Characterization of Lysophospholipase D Activity in Mammalian Cell Membranes.

Lysophosphatidic acid (LPA) is a lipid mediator that binds to G-protein-coupled receptors, eliciting a wide variety of responses in mammalian cells. Lyso-phospholipids generated via phospholipase A2 (PLA2) can be converted to LPA by a lysophospholipase D (lyso-PLD). Secreted lyso-PLDs have been studied in more detail than membrane-localized lyso-PLDs. This study utilized in vitro enzyme assays with fluorescent substrates to examine LPA generation in membranes from multiple mammalian cell lines (PC12, rat pheochromocytoma; A7r5, rat vascular smooth muscle; Rat-1, rat fibroblast; PC-3, human prostate carcinoma; and SKOV-3 and OVCAR-3, human ovarian carcinoma). The results show that membranes contain a lyso-PLD activity that generates LPA from a fluorescent alkyl-lyso-phosphatidylcholine, as well as from naturally occurring acyl-linked lysophospholipids. Membrane lyso-PLD and PLD activities were distinguished by multiple criteria, including lack of effect of PLD2 over-expression on lyso-PLD activity and differential sensitivities to vanadate (PLD inhibitor) and iodate (lyso-PLD inhibitor). Based on several lines of evidence, including siRNA knockdown, membrane lyso-PLD is distinct from autotaxin, a secreted lyso-PLD. PC-3 cells express GDE4 and GDE7, recently described lyso-PLDs that localize to membranes. These findings demonstrate that membrane-associated lyso-D activity, expressed by multiple mammalian cell lines, can contribute to LPA production.

Superconducting nonlinear transport in optically driven high-temperature K3C60.

Optically driven quantum materials exhibit a variety of non-equilibrium functional phenomena, which to date have been primarily studied with ultrafast optical, X-Ray and photo-emission spectroscopy. However, little has been done to characterize their transient electrical responses, which are directly associated with the functionality of these materials. Especially interesting are linear and nonlinear current-voltage characteristics at frequencies below 1 THz, which are not easily measured at picosecond temporal resolution. Here, we report on ultrafast transport measurements in photo-excited K3C60. Thin films of this compound were connected to photo-conductive switches with co-planar waveguides. We observe characteristic nonlinear current-voltage responses, which in these films point to photo-induced granular superconductivity. Although these dynamics are not necessarily identical to those reported for the powder samples studied so far, they provide valuable new information on the nature of the light-induced superconducting-like state above equilibrium Tc. Furthermore, integration of non-equilibrium superconductivity into optoelectronic platforms may lead to integration in high-speed devices based on this effect.

A motion model-guided 4D dose reconstruction for pencil beam scanned proton therapy.

Objective.4D dose reconstruction in proton therapy with pencil beam scanning (PBS) typically relies on a single pre-treatment 4DCT (p4DCT). However, breathing motion during the fractionated treatment can vary considerably in both amplitude and frequency. We present a novel 4D dose reconstruction method combining delivery log files with patient-specific motion models, to account for the dosimetric effect of intra- and inter-fractional breathing variability.Approach.Correlation between an external breathing surrogate and anatomical deformations of the p4DCT is established using principal component analysis. Using motion trajectories of a surface marker acquired during the dose delivery by an optical tracking system, deformable motion fields are retrospectively reconstructed and used to generate time-resolved synthetic 4DCTs ('5DCTs') by warping a reference CT. For three abdominal/thoracic patients, treated with respiratory gating and rescanning, example fraction doses were reconstructed using the resulting 5DCTs and delivery log files. The motion model was validated beforehand using leave-one-out cross-validation (LOOCV) with subsequent 4D dose evaluations. Moreover, besides fractional motion, fractional anatomical changes were incorporated as proof of concept.Main results.For motion model validation, the comparison of 4D dose distributions for the original 4DCT and predicted LOOCV resulted in 3%/3 mm gamma pass rates above 96.2%. Prospective gating simulations on the p4DCT can overestimate the target dose coverage V95%by up to 2.1% compared to 4D dose reconstruction based on observed surrogate trajectories. Nevertheless, for the studied clinical cases treated with respiratory-gating and rescanning, an acceptable target coverage was maintained with V95%remaining above 98.8% for all studied fractions. For these gated treatments, larger dosimetric differences occurred due to CT changes than due to breathing variations.Significance.To gain a better estimate of the delivered dose, a retrospective 4D dose reconstruction workflow based on motion data acquired during PBS proton treatments was implemented and validated, thus considering both intra- and inter-fractional motion and anatomy changes.

Light-induced anomalous Hall effect in graphene.

Many non-equilibrium phenomena have been discovered or predicted in optically-driven quantum solids1. Examples include light-induced superconductivity2,3 and Floquet-engineered topological phases4-8. These are short lived effects that should lead to measurable changes in electrical transport, which can be characterized using an ultrafast device architecture based on photoconductive switches9. Here, we report the observation of a light-induced anomalous Hall effect in monolayer graphene driven by a femtosecond pulse of circularly polarized light. The dependence of the effect on a gate potential used to tune the Fermi level reveals multiple features that reflect a Floquet-engineered topological band structure4,5, similar to the band structure originally proposed by Haldane10. This includes an approximately 60 meV wide conductance plateau centered at the Dirac point, where a gap of equal magnitude is predicted to open. We find that when the Fermi level lies within this plateau, the estimated anomalous Hall conductance saturates around 1.8±0.4 e2/h.

Usability of daily SF36 questionnaires to capture the QALD variation experienced after vaccination with AS03A-adjuvanted monovalent influenza A (H5N1) vaccine in a safety and tolerability study.

BACKGROUND: This study aims to describe the short-term reactogenicity of the AS03-adjuvanted H5N1 vaccine expressed through adverse events (AEs) and quality-adjusted life-day (QALD) scores. The AEs are likely to be short-term and therefore the quality of life (QoL) questionnaire, SF-36v2, was administered daily to record changes over seven days. A more sensitive application of this instrument should allow for a better understanding of short-term tolerability of adjuvanted vaccines. METHODS: Participants (N = 50) received a 2-dose vaccination schedule. Solicited (collected daily: days 0 to 7 [post dose 1] and 21 to 28 [post dose 2]) and unsolicited (collected weekly until day 21) AEs were collected via diary cards. The QoL questionnaires were completed daily (days 0-6) and weekly (days 0, 6, 21, 27) after dose one. Questionnaire data were transformed into SF-6D scores to report QALDs. It was hypothesized post-hoc that the QALD and daily AEs scores should correlate if discrete QoL-changes were captured. RESULTS: Pain (92%) and muscle ache (66%) were the most commonly reported solicited local and general AEs respectively, neither increased in intensity nor in frequency after dose 2. No safety concerns were identified during the study. A correlation between the daily AEs and QALD scores existed (correlation coefficient, - 0.97 (p < 0.001)). The impact of the AEs scores on the QALD was marginal (- 0.02 max for one day). CONCLUSION: Similarly with other H5N1 studies, no safety concern was identified throughout the study. Some time-limited variations in QALD-scores were reported. Our results imply that daily administration of the SF-36v2 captures changes in QALD-scores. TRIAL REGISTRATION: ClinicalTrials.gov . NCT01788228. Registered 11 February 2013.

Alternatives to patient specific verification measurements in proton therapy: a comparative experimental study with intentional errors.

Patient specific verification (PSV) measurements for pencil beam scanning (PBS) proton therapy are resource-consuming and necessitate substantial beam time outside of clinical hours. As such, efforts to safely reduce the PSV-bottleneck in the clinical work-flow are of great interest. Here, capabilities of current PSV methods to ensure the treatment integrity were investigated and compared to an alternative approach of reconstructing the dose distribution directly from the machine control- or delivery log files with the help of an independent dose calculation (IDC). Scenarios representing a wide range of delivery or work-flow failures were identified (e.g. error in spot position, air gap or pre-absorber setting) and machine files were altered accordingly. This yielded 21 corrupted treatment files, which were delivered and measured with our clinical PSV protocol. IDC machine- and log file checks were also conducted and their sensitivity at detecting the errors compared to the measurements. Although some of the failure scenarios induced clinically relevant dose deviations in the patient geometry, the PSV measurement protocol only detected one out of 21 error scenarios. However, 11 and all 21 error scenarios were detected using dose reconstructions based on the log and machine files respectively. Our data suggests that, although commonly used in particle therapy centers, PSV measurements do a poor job detecting data transfer failures and imperfect delivery machine performance. Machine- and log-file IDCs have been shown to successfully detect erroneous work-flows and to represent a reliable addition to the QA procedure, with the potential to replace PSV.

Validating a Monte Carlo approach to absolute dose quality assurance for proton pencil beam scanning.

For radiotherapy, it is crucial to guarantee that the delivered dose matches the planned dose. Therefore, patient specific quality assurance (QA) of absolute dose distributions is necessary. Here, we investigate the potential of replacing patient specific QA for pencil beam scanned proton therapy with Monte Carlo simulations. First, the set-up of the automated Monte Carlo model is presented with an emphasis on the absolute dose validation. Second, the absolute dose results obtained from the Monte Carlo simulation for a comprehensive set of patient fields are compared to patient specific QA measurements. Absolute doses measured with the Farmer chamber are shown to be 1.4% higher than the doses measured with the Semiflex chamber. For single energy layers, Monte Carlo simulated doses are 2.1% ± 0.4% lower than the ones measured with the ionization chamber and 1.1% ± 1.0% lower than measurements compared to patient field verification measurements. After rescaling to account for this 1.1% discrepancy, 98 fields (94.2%) agree within 2% to measurements, the maximum difference being 2.3%. In conclusion, an automated, easy-to-use Monte Carlo calculation system has been set up. This system reproduced patient specific QA results over a wide range of cases, showing that the time consuming measurements could be reduced or even replaced using Monte Carlo simulations without jeopardizing treatment quality.

Contour scanning for penumbra improvement in pencil beam scanned proton therapy.

Proton therapy, especially in the form of pencil beam scanning (PBS), allows for the delivery of highly conformal dose distributions for complex tumor geometries. However, due to scattering of protons inside the patient, lateral dose gradients cannot be arbitrarily steep, which is of importance in cases with organs at risk (OARs) in close proximity to, or overlapping with, planning target volumes (PTVs). In the PBS approach, physical pencil beams are planned using a regular grid orthogonal to the beam direction. In this work, we propose an alternative to this commonly used approach where pencil beams are placed on an irregular grid along concentric paths based on the target contour. Contour driven pencil beam placement is expected to improve dose confirmation by allowing the optimizer to best enhance the penumbra of irregularly shaped targets using edge enhancement. Its effectiveness has been shown to improve dose confirmation to the target volume and reduce doses to OARs in head-and-neck planning studies. Furthermore, the deliverability of such plans, as well as the dosimetric improvements over conventional grid-based plans, have been confirmed in first phantom based verifications.

Expression of a mutant prohibitin from the aP2 gene promoter leads to obesity-linked tumor development in insulin resistance-dependent manner.

A critical unmet need for the study of obesity-linked cancer is the lack of preclinical models that spontaneously develop obesity and cancer sequentially. Prohibitin (PHB) is a pleiotropic protein that has a role in adipose and immune functions. We capitalized on this attribute of PHB to develop a mouse model for obesity-linked tumor. We achieved this by expressing Y114F-PHB (m-PHB) from the aP2 gene promoter for simultaneous manipulation of adipogenic and immune signaling functions. The m-PHB mice develop obesity in a sex-neutral manner, but only male mice develop impaired glucose homeostasis and hyperinsulinemia similar to transgenic mice expressing PHB. Interestingly, only male m-PHB mice develop histiocytosis with lymphadenopathy, suggesting that metabolic dysregulation or m-PHB alone is not sufficient for the tumor development and that both are required for tumorigenesis. Moreover, ovariectomy in female m-PHB mice resulted in impaired glucose homeostasis, hyperinsulinemia and consequently tumor development similar to male m-PHB mice. These changes were not observed in sham-operated control m-Mito-Ob mice, further confirming the role of obesity-related metabolic dysregulation in tumor development in m-PHB mice. Our data provide a proof-of-concept that obesity-associated hyperinsulinemia promotes tumor development by facilitating dormant mutant to manifest and reveals a sex-dimorphic role of PHB in adipose-immune interaction or immunometabolism. Targeting PHB may provide a unique opportunity for the modulation of immunometabolism in obesity, cancer and in immune diseases.

Assessing the quality of proton PBS treatment delivery using machine log files: comprehensive analysis of clinical treatments delivered at PSI Gantry 2.

Pencil beam scanning (PBS) proton therapy requires the delivery of many thousand proton beams, each modulated for position, energy and monitor units, to provide a highly conformal patient treatment. The quality of the treatment is dependent on the delivery accuracy of each beam and at each fraction. In this work we describe the use of treatment log files, which are a record of the machine parameters for a given field delivery on a given fraction, to investigate the integrity of treatment delivery compared to the nominal planned dose. The dosimetry-relevant log file parameters are used to reconstruct the 3D dose distribution on the patient anatomy, using a TPS-independent dose calculation system. The analysis was performed for patients treated at Paul Scherrer Institute on Gantry 2, both for individual fields and per series (or plan), and delivery quality was assessed by determining the percentage of voxels in the log file dose distribution within +/- 1% of the nominal dose. It was seen that, for all series delivered, the mean pass rate is 96.4%. Furthermore, this work establishes a correlation between the delivery quality of a field and the beam position accuracy. This correlation is evident for all delivered fields regardless of individual patient or plan characteristics. We have also detailed further usefulness of log file analysis within our clinical workflow. In summary, we have highlighted that the integrity of PBS treatment delivery is dependent on daily machine performance and is specifically highly correlated with the accuracy of beam position. We believe this information will be useful for driving machine performance improvements in the PBS field.

Stabilization of magnetic helix in exchange-coupled thin films.

Based on micromagnetic simulations, we report on a novel magnetic helix in a soft magnetic film that is sandwiched between and exchange-coupled to two hard magnetic layers with different anisotropies. We show that such a confined helix stays stable without the presence of an external magnetic field. The magnetic stability is determined by the energy minimization and is a result of an internal magnetic field created by the exchange interaction. We show that this internal field stores a magnetic energy density of a few kJ/m(3). We also find that it dramatically modifies ferromagnetic resonances, such that the helix can be used as a ferromagnetic resonance filter and a fast acting attenuator.

Cost-effectiveness analysis of quadrivalent influenza vaccination in at-risk adults and the elderly: an updated analysis in the U.K.

OBJECTIVE: To update an earlier evaluation estimating the cost-effectiveness of quadrivalent influenza vaccination (QIV) compared with trivalent influenza vaccination (TIV) in the adult population currently recommended for influenza vaccination in the UK (all people aged ≥65 years and people aged 18-64 years with clinical risk conditions). METHODS: This analysis takes into account updated vaccine prices, reference costs, influenza strain circulation, and burden of illness data. A lifetime, multi-cohort, static Markov model was constructed with seven age groups. The model was run in 1-year cycles for a lifetime, i.e., until the youngest patients at entry reached the age of 100 years. The base-case analysis was from the perspective of the UK National Health Service, with a secondary analysis from the societal perspective. Costs and benefits were discounted at 3.5%. Herd effects were not included. Inputs were derived from systematic reviews, peer-reviewed articles, and government publications and databases. One-way and probabilistic sensitivity analyses were performed. RESULTS: In the base-case, QIV would be expected to avoid 1,413,392 influenza cases, 41,780 hospitalizations, and 19,906 deaths over the lifetime horizon, compared with TIV. The estimated incremental cost-effectiveness ratio (ICER) was £14,645 per quality-adjusted life-year (QALY) gained. From the societal perspective, the estimated ICER was £13,497/QALY. A strategy of vaccinating only people aged ≥65 years had an estimated ICER of £11,998/QALY. Sensitivity analysis indicated that only two parameters, seasonal variation in influenza B matching and influenza A circulation, had a substantial effect on the ICER. QIV would be likely to be cost-effective compared with TIV in 68% of simulations with a willingness-to-pay threshold of <£20,000/QALY and 87% with a willingness-to-pay threshold of <£30,000/QALY. CONCLUSIONS: In this updated analysis, QIV was estimated to be cost-effective compared with TIV in the U.K.

Independent dose calculations for commissioning, quality assurance and dose reconstruction of PBS proton therapy.

Pencil beam scanning proton therapy allows the delivery of highly conformal dose distributions by delivering several thousand pencil beams. These beams have to be individually optimised and accurately delivered requiring a significant quality assurance workload. In this work we describe a toolkit for independent dose calculations developed at Paul Scherrer Institut which allows for dose reconstructions at several points in the treatment workflow. Quality assurance based on reconstructed dose distributions was shown to be favourable to pencil beam by pencil beam comparisons for the detection of delivery uncertainties and estimation of their effects. Furthermore the dose reconstructions were shown to have a sensitivity of the order of or higher than the measurements currently employed in the clinical verification procedures. The design of the independent dose calculation tool allows for a high modifiability of the dose calculation parameters (e.g. depth dose profiles, angular spatial distributions) allowing for a safe environment outside of the clinical treatment planning system for investigating the effect of such parameters on the resulting dose distributions and thus distinguishing between different contributions to measured dose deviations. The presented system could potentially reduce the amount of patient-specific quality assurance measurements which currently constitute a bottleneck in the clinical workflow.

Assessment of posttranslational modification of mitochondrial proteins.

Mitochondria play vital roles in the maintenance of cellular homeostasis. They are a storehouse of cellular energy and antioxidative enzymes. Because of its immense role and function in the development of an organism, this organelle is required for the survival. Defects in mitochondrial proteins lead to complex mitochondrial disorders and heterogeneous diseases such as cancer, type 2 diabetes, and cardiovascular and neurodegenerative diseases. It is widely known in the literature that some of the mitochondrial proteins are regulated by posttranslational modifications. Hence, designing methods to assess these modifications in mitochondria will be an important way to study the regulatory roles of mitochondrial proteins in greater detail. In this chapter, we outlined procedures to isolate mitochondria from cells and separate the mitochondrial proteins by two-dimensional gel electrophoresis and identify the different posttranslational modifications in them by using antibodies specific to each posttranslational modification.

Examining Ontario's universal influenza immunization program with a multi-strain dynamic model.

Seasonal influenza imposes a significant worldwide health burden each year. Mathematical models help us to understand how changes in vaccination affect this burden. Here, we develop a new dynamic transmission model which directly tracks the four dominant seasonal influenza strains/lineages, and use it to retrospectively examine the impact of the switch from a targeted to a universal influenza immunization program (UIIP) in the Canadian province of Ontario in 2000. According to our model results, averaged over the first four seasons post-UIIP, the rates of influenza-associated health outcomes in Ontario were reduced to about half of their pre-UIIP values. This is conservative compared to the results of a study estimating the UIIP impact from administrative data, though that study finds age-specific trends similar to those presented here. The strain interaction in our model, together with its flexible parameter calibration scheme, make it readily extensible to studying scenarios beyond the one explored here.

Prohibitin overexpression in adipocytes induces mitochondrial biogenesis, leads to obesity development, and affects glucose homeostasis in a sex-specific manner.

Adipocytes are the primary cells in the body that store excess energy as triglycerides. To perform this specialized function, adipocytes rely on their mitochondria; however, the role of adipocyte mitochondria in the regulation of adipose tissue homeostasis and its impact on metabolic regulation is not understood. We developed a transgenic mouse model, Mito-Ob, overexpressing prohibitin (PHB) in adipocytes. Mito-Ob mice developed obesity due to upregulation of mitochondrial biogenesis in adipocytes. Of note, Mito-Ob female mice developed more visceral fat than male mice. However, female mice exhibited no change in glucose homeostasis and had normal insulin and high adiponectin levels, whereas male mice had impaired glucose homeostasis, compromised brown adipose tissue structure, and high insulin and low adiponectin levels. Mechanistically, we found that PHB overexpression enhances the cross talk between the mitochondria and the nucleus and facilitates mitochondrial biogenesis. The data suggest a critical role of PHB and adipocyte mitochondria in adipose tissue homeostasis and reveal sex differences in the effect of PHB-induced adipocyte mitochondrial remodeling on whole-body metabolism. Targeting adipocyte mitochondria may provide new therapeutic opportunities for the treatment of obesity, a major risk factor for type 2 diabetes.

Synthesis and anticancer activity of new flavonoid analogs and inconsistencies in assays related to proliferation and viability measurements.

Flavonoids have been studied intensely for their ability to act as anti-carcinogenic, anti-inflammatory, anti-viral and anti-aging agents and are often marketed as supplements related to their anti-inflammatory activity. Previous studies have primarily focused on the effects of polar natural flavonoids. We examined the activity of novel hydrophobic and lipophilic flavonols against human DU-145 and PC-3 prostate cancer cell lines. All flavonol analogs were more active than the naturally occurring flavonols quercetin, kaempferol, kaempferide and galangin. The most potent analogs were 6.5-fold more active against DU-145 and PC-3 cells than quercetin and fell within the biologically relevant concentration range (low micromolar). We also evaluated the potential toxic effects of flavonol analogs on normal cells, an assessment that has frequently been ignored when studying the anticancer effects of flavonoids. During these analyses, we discovered that various metabolic and DNA staining assays were unreliable methods for assessing cell viability of flavonoids. Flavonoids reduce colorimetric dyes such as MTT and Alamar Blue in the absence of cells. We showed that flavonol-treated prostate cancer cells were stained less intensely with crystal violet than untreated cells at non-toxic concentrations. The trypan blue exclusion assay was selected as a reliable alternative for measuring cell viability.

Current-driven domain wall depinning from an anisotropy boundary in nanowires.

The interaction of a current-driven domain wall with an anisotropy boundary in nanowires with perpendicular magnetic anisotropy is investigated. A local reduction of the anisotropy constant is used to create an artificial boundary where the domain wall gets pinned. Micromagnetic simulations and analytical calculations, based on a one-dimensional model, are employed to describe the interaction of the domain wall and the anisotropy boundary and to determine the depinning current densities. Two different pinning regimes-an intrinsic and an extrinsic-can be identified in dependence with the characteristic of the boundary. A very good agreement between simulated and analytically obtained data is achieved.

Structure-activity relationship studies of flavonol analogues on pollen germination.

Flavonoids are polyphenolic compounds required in the fertilization process in many, if not all, plants. However, the exact biological mechanism(s) and the interacting proteins are unknown. To determine the characteristics important in activating or inhibiting the pollination sequence, a structure-activity relationship analysis of natural and synthetic flavonols was conducted. Flavonol analogues were synthesized through a modified "one-pot" procedure that utilized a Baker-Venkataraman type rearrangement and a Suzuki-Miyaura cross-coupling of a halo-flavonol with an organotrifluoroborate. Of the flavonols tested, kaempferol was the only compound to act as a full agonist. The other smaller, less sterically hindered flavonols (galangin, kaempferide, and 4'-methyl flavonol) acted as partial agonists. Larger more hydrophobic flavonol analogues (3'- and 4'-benzoyl, 3'- and 4'-phenyl, and 3'- and 4'-iodo flavonols) had minimal or no agonist activity. Competition assays between kaempferol and these minimally activating flavonols showed that these analogues inhibited the action of kaempferol in a manner consistent with noncompetitive antagonism. The results suggest that steric hindrance is the most important factor in determining a good agonist. Hydrogen bonding also had a positive effect as long as the substituent did not cause any steric hindrance.