Graduate Recital:Ann M. Polishinski, Violin

Kemp Recital Hall Thursday Evening April 26, 2001 6:00 p.m

Linear approaches to intramolecular Förster Resonance Energy Transfer probe measurements for quantitative modeling

Numerous unimolecular, genetically-encoded Forster Resonance Energy Transfer (FRET) probes for monitoring biochemical activities in live cells have been developed over the past decade. As these probes allow for collection of high frequency, spatially resolved data on signaling events in live cells and tissues, they are an attractive technology for obtaining data to develop quantitative, mathematical models of spatiotemporal signaling dynamics. However, to be useful for such purposes the observed FRET from such probes should be related to a biological quantity of interest through a defined mathematical relationship, which is straightforward when this relationship is linear, and can be difficult otherwise. First, we show that only in rare circumstances is the observed FRET linearly proportional to a biochemical activity. Therefore in most cases FRET measurements should only be compared either to explicitly modeled probes or to concentrations of products of the biochemical activity, but not to activities themselves. Importantly, we find that FRET measured by standard intensity-based, ratiometric methods is inherently non-linear with respect to the fraction of probes undergoing FRET. Alternatively, we find that quantifying FRET either via (1) fluorescence lifetime imaging (FLIM) or (2) ratiometric methods where the donor emission intensity is divided by the directly-excited acceptor emission intensity (denoted R<sub>alt</sub>) is linear with respect to the fraction of probes undergoing FRET. This linearity property allows one to calculate the fraction of active probes based on the FRET measurement. Thus, our results suggest that either FLIM or ratiometric methods based on R<sub>alt</sub> are the preferred techniques for obtaining quantitative data from FRET probe experiments for mathematical modeling purpose

Changing patterns in HIV-1 non-B clade prevalence and diversity in Italy over three decades

n/

Photoswitchable diacylglycerols enable optical control of protein kinase C.

Increased levels of the second messenger lipid diacylglycerol (DAG) induce downstream signaling events including the translocation of C1-domain-containing proteins toward the plasma membrane. Here, we introduce three light-sensitive DAGs, termed PhoDAGs, which feature a photoswitchable acyl chain. The PhoDAGs are inactive in the dark and promote the translocation of proteins that feature C1 domains toward the plasma membrane upon a flash of UV-A light. This effect is quickly reversed after the termination of photostimulation or by irradiation with blue light, permitting the generation of oscillation patterns. Both protein kinase C and Munc13 can thus be put under optical control. PhoDAGs control vesicle release in excitable cells, such as mouse pancreatic islets and hippocampal neurons, and modulate synaptic transmission in Caenorhabditis elegans. As such, the PhoDAGs afford an unprecedented degree of spatiotemporal control and are broadly applicable tools to study DAG signaling

The marine fisheries in Bulgaria's Exclusive Economic Zone, 1950-2013

The aim of this study is to reconstruct the total historic catch of Bulgarian marine fisheries in their Exclusive Economic Zone for the time period 1950-2013, including unreported landings, discards, recreational and subsistence catches from the ecosystem. The landings data officially reported by Bulgaria to the Food and Agriculture Organization of the United Nations for the Mediterranean and Black Seas (FAO Area 37) were revised in line with all available information. The reconstructed total catch for 1950-2013 was 1.7 times the (adjusted) baseline data reported by Bulgaria to FAO and 1.5 times the unadjusted data as reported by FAO. This study revealed major deficiencies which exist in the officially reported Bulgarian catch data, foremost being the large amount of unreported industrial catches, especially for the last two decades. Furthermore, the exclusion of some fisheries sectors, notably the absence of data on the subsistence and recreational fisheries in reported data are also noteworthy

β 2 -Adrenergic Receptor Signaling and Desensitization Elucidated by Quantitative Modeling of Real Time cAMP Dynamics

G protein-coupled receptor signaling is dynamically regulated by multiple feedback mechanisms, which rapidly attenuate signals elicited by ligand stimulation, causing desensitization. The individual contributions of these mechanisms, however, are poorly understood. Here, we use an improved fluorescent biosensor for cAMP to measure second messenger dynamics stimulated by endogenous beta(2)-adrenergic receptor (beta(2)AR) in living cells. beta(2)AR stimulation with isoproterenol results in a transient pulse of cAMP, reaching a maximal concentration of approximately 10 microm and persisting for less than 5 min. We investigated the contributions of cAMP-dependent kinase, G protein-coupled receptor kinases, and beta-arrestin to the regulation of beta(2)AR signal kinetics by using small molecule inhibitors, small interfering RNAs, and mouse embryonic fibroblasts. We found that the cAMP response is restricted in duration by two distinct mechanisms in HEK-293 cells: G protein-coupled receptor kinase (GRK6)-mediated receptor phosphorylation leading to beta-arrestin mediated receptor inactivation and cAMP-dependent kinase-mediated induction of cAMP metabolism by phosphodiesterases. A mathematical model of beta(2)AR signal kinetics, fit to these data, revealed that direct receptor inactivation by cAMP-dependent kinase is insignificant but that GRK6/beta-arrestin-mediated inactivation is rapid and profound, occurring with a half-time of 70 s. This quantitative system analysis represents an important advance toward quantifying mechanisms contributing to the physiological regulation of receptor signaling

Deliverable D4.4-5: Precision and behaviour of fish-based ecological quality metrics in relation to natural and anthropogenic pressure gradients in European estuaries and lagoons

This report summarises the work conducted in Work Package 4.4 – BQE fish in transitional (i.e. estuarine and lagoon) waters (TW) within the project WISER under the sponsorship of the European Commission. It omits most technical details of the analyses given in the four previous Work Package reports, but still provides the necessary information to understand the rationale, approach and underlying assumptions necessary to discuss the results. The focus is therefore to discuss and integrate the results obtained within Work Package 4.4 and with this, make recommendations to improve fish-based ecological assessments in TW, principally estuaries and lagoons. In addition, and to assist with the WFD implementation which is the overarching theme of WISER, the deliverable includes, where appropriate, case studies where we have used multi-metric fish indices currently under development, or already in use for WFD compliance monitoring across Europe. Furthermore, results of the work package have been shared with relevant Geographical Intercalibration Groups (GIGs) supporting the harmonization and equalization process across transitional fish indices in Europe. Development strategies for fish indices in TW vary but generally include: (1) the calibration of metrics to anthropogenic pressures, (2) the development of reference conditions, (3) the calculation of ecological quality ratios, and (4) the designation of thresholds for Ecological Status (ES) class. New fish indices are developed for a defined geographical area, using specific sampling method and under locally relevant pressure fields. The former two factors, area and sampling methods, define the relevant reference condition in the calculation of Ecological Quality Ratios (EQR) and the latter factor, human pressures, define the índex structure and especially the fish metric selection. To assess index relevance across areas, we calculated a suite of transitional fish indices on a standardized WISER dataset and then compared the agreement of the outcomes (using correlation analysis). The application of current indices to areas (or countries) different from the area in which it was originally developed leads to inconclusive or spurious results. The failure to accommodate the diferente indices to a standardized dataset in this work clearly demonstrates the fundamental reliance of current fish indices on the sampling methods and design of monitoring programmes used in the development of the index. Despite this, for some indices, correlations although weaker are statistically significant, also indicating the possible agreement in successful intercalibration between these indices. Harmonization of BQE fish methodologies across Europe (common metrics) is unlikely by adapting or creating new fish indices but inter-comparison assessments are possible and valid using a common pressure index to harmonise diferente indices on a common scale. We found a negative response of fish quality features to pressure gradients which make BQE fish in TW suitable for greater ecological integration than other BQEs. However, successful assessment of Ecological Status (ES) require a matching combination of fish index, reference values and local dataset gathered with compatible sampling methods. Whole indices provide more consistent overall ES assessments but fish metrics considered individually may be more useful as a means to focus restoration measures. Future work is needed to identify those specific pressures affecting fish assemblages providing targets for minimising the effects of stress in mitigation and restoration plans. In order to achieve this, and although the interpretation of outcomes is still difficult, more recent transitional fish indices are leading in the use of comprehensive appraisal and validation exercises to test the responsiveness of BQEs for the assessment of ES. Here we proposed for the first time a simple sensitivity exercise under realistic scenarios of metric change to explore the expected inertia (i.e. the tendency to buffer ES change after quality alterations), dynamic range (i.e. the ratio between the largest and smallest possible ES values) and most relevant metric components (i.e. the those driving the most likely scenarios leading to ES change) from a multi-metric fish índex under relevant human pressure gradients. Overall, the behaviour of multi-metric índices under manipulations of metric scores clearly indicated that metric type, number of metrics used and correlations between metrics are important in determining the index performance, with indices including more and/or uncorrelated metrics or metrics with skewed distribution being less affected by extreme metric manipulations. Results of this analysis may be used to set realistic management targets and also to identify the aspects of the indices that are more likely to affect the outcomes leading to more robust and responsive indices. Further improvements of fish indices may be attained by reducing the variability confounding biological quality metrics. This variability is undesirable noise in assessments and can be technical (i.e. linked to the method of assessment including sampling effort) or natural (physicochemical and biological). The implication for assessments is that different facts might then confound the metric-pressure correlation (the ‘signal’ in the signal-to-noise ratio in the assessments) increasing uncertainty in ES assignment. Models showed that salinity class, depth, season, time of fishing (day vs. night) and year of fishing may influence the values of the fish metrics. The modelling exercise also demonstrated that unexplained variance remains generally much higher within-systems than between-systems suggesting a higher importance of sources of variability acting at the WB level. Modelling and improved standardization in monitoring campaigns should reduce uncertainty in ES assignment. One important factor that was assessed further was the effect of sampling effort. The results suggest that richness-based metrics require larger sampling efforts although a similar effortrelated bias may be an issue for density-based metrics if fish distribution is very patchy (i.e. schooling fish or those aggregated in specific habitats) and insufficient replicates are taken to fully characterise the patchiness in their distribution. It is apparent that to overcome a potential large source of error, the Reference Conditions must be defined according to the level of effort used in the monitoring programme or, conversely, the monitoring must be carried out at the same level of effort used to derive the Reference Condition. The WP finally explored the use of a predictive linear modelling approach to define reference conditions for fish metrics in transitional waters. The fish response data was modelled together with Corine Land Cover (CLC)-derived pressure proxies (% agricultural, urban and natural land coverage). Based on the obtained models, the expected metric score was predicted by setting pressure levels either to the lowest observed pressure in the dataset or to zero in order to define the sample and theoretical reference condition, respectively. Even when significant, the effect of pressures on fish metrics was generally very weak, probably reflecting the use of too-generic pressure indicators (such as land cover data instead of more relevant estuarine proxies such as dredging, port development, waterborne pollutants, etc). The best explanatory models included sampling factors and natural characteristics considered important discriminant features in the definition of water body types. In particular, the present work argues for considering not only estuaries and lagoons as different typologies but also other natural and design characteristic such as the gear type, the sampling season and the salinity class. Furthermore, a relevant reference needs to account for survey design bias, including rare species contribution to assessment datasets, patchiness, choice of pressure proxies or sampling gear. The modelling approach of fish metrics against the physicochemical variables has proved useful to derive Reference Conditions. This is important for the computation of relevant EQRs in Europe where there is a general lack of pristine areas or historical data on fish BQE and it provides an alternative to best professional judgment. Taking all WP analysis and case studies together, the work conducted has highlighted the following key messages and linked research needs necessary to optimize BQE fish for the quality assessment of transitional waters: Key Message 01: Harmonization of BQE fish methodologies across Europe (common metrics) is unlikely by adapting or creating new fish indices but inter-comparison assessments are possible and valid using a common pressure index to harmonise diferente indices on a common scale. Research needs to be focused on more widely-applicable fish indices will require the formulation of completely new indices based on a more flexible use of fish metrics according to system typologies, relevance and, probably, an increased use of functional traits. For current indices, further research on a method of intercalibration is needed. Key Message 02: BQE Fish in TW respond consistently to human pressure gradients across transitional waters providing the means to assess Ecological Status (ES). Further work will be needed to identify those specific pressures affecting fish assemblages providing targets for minimising the effects of stress in mitigation and restoration plans. Key Message 03 Although the interpretation of outcomes is still difficult, more recente transitional fish indices are leading in the use of comprehensive appraisal and validation exercises to test the performance of BQEs in the assessment of Ecological Status (ES). Further appraisal of fish indices behaviour is needed to understand the meaning of the quality outcomes, to set realistic management targets and also to identify the aspects of the índices that are more likely to affect the outcomes leading to more robust and responsive indices Key Message 04 Uncertainty levels associated with metric variability in multi-metric fish indices can be managed to increase the confidence in Ecological Status (ES) class assignment. Further research is needed to include knowledge of habitat partition within systems, to understand metrics behaviour and precision, to test new combination rules allowing metric weighting by robustness and importantly to evaluate more robust sampling tools and methods. Key Message 05 Reference conditions for BQE fish-based quality assessments can be objectively estimated using predictive modelling. Further refinements will require the use of better pressure proxies, robust metrics amenable to modelling and to account for survey design bias (effort & choice of sampling gear) at the relevant scales used in monitoring programmes.info:eu-repo/semantics/publishedVersio

Trends and correlates of HIV-1 resistance among subjects failing an antiretroviral treatment over the 2003-2012 decade in Italy

BACKGROUND: Despite a substantial reduction in virological failures following introduction of new potent antiretroviral therapies in the latest years, drug resistance remains a limitation for the control of HIV-1 infection. We evaluated trends and correlates of resistance in treatment failing patients in a comprehensive database over a time period of relevant changes in prescription attitudes and treatment guidelines. METHODS: We analyzed 6,796 HIV-1 pol sequences from 49 centres stored in the Italian ARCA database during the 2003-2012 period. Patients (n = 5,246) with viremia > 200 copies/mL received a genotypic test while on treatment. Mutations were identified from IAS-USA 2013 tables. Class resistance was evaluated according to antiretroviral regimens in use at failure. Time trends and correlates of resistance were analyzed by Cochran-Armitage test and logistic regression models. RESULTS: The use of NRTI backbone regimens slightly decreased from 99.7% in 2003-2004 to 97.4% in 2010-2012. NNRTI-based combinations dropped from 46.7% to 24.1%. PI-containing regimens rose from 56.6% to 81.7%, with an increase of boosted PI from 36.5% to 68.9% overtime. In the same reference periods, Resistance to NRTIs, NNRTIs and PIs declined from 79.1% to 40.8%, from 77.8% to 53.8% and from 59.8% to 18.9%, respectively (p < .0001 for all comparisons). Dual NRTI +\u2009NNRTI and NRTI + PI resistance decreased from 56.4% to 33.3% and from 36.1% to 10.5%, respectively. Reduced risk of resistance over time periods was confirmed by a multivariate analysis. CONCLUSIONS: Mutations associated with NRTIs, NNRTIs and PIs at treatment failure declined overtime regardless of specific class combinations and epidemiological characteristics of treated population. This is likely due to the improvement of HIV treatment, including both last generation drug combinations and prescription guidelines

Functional divergence in the role of N-linked glycosylation in smoothened signaling

The G protein-coupled receptor (GPCR) Smoothened (Smo) is the requisite signal transducer of the evolutionarily conserved Hedgehog (Hh) pathway. Although aspects of Smo signaling are conserved from Drosophila to vertebrates, significant differences have evolved. These include changes in its active sub-cellular localization, and the ability of vertebrate Smo to induce distinct G protein-dependent and independent signals in response to ligand. Whereas the canonical Smo signal to Gli transcriptional effectors occurs in a G protein-independent manner, its non-canonical signal employs Gαi. Whether vertebrate Smo can selectively bias its signal between these routes is not yet known. N-linked glycosylation is a post-translational modification that can influence GPCR trafficking, ligand responsiveness and signal output. Smo proteins in Drosophila and vertebrate systems harbor N-linked glycans, but their role in Smo signaling has not been established. Herein, we present a comprehensive analysis of Drosophila and murine Smo glycosylation that supports a functional divergence in the contribution of N-linked glycans to signaling. Of the seven predicted glycan acceptor sites in Drosophila Smo, one is essential. Loss of N-glycosylation at this site disrupted Smo trafficking and attenuated its signaling capability. In stark contrast, we found that all four predicted N-glycosylation sites on murine Smo were dispensable for proper trafficking, agonist binding and canonical signal induction. However, the under-glycosylated protein was compromised in its ability to induce a non-canonical signal through Gαi, providing for the first time evidence that Smo can bias its signal and that a post-translational modification can impact this process. As such, we postulate a profound shift in N-glycan function from affecting Smo ER exit in flies to influencing its signal output in mice