Accurate calculation of affinity changes to the close state of influenza A M2 transmembrane domain in response to subtle structural changes of adamantyl amines using free energy perturbation methods in different lipid bilayers.

Experimental binding free energies of 27 adamantyl amines against the influenza M2(22-46) WT tetramer, in its closed form at pH 8, were measured by ITC in DPC micelles. The measured Kd's range is ~44 while the antiviral potencies (IC50) range is ~750 with a good correlation between binding free energies computed with Kd and IC50 values (r = 0.76). We explored with MD simulations (ff19sb, CHARMM36m) the binding profile of complexes with strong, moderate and weak binders embedded in DMPC, DPPC, POPC or a viral mimetic membrane and using different experimental starting structures of M2. To predict accurately differences in binding free energy in response to subtle changes in the structure of the ligands, we performed 18 alchemical perturbative single topology FEP/MD NPT simulations (OPLS2005) using the BAR estimator (Desmond software) and 20 dual topology calculations TI/MD NVT simulations (ff19sb) using the MBAR estimator (Amber software) for adamantyl amines in complex with M2(22-46) WT in DMPC, DPPC, POPC. We observed that both methods with all lipids show a very good correlation between the experimental and calculated relative binding free energies (r = 0.77-0.87, mue = 0.36-0.92 kcal mol-1) with the highest performance achieved with TI/MBAR and lowest performance with FEP/BAR in DMPC bilayers. When antiviral potencies are used instead of the Kd values for computing the experimental binding free energies we obtained also good performance with both FEP/BAR (r = 0.83, mue = 0.75 kcal mol-1) and TI/MBAR (r = 0.69, mue = 0.77 kcal mol-1).

A Study of the Activity of Adamantyl Amines against Mutant Influenza A M2 Channels Identified a Polycyclic Cage Amine Triple Blocker, Explored by Molecular Dynamics Simulations and Solid-State NMR.

We compared the anti-influenza potencies of 57 adamantyl amines and analogs against influenza A virus with serine-31 M2 proton channel, usually termed as WT M2 channel, which is amantadine sensitive. We also tested a subset of these compounds against viruses with the amantadine-resistant L26F, V27A, A30T, G34E M2 mutant channels. Four compounds inhibited WT M2 virus in vitro with mid-nanomolar potency, with 27 compounds showing sub-micromolar to low micromolar potency. Several compounds inhibited L26F M2 virus in vitro with sub-micromolar to low micromolar potency, but only three compounds blocked L26F M2-mediated proton current as determined by electrophysiology (EP). One compound was found to be a triple blocker of WT, L26F, V27A M2 channels by EP assays, but did not inhibit V27A M2 virus in vitro, and one compound inhibited WT, L26F, V27A M2 in vitro without blocking V27A M2 channel. One compound blocked only L26F M2 channel by EP, but did not inhibit virus replication. The triple blocker compound is as long as rimantadine, but could bind and block V27A M2 channel due to its larger girth as revealed by molecular dynamics simulations, while MAS NMR informed on the interaction of the compound with M2(18-60) WT or L26F or V27A.

Heat-related mortality in Cyprus for current and future climate scenarios.

Extreme temperatures have long been associated with adverse health impacts, ranging from minor illness, to increased hospitalizations and mortality. Heat-related mortality during summer months is likely to become an increasing public health problem in future due to the effects of climate change. We performed a health impact assessment for heat-related mortality for the warm months of April-September for the years 2004 to 2009 inclusive, for the city of Nicosia and for Cyprus as a whole, based on separately derived exposure-response functions. We further estimated the potential future heat-related mortality by including climate projections for southern Europe, which suggest changes in temperature of between 1°C and 5°C over the next century. There were 32 heat-related deaths per year in Cyprus over the study period. When adding the projected increase in temperature due to climate change, there was a substantial increase in mortality: for a 1°C increase in temperature, heat related mortality in Cyprus was estimated to double to 64 per year, and for a 5°C increase, heat-related mortality was expected to be 8 times the baseline rate for the warm season (281 compared with 32). This analysis highlights the importance of preparing for potential health impacts due to heat in Cyprus, particularly under a changing climate.

Rats as indicators of the presence and dispersal of pathogens in Cyprus: ectoparasites, parasitic helminths, enteric bacteria, and encephalomyocarditis virus.

Rodents play an active role in the transmission of a number of zoonoses by harboring and disseminating the pathogens involved, either through their biological materials or via their ectoparasites. Hence, the spatial and seasonal distribution of potential agents of zoonoses may be studied by examining their distribution in the rodent vectors and their ectoparasites. A surveillance was conducted in wild rodents in 51 different areas of Cyprus, an island ecosystem, to monitor the reemergence of Echinococcus granulosus and Encephalomyocarditis virus (pathogens that have been eradicated from Cyprus), to study the presence and dispersal of Salmonella spp. (a bacterium found in patients and poultry in the island), as well as to investigate the presence of helminth parasites and rodent ectoparasites. Biological material collected from 625 rodents, examined macroscopically, microscopically, and after culture, showed that the most widespread pathogens encountered in wild rats (Rattus rattus and Rattus norvegicus) were helminth parasites, found in 92 animals (three helminth species found for the first time in Cyprus: Cysticercus fasciolaris, Hymenolepis diminuta, and Physalloptera spp.), and Salmonella spp., detected in the intestine of 56 rats (12 different Salmonella spp. and serotypes). None of the rodents were found infected with the cestode Echinococcus or Encephalomyocarditis virus, indicating that the control measures taken by the Veterinary Services on the island prevented its reestablishment despite changing conditions. The rodents were also free of the nematode Trichinella. Over 40% of the rats collected were infested with fleas, mainly Xenopsylla cheopis. The results, analyzed using the Geographical Information System technology, revealed two of the areas studied as high risk for public health.