A 28,000 years old Cro-Magnon mtDNA sequence differs from all potentially contaminating modern sequences

Background

DNA sequences from ancient speciments may in fact result from undetected contamination of the ancient specimens by modern DNA, and the problem is particularly challenging in studies of human fossils. Doubts on the authenticity of the available sequences have so far hampered genetic comparisons between anatomically archaic (Neandertal) and early modern (Cro-Magnoid) Europeans.

Methodology/Principal Findings

We typed the mitochondrial DNA (mtDNA) hypervariable region I in a 28,000 years old Cro-Magnoid individual from the Paglicci cave, in Italy (Paglicci 23) and in all the people who had contact with the sample since its discovery in 2003. The Paglicci 23 sequence, determined through the analysis of 152 clones, is the Cambridge reference sequence, and cannot possibly reflect contamination because it differs from all potentially contaminating modern sequences.

Conclusions/Significance:

The Paglicci 23 individual carried a mtDNA sequence that is still common in Europe, and which radically differs from those of the almost contemporary Neandertals, demonstrating a genealogical continuity across 28,000 years, from Cro-Magnoid to modern Europeans. Because all potential sources of modern DNA contamination are known, the Paglicci 23 sample will offer a unique opportunity to get insight for the first time into the nuclear genes of early modern Europeans.     

Temporin A is effective in MRSA-infected wounds through bactericidal activity and acceleration of wound repair in a murine model

We investigated the effect of topical temporin A in the management of methicillin-resistant strain of Staphylococcus aureus (MRSA)-infected experimental surgical wounds in mice. The wound, cut through the panniculus carnosus of BALB/c mice, was inoculated with 5x10(7) colony-forming units of MRSA. Mice were treated with Allevyn, temporin A-soaked Allevyn, Allevyn and daily intraperitoneal teicoplanin (7mg/kg), temporin A-soaked Allevyn and daily intraperitoneal teicoplanin. Main outcome measurements were: quantitative bacterial culture, histological examination with assessment of micro-vessel density and of vascular endothelial growth factor (VEGF) expression in tissue sections, and VEGF plasma levels alike. Treatment with temporin-A associated with teicoplanin injection significantly reduced bacterial load to 0.85 x 10(1)+/-0.1 x 10(1)CFU/ml. Histological examination showed that infected mice receiving temporin A-soaked Allevyn (with or without teicoplanin) had a higher degree of granulation tissue formation and collagen deposition compared to the other treated groups. A significant increase in serum VEGF expression was observed in mice receiving temporin A topically and temporin A topically associated with intraperitoneal teicoplanin. In conclusion our results demonstrated that temporin A is effective in the management of infected wounds, by a significant bacterial growth inhibition and acceleration of wound repair process.     

GJB2 Gene Mutations in Syndromic Skin Diseases with Sensorineural Hearing Loss

The GJB2 gene is located on chromosome 13q12 and it encodes the connexin 26, a transmembrane protein involved in cell-cell attachment of almost all tissues. GJB2 mutations cause autosomal recessive (DFNB1) and sometimes dominant (DFNA3) non-syndromic sensorineural hearing loss. Moreover, it has been demonstrated that connexins are involved in regulation of growth and differentiation of epidermis and, in fact, GJB2 mutations have also been identified in syndromic disorders with hearing loss associated with various skin disease phenotypes. GJB2 mutations associated with skin disease are, in general, transmitted with a dominant inheritance pattern. Nonsyndromic deafness is caused prevalently by a loss-of-function, while literature evidences suggest for syndromic deafness a mechanism based on gain-of-function. The spectrum of skin manifestations associated with some mutations seems to have a very high phenotypic variability. Why some mutations can lead to widely varying cutaneous manifestations is poorly understood and in particular, the reason why the skin disease-deafness phenotypes differ from each other thus remains unclear. This review provides an overview of recent findings concerning pathogenesis of syndromic deafness imputable to GJB2 mutations with an emphasis on relevant clinical genotype-phenotype correlations. After describing connexin 26 fundamental characteristics, the most relevant and recent information about its known mutations involved in the syndromic forms causing hearing loss and skin problems are summarized. The possible effects of the mutations on channel expression and function are discussed.     

Functional characterization of residues within the carnitine/acylcarnitine translocase RX2PANAAXF distinct motif

The mitochondrial carnitine/acylcarnitine carrier (CAC) is characterized by the presence of a distinct motif, RXXPANAAXF, within its sixth transmembrane α-helix. In this study, we analysed the role of the amino acids of this motif in the structure-function relationships of the human CAC by using two complementary approaches. First, we performed functional analysis in the model fungus Aspergillus nidulans of selected mutations with structural and functional relevance. Second, similar mutant human CACs were biochemically characterized after their reconstitution into liposomes. Both analyses have provided relevant information on the importance and role of the CAC motif residues in the activity and metabolic function of CAC. Only the two adjacent alanines, Ala281 and Ala282 in the human CAC, have been found not to be crucial for transport activity and in vivo function. Results obtained from amino acid substitutions of residues Arg275, Asn280 and Phe284 of human CAC together with structural analysis using molecular modelling of the carrier suggest that R275, N280 and F284 are involved in substrate binding during acylcarnitine/carnitine translocation. Furthermore, functional analysis of mutations of residues Pro278 and Ala279 in A. nidulans, together with kinetic data in reconstituted liposomes, suggest a predominant structural role for these amino acids.     

Phenotypic and histochemical traits of the interaction between Plasmopara viticola and resistant or susceptible grapevine varieties

ABSTRACT: BACKGROUND: Grapevine downy mildew, caused by Plasmopara viticola, is a very serious disease affectingmainly Vitis vinifera cultivated varieties around the world. Breeding for resistance throughthe crossing with less susceptible species is one of the possible means to reduce the diseaseincidence and the application of fungicides. The hybrid Bianca and some of its siblings areconsidered very promising but their resistance level can vary depending on the pathogenstrain. Moreover, virulent strains characterized by high fitness can represent a potential threatto the hybrid cultivation. RESULTS: The host response and the pathogen virulence were quantitatively assessed by artificiallyinoculating cv Chardonnay, cv Bianca and their siblings with P. viticola isolates derived fromsingle germinating oospores collected in various Italian viticultural areas. The hostphenotypes were classified as susceptible, intermediate and resistant, according to the AreaUnder the Disease Progress Curve caused by the inoculated strain. Host responses in cvBianca and its siblings significantly varied depending on the P. viticola isolates, which in turndiffered in their virulence levels. The fitness of the most virulent strain did not significantlyvary on the different hybrids including Bianca in comparison with the susceptible cvChardonnay, suggesting that no costs are associated with virulence. Among the individualfitness components, only sporangia production was significantly reduced in cv Bianca and insome hybrids. Comparative histological analysis revealed differences between susceptibleand resistant plants in the pathogen diffusion and cytology from 48 h after inoculation onwards. Defence mechanisms included callose depositions in the infected stomata, increasein peroxidase activity, synthesis of phenolic compounds and flavonoids and the necrosis ofstomata and cells immediately surrounding the point of invasion and determined alterations inthe size of the infected areas and in the number of sporangia differentiated. CONCLUSIONS: Some hybrids were able to maintain an intermediate-resistant behaviour even wheninoculated with the most virulent strain. Such hybrids should be considered for further fieldtrials.     

Characterization of lactic acid bacteria isolated from sourdoughs for Cornetto, a traditional bread produced in Basilicata (Southern Italy)

A total of 41 strains of lactic acid bacteria (LAB) isolated from durum wheat sourdoughs used to produce Cornetto di Matera bread, were identified by SDS-PAGE of whole cell proteins (WCP) and screened for acid production ability, antimicrobial activity and exopolysaccharide (EPS) production. The isolates were identified as Lactobacillus plantarum (49%), Leuconostoc mesenteroides (17%), Lactobacillus curvatus (15%), Lactobacillus paraplantarum (12%), Weissella cibaria (5%) and Lactobacillus pentosus (2%). Several strains of Lb. plantarum and Leuc. mesenteroides showed a high acid production ability. The antagonistic activity was tested using an agar-spot deferred antagonism assay against a set of five indicators. The species had different profiles of inhibition. Lb. plantarum had the largest spectrum of inhibition, while no isolates of W. cibaria and Leuc. mesenteroides showed antimicrobial activity. No strains had antimicrobial activity against Bacillus cereus. The inhibitory activity of five strains was confirmed to be sensitive to proteolytic enzymes and thus potentially due to bacteriocin production. All Leuc. mesenteroides and W. cibaria strains produced EPS from sucrose. Some Lb. plantarum and Lb. paraplantarum strains produced EPS from different sugars in solid media. EPS production in liquid media was different within the species, with the highest production in liquid media containing glucose and maltose. A defined strain starter culture (W. cibaria DBPZ1006, Lb. plantarum DBPZ1015 and S. cerevisiae MTG10) was selected on the basis of technological properties and tested in model sourdough fermentations.     

Optimization and preclinical evaluation of novel histamine H3 receptor ligands: Acetyl and propionyl phenoxyalkyl piperazine derivatives

As a continuation of our search for novel histamine H₃ receptor ligands, a series of new acetyl and propionyl phenoxyalkylamine derivatives (2–25) was synthesized. Compounds with three to four carbon atoms alkyl chain spacer, composed of six various 4N-substituted piperazine moieties were evaluated for their binding properties at human histamine H₃ receptors (hH₃R). In vitro test results proved the 4-pyridylpiperazine moiety as crucial element for high hH₃R affinity (hH₃R K_i?=?5.2–115?nM). Moreover introduction of carbonyl group containing residues in the lipophilic part of molecules instead of branched alkyl substituents resulted in increased affinity in correlation to previously described series, whereas propionyl derivatives showed slightly higher affinities than those of acetyl (16 and 22vs.4 and 10; hH₃R K_i?=?5.2 and 15.4?nM vs. 10.2 and 115?nM, respectively). These findings were confirmed by molecular modelling studies, demonstrating multiple ligand-receptor interactions. Furthermore, pharmacological in vivo test results of compound 4 clearly indicate that it may affect the amount of calories consumed, thus act as an anorectic compound. Likewise, its protective action against hyperglycemia and the development of overweight has been shown. In order to estimate drug-likeness of compound 4, in silico and experimental evaluation of metabolic stability in human liver microsomes was performed.     

Blockade by cAMP of native sodium channels of adult rat skeletal muscle fibers

Although theskeletal muscle sodium channel is a good substrate for cAMP-dependentprotein kinase (PKA), no functional consequence was observed for thischannel expressed in heterologous systems. Therefore, we investigatedthe effect of 8-(4-chlorophenylthio)adenosine 3',5'-cyclicmonophosphate (CPT-cAMP), a membrane-permeable cAMP analog, on thenative sodium channels of freshly dissociated rat skeletal musclefibers by means of the cell-attached patch-clamp technique. Externallyapplied CPT-cAMP (0.5 mM) reduced peak ensemble average currents by~75% with no change in kinetics. Single-channel conductance andnormalized activation curves were unchanged by CPT-cAMP. In contrast,steady-state inactivation curves showed a reduction of the maximalavailable current and a negative shift of the half-inactivationpotential. Similar effects were observed with dibutyryl adenosine3',5'-cyclic monophosphate but not with cAMP, which doesnot easily permeate the cell membrane. Incubation of fibers for 1 hwith 10 µM H-89, a PKA inhibitor, did not prevent the effect ofCPT-cAMP. Finally, the -adrenoreceptor agonist isoproterenolmimicked CPT-cAMP when applied at 0.5 mM but had no effect at 0.1 mM.These results indicate that cAMP inhibits native skeletal muscle sodiumchannels by acting within the fiber, independently of PKA activation.