Trait‐based diatom functional diversity as an appropriate tool for understanding the effects of environmental changes in soda pans

Saline lakes, among the most seriously endangered ecosystems, are threatened due to climate change and human activities. One valuable feature of these environments is that they constitute areas of high biodiversity. Ecologists are, therefore, under great pressure to improve their understanding of the effects of natural and anthropogenic disturbances on the biodiversity of saline lakes. In this study, a total of 257 samples from 32 soda pans in Central Europe between 2006 and 2015 were examined. The effects of environmental variables and of geographical and limnoecological factors on functional diversity were analyzed. Furthermore, the explanatory power of the trait‐based approach was assessed, and the applicability of the indices for biomonitoring purposes was determined. It was found that low habitat heterogeneity and harsh environments lead to the selection of a small number of suitable traits, and consequently, to a naturally low level of functional diversity. Anthropogenic activities enhance diversity at functional level due to the shift toward freshwater characteristics. On the regional scale, the effects of the region and status (natural, degraded, reconstructed) on diatom functional diversity were significant and more pronounced than that of the environmental and other limnoecological factors. The degree of variance found in functional diversity ascribed to environmental variables is five times greater in the case of the application of a trait‐based approach, than when a taxonomic one is employed in the literature. Each of the tested functional diversity indices was sensitive to the most important environmental variables. Furthermore, these were type‐specific and proved to be more complex indicators than taxonomic metrics. It is possible to suggest four functional diversity indices (FGR, FRic, FDis, and FDiv) which emphasize their independence from substrate and seasonal variations for ecological status assessment and conservation planning.     

Different Patterns of Akt and ERK Feedback Activation in Response to Rapamycin,Active-Site mTOR Inhibitors and Metformin in Pancreatic Cancer Cells

The mTOR pathway is aberrantly stimulated in many cancer cells, including pancreatic ductal adenocarcinoma (PDAC), and thus it is a potential target for therapy. However, the mTORC1/S6K axis also mediates negative feedback loops that attenuate signaling via insulin/IGF receptor and other tyrosine kinase receptors. Suppression of these feed-back loops unleashes over-activation of upstream pathways that potentially counterbalance the antiproliferative effects of mTOR inhibitors. Here, we demonstrate that treatment of PANC-1 or MiaPaCa-2 pancreatic cancer cells with either rapamycin or active-site mTOR inhibitors suppressed S6K and S6 phosphorylation induced by insulin and the GPCR agonist neurotensin. Rapamycin caused a striking increase in Akt phosphorylation at Ser⁴⁷³ while the active-site inhibitors of mTOR (KU63794 and PP242) completely abrogated Akt phosphorylation at this site. Conversely, active-site inhibitors of mTOR cause a marked increase in ERK activation whereas rapamycin did not have any stimulatory effect on ERK activation. The results imply that first and second generation of mTOR inhibitors promote over-activation of different pro-oncogenic pathways in PDAC cells, suggesting that suppression of feed-back loops should be a major consideration in the use of these inhibitors for PDAC therapy. In contrast, metformin abolished mTORC1 activation without over-stimulating Akt phosphorylation on Ser⁴⁷³ and prevented mitogen-stimulated ERK activation in PDAC cells. Metformin induced a more pronounced inhibition of proliferation than either KU63794 or rapamycin while, the active-site mTOR inhibitor was more effective than rapamycin. Thus, the effects of metformin on Akt and ERK activation are strikingly different from allosteric or active-site mTOR inhibitors in PDAC cells, though all these agents potently inhibited the mTORC1/S6K axis.     

Poxvirus Antigen Staining of Immune Cells as a Biomarker to Predict Disease Outcome in Monkeypox and Cowpox Virus Infection in Non-Human Primates

Infection of non-human primates (NHPs) such as rhesus and cynomolgus macaques with monkeypox virus (MPXV) or cowpox virus (CPXV) serve as models to study poxvirus pathogenesis and to evaluate vaccines and anti-orthopox therapeutics. Intravenous inoculation of macaques with high dose of MPXV (>1–2×10⁷ PFU) or CPXV (>10² PFU) results in 80% to 100% mortality and 66 to 100% mortality respectively. Here we report that NHPs with positive detection of poxvirus antigens in immune cells by flow cytometric staining, especially in monocytes and granulocytes succumbed to virus infection and that early positive pox staining is a strong predictor for lethality. Samples from four independent studies were analyzed. Eighteen NHPs from three different experiments were inoculated with two different MPXV strains at lethal doses. Ten NHPs displayed positive pox-staining and all 10 NHPs reached moribund endpoint. In contrast, none of the three NHPs that survived anticipated lethal virus dose showed apparent virus staining in the monocytes and granulocytes. In addition, three NHPs that were challenged with a lethal dose of MPXV and received cidofovir treatment were pox-antigen negative and all three NHPs survived. Furthermore, data from a CPXV study also demonstrated that 6/9 NHPs were pox-antigen staining positive and all 6 NHPs reached euthanasia endpoint, while the three survivors were pox-antigen staining negative. Thus, we conclude that monitoring pox-antigen staining in immune cells can be used as a biomarker to predict the prognosis of virus infection. Future studies should focus on the mechanisms and implications of the pox-infection of immune cells and the correlation between pox-antigen detection in immune cells and disease progression in human poxviral infection.     

Nonhuman Transferrin Receptor 1 Is an Efficient Cell Entry Receptor for Ocozocoautla de Espinosa Virus

Ocozocoautla de Espinosa virus (OCEV) is a novel, uncultured arenavirus. We found that the OCEV glycoprotein mediates entry into grivet and bat cells through transferrin receptor 1 (TfR1) binding but that OCEV glycoprotein precursor (GPC)-pseudotyped retroviruses poorly entered 53 human cancer cell lines. Interestingly, OCEV and Tacaribe virus could use bat, but not human, TfR1. Replacing three human TfR1 amino acids with their bat ortholog counterparts transformed human TfR1 into an efficient OCEV and Tacaribe virus receptor.     

Neuron‐type specific cannabinoid‐mediated G protein signalling in mouse hippocampus

Type 1 cannabinoid receptor (CB1) is expressed in different neuronal populations in the mammalian brain. In particular, CB1 on GABAergic or glutamatergic neurons exerts different functions and display different pharmacological properties in vivo. This suggests the existence of neuron‐type specific signalling pathways activated by different subpopulations of CB1. In this study, we analysed CB1 expression, binding and signalling in the hippocampus of conditional mutant mice, bearing CB1 deletion in GABAergic (GABA‐CB1‐KO mice) or cortical glutamatergic neurons (Glu‐CB1‐KO mice). Compared to their wild‐type littermates, Glu‐CB1‐KO displayed a small decrease of CB1 mRNA amount, immunoreactivity and [³H]CP55,940 binding. Conversely, GABA‐CB1‐KO mice showed a drastic reduction of these parameters, confirming that CB1 is present at much higher density on hippocampal GABAergic interneurons than glutamatergic neurons. Surprisingly, however, saturation analysis of HU210‐stimulated [³⁵S]GTPγS binding demonstrated that ‘glutamatergic’ CB1 is more efficiently coupled to G protein signalling than ‘GABAergic’ CB1. Thus, the minority of CB1 on glutamatergic neurons is paradoxically several fold more strongly coupled to G protein signalling than ‘GABAergic’ CB1. This selective signalling mechanism raises the possibility of designing novel cannabinoid ligands that differentially activate only a subset of physiological effects of CB1 stimulation, thereby optimizing therapeutic action.     

A Self-compartmentalizing Hexamer Serine Protease from Pyrococcus Horikoshii: SUBSTRATE SELECTION ACHIEVED THROUGH MULTIMERIZATION*

Oligopeptidases impose a size limitation on their substrates, the mechanism of which has long been under debate. Here we present the structure of a hexameric serine protease, an oligopeptidase from Pyrococcus horikoshii (PhAAP), revealing a complex, self-compartmentalized inner space, where substrates may access the monomer active sites passing through a double-gated “check-in” system, first passing through a pore on the hexamer surface and then turning to enter through an even smaller opening at the monomers'' domain interface. This substrate screening strategy is unique within the family. We found that among oligopeptidases, a residue of the catalytic apparatus is positioned near an amylogenic β-edge, which needs to be protected to prevent aggregation, and we found that different oligopeptidases use different strategies to achieve such an end. We propose that self-assembly within the family results in characteristically different substrate selection mechanisms coupled to different multimerization states.     

Insights into β-Lactamases from Burkholderia Species,Two Phylogenetically Related yet Distinct Resistance Determinants

Burkholderia cepacia complex and Burkholderia pseudomallei are opportunistic human pathogens. Resistance to β-lactams among Burkholderia spp. is attributable to expression of β-lactamases (e.g. PenA in B. cepacia complex and PenI in B. pseudomallei). Phylogenetic comparisons reveal that PenA and PenI are highly related. However, the analyses presented here reveal that PenA is an inhibitor-resistant carbapenemase, most similar to KPC-2 (the most clinically significant serine carbapenemase), whereas PenI is an extended spectrum β-lactamase. PenA hydrolyzes β-lactams with k_cat values ranging from 0.38 ± 0.04 to 460 ± 46 s⁻¹ and possesses high k_cat/k_inact values of 2000, 1500, and 75 for β-lactamase inhibitors. PenI demonstrates the highest k_cat value for cefotaxime of 9.0 ± 0.9 s⁻¹. Crystal structure determination of PenA and PenI reveals important differences that aid in understanding their contrasting phenotypes. Changes in the positioning of conserved catalytic residues (e.g. Lys-73, Ser-130, and Tyr-105) as well as altered anchoring and decreased occupancy of the deacylation water explain the lower k_cat values of PenI. The crystal structure of PenA with imipenem docked into the active site suggests why this carbapenem is hydrolyzed and the important role of Arg-220, which was functionally confirmed by mutagenesis and biochemical characterization. Conversely, the conformation of Tyr-105 hindered docking of imipenem into the active site of PenI. The structural and biochemical analyses of PenA and PenI provide key insights into the hydrolytic mechanisms of β-lactamases, which can lead to the rational design of novel agents against these pathogens.     

Ghrelin amplifies the nicotine-induced dopamine release in the rat striatum

The orexigenic peptide ghrelin plays a prominent role in the regulation of energy balance and in the mediation of reward mechanisms and reinforcement for addictive drugs, such as nicotine. Nicotine is the principal psychoactive component in tobacco, which is responsible for addiction and relapse of smokers. Nicotine activates the mesencephalic dopaminergic neurons via nicotinic acetylcholine receptors (nAchR). Ghrelin stimulates the dopaminergic neurons via growth hormone secretagogue receptors (GHS-R1A) in the ventral tegmental area and the substantia nigra pars compacta resulting in the release of dopamine in the ventral and dorsal striatum, respectively. In the present study an in vitro superfusion of rat striatal slices was performed, in order to investigate the direct action of ghrelin on the striatal dopamine release and the interaction of ghrelin with nicotine through this neurotransmitter release. Ghrelin increased significantly the dopamine release from the rat striatum following electrical stimulation. This stimulatory effect was reversed by both the selective nAchR antagonist mecamylamine and the selective GHS-R1A antagonist GHRP-6. Nicotine also increased significantly the dopamine release under the same conditions. This stimulatory effect was antagonized by mecamylamine, but not by GHRP-6. Ghrelin further stimulated the nicotine-induced dopamine release and this effect was abolished by mecamylamine and was partially inhibited by GHRP-6. The present results demonstrate that ghrelin stimulates directly the dopamine release and amplifies the nicotine-induced dopamine release in the rat striatum. We presume that striatal cholinergic interneurons also express GHS-R1A, through which ghrelin can amplify the nicotine-induced dopamine release in the striatum. This study provides further evidence of the impact of ghrelin on the mesolimbic and nigrostriatal dopaminergic pathways. It also suggests that ghrelin signaling may serve as a novel pharmacological target for treatment of addictive and neurodegenerative disorders.     

Fluorescent ligands for adenosine receptors

Interest is increasing in developing fluorescent ligands for characterization of adenosine receptors (ARs), which hold a promise of usefulness in the drug discovery process. The size of a strategically labeled AR ligand can be greatly increased after the attachment of a fluorophore. The choice of dye moiety (e.g. Alexa Fluor 488), attachment point and linker length can alter the selectivity and potency of the parent molecule. Fluorescent derivatives of adenosine agonists and antagonists (e.g. XAC and other heterocyclic antagonist scaffolds) have been synthesized and characterized pharmacologically. Some are useful AR probes for flow cytometry, fluorescence correlation spectroscopy, fluorescence microscopy, fluorescence polarization, fluorescence resonance energy transfer, and scanning confocal microscopy. Thus, the approach of fluorescent labeled GPCR ligands, including those for ARs, is a growing dynamic research field.