Study on the Influence of Chirality in the Threading of Calix[6]arene Hosts with Dialkylammonium Axles

The influence of chirality in calixarene threading has been studied by exploiting the “superweak anion approach”. In particular, the formation of chiral pseudo[2]rotaxanes bearing a classical stereogenic center in their axle and/or wheel components has been considered. Two kind of pseudo[2]rotaxane stereoadducts, the “endo-chiral” and “exo-chiral” ones, having the stereogenic center of a cationic axle inside or outside, respectively, the calix-cavity of a chiral calixarene were preferentially formed with specifically designed chiral axles by a fine exploitation of the so-called “endo-alkyl rule” and a newly defined “endo-α-methyl-benzyl rule” (threading of a hexaalkoxycalix[6]arene with a directional (α-methyl-benzyl)benzylammonium axle occurs with an endo-α-methyl-benzyl preference). The obtained pseudorotaxanes were studied in solution by 1D and 2D NMR, and in the gas-phase by means of the enantiomer-labeled (EL) mass spectrometry method, by combining enantiopure hosts with pseudoracemates of one deuterated and one unlabeled chiral axle enantiomer. In both instances, there was not a clear enantiodiscrimination in the threading process with the studied host/guest systems. Possible rationales are given to explain the scarce reciprocal influence between the guest and host chiral centers

Fast Detection of Two Smenamide Family Members Using Molecular Networking.

Caribbean sponges of the genus Smenospongia are a prolific source of chlorinated secondary metabolites. The use of molecular networking as a powerful dereplication tool revealed in the metabolome of S. aurea two new members of the smenamide family, namely smenamide F (1) and G (2). The structure of smenamide F (1) and G (2) was determined by spectroscopic analysis (NMR, MS, ECD). The relative and the absolute configuration at C-13, C-15, and C-16 was determined on the basis of the conformational rigidity of a 1,3-disubstituted alkyl chain system (i.e., the C-12/C-18 segment of compound (1). Smenamide F (1) and G (2) were shown to exert a selective moderate antiproliferative activity against cancer cell lines MCF-7 and MDA-MB-231, while being inactive against MG-63

Chirality Transfer in a Calixarene-Based Directional Pseudorotaxane Complex

Hexamethoxycalix[6]arene 3 forms a directional pseudorotaxane complex with the chiral axle (S)-(alpha-methyl-benzyl)benzylammonium 2(+). Between the two (endo-chiral)-2(+)@3 and (exo-chiral)-2(+)@3 pseudorotaxane stereoisomers, the former is preferentially formed. This result confirms the validity of the "endo-alpha-methyl-benzyl rule", previously reported by us. DFT calculations suggest that C-H horizontal ellipsis pi interactions between the methyl group of 2(+) and the calixarene aromatic rings, determine the stereoselectivity of the threading process toward the "endo-alpha-methyl-benzyl preference". An amplification of optical rotation is observed upon formation of the pseudorotaxane complex (endo-chiral)-2(+)@3 with respect to free axle 2(+). Thus, the specifical rotation of the 1:1 mixture of chiral 2(+)center dot B(ArF)(4)(-) salt and achiral 3 was augmented upon formation of the pseudorotaxane and DFT calculations were used to rationalize this result

Cyanobacteria as indicators of water quality in Campania coasts, Italy: a monitoring strategy combining remote/proximal sensing andin situdata

Cyanobacterial blooms (CBs) are generally triggered by eutrophic conditions due to anthropogenic nutrient inputs to local waters (wastewater or contaminated waters). During the bloom, some species produce toxic secondary metabolites (cyanotoxins) that are dangerous for humans and animals. Here, a multidisciplinary strategy for an early detection and constant monitoring is proposed. This strategy combines remote/proximal sensing technology with analytical/biotechnological analyses. To demonstrate the applicability of this strategy, four anthropogenically-impacted sites were selected along the Campania coast of southwestern Italy, in the so called 'Land of Fires'. The sites were observed using satellite and aircraft images during summer, 2015. Algal community composition was determined using spectrophotometric analysis for the detection of the cyanobacterial pigment phycocyanin (PC). Complementary metagenomic analysis revealed the taxonomic presence of cyanobacteria belonging to genera associated with strong eutrophic conditions. Key elements of this strategy are the combination and integration of applying different methodological approaches such as the parallel and combined use of satellite, aerial and in-situ data, the simplified multispectral image indexing and classification for a truly efficient method in detecting early blooms of cyanobacteria. The effectiveness of the strategy has been validated also by the specific taxa of cyanobacteria found in the examined areas that confirm the assumption that cyanobacterial blooms may serve as useful bioindicators of degraded water quality in coastal ecosystems. To our knowledge this is the first time that the presence of cyanobacteria has been observed in water bodies along the Campania coast

Rationale and design of an independent randomised controlled trial evaluating the effectiveness of aripiprazole or haloperidol in combination with clozapine for treatment-resistant schizophrenia

<p>Abstract</p> <p>Background</p> <p>One third to two thirds of people with schizophrenia have persistent psychotic symptoms despite clozapine treatment. Under real-world circumstances, the need to provide effective therapeutic interventions to patients who do not have an optimal response to clozapine has been cited as the most common reason for simultaneously prescribing a second antipsychotic drug in combination treatment strategies. In a clinical area where the pressing need of providing therapeutic answers has progressively increased the occurrence of antipsychotic polypharmacy, despite the lack of robust evidence of its efficacy, we sought to implement a pre-planned protocol where two alternative therapeutic answers are systematically provided and evaluated within the context of a pragmatic, multicentre, independent randomised study.</p> <p>Methods/Design</p> <p>The principal clinical question to be answered by the present project is the relative efficacy and tolerability of combination treatment with clozapine plus aripiprazole compared with combination treatment with clozapine plus haloperidol in patients with an incomplete response to treatment with clozapine over an appropriate period of time. This project is a prospective, multicentre, randomized, parallel-group, superiority trial that follow patients over a period of 12 months. Withdrawal from allocated treatment within 3 months is the primary outcome.</p> <p>Discussion</p> <p>The implementation of the protocol presented here shows that it is possible to create a network of community psychiatric services that accept the idea of using their everyday clinical practice to produce randomised knowledge. The employed pragmatic attitude allowed to randomly allocate more than 100 individuals, which means that this study is the largest antipsychotic combination trial conducted so far in Western countries. We expect that the current project, by generating evidence on whether it is clinically useful to combine clozapine with aripiprazole rather than with haloperidol, provides physicians with a solid evidence base to be directly applied in the routine care of patients with schizophrenia.</p> <p>Trial Registration</p> <p><b>Clincaltrials.gov Identifier</b>: NCT00395915</p

Albiglutide and cardiovascular outcomes in patients with type 2 diabetes and cardiovascular disease (Harmony Outcomes): a double-blind, randomised placebo-controlled trial

Background: Glucagon-like peptide 1 receptor agonists differ in chemical structure, duration of action, and in their effects on clinical outcomes. The cardiovascular effects of once-weekly albiglutide in type 2 diabetes are unknown. We aimed to determine the safety and efficacy of albiglutide in preventing cardiovascular death, myocardial infarction, or stroke. Methods: We did a double-blind, randomised, placebo-controlled trial in 610 sites across 28 countries. We randomly assigned patients aged 40 years and older with type 2 diabetes and cardiovascular disease (at a 1:1 ratio) to groups that either received a subcutaneous injection of albiglutide (30–50 mg, based on glycaemic response and tolerability) or of a matched volume of placebo once a week, in addition to their standard care. Investigators used an interactive voice or web response system to obtain treatment assignment, and patients and all study investigators were masked to their treatment allocation. We hypothesised that albiglutide would be non-inferior to placebo for the primary outcome of the first occurrence of cardiovascular death, myocardial infarction, or stroke, which was assessed in the intention-to-treat population. If non-inferiority was confirmed by an upper limit of the 95% CI for a hazard ratio of less than 1·30, closed testing for superiority was prespecified. This study is registered with ClinicalTrials.gov, number NCT02465515. Findings: Patients were screened between July 1, 2015, and Nov 24, 2016. 10 793 patients were screened and 9463 participants were enrolled and randomly assigned to groups: 4731 patients were assigned to receive albiglutide and 4732 patients to receive placebo. On Nov 8, 2017, it was determined that 611 primary endpoints and a median follow-up of at least 1·5 years had accrued, and participants returned for a final visit and discontinuation from study treatment; the last patient visit was on March 12, 2018. These 9463 patients, the intention-to-treat population, were evaluated for a median duration of 1·6 years and were assessed for the primary outcome. The primary composite outcome occurred in 338 (7%) of 4731 patients at an incidence rate of 4·6 events per 100 person-years in the albiglutide group and in 428 (9%) of 4732 patients at an incidence rate of 5·9 events per 100 person-years in the placebo group (hazard ratio 0·78, 95% CI 0·68–0·90), which indicated that albiglutide was superior to placebo (p&lt;0·0001 for non-inferiority; p=0·0006 for superiority). The incidence of acute pancreatitis (ten patients in the albiglutide group and seven patients in the placebo group), pancreatic cancer (six patients in the albiglutide group and five patients in the placebo group), medullary thyroid carcinoma (zero patients in both groups), and other serious adverse events did not differ between the two groups. There were three (&lt;1%) deaths in the placebo group that were assessed by investigators, who were masked to study drug assignment, to be treatment-related and two (&lt;1%) deaths in the albiglutide group. Interpretation: In patients with type 2 diabetes and cardiovascular disease, albiglutide was superior to placebo with respect to major adverse cardiovascular events. Evidence-based glucagon-like peptide 1 receptor agonists should therefore be considered as part of a comprehensive strategy to reduce the risk of cardiovascular events in patients with type 2 diabetes. Funding: GlaxoSmithKline

Reducing the environmental impact of surgery on a global scale: systematic review and co-prioritization with healthcare workers in 132 countries

Abstract Background Healthcare cannot achieve net-zero carbon without addressing operating theatres. The aim of this study was to prioritize feasible interventions to reduce the environmental impact of operating theatres. Methods This study adopted a four-phase Delphi consensus co-prioritization methodology. In phase 1, a systematic review of published interventions and global consultation of perioperative healthcare professionals were used to longlist interventions. In phase 2, iterative thematic analysis consolidated comparable interventions into a shortlist. In phase 3, the shortlist was co-prioritized based on patient and clinician views on acceptability, feasibility, and safety. In phase 4, ranked lists of interventions were presented by their relevance to high-income countries and low–middle-income countries. Results In phase 1, 43 interventions were identified, which had low uptake in practice according to 3042 professionals globally. In phase 2, a shortlist of 15 intervention domains was generated. In phase 3, interventions were deemed acceptable for more than 90 per cent of patients except for reducing general anaesthesia (84 per cent) and re-sterilization of ‘single-use’ consumables (86 per cent). In phase 4, the top three shortlisted interventions for high-income countries were: introducing recycling; reducing use of anaesthetic gases; and appropriate clinical waste processing. In phase 4, the top three shortlisted interventions for low–middle-income countries were: introducing reusable surgical devices; reducing use of consumables; and reducing the use of general anaesthesia. Conclusion This is a step toward environmentally sustainable operating environments with actionable interventions applicable to both high– and low–middle–income countries

Zeamide, a Glycosylinositol Phosphorylceramide with the Novel Core Arap(1β→6)Ins Motif from the Marine Sponge Svenzea zeai

Glycosylinositol phosphorylceramides (GIPCs) show a great structural diversity, but all share a small number of core structures, with a glucosamine, a mannose, or a glucuronic acid as the first sugar linked to the inositol. The Caribbean sponge Svenzea zeai was shown to consistently contain zeamide (1), the first example of a new class of GIPCs, in which the inositol is glycosylated by a d-arabinose. The structure of zeamide was determined by spectroscopic analysis (NMR, MS, ECD) and microscale chemical degradation. The 6-O-β-d-arabinopyranosyl-myo-inositol (d-Arap(1β→6)Ins) core motif of zeamide is unprecedented not only among GIPCs, but also in any natural glycoconjugate

Exploiting the potential of marine natural products: structure elucidation and metagenomic approaches to biotechnological production

Sponges represent the most prolific producers of novel marine bioactive secondary metabolites. In the last years, several drugs derived from marine natural products have appeared in the market, and others are in clinical trials. The aim of my research project was to exploit the unusual and often surprising chemistry of marine sponges, in the frame of the more general purpose of discovering and developing new drugs from natural products. The research work presented in this PhD Thesis was directed to two different aspects of the study of marine secondary metabolites. On one hand, in parallel with the advent of environmental genomics from a drug discovery perspective, the largest part of my research activity focused on the metagenomic analysis of the Caribbean sponge P. simplex, and was aimed at the identification of new genes coding for polyketide synthases (PKSs), the giant enzyme complexes that produce polyketides, a large class of secondary metabolites that include many antibiotic and antitumor compounds. On the other hand, the remaining part of the research described in this PhD Thesis was more related to the “core activity” of natural product chemistry, and directed to the isolation and structure elucidation of new bioactive compounds from different specimens of sponges living in tropical oceans, wonderful sources of unusual molecular architectures to be used as leads and scaffolds for the elaboration of new drugs. Metagenomic investigations on Plakortis simplex (Demospongiae, Homosclerophorida, Plakinidae) was started because the sponge is known for the production of large amounts of polyketide peroxides, of which plakortin is the most abundant. Plakortin is of special interest due to its anti-malarial activity, which is retained also against chloroquine-resistant strains of Plasmodium falciparum. Therefore, a study of the biosynthesis of plakortin was undertaken, with the final aim of its biotechnological production. Most non-aromatic polyketides are synthesized by type I polyketide synthases (type I PKSs), produced in a number of cases by bacterial symbionts. The bacterial origin of plakortin is therefore a reasonable hypothesis, and indeed cell fractionation of P. simplex has shown that plakortin is mainly present in the bacterial cells. Since cultivation of true sponge symbionts failed in most cases, the search for the plakortin genes had to rely on cultivation-independent techniques, such as the study of the sponge metagenome (collective genome of the sponge and its symbionts). While the putative genes implied in plakortin biosynthesis could not be identified, an unexpected result from the metagenomic library screening was the discovery of Swf, a new group of mono-modular type I PKS/FAS (“hybrid polyketide synthase/fatty acid synthase”), which appears to be specifically associated to sponge symbionts. The putative swf operon consists of swfA (FAS/PKS I), swfB (R and ST domains), and swfC (radical SAM). SwfA contains a single PKS module, which builds the backbone of the acyl chain by recruiting iteratively malonyl units according to the substrate determining motif of its AT domain. The domain organization of SwfA is KS-AT-DH-ER-KR-ACP and from this architecture a saturated fatty acyl chain is expected, although a (poly)unsaturated and/or (poly)hydroxylated acyl chain cannot be excluded, because in iterative PKSs the reduction domains can be optionally used during each of the elongation steps. SwfB [composed of R (thioester reductase) and ST (sulfotransferase) domains] and SwfC (a radical SAM), are expected to modify the acyl chain produced by SwfA in unknown ways. As the R and ST domains are contiguous in SwfB, the expected product of elaboration of an acyl chain by SwfB would possibly be an alkyl sulphate or an alkylaminosulphonate: while the R domain can reductively release the assembled chain as a primary alcohol or amine, the ST domain can transfer the sulfate group to the hydroxyl or amino group. SwfC represents a radical SAM enzyme which can catalyze methylation of the substrate through a radical mechanism. Two different examples of the swf cluster were found in the metagenome of P. simplex, PS11G3 and PSA11D7 (PSA11D7 lacks the swfC gene). In addition, PCR amplification of metagenomic DNA from three different and taxonomically distant “high microbial abundance” sponges, Aplysina fulva, Smenospongia aurea and Pseudoceratina crassa, with primers designed for swf , produced amplicons which showed high sequence similarities to the AT domain of swfA. Therefore, the swf cluster is widespread in marine sponges and presumably associated to ubiquitous sponge symbionts. It represents the second group of mono-modular PKS, after the supA family, to be ubiquitously present in marine sponges. Preliminary studies of heterologous expression of swf genes were undertaken with the final aim of characterizing the unknown metabolite produced by the cluster. Activation of the ACP domain of the SwfA protein to its holo-form by co-expression with the phosphopantetheinyl transferase Svp was the first functional proof of swf type genes in marine sponges. Furthermore, applying homologous recombination for expression vector engineering, swfA was clearly expressed at the protein level in E.coli BL21-CodonPlus®(DE3)-RIPL cells by coexpression with the chaperone plasmid pTf16, which encodes for the molecular chaperone Trigger factor aiding the protein folding process. After cloning the whole swf operon into the expression vector pHIS8-Svp by homologous recombination, the new recombinant construct was used for heterologous expression trials of the whole cluster in E. coli BL21-(DE3) BAP1. Methanol extracts of transformants and their culture broths were analysed by LC-HR-ESI-MS, but no compounds which were present in all the transformants and absent in all the negative controls could be detected. In addition, fatty acid composition of transformants and their culture broths was characterized by saponification of the lipid extract and derivatization to fatty acid methyl esters (FAMEs) followed by GC/MS analysis. Even in this case, no new metabolite was detected, suggesting that the swf pathway is not functional in this expression system. As a consequence, the biosynthetic function of the swf cluster remains unknown at present. In parallel, metagenomic investigations conducted using high-throughput sequencing based on massively parallel 454 pyrosequencing led to a comprehensive overview of the polyketide metabolism of P.simplex and its symbionts, shedding light on the existence of novel polyketide synthase pathways potentially involved in bioactive compound biosynthesis. 454 pyrosequencing was performed on complex and heterogeneous PCR products amplified from the metagenomic DNA of P.simplex with degenerate probes targeting ketosynthase and acyltransferase domains of type I PKSs. Next generation sequencing of AT amplicon mixture generated 8995 reads; applying this modern approach, no PKS/FAS other than known SupA and SwfA could be found. Almost 51% of the total reads belonged to the Swf enzymes, while only the 4% was represented by AT belonging to SupA enzymes (the remaining reads appear not to be related to AT domains). On the other hand, 454 pyrosequencing of KS amplicon PCR mixture generated 19333 reads. Besides the expected huge presence of KS forming parts of SupA enzymes (~ 80% of the total reads), BLASTx analyses led to the detection of 8 new KS fragments, not reported in genbank database. All the eight putative KS fragments (which based on phylogenetic analysis appeared to be part of one hybrid NRPS-cis-AT PKS and seven cis-AT PKSs) are significantly different (E values ≥ 10-6) to each other, and BLASTx analysis as well as the rebuilt phylogenetic taxonomy revealed that they are only distantly related to PKSs of characterized function. In addition, phylogenetic analyses suggest that these KS fragments are mainly related to PKSs from Cyanobacteria, Actinomycetes and Myxobacteria, commonly known as precious sources of bioactive polyketides. These fragments may represent important starting points for further research towards the isolation of new PKS genes. The second line of research of my project was directed to the isolation and structure elucidation of new secondary metabolites from two tropical sponges, Chalinula molitba and Plakortis cf. lita. Stereo structure determination of the new compounds was determined by a combination of mass spectrometry, mono- and bidimensional NMR experiments and micro-scale chemical degradation. The analysis of the organic extract of the Caribbean sponge C. molitba led to the identification of chalinulasterol, a new C-24 chlorinated sterol disulfate. On the basis of the structural similarity with the PXR agonist solomonsterol A, the possible role of chalinulasterol as modulator of the pregnane-X receptor activity was evaluated by carrying out a transactivation assay (luciferase assay) on HepG2 cells, a human hepatocarcinoma cell line. Despite the structural similarity, chalinulasterol failed in transactivating PXR. The possibility that chalinulasterol could act as potential PXR antagonist was investigated, thus, also in this case, failed to reverse the induction of luciferase caused by rifaximin. Although negative, these results have an important implication in terms of structure-activity relationship, because they suggest that the sulfate group (absent in chalinulasterol) present in the side chain at position C-24 of solomonsterol A is essential in the ligand-receptor binding and receptor transactivation, confirming a proposed binding model where a clear interaction of the 24-sulfate with the positively charged Lys210 is hypothesized. Chemical analysis of the Indonesian sponge P. lita revealed, as first remarkable result, a secondary metabolite profile (glycolipids, hopanoids, polyketides) that, in spite of the geographical distance, was very similar to that of the Caribbean sponges of the genus Plakortis. Taking into account that secondary metabolites are often of bacterial origin, this indicates that bacterial communities associated to many species of Plakortis sponges are highly specific and consistently conserved in specimens collected in different times and geographical areas, suggesting vertical transmission within their hosts. In addition, among the many known compounds, plakohopanoid, a novel type of hopanoid, was isolated. Plakohopanoid is composed of a C32 hopanoid acyl ester-linked to a mannosyl-myo-inositol unit. It is interesting to note that C32 hopanoic derivatives are commonly considered as geohopanoids, i.e. diagenetic products formed through abiotic degradation of the hopanoids biosynthesized by bacteria (biohopanoids). As a consequence, the presence of plakohopanoid in a marine living organism is worthy of note, because it shows that there is a biosynthetic pathway to C32 hopanoic acids, which therefore should not be classified anymore as sure geohopanoids