Antifungal activity of the ribosome-inactivating protein BE27 from sugar beet (Beta vulgaris†L.) against the green mould Penicillium digitatum.

The ribosome-inactivating protein BE27 from sugar beet (Beta vulgaris L.) leaves is an apoplastic protein induced by signalling compounds, such as hydrogen peroxide and salicylic acid, which has been reported to be involved in defence against viruses. Here, we report that, at a concentration much lower than that present in the apoplast, BE27 displays antifungal activity against the green mould Penicillium digitatum, a necrotrophic fungus that colonizes wounds and grows in the inter- and intracellular spaces of the tissues of several edible plants. BE27 is able to enter into the cytosol and kill fungal cells, thus arresting the growth of the fungus. The mechanism of action seems to involve ribosomal RNA (rRNA) N-glycosylase activity on the sarcin-ricin loop of the major rRNA which inactivates irreversibly the fungal ribosomes, thus inhibiting protein synthesis. We compared the C-terminus of the BE27 structure with antifungal plant defensins and hypothesize that a structural motif composed of an α-helix and a ß-hairpin, similar to the γ-core motif of defensins, might contribute to the specific interaction with the fungal plasma membranes, allowing the protein to enter into the cell.

Snake Venomics and Antivenomics of Bothrops diporus, a Medically Important Pitviper in Northeastern Argentina.

Snake species within genus Bothrops are responsible for more than 80% of the snakebites occurring in South America. The species that cause most envenomings in Argentina, B. diporus, is widely distributed throughout the country, but principally found in the Northeast, the region with the highest rates of snakebites. The venom proteome of this medically relevant snake was unveiled using a venomic approach. It comprises toxins belonging to fourteen protein families, being dominated by PI- and PIII-SVMPs, PLA2 molecules, BPP-like peptides, L-amino acid oxidase and serine proteinases. This toxin profile largely explains the characteristic pathophysiological effects of bothropic snakebites observed in patients envenomed by B. diporus. Antivenomic analysis of the SAB antivenom (Instituto Vital Brazil) against the venom of B. diporus showed that this pentabothropic antivenom efficiently recognized all the venom proteins and exhibited poor affinity towards the small peptide (BPPs and tripeptide inhibitors of PIII-SVMPs) components of the venom.

Phospholipase A(2) enhances the endothelial cell detachment effect of a snake venom metalloproteinase in the absence of catalysis.

Microvessel disruption leading to hemorrhage stands among the most dangerous consequences of envenomings by snakes of the family Viperidae. A PIII metalloproteinase (SVMP), balteragin, purified from the venom of the snake Bothrops alternatus, displays a potent hemorrhagic effect, and a moderate myotoxicity in vivo. Previous studies described the ability of this SVMP to induce the detachment of C2C12 myoblasts in culture, without causing cytolysis. Surprisingly, a purified acidic phospholipase A2 (PLA2) from the same venom was found to increase this detaching activity of the SVMP on myoblasts. Since endothelial cells are a natural target of SVMPs in vivo, the possibility that this synergistic effect is also observed on this cell type was explored in the present work. In addition, a first approach of the mechanism of action of this effect was studied. Results clearly confirm that the acidic PLA2, despite lacking toxicity towards endothelial cells, significantly enhances the detaching effect of the SVMP even at a concentration as low as 1 µg/mL. Inhibition of enzymatic activity of the PLA2 by chemical modification with p-bromophenacyl bromide did not affect the synergistic activity, suggesting that this effect is not dependent on phospholipase enzymatic activity and may instead be the consequence of an interaction of the PLA2 with endothelial cell plasma membrane. To our knowledge, this is the first report of a synergistic action of a non toxic PLA2 in enhancing the detachment of endothelial cells induced by a metalloproteinase.

Combatting resistance in intensive care: the multimodal approach of the Spanish ICU "Zero Resistance" program.

This article is one of ten reviews selected from the Annual Update in Intensive Care and Emergency Medicine 2015 and co-published as a series in Critical Care. Other articles in the series can be found online at http://ccforum.com/series/annualupdate2015. Further information about the Annual Update in Intensive Care and Emergency Medicine is available from http://www.springer.com/series/8901.

[cycEVA study: case control study measuring influenza vaccine effectiveness in Spain, 2008-2013]. / Estudio cycEVA: casos y controles para la estimación de la efectividad de la vacuna antigripal en España, 2008-2013.

BACKGROUND: In Spain, influenza vaccine effectiveness (EV) is estimated since 2008-09 season through the cycEVA case-control study, the Spanish component of the European I-MOVE (Monitoring Influenza Vaccine Effectiveness in the EU/EEA) network. We aimed at describing cycEVA performance in its five consolidated editions 2008/09 -; 2012/13. METHODS: During the study period the following indicators were analysed: 1) the participation of sentinel general practitioners and pediatricians (MP), 2) the population studied and the study period, 3) the data quality and 4) the dissemination of the cycEVA results. Trend analysis of the indicators was done using the Cochran-Armitage test to compute the Annual Percentage Change (PCA). RESULTS: The number of participating MP increased from 164 in 2008-09 to 246 in the following editions. The percentage of MP recruiting at least one patient increased significantly annually (PCA = 15.33%). The percentage of recruited patients included into the analysis increased (PCA=5.91%) from 77% in 2008-09 to more than 95% in the following editions. The percentage of cycEVA patients contributing to the I-MOVE study ranged between 23% and 30% in the pilot and 2011-12 editions respectively.. Final results were disseminated in quartile 2 peer-reviewed journals and 2010-11 and 2011-12 preliminary EV estimates were published in quartile 1 journals. cycEVA publications received 97 citations. CONCLUSION: cycEVA study achieved more quality information, timely EV estimates and a higher impact of the results.

Effect of monospecific antibodies against baltergin in myotoxicity induced by Bothrops alternatus venom from northeast of Argentina. Role of metalloproteinases in muscle damage.

Myotoxicity, one of the most relevant local manifestations in envenomation by Bothrops genus, may result from a direct action of myotoxins or be due to an indirect vascular degeneration and ischemia. Baltergin, a snake venom metalloproteinase (SVMP), isolated from Bothrops alternatus venom has been used to obtain monospecific IgG, in order to determine the relative role of toxin in myotoxicity induced by whole venom. Bothrops diporus venom, another medical relevant genus of the northeastern region of Argentina, was also studied. Anti-baltergin IgG was able to neutralize completely the hemorrhagic activity of B. alternatus venom at an antibodies:venom ratio of 30:1 (w:w). However, mice injected with B. diporus venom showed a small spot remaining even at the highest ratio of IgG:venom assayed (50:1; w:w). Specific antibodies were efficient to neutralize the myotoxicity of B. alternatus venom at ratio 30:1 (w:w) but did not neutralize the same effects in B. diporus venom. Anti-baltergin polyclonal antibodies were useful tools for revealing the central role of SVMPs in the development of myotoxicity of B. alternatus venom, as well as, helping to suggest indirectly presence of potent myotoxic phospholipases A2 (PLA2s) in B. diporus venom.

Edematogenic and myotoxic activities of the Duvernoy's gland secretion of Philodryas olfersii from the north-east region of Argentina.

Philodryas olfersii is found in South America, from Amazonas to Patagonia. It is important to characterize the venom of P. olfersii, who inhabits the North-East region of Argentina, since snake venoms are known to exhibit considerable variability in composition and biological activities. In this work, mice weighing 18-20 g (n = 4 for each experimental group) were used. For the edematogenic activity mice were injected s.c. in the right foot pad with 50 microl of solutions containing different amounts of venom, whereas the left foot pad was injected with 50 microl of PBS. Two hours after injection mice were killed by cervical dislocation and both feet were cut off and weighed individually. For the myotoxic activity mice were injected i.m. with 100 microl of solutions containing 40 microg of venom. Blood samples were extracted after 1, 3, 6, 8, 10, 12, 14, 16 and 24 h of venom injection to determinate serum CPK activity and mice were sacrificed at the same time intervals to obtain the inoculated gastrocnemius muscle. They were fixed with Bouin solution and stained with Hematoxylin-Eosin. Results showed that P. olfersii venom exhibits a high edematogenic activity (MED = 0.31 microg) and a moderate myotoxic activity. Myonecrosis reached its highest level after 12 h of venom injection as shown by plasmatic CPK levels (5,401 +/- 330 IU/l) and microscopic assay. It demonstrates the potential toxicity of the venom of P. olfersii, who inhabits the North-East region of Argentina. It also reinforces the original warning concerning the potential danger of bites by colubrids.