The importance of being kinked: role of Pro residues in the selectivity of the helical antimicrobial peptide P5

Antimicrobial peptides (AMPs) are promising compounds for developing new antibiotic drugs against drug‐resistant bacteria. Many of them kill bacteria by perturbing their membranes but exhibit no significant toxicity towards eukaryotic cells. The identification of the features responsible for this selectivity is essential for their pharmacological development. AMPs exhibit few conserved features, but a statistical analysis of an AMP sequence database indicated that many α‐helical AMPs surprisingly have a helix‐breaking Pro residue in the middle of their sequence. To discriminate among the different possible hypotheses for the functional role of this feature, we designed an analogue of the antimicrobial peptide P5, in which the central Pro was deleted (analogue P5Del). Pro removal resulted in a dramatic increase of toxicity. This was explained by the observation that P5Del binds both charged and neutral membranes, whereas P5 has no appreciable affinity towards neutral bilayers. CD and simulative data provided a rationalization of this behavior. In solution P5, due to the presence of Pro, attains compact conformations, in which its apolar residues are partially shielded from the solvent, whereas P5Del is more helical. These structural differences reduce the hydrophobic driving force for association of P5 to neutral membranes, whereas its binding to anionic bilayers can still take place because of electrostatic attraction. After membrane binding, the Pro residue does not preclude the attainment of a membrane‐active amphiphilic helical conformation. These findings shed light on the role of Pro residues in the selectivity of AMPs and provide hints for the design of new, highly selective compounds. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

The desire for variety: Italian wall lizard (Podarcis siculus) populations introduced to the United States via the pet trade are derived from multiple native-range sources

Tests of invasion success often require comparisons between introduced and native populations, but determining the native-range sources for introduced populations can be difficult. Molecular markers can help clarify the geographic extent of native-range sources, helping to identify which populations are appropriate for comparative studies. The Italian Wall Lizard (Podarcis siculus) was introduced multiple times to the United States with extant populations in California, Kansas, New Jersey, and New York. We used phylogeographic analysis of mtDNA sequences (cytb gene) for individuals sampled from these introduced populations and across the native range to identify the number of independent introductions and the location of the source populations. Haplotypes sampled from introduced populations were nested within three geographically distinct, well-supported clades that together encompassed a large portion of the native range. Combining these phylogeographic results with documentation of the introductions revealed putative sources: California individuals are derived from Sicily; Kansas and New York populations are from Tuscany near Florence; and the New Jersey population is likely from the Adriatic coastal region, but a more specific locality is not possible. The pet trade dominates the invasion pathway for P. siculus introductions to the US. The genetically and geographically diverse sampling of its native range may be driven by the desire for phenotypic variety in the pet trade, a hypothesis that needs future testing.     

EGFR inhibitors exacerbate differentiation and cell cycle arrest induced by retinoic acid and vitamin D3 in acute myeloid leukemia cells

By means of an unbiased, automated fluorescence microscopy-based screen, we identified the epidermal growth factor receptor (EGFR) inhibitors erlotinib and gefitinib as potent enhancers of the differentiation of HL-60 acute myeloid leukemia (AML) cells exposed to suboptimal concentrations of vitamin A (all-trans retinoic acid, ATRA) or vitamin D (1α,25-hydroxycholecalciferol, VD). Erlotinib and gefitinib alone did not promote differentiation, yet stimulated the acquisition of morphological and biochemical maturation markers (including the expression of CD11b and CD14 as well as increased NADPH oxidase activity) when combined with either ATRA or VD. Moreover, the combination of erlotinib and ATRA or VD synergistically induced all the processes that are normally linked to terminal hematopoietic differentiation, namely, a delayed proliferation arrest in the G₀/G₁ phase of the cell cycle, cellular senescence, and apoptosis. Erlotinib potently inhibited the (auto)phosphorylation of mitogen-activated protein kinase 14 (MAPK14, best known as p38^MAPK) and SRC family kinases (SFKs). If combined with the administration of ATRA or VD, the inhibition of p38^MAPK or SFKs with specific pharmacological agents mimicked the pro-differentiation activity of erlotinib. These data were obtained with 2 distinct AML cell lines (HL-60 and MOLM-13 cells) and could be confirmed on primary leukemic blasts isolated from the circulation of AML patients. Altogether, these findings point to a new regimen for the treatment of AML, in which naturally occurring pro-differentiation agents (ATRA or VD) may be combined with EGFR inhibitors.     

How Skill Expertise Shapes the Brain Functional Architecture: An fMRI Study of Visuo-Spatial and Motor Processing in Professional Racing-Car and Na?ve Drivers

The present study was designed to investigate the brain functional architecture that subserves visuo-spatial and motor processing in highly skilled individuals. By using functional magnetic resonance imaging (fMRI), we measured brain activity while eleven Formula racing-car drivers and eleven ‘naïve’ volunteers performed a motor reaction and a visuo-spatial task. Tasks were set at a relatively low level of difficulty such to ensure a similar performance in the two groups and thus avoid any potential confounding effects on brain activity due to discrepancies in task execution. The brain functional organization was analyzed in terms of regional brain response, inter-regional interactions and blood oxygen level dependent (BOLD) signal variability. While performance levels were equal in the two groups, as compared to naïve drivers, professional drivers showed a smaller volume recruitment of task-related regions, stronger connections among task-related areas, and an increased information integration as reflected by a higher signal temporal variability. In conclusion, our results demonstrate that, as compared to naïve subjects, the brain functional architecture sustaining visuo-motor processing in professional racing-car drivers, trained to perform at the highest levels under extremely demanding conditions, undergoes both ‘quantitative’ and ‘qualitative’ modifications that are evident even when the brain is engaged in relatively simple, non-demanding tasks. These results provide novel evidence in favor of an increased ‘neural efficiency’ in the brain of highly skilled individuals.     

Roux-en-Y Gastric Bypass Increases Intravenous Ethanol Self-Administration in Dietary Obese Rats

Roux-en-Y gastric bypass surgery (RYGB) is an effective treatment for severe obesity. Clinical studies however have reported susceptibility to increased alcohol use after RYGB, and preclinical studies have shown increased alcohol intake in obese rats after RYGB. This could reflect a direct enhancement of alcohol’s rewarding effects in the brain or an indirect effect due to increased alcohol absorption after RGYB. To rule out the contribution that changes in alcohol absorption have on its rewarding effects, here we assessed the effects of RYGB on intravenously (IV) administered ethanol (1%). For this purpose, high fat (60% kcal from fat) diet-induced obese male Sprague Dawley rats were tested ∼2 months after RYGB or sham surgery (SHAM) using both fixed and progressive ratio schedules of reinforcement to evaluate if RGYB modified the reinforcing effects of IV ethanol. Compared to SHAM, RYGB rats made significantly more active spout responses to earn IV ethanol during the fixed ratio schedule, and achieved higher breakpoints during the progressive ratio schedule. Although additional studies are needed, our results provide preliminary evidence that RYGB increases the rewarding effects of alcohol independent of its effects on alcohol absorption.     

Manufacturing Devices and Instruments for Easier Rat Liver Transplantation

Orthotopic rat liver transplantation is a popular model, which has been shown in a recent JoVE paper with the use of the "quick-linker" device. This technique allows for easier venous cuff-anatomoses after a reasonable learning curve. The device is composed of two handles, which are carved out from scalpel blades, one approximator, which is obtained by modifying Kocher''s forceps, and cuffs designed from fine-bore polyethylene tubing. The whole process can be performed at a low-cost using common laboratory material. The present report provides a step-by-step protocol for the design of the required pieces and includes stencils.     

A comparative physiological and transcriptional study of carotenoid biosynthesis in white and red grapefruit (Citrus paradisi Macf.)

Accumulation of lycopene in citrus fruits is an unusual feature restricted to selected mutants. Grapefruit (Citrus paradisi Macf.) is the Citrus specie with greater number of red-fleshed mutants, but the molecular bases of this alteration are not fully understood. To gain knowledge into the mechanisms implicated in this alteration, we conducted a comparative analysis of carotenoid profile and of the expression of genes related to carotenoid biosynthesis and catabolism in flavedo and pulp of two grapefruit cultivars with marked differences in colouration: the white Marsh and the red Star Ruby. Mature green fruit of Marsh accumulated chloroplastic carotenoids, while mature tissues lacked carotenoids. However, accumulation of downstream products such as abscisic acid (ABA) and expression of its biosynthetic genes, 9-cis-epoxycarotenoid dioxygenase (NCED1 and NCED2), increased after the onset of colouration. In contrast, red grapefruit accumulated lycopene, phytoene and phytofluene, while ABA content and NCED gene expression were lower than in Marsh, suggesting a blockage in the carotenoid biosynthetic pathway. Expression analysis of three genes of the isoprenoid pathway and nine of the carotenoid biosynthetic pathway revealed virtually no differences in flavedo and pulp between both genotypes, except for the chromoplast-specific lycopene cyclase 2 (β-LCY2) which was lower in the pulp of the red grapefruit. The proportion in the expression of the allele with high (β-LCY2a) and low (β-LCY2b) activity was also similar in the pulp of both genotypes. Therefore, results suggest that reduced expression of β-LCY2 appears to be responsible of lycopene accumulation in the red Star Ruby grapefruit.