Transcriptomic insights into the dominance of two phototrophs throughout the water column of a tropical hypersaline-alkaline crater lake (Dziani Dzaha, Mayotte).

Saline-alkaline lakes often shelter high biomasses despite challenging conditions, owing to the occurrence of highly adapted phototrophs. Dziani Dzaha (Mayotte) is one such lake characterized by the stable co-dominance of the cyanobacterium Limnospira platensis and the picoeukaryote Picocystis salinarum throughout its water column. Despite light penetrating only into the uppermost meter, the prevailing co-dominance of these species persists even in light- and oxygen-deprived zones. Here, a depth profile of phototrophs metatranscriptomes, annotated using genomic data from isolated strains, is employed to identify expression patterns of genes related to carbon processing pathways including photosynthesis, transporters and fermentation. The findings indicate a prominence of gene expression associated with photosynthesis, with a peak of expression around 1 m below the surface, although the light intensity is very low and only red and dark red wavelengths can reach it, given the very high turbidity linked to the high biomass of L. platensis. Experiments on strains confirmed that both species do grow under these wavelengths, at rates comparable to those obtained under white light. A decrease in the expression of photosynthesis-related genes was observed in L. platensis with increasing depth, whereas P. salinarum maintained a very high pool of psbA transcripts down to the deepest point as a possible adaptation against photodamage, in the absence and/or very low levels of expression of genes involved in protection. In the aphotic/anoxic zone, expression of genes involved in fermentation pathways suggests active metabolism of reserve or available dissolved carbon compounds. Overall, L. platensis seems to be adapted to the uppermost water layer, where it is probably maintained thanks to gas vesicles, as evidenced by high expression of the gvpA gene. In contrast, P. salinarum occurs at similar densities throughout the water column, with a peak in abundance and gene expression levels which suggests a better adaptation to lower light intensities. These slight differences may contribute to limited inter-specific competition, favoring stable co-dominance of these two phototrophs.

Bacterial Microbiota of Ostreobium, the Coral-Isolated Chlorophyte Ectosymbiont, at Contrasted Salinities.

Microscopic filaments of the siphonous green algae Ostreobium (Ulvophyceae, Bryopsidales) colonize and dissolve the calcium carbonate skeletons of coral colonies in reefs of contrasted salinities. Here, we analyzed their bacterial community's composition and plasticity in response to salinity. Multiple cultures of Pocillopora coral-isolated Ostreobium strains from two distinct rbcL lineages representative of IndoPacific environmental phylotypes were pre-acclimatized (>9 months) to three ecologically relevant reef salinities: 32.9, 35.1, and 40.2 psu. Bacterial phylotypes were visualized for the first time at filament scale by CARD-FISH in algal tissue sections, within siphons, at their surface or in their mucilage. Ostreobium-associated microbiota, characterized by bacterial 16S rDNA metabarcoding of cultured thalli and their corresponding supernatants, were structured by host genotype (Ostreobium strain lineage), with dominant Kiloniellaceae or Rhodospirillaceae (Alphaproteobacteria, Rhodospirillales) depending on Ostreobium lineage, and shifted Rhizobiales' abundances in response to the salinity increase. A small core microbiota composed of seven ASVs (~1.5% of thalli ASVs, 19-36% cumulated proportions) was persistent across three salinities in both genotypes, with putative intracellular Amoebophilaceae and Rickettsiales_AB1, as well as Hyphomonadaceae and Rhodospirillaceae also detected within environmental (Ostreobium-colonized) Pocillopora coral skeletons. This novel knowledge on the taxonomic diversity of Ostreobium bacteria paves the way to functional interaction studies within the coral holobiont.

Discovery of a Highly Selective ß2-Adrenoceptor Agonist with a 2-Amino-2-phenylethanol Scaffold as an Oral Antiasthmatic Agent.

Asthma patients in resource-poor countries cannot obtain adequate basic asthma medications because most asthma medications are supplied as inhalants. An alternative approach is to create oral antiasthmatic drugs with high ß2/ß1-selectivity, which should reduce treatment costs. In this study, we designed a cohort of compounds 1 using 2-(4-amino-3-chloro-5-(trifluoromethyl)phenyl)-2-(tert-butylamino)ethan-1-ol hydrogen chloride (1a) as the lead compound with an aim to expand the library of compounds possessing the 2-amino-2-phenylethanol scaffold. Structure-activity relationship studies on these compounds revealed that compounds created showed remarkable ß2 selectivity compared to isoproterenol and gave additional insights on the rational design of ß2-adrenoceptor agonists. Moreover, 1a was found as the best candidate compound showing the greatest potential for drug development. Cell-based assays showed that 1a was about 10 times more selective than salbutamol toward the ß2-adrenoceptor. Moreover, 1a exhibited good oral bioavailability and low acute oral toxicity. These data reveal 1a as an oral antiasthmatic agent.

Phylogeny of the Varidnaviria Morphogenesis Module: Congruence and Incongruence With the Tree of Life and Viral Taxonomy.

Double-stranded DNA viruses of the realm Varidnaviria (formerly PRD1-adenovirus lineage) are characterized by homologous major capsid proteins (MCPs) containing one (kingdom: Helvetiavirae) or two ß-barrel domains (kingdom: Bamfordvirae) known as the jelly roll folds. Most of them also share homologous packaging ATPases (pATPases). Remarkably, Varidnaviria infect hosts from the three domains of life, suggesting that these viruses could be very ancient and share a common ancestor. Here, we analyzed the evolutionary history of Varidnaviria based on single and concatenated phylogenies of their MCPs and pATPases. We excluded Adenoviridae from our analysis as their MCPs and pATPases are too divergent. Sphaerolipoviridae, the only family in the kingdom Helvetiavirae, exhibit a complex history: their MCPs are very divergent from those of other Varidnaviria, as expected, but their pATPases groups them with Bamfordvirae. In single and concatenated trees, Bamfordvirae infecting archaea were grouped with those infecting bacteria, in contradiction with the cellular tree of life, whereas those infecting eukaryotes were organized into three monophyletic groups: the Nucleocytoviricota phylum, formerly known as the Nucleo-Cytoplasmic Large DNA Viruses (NCLDVs), Lavidaviridae (virophages) and Polintoviruses. Although our analysis mostly supports the recent classification proposed by the International Committee on Taxonomy of Viruses (ICTV), it also raises questions, such as the validity of the Adenoviridae and Helvetiavirae ranking. Based on our phylogeny, we discuss current hypotheses on the origin and evolution of Varidnaviria and suggest new ones to reconcile the viral and cellular trees.

8-Hydroxyquinolin-2(1H)-one analogues as potential ß2-agonists: Design, synthesis and activity study.

ß2-Agonists that bind to plasmalemmal ß2-adrenoceptors causing cAMP accumulation are widely used as bronchodilators in chronic respiratory diseases. Here, we designed and synthesized a group of 8-hydroxyquinolin-2(1H)-one analogues and studied their ß2-agonistic activities with a cellular cAMP assay. Compounds B05 and C08 were identified as potent (EC50 < 20 pM) and selective ß2-agonists among the compounds tested. They behaved as partial ß2-agonists in non-overexpressed HEK293 cells, and possessed rapid smooth muscle relaxant actions and long duration of action in isolated guinea pig tracheal strip preparations. In summary, B05 and C08 are ß2-agonists with potential applicability in chronic respiratory diseases.

Heterodimerization With 5-HT2BR Is Indispensable for ß2AR-Mediated Cardioprotection.

RATIONALE: The ß2-adrenoceptor (ß2-AR), a prototypical GPCR (G protein-coupled receptor), couples to both Gs and Gi proteins. Stimulation of the ß2-AR is beneficial to humans and animals with heart failure presumably because it activates the downstream Gi-PI3K-Akt cell survival pathway. Cardiac ß2-AR signaling can be regulated by crosstalk or heterodimerization with other GPCRs, but the physiological and pathophysiological significance of this type of regulation has not been sufficiently demonstrated. OBJECTIVE: Here, we aim to investigate the potential cardioprotective effect of ß2-adrenergic stimulation with a subtype-selective agonist, (R,R')-4-methoxy-1-naphthylfenoterol (MNF), and to decipher the underlying mechanism with a particular emphasis on the role of heterodimerization of ß2-ARs with another GPCR, 5-hydroxytryptamine receptors 2B (5-HT2BRs). METHODS AND RESULTS: Using pharmacological, genetic and biophysical protein-protein interaction approaches, we studied the cardioprotective effect of the ß2-agonist, MNF, and explored the underlying mechanism in both in vivo in mice and cultured rodent cardiomyocytes insulted with doxorubicin, hydrogen peroxide (H2O2) or ischemia/reperfusion. In doxorubicin (Dox)-treated mice, MNF reduced mortality and body weight loss, while improving cardiac function and cardiomyocyte viability. MNF also alleviated myocardial ischemia/reperfusion injury. In cultured rodent cardiomyocytes, MNF inhibited DNA damage and cell death caused by Dox, H2O2 or hypoxia/reoxygenation. Mechanistically, we found that MNF or another ß2-agonist zinterol markedly promoted heterodimerization of ß2-ARs with 5-HT2BRs. Upregulation of the heterodimerized 5-HT2BRs and ß2-ARs enhanced ß2-AR-stimulated Gi-Akt signaling and cardioprotection while knockdown or pharmacological inhibition of the 5-HT2BR attenuated ß2-AR-stimulated Gi signaling and cardioprotection. CONCLUSIONS: These data demonstrate that the ß2-AR-stimulated cardioprotective Gi signaling depends on the heterodimerization of ß2-ARs and 5-HT2BRs.

Recent Advances in ß2-Agonists for Treatment of Chronic Respiratory Diseases and Heart Failure.

ß2-Adrenoceptor (ß2-AR) agonists are widely used as bronchodilators. The emerge of ultralong acting ß2-agonists is an important breakthrough in pulmonary medicine. In this review, we will provide mechanistic insights into the application of ß2-agonists in asthma, chronic obstructive pulmonary disease (COPD), and heart failure (HF). Recent studies in ß-AR signal transduction have revealed opposing functions of the ß1-AR and the ß2-AR on cardiomyocyte survival. Thus, ß2-agonists and ß-blockers in combination may represent a novel strategy for HF management. Allosteric modulation and biased agonism at the ß2-AR also provide a theoretical basis for developing drugs with novel mechanisms of action and pharmacological profiles. Overlap of COPD and HF presents a substantial clinical challenge but also a unique opportunity for evaluation of the cardiovascular safety of ß2-agonists. Further basic and clinical research along these lines can help us develop better drugs and innovative strategies for the management of these difficult-to-treat diseases.

Design, synthesis and biological evaluation of 8-(2-amino-1-hydroxyethyl)-6-hydroxy-1,4-benzoxazine-3(4H)-one derivatives as potent ß2-adrenoceptor agonists.

A series of ß2-adrenoceptor agonists with an 8-(2-amino-1-hydroxyethyl)-6-hydroxy-1,4-benzoxazine-3(4H)-one moiety is presented. The stimulatory effects of the compounds on human ß2-adrenoceptor and ß1-adrenoceptor were characterized by a cell-based assay. Their smooth muscle relaxant activities were tested on isolated guinea pig trachea. Most of the compounds were found to be potent and selective agonists of the ß2-adrenoceptor. One of the compounds, (R)-18c, possessed a strong ß2-adrenoceptor agonistic effect with an EC50 value of 24 pM. It produced a full and potent airway smooth muscle relaxant effect same as olodaterol. Its onset of action was 3.5 min and its duration of action was more than 12 h in an in vitro guinea pig trachea model of bronchodilation. These results suggest that (R)-18c is a potential candidate for long-acting ß2-AR agonists.

Diversification of giant and large eukaryotic dsDNA viruses predated the origin of modern eukaryotes.

Giant and large eukaryotic double-stranded DNA viruses from the Nucleo-Cytoplasmic Large DNA Virus (NCLDV) assemblage represent a remarkably diverse and potentially ancient component of the eukaryotic virome. However, their origin(s), evolution, and potential roles in the emergence of modern eukaryotes remain subjects of intense debate. Here we present robust phylogenetic trees of NCLDVs, based on the 8 most conserved proteins responsible for virion morphogenesis and informational processes. Our results uncover the evolutionary relationships between different NCLDV families and support the existence of 2 superclades of NCLDVs, each encompassing several families. We present evidence strongly suggesting that the NCLDV core genes, which are involved in both informational processes and virion formation, were acquired vertically from a common ancestor. Among them, the largest subunits of the DNA-dependent RNA polymerase were transferred between 2 clades of NCLDVs and proto-eukaryotes, giving rise to 2 of the 3 eukaryotic DNA-dependent RNA polymerases. Our results strongly suggest that these transfers and the diversification of NCLDVs predated the emergence of modern eukaryotes, emphasizing the major role of viruses in the evolution of cellular domains.

Discovery of ß-arrestin-biased ß2-adrenoceptor agonists from 2-amino-2-phenylethanol derivatives.

ß-Arrestins are a small family of proteins important for signal transduction at G protein-coupled receptors (GPCRs). ß-Arrestins are involved in the desensitization of GPCRs. Recently, biased ligands possessing different efficacies in activating the G protein- versus the ß-arrestin-dependent signals downstream of a single GPCR have emerged, which can be used to selectively modulate GPCR signal transduction in such a way that desirable signals are enhanced to produce therapeutic effects while undesirable signals of the same GPCR are suppressed to avoid side effects. In the present study, we evaluated agonist bias for compounds developed along a drug discovery project of ß2-adrenoceptor agonists. About 150 compounds, including derivatives of fenoterol, 2-amino-1-phenylethanol and 2-amino-2-phenylethanol, were obtained or synthesized, and initially screened for their ß-adrenoceptor-mediated activities in the guinea pig tracheal smooth muscle relaxation assay or the cardiomyocyte contractility assay. Nineteen bioactive compounds were further assessed using both the HTRF cAMP assay and the PathHunter ß-arrestin assay. Their concentration-response data in stimulating cAMP synthesis and ß-arrestin recruitment were applied to the Black-Leff operational model for ligand bias quantitation. As a result, three compounds (L-2, L-4, and L-12) with the core structure of 5-(1-amino-2-hydroxyethyl)-8-hydroxyquinolin-2(1H)-one were identified as a new series of ß-arrestin-biased ß2-adrenoceptor agonists, whereas salmeterol was found to be Gs-biased. These findings would facilitate the development of novel drugs for the treatment of both heart failure and asthma.

Design, synthesis and biological evaluation of 5-(2-amino-1-hydroxyethyl)-8-hydroxyquinolin-2(1H)-one derivatives as potent ß2-adrenoceptor agonists.

A series of novel ß2-adrenoceptor agonists with a 5-(2-amino-1-hydroxyethyl)-8-hydroxyquinolin-2(1H)-one moiety was designed, synthesized and evaluated for biological activity in human embryonic kidney 293 cells and isolated guinea pig trachea. Compounds 9g and (R)-18c exhibited the most excellent ß2-adrenoceptor agonistic effects and high ß2/ß1-selectivity with EC50 values of 36 pM for 9g and 21 pM for (R)-18c. They produced potent airway smooth muscle relaxant effects with fast onset of action and long duration of action in an in vitro guinea pig trachea model of bronchodilation. These results support further development of the two compounds into drug candidates.

ADRB2 polymorphism Arg16Gly modifies the natural outcome of heart failure and dictates therapeutic response to ß-blockers in patients with heart failure.

We sought to investigate the association of single nucleotide polymorphisms (SNPs) of the genes involved in ßAR signaling with the response of patients to ßAR blockers. A total of 2403 hospitalized patients with chronic heart failure (HF) were enrolled in a multicenter observational study as the first cohort and followed up for a mean period of 20 months. Genes for ß1AR, ß2AR, and the major cardiac G-protein-coupled receptor kinases (GRKs) GRK2 and GRK5 were analyzed to identify SNPs, and patients were stratified according to genotypes. A second independent cohort enrolling 919 patients with chronic HF was applied to validate the observed associations. The signaling properties of the key identified SNPs were assessed in vitro. Our data showed that HF patients harboring the Gly16 allele in the gene for ß2AR (ADRB2) had an increased risk of the composite end point relative to patients who were homozygous for Arg16. Notably, these patients showed a beneficial response to ßAR-blocker treatment in a G allele-dose-dependent manner, whereas Arg16 homozygotes had no response to ßAR-blocker therapy. This Arg16Gly genotype-dependent heterogeneity in clinical outcomes of HF was successfully validated in the second independent population. Besides, the in vitro experiments revealed that G allele carriers were defective in ß2AR-coupled inhibitory adenylate cyclase g (Gi) protein signaling.

Heterogeneity of spontaneous DNA replication errors in single isogenic Escherichia coli cells.

Despite extensive knowledge of the molecular mechanisms that control mutagenesis, it is not known how spontaneous mutations are produced in cells with fully operative mutation-prevention systems. By using a mutation assay that allows visualization of DNA replication errors and stress response transcriptional reporters, we examined populations of isogenic Escherichia coli cells growing under optimal conditions without exogenous stress. We found that spontaneous DNA replication errors in proliferating cells arose more frequently in subpopulations experiencing endogenous stresses, such as problems with proteostasis, genome maintenance, and reactive oxidative species production. The presence of these subpopulations of phenotypic mutators is not expected to affect the average mutation frequency or to reduce the mean population fitness in a stable environment. However, these subpopulations can contribute to overall population adaptability in fluctuating environments by serving as a reservoir of increased genetic variability.

Synthesis and biological evaluation of ß2-adrenoceptor agonists bearing the 2-amino-2-phenylethanol scaffold.

A new series of ß2-adrenoceptor agonists bearing the 2-amino-2-phenylethanol scaffold was synthesized. Evaluation of the compounds using cell assays and an in vitro guinea pig trachea relaxation assay showed that 8-hydroxy-5-(2-hydroxy-1-((4-hydroxyphenethyl)amino)ethyl)quinolin-2(1H)-one (compound 5j) has the best pharmacological profile among all the evaluated compounds. The (S)-isomer of 5j was subsequently found to be the active enantiomer with a promising EC50 value of 1.26 nM in stimulating ß2-adrenoceptor-mediated cAMP accumulation and a substantially higher selectivity for the ß2 than for the ß1 subtype. The putative binding mode of (S)-5j revealed by molecular docking of the ß2-adrenoceptor resembles that in agonist binding. Taken together, these results showed that compound (S)-5j is a promising compound worthy of further study for the development of ß2-adrenoceptor agonists.

Synthesis, biological evaluation and molecular modeling of 2-amino-2-phenylethanol derivatives as novel ß2-adrenoceptor agonists.

A novel series of 2-amino-2-phenylethanol derivatives were developed as ß2-adrenoceptor agonists. Among them, 2-amino-3-fluoro-5-(2-hydroxy-1-(isopropylamino)ethyl)benzonitrile (compound 2f) exhibited the highest activity (EC50 = 0.25 nM) in stimulating ß2-adrenoceptor-mediated cellular cAMP production with a 763.6-fold selectivity over the ß1-adrenoceptor. The (S)-isomer of 2f was subsequently found to be 8.5-fold more active than the (R)-isomer. Molecular docking was performed to determine the putative binding modes of this new class of ß2-adrenoceptor agonists. Taken together, these data show that compound 2f is a promising lead compound worthy of further study for the development of ß2-adrenoceptor agonists.

GRK2/ß-arrestin mediates arginine vasopressin-induced cardiac fibroblast proliferation.

Cardiac fibrosis is a pathological feature commonly found in hearts exposed to haemodynamic orneurohormonal stress. Elevated levels of arginine vasopressin (AVP) are closely associated with the progression of heart failure and could be an underlying cause of cardiac fibrosis. The aim of this study is to characterize the effect of AVP on neonatal rat cardiac fibroblasts (NRCFs) and to illustrate its signalling mechanism. The proliferative effect of AVP was assessed by methylthiazolyldiphenyl-tetrazolium assay and 5-bromo-2'-deoxyuridine (BrdU) incorporation assay, and the amounts of cellular signalling proteins α-smooth muscle actin (α-SMA), matrix metalloproteinase (MMP) 2, MMP9, and phosphorylated ERK1/2 were determined by western blotting. AVP, in a time- and concentration-dependent manner, promoted NRCF proliferation and the expression of MMP2 and MMP9. Inhibition of G protein-coupled receptor kinase2 (GRK2) by the inhibitory peptide GRK2-Ct or knock-down of GRK2 suppressed AVP-induced BrdU incorporation and the expression of MMP2 and α-SMA in NRCFs. Moreover, shRNA-mediated silencing of ß-arrestin1 or ß-arrestin 2 abolished AVP-induced BrdU incorporation and MMP2 expression. AVP-induced NRCF proliferation depended on the phosphorylation of ERK1/2 , and inhibition of GRK2 or silencing of ß-arrestins blocked AVP-induced ERK1/2 phosphorylation. The effects of AVP on NRCF proliferation and α-SMA expression were blocked by SR45059, a vasopressin receptor type1A (V1A R) selective antagonist. In conclusion, AVP promotes NRCF proliferation through V1A R-mediated GRK2/ß-arrestin/ERK1/2 signalling.

Vasopressin V1A receptor mediates cell proliferation through GRK2-EGFR-ERK1/2 pathway in A7r5 cells.

Abnormal proliferation and hypertrophy of vascular smooth muscle (VSMC), as the main structural component of the vasculature, is an important pathological mechanism of hypertension. Recently, increased levels of arginine vasopressin (AVP) and copeptin, the C-terminal fragment of provasopressin, have been shown to correlate with the development of preeclampsia. AVP targets on the Gq-coupled vasopressin V1A receptor and the Gs-coupled V2 receptor in VSMC and the kidneys to regulate vascular tone and water homeostasis. However, the role of the vasopressin receptor on VSM cell proliferation during vascular remodeling is unclear. Here, we studied the effects of AVP on the proliferation of the rat VSMC-derived A7r5 cells. AVP, in a time- and concentration-dependent manner, promoted A7r5 cell proliferation as indicated by the induction of proliferating cell nuclear antigen expression, methylthiazolyldiphenyl-tetrazolium reduction and incorporation of 5'-bromodeoxyuridine into cellular DNA. These effects, coupled with the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), were blocked by a V1A receptor antagonist SR45059 but not by a V2 receptor antagonist lixivaptan. Although acute activation of V1A receptor induced ERK1/2 phosphorylation via a protein kinase C-dependent pathway, this effect was not involved in cell proliferation. Cell proliferation and ERK1/2 phosphorylation in response to prolonged stimulation with AVP were abolished by inhibition of G protein-coupled receptor kinase 2 (GRK2) and epidermal growth factor receptor (EGFR) using specific inhibitors or small hairpin RNA knock-down. These results suggest that activation of V1A, but not V2 receptor, produces a cell proliferative signal in A7r5 cells via a GRK2/EGFR/ERK1/2-dependent mechanism.

Interaction of ß1-adrenoceptor with RAGE mediates cardiomyopathy via CaMKII signaling.

Stimulation of ß1-adrenergic receptor (ß1AR), a GPCR, and the receptor for advanced glycation end-products (RAGE), a pattern recognition receptor (PRR), have been independently implicated in the pathogenesis of cardiomyopathy caused by various etiologies, including myocardial infarction, ischemia/reperfusion injury, and metabolic stress. Here, we show that the two distinctly different receptors, ß1AR and RAGE, are mutually dependent in mediating myocardial injury and the sequelae of cardiomyopathy. Deficiency or inhibition of RAGE blocks ß1AR- and RAGE-mediated myocardial cell death and maladaptive remodeling. Ablation or blockade of ß1AR fully abolishes RAGE-induced detrimental effects. Mechanistically, RAGE and ß1AR form a complex, which in turn activates Ca2+/calmodulin-dependent kinase II (CaMKII), resulting in loss of cardiomyocytes and myocardial remodeling. These results indicate that RAGE and ß1AR not only physically crosstalk at the receptor level, but also functionally converge at the common mediator, CaMKII, highlighting a combined inhibition of RAGE and ß1AR as a more effective therapy to treat diverse cardiovascular diseases, such as myocardial infarction, ischemia/reperfusion injury, and diabetic cardiovascular complications.