Role of the N- and C-lobes of calmodulin in the activation of Ca(2+)/calmodulin-dependent protein kinase II.

Understanding the principles of calmodulin (CaM) activation of target enzymes will help delineate how this seemingly simple molecule can play such a complex role in transducing Ca (2+)-signals to a variety of downstream pathways. In the work reported here, we use biochemical and biophysical tools and a panel of CaM constructs to examine the lobe specific interactions between CaM and CaMKII necessary for the activation and autophosphorylation of the enzyme. Interestingly, the N-terminal lobe of CaM by itself was able to partially activate and allow autophosphorylation of CaMKII while the C-terminal lobe was inactive. When used together, CaMN and CaMC produced maximal CaMKII activation and autophosphorylation. Moreover, CaMNN and CaMCC (chimeras of the two N- or C-terminal lobes) both activated the kinase but with greater K act than for wtCaM. Isothermal titration calorimetry experiments showed the same rank order of affinities of wtCaM > CaMNN > CaMCC as those determined in the activity assay and that the CaM to CaMKII subunit binding ratio was 1:1. Together, our results lead to a proposed sequential mechanism to describe the activation pathway of CaMKII led by binding of the N-lobe followed by the C-lobe. This mechanism contrasts the typical sequential binding mode of CaM with other CaM-dependent enzymes, where the C-lobe of CaM binds first. The consequence of such lobe specific binding mechanisms is discussed in relation to the differential rates of Ca (2+)-binding to each lobe of CaM during intracellular Ca (2+) oscillations.

Clinical Impact of Couch Top and Rails on IMRT and Arc Therapy

Purpose: To evaluate the clinical impact of the Varian Exact Couch on dose and volume coverage to targets and critical structures and tumor control probability (TCP) for 6-MV IMRT and Arc Therapy. Methods: Five clinical prostate patients were planned with both, 6-MV 8-field IMRT and 6-MV 2-field RapidArc using the Eclipse treatment planning system (TPS). These plans neglected treatment couch attenuation, as is standard clinical practice. Dose distributions were then recalculated in Eclipse with the inclusion of the Varian Exact Couch (imaging couch top) and the rails in varying configurations. The changes in dose and coverage were evaluated using the DVHs from each plan iteration. We used a tumor control probability (TCP) model to calculate losses in tumor control resulting from not accounting for the couch top and rails. We also verified dose measurements in a phantom. Results: Failure to account for the treatment couch and rails resulted in clinically unacceptable dose and volume coverage losses to the target for both IMRT and RapidArc. The couch caused average dose losses (relative to plans that ignored the couch) to the prostate of 4.2% and 2.0% for IMRT with the rails out and in, respectively, and 3.2% and 2.9% for RapidArc with the rails out and in, respectively. On average, the percentage of the target covered by the prescribed dose dropped to 35% and 84% for IMRT (rails out and in, respectively) and to 18% and 17% for RapidArc (rails out and in, respectively). The TCP was also reduced by as much as 10.5% (6.3% on average). Dose and volume coverage losses for IMRT plans were primarily due to the rails, while the imaging couch top contributed most to losses for RapidArc. Both the couch top and rails contribute to dose and coverage losses that can render plans clinically unacceptable. A follow-up study we performed found that the less attenuating unipanel mesh couch top available with the Varian Exact couch does not cause a clinically impactful loss of dose or coverage for IMRT but still causes an unacceptable loss for RapidArc. Conclusions: Both the imaging couch top and rails contribute to dose and coverage loss to a degree that, if included, would prevent the plan from meeting clinical planning criteria. Therefore, the imaging and mesh couch tops and rails should be accounted for in Arc Therapy and the imaging couch and rails only in IMRT treatment planning.

The effect of shoulder variation on IMRT and SmartArc for Head and Neck Cancer

Purpose: First, to determine an average and maximum displacement of the shoulder relative to isocenter over the course of treatment. Second, to establish the dosimetric effect of shoulder displacements relative to correct isocenter alignment on the dose delivered to the target and the surrounding structures for head and neck cancer patients. Method and Materials: The frequency of shoulder shifts of various magnitudes relative to isocenter was assessed for 4 patients using image registration software. The location of the center of the right and left humeral head relative to isocenter (usually C2) was found daily from CT on rails scans, and was compared to the location of the humeral heads relative to isocenter on the initial simulation CT. Three Baseline head and neck IMRT and SmartArc plans were generated in Pinnacle based on simulation CTs. The CT datasets (external contour and boney structures) were then modified to represent shifts of the shoulder (relative to isocenter) between 3 mm and 15 mm in the SI, AP, and LR directions. The initial plans were recalculated on the image sets with shifted shoulders. Results: On average, shoulder variation was 2-5 mm in each direction, although displacements of over 1 cm in the inferior and posterior directions occurred. Shoulder shifts induced perturbations in the dose distribution, although generally only for large shifts. Most substantially, large, superior shifts resulted in coverage loss by the 95% isodose line for targets in the lower neck. Inferior shifts elevated the dose to the brachial plexus by 0.6-4.1 Gy. SmartArc plans showed similar loss of target coverage as IMRT plans. Conclusions: The position of the shoulder can have an impact on target coverage and critical structure dose. Shoulder position may need to be considered for setup of head and neck patients depending on target location.

The Development and Implementation of an Anthropomorphic Head Phantom for the Assessment of Proton Therapy Treatment Procedures

Proton therapy has become an increasingly more common method of radiation therapy, with the dose sparing to distal tissue making it an appealing option, particularly for treatment of brain tumors. This study sought to develop a head phantom for the Radiological Physics Center (RPC), the first to be used for credentialing of institutions wishing to participate in clinical trials involving brain tumor treatment of proton therapy. It was hypothesized that a head phantom could be created for the evaluation of proton therapy treatment procedures (treatment simulation, planning, and delivery) to assure agreement between the measured dose and calculated dose within ±5%/3mm with a reproducibility of ±3%. The relative stopping power (RSP) and Hounsfield Units (HU) were measured for potential phantom materials and a human skull was cast in tissue-equivalent Alderson material (RLSP 1.00, HU 16) with anatomical airways and a cylindrical hole for imaging and dosimetry inserts drilled into the phantom material. Two treatment plans, proton passive scattering and proton spot scanning, were created. Thermoluminescent dosimeters (TLDs) and film were loaded into the phantom dosimetry insert. Each treatment plan was delivered three separate times. Each treatment plan passed our 5%/3mm criteria, with a reproducibility of ±3%. The hypothesis was accepted and the phantom was found to be suitable for remote audits of proton therapy treatment facilities.

EVALUATION OF INTENSITY MODULATED RADIATION THERAPY (IMRT) DELIVERY ERROR DUE TO IMRT TREATMENT PLAN COMPLEXITY AND IMPROPERLY MATCHED DOSIMETRY DATA

Intensity modulated radiation therapy (IMRT) is a technique that delivers a highly conformal dose distribution to a target volume while attempting to maximally spare the surrounding normal tissues. IMRT is a common treatment modality used for treating head and neck (H&N) cancers, and the presence of many critical structures in this region requires accurate treatment delivery. The Radiological Physics Center (RPC) acts as both a remote and on-site quality assurance agency that credentials institutions participating in clinical trials. To date, about 30% of all IMRT participants have failed the RPC’s remote audit using the IMRT H&N phantom. The purpose of this project is to evaluate possible causes of H&N IMRT delivery errors observed by the RPC, specifically IMRT treatment plan complexity and the use of improper dosimetry data from machines that were thought to be matched but in reality were not. Eight H&N IMRT plans with a range of complexity defined by total MU (1460-3466), number of segments (54-225), and modulation complexity scores (MCS) (0.181-0.609) were created in Pinnacle v.8m. These plans were delivered to the RPC’s H&N phantom on a single Varian Clinac. One of the IMRT plans (1851 MU, 88 segments, and MCS=0.469) was equivalent to the median H&N plan from 130 previous RPC H&N phantom irradiations. This average IMRT plan was also delivered on four matched Varian Clinac machines and the dose distribution calculated using a different 6MV beam model. Radiochromic film and TLD within the phantom were used to analyze the dose profiles and absolute doses, respectively. The measured and calculated were compared to evaluate the dosimetric accuracy. All deliveries met the RPC acceptance criteria of ±7% absolute dose difference and 4 mm distance-to-agreement (DTA). Additionally, gamma index analysis was performed for all deliveries using a ±7%/4mm and ±5%/3mm criteria. Increasing the treatment plan complexity by varying the MU, number of segments, or varying the MCS resulted in no clear trend toward an increase in dosimetric error determined by the absolute dose difference, DTA, or gamma index. Varying the delivery machines as well as the beam model (use of a Clinac 6EX 6MV beam model vs. Clinac 21EX 6MV model), also did not show any clear trend towards an increased dosimetric error using the same criteria indicated above.

Annexin A5-Conjugated Polymeric Micelles For Dual SPECT and Optical Detection of Apoptosis

Apoptosis, a form of programmed cell death, is critical to homoeostasis, normal development, and physiology. Dysregulation of apoptosis can lead to the accumulation of unwanted cells, such as occurs in cancer, and the removal of needed cells or disorders of normal tissues, such as heart, neurodegenerative, and autoimmune diseases. Noninvasive detection of apoptosis may play an important role in the evaluation of disease states and response to therapeutic intervention for a variety of diseases. It is desirable to have an imaging method to accurately detect and monitor this process in patients. In this study, we developed annexin A5-conjugated polymeric micellar nanoparticles dual-labeled with a near-infrared fluorescence fluorophores (Cy7) and a radioisotope (111In), named as 111In-labeled annexin A5-CCPM. In vitro studies demonstrated that annexin A5-CCPM could strongly and specifically bind to apoptotic cells. In vivo studies showed that apoptotic tissues could be clearly visualized by both single photon emission computed tomography (SPECT) and fluorescence molecular tomography (FMT) after intravenous injection of 111In-labeled Annexin A5-CCPM in 6 different apoptosis models. In contrast, there was little signal in respective healthy tissues. All the biodistribution data confirmed imaging results. Moreover, histological analysis revealed that radioactivity count correlated with fluorescence signal from the nanoparticles, and both signals co-localized with the region of apoptosis. In sum, 111In-labeled annexin A5-CCPM allowed visualization of apoptosis by both nuclear and optical imaging techniques. The complementary information acquired with multiple imaging techniques should be advantageous in improving diagnostics and management of patients.

TP53 as a Biomarker in Head and Neck Squamous Cell Carcinoma

Currently, there are no molecular biomarkers that guide treatment decisions for patients with head and neck squamous cell carcinoma (HNSCC). Several retrospective studies have evaluated TP53 in HNSCC, and results have suggested that specific mutations are associated with poor outcome. However, there exists heterogeneity among these studies in the site and stage of disease of the patients reviewed, the treatments rendered, and methods of evaluating TP53 mutation. Thus, it remains unclear as to which patients and in which clinical settings TP53 mutation is most useful in predicting treatment failure. In the current study, we reviewed the records of a cohort of patients with advanced, resectable HNSCC who received surgery and post-operative radiation (PORT) and had DNA isolated from fresh tumor tissue obtained at the time of surgery. TP53 mutations were identified using Sanger sequencing of exons 2-11 and the associated splice regions of the TP53 gene. We have found that the group of patients with either non-disruptive or disruptive TP53 mutations had decreased overall survival, disease-free survival, and an increased rate of distant metastasis. When examined as an independent factor, disruptive mutation was strongly associated with the development of distant metastasis. As a second aim of this project, we performed a pilot study examining the utility of the AmpliChip® p53 test as a practical method for TP53 sequencing in the clinical setting. AmpliChip® testing and Sanger sequencing was performed on a separate cohort of patients with HNSCC. Our study demonstrated the ablity of the AmpliChip® to call TP53 mutation from a single formalin-fixed paraffin-embedded slide. The results from AmpliChip® testing were identical with the Sanger method in 11 of 19 cases, with a higher rate of mutation calls using the AmpliChip® test. TP53 mutation is a potential prognostic biomarker among patients with advanced, resectable HNSCC treated with surgery and PORT. Whether this subgroup of patients could benefit from the addition of concurrent or induction chemotherapy remains to be evaluated in prospective clinical trials. Our pilot study of the p53 AmpliChip® suggests this could be a practical and reliable method of TP53 analysis in the clinical setting.

Intellectual Property and Traditional Cultural Expressions in a Digital Environment. Preface

In the face of increasing globalisation, there is a pressing need for innovative trans-disciplinary analyses of the value of traditional cultural expressions (TCE) that also suggest appropriate protection mechanisms for them. The book to which this preface belongs combines approaches from history, philosophy, anthropology, sociology and law, and charts previously untravelled paths for developing new policy tools and legal designs that go beyond conventional copyright models. It reflects also upon the specific features of the digital environment, which, despite enhancing the risks of misappropriation of traditional knowledge and creativity, may equally offer some opportunities for revitalising indigenous peoples' values and provide for the sustainability of TCE.

Quantitative morphometric analysis of lakes using GIS: rectangularity R , ellipticity E , orientation O , and the rectangularity vs. ellipticity index, REi

Quantitative measures of polygon shapes and orientation are important elements of geospatial analysis. These kinds of measures are particularly valuable in the case of lakes, where shape and orientation patterns can help identifying the geomorphological agents behind lake formation and evolution. However, the lack of built-in tools in commercial geographic information system (GIS) software packages designed for this kind of analysis has meant that many researchers often must rely on tools and workarounds that are not always accurate. Here, an easy-to-use method to measure rectangularity R, ellipticity E, and orientation O is developed. In addition, a new rectangularity vs. ellipticity index, REi, is defined. Following a step-by-step process, it is shown how these measures and index can be easily calculated using a combination of GIS built-in functions. The identification of shapes and estimation of orientations performed by this method is applied to the case study of the geometric and oriented lakes of the Llanos de Moxos, in the Bolivian Amazon, where shape and orientation have been the two most important elements studied to infer possible formation mechanisms. It is shown that, thanks to these new indexes, shape and orientation patterns are unveiled, which would have been hard to identify otherwise.

Digital Data Collection and Processing in Field and Lab: The Approach of Kinneret Regional Project

The paper showcases the field- and lab-documentation system developed for Kinneret Regional Project, an international archaeological expedition to the Northwestern shore of the Sea of Galilee (Israel) under the auspices of the University of Bern, the University of Helsinki, Leiden University and Wofford College. The core of the data management system is a fully relational, server-based database framework, which also includes time-based and static GIS services, stratigraphic analysis tools and fully indexed document/digital image archives. Data collection in the field is based on mobile, hand-held devices equipped with a custom-tailored stand-alone application. Comprehensive three-dimensional documentation of all finds and findings is achieved by means of total stations and/or high-precision GPS devices. All archaeological information retrieved in the field – including tachymetric data – is synched with the core system on the fly and thus immediately available for further processing in the field lab (within the local network) or for post-excavation analysis at remote institutions (via the WWW). Besides a short demonstration of the main functionalities, the paper also presents some of the key technologies used and illustrates usability aspects of the system’s individual components.

Patient and physician gender concordance in preventive care in university primary care settings.

OBJECTIVE To assess the quality of preventive care according to physician and patient gender in a country with universal health care coverage. METHODS We assessed a retrospective cohort study of 1001 randomly selected patients aged 50-80 years followed over 2 years (2005-2006) in 4 Swiss university primary care settings (Basel, Geneva, Lausanne, Zürich). We used indicators derived from RAND's Quality Assessment Tools and examined percentages of recommended preventive care. Results were adjusted using hierarchical multivariate logistic regression models. RESULTS 1001 patients (44% women) were followed by 189 physicians (52% women). Female patients received less preventive care than male patients (65.2% vs. 72.1%, p<0.001). Female physicians provided significantly more preventive care than male physicians (p=0.01) to both female (66.7% vs. 63.6%) and male patients (73.4% vs. 70.7%). After multivariate adjustment, differences according to physician (p=0.02) and patient gender (p<0.001) remained statistically significant. Female physicians provided more recommended cancer screening than male physicians (78.4 vs. 71.9%, p=0.01). CONCLUSIONS In Swiss university primary care settings, female patients receive less preventive care than male patients, with female physicians providing more preventive care than male physicians. Greater attention should be paid to female patients in preventive care and to why female physicians tend to provide better preventive care.

Structural analogues of the natural products magnolol and honokiol as potent allosteric potentiators of GABA(A) receptors.

Biphenylic compounds related to the natural products magnolol and 4'-O-methylhonokiol were synthesized, evaluated and optimized as positive allosteric modulators (PAMs) of GABA(A) receptors. The most efficacious compounds were the magnolol analog 5-ethyl-5'-hexylbiphenyl-2,2'-diol (45) and the honokiol analogs 4'-methoxy-5-propylbiphenyl-2-ol (61), 5-butyl-4'-methoxybiphenyl-2-ol (62) and 5-hexyl-4'-methoxybiphenyl-2-ol (64), which showed a most powerful potentiation of GABA-induced currents (up to 20-fold at a GABA concentration of 3μM). They were found not to interfere with the allosteric sites occupied by known allosteric modulators, such as benzodiazepines and N-arachidonoylglycerol. These new PAMs will be useful as pharmacological tools and may have therapeutic potential for mono-therapy, or in combination, for example, with GABA(A) receptor agonists.

Capacity Building in the Use of Geospatial Tools for Natural Resource Management in Tajikistan: Inception report

The sustainable management of natural resources is a key issue for sustainable development of a poor, mountainous country such as Tajikistan. In order to strengthen its agricultural and infrastructural development efforts and alleviate poverty in rural areas, spatial information and analysis are of crucial importance to improve priority setting and decision making efficiency. However, poor access to geospatial data and tools, and limited capacity in their use has greatly constrained the ability of governmental institutions to effectively assess, plan, and monitor natural resources management. The Centre for Development and Environment (CDE) has thus been mandated by the World Bank Group to provide adequate technical support to the Community Agriculture and Watershed Management Project (CAWMP). This support consists of a spatial database on soil degradation trends in 4 watersheds, capacity development in and awareness creation about geographic information technology and a spatial data exchange hub for natural resources management in Tajikistan. CDE’s support has started in July 2007 and will last until December 2007 with a possible extension in 2008.

Sudden cardiac death among general population and sport related population in forensic experience

PURPOSE: The goal of the study was to assess the causes and analyze the cases of sudden cardiac death (SCD) victims referred to the department of forensic medicine in Lausanne, with a particular focus on sports-related fatalities including also leisure sporting activities. To date, no such published assessment has been done nor for Switzerland nor for the central Europe. METHODS: This is a retrospective study based on autopsy records of SCD victims, from 10 to 50 years of age, performed at the University Centre of Legal Medicine in Lausanne from 1995 to 2010. The study population was divided into two groups: sport-related (SR) and not sport-related (NSR) SCDs. RESULTS: During the study period, 188 cases of SCD were recorded: 166 (88%) were NSR and 22 (12%) SR. The mean age of the 188 victims was 37.3 +/- 10.1 years, with the majority of the cases being male (79%). A cause of death was established in 84%, and the pathology responsible for death varied according to the age of the victims. In the NSR group, the mean age was 38.2 +/- 9.2 years and there was 82% of male. Coronary artery disease (CAD) was the main diagnosis in the victims aged 30-50 years. The majority of morphologically normal hearts were observed in the 15-29 year age range. There was no case in the 10-14 year age range. In the SR group, 91% of victims died during leisure sporting activities. In this group the mean age was 30.5 +/- 13.5 years, with the majority being male (82%). The main cause of death was CAD, with 6 cases (27%) and a mean age of 40.8 +/- 5.5 years. The youngest victim with CAD was 33 years old. A morphologically normal heart was observed in 5 cases (23%), with a mean age of 24.4 +/- 14.9 years. The most frequently implicated sporting activities were hiking (26%) and swimming (17%). CONCLUSION: In this study, CAD was the most common cause of death in both groups. Although this pathology most often affects adults over 35 years of age, there were also some victims under 35 years of age in both groups. SCDs during sport are mostly related to leisure sporting activities, for which preventive measures are not yet usually established. This study highlights also the need to inform both athletes and non athletes of the cardiovascular risks during sport activities and the role of a forensic autopsy and registries involving forensic pathologists for SR SCD.

Dose-painting intensity-modulated proton therapy for intermediate- and high-risk meningioma.

BACKGROUND Newly diagnosed WHO grade II-III or any WHO grade recurrent meningioma exhibit an aggressive behavior and thus are considered as high- or intermediate risk tumors. Given the unsatisfactory rates of disease control and survival after primary or adjuvant radiation therapy, optimization of treatment strategies is needed. We investigated the potential of dose-painting intensity-modulated proton beam-therapy (IMPT) for intermediate- and high-risk meningioma. MATERIAL AND METHODS Imaging data from five patients undergoing proton beam-therapy were used. The dose-painting target was defined using [68]Ga-[1,4,7,10-tetraazacyclododecane tetraacetic acid]- d-Phe(1),Tyr(3)-octreotate ([68]Ga-DOTATATE)-positron emission tomography (PET) in target delineation. IMPT and photon intensity-modulated radiation therapy (IMRT) treatment plans were generated for each patient using an in-house developed treatment planning system (TPS) supporting spot-scanning technology and a commercial TPS, respectively. Doses of 66 Gy (2.2 Gy/fraction) and 54 Gy (1.8 Gy/fraction) were prescribed to the PET-based planning target volume (PTVPET) and the union of PET- and anatomical imaging-based PTV, respectively, in 30 fractions, using simultaneous integrated boost. RESULTS Dose coverage of the PTVsPET was equally good or slightly better in IMPT plans: dose inhomogeneity was 10 ± 3% in the IMPT plans vs. 13 ± 1% in the IMRT plans (p = 0.33). The brain Dmean and brainstem D50 were small in the IMPT plans: 26.5 ± 1.5 Gy(RBE) and 0.002 ± 0.0 Gy(RBE), respectively, vs. 29.5 ± 1.5 Gy (p = 0.001) and 7.5 ± 11.1 Gy (p = 0.02) for the IMRT plans, respectively. The doses delivered to the optic structures were also decreased with IMPT. CONCLUSIONS Dose-painting IMPT is technically feasible using currently available planning tools and resulted in dose conformity of the dose-painted target comparable to IMRT with a significant reduction of radiation dose delivered to the brain, brainstem and optic apparatus. Dose escalation with IMPT may improve tumor control and decrease radiation-induced toxicity.