Targeting RORs nuclear receptors by novel synthetic steroidal inverse agonists for autoimmune disorders

Designing novel inverse agonists of NR RORγt still represents a challenge for the pharmaceutical community to develop therapeutics for treating immune diseases. By exploring the structure of NRs natural ligands, the representative arotenoid ligands and RORs specific ligands share some chemical homologies which can be exploited to design a novel molecular structure characterized by a polycyclic core bearing a polar head and a hydrophobic tail. Compound MG 2778 (8), a cyclopenta[a]phenantrene derivative, was identified as lead compound which was chemically modified at position 2 in order to obtain a small library for preliminary SARs. Cell viability and estrogenic activity of compounds 7, 8, 19a, 30, 31 and 32 were evaluated to attest selectivity. The selected 7, 8, 19a and 31 compounds were assayed in a Gal4 UAS-Luc co-transfection system in order to determine their ability to modulate RORγt activity in a cellular environment. They were evaluated as inverse agonists taken ursolic acid as reference compound. The potency of compounds was lower than that of ursolic acid, but their efficacy was similar. Compound 19a was the most active, significantly reducing RORγt activity at low micromolar concentrations.     

How to meet the 2020 GSPC target 8 in Europe: priority-setting for seed banking of native threatened plants

The contribution of the European Native Seed Conservation Network (ENSCONET, 2004-2009) and the ENSCONET Consortium (since 2010) towards meeting the 2020 Global Strategy for Plant Conservation (GSPC) target 8 was assessed in 2017. While the outcome was positive (62.7% of European threatened species already conserved ex situ in seed banks), the analysis showed that it was essential to provide guidance on which European native threatened species should be collected as a priority if the target was to be reached by 2020. In this paper we present a priority-setting method and its result, designed to guide collecting strategies across Europe to meet the 2020 GSPC target 8. The result of our study is a country-based checklist of European threatened taxa to be collected and stored ex situ across the seed banks of the ENSCONET Consortium by 2020. After discussing the results of the applied method, the ENSCONET Consortium Steering Committee has identified some key action points to support the implementation of such a collecting strategy across Europe in order to meet the 2020 GSPC target 8 for Europe.     

Carbonic anhydrase inhibitors. Inhibition of the Rv1284 and Rv3273 β-carbonic anhydrases from Mycobacterium tuberculosis with diazenylbenzenesulfonamides

A series of diazenylbenzenesulfonamides obtained from sulfanilamide or metanilamide by diazotization followed by coupling with phenols or amines, was tested for the inhibition of the β-carbonic anhydrases (CAs, EC 4.2.1.1) encoded by the genes Rv1284 and Rv3273 of Mycobacterium tuberculosis. Several low micromolar inhibitors of the two enzymes were detected, with prontosil being the best inhibitor (K_Is of 126–148 nM). Inhibition of pathogenic β-CAs may lead to the development of antiinfectives with a new mechanism of action, devoid of resistance problems encountered with classical antibiotics.     

Effect of hydrostatic pressure on morphological and ultrastructural aspects of normal and osteoarthritic human articular chondrocytes.

In this work we studied the morphological and ultrastructural aspects of normal and osteoarthritic (OA) human articular chondrocytes cultivated in alginate gel for 48 hours. After this period the chondrocytes in Petri dishes were exposed to cyclic pressurization (minimum pressure 1 MPa and maximum pressure 5 MPa) at 0.25 Hz frequency for three hours. In other loading procedures the cells were exposed to continuous pressure (24 MPa) for three hours. Some dishes were not pressurised and these served as controls. The cells were then fixed for transmission electron microscopy (T.E.M.) and for scanning electron microscopy (S.E.M.). No ultrastructural changes were observed in normal chondrocytes exposed at physiological pressure. OA cells placed under physiological pressure showed a partial recovery on morphological and ultrastructural aspects. Normal and OA samples exposed to continuous pressure (24 MPa) showed a morphological worsening in both T.E.M. and S.E.M. studies.     

Heterologous expression and biochemical characterisation of the recombinant β-carbonic anhydrase (MpaCA) from the warm-blooded vertebrate pathogen malassezia pachydermatis

Warm-blooded animals may have Malassezia pachydermatis on healthy skin, but changes in the skin microenvironment or host defences induce this opportunistic commensal to become pathogenic. Malassezia infections in humans and animals are commonly treated with azole antifungals. Fungistatic treatments, together with their long-term use, contribute to the selection and the establishment of drug-resistant fungi. To counteract this rising problem, researchers must find new antifungal drugs and enhance drug resistance management strategies. Cyclic adenosine monophosphate, adenylyl cyclase, and bicarbonate have been found to promote fungal virulence, adhesion, hydrolase synthesis, and host cell death. The CO₂/HCO₃^-/pH-sensing in fungi is triggered by HCO₃^- produced by metalloenzymes carbonic anhydrases (CAs, EC 4.2.1.1). It has been demonstrated that the growth of M. globosa can be inhibited in vivo by primary sulphonamides, which are the typical CA inhibitors. Here, we report the cloning, purification, and characterisation of the β-CA (MpaCA) from the pathogenic fungus M. pachydermatis, which is homologous to the enzyme encoded in the genome of M. globosa and M. restricta, that are responsible for dandruff and seborrhoeic dermatitis. Fungal CAs could be thus considered a new pharmacological target for combating fungal infections and drug resistance developed by most fungi to the already used drugs.     

Dithiocarbamates effectively inhibit the α-carbonic anhydrase from Neisseria gonorrhoeae

Recently, inorganic anions and sulphonamides, two of the main classes of zinc-binding carbonic anhydrase inhibitors (CAIs), were investigated for inhibition of the α-class carbonic anhydrase (CA, EC 4.2.1.1) from Neisseria gonorrhoeae, NgCA. As an extension to our previous studies, we report that dithiocarbamates (DTCs) derived from primary or secondary amines constitute a class of efficient inhibitors of NgCA. K_Is ranging between 83.7 and 827 nM were measured for a series of 31 DTCs that incorporated various aliphatic, aromatic, and heterocyclic scaffolds. A subset of DTCs were selected for antimicrobial testing against N. gonorrhoeae, and three molecules displayed minimum inhibitory concentration (MIC) values less than or equal to 8 µg/mL. As NgCA was recently validated as an antibacterial drug target, the DTCs may lead to development of novel antigonococcal agents.     

Lack of evidence of paratuberculosis in wild canids from Southwestern Europe

Wild carnivores are at the top of the trophic chain. They are predators and carrion consumers, and thus, prone to come in contact with disease agents contaminating the environment or infecting live or dead animals. We hypothesized that wild canids could be used as sentinels for the detection of regions with higher Mycobacterium avium paratuberculosis (MAP) prevalence in wild and domestic animals. To test this hypothesis, we set up an ELISA to test 262 wolf (Canis lupus) and fox (Vulpes vulpes) sera for MAP-specific antibodies and processed a subset of samples for culture (n = 61), MAP-specific PCR (15) and histopathology (14). In wolves, the optical density (OD) values in the ELISA were continuously distributed. Ten fox sera (4%) had OD readings of over twice the mean, suggesting contact with mycobacteria. However, all samples tested by PCR were negative for both IS900 and ISMAP02 sequences, and samples cultured for MAP yielded no growth. No visible paratuberculosis or tuberculosis-compatible lesions were recorded. On histopathological examination, no lesions compatible with mycobacterial diseases were observed. These results suggest that wild canids show little or no evidence of paratuberculosis and are unlikely to be useful sentinels for the detection of MAP in Southwestern Europe.

     

Comparative seed dormancy and germination of eight annual species of ephemeral wetland vegetation in a Mediterranean climate

Ephemeral wetland vegetation (EWV) in the Mediterranean Basin appears in temporary wetlands where favourable hydrological conditions exist only for a short time and year-to-year variability is high. Here, we report results of the seed germination, dormancy and desiccation tolerance of eight annual species living in this vulnerable habitat. Experiments were performed in laboratory conditions under constant and alternating temperatures and using a 12-h daily photoperiod or continuous darkness. Whilst germination and dormancy differed between the species, seeds demonstrated an absolute light requirement and prefer cool temperatures to germinate (mean ≤15 °C). Logistic regression analysis showed significant effects of alternating temperature in all the species except in Tillaea vaillantii whose germination was stimulated by constant temperature. Mean temperature was a significant term in the logistic models for the dormant species Cicendia filiformis, Linum radiola and T. vaillantii for which after-ripening was an effective dormancy-breaking treatment. From these results we infer three strategies of regeneration by seeds: (1) species germinating during the whole vegetative season (2) species germinating in a narrow temperature niche and (3) species requiring flooding (T. vaillantii). Seeds possessed orthodox storage behaviour (tolerating drying to 15 % relative humidity) and may be amenable to seed banking as a means of ex-situ conservation. We conclude that EWV species are adapted to the irregular presence of water with characteristics that are typical of neither truly aquatic nor wetland plants. These EWV species showed a more plastic germination response based on alternating and constant temperature sensitivity and a low proportion of dormant seeds.     

Synthesis of esters using a nylon-immobilized lipase in batch and continuous reactors.

Direct esterifications using a nylon-immobilized lipase from Candida cylindracea were carried out in batch and continuous-flow reactors. The immobilized enzyme was effective in catalyzing the synthesis of ethylpropionate, isoamylpropionate, and isoamylbutyrate. With ethanol dissolved in hexane as a substrate, the maximum initial esterification rate was 0.02 mole/(h x g of immobilized protein), but the enzyme was stable only when the substrate concentrations were lower than 0.2 M. With isoamyl alcohol in hexane as a substrate, esterification rates as high as 0.085 mole/(h x g of immobilized protein) were observed and the immobilized enzyme was stable over a much broader concentration range. However, in this case, the use of a solvent, such as hexane, was not necessary for esterification, and the enzyme could be employed in equimolar acid/alcohol mixtures. A packed-bed reactor was operated successfully for the continuous synthesis of esters. The reactor was stable for long periods of time, and the steady-state performance could be accurately predicted on the basis of batch reaction experiments.