Staggered molecular packing in crystals of a collagen-like peptide with a single charged pair

The crystal structure of the triple-helical peptide, (Pro-Hyp-Gly)(4)-Glu-Lys-Gly-(Pro-Hyp-Gly)(5) has been determined to 1.75 A resolution. This peptide was designed to examine the effect of a pair of adjacent, oppositely charged residues on collagen triple-helical conformation and intermolecular interactions. The molecular conformation (a 7(5) triple helix) and hydrogen bonding schemes are similar to those previously reported for collagen triple helices and provides a second instance of water mediated N--H . . . O==C interchain hydrogen bonds for the amide group of the residue following Gly. Although stereochemically capable of forming intramolecular or intermolecular ion pairs, the lysine and glutamic acid side-chains instead display direct interactions with carbonyl groups and hydroxyproline hydroxyl groups or interactions mediated by water molecules. Solution studies on the EKG peptide indicate stabilization at neutral pH values, where both Glu and Lys are ionized, but suggest that this occurs because of the effects of ionization on the individual residues, rather than ion pair formation. The EKG structure suggests a molecular mechanism for such stabilization through indirect hydrogen bonding. The molecular packing in the crystal includes an axial stagger between molecules, reminiscent of that observed in D-periodic collagen fibrils. The presence of a Glu-Lys-Gly triplet in the middle of the sequence appears to mediate this staggered molecular packing through its indirect water-mediated interactions with backbone C==O groups and side chains.     

Formation of supramolecular structures of a native-like protein in the presence of amphiphilic peptides: Variations in aggregate morphology

A striking potential of the amphiphilic dipeptides, Arg-Phe or Asp-Phe, to induce aggregation of a model protein, alcohol dehydrogenase in its native-like state, has been demonstrated under physiologically relevant conditions, using dynamic light scattering, fluorescence spectroscopy, circular dichroism, transmission electron- and atomic force microscopy. The peptide action resulted in accumulation of a variety of morphologically distinct supramolecular structures profoundly differing from those generated by the heat-induced aggregation at the early stages of the process, when amyloid fibril assemblies were not detectable. The biogenic amphiphilic agents are suggested to act as regulators of structural transformations of native-like proteins.     

Assessing the potential of wildfires as a sustainable bioenergy opportunity

As the environmental and economic consequences of fossil‐fuel use become clear, land is increasingly targeted as a source of bioenergy. We explore the potential for generating electricity from biomass vulnerable to fires as an ecologic and socioeconomic opportunity that can reduce the risk of greenhouse gas generation from wildfires and help to create incentives to preserve natural and seminatural vegetation and prevent its conversion to agriculture, including biofuel crops. On the basis of a global analysis of the energy generation and spatial distribution of fires, we show that between 2003 and 2010, global fires consumed ~8300 ± 592 PJ yr^?1 of energy, equivalent to ~36–44% of the global electricity consumption in 2008 and >100% national consumption in 57 countries. Forests/woodlands, cultivated areas, shrublands, and grasslands contributed 53%, 19%, 16%, and 3.5% of the global energy released by fires. Although many agroecological, socioeconomic, and engineering challenges need to be overcome before diverting the energy lost in fires into more useable forms, done cautiously it could reconcile habitat preservation with economic yields in natural systems.     

Potential Role of M. tuberculosis Specific IFN-γ and IL-2 ELISPOT Assays in Discriminating Children with Active or Latent Tuberculosis

Background

Although currently available IGRA have been reported to be promising markers for TB infection, they cannot distinguish active tuberculosis (TB) from latent infection (LTBI).

Objective

Children with LTBI, active TB disease or uninfected were prospectively evaluated by an in-house ELISPOT assay in order to investigate possible immunological markers for a differential diagnosis between LTBI and active TB.

Methods

Children at risk for TB infection prospectively enrolled in our infectious disease unit were evaluated by in-house IFN-γ and IL-2 based ELISPOT assays using a panel of Mycobacterium tuberculosis antigens.

Results

Twenty-nine children were classified as uninfected, 21 as LTBI and 25 as active TB cases (including 5 definite and 20 probable cases). Significantly higher IFN-γ ELISPOT responses were observed in infected vs. uninfected children for ESAT-6 (p<0.0001), CFP-10 (p<0.0001), TB 10.3 (p = 0.003), and AlaDH (p = 0.001), while differences were not significant considering Ag85B (p = 0.063), PstS1 (p = 0.512), and HspX (16 kDa) (p = 0.139). IL-2 ELISPOT assay responses were different for ESAT-6 (p<0.0001), CFP-10 (p<0.0001), TB 10.3 (p<0.0001), HspX (16 kDa) (p<0.0001), PstS1 (p<0.0001) and AlaDH (p = 0.001); but not for Ag85B (p = 0.063). Comparing results between children with LTBI and those with TB disease differences were significant for IFN-γ ELISPOT only for AlaDH antigen (p = 0.021) and for IL-2 ELISPOT assay for AlaDH (p<0.0001) and TB 10.3 antigen (p = 0.043). ROC analyses demonstrated sensitivity of 100% and specificity of 81% of AlaDH-IL-2 ELISPOT assay in discriminating between latent and active TB using a cut off of 12.5 SCF per million PBMCs.

Conclusion

Our data suggest that IL-2 based ELISPOT with AlaDH antigen may be of help in discriminating children with active from those with latent TB.     

Collagen-like proteins in pathogenic E. coli strains

The genome sequences of enterohaemorrhagic E. coli O157:H7 strains show multiple open-reading frames with collagen-like sequences that are absent from the common laboratory strain K-12. These putative collagens are included in prophages embedded in O157:H7 genomes. These prophages carry numerous genes related to strain virulence and have been shown to be inducible and capable of disseminating virulence factors by horizontal gene transfer. We have cloned two collagen-like proteins from E. coli O157:H7 into a laboratory strain and analysed the structure and conformation of the recombinant proteins and several of their constituting domains by a variety of spectroscopic, biophysical, and electron microscopy techniques. We show that these molecules exhibit many of the characteristics of vertebrate collagens, including trimer formation and the presence of a collagen triple helical domain. They also contain a C-terminal trimerization domain, and a trimeric α-helical coiled-coil domain with an unusual amino acid sequence almost completely lacking leucine, valine or isoleucine residues. Intriguingly, these molecules show high thermal stability, with the collagen domain being more stable than those of vertebrate fibrillar collagens, which are much longer and post-translationally modified. Under the electron microscope, collagen-like proteins from E. coli O157:H7 show a dumbbell shape, with two globular domains joined by a hinged stalk. This morphology is consistent with their likely role as trimeric phage side-tail proteins that participate in the attachment of phage particles to E. coli target cells, either directly or through assembly with other phage tail proteins. Thus, collagen-like proteins in enterohaemorrhagic E. coli genomes may have a direct role in the dissemination of virulence-related genes through infection of harmless strains by induced bacteriophages.     

Laudatio for centenary of the birth of Luigi Di Bella, MD, PhD

On the centennial of the birth of Luigi Di Bella, the desire to memorialise, at least in part, his thoughts has prompted us to write this tribute, in the hope that one day his dreams may come true. Throughout his research for a treatment of cancer, he deemed it necessary to employ a complex array of substances that, by acting centripetally on neoplastic cells, could in turn be capable of affecting, either simultaneously or sequentially, the myriad of biological reactions supporting their lives. Hence, not a substance but a method (Di Bella Method, DBM). These brief hints at some aspects of Prof. Di Bella's multifaceted scientific vision are aimed not only at reasserting the truth, but also at giving a modest contribution to a novel and free direction in experimental and clinical science.     

A Two‐Year Bio‐Agronomic and Chemotaxonomic Evaluation of Wild Sicilian Myrtle (Myrtus communis L.) Berries and Leaves

A collection of nine Myrtus communis samples from different localities of Sicily was evaluated. Morphological traits and production characteristics have been chosen as parameters to arrange the samples into homogeneous groups and to identify the best biotypes for possible future agro‐industrial exploitation. The plant material has been subjected to taxonomic characterization from biometric and phytochemical perspectives. Myrtle berries and leaves have been analyzed for their content in metabolites, applying a cascade extraction protocol for M. communis leaves and a single hydroalcoholic extraction for berries, whereas hydrodistillation procedures have been applied to obtain the essential oils from berries and leaves. The analyses of non‐volatile components were carried out by LC‐UV‐DAD‐ESI‐MS. All the extracts were characterized by the presence of numerous polyphenols, namely highly hydroxylated flavonols such as quercetin and myricetin; and ellagic acid detected in all samples. In addition, myrtle berries contained nine different anthocyanins, namely delphinidin, petunidin, cyanidin and malvidin derivatives. The essential oils (EOs) were analyzed by a combination of GC‐FID and GC/MS. A total of 33 and 34 components were fully characterized with the predominance of α‐pinene, myrtenyl acetate, linalool, 1,8‐cineole and linalyl acetate. All phytochemical profiles were subjected to cluster analyses, which allowed subdividing the myrtle samples in different chemical groups.     

Antibacterial activity of extract, fractions and four compounds extracted from Piper solmsianum C. DC. VAR. solmsianum (Piperaceae)

Piper solmsianum C. DC. var. solmsianum (Piperaceae) is a shrub commonly found in areas with wet tropical soils. Other Piper species have been used in folk medicine as antitumoral and antiseptic agents. We studied the crude methanolic extract, some organic fractions and compounds isolated from this plant for possible antimicrobial activity against Gram-positive and Gram-negative bacteria. The bioautographic assays disclosed three inhibition zones. The minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) were determined showing excellent activity, particularly against the Gram-positive bacteria (Bacillus cereus, Staphylococcus aureus, Staphylococcus saprophyticus and Streptococcus agalactiae). It appears that the antimicrobial activity of Piper solmsianum is related mainly to the presence of conocarpan and eupomatenoid-5 (neolignans). However another, as yet unidentified, active compound could also be extracted from the plant.     

Effect of cattle grazing on soil salinity and vegetation composition along an elevation gradient in a temperate coastal salt marsh of Samborombón Bay (Argentina)

Salt marshes of Samborombón Bay (Argentina) have been grazed sporadically at very low stocking rates, but in the last decade, grazing intensity increased due to agriculture expansion. We investigated the effect of cattle grazing on vegetation and soil salinity on the most extended Spartina densiflora community. This community develops along an elevation gradient where the frequency and duration of tidal flooding and soil salinity increased as elevation decreased. Vegetation and soil data were collected from a national park excluded to cattle grazing for 30 years and from an adjacent commercial livestock farm continuously grazed by cattle. As elevation level decreased, plant cover, richness and diversity of functional groups and species decreased. As we expected, grazing altered soil salinity and vegetation composition in different extent along the elevation gradient. Grazing changed vegetation structure more intensively in the high elevation level because it reduced the competitive exclusion exerted by S. densiflora, allowing the increase in floristic richness. Grazing increased soil salinity and the contribution of salt-tolerant species only in the medium but not in the low elevation level probably because the higher frequency and duration of tidal flooding counterbalanced the increase in evaporation promoted by biomass removal in the low respect to the medium elevation level. While grazing may cause positive impacts for plant conservation in the high elevation level, it may cause negative consequence for livestock production because of the reduction in forage quality along the entire elevation gradient.