Exploration of natural compounds as sources of new bifunctional scaffolds targeting cholinesterases and beta amyloid aggregation: The case of chelerythrine

The presented project started by screening a library consisting of natural and natural based compounds for their acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitory activity. Active compounds were chemically clustered into groups and further tested on the human cholinesterases isoforms. The aim of the presented study was to identify compounds that could be used as leads to target two key mechanisms associated with the AD’s pathogenesis simultaneously: cholinergic depletion and beta amyloid (Aβ) aggregation. Berberin, palmatine and chelerythrine, chemically clustered in the so-called isoquinoline group, showed promising cholinesterase inhibitory activity and were therefore further investigated. Moreover, the compounds demonstrated moderate to good inhibition of Aβ aggregation as well as the ability to disaggregate already preformed Aβ aggregates in an experimental set-up using HFIP as promotor of Aβ aggregates. Analysis of the kinetic mechanism of the AChE inhibition revealed chelerythrine as a mixed inhibitor. Using molecular docking studies, it was further proven that chelerythrine binds on both the catalytic site and the peripheral anionic site (PAS) of the AChE. In view of this, we went on to investigate its effect on inhibiting Aβ aggregation stimulated by AChE. Chelerythrine showed inhibition of fibril formation in the same range as propidium iodide. This approach enabled for the first time to identify a cholinesterase inhibitor of natural origin—chelerythrine—acting on AChE and BChE with a dual ability to inhibit Aβ aggregation as well as to disaggregate preformed Aβ aggregates. This compound could be an excellent starting point paving the way to develop more successful anti-AD drugs.     

Immediate Effects of Experimental Human Trampling on Mid-Upper Intertidal benthic Invertebrates at the Asinara Island MPA (NW Mediterranean)

To assess potential risks of human visitation to ecological communities, the immediate effects of human trampling were investigated experimentally on small invertebrates inhabiting mid-upper intertidal hard bottoms covered by algae. Two different experimental intensities of trampling (60 and 120 footsteps) and controls (with no trampling) were applied to quadrats 20×20 cm in size (experimental area), within the two ‘no-entry, no-take’ zones of the Asinara Island MPA (Italy, Mediterranean Sea). One day after trampling ended, samples of benthic fauna were collected and the animals attributed to macrofaunal and meiofaunal components. Analyses of variance on the nine most common taxa of macrofauna identified significant higher abundance of bivalves, gammarid amphipods, polychaetes, isopods, oligochaetes in controls than in trampled plots. For nematodes, polychaetes, ostracods, oligochaetes, bivalves, acari, caprellid amphipods and tanaids a significant higher abundance of meiofaunal animals was found in controls than in trampled areas. Although no information on recovery is available, these results suggest that macrofaunal and meiofaunal taxa are vulnerable to this type of disturbance.     

Comparable Long-Term Efficacy of Lopinavir/Ritonavir and Similar Drug-Resistance Profiles in Different HIV-1 Subtypes

Background

Analysis of potentially different impact of Lopinavir/Ritonavir (LPV/r) on non-B subtypes is confounded by dissimilarities in the conditions existing in different countries. We retrospectively compared its impact on populations infected with subtypes B and C in Israel, where patients infected with different subtypes receive the same treatment.

Methods

Clinical and demographic data were reported by physicians. Resistance was tested after treatment failure. Statistical analyses were conducted using SPSS.

Results

607 LPV/r treated patients (365 male) were included. 139 had HIV subtype B, 391 C, and 77 other subtypes. At study end 429 (71%) were receiving LPV/r. No significant differences in PI treatment history and in median viral-load (VL) at treatment initiation and termination existed between subtypes. MSM discontinued LPV/r more often than others even when the virologic outcome was good (p = 0.001). VL was below detection level in 81% of patients for whom LPV/r was first PI and in 67% when it was second (P = 0.001). Median VL decrease from baseline was 1.9±0.1 logs and was not significantly associated with subtype. Median CD4 increase was: 162 and 92cells/µl, respectively, for patients receiving LPV/r as first and second PI (P = 0.001), and 175 and 98, respectively, for subtypes B and C (P<0.001). Only 52 (22%) of 237 patients genotyped while under LPV/r were fully resistant to the drug; 12(5%) were partially resistant. In48%, population sequencing did not reveal resistance to any drug notwithstanding the virologic failure. No difference was found in the rates of resistance development between B and C (p = 0.16).

Conclusions

Treatment with LPV/r appeared efficient and tolerable in both subtypes, B and C, but CD4 recovery was significantly better in virologically suppressed subtype-B patients. In both subtypes, LPV/r was more beneficial when given as first PI. Mostly, reasons other than resistance development caused discontinuation of treatment.     

Identification of novel thiazolo[5,4-d]pyrimidine derivatives as human A1 and A2A adenosine receptor antagonists/inverse agonists

In this study a new set of thiazolo[5,4-d]pyrimidine derivatives was synthesized. These derivatives bear different substituents at positions 2 and 5 of the thiazolopyrimidine core while maintaining a free amino group at position-7. The new compounds were tested for their affinity and potency at human (h) A₁, A_2A, A_2B and A₃ adenosine receptors expressed in CHO cells. The results reveal that the higher affinity of these new set of thiazolopyrimidines is toward the hA₁ and hA_2A adenosine receptors subtypes and is tuned by the substitution pattern at both the 2 and 5 positions of the thiazolopyrimidine nucleus. Functional studies evidenced that the compounds behaved as dual A₁/A_2A antagonists/inverse agonists. Compound 3, bearing a 5-((2-methoxyphenyl) methylamino) group and a phenyl moiety at position 2, displayed the highest affinity (hA₁ K_i?=?10.2?nM; hA_2A K_i?=?4.72?nM) and behaved as a potent A₁/A_2A antagonist/inverse agonist (hA₁ IC₅₀?=?13.4?nM; hA_2A IC₅₀?=?5.34?nM).     

Plant sex and phenological stage affect interactions with rhizosphere nematode communities

Background: In dioecious plants, females and males associate differently with mycorrhizal fungi, but interactions with other rhizosphere organisms are less well studied.

Aims: We investigated the effect of plant sex on rhizosphere nematode communities associated with Corema album, a dioecious shrub occurring in coastal habitats.

Methods: Rhizosphere samples were collected from males and females in three populations (150 plants), during fruiting and flowering. Nematode communities were characterised and compared between plant sexes through statistical analyses of the abundance of trophic groups, plant parasitic nematode (PPN) genera and ecological indices.

Results: Free-living nematodes showed no statistically significant differences owing to plant sex. Conversely, PPN community composition was significantly different between plant sexes during fruiting but not flowering, suggesting that physiological requirements over the annual phenological cycle of the plant influence ecological interactions with the rhizosphere.

Of the 13 PPN genera identified, the ectoparasitic Criconema and Hemicriconemellawere more abundant in the rhizosphere of males during fruiting, whereas the endoparasitic Meloidogyne associated more frequently with females, suggesting that plant host suitability is related to PPN feeding strategy.

Conclusions: It appears that interactions of individuals of different sexes of C. album with the rhizosphere nematode community vary with phenological stage, especially for PPN.     

Molecular and morphological characterization of the predatory mite <Emphasis Type="Italic">Amblyseius largoensis</Emphasis> (Acari: Phytoseiidae): surprising similarity between an Asian and American populations

The accurate characterization of biological control agents is a key step in control programs. Recently, Amblyseius largoensis from Thailand were introduced in Brazil to evaluate their efficiency for the control of the red palm mite, Raoiella indica. The aim of this study was to confirm their identification and to characterize the population from Thailand, comparing it to populations of the Americas and Indian Ocean islands. In addition, a population of A. largoensis from New Caledonia, Oceania, of which DNA sequences were available, was included in phylogenetic analyses. Morphometric data obtained for the population of A. largoensis from Thailand were compared to those of populations from Reunion Island and the Americas through univariate and multivariate analyses. Two DNA fragments were amplified and sequenced: the nuclear ribosomal region ITSS and the mitochondrial 12S rRNA. Haplotypes (12S rRNA) and genotypes (ITSS) were identified and phylogenetic analyses using both fragments were conducted separately and combined using maximum likelihood and the Bayesian information criterion. The integrative approach reveals morphometric and molecular variabilities among populations of A. largoensis and shows that the population identified as A. largoensis collected in Thailand, as well as that from New Caledonia, are conspecific to the populations of the Americas and Indian Ocean islands. Populations from the Americas and Asia are more related to each other than with that from the Indian Ocean islands. Hypotheses to explain this clustering are proposed. Data on the molecular intraspecific variability of this predatory mite from remote areas will be helpful for the development of molecular diagnosis.     

Meiotic transmission of T-DNA genes inArabidopsis thaliana plants and their expression after 5-azacytidine treatment

Arabidopsis thaliana tumors were induced by octopine strain ofAgrobacterium tumefaciens B6S3 and its derivatives with modified T-DNA. Flowering shoots appeared sponta-neously onin vitro cultivated tumors and set seeds. R₁ and R₂ progeny of octopine synthesizing plants segregated in opine synthesis activities 3:1 and 15:1. Octopine synthase activity showed absolute linkage with agropine synthesis in most lines. In R₃ and R₄ progenies, the fraction of octopine synthase and agropine synthesis positive plants was lower than expected, but Mendelian segregation was restored if plants were cultivated on medium with 5-azacytidine. The most probable mechanism of disapearance of opine synthesis is cytosine methylation. The effect of 5-azacytidine lasted for at least next two generations.     

Crystal structure of Plasmodium falciparum thioredoxin reductase, a validated drug target

Plasmodium falciparum is the vector of the most prevalent and deadly form of malaria, and, among the Plasmodium species, it is the one with the highest rate of drug resistance. At the basis of a rational drug design project there is the selection and characterization of suitable target(s). Thioredoxin reductase, the first protection against reactive oxygen species in the erythrocytic phase of the parasite, is essential for its survival. Hence it represents a good target for the design of new anti-malarial active compounds. In this paper we present the first crystal structure of recombinant P. falciparum thioredoxin reductase (PfTrxR) at 2.9 Å and discuss its differences with respect to the human orthologue. The most important one resides in the dimer interface, which offers a good binding site for selective non competitive inhibitors. The striking conservation of this feature among the Plasmodium parasites, but not among other Apicomplexa parasites neither in mammals, boosts its exploitability.