Oxidative stress and inflammation response after nanoparticle exposure: differences between human lung cell monocultures and an advanced three-dimensional model of the human epithelial airways

Combustion-derived and manufactured nanoparticles (NPs) are known to provoke oxidative stress and inflammatory responses in human lung cells; therefore, they play an important role during the development of adverse health effects. As the lungs are composed of more than 40 different cell types, it is of particular interest to perform toxicological studies with co-cultures systems, rather than with monocultures of only one cell type, to gain a better understanding of complex cellular reactions upon exposure to toxic substances. Monocultures of A549 human epithelial lung cells, human monocyte-derived macrophages and monocyte-derived dendritic cells (MDDCs) as well as triple cell co-cultures consisting of all three cell types were exposed to combustion-derived NPs (diesel exhaust particles) and to manufactured NPs (titanium dioxide and single-walled carbon nanotubes). The penetration of particles into cells was analysed by transmission electron microscopy. The amount of intracellular reactive oxygen species (ROS), the total antioxidant capacity (TAC) and the production of tumour necrosis factor (TNF)-alpha and interleukin (IL)-8 were quantified. The results of the monocultures were summed with an adjustment for the number of each single cell type in the triple cell co-culture. All three particle types were found in all cell and culture types. The production of ROS was induced by all particle types in all cell cultures except in monocultures of MDDCs. The TAC and the (pro-)inflammatory reactions were not statistically significantly increased by particle exposure in any of the cell cultures. Interestingly, in the triple cell co-cultures, the TAC and IL-8 concentrations were lower and the TNF-alpha concentrations were higher than the expected values calculated from the monocultures. The interplay of different lung cell types seems to substantially modulate the oxidative stress and the inflammatory responses after NP exposure.

Is the acetabular cup orientation after total hip arthroplasty on a two dimension or three dimension model accurate?

Purpose Malposition of the acetabular component in total hip arthroplasty (THA) is a common surgical problem that can lead to hip dislocation, reduced range of motion and may result in early loosening. The aim of this study is to validate the accuracy and reproducibility of a single x-ray image based 2D/3D reconstruction technique in determining cup inclination and anteversion against two different computer tomography (CT)-based measurement techniques. Methods Cup anteversion and inclination of 20 patients after cementless primary THA was measured on standard anteroposterior (AP) radiographs with the help of the single x-ray 2D/3D reconstruction program and compared with two different 3D CT-based analyses [Ground Truth (GT) and MeVis (MV) reconstruction model]. Results The measurements from the single x-ray 2D/3D reconstruction technique were strongly correlated with both types of CT image-processing protocols for both cup inclination [R²=0.69 (GT); R²=0.59 (MV)] and anteversion [R²=0.89 (GT); R²=0.80 (MV)]. Conclusions The single x-ray image based 2D/3D reconstruction technique is a feasible method to assess cup position on postoperative x-rays. CTscans remain the golden standard for a more complex biomechanical evaluation when a lower tolerance limit (+/-2 degrees) is required.

Evaluation of Three State-of-the-Art Classifiers for Recognition of Activities of Daily Living from Smart Home Ambient Data

Smart homes for the aging population have recently started attracting the attention of the research community. The "health state" of smart homes is comprised of many different levels; starting with the physical health of citizens, it also includes longer-term health norms and outcomes, as well as the arena of positive behavior changes. One of the problems of interest is to monitor the activities of daily living (ADL) of the elderly, aiming at their protection and well-being. For this purpose, we installed passive infrared (PIR) sensors to detect motion in a specific area inside a smart apartment and used them to collect a set of ADL. In a novel approach, we describe a technology that allows the ground truth collected in one smart home to train activity recognition systems for other smart homes. We asked the users to label all instances of all ADL only once and subsequently applied data mining techniques to cluster in-home sensor firings. Each cluster would therefore represent the instances of the same activity. Once the clusters were associated to their corresponding activities, our system was able to recognize future activities. To improve the activity recognition accuracy, our system preprocessed raw sensor data by identifying overlapping activities. To evaluate the recognition performance from a 200-day dataset, we implemented three different active learning classification algorithms and compared their performance: naive Bayesian (NB), support vector machine (SVM) and random forest (RF). Based on our results, the RF classifier recognized activities with an average specificity of 96.53%, a sensitivity of 68.49%, a precision of 74.41% and an F-measure of 71.33%, outperforming both the NB and SVM classifiers. Further clustering markedly improved the results of the RF classifier. An activity recognition system based on PIR sensors in conjunction with a clustering classification approach was able to detect ADL from datasets collected from different homes. Thus, our PIR-based smart home technology could improve care and provide valuable information to better understand the functioning of our societies, as well as to inform both individual and collective action in a smart city scenario.

Three-State Single-Molecule Naphthalenediimide Switch: Integration of a Pendant Redox Unit for Conductance Tuning

We studied charge transport through core-substituted naphthalenediimide (NDI) single-molecule junctions using the electrochemical STM-based break-junction technique in combination with DFT calculations. Conductance switching among three well-defined states was demonstrated by electrochemically controlling the redox state of the pendent diimide unit of the molecule in an ionic liquid. The electrical conductances of the dianion and neutral states differ by more than one order of magnitude. The potential-dependence of the charge-transport characteristics of the NDI molecules was confirmed by DFT calculations, which account for electrochemical double-layer effects on the conductance of the NDI junctions. This study suggests that integration of a pendant redox unit with strong coupling to a molecular backbone enables the tuning of charge transport through single-molecule devices by controlling their redox states.

Laser scanning microscopy combined with image restoration to analyse a 3D model of the human epithelial airway barrier

A laser scanning microscope collects information from a thin, focal plane and ignores out of focus information. During the past few years it has become the standard imaging method to characterise cellular morphology and structures in static as well as in living samples. Laser scanning microscopy combined with digital image restoration is an excellent tool for analysing the cellular cytoarchitecture, expression of specific proteins and interactions of various cell types, thus defining valid criteria for the optimisation of cell culture models. We have used this tool to establish and evaluate a three dimensional model of the human epithelial airway wall.

Clinical and biochemical description of a novel CYP21A2 gene mutation 962_963insA using a new 3D model for the P450c21 protein

OBJECTIVE: A severely virilized 46, XX newborn girl was referred to our center for evaluation and treatment of congenital adrenal hyperplasia (CAH) because of highly elevated 17alpha-hydroxyprogesterone levels at newborn screening; biochemical tests confirmed the diagnosis of salt-wasting CAH. Genetic analysis revealed that the girl was compound heterozygote for a previously reported Q318X mutation in exon 8 and a novel insertion of an adenine between nucleotides 962 and 963 in exon 4 of the CYP21A2 gene. This 962_963insA mutation created a frameshift leading to a stop codon at amino acid 161 of the P450c21 protein. AIM AND METHODS: To better understand structure-function relationships of mutant P450c21 proteins, we performed multiple sequence alignments of P450c21 with three mammalian P450s (P450 2C8, 2C9 and 2B4) with known structures as well as with human P450c17. Comparative molecular modeling of human P450c21 was then performed by MODELLER using the X-ray crystal structure of rabbit P450 2B4 as a template. RESULTS: The new three dimensional model of human P450c21 and the sequence alignment were found to be helpful in predicting the role of various amino acids in P450c21, especially those involved in heme binding and interaction with P450 oxidoreductase, the obligate electron donor. CONCLUSION: Our model will help in analyzing the genotype-phenotype relationship of P450c21 mutations which have not been tested for their functional activity in an in vitro assay.

Structural model of the CopA copper ATPase of Enterococcus hirae based on chemical cross-linking

The CopA copper ATPase of Enterococcus hirae belongs to the family of heavy metal pumping CPx-type ATPases and shares 43% sequence similarity with the human Menkes and Wilson copper ATPases. Due to a lack of suitable protein crystals, only partial three-dimensional structures have so far been obtained for this family of ion pumps. We present a structural model of CopA derived by combining topological information obtained by intramolecular cross-linking with molecular modeling. Purified CopA was cross-linked with different bivalent reagents, followed by tryptic digestion and identification of cross-linked peptides by mass spectrometry. The structural proximity of tryptic fragments provided information about the structural arrangement of the hydrophilic protein domains, which was integrated into a three-dimensional model of CopA. Comparative modeling of CopA was guided by the sequence similarity to the calcium ATPase of the sarcoplasmic reticulum, Serca1, for which detailed structures are available. In addition, known partial structures of CPx-ATPase homologous to CopA were used as modeling templates. A docking approach was used to predict the orientation of the heavy metal binding domain of CopA relative to the core structure, which was verified by distance constraints derived from cross-links. The overall structural model of CopA resembles the Serca1 structure, but reveals distinctive features of CPx-type ATPases. A prominent feature is the positioning of the heavy metal binding domain. It features an orientation of the Cu binding ligands which is appropriate for the interaction with Cu-loaded metallochaperones in solution. Moreover, a novel model of the architecture of the intramembranous Cu binding sites could be derived.

Statistical model-based segmentation of the proximal femur in digital antero-posterior (AP) pelvic radiographs.

PURPOSE    Segmentation of the proximal femur in digital antero-posterior (AP) pelvic radiographs is required to create a three-dimensional model of the hip joint for use in planning and treatment. However, manually extracting the femoral contour is tedious and prone to subjective bias, while automatic segmentation must accommodate poor image quality, anatomical structure overlap, and femur deformity. A new method was developed for femur segmentation in AP pelvic radiographs. METHODS    Using manual annotations on 100 AP pelvic radiographs, a statistical shape model (SSM) and a statistical appearance model (SAM) of the femur contour were constructed. The SSM and SAM were used to segment new AP pelvic radiographs with a three-stage approach. At initialization, the mean SSM model is coarsely registered to the femur in the AP radiograph through a scaled rigid registration. Mahalanobis distance defined on the SAM is employed as the search criteria for each annotated suggested landmark location. Dynamic programming was used to eliminate ambiguities. After all landmarks are assigned, a regularized non-rigid registration method deforms the current mean shape of SSM to produce a new segmentation of proximal femur. The second and third stages are iteratively executed to convergence. RESULTS    A set of 100 clinical AP pelvic radiographs (not used for training) were evaluated. The mean segmentation error was [Formula: see text], requiring [Formula: see text] s per case when implemented with Matlab. The influence of the initialization on segmentation results was tested by six clinicians, demonstrating no significance difference. CONCLUSIONS    A fast, robust and accurate method for femur segmentation in digital AP pelvic radiographs was developed by combining SSM and SAM with dynamic programming. This method can be extended to segmentation of other bony structures such as the pelvis.

Additive effect of anemia and renal impairment on long-term outcome after percutaneous coronary intervention

INTRODUCTION Anemia and renal impairment are important co-morbidities among patients with coronary artery disease undergoing Percutaneous Coronary Intervention (PCI). Disease progression to eventual death can be understood as the combined effect of baseline characteristics and intermediate outcomes. METHODS Using data from a prospective cohort study, we investigated clinical pathways reflecting the transitions from PCI through intermediate ischemic or hemorrhagic events to all-cause mortality in a multi-state analysis as a function of anemia (hemoglobin concentration <120 g/l and <130 g/l, for women and men, respectively) and renal impairment (creatinine clearance <60 ml/min) at baseline. RESULTS Among 6029 patients undergoing PCI, anemia and renal impairment were observed isolated or in combination in 990 (16.4%), 384 (6.4%), and 309 (5.1%) patients, respectively. The most frequent transition was from PCI to death (6.7%, 95% CI 6.1-7.3), followed by ischemic events (4.8%, 95 CI 4.3-5.4) and bleeding (3.4%, 95% CI 3.0-3.9). Among patients with both anemia and renal impairment, the risk of death was increased 4-fold as compared to the reference group (HR 3.9, 95% CI 2.9-5.4) and roughly doubled as compared to patients with either anemia (HR 1.7, 95% CI 1.3-2.2) or renal impairment (HR 2.1, 95% CI 1.5-2.9) alone. Hazard ratios indicated an increased risk of bleeding in all three groups compared to patients with neither anemia nor renal impairment. CONCLUSIONS Applying a multi-state model we found evidence for a gradient of risk for the composite of bleeding, ischemic events, or death as a function of hemoglobin value and estimated glomerular filtration rate at baseline.

Three-dimensional printing for perioperative planning of complex aortic arch surgery

Purpose In this study, we show the use of three-dimensional printing models for preoperative planning of surgery for patients with complex aortic arch anomalies. Description A 70-year-old man with an extensively arteriosclerotic aneurysm reaching from the ascending aorta to the descending aorta was referred to our center for complete aortic arch replacement. We visualized and reconstructed computed tomography data of the patient and fabricated a flexible three-dimensional model of the aortic arch including the aneurysm. Evaluation This model was very helpful for the preoperative decision making and planning of the frozen elephant trunk procedure owing to the exact and lifelike illustration of the native aortic arch. Conclusions Three-dimensional models are helpful in preoperative planning and postoperative evaluation of frozen elephant trunk procedures in patients with complex aortic anatomy.

Firearm legislation and firearm mortality in the USA: a cross-sectional, state-level study.

BACKGROUND In an effort to reduce firearm mortality rates in the USA, US states have enacted a range of firearm laws to either strengthen or deregulate the existing main federal gun control law, the Brady Law. We set out to determine the independent association of different firearm laws with overall firearm mortality, homicide firearm mortality, and suicide firearm mortality across all US states. We also projected the potential reduction of firearm mortality if the three most strongly associated firearm laws were enacted at the federal level. METHODS We constructed a cross-sectional, state-level dataset from Nov 1, 2014, to May 15, 2015, using counts of firearm-related deaths in each US state for the years 2008-10 (stratified by intent [homicide and suicide]) from the US Centers for Disease Control and Prevention's Web-based Injury Statistics Query and Reporting System, data about 25 firearm state laws implemented in 2009, and state-specific characteristics such as firearm ownership for 2013, firearm export rates, and non-firearm homicide rates for 2009, and unemployment rates for 2010. Our primary outcome measure was overall firearm-related mortality per 100 000 people in the USA in 2010. We used Poisson regression with robust variances to derive incidence rate ratios (IRRs) and 95% CIs. FINDINGS 31 672 firearm-related deaths occurred in 2010 in the USA (10·1 per 100 000 people; mean state-specific count 631·5 [SD 629·1]). Of 25 firearm laws, nine were associated with reduced firearm mortality, nine were associated with increased firearm mortality, and seven had an inconclusive association. After adjustment for relevant covariates, the three state laws most strongly associated with reduced overall firearm mortality were universal background checks for firearm purchase (multivariable IRR 0·39 [95% CI 0·23-0·67]; p=0·001), ammunition background checks (0·18 [0·09-0·36]; p<0·0001), and identification requirement for firearms (0·16 [0·09-0·29]; p<0·0001). Projected federal-level implementation of universal background checks for firearm purchase could reduce national firearm mortality from 10·35 to 4·46 deaths per 100 000 people, background checks for ammunition purchase could reduce it to 1·99 per 100 000, and firearm identification to 1·81 per 100 000. INTERPRETATION Very few of the existing state-specific firearm laws are associated with reduced firearm mortality, and this evidence underscores the importance of focusing on relevant and effective firearms legislation. Implementation of universal background checks for the purchase of firearms or ammunition, and firearm identification nationally could substantially reduce firearm mortality in the USA. FUNDING None.

Block of the hERG channel by bupivacaine: Electrophysiological and modeling insights towards stereochemical optimization

The hERG voltage-gated potassium channel mediates the cardiac I(Kr) current, which is crucial for the duration of the cardiac action potential. Undesired block of the channel by certain drugs may prolong the QT interval and increase the risk of malignant ventricular arrhythmias. Although the molecular determinants of hERG block have been intensively studied, not much is known about its stereoselectivity. Levo-(S)-bupivacaine was the first drug reported to have a higher affinity to block hERG than its enantiomer. This study strives to understand the principles underlying the stereoselectivity of bupivacaine block with the help of mutagenesis analyses and molecular modeling simulations. Electrophysiological measurements of mutated hERG channels allowed for the identification of residues involved in bupivacaine binding and stereoselectivity. Docking and molecular mechanics simulations for both enantiomers of bupivacaine and terfenadine (a non-stereoselective blocker) were performed inside an open-state model of the hERG channel. The predicted binding modes enabled a clear depiction of ligand-protein interactions. Estimated binding affinities for both enantiomers were consistent with electrophysiological measurements. A similar computational procedure was applied to bupivacaine enantiomers towards two mutated hERG channels (Tyr652Ala and Phe656Ala). This study confirmed, at the molecular level, that bupivacaine stereoselectively binds the hERG channel. These results help to lay the foundation for structural guidelines to optimize the cardiotoxic profile of drug candidates in silico.

Behavioral Assessment of Core ADHD Symptoms Using the QbTest

Objective: Hyperactivity, one of the core symptoms of ADHD, has been mostly neglected in neuropsychological assessment of childhood ADHD. The neuropsychological Quantified behavior Test (QbTest) separately assesses all three core symptoms of ADHD on a behavioral level. Factor structure of the QbTest and its concurrent and discriminant validity are presented. Method: An exploratory factor analysis (n = 828 children) was performed. In a second sample (n = 102 children) a Multi-Trait-Multi-Method (MTMM) approach was used for validity analyses. Results: A three factorial model explained 76 % of the total variance, with the resulting QbTest factors significantly influenced by age and gender. The MTMM approach yielded promising results for discriminant, yet inconsistent findings for concurrent validity between the QbTest and another attention test as well as for Conners' Parent and Teacher Rating Scales. Conclusion: Results indicate that the QbTest may be helpful for the behavioral assessment of childhood ADHD, yet further studies on its psychometric quality and clinical utility are needed. (J. of Att. Dis. 2012; XX(X) 1-XX).

Biochemical analysis of mutations in P450 oxidoreductase

All microsomal P450s require POR (cytochrome P450 reductase) for catalytic activity. Most of the clinically used drugs are metabolized by a small number of P450s and polymorphisms in the cytochrome P450s are known to cause changes in drug metabolism. We have recently found a number of POR missense mutations in the patients with disordered steroidogenesis. Our initial report described five missense mutations (A284P, R454H, V489E, C566Y and V605F) identified in four patients. We built bacterial expression vectors for each POR variant, purified the membranes expressing normal or variant POR and characterized their activities with cytochrome c and P450c17 assays. We have recently completed an extensive study of the range of POR mutations and characterized the mutants/polymorphisms A112V, T139A, M260V, Y456H, A500V, G536R, L562P, R613X, V628I and F643del from sequencing of patient DNA. We also studied POR variants Y179D, P225L, R313W, G410S and G501R that were available in databases or the published literature. We analysed the mutations with a three-dimensional model of human POR that was based on an essentially similar rat POR with known crystal structure. The missense mutations found in patients with disordered steroidogenesis mapped to functionally important domains of POR and the apparent polymorphisms mapped to less crucial regions. Since a variation in POR can alter the activity of all microsomal P450s, it can also affect the drug metabolism even with a normal P450. Understanding the genetic and biochemical basis of POR-mediated drug metabolism will provide valuable information about possible differences in P450-mediated reactions among the individuals carrying a variant or polymorphic form of POR.