Probing intraligand and charge transfer excited states of fac-[Re(R)(CO)(3)(CO(2)Et-dppz)](+) (R = py, 4-Me(2)N-py; CO(2)Et-dppz = dipyrido[3,2a:2',3'c]phenazine-11-carboxylic ethyl ester) using time-resolved infrared spectroscopy.

The photophysics of fac-[Re(R)(CO)(3)(CO(2)Et-dppz)](+) (R = py (), 4-Me(2)N-py (); CO(2)Et-dppz = dipyrido[3,2a:2',3'c]phenazine-11-carboxylic ethyl ester) was studied with luminescence spectroscopy and time-resolved infrared (TRIR) spectroscopy in the metal carbonyl (2,100-1,800 cm(-1)) and organic ester (1,800-1,600 cm(-1)) regions. For 1, the picosecond TRIR spectra in the metal carbonyl region provided evidence for the formation of an intra-ligand IL (pi-pi) excited state, which partially decays to an equilibrium with the metal-to-ligand charge transfer (MLCT) excited state. For 2 it is evident that both IL (pi-pi) and MLCT excited states are formed within 2 ps of excitation. The magnitude of the nu(CO) shift in the metal carbonyl region following excitation allows the MLCT excited states to be described more precisely as a dpi(Re) -->pi (phenazine) (3)MLCT state for 1 and as a dpi(Re) -->pi (phenanthroline) (3)MLCT state for 2.     

Acyldepsipeptide antibiotics kill mycobacteria by preventing the physiological functions of the ClpP1P2 protease

The Clp protease complex in Mycobacterium tuberculosis is unusual in its composition, functional importance and activation mechanism. Whilst most bacterial species contain a single ClpP protein that is dispensable for normal growth, mycobacteria have two ClpPs, ClpP1 and ClpP2, which are essential for viability and together form the ClpP1P2 tetradecamer. Acyldepsipeptide antibiotics of the ADEP class inhibit the growth of Gram‐positive firmicutes by activating ClpP and causing unregulated protein degradation. Here we show that, in contrast, mycobacteria are killed by ADEP through inhibition of ClpP function. Although ADEPs can stimulate purified M. tuberculosis ClpP1P2 to degrade larger peptides and unstructured proteins, this effect is weaker than for ClpP from other bacteria and depends on the presence of an additional activating factor (e.g. the dipeptide benzyloxycarbonyl‐leucyl‐leucine in vitro) to form the active ClpP1P2 tetradecamer. The cell division protein FtsZ, which is a particularly sensitive target for ADEP‐activated ClpP in firmicutes, is not degraded in mycobacteria. Depletion of the ClpP1P2 level in a conditional Mycobacterium bovis BCG mutant enhanced killing by ADEP unlike in other bacteria. In summary, ADEPs kill mycobacteria by preventing interaction of ClpP1P2 with the regulatory ATPases, ClpX or ClpC1, thus inhibiting essential ATP‐dependent protein degradation.     

The acylation state of surface lipoproteins of mollicute Acholeplasma laidlawii

Acylation of the N-terminal Cys residue is an essential, ubiquitous, and uniquely bacterial posttranslational modification that allows anchoring of proteins to the lipid membrane. In gram-negative bacteria, acylation proceeds through three sequential steps requiring lipoprotein diacylglyceryltransferase, lipoprotein signal peptidase, and finally lipoprotein N-acyltransferase. The apparent lack of genes coding for recognizable homologs of lipoprotein N-acyltransferase in gram-positive bacteria and Mollicutes suggests that the final step of the protein acylation process may be absent in these organisms. In this work, we monitored the acylation state of eight major lipoproteins of the mollicute Acholeplasma laidlawii using a combination of standard two-dimensional gel electrophoresis protein separation, blotting to nitrocellulose membranes, and MALDI-MS identification of modified N-terminal tryptic peptides. We show that for each A. laidlawii lipoprotein studied a third fatty acid in an amide linkage on the N-terminal Cys residue is present, whereas diacylated species were not detected. The result thus proves that A. laidlawii encodes a lipoprotein N-acyltransferase activity. We hypothesize that N-acyltransferases encoded by genes non-homologous to N-acyltransferases of gram-negative bacteria are also present in other mollicutes and gram-positive bacteria.     

Genetics of VEGF serum variation in human isolated populations of cilento: importance of VEGF polymorphisms

Vascular Endothelial Growth Factor (VEGF) is the main player in angiogenesis. Because of its crucial role in this process, the study of the genetic factors controlling VEGF variability may be of particular interest for many angiogenesis-associated diseases. Although some polymorphisms in the VEGF gene have been associated with a susceptibility to several disorders, no genome-wide search on VEGF serum levels has been reported so far. We carried out a genome-wide linkage analysis in three isolated populations and we detected a strong linkage between VEGF serum levels and the 6p21.1 VEGF region in all samples. A new locus on chromosome 3p26.3 significantly linked to VEGF serum levels was also detected in a combined population sample. A sequencing of the gene followed by an association study identified three common single nucleotide polymorphisms (SNPs) influencing VEGF serum levels in one population (Campora), two already reported in the literature (rs3025039, rs25648) and one new signal (rs3025020). A fourth SNP (rs41282644) was found to affect VEGF serum levels in another population (Cardile). All the identified SNPs contribute to the related population linkages (35% of the linkage explained in Campora and 15% in Cardile). Interestingly, none of the SNPs influencing VEGF serum levels in one population was found to be associated in the two other populations. These results allow us to exclude the hypothesis that the common variants located in the exons, intron-exon junctions, promoter and regulative regions of the VEGF gene may have a causal effect on the VEGF variation. The data support the alternative hypothesis of a multiple rare variant model, possibly consisting in distinct variants in different populations, influencing VEGF serum levels.     

Antimicrobial benzodiazepine‐based short cationic peptidomimetics

Antimicrobial peptides (AMPs) appear to be good candidates for the development of new antibiotic drugs. We describe here the synthesis of peptidomimetic compounds that are based on a benzodiazepine scaffold flanked with positively charged and hydrophobic amino acids. These compounds mimic the essential properties of cationic AMPs. The new design possesses the benzodiazepine scaffold that is comprised of two glycine amino acids and which confers flexibility and aromatic hydrophobic ‘back’, and two arms used for further synthesis on solid phase for incorporation of charged and hydrophobic amino acids. This approach allowed us a better understanding of the influence of these features on the antimicrobial activity and selectivity. A novel compound was discovered which has MICs of 12.5 µg/ml against Staphylococcus aureus and 25 µg/ml against Escherichia coli, similar to the well‐known antimicrobial peptide MSI‐78. In contrast to MSI‐78, the above mentioned compound has lower lytic effect against mammalian red blood cells. These peptidomimetic compounds will pave the way for future design of potent synthetic mimics of AMPs for therapeutic and biomedical applications. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

Modulation of amyloid-beta-induced and age-associated changes in rat hippocampus by eicosapentaenoic acid

The age-related deficit in long-term potentiation (LTP) in the dentate gyrus is positively correlated with hippocampal concentration of the pro-inflammatory cytokine, interleukin-1beta (IL-1beta). Previous evidence also indicates that the inhibition of LTP induced by intracerebroventricular injection of amyloid-beta(1-40) (Abeta) is accompanied by increased hippocampal IL-1beta concentration and IL-1beta-stimulated signalling, specifically activation of the stress-activated protein kinase, c-jun N-terminal kinase (JNK). We considered that the underlying age-related neuroinflammation may render older rats more susceptible to Abeta administration and, to investigate this, young, middle-aged and aged rats were injected intracerebroventricularly with Abeta or vehicle. Hippocampal IL-1beta concentration, JNK phosphorylation, expression of the putative Abeta receptor, Receptor for advanced glycation end products (RAGE) and the microglial cell surface marker, CD40 were assessed. We report that Abeta inhibited LTP in a concentration-dependent manner in young rats and that this was accompanied by concentration-dependent increases in hippocampal IL-1beta and expression of phosphorylated JNK, RAGE and CD40. While 20 micromol/L Abeta exerted no significant effect on LTP in young rats, it inhibited LTP in middle-aged and aged rats and the increased vulnerability of aged rats was associated with increased IL-1beta concentration. Treatment of rats with eicosapentaenoic acid attenuated the inhibitory effect of 60 micromol/L Abeta on LTP in young rats and the effect of 20 micromol/L Abeta in middle-aged and aged rats. We present evidence which indicates that the effect of eicosapentaenoic acid may be linked with its ability to stimulate activation of peroxisome proliferator-activated receptor gamma.     

Enhanced control of pathogenic Simian immunodeficiency virus SIVmac239 replication in macaques immunized with an interleukin-12 plasmid and a DNA prime-viral vector boost vaccine regimen

DNA priming has previously been shown to elicit augmented immune responses when administered by electroporation (EP) or codelivered with a plasmid encoding interleukin-12 (pIL-12). We hypothesized that the efficacy of a DNA prime and recombinant adenovirus 5 boost vaccination regimen (DNA/rAd5) would be improved when incorporating these vaccination strategies into the DNA priming phase, as determined by pathogenic simian immunodeficiency virus SIVmac239 challenge outcome. The whole SIVmac239 proteome was delivered in 5 separate DNA plasmids (pDNA-SIV) by EP with or without pIL-12, followed by boosting 4 months later with corresponding rAd5-SIV vaccine vectors. Remarkably, after repeated low-dose SIVmac239 mucosal challenge, we demonstrate 2.6 and 4.4 log reductions of the median SIV peak and set point viral loads in rhesus macaques (RMs) that received pDNA-SIV by EP with pIL-12 compared to the median peak and set point viral loads in mock-immunized controls (P < 0.01). In 5 out of 6 infected RMs, strong suppression of viremia was observed, with intermittent "blips" in virus replication. In 2 RMs, we could not detect the presence of SIV RNA in tissue and lymph nodes, even after 13 viral challenges. RMs immunized without pIL-12 demonstrated a typical maximum of 1.5 log reduction in virus load. There was no significant difference in the overall magnitude of SIV-specific antibodies or CD8 T-cell responses between groups; however, pDNA delivery by EP with pIL-12 induced a greater magnitude of SIV-specific CD4 T cells that produced multiple cytokines. This vaccine strategy is relevant for existing vaccine candidates entering clinical evaluation, and this model may provide insights into control of retrovirus replication.     

Airborne pollen in Padua (NE-Italy): A comparison between two pollen samplers

During recent years a gradual decrease inallergenic airborne pollen concentration hasbeen observed in the monitoring station ofPadua (Italy). Because technical checks of thesampler were not able to explain this trend,the results obtained from two twinpollen-samplers (Lanzoni VPPS 2000), placed twometres apart, were compared.An eight-week sampling was carried out duringthe year 2000 from July to September.Subsequent analysis revealed no statisticallysignificant difference between the dataobtained with the two instruments. On the otherhand, both samplers captured high levels offungal spores. We conclude that the observednegative trend in pollen count is real and notrelated to technical biases.     

Shifts in phenology of Bythotrephes longimanus and its modern success in Lake Maggiore as a result of changes in climate and trophy

In Lake Maggiore, the density of the invertebrate predator Bythotrepheslongimanus increased following lake re-oligotrophication inthe late 1980s. This "invasion" was followed by dramatic changesin the pelagic food web, consistent with those that followedthe establishment of B. longimanus in North American lakes whereit is not native. In this contribution, we explore the modernsuccess of B. longimanus in Lake Maggiore by investigating itsphenology and population density, and their correlations withabiotic and biotic factors during the period from 1981 to 2003.A 10-fold increase in the abundance of B. longimanus followedan earlier start and longer duration of annual population growth.Increased prey resources and decreased predation pressure werenot observed during the B. longimanus density increase. Instead,a rise in lake temperature may have altered the reproductivecycle of this species. Furthermore, the depth and duration ofa refuge from visually orienting fish predators increased duringthese 20 years, as a result of changes in the thermal stratificationregime of the lake. This case study provides a timely exampleof how climatic changes may interact with biotic drivers (e.g.fish predation) to influence the density and phenology of aninvertebrate predator.