Genetically encoded intrabody sensors report the interaction and trafficking of β-arrestin 1 upon activation of G protein-coupled receptors

Agonist stimulation of G protein-coupled receptors (GPCRs) typically leads to phosphorylation of GPCRs and binding to multifunctional proteins called β-arrestins (βarrs). The GPCR-βarr interaction critically contributes to GPCR desensitization, endocytosis, and downstream signaling, and GPCR-βarr complex formation can be used as a generic readout of GPCR and βarr activation. Although several methods are currently available to monitor GPCR-βarr interactions, additional sensors to visualize them may expand the toolbox and complement existing methods. We have previously described antibody fragments (FABs) that recognize activated βarr1 upon its interaction with the vasopressin V2 receptor C-terminal phosphopeptide (V2Rpp). Here, we demonstrate that these FABs efficiently report the formation of a GPCR-βarr1 complex for a broad set of chimeric GPCRs harboring the V2R C terminus. We adapted these FABs to an intrabody format by converting them to single-chain variable fragments (ScFvs) and used them to monitor the localization and trafficking of βarr1 in live cells. We observed that upon agonist simulation of cells expressing chimeric GPCRs, these intrabodies first translocate to the cell surface, followed by trafficking into intracellular vesicles. The translocation pattern of intrabodies mirrored that of βarr1, and the intrabodies co-localized with βarr1 at the cell surface and in intracellular vesicles. Interestingly, we discovered that intrabody sensors can also report βarr1 recruitment and trafficking for several unmodified GPCRs. Our characterization of intrabody sensors for βarr1 recruitment and trafficking expands currently available approaches to visualize GPCR-βarr1 binding, which may help decipher additional aspects of GPCR signaling and regulation

Variations in the SDN Loop of Class A Beta-Lactamases: A Study of the Molecular Mechanism of BlaC (Mycobacterium tuberculosis) to Alter the Stability and Catalytic Activity Towards Antibiotic Resistance of MBIs

The emergence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis calls for an immediate search for novel treatment strategies. Recently, BlaC, the principal beta-lactamase of Mycobacterium tuberculosis, was recognized as a potential therapeutic target. BlaC belongs to Ambler class A, which is generally susceptible to the beta-lactamase inhibitors currently used in clinics: tazobactam, sulbactam, and clavulanate. Alterations at Ser130 in conserved SDN loop confer resistance to mechanism-based inhibitors (MBIs) commonly observed in various clinical isolates. The absence of clinical evidence of S130G conversion in M. tuberculosis draws our attention to build laboratory mutants of S130G and S130A of BlaC. The study involving steady state, inhibition kinetics, and fluorescence microscopy shows the emergence of resistance against MBIs to the mutants expressing S130G and S130A. To understand the molecular reasoning behind the unavailability of such mutation in real life, we have used circular dichroism (CD) spectroscopy, differential scanning calorimetry (DSC), molecular dynamics (MD) simulation, and stability-based enzyme activity to compare the stability and dynamic behaviors of native and S130G/A mutant form of BlaC. A significant decrease in melting temperature (BlaC T M 60°C, S130A T M 50°C, and S130G T M 45°C), kinetic instability at higher temperature, and comparative dynamic instability correlate the fact that resistance to beta-lactam/beta-lactamase inhibitor combinations will likely not arise from the structural alteration of BlaC, therefore establishing confidence that this therapeutic modality can be potentially applied as a part of a successful treatment regimen against M. tuberculosis

Theoretical studies of 31P NMR spectral properties of phosphanes and related compounds in solution

Selected theoretical methods, basis sets and solvation models have been tested in their ability to predict 31P NMR chemical shifts of large phosphorous-containing molecular systems in solution. The most efficient strategy was found to involve NMR shift calculations at the GIAO-MPW1K/6-311++G(2d,2p)//MPW1K/6-31G(d) level in combination with a dual solvation model including the explicit consideration of single solvent molecules and a continuum (PCM) solvation model. For larger systems it has also been established that reliable 31P shift predictions require Boltzmann averaging over all accessible conformations in solution

Impacts of surface heterogeneity on dry planetary boundary layers in an urban-rural setting

Understanding the impacts of land use and land-cover change such as urbanization is essential in many disciplines. This study investigates the impacts of urban-rural contrasts in terms of momentum roughness length (z0) and aerodynamic surface temperature (TSK) on dry planetary boundary layers (PBLs) using large-eddy simulations (LES) with the Weather Research and Forecasting (WRF) model. In addition, the impacts of small-scale heterogeneities within urban areas are also examined. The original WRF-LES is modified in order to use prescribed TSK as surface boundary conditions. Numerical simulations are then conducted to examine turbulence characteristics and mesoscale circulations resulting from large-scale urban-rural contrasts as well as small-scale heterogeneities in urban areas. The results indicate that (1) the urban-rural contrasts in z0 and TSK have significant but different impacts on surface heat fluxes, mesoscale circulations, and the wind and potential temperature profiles. Compared to the case where the whole domain is homogeneous, increases in z0 and/or TSK in urban areas in the center of domain induce stronger sensible heat fluxes, stronger urban circulations, and weaker inversions at the top of the PBL. (2) When the patch size that characterizes the urban heterogeneity scale is comparable to the size of the whole urban area, the simulated results are strongly dependent on both the heterogeneity scale and the specified surface temperature values. As the patch size decreases, the simulated results become more similar to those over a homogeneous urban surface

Regional carbon fluxes and the effect of topography on the variability of atmospheric CO2.

Using a mesoscale atmospheric circulation model, it is shown that relatively modest topography height differences of ∼500 m over 200 km near Zotino (60°N, 89°E) in central Siberia may generate horizontal gradients in CO<inf>2</inf> concentration in the order of 30 ppm. In a case study for 15 and 16 July 1996, when Lloyd et al. (2001) conducted a convective boundary layer budget experiment in the area, we show that advection of these gradients disturbs the relation between diurnal concentration changes in the boundary layer and the surface fluxes. This demonstrates that mesoscale atmospheric heterogeneity may have severe impact on the applicability of methods to derive the regional-scale fluxes from CO<inf>2</inf> concentrations measurements, such as the convective boundary layer budget method or inverse modeling. It is shown that similar mesoscale gradients are likely to occur at many long-term observation stations and tall towers. We use the modeled concentration fields to quantify the horizontal and vertical variability of carbon dioxide in the atmosphere. In future observation campaigns, mesoscale processes may be best accounted for by measuring horizontal variability over a few hundred kilometers and by attempting to quantify the representation errors as a function of mesoscale conditions. Copyright 2007 by the American Geophysical Union

Exploring the influence of precipitation extremes and human water use on total water storage (TWS) changes in the Ganges-Brahmaputra-Meghna River Basin

Climate extremes such as droughts and intense rainfall events are expected to strongly influence global/regional water resources in addition to the growing demands for freshwater. This study examines the impacts of precipitation extremes and human water usage on total water storage (TWS) over the Ganges-Brahmaputra-Meghna (GBM) River Basin in South Asia. Monthly TWS changes derived from the Gravity Recovery And Climate Experiment (GRACE) (2002–2014) and soil moisture from three reanalyses (1979–2014) are used to estimate new extreme indices. These indices are applied in conjunction with standardized precipitation indices (SPI) to explore the impacts of precipitation extremes on TWS in the region. The results indicate that although long-term precipitation do not indicate any significant trends over the two subbasins (Ganges and Brahmaputra-Meghna), there is significant decline in rainfall (9.0 ± 4.0 mm/decade) over the Brahmaputra-Meghna River Basin from 1998 to 2014. Both river basins exhibit a rapid decline of TWS from 2002 to 2014 (Ganges: 12.2 ± 3.4 km3/yr and Brahmaputra-Meghna: 9.1 ± 2.7 km3/yr). While the Ganges River Basin has been regaining TWS (5.4 ± 2.2 km3/yr) from 2010 onward, the Brahmaputra-Meghna River Basin exhibits a further decline (13.0 ± 3.2 km3/yr) in TWS from 2011 onward. The impact of human water consumption on TWS appears to be considerably higher in Ganges compared to Brahmaputra-Meghna, where it is mainly concentrated over Bangladesh. The interannual water storage dynamics are found to be strongly associated with meteorological forcing data such as precipitation. In particular, extreme drought conditions, such as those of 2006 and 2009, had profound negative impacts on the TWS, where groundwater resources are already being unsustainably exploited

Simulations of the 2004 North American Monsoon: NAMAP2

The second phase of the North American Monsoon Experiment (NAME) Model Assessment Project (NAMAP2) was carried out to provide a coordinated set of simulations from global and regional models of the 2004 warm season across the North American monsoon domain. This project follows an earlier assessment, called NAMAP, that preceded the 2004 field season of the North American Monsoon Experiment. Six global and four regional models are all forced with prescribed, time-varying ocean surface temperatures. Metrics for model simulation of warm season precipitation processes developed in NAMAP are examined that pertain to the seasonal progression and diurnal cycle of precipitation, monsoon onset, surface turbulent fluxes, and simulation of the low-level jet circulation over the Gulf of California. Assessment of the metrics is shown to be limited by continuing uncertainties in spatially averaged observations, demonstrating that modeling and observational analysis capabilities need to be developed concurrently. Simulations of the core subregion (CORE) of monsoonal precipitation in global models have improved since NAMAP, despite the lack of a proper low-level jet circulation in these simulations. Some regional models run at higher resolution still exhibit the tendency observed in NAMAP to overestimate precipitation in the CORE subregion; this is shown to involve both convective and resolved components of the total precipitation. The variability of precipitation in the Arizona/New Mexico (AZNM) subregion is simulated much better by the regional models compared with the global models, illustrating the importance of transient circulation anomalies (prescribed as lateral boundary conditions) for simulating precipitation in the northern part of the monsoon domain. This suggests that seasonal predictability derivable from lower boundary conditions may be limited in the AZNM subregion.open131

The climatic impacts of land surface change and carbon management, and the implications for climate-change mitigation policy

http://www.sciencedirect.com/science/journal/14693062Strategies to mitigate anthropogenic climate change recognize that carbon sequestration in the terrestrial biosphere can reduce the build-up of carbon dioxide in the Earth’s atmosphere. However, climate mitigation policies do not generally incorporate the effects of these changes in the land surface on the surface albedo, the fluxes of sensible and latent heat to the atmosphere, and the distribution of energy within the climate system. Changes in these components of the surface energy budget can affect the local, regional, and global climate. Given the goal of mitigating climate change, it is important to consider all of the effects of changes in terrestrial vegetation and to work toward a better understanding of the full climate system. Acknowledging the importance of land surface change as a component of climate change makes it more challenging to create a system of credits and debits wherein emission or sequestration of carbon in the biosphere is equated with emission of carbon from fossil fuels. Recognition of the complexity of human-caused changes in climate does not, however, weaken the importance of actions that would seek to minimize our disturbance of the Earth’s environmental system and that would reduce societal and ecological vulnerability to environmental change and variability