From diabetes to heart disease : studies on dyslipidemia-induced B-cell dysfunction, immunomodulation in heart transplantation, and cardiac stem cell therapy

Diabetes is a growing epidemic with devastating human, social and economic impact. It is associated with significant changes in plasma concentrations of lipoproteins. We tested the hypothesis that lipoproteins modulate the function and survival of insulin-secreting cells. We first detected the presence of several receptors that participate in the binding and processing of plasma lipoproteins and confirmed the internalization of fluorescent LDL and HDL particles in insulin-secreting β-cells. Purified human VLDL and LDL particles reduced insulin mRNA levels and β-cell proliferation, and induced a dose-dependent increase in the rate of apoptosis. In mice lacking the LDL receptor, islets showed a dramatic decrease in LDL uptake and were partially resistant to apoptosis caused by LDL. VLDL-induced apoptosis of β-cells involved caspase-3 cleavage and reduction in levels of the c-Jun N-terminal (JNK) Interacting Protein-1 (IB1/JIP-1). In contrast, the pro-apoptotic signaling of lipoproteins was antagonized by HDL particles or by a small peptide inhibitor of JNK. The protective effects of HDL were mediated, in part, by inhibition of caspase-3 cleavage and activation of the protein kinase Akt/PKB. Heart disease is a major cause of morbidity and mortality among patients with diabetes. When heart failure is refractory to medical therapy and cannot be improved by electrical resynchronization, percutaneous angioplasty or coronary graft bypass surgery, heart transplantation remains a "last resort" therapy. Nevertheless, it is limited by the side effects of immunosuppressive drugs and chronic rejection. Localized expression of immunomodulatory genes in the donor organ can create a state of immune privilege within the graft, and was performed in rodent hearts by infecting cells with an adenovirus encoding indoleamine 2,3-dioxygenase (IDO), the rate-limiting enzyme in the catabolism of tryptophane. Other strategies are based on genetic manipulation of dendritic cells (DCs) with immunosuppressive genes and in vitro exposure of DCs to agents that prevent their maturation by inflammatory cytokines. Finally, we used 5-bromo-2'-deoxyuridine, which is incorporated into DNA and diluted with cell division, to identify long-term label retaining cells in the adult rodent heart. The majority of these cells were positive for the stem cell antigen-1 (Sca-1) and negative for the endothelial precursor marker CD31. They formed cardiospheres in vitro and showed differentiation potential into mesenchymal cell lineages. When cultured in cardiomyogenic differentiation medium, they expressed cardiac-specific genes. Taken together, these data provide evidence of slow-cycling stem cells in the rodent heart. Chronic shortage of donor organs opens the way to cardiac stem cell therapy in humans, although the long way from animal experimentation to routine therapy in patients may still take several years. - Du diabète de type 2 à la maladie coronarienne : trois études sur les dysfonctions de la cellule sécrétrice d'insuline induites par les dyslipidémies, l'immunomodulation dans la transplantation cardiaque, et la thérapie par des cellules souches myocardiques. Le diabète de type 2 a pris les dimensions d'une épidémie, avec des conséquences sociales et économiques dont nous n'avons pas encore pris toute la mesure. La maladie s'accompagne souvent d'une dyslipidémie caractérisée par une hypertriglycéridémie, des taux abaissés de cholestérol HDL, et des concentrations de cholestérol LDL à la limite supérieure de ce qui est considéré comme acceptable. L'hypothèse à la base de cette étude est qu'une modification des taux plasmatiques de lipoprotéines pourrait avoir une influence directe sur la cellule β sécrétrice d'insuline en modifiant sa fonction, sa durée de vie et son taux de régénération. Dans un premier temps, nous avons mis en évidence, sur la cellule β, la présence de plusieurs récepteurs impliqués dans la captation des lipoprotéines. Nous avons confirmé la fonctionnalité de ces récepteurs en suivant l'internalisation de LDL et de HDL marqués. En présence de VLDL ou de LDL humains, nous avons observé une diminution de la transcription du gène de l'insuline, une prolifération cellulaire réduite, et une augmentation de l'apoptose, toutes fonctions de la dose et du temps d'exposition. L'apoptose induite par les VLDL passe par une activation de la caspase-3 et une réduction du taux de la protéine IB1/JIP-1 (Islet Brain1/JNK Interacting Protein 1), dont une mutation est associée à une forme monogénique de diabète de type 2. Par opposition, les HDL, ainsi que des peptides inhibiteurs de JNK, sont capables de contrer la cascade pro-apoptotique déclenchée, respectivement, par les LDL et les VLDL. Ces effets protecteurs comprennent l'inhibition du clivage de la caspase-3 et l'activation de la protéine kinase Akt/PKB. En conclusion, les lipoprotéines sont des éléments clés de la survie de la cellule β, et pourraient contribuer au dysfonctionnement observé dans le pancréas endocrine au cours du développement du diabète. La maladie cardiaque, et plus particulièrement la maladie coronarienne, est une cause majeure de morbidité et de mortalité chez les patients atteints de diabète. Plusieurs stratégies sont utilisées quotidiennement pour pallier les atteintes cardiaques: traitements médicamenteux, électromécaniques par resynchronisation électrique, ou communément appelés « interventionnels » lorsqu'ils font appel à l'angioplastie percutanée. La revascularisation du myocarde par des pontages coronariens donne également de très bons résultats dans certaines situations. Il existe toutefois des cas où plus aucune de ces approches n'est suffisante. La transplantation cardiaque est alors la thérapie de choix pour un nombre restreint de patients. La thérapie génique, en permettant l'expression locale de gènes immunomodulateurs dans l'organe greffé, permet de diminuer les réactions de rejet inhérentes à toute transplantation (à l'exception de celles réalisées entre deux jumeaux homozygotes). Nous avons appliqué chez des rongeurs cette stratégie en infectant le coeur greffé avec un adénovirus codant pour l'enzyme indoleamine 2,3-dioxygénase (IDO), une enzyme clé dans le catabolisme du tryptophane. Nous avons procédé de manière identique in vitro en surexprimant IDO dans les cellules dendritiques, dont le rôle est de présenter les antigènes aux lymphocytes Τ du receveur. Des expériences similaires ont été réalisées en traitant les cellules dendritiques avec des substances capables de prévenir, en partie du moins, leur maturation par des agents pro-inflammatoires. Finalement, nous avons exploré une stratégie utilisée couramment en hématologie, mais qui n'en est encore qu'à ses débuts au niveau cardiaque : la thérapie par des cellules souches. En traitant des rongeurs avec un marqueur qui s'incorpore dans l'ADN nucléaire, le 5-bromo- 2'-deoxyuridine, nous avons identifié une population cellulaire se divisant rarement, positive en grande partie pour l'antigène embryonnaire Sca-1 et négative pour le marqueur endothélial CD31. En culture, ces cellules forment des cardiosphères et sont capables de se différencier dans les principaux types tissulaires mésenchymateux. Dans un milieu de differentiation adéquat, ces cellules expriment des gènes cardiomyocytaires. En résumé, ces données confirment la présence chez le rongeur d'une population résidente de précurseurs myocardiques. En addenda, on trouvera deux publications relatives à la cellule β productrice d'insuline. Le premier article démontre le rôle essentiel joué par la complexine dans l'insulino-sécrétion, tandis que le second souligne l'importance de la protéine IB1/JIP-1 dans la protection contre l'apoptose de la cellule β induite par certaines cytokines.

Distinct neuronal circuits underlying the bahavioral and physiological components of the fear response

Abstract : Expression of fear involves changes in a number of behavioral and physiological parameters that are triggered by the central amygdala (CeA). The fear circuit also includes a series of brain stem nuclei that are the final effectors of the changes induced by the fear reaction. The CeA expresses many different neuropeptide receptors that can modulate fear responses. Today, the precise organization and the modulation of projections from the amygdala to the brain stem are still poorly understood. The aim of this project was to better understand the organization and the modulation of the fear circuit. To investigate this we first determined whether the CeA is composed of separate neuronal populations, where each one projects to specific brain stem nuclei, or whether single CeA neurons project to several nuclei. For this purpose, we first selected two brain stem nuclei implicated in the modulation of different components of the fear reactions, the periaqueductal gray (implicated in freezing) and the nucleus of solitary tract (implicated in heart rate modulation). We then performed double injections of two different retrograde tracers in these two nuclei and we quantified the subsequent presence of co-labelling in the CeA. We found that neurons projecting to the PAG and to the NTS are organized in separate populations. Subsequent electrophysiological recordings of the two populations revealed that PAG and NTS projecting neurons also have different electrophysiological characteristics. We then verified in vitro whether the neurons projecting to different brain stem nuclei express specific combinations of neuropeptide receptors, and whether a neuropeptide acting pre-synaptically (oxytocin) specifically modulates one of these two projections. We did not find differences at the level of expression of neurópeptide receptors, but we observed that oxytocin, a neuropeptide with anxiolytic properties, modulates PAG projecting neurons without affecting NTS projecting neurons. As oxytocin appeared to specifically modulate projections to the PAG, involved in the modulation of the freezing reaction, but did not affect the projections to the NTS, implicated in the modulation of cardiovascular parameters, we verified how this modulation translates in living animals. We investigated the effects of infra-amygdala injection of oxytocin on cardiovascular and behavioral changes induced by contextual fear conditioning. We found that oxytocin decreased the freezing response without affecting the cardiovascular system. Finally, as neuropeptides are considered potential future anxiolytics, we investigated whether diazepam and oxytocin, acting on the same circuit, had additive effects. This question was addressed exclusively with an in vitro electrophysiological approach. We obtained that oxytocin and diazepam, when co-applied, had an additive effect on both synaptic transmission and neuronal activity. These results open new perspectives for the possible clinical applications of oxytocin. Résumé : L'expression de la peur est accompagnée par de nombreux changements physiologiques et comportementaux qui sont déclenchés par l'amygdale centrale (CeA). Le circuit inclue aussi une série de noyaux du tronc cérébrale qui sont les effecteurs des différentes composantes de la réaction de peur. On sait que CeA envoie des projections aux noyaux du tronc cérébral et que ces neurones expriment une grande variété de récepteurs aux neuropeptides. Par contre, l'organisation des projections, ainsi que la modulation de ces projections par les neuropeptides reste encore peu connue. Avec ce projet, on premièrement voulu déterminer si CeA est composée de populations neuronales séparées qui projettent vers un noyau spécifique, ou bien si chaque neurones envoie des projections vers plusieurs noyaux. A ce propos, on a effectué des doubles injections de deux traceurs rétrogrades différentes dans deux noyaux du tronc cérébral impliqués dans des différentes composantes des réactions de peur. On a injecté la substance grise périaqueducale (PAG), qui est impliquée dans la réponse d'immobilisation, ainsi que le noyau du tractus solitaire (NTS) qui est responsable des changements cardiovasculaires. On a ensuite quantifié la présence de neurones contenant les deux traceurs dans CeA. On a trouvé que la plupart des neurones de l'amygdale centrale projettent vers un noyau spécifique, et on peut donc dire que l'amygdale semble être composée de populations neuronales séparées. On a ensuite mesuré les caractéristiques électrophysiologiques de ces deux projections et on a trouvé des différences substantielles concernant la résistance membranaire, la capacitance, le potentiel membranaire de repos ainsi que la fréquence des potentiels d'action spontanés. Puis, comme beaucoup de neuropéptides dans l'amygdale exercent un effet modulatoire sûr les réactions de peur et sur l'anxiété, on a étudié les effets directs et indirects d'une série de neuropeptides sur les différentes projections pour évaluer s'il y a des neuropeptides qui agissent spécifiquement sur une. On n'a pas trouvé de différences entre neurones qui projettent vers le PAG et neurones qui projettent vers le NTS concernant les effets de neuropeptides qui agissent directement sur ces cellules. Par contre, on a trouvé que l'ocytocine, un neuropeptide qui se lie à des récepteurs dans la partie latérale de l'amygdale centrale et inhibe de façon indirecte les neurones de l'amygdala centrale médiale, module les projections vers le PAG sans affecter celles qui vont vers le NTS. Comme le PAG est impliqué dans la réponse d'immobilisation, alors que le NTS est impliqué dans la modulation cardiovasculaire, on a ensuite étudié les effets de l'ocytocine injectée dans l'amygdale de rat vivants sur les réactions de peur conditionnées. On a trouvé que l'ocytocine diminue la réponse d'immobilisation sans par contre affecter la réponse cardiovasculaire. Pour terminer, on a vérifié si l'ocytocine potentialise les effets d'un médicament anxiolytique, le diazeparn. Avec une étude in vitro on a trouvé qu'une co-application d'ocytocine et diazeparn résulte en un effet additionnel à la fois sur la transmission synaptique ainsi que sur l'activité neuronale des neurones de l'amygdale centrale médiale. Ces résultats ouvrent des nouvelles perspectives pour une potentielle utilisation clinique de l'ocytocine.

Exogenous mRNA delivery and bioavailability in gene transfer mediated by piggyBac transposition.

BACKGROUND: Up to now, the different uptake pathways and the subsequent intracellular trafficking of plasmid DNA have been largely explored. By contrast, the mode of internalization and the intracellular routing of an exogenous mRNA in transfected cells are poorly investigated and remain to be elucidated. The bioavailability of internalized mRNA depends on its intracellular routing and its potential accumulation in dynamic sorting sites for storage: stress granules and processing bodies. This question is of particular significance when a secure transposon-based system able to integrate a therapeutic transgene into the genome is used. Transposon vectors usually require two components: a plasmid DNA, carrying the gene of interest, and a source of transposase allowing the integration of the transgene. The principal drawback is the lasting presence of the transposase, which could remobilize the transgene once it has been inserted. Our study focused on the pharmacokinetics of the transposition process mediated by the piggyBac transposase mRNA transfection. Exogenous mRNA internalization and trafficking were investigated towards a better apprehension and fine control of the piggyBac transposase bioavailability. RESULTS: The mRNA prototype designed in this study provides a very narrow expression window of transposase, which allows high efficiency transposition with no cytotoxicity. Our data reveal that exogenous transposase mRNA enters cells by clathrin and caveolae-mediated endocytosis, before finishing in late endosomes 3 h after transfection. At this point, the mRNA is dissociated from its carrier and localized in stress granules, but not in cytoplasmic processing bodies. Some weaker signals have been observed in stress granules at 18 h and 48 h without causing prolonged production of the transposase. So, we designed an mRNA that is efficiently translated with a peak of transposase production 18 h post-transfection without additional release of the molecule. This confines the integration of the transgene in a very small time window. CONCLUSION: Our results shed light on processes of exogenous mRNA trafficking, which are crucial to estimate the mRNA bioavailability, and increase the biosafety of transgene integration mediated by transposition. This approach provides a new way for limiting the transgene copy in the genome and their remobilization by mRNA engineering and trafficking.

Activity assays and immunoassays for plasma Renin and prorenin: information provided and precautions necessary for accurate measurement.

BACKGROUND: Measurement of plasma renin is important for the clinical assessment of hypertensive patients. The most common methods for measuring plasma renin are the plasma renin activity (PRA) assay and the renin immunoassay. The clinical application of renin inhibitor therapy has thrown into focus the differences in information provided by activity assays and immunoassays for renin and prorenin measurement and has drawn attention to the need for precautions to ensure their accurate measurement. CONTENT: Renin activity assays and immunoassays provide related but different information. Whereas activity assays measure only active renin, immunoassays measure both active and inhibited renin. Particular care must be taken in the collection and processing of blood samples and in the performance of these assays to avoid errors in renin measurement. Both activity assays and immunoassays are susceptible to renin overestimation due to prorenin activation. In addition, activity assays performed with peptidase inhibitors may overestimate the degree of inhibition of PRA by renin inhibitor therapy. Moreover, immunoassays may overestimate the reactive increase in plasma renin concentration in response to renin inhibitor therapy, owing to the inhibitor promoting conversion of prorenin to an open conformation that is recognized by renin immunoassays. CONCLUSIONS: The successful application of renin assays to patient care requires that the clinician and the clinical chemist understand the information provided by these assays and of the precautions necessary to ensure their accuracy.

Offshore work and the work of nurses on board: an integrative review

OBJECTIVETo know the production of theoretical approaches on issues related to offshore work and the work of offshore nurses.METHODIntegrative literature review conducted in the databases of LILACS, BDENF, MEDLINE, SciELO and Index PSI.RESULTSWe selected 33 studies published in national and international journals between 1997 and 2014. The thematic analysis corpus resulted in four central themes: offshore work environment; amid work adversities, an escape; structuring of offshore health and safety services; in search of safe practices.CONCLUSIONThis study contributes to the offshore work of nurses in relation to the nature of work, acting amid adversities and the restless search for safe practices in the open sea.

Current approaches and challenges for the metabolite profiling of complex natural extracts.

Metabolite profiling is critical in many aspects of the life sciences, particularly natural product research. Obtaining precise information on the chemical composition of complex natural extracts (metabolomes) that are primarily obtained from plants or microorganisms is a challenging task that requires sophisticated, advanced analytical methods. In this respect, significant advances in hyphenated chromatographic techniques (LC-MS, GC-MS and LC-NMR in particular), as well as data mining and processing methods, have occurred over the last decade. Together, these tools, in combination with bioassay profiling methods, serve an important role in metabolomics for the purposes of both peak annotation and dereplication in natural product research. In this review, a survey of the techniques that are used for generic and comprehensive profiling of secondary metabolites in natural extracts is provided. The various approaches (chromatographic methods: LC-MS, GC-MS, and LC-NMR and direct spectroscopic methods: NMR and DIMS) are discussed with respect to their resolution and sensitivity for extract profiling. In addition the structural information that can be generated through these techniques or in combination, is compared in relation to the identification of metabolites in complex mixtures. Analytical strategies with applications to natural extracts and novel methods that have strong potential, regardless of how often they are used, are discussed with respect to their potential applications and future trends.

Crescent and star shapes of members of the Chlamydiales order: impact of fixative methods.

Members of the Chlamydiales order all share a biphasic lifecycle alternating between small infectious particles, the elementary bodies (EBs) and larger intracellular forms able to replicate, the reticulate bodies. Whereas the classical Chlamydia usually harbours round-shaped EBs, some members of the Chlamydia-related families display crescent and star-shaped morphologies by electron microscopy. To determine the impact of fixative methods on the shape of the bacterial cells, different buffer and fixative combinations were tested on purified EBs of Criblamydia sequanensis, Estrella lausannensis, Parachlamydia acanthamoebae, and Waddlia chondrophila. A linear discriminant analysis was performed on particle metrics extracted from electron microscopy images to recognize crescent, round, star and intermediary forms. Depending on the buffer and fixatives used, a mixture of alternative shapes were observed in varying proportions with stars and crescents being more frequent in C. sequanensis and P. acanthamoebae, respectively. No tested buffer and chemical fixative preserved ideally the round shape of a majority of bacteria and other methods such as deep-freezing and cryofixation should be applied. Although crescent and star shapes could represent a fixation artifact, they certainly point towards a diverse composition and organization of membrane proteins or intracellular structures rather than being a distinct developmental stage.

On-shoe wearable sensors for gait and turning assessment of patients with Parkinson's disease.

Assessment of locomotion through simple tests such as timed up and go (TUG) or walking trials can provide valuable information for the evaluation of treatment and the early diagnosis of people with Parkinson's disease (PD). Common methods used in clinics are either based on complex motion laboratory settings or simple timing outcomes using stop watches. The goal of this paper is to present an innovative technology based on wearable sensors on-shoe and processing algorithm, which provides outcome measures characterizing PD motor symptoms during TUG and gait tests. Our results on ten PD patients and ten age-matched elderly subjects indicate an accuracy ± precision of 2.8 ± 2.4 cm/s and 1.3 ± 3.0 cm for stride velocity and stride length estimation compared to optical motion capture, with the advantage of being practical to use in home or clinics without any discomfort for the subject. In addition, the use of novel spatio-temporal parameters, including turning, swing width, path length, and their intercycle variability, was also validated and showed interesting tendencies for discriminating patients in ON and OFF states and control subjects.

Concrete Pavement Mixture Design and Analysis (MDA): Assessment of Air Void System Requirements for Durable Concrete, TPF-5(205), 2012

Concrete will suffer frost damage when saturated and subjected to freezing temperatures. Frost-durable concrete can be produced if a specialized surfactant, also known as an air-entraining admixture (AEA), is added during mixing to stabilize microscopic air voids. Small and well-dispersed air voids are critical to produce frost-resistant concrete. Work completed by Klieger in 1952 found the minimum volume of air required to consistently ensure frost durability in a concrete mixture subjected to rapid freezing and thawing cycles. He suggested that frost durability was provided if 18 percent air was created in the paste. This is the basis of current practice despite the tests being conducted on materials that are no longer available using tests that are different from those in use today. Based on the data presented, it was found that a minimum air content of 3.5 percent in the concrete and 11.0 percent in the paste should yield concrete durable in the ASTM C 666 with modern AEAs and low or no lignosulfonate water reducers (WRs). Limited data suggests that mixtures with a higher dosage of lignosulfonate will need about 1 percent more air in the concrete or 3 percent more air in the paste for the materials and procedures used. A spacing factor of 0.008 in. was still found to be necessary to provide frost durability for the mixtures investigated.

Contribution of microbial and invertebrate communities to leaf litter colonization in a Mediterranean stream

Leaf litter inputs and retention play an important role in ecosystem functioning in forested streams. We examined colonization of leaves by microbes (bacteria, fungi, and protozoa) and fauna in Fuirosos, an intermittent forested Mediterranean stream. Black poplar (Populus nigra) and plane (Platanus acerifolia) leaf packs were placed in the stream for 4 mo. We measured the biomasses and calculated the densities of bacteria, fungi, protozoa, meiofauna, and macroinvertebrates to determine their dynamics and potential interactions throughout the colonization process. Colonization was strongly correlated with hydrological variability (defined mainly by water temperature and discharge). The 1st week of colonization was characterized by hydrological stability and warm water temperatures, and allocation of C from microbial to invertebrate compartments on the leaf packs was rapid. Clumps of fine particulate organic matter (FPOM) were retained by the leaf packs, and enhanced rapid colonization by microfauna and meiofaunal collector-gatherers (ostracods and copepods). After 2 wk, an autumnal flood caused a 20-fold increase in water flow. Higher discharge and lower water temperature caused FPOM-related fauna to drift away from the packs and modified the subsequent colonization sequence. Fungi showed the highest biomass, with similar values to those recorded at the beginning of the experiment. After 70 d of postflood colonization, fungi decreased to nearly 40% of the total C in the leaf packs, whereas invertebrates became more abundant and accounted for 60% of the C. Natural flood occurrence in Mediterranean streams could be a key factor in the colonization and processing of organic matter.

Low-Cost Rural Surface Alternatives: Literature Review and Recommendations, TR-632, 2013

Freezing and thawing action induces damage to unbound gravel roads in Iowa resulting in maintenance costs for secondary road departments. Some approaches currently used by County Engineers to deal with this problem include temporarily spreading rock on the affected areas, lowering or improving drainage ditches, tiling, bridging the area with stone and geosynthetic covered by a top course of aggregate or gravel, coring boreholes and filling them with calcium chloride to melt lenses and provide drainage, and re-grading the crown to a slope of 4% to 6% to maximize spring drainage. However, most of these maintenance solutions are aimed at dealing with conditions after they occur. This study was tasked with identifying alternative approaches in the literature to mitigate the problem. An annotated bibliographic record of literature on the topic of frost-heave and thaw-weakening of gravel roads was generated and organized by topic, and all documents were assessed in terms of a suitable rating for mitigating the problem in Iowa. Over 300 technical articles were collected and selected down to about 150 relevant articles for a full assessment. The documents collected have been organized in an electronic database, which can be used as a tool by practitioners to search for information regarding the various repair and mitigation solutions, measurement technologies, and experiences that have been documented by selected domestic and international researchers and practitioners. Out of the 150+ articles, 71 articles were ranked as highly applicable to conditions in Iowa. The primary mitigation methods identified in this study included chemical and mechanical stabilization; scarification, blending, and recompaction; removal and replacement; separation, and reinforcement; geogrids and cellular confinement; drainage control and capillary barriers, and use of alternative materials. It is recommended that demonstration research projects be established to examine a range of construction methods and materials for treating granular surfaced roadways to mitigate frost-heave and thaw-weakening problems. Preliminary frost-susceptibility test results from ASTM D5916 are included for a range of Iowa materials.

Intelligence-led crime scene processing. Part I : Forensic intelligence.

Forensic science is generally defined as the application of science to address questions related to the law. Too often, this view restricts the contribution of science to one single process which eventually aims at bringing individuals to court while minimising risk of miscarriage of justice. In order to go beyond this paradigm, we propose to refocus the attention towards traces themselves, as remnants of a criminal activity, and their information content. We postulate that traces contribute effectively to a wide variety of other informational processes that support decision making inmany situations. In particular, they inform actors of new policing strategies who place the treatment of information and intelligence at the centre of their systems. This contribution of forensic science to these security oriented models is still not well identified and captured. In order to create the best condition for the development of forensic intelligence, we suggest a framework that connects forensic science to intelligence-led policing (part I). Crime scene attendance and processing can be envisaged within this view. This approach gives indications abouthowto structure knowledge used by crime scene examiners in their effective practice (part II).

Perception and Interpretation of Advance Warning Signs on County Roads, HR-256, 1984

This research consisted of five laboratory experiments designed to address the following two objectives in an integrated analysis: (1) To discriminate between the symbol Stop Ahead warning sign and a small set of other signs (which included the word-legend Stop Ahead sign); and (2) To analyze sign detection, recognizability, and processing characteristics by drivers. A set of 16 signs was used in each of three experiments. A tachistoscope was used to display each sign image to a respondent for a brief interval in a controlled viewing experiment. The first experiment was designed to test detection of a sign in the driver's visual field; the second experiment was designed to test the driver's ability to recognize a given sign in the visual field; and the third experiment was designed to test the speed and accuracy of a driver's response to each sign as a command to perform a driving action. A fourth experiment tested the meanings drivers associated with an eight-sign subset of the 16 signs used in the first three experiments. A fifth experiment required all persons to select which (if any) signs they considered to be appropriate for use on two scale model county road intersections. The conclusions are that word-legend Stop Ahead signs are more effective driver communication devices than symbol stop-ahead signs; that it is helpful to drivers to have a word plate supplementing the symbol sign if a symbol sign is used; and that the guidance in the Manual on Uniform Traffic Control Devices on the placement of advance warning signs should not supplant engineering judgment in providing proper sign communication at an intersection.

CHO cell engineering to prevent polypeptide aggregation and improve therapeutic protein secretion.

The ability to efficiently produce recombinant proteins in a secreted form is highly desirable and cultured mammalian cells such as CHO cells have become the preferred host as they secrete proteins with human-like post-translational modifications. However, attempts to express high levels of particular proteins in CHO cells may consistently result in low yields, even for non-engineered proteins such as immunoglobulins. In this study, we identified the responsible faulty step at the stage of translational arrest, translocation and early processing for such a "difficult-to-express" immunoglobulin, resulting in improper cleavage of the light chain and its precipitation in an insoluble cellular fraction unable to contribute to immunoglobulin assembly. We further show that proper processing and secretion were restored by over-expressing human signal receptor protein SRP14 and other components of the secretion pathway. This allowed the expression of the difficult-to-express protein to high yields, and it also increased the production of an easy-to-express protein. Our results demonstrate that components of the secretory and processing pathways can be limiting, and that engineering of the secretory pathway may be used to improve the secretion efficiency of therapeutic proteins from CHO cells.

A Study of the Reliability of the ASTM C-666 Freeze-Thaw Test, MLR-72-01 and R-252, 1972

The Iowa State Highway Commission purchased a Conrad automatic freeze and thaw machine and placed it in operation during October 1961. There were a few problems, but considering, the many electrical and mechanical devices used in the automatic system it has always functioned quite well. Rapid freezing and thawing of 4"x4"xl8" concrete beams has been conducted primarily in accordance with ASTM C-29l (now ASTM C-666 procedure B) at the rate of one beam per day. Over 4000 beams have been tested since 1961, with determination of the resulting durability factors. Various methods of curing were used and a standard 90 day moist cure was selected. This cure seemed to yield durability factors that correlated very well with ratings of coarse aggregates based on service records. Some concrete beams had been made using the same coarse aggregate and the durability factors compared relatively well with previous tests. Durability factors seemed to yield reasonable results until large variations in durability factors were noted from beams of identical concrete mix proportions in research projects R-234 and R-247. This then presents the question "How reliable is the durability as determined by ASTM C-666?" This question became increasingly more important when a specification requiring a minimum durability factor for P.C. concrete made from coarse aggregates was incorporated into the 1972 Standard Specification for coarse aggregates for concrete.