces from genetic patterns. Mycelis muralis has the potential for long-distance seed dispersal by wind, is mainly self-pollinated, and has patchily distributed populations, some of which may show metapopulation dynamics. A total of 359 individuals were sampled from 17 populations located in three regions, designated southern Europe (Spain and France), the Netherlands, and Sweden. At this within-region scale, contemporary evolutionary forces (selfing and metapopulation dynamics) are responsible for high differentiation between populations (0.34 < F(ST) < 0.60) but, contrary to expectation, levels of within-population diversity, estimated by Nei's unbiased expected heterozygosity (H(E)) (0.24 < H(E) < 0.68) or analyses of molecular variance (50% of the variation found within-populations), were not low. We suggest that the latter results, which are unusual in selfing species, arise from efficient seed dispersal that counteracts population turnover and thus maintains genetic diversity within populations. At the European scale, northern regions showed lower allelic richness (A = 2.38) than populations from southern Europe (A = 3.34). In light of postglacial colonization hypotheses, these results suggest that rare alleles may have been lost during recolonization northwards. Our results further suggest that mutation has contributed to genetic differentiation between southern and northern Europe, and that Sweden may have been colonized by dispersers originating from at least two different refugia.


Reliability of lower extremity functional performance tests.
Clinicians routinely have used functional performance tests as an evaluation tool in deciding when an athlete can safely return to unrestricted sporting activities. These practitioners assumed that these tests provide a reliable measure of lower extremity performance; however, little research has been reported on the reliability of these measures. The purpose of this investigation was to determine the reliability of lower extremity functional performance tests. Five male and 15 female volunteers were evaluated using the single hop for distance, triple hop for distance, 6-m timed hop, and cross-over hop for distance as described by Noyes (10). One clinician measured each subject's performance using a standardized protocol and retested subjects in the same manner approximately 48 hours later. The order of testing was randomly determined. Subjects' average and individual scores on each functional performance test were used for statistical analysis. Intraclass correlation coefficients (ICCs) and standard error of measurement (SEM) values based on average day 1 and day 2 scores were used to estimate the reliability of each functional performance test. Intraclass correlation coefficients were .96, .95, and .96, and SEMs were 4.56 cm, 15.44 cm, and 15.95 cm, respectively, for the single hop, triple hop, and cross-over hop for distance tests. An ICC of .66 and SEM of .13 seconds for the 6-m timed hop resulted from limited variability between measurements; however, its small SEM value inferred that the inconsistency of measurement would occur in an acceptably small range. A repeated measures analysis of variance revealed no significant difference ( p > .05) between individual trial scores except for the single hop for distance. We concluded that this difference represented a learning effect not found with the other tests. The results of this investigation demonstrate that clinicians can use functional performance testing to obtain reliable measures of lower extremity performance when using a standardized protocol.


Utility of the pooling approach as applied to whole genome association scans with high-density Affymetrix microarrays.
We report an attempt to extend the previously successful approach of combining SNP (single nucleotide polymorphism) microarrays and DNA pooling (SNP-MaP) employing high-density microarrays. Whereas earlier studies employed a range of Affymetrix SNP microarrays comprising from 10 K to 500 K SNPs, this most recent investigation used the 6.0 chip which displays 906,600 SNP probes and 946,000 probes for the interrogation of CNVs (copy number variations). The genotyping assay using the Affymetrix SNP 6.0 array is highly demanding on sample quality due to the small feature size, low redundancy, and lack of mismatch probes. In the first study published so far using this microarray on pooled DNA, we found that pooled cheek swab DNA could not accurately predict real allele frequencies of the samples that comprised the pools. In contrast, the allele frequency estimates using blood DNA pools were reasonable, although inferior compared to those obtained with previously employed Affymetrix microarrays. However, it might be possible to improve performance by developing improved analysis methods. Despite the decreasing costs of genome-wide individual genotyping, the pooling approach may have applications in very large-scale case-control association studies. In such cases, our study suggests that high-quality DNA preparations and lower density platforms should be preferred.


Assessment of Quality of Life and Urinary and Sexual Function After Radical Hysterectomy in Long-Term Cervical Cancer Survivors.
The aim of this study was to evaluate long-term quality of life and urinary and sexual function in long-term cervical cancer survivors previously treated with radical hysterectomy (RH) type C2/type III. All patients who presented at Campus Bio-Medico of Rome for RH type C2/type III for cervical cancer were considered eligible for this retrospective study protocol. We included exclusively patients with complete response to primary treatment with at least 36 months of follow up. Included subjects were interviewed with the European Organization for Research and Treatment of Cancer QLQ-CX24 Questionnaire, European Organization for Research and Treatment of Cancer QLQ-C30, and an Incontinence Impact Questionnaire 7. From January 2004 to June 2014, 251 patients affected by locally advanced cervical cancer were treated at Campus Bio-Medico of Rome treated with type C2/type III RH. At time point of March 2017, 90 patients were included with a mean age of 55.6 ± 8.5 years. The questionnaires were administered after a median follow-up of 49 months after the end of therapy. The symptoms of fatigue, nausea and vomiting, appetite loss, pain, insomnia, and dyspnea, as well as a negative financial impact, were reported as not frequent and rarely disabling. On the contrary, patients frequently reported gastrointestinal complaints. Diarrhea was present in 6% of patients and was referred as mild; constipation was present in 75% of women and was reported as mild in 30% of cases, moderate in 30%, and severe in 15%. Concerning sexual activity, data indicated a good level of sexual enjoyment with a slight worsening of sexual activity. Incontinence was reported in 28% of cases and appeared to be mild and rarely disabling (all mean values <2). Waiting for ongoing randomized controlled trials, this study confirmed that RH may be considered as a useful treatment plan, according to its negligible long-term impact on quality of life, urinary dysfunction, and sexual function.


Investigation of folate pathway gene polymorphisms and the incidence of neural tube defects in a Texas hispanic population.
Neural tube defects (NTDs) are multifactorial in their etiology, having both genetic and environmental factors contributing to their development. Recent evidence demonstrates that periconceptional supplementation of the maternal diet with a multivitamin containing folic acid significantly reduces the occurrence and recurrence risk for having a pregnancy complicated by NTDs. Unfortunately, the mechanism underlying the beneficial effects of folic acid remains unknown. NTD surveillance data from the Texas-Mexico border show that the high NTD rate (28/10,000 live births) noted during the 1990-1991 Cameron county NTD cluster was superimposed on a background Cameron county NTD rate (16/10,000 live births) which is considerably higher than that generally noted in the United States (8-10/10,000 live births). These data suggest that genetic factors as well as transient environmental factors may contribute to the etiology of the NTDs. Furthermore, clinical and experimental evidence imply that allelic forms of genes involved with folate metabolism and/or transport may explain some of the observed variation in the NTD rates found across different populations. Two folate pathway genes were selected for evaluation in this study. The loci investigated included two known alleles of the 5, 10-methylenetetrahydrofolate reductase (MTHFR) gene, as well as the promoter region of the folate receptor-alpha (FR-alpha) gene. Odds ratios (ORs) for the C677T polymorphism in the MTHFR gene were 1.8 (CI 0.47-6.8) for heterozygosity and 1.8 (CI 0.35-9.4) for homozygosity for the mutant 677T allele, relative to wildtype homozygotes. The odds ratio for the heterozygosity for the A1298C polymorphism in the same gene was 1.1 (CI 0.09-14). No individuals homozygous for the 1298C allele were observed. The OR for heterozygosity of FR-alpha gene polymorphisms detected at nucleotide 762 and at nucleotides 610/631 was 1.4 and 0.7, respectively. Neither of the FR-alpha polymorphisms was observed in the homozygous condition. No statistically significant associations were observed for any of the polymorphisms examined, as the 95% confidence intervals for all of the ORs included one. However, the frequency of the MTHFR 677T allele in the largely Hispanic control group from Texas was significantly different from other populations (P < 0.005), and among the highest reported for any control populations examined.


Durable adoptive immunotherapy for leukemia produced by manipulation of multiple regulatory pathways of CD8+ T-cell tolerance.
Tolerizing mechanisms within the host and tumor microenvironment inhibit T-cell effector functions that can control cancer. These mechanisms blunt adoptive immunotherapy with infused T-cells due to a complex array of signals that determine T-cell tolerance, survival, or deletion. Ligation of the negative regulatory receptors CTLA4, PD-1(PDCD1), or LAG3 on T-cells normally hinders their response to antigen through nonredundant biochemical processes that interfere with stimulatory pathways. In this study, we used an established mouse model of T-cell tolerance to define the roles of these inhibitory receptors in regulating CD8(+) T-cell tolerance during adoptive immunotherapy to treat leukemia. Blocking CTLA4 and PD-1 in vivo combined to promote survival of transferred T-cells despite powerful deletional signals that mediate Bim (BCL2L11)-dependent apoptosis. However, this dual blockade was not optimal for stimulating effector function by responding T-cells, which required the additional blockade of LAG3 to induce full expansion and allow the acquisition of robust cytolytic activity. Thus, the cooperation of multiple distinct regulatory pathways was needed for the survival and effector differentiation of adoptively transferred tumor-reactive CD8(+) T-cells. Our work defines the immune escape pathways in which simultaneous blockade could yield durable immunotherapeutic responses that can eradicate disseminated leukemia.


SNP markers-based map construction and genome-wide linkage analysis in Brassica napus.
An Illumina Infinium array comprising 5306 single nucleotide polymorphism (SNP) markers was used to genotype 175 individuals of a doubled haploid population derived from a cross between Skipton and Ag-Spectrum, two Australian cultivars of rapeseed (Brassica napus L.). A genetic linkage map based on 613 SNP and 228 non-SNP (DArT, SSR, SRAP and candidate gene markers) covering 2514.8 cM was constructed and further utilized to identify loci associated with flowering time and resistance to blackleg, a disease caused by the fungus Leptosphaeria maculans. Comparison between genetic map positions of SNP markers and the sequenced Brassica rapa (A) and Brassica oleracea (C) genome scaffolds showed several genomic rearrangements in the B. napus genome. A major locus controlling resistance to L. maculans was identified at both seedling and adult plant stages on chromosome A07. QTL analyses revealed that up to 40.2% of genetic variation for flowering time was accounted for by loci having quantitative effects. Comparative mapping showed Arabidopsis and Brassica flowering genes such as Phytochrome A/D, Flowering Locus C and agamous-Like MADS box gene AGL1 map within marker intervals associated with flowering time in a DH population from Skipton/Ag-Spectrum. Genomic regions associated with flowering time and resistance to L. maculans had several SNP markers mapped within 10 cM. Our results suggest that SNP markers will be suitable for various applications such as trait introgression, comparative mapping and high-resolution mapping of loci in B. napus.


Experiences of Dutch students growing up with a family member with a chronic illness: A qualitative study.
Students growing up with a family member with a chronic illness often delay or drop out of school when the combination of family care and their educational programme is too demanding. This study aims to describe the themes experienced by students growing up in this situation. A qualitative focus group design was used. The recruitment for this study was done through an online survey where students could indicate whether they also wanted to participate in a focus group. Twenty-five Dutch students (age 18-25 years) who grew up with a family member with a chronic illness participated in one of the total of seven focus groups. A qualitative inductive analysis was used to identify codes and main themes. Students growing up with a family member with a chronic illness experience responsibility issues, loyalty conflicts, peer alienation, role reversal, negative influence on personal development, daily worries and confronting situations. They reported substantial impact in terms of their general well-being, their family relationships and in their choices for their future. Students articulated that growing up in this situation has a serious and substantial impact on their lives. Further research is required in order to gain comprehensive insights into the requests of these students regarding personal and family support.


Informing Patients About Placebo Effects: Using Evidence, Theory, and Qualitative Methods to Develop a New Website.
According to established ethical principles and guidelines, patients in clinical trials should be fully informed about the interventions they might receive. However, information about placebo-controlled clinical trials typically focuses on the new intervention being tested and provides limited and at times misleading information about placebos. We aimed to create an informative, scientifically accurate, and engaging website that could be used to improve understanding of placebo effects among patients who might be considering taking part in a placebo-controlled clinical trial. Our approach drew on evidence-, theory-, and person-based intervention development. We used existing evidence and theory about placebo effects to develop content that was scientifically accurate. We used existing evidence and theory of health behavior to ensure our content would be communicated persuasively, to an audience who might currently be ignorant or misinformed about placebo effects. A qualitative 'think aloud' study was conducted in which 10 participants viewed prototypes of the website and spoke their thoughts out loud in the presence of a researcher. The website provides information about 10 key topics and uses text, evidence summaries, quizzes, audio clips of patients' stories, and a short film to convey key messages. Comments from participants in the think aloud study highlighted occasional misunderstandings and off-putting/confusing features. These were addressed by modifying elements of content, style, and navigation to improve participants' experiences of using the website. We have developed an evidence-based website that incorporates theory-based techniques to inform members of the public about placebos and placebo effects. Qualitative research ensured our website was engaging and convincing for our target audience who might not perceive a need to learn about placebo effects. Before using the website in clinical trials, it is necessary to test its effects on key outcomes including patients' knowledge and capacity for making informed choices about placebos.


The genetic epidemiology of cancer: interpreting family and twin studies and their implications for molecular genetic approaches.
The recent completion of a rough draft of the human genome sequence has ushered in a new era of molecular genetics research into the inherited basis of a number of complex diseases such as cancer. At the same time, recent twin studies have suggested a limited role of genetic susceptibility to many neoplasms. A reappraisal of family and twin studies for many cancer sites suggests the following general conclusions: (a) all cancers are familial to approximately the same degree, with only a few exceptions (both high and low); (b) early age of diagnosis is generally associated with increased familiality; (c) familiality does not decrease with decreasing prevalence of the tumor-in fact, the trend is toward increasing familiality with decreasing prevalence; (d) a multifactorial (polygenic) threshold model fits the twin data for most cancers less well than single gene or genetic heterogeneity-type models; (e) recessive inheritance is less likely generally than dominant or additive models; (f) heritability decreases for rarer tumors only in the context of the polygenic model but not in the context of single-locus or heterogeneity models; (g) although the family and twin data do not account for gene-environment interactions or confounding, they are still consistent with genes contributing high attributable risks for most cancer sites. These results support continued search for genetic and environmental factors in cancer susceptibility for all tumor types. Suggestions are given for optimal study designs depending on the underlying architecture of genetic predisposition.


Exact confidence intervals for the relative risk and the odds ratio.
For comparison of proportions, there are three commonly used measurements: the difference, the relative risk, and the odds ratio. Significant effort has been spent on exact confidence intervals for the difference. In this article, we focus on the relative risk and the odds ratio when data are collected from a matched-pairs design or a two-arm independent binomial experiment. Exact one-sided and two-sided confidence intervals are proposed for each configuration of two measurements and two types of data. The one-sided intervals are constructed using an inductive order, they are the smallest under the order, and are admissible under the set inclusion criterion. The two-sided intervals are the intersection of two one-sided intervals. R codes are developed to implement the intervals. Supplementary materials for this article are available online.