Development and applications of hollow fiber flow field-flow fractionation in the bioanalytical field. Studies of aggregation phenomena in complex protein samples

Recent advances in the fast growing area of therapeutic/diagnostic proteins and antibodies - novel and highly specific drugs - as well as the progress in the field of functional proteomics regarding the correlation between the aggregation of damaged proteins and (immuno) senescence or aging-related pathologies, underline the need for adequate analytical methods for the detection, separation, characterization and quantification of protein aggregates, regardless of the their origin or formation mechanism. Hollow fiber flow field-flow fractionation (HF5), the miniaturized version of FlowFFF and integral part of the Eclipse DUALTEC FFF separation system, was the focus of this research; this flow-based separation technique proved to be uniquely suited for the hydrodynamic size-based separation of proteins and protein aggregates in a very broad size and molecular weight (MW) range, often present at trace levels. HF5 has shown to be (a) highly selective in terms of protein diffusion coefficients, (b) versatile in terms of bio-compatible carrier solution choice, (c) able to preserve the biophysical properties/molecular conformation of the proteins/protein aggregates and (d) able to discriminate between different types of protein aggregates. Thanks to the miniaturization advantages and the online coupling with highly sensitive detection techniques (UV/Vis, intrinsic fluorescence and multi-angle light scattering), HF5 had very low detection/quantification limits for protein aggregates. Compared to size-exclusion chromatography (SEC), HF5 demonstrated superior selectivity and potential as orthogonal analytical method in the extended characterization assays, often required by therapeutic protein formulations. In addition, the developed HF5 methods have proven to be rapid, highly selective, sensitive and repeatable. HF5 was ideally suitable as first dimension of separation of aging-related protein aggregates from whole cell lysates (proteome pre-fractionation method) and, by HF5-(UV)-MALS online coupling, important biophysical information on the fractionated proteins and protein aggregates was gathered: size (rms radius and hydrodynamic radius), absolute MW and conformation.

Advanced aspects of radiation protection in the use of particle accelerators in the medical field

In this work, the well-known MC code FLUKA was used to simulate the GE PETrace cyclotron (16.5 MeV) installed at “S. Orsola-Malpighi” University Hospital (Bologna, IT) and routinely used in the production of positron emitting radionuclides. Simulations yielded estimates of various quantities of interest, including: the effective dose distribution around the equipment; the effective number of neutron produced per incident proton and their spectral distribution; the activation of the structure of the cyclotron and the vault walls; the activation of the ambient air, in particular the production of 41Ar, the assessment of the saturation yield of radionuclides used in nuclear medicine. The simulations were validated against experimental measurements in terms of physical and transport parameters to be used at the energy range of interest in the medical field. The validated model was also extensively used in several practical applications uncluding the direct cyclotron production of non-standard radionuclides such as 99mTc, the production of medical radionuclides at TRIUMF (Vancouver, CA) TR13 cyclotron (13 MeV), the complete design of the new PET facility of “Sacro Cuore – Don Calabria” Hospital (Negrar, IT), including the ACSI TR19 (19 MeV) cyclotron, the dose field around the energy selection system (degrader) of a proton therapy cyclotron, the design of plug-doors for a new cyclotron facility, in which a 70 MeV cyclotron will be installed, and the partial decommissioning of a PET facility, including the replacement of a Scanditronix MC17 cyclotron with a new TR19 cyclotron.

A model for radiofrequency electromagnetic field predictions at outdoor and indoor locations in the context of epidemiological research

We present a geospatial model to predict the radiofrequency electromagnetic field from fixed site transmitters for use in epidemiological exposure assessment. The proposed model extends an existing model toward the prediction of indoor exposure, that is, at the homes of potential study participants. The model is based on accurate operation parameters of all stationary transmitters of mobile communication base stations, and radio broadcast and television transmitters for an extended urban and suburban region in the Basel area (Switzerland). The model was evaluated by calculating Spearman rank correlations and weighted Cohen's kappa (kappa) statistics between the model predictions and measurements obtained at street level, in the homes of volunteers, and in front of the windows of these homes. The correlation coefficients of the numerical predictions with street level measurements were 0.64, with indoor measurements 0.66, and with window measurements 0.67. The kappa coefficients were 0.48 (95%-confidence interval: 0.35-0.61) for street level measurements, 0.44 (95%-CI: 0.32-0.57) for indoor measurements, and 0.53 (95%-CI: 0.42-0.65) for window measurements. Although the modeling of shielding effects by walls and roofs requires considerable simplifications of a complex environment, we found a comparable accuracy of the model for indoor and outdoor points.

Radiation dose optimized lateral expansion of the field of view in synchrotron radiation X-ray tomographic microscopy

Volumetric data at micrometer level resolution can be acquired within a few minutes using synchrotron-radiation-based tomographic microscopy. The field of view along the rotation axis of the sample can easily be increased by stacking several tomograms, allowing the investigation of long and thin objects at high resolution. On the contrary, an extension of the field of view in the perpendicular direction is non-trivial. This paper presents an acquisition protocol which increases the field of view of the tomographic dataset perpendicular to its rotation axis. The acquisition protocol can be tuned as a function of the reconstruction quality and scanning time. Since the scanning time is proportional to the radiation dose imparted to the sample, this method can be used to increase the field of view of tomographic microscopy instruments while optimizing the radiation dose for radiation-sensitive samples and keeping the quality of the tomographic dataset on the required level. This approach, dubbed wide-field synchrotron radiation tomographic microscopy, can increase the lateral field of view up to five times. The method has been successfully applied for the three-dimensional imaging of entire rat lung acini with a diameter of 4.1 mm at a voxel size of 1.48 microm.

Does Israel Follow the Law? A Critical Analysis of Israel's Application of the International Law of Belligerent Occupation in the Occupied Territories

Israel's occupation of territories it captured in 1967 has become one of the longest and most controversial occupations of the last fifty years. Eschewing the traditional political analysis of the Israeli-Palestinian conflict, this paper aims to explore whether Israel has adequately applied international law in the occupied territories, in particular, the law of belligerent occupation. The two actors under assessment are the Israeli government, particularly its military which enforces and maintains the law in the territories, and the Supreme Court of Israel, which has the power of review over military actions in the territories. The particular issues of the occupation that are critically analyzed are the general legal framework that Israel established in the territories, Israel's civilian settlement policy in territories, and Israel's construction of a barrier in the West Bank. This paper concludes that Israel has incorrectly applied the legal framework of belligerent occupation by refusing to apply the Fourth Geneva Convention; it has wrongly concluded that the establishment of civilian settlements in the territories conform with international law; yet it has rightly concluded that the construction of the barrier in the West Bank is permissible under international law, in contrast to the conclusion of the much publicized International Court of Justice's Advisory Opinion on the 'Wall.' Along with these general assessments, the author will also provide some historical and political insight into why the Israeli government and the Supreme Court may have applied the law in the way that they did.

A field study on characteristics and diversity of gene expression in the liver of dairy cows during the transition period

Metabolic and endocrine adaptations to support milk production during the transition period vary between individual cows. This variation between cows to adapt to lactation may have a genetic basis. The present field study was carried out to determine hepatic adaptations occurring from late pregnancy through early lactation by measuring mRNA abundance of candidate genes in dairy cows on-farm. Additionally, the objective was to observe the diversity in inter-individual variation for the candidate genes that may give indications where individual adaptations at a molecular level can be found. This study was carried out on-farm including 232 dairy cows (parity >3) from 64 farms in Switzerland. Blood and liver samples were collected on d 20+/-7 before parturition, on d 24+/-2, and on d 89+/-4 after parturition. Blood plasma was assayed for concentrations of glucose, nonesterified fatty acids, beta-hydroxybutyrate, cholesterol, triglycerides, urea, albumin, protein, insulin, insulin-like growth factor-1, leptin, 3,5,3'-triiodothyronine, and thyroxine. Liver samples were obtained at the same time points and were measured for mRNA abundance of 26 candidate genes encoding enzymes and nuclear receptors involved in gluconeogenesis, fatty acid beta-oxidation, fatty acid and triglyceride synthesis, ketogenesis, citric acid cycle, cholesterol synthesis, and the urea cycle. The cows in the present study experienced a marked metabolic load in early lactation, as presented by changes in plasma metabolites and hormones, and responded accordingly with upregulation and downregulation of almost all candidate genes involved in metabolic processes in the liver. The observed inter-individual variation for the candidate genes, which was highest for acetyl-CoA-carboxylase and glycerol-3-phosphate dehydrogenase 2, should be further investigated to unravel the regulation at molecular level for optimal adaptive performance in dairy cows.

Pathogenesis and classification of eosinophil disorders: a review of recent developments in the field

Eosinophils and their products play an essential role in the pathogenesis of various reactive and neoplastic disorders. Depending on the underlying disease, molecular defect and involved cytokines, hypereosinophilia may develop and may lead to organ damage. In other patients, persistent eosinophilia is accompanied by typical clinical findings, but the causative role and impact of eosinophilia remain uncertain. For patients with eosinophil-mediated organ pathology, early therapeutic intervention with agents reducing eosinophil counts can be effective in limiting or preventing irreversible organ damage. Therefore, it is important to approach eosinophil disorders and related syndromes early by using established criteria, to perform all appropriate staging investigations, and to search for molecular targets of therapy. In this article, we review current concepts in the pathogenesis and evolution of eosinophilia and eosinophil-related organ damage in neoplastic and non-neoplastic conditions. In addition, we discuss classifications of eosinophil disorders and related syndromes as well as diagnostic algorithms and standard treatment for various eosinophil-related disorders.

Virtopsy - the concept of a centralized database in forensic medicine for analysis and comparison of radiological and autopsy data

Recent developments in clinical radiology have resulted in additional developments in the field of forensic radiology. After implementation of cross-sectional radiology and optical surface documentation in forensic medicine, difficulties in the validation and analysis of the acquired data was experienced. To address this problem and for the comparison of autopsy and radiological data a centralized database with internet technology for forensic cases was created. The main goals of the database are (1) creation of a digital and standardized documentation tool for forensic-radiological and pathological findings; (2) establishing a basis for validation of forensic cross-sectional radiology as a non-invasive examination method in forensic medicine that means comparing and evaluating the radiological and autopsy data and analyzing the accuracy of such data; and (3) providing a conduit for continuing research and education in forensic medicine. Considering the infrequent availability of CT or MRI for forensic institutions and the heterogeneous nature of case material in forensic medicine an evaluation of benefits and limitations of cross-sectional imaging concerning certain forensic features by a single institution may be of limited value. A centralized database permitting international forensic and cross disciplinary collaborations may provide important support for forensic-radiological casework and research.

Executive Reorganization: Building a State Government That Worked for People -- Tom Harrison, Diana Dowling & Sheena Wilson “In the Crucible of Change”

One of the primary accomplishments of Governor Forrest Anderson in 1969-71 was the reorganization of the Executive Branch of Montana government, something that had been attempted six different times between 1919 and 1962 as state government had grown from twenty agencies to almost 200 uncontrolled boards, bureaus and commissions. The chaotic structure of the executive branch disempowered governors of both parties and empowered the private corporations and organizations that were the power structure of Montana. With remarkable political acumen, Governor Anderson figured out how to get that near impossible job done. Central to his efforts was the creation of an Executive Reorganization Commission, including eight legislators and the Governor, the adoption of a Constitutional Amendment that limited the executive branch to no more than twenty departments under the Governor, and the timely completion of a massive research effort to delineate the actual structure of the twenty departments. That story is told in this episode by three major players in the effort, all involved directly with the Executive Reorganization Commission: Tom Harrison, Diana Dowling and Sheena Wilson. Their recollections reflect an insider’s perspective of this significant accomplishment that helped change Montana “In the Crucible of Change.” Tom Harrison is a former Republican State Representative and State Senator from Helena, who was a member of the Executive Reorganization Commission. As Majority Leader in the Montana House of Representatives in 1971, he was the primary sponsor of the House’s executive reorganization bill and helped shepherd the Senate’s version to passage. Harrison was the Republican candidate for Attorney General in 1976 after which he practiced private law for 3 more decades. He served in the Montana Army National Guard for almost 34 years, rising to the rank of Colonel in the position of Judge Advocate General. He was a founding Director of Federal Defenders of Montana (legal representation for indigents accused within the Federal Judicial System); appointed Chairman of the original Montana State Fund (workers' compensation insurance) by Gov. Stephens; served as President of the Montana Trial Lawyers Association, Helena Kiwanis Club and St. Peter's Community Hospital Foundation, as well as Chairman and Director of AAA MountainWest; and was a founder, first Chairman and Director of the Valley Bank of Helena for over 25 years. Diana Dowling was an attorney for the Executive Reorganization Commission and helped draft the legislation that was passed. She also worked for Governor Forrest Anderson and for the 1972 Constitutional Convention where she prepared and directed publication of official explanation of the new Constitution that was mailed to all Montana voters. Diana was Executive Director of the Montana Bar Association and for 20 years held various legal positions with the Montana Legislative Council. For 12 years she was a commissioner on the National Conference of Commissioners on Uniform State Laws and for 7 years was a member of Montana State Board of Bar Examiners. Diana was the first director of the Montana Lottery, an adjunct professor at both Carroll College and the UM Law School, and an administrative officer for Falcon Press Publishing Co. Diana is currently - and intends to continue being - a perpetual college student. Sheena Wilson came fresh out of the University of Montana to become a Research Assistant for the Executive Reorganization Commission. Later she worked for seven years as a field representative in Idaho and Montana for the Mountain Plains Family Education Program, for thirteen years with Congressman Pat Williams as Executive Assistant in Washington and Field Assistant here in Montana, owned and managed a Helena restaurant for seven years, worked as Executive Assistant for State Auditor John Morrison and was Deputy Chief of Staff for Governor Brian Schweitzer his full 8 years in the Governorship. Though currently “retired”, Sheena serves on the Montana Board of Investments, the Public Employees Retirement Board and the Capitol Complex Advisory Council and is a partner in a dry-land wheat farm in Teton County that was homesteaded by her great uncle.

Present at the Creation: Montana Higher Education Reforms from 1972 Constitution -- Dr. Lawrence K. Pettit “In the Crucible of Change”

Prior to the passage of the 1972 Constitution, Montana’s higher education system was both controlled and victimized by Montana’s politics. Alternatingly, Governors or the Legislature tried to control and/or impose political ideology upon the management and teaching/content within the University System. Political favoritism and power-broking were the hallmark of the legislative appropriation process. Under the new Constitution, a newly empowered Board of Regents, and a new Commissioner of Higher Education managed the system and controlled the allocation of the legislative appropriations, but not without a major battle before the Montana Supreme Court. Dr. Lawrence K. Pettit (Larry Pettit) (b. 5/2/1937) was present at the creation of this newly structured higher education system as the first Commissioner of Higher Education in Montana after his appointment by the Board of Regents of the University System in 1973. Larry Pettit has had a dual career in politics and higher education. Pettit, of Lewistown, served as legislative assistant to U.S. Senators James E. Murray and Lee Metcalf, campaign manager, head of transition team and assistant to Montana Governor Thomas L. Judge, taught political science at The Pennsylvania State University (main campus), was chair of political science at Montana State University, Deputy Commissioner for Academic Programs at the Texas Higher Education Coordinating Board, Chancellor of the University System of South Texas (since merged with Texas A&M University), President of Southern Illinois University, and President of Indiana University of Pennsylvania from where he retired in 2003. He has served as chair of the Commission on Leadership for the American Council on Education, president of the National Association of (University) System Heads, and on many national and state boards and commissions in higher education. Pettit is author of “If You Live by the Sword: Politics in the Making and Unmaking of a University President.” More about Pettit is found at http://www.lawrencekpettit.com