Differential Effects of AGS3 Expression on D2L Dopamine Receptor-Mediated Adenylyl Cyclase Signaling

Activator of G protein signaling 3 (AGS3) binds Gα_i subunits in the GDP-bound state, implicating AGS3 as an important regulator of Gα_i-linked receptor (e.g., D2 dopamine and μ-opioid) signaling. We examined the ability of AGS3 to modulate recombinant adenylyl cyclase (AC) type 1 and 2 signaling in HEK293 cells following both acute and persistent activation of the D_2L dopamine receptor (D_2LDR). AGS3 expression modestly enhanced the potency of acute quinpirole-induced D_2LDR modulation of AC1 or AC2 activity. AGS3 also promoted desensitization of D_2LDR-mediated inhibition of AC1, whereas desensitization of D_2LDR-mediated AC2 activation was significantly attenuated. Additionally, AGS3 reduced D_2LDR-mediated sensitization of AC1 and AC2. These data suggest that AGS3 is involved in altering G protein signaling in a complex fashion that is effector-specific and dependent on the duration of receptor activation.     

High CO2 concentration and iron availability determine the metabolic inventory in an Emiliania huxleyi-dominated phytoplankton community

Ocean acidification (OA), a consequence of anthropogenic carbon dioxide (CO₂) emissions, strongly impacts marine ecosystems. OA also influences iron (Fe) solubility, affecting biogeochemical and ecological processes. We investigated the interactive effects of CO₂ and Fe availability on the metabolome response of a natural phytoplankton community. Using mesocosms we exposed phytoplankton to ambient (390 μatm) or future CO₂ levels predicted for the year 2100 (900 μatm), combined with ambient (4.5 nM) or high (12 nM) dissolved iron (dFe). By integrating over the whole phytoplankton community, we assigned functional changes based on altered metabolite concentrations. Our study revealed the complexity of phytoplankton metabolism. Metabolic profiles showed three stages in response to treatments and phytoplankton dynamics. Metabolome changes were related to the plankton group contributing respective metabolites, explaining bloom decline and community succession. CO₂ and Fe affected metabolic profiles. Most saccharides, fatty acids, amino acids and many sterols significantly correlated with the high dFe treatment at ambient pCO₂. High CO₂ lowered the abundance of many metabolites irrespective of Fe. However, sugar alcohols accumulated, indicating potential stress. We demonstrate that not only altered species composition but also changes in the metabolic landscape affecting the plankton community may change as a consequence of future high-CO₂ oceans.     

Neuronal control of lipid metabolism by STR‐2 G protein‐coupled receptor promotes longevity in Caenorhabditis elegans

The G protein‐coupled receptor (GPCR) encoding family of genes constitutes more than 6% of genes in Caenorhabditis elegans genome. GPCRs control behavior, innate immunity, chemotaxis, and food search behavior. Here, we show that C. elegans longevity is regulated by a chemosensory GPCR STR‐2, expressed in AWC and ASI amphid sensory neurons. STR‐2 function is required at temperatures of 20°C and higher on standard Escherichia coli OP50 diet. Under these conditions, this neuronal receptor also controls health span parameters and lipid droplet (LD) homeostasis in the intestine. We show that STR‐2 regulates expression of delta‐9 desaturases, fat‐5, fat‐6 and fat‐7, and of diacylglycerol acyltransferase dgat‐2. Rescue of the STR‐2 function in either AWC and ASI, or ASI sensory neurons alone, restores expression of fat‐5, dgat‐2 and restores LD stores and longevity. Rescue of stored fat levels of GPCR mutant animals to wild‐type levels, with low concentration of glucose, rescues its lifespan phenotype. In all, we show that neuronal STR‐2 GPCR facilitates control of neutral lipid levels and longevity in C. elegans.     

Increased high‐latitude photosynthetic carbon gain offset by respiration carbon loss during an anomalous warm winter to spring transition

Arctic and boreal ecosystems play an important role in the global carbon (C) budget, and whether they act as a future net C sink or source depends on climate and environmental change. Here, we used complementary in situ measurements, model simulations, and satellite observations to investigate the net carbon dioxide (CO₂) seasonal cycle and its climatic and environmental controls across Alaska and northwestern Canada during the anomalously warm winter to spring conditions of 2015 and 2016 (relative to 2010–2014). In the warm spring, we found that photosynthesis was enhanced more than respiration, leading to greater CO₂ uptake. However, photosynthetic enhancement from spring warming was partially offset by greater ecosystem respiration during the preceding anomalously warm winter, resulting in nearly neutral effects on the annual net CO₂ balance. Eddy covariance CO₂ flux measurements showed that air temperature has a primary influence on net CO₂ exchange in winter and spring, while soil moisture has a primary control on net CO₂ exchange in the fall. The net CO₂ exchange was generally more moisture limited in the boreal region than in the Arctic tundra. Our analysis indicates complex seasonal interactions of underlying C cycle processes in response to changing climate and hydrology that may not manifest in changes in net annual CO₂ exchange. Therefore, a better understanding of the seasonal response of C cycle processes may provide important insights for predicting future carbon–climate feedbacks and their consequences on atmospheric CO₂ dynamics in the northern high latitudes.     

Sustainable intensification of crop residue exploitation for bioenergy: Opportunities and challenges

Crop residue exploitation for bioenergy can play an important role in climate change mitigation without jeopardizing food security, but it may be constrained by impacts on soil organic carbon (SOC) stocks, and market, logistic and conversion challenges. We explore opportunities to increase bioenergy potentials from residues while reducing environmental impacts, in line with sustainable intensification. Using the case study of North Rhine‐Westphalia in Germany, we employ a spatiotemporally explicit approach combined with stakeholder interviews. First, the interviews identify agronomic and environmental impacts due to the potential reduction in SOC as the most critical challenge associated with enhanced crop residue exploitation. Market and technological challenges and competition with other residue uses are also identified as significant barriers. Second, with the use of agroecosystem modelling and estimations of bioenergy potentials and greenhouse gas emissions till mid‐century, we evaluate the ability of agricultural management to tackle the identified agronomic and environmental challenges. Integrated site‐specific management based on (a) humus balancing, (b) optimized fertilization and (c) winter soil cover performs better than our reference scenario with respect to all investigated variables. At the regional level, we estimate (a) a 5% increase in technical residue potentials and displaced emissions from substituting fossil fuels by bioethanol, (b) an 8% decrease in SOC losses and associated emissions, (c) an 18% decrease in nitrous oxide emissions, (d) a 37% decrease in mineral fertilizer requirements and emissions from their production and (e) a 16% decrease in nitrate leaching. Results are spatially variable and, despite improvements induced by management, limited amounts of crop residues are exploitable for bioenergy in areas prone to SOC decline. In order to sustainably intensify crop residue exploitation for bioenergy and reconcile climate change mitigation with other sustainability objectives, such as those on soil and water quality, residue management needs to be designed in an integrated and site‐specific manner.     

Recognition of RNA by the S9.6 antibody creates pervasive artifacts when imaging RNA:DNA hybrids

The S9.6 antibody is broadly used to detect RNA:DNA hybrids but has significant affinity for double-stranded RNA. The impact of this off-target RNA binding activity has not been thoroughly investigated, especially in the context of immunofluorescence microscopy. We report that S9.6 immunofluorescence signal observed in fixed human cells arises predominantly from ribosomal RNA, not RNA:DNA hybrids. S9.6 staining was unchanged by pretreatment with the RNA:DNA hybrid–specific nuclease RNase H1, despite verification in situ that S9.6 recognized RNA:DNA hybrids and that RNase H1 was active. S9.6 staining was, however, significantly sensitive to RNase T1, which specifically degrades RNA. Additional imaging and biochemical data indicate that the prominent cytoplasmic and nucleolar S9.6 signal primarily derives from ribosomal RNA. Importantly, genome-wide maps obtained by DNA sequencing after S9.6-mediated DNA:RNA immunoprecipitation (DRIP) are RNase H1 sensitive and RNase T1 insensitive. Altogether, these data demonstrate that imaging using S9.6 is subject to pervasive artifacts without pretreatments and controls that mitigate its promiscuous recognition of cellular RNAs.     

Performance evaluation of Baermann techniques: The quest for developing a microscopy reference standard for the diagnosis of Strongyloides stercoralis

BackgroundSoil-transmitted helminths (STH) are common in low and middle income countries where there is lack of access to clean water and sanitation. Effective diagnosis and treatment are essential for the control of STH infections. However, among STH parasites, Strongyloides stercoralis is the most neglected species, both in diagnostics and control strategies. Diagnostic methods cover different approaches, each with different sensitivities and specificities, such as serology, molecular techniques and microscopy based techniques. Of the later, the Baermann technique is the most commonly used procedure. In the literature, several ways have been described to perform the Baermann method, which illustrates the overall lack of a ‘(gold) reference standard’ method for the diagnosis of S. stercoralis infection. In this study we have evaluated the performance of three Baermann techniques in order to improve the reference standard for the microscopic diagnosis of S. stercoralis infection thereby facilitating individual case detection, mapping of the disease and proper evaluation of treatment responses.Methods/Principal findingsA community based cross sectional study was conducted at Zenzelima, Bahir Dar Zuria Ethiopia. A total of 437 stool samples were collected and analyzed by the following procedures: conventional Baermann (CB), modified Baermann (MB), and modified Baermann with charcoal pre-incubation (MBCI). The diagnostic sensitivity and Negative Predictive Value (NPV) of each technique was calculated using the combination of all the three techniques as a composite reference standard. Our result indicated that larvae of S. stercoralis were detected in 151 (34.6%) stool samples. The prevalence of S. stercoralis infection based on the three diagnostic methods was 9.6%, 8.0%, and 31.3% by CB, MB, and MBCI respectively. The sensitivity and NPV for CB, MB, and MBCI were 26.7% and 70.8%, 22.1% and 69.6%, and 87.0% and 93.2%, respectively. The MBCI showed significant difference (P- value = <0.001) in the sensitivity and NPV values when compared with CB and MB values. The agreement between CB, MB, and MBCI with the composite reference standard was 31.8%, 26.7%, 89.6%, respectively.Conclusion/SignificanceOur results suggest the superior performance of MBCI. It is relatively easy to implement, simple to perform and comparatively cheaper. The CB is by far the commonly used method in routine diagnostic although this technique significantly underestimates the true burden of the disease and thereby contributing to the exclusion of S. stercoralis from the control strategies. Therefore, MBCI is recommended as a routine microscopy-based diagnostic test for S. stercoralis infection, particularly in settings where molecular procedures are not available.     

A Method for Screening Baeyer-Villiger Monooxygenase Activity Against Monocyclic Ketones

The assay for Baeyer-Villiger monooxygenase (BVMO) enzyme activity has relied to date on the spectrophotometric change observed on the oxidation of the nicotinamide cofactor during the enzymatic reaction. By analogy to the cyclohexanol catabolic pathway of Acinetobacter calcoaceticus NCIMB 9871, we have developed a specific colorimetric screening method that utilises an esterase to cleave the lactone that is formed in the BVMO reaction. When carried out in a non-buffered or weakly buffered system the resultant change in pH can be visually detected. This allows the rapid assaying and screening of BVMO enzymes. This has been demonstrated with cyclohexanone monooxygenase from A. calcoaceticus. The resultant colour change has been visualised with washed cell suspensions, individual bacterial colonies on Petri dishes and with semi-purified recombinant enzyme utilising Linbro dishes.