Protocetraric and Salazinic Acids as Potential Inhibitors of SARS-CoV-2 3CL Protease: Biochemical, Cytotoxic, and Computational Characterization of Depsidones as Slow-Binding Inactivators

The study investigated the inhibitory activity of protocetraric and salazinic acids against SARS-CoV-2 3CL(pro). The kinetic parameters were determined by microtiter plate-reading fluorimeter using a fluorogenic substrate. The cytotoxic activity was tested on murine Sertoli TM4 cells. In silico analysis was performed to ascertain the nature of the binding with the 3CL(pro). The compounds are slow-binding inactivators of 3CL(pro) with a K(i) of 3.95 μM and 3.77 μM for protocetraric and salazinic acid, respectively, and inhibitory efficiency k(inact)/K(i) at about 3 × 10(−5) s(−1)µM(−1). The mechanism of inhibition shows that both compounds act as competitive inhibitors with the formation of a stable covalent adduct. The viability assay on epithelial cells revealed that none of them shows cytotoxicity up to 80 μM, which is well below the K(i) values. By molecular modelling, we predicted that the catalytic Cys145 makes a nucleophilic attack on the carbonyl carbon of the cyclic ester common to both inhibitors, forming a stably acyl-enzyme complex. The computational and kinetic analyses confirm the formation of a stable acyl-enzyme complex with 3CL(pro). The results obtained enrich the knowledge of the already numerous biological activities exhibited by lichen secondary metabolites, paving the way for developing promising scaffolds for the design of cysteine enzyme inhibitors

A global threats overview for Numeniini populations: synthesising expert knowledge for a group of declining migratory birds

The Numeniini is a tribe of thirteen wader species (Scolopacidae, Charadriiformes) of which seven are near-threatened or globally threatened, including two critically endangered. To help inform conservation management and policy responses, we present the results of an expert assessment of the threats that members of this taxonomic group face across migratory flyways. Most threats are increasing in intensity, particularly in non-breeding areas, where habitat loss resulting from residential and commercial development, aquaculture, mining, transport, disturbance, problematic invasive species, pollution and climate change were regarded as having the greatest detrimental impact. Fewer threats (mining, disturbance, problematic native species and climate change) were identified as widely affecting breeding areas. Numeniini populations face the greatest number of non-breeding threats in the East Asian-Australasian Flyway, especially those associated with coastal reclamation; related threats were also identified across the Central and Atlantic Americas, and East Atlantic flyways. Threats on the breeding grounds were greatest in Central and Atlantic Americas, East Atlantic and West Asian flyways. Three priority actions were associated with monitoring and research: to monitor breeding population trends (which for species breeding in remote areas may best be achieved through surveys at key non-breeding sites), to deploy tracking technologies to identify migratory connectivity, and to monitor land-cover change across breeding and non-breeding areas. Two priority actions were focused on conservation and policy responses: to identify and effectively protect key non-breeding sites across all flyways (particularly in the East Asian - Australasian Flyway), and to implement successful conservation interventions at a sufficient scale across human-dominated landscapes for species’ recovery to be achieved. If implemented urgently, these measures in combination have the potential to alter the current population declines of many Numeniini species and provide a template for the conservation of other groups of threatened species

Challenges faced by shorebird species using the inland wetlands of the East Asian-Australasian Flyway : The little curlew example

Asia is experiencing an alarming rate of inland wetlands loss, posing a risk to the future long-term survival of many species depending on these ecosystems. This review on the status and conservation of the little curlew (Numenius minutus) aims to draw attention to the conservation challenges faced by migratory shorebird species using the inland wetlands of the East Asian-Australasian Flyway (EAAF). Extensive and systematic research survey efforts along the EAAF have focused on species using coastal and tidal areas rather than on species using inland wetlands. Knowledge gaps include functional ecology and physiological responses to quality of food resources, population trends, migratory strategy and the role species play in supporting ecosystems resilience. Studies using remote sensing and geographic information system techniques to track the movements of birds along the flyway and to map habitat condition will prove essential in the future to allow a better understanding of the dynamics occurring at the stopover areas, how birds use resources and what competition pressures exist among species. Ultimately, these studies will contribute to our ability to predict changes and establish management practices for the long-term protection and conservation of the stopover areas for a suite of shorebird species using inland wetlands along the flyway. © CSIRO 2017

The influence of a mindfulness-based intervention on job satisfaction and work-related stress and anxiety

Workplace mindfulness is a recognised tool for enhancing health and well-being of university staff and may result in better task performance and satisfaction in the workplace. The study examined the beneficial effects of mindfulness meditation on job satisfaction, mindful awareness and anxiety levels in university personnel. Mixed methods with a quasi-experiment and in-depth interviews were used. Fifteen participants engaged in a mindfulness meditation approach and five volunteers were interviewed. The findings showed a significant increase in awareness, with staying focused (t(14) = –3.09, p =.00), noticing feelings of physical tension (t(14) = –4.00, p =.00), being aware of running automatically (t(14) = –3.55, p =.00) and not being preoccupied with the future or the past (t(14) = –2.69, p =.01), respectively. Mindfulness was also effective in reducing sleep disturbance. Qualitative results demonstrated the mindfulness approach contributed to calmness and relaxation, and increased ability to handle difficult matters in the workplace. Apart from helping participants to better manage emotions, the mindfulness intervention could promote better relationships towards family members and reduce blood pressure to normal levels. Therefore, mindfulness meditation should be promoted across academic settings to enhance job performance and satisfaction and reduce work-related stress

A multidisciplinary approach to the design of novel inhibitors for KPC-2

Background. The emergence and dissemination of multi drug resistant (MDR) Gram-negative pathogens resistant to all available antibiotics poses a significant threat in clinical therapy. Among them, Klebsiella Pneumoniae clinical isolates overexpressing KPC-2 carbapenemase are the most worrisome, extending bacterial resistance to last resort carbapenems. [1-2] Materials/methods. Four boronic acid derivatives were designed and tested in vitro vs KPC-2.[3] In biological assays their ability to synergize last generation antibiotics was evaluated. X-ray crystallography was applied to confirm binding orientation and new compounds ability to reach consensus-binding sites in carbapenemases (Figure 1). Results. For the most actives active compounds nanomolar affinity was achieved. The best inhibitor has nanomolar affinity for the enzyme, a ligand efficiency of 0.78 kcal mol–1 and a molecular weight of 158 Da validating it as lead-like molecule. In biological assays against Escherichia coli overexpressing KPC-2 new derivatives restored susceptibility to cefotaxime, aztreonam and last resort carbapenems. Two crystallographic binary complexes of the best inhibitors binding KPC-2 were obtained at high resolution. Conclusion. We investigate the molecular recognition requirements in KPC-2 active site by boronic acid derivatives. Kinetic descriptions of slow binding, time dependent inhibition and interactions geometries in KPC-2 were fully investigated. This study will guide further lead optimization and development of more effective KPC-2 inhibitors. Figure 1 References [1] Jean-Marie Frère, Eric Sauvage and Frédéric Kerff. “From “An enzyme able to destroy penicillin » to carbapenemases: 70 years of beta-lactamase misbehavior” Current Drug Targets, (2016). Volume 16. (E-pub ahead of print). [2] Tondi, D.; Cross, S.; Venturelli, A.; Costi, MP; Cruciani, G.; Spyrakis, F. Current Drug Targets 2016 17, no. 9 (2016) [3] Tondi,D.; Venturelli,A.; Bonnet,R.; Pozzi, C.; Shoichet, BK.; Costi, M.P. JMC. 2014. 57 (12), pp 5449–5458

Structure based drug design: the discovery of novel inhibitors active against New Delhi Metallo-β-lactamase-1

Background. The emergence of bacterial strains resistant to available antibiotic armamentarium is nowadays a pressing issue in clinical therapy. Among the several mechanisms of bacterial resistance, the rapid evolution and spread of carbapenemases, β-lactamases (BLs) with versatile hydrolytic capacities able to inactivate last resort carbapenems, represents a menace in treating highly resistant infections. [1-3] Carbapenem-resistant Enterobacteriaceae (CREs), reported with increased frequency, are progressively spreading throughout the World thus leaving no effective antibiotics. [1-3] In particular CREs overexpressing Metallo beta-lactamases (MBLs; i.e. NDM-1, VIM-2, IMP-1) are high-priority target pathogens for the development of novel antibacterials. Our studies focused on NDM-1: its substrate promiscuity, its resistance to available drugs, the easiness of variants appearance and transferability make NDM-1 one of the most worrisome BLs. [2] Moreover, respect to serine β-lactamases, no inhibitors for any MBLs are currently available in therapy. Materials/methods. Looking for new potential MBLs inhibitors we performed a structure-based in silico screening of commercially available library using FLAP, and identified several non β-lactam derivatives as promising candidates active against our target New Delhi metallo-beta-lactamase-1 NDM-1. Candidates were validated in vitro and investigated for their mechanism of inhibition. Results. The binding affinities of the highest scoring hits revealed, for several of them, micromolar inhibitory activity towards NDM-1. Among molecules selected for targeting NDM-1 few demonstrated an activity comparable to that provided by known inhibitors (Ki for the best-selected inhibitor equal to 0.72 μM; ). The identified inhibitors all share common non-covalent, competitive inhibition mechanism vs NDM-1. Conclusion. For the best ones, studies for improving their affinity and to investigate their potential to synergize beta-lactam antibiotics are ongoing. X-ray crystallography studies are now in progress to confirm our docking prediction and to deeply investigate the structural requirement necessary for proficuous hit to lead generation. Figure 1 References [1] Jean-Marie Frère, Eric Sauvage and Frédéric Kerff. “From “An enzyme able to destroy penicillin » to carbapenemases: 70 years of beta-lactamase misbehavior” Current Drug Targets, (2016). Volume 16. (E-pub ahead of print). [2] Robert A. Bonomo. “New Delhi Metallo-β-Lactamase and Multidrug Resistance: A Global SOS?”Clin Infect Dis. (2011) 52 (4): 485-487 [3] Donatella Tondi, Alberto Venturelli, Richard Bonnet, Cecilia Pozzi, Brian K Shoichet and Maria Paola Costi. “Targeting Serine Beta lactamases with a novel broad spectrum boronic acid”. Journal Medicinal Chemistry. (2014

Protolichesterinic acid enhances doxorubicin-induced apoptosis in HeLa cells in vitro

Aim The aim of this study was to investigate the effect of protolichesterinic acid, a lichen secondary metabolite, on anti-proliferative activity of doxorubicin in three human cancer cell lines, HeLa, SH-SY5Y and K562 cells. Main methods The data obtained from MTT assays, performed on cells treated with protolichesterinic acid and doxorubicin alone and in combination, were analysed by the median-effect method as proposed by Chou and Talalay and the Bliss independence model. Apoptosis rate was evaluated by fluorescence microscopy, caspase-3, 8 and 9 activities were detected by spectrofluorimetric analysis and protein expression of Bim, Bid, Bax and Mcl-2 was analysed by Western blotting. The interaction of protolichesterinic acid with thioesterase domain of human fatty acid synthase (hFAS) was investigated by a molecular docking study. Key findings The in vitro activity of doxorubicin against HeLa cancer cell line, but not against SH-SY5Y and K562 cells, was synergically increased by protolichesterinic acid. The increased cytotoxicity caused by protolichesterinic acid in HeLa cells was due to a pro-apoptotic effect and was associated to caspase-3, 8 and 9 activation. The simultaneous treatment for 24 h with protolichesterinic acid plus doxorubicin caused an increase of Bim protein expression and the appearance of cleaved form of Bid protein. The molecular modelling analysis showed that protolichesterinic acid seemed to behave as a competitive inhibitor of hFAS. Significance These results suggest that protolichesterinic acid could be envisaged as an useful tool against certain types of tumor cells in combination with anticancer drugs

Evaluation of the Analytical Performances of the Biolabo SOLEA 100 Optical Coagulometer and Comparison with the Stago STA-R MAX Analyser in the Determination of PT, APTT, and Fibrinogen

Introduction. The Biolabo Solea 100 is a fully automated coagulation analyser using an optical system to detect coagulation designed to meet the needs of small- and medium-sized laboratories. This study aimed to evaluate the analytical performance in terms of bias, precision, and interference of the Biolabo Solea 100 coagulometer under routine laboratory conditions. In addition, a comparison was made with Stago STA-R MAX. Materials and Methods. Imprecision and bias were evaluated for activated partial thromboplastin time (APTT), fibrinogen (FIB), and prothrombin time (PT) at the medical decision levels. The results of 200, 181, and 206 plasma samples for APTT, FIB, and PT, respectively, were compared with those obtained by Stago STA-R MAX. In addition, the interference level of bilirubin, haemoglobin, triglycerides, and fractionated heparin was evaluated. Results. Repeatability, intermediate imprecision, bias, and total error are overall below the defined limits of acceptability. Of interest is the high degree of agreement between Solea 100 and STA-R MAX with respect to PT (s), which fits perfectly with the theoretical line of identity (y = 0 + 1.00x). No interferences were found within the limits stated by the manufacturer, with some exceptions for APTT with heparin and APTT and PT for higher bilirubin concentrations. Conclusions. In conclusion, the performance of the Solea 100 optical analyser is satisfactory and adequate for the determination of routine coagulation tests. Moreover, they are perfectly comparable to mechanical systems, such as STA-R MAX and other upper-level analysers, even considering the low interference levels under routine conditions

Evaluation of the Analytical Performances of the Biolabo SOLEA 100 Optical Coagulometer and Comparison with the Stago STA-R MAX Analyser in the Determination of PT, APTT, and Fibrinogen

Introduction. The Biolabo Solea 100 is a fully automated coagulation analyser using an optical system to detect coagulation designed to meet the needs of small- and medium-sized laboratories. This study aimed to evaluate the analytical performance in terms of bias, precision, and interference of the Biolabo Solea 100 coagulometer under routine laboratory conditions. In addition, a comparison was made with Stago STA-R MAX. Materials and Methods. Imprecision and bias were evaluated for activated partial thromboplastin time (APTT), fibrinogen (FIB), and prothrombin time (PT) at the medical decision levels. The results of 200, 181, and 206 plasma samples for APTT, FIB, and PT, respectively, were compared with those obtained by Stago STA-R MAX. In addition, the interference level of bilirubin, haemoglobin, triglycerides, and fractionated heparin was evaluated. Results. Repeatability, intermediate imprecision, bias, and total error are overall below the defined limits of acceptability. Of interest is the high degree of agreement between Solea 100 and STA-R MAX with respect to PT (s), which fits perfectly with the theoretical line of identity (y = 0 + 1.00x). No interferences were found within the limits stated by the manufacturer, with some exceptions for APTT with heparin and APTT and PT for higher bilirubin concentrations. Conclusions. In conclusion, the performance of the Solea 100 optical analyser is satisfactory and adequate for the determination of routine coagulation tests. Moreover, they are perfectly comparable to mechanical systems, such as STA-R MAX and other upper-level analysers, even considering the low interference levels under routine conditions

Structure-based virtual screening for the discovery of novel inhibitors of New Delhi Metallo-β-lactamase-1

Bacterial resistance has become a worldwide concern after the emergence of metallo β-lactamases MBLs. They represent one of the major mechanisms of bacterial resistance against beta-lactam antibiotics. Among MBLs, New Delhi metallo-β-lactamase-1 NDM-1, the most prevalent type, is extremely efficient in inactivating nearly all-available antibiotics including last resort carbapenems. No inhibitors for NDM-1 are currently available in therapy, making the spread of NDM-1 producing bacterial strains a serious menace. In this perspective, we performed a structure-based in silico screening of a commercially available library using FLAPdock and identified several, non β-lactam derivatives as promising candidates active against NDM-1. The binding affinities of the highest scoring hits were measured in vitro revealing, for some of them, low micromolar affinity towards NDM-1. For the best inhibitors, efficacy against resistant bacterial strains overexpressing NDM-1 was validated, confirming their favorable synergistic effect in combination with the carbapenem MeropenemBacterial resistance has become a worldwide concern after the emergence of metallo-β-lactamases (MBLs). They represent one of the major mechanisms of bacterial resistance against beta-lactam antibiotics. Among MBLs, New Delhi metallo-β-lactamase-1 NDM-1, the most prevalent type, is extremely efficient in inactivating nearly all-available antibiotics including last resort carbapenems. No inhibitors for NDM-1 are currently available in therapy, making the spread of NDM-1 producing bacterial strains a serious menace. With this perspective, we performed a structure-based in silico screening of a commercially available library using FLAPdock and identified several, non-β-lactam derivatives as promising candidates active against NDM-1. The binding affinities of the highest scoring hits were measured in vitro revealing, for some of them, low micromolar affinity toward NDM-1. For the best inhibitors, efficacy against resistant bacterial strains overexpressing NDM-1 was validated, confirming their favorable synergistic effect in combination with the carbapenem Meropenem