Activity and isoenzyme composition of peroxidase and esterase in fertile and male-sterile lines of tomatoes (Lycopersicon esculentum Mill.)

A comparative study of the isoenzyme patterns of esterase and peroxidase and overall peroxidase activity in stamens of male-sterile (MS) lines of Pearson ms-35 and P ms-35aa and of the respective male-fertile (MF) tomato plants have been conducted. The study has been made at two stages of stamens development — tetrad and pollen. Higher activities of the esterase isoenzymes in the MF stamens than that of MS in both ontogeny stages have been found. The slow moving esterase isoenzymes both of the MF and the MS stamens are the major isoenzymes in the early stage and are connected with tapetum development while the fast moving esterase isoenzymes are connected with pollen formation in the later ontogeny stage. Overall peroxidase levels in the MS stamens were higher than those of MF. The peroxidase patterns of the MS lines are also characterized by the greater number of isoenzymes and also the presence of specific isoenzymes, the contrast between the MF and the MS stamens being more strongly expressed at the later stage of development. A strong similitude between esterase and peroxidase patterns behaviour in both MS lines has been found.     

DNA barcoding survey of Trichoderma diversity in soil and litter of the Colombian lowland Amazonian rainforest reveals Trichoderma strigosellum sp. nov. and other species

The diversity of Trichoderma (Hypocreales, Ascomycota) colonizing leaf litter as well as the rhizosphere of Garcinia macrophylla (Clusiaceae) was investigated in primary and secondary rain forests in Colombian Amazonia. DNA barcoding of 107 strains based on the internal transcribed spacers 1 and 2 (ITS1 and 2) of the ribosomal RNA gene cluster and the partial sequence of the translation elongation factor 1 alpha (tef1) gene revealed that the diversity of Trichoderma was dominated (71 %) by three common cosmopolitan species, namely Trichoderma harzianum sensu lato (41 %), Trichoderma spirale (17 %) and Trichoderma koningiopsis (13 %). Four ITS 1 and 2 phylotypes (13 strains) could not be identified with certainty. Multigene phylogenetic analysis and phenotype profiling of four strains with an ITS1 and 2 phylotype similar to Trichoderma strigosum revealed a new sister species of the latter that is described here as Trichoderma strigosellum sp. nov. Sequence similarity searches revealed that this species also occurs in soils of Malaysia and Cameroon, suggesting a pantropical distribution.     

Global genetic diversity,lineage distribution,and Wolbachia infection of the alfalfa weevil Hypera postica (Coleoptera: Curculionidae)

The alfalfa weevil (Hypera postica) is a well‐known example of a worldwide‐distributed pest with high genetic variation. Based on the mitochondrial genes, the alfalfa weevil clusters into two main mitochondrial lineages. However, there is no clear picture of the global diversity and distribution of these lineages; neither the drivers of its diversification are known. However, it appears likely that historic demographic events including founder effects played a role. In addition, Wolbachia, a widespread intracellular parasite/symbiont, likely played an important role in the evolution of the species. Wolbachia infection so far was only detected in the Western lineage of H. postica with no information on the infecting strain, its frequency, and its consequences on the genetic diversity of the host. We here used a combination of mitochondrial and nuclear sequences of the host and sequence information on Wolbachia to document the distribution of strains and the degree of infection. The Eastern lineage has a higher genetic diversity and is found in the Mediterranean, the Middle East, Eastern Europe, and eastern America, whereas the less diverse Western lineage is found in Central Europe and the western America. Both lineages are infected with the same common strain of Wolbachia belonging to Supergroup B. Based on neutrality tests, selection tests, and the current distribution and diversification of Wolbachia in H. postica, we suggested the Wolbachia infection did not shape genetic diversity of the host. The introduced populations in the United States are generally genetically less diverse, which is in line with founder effects.     

Development of a chitosan nanofibrillar scaffold for skin repair and regeneration

The final goal of the present study was the development of a 3-D chitosan dressing that would shorten the healing time of skin wounds by stimulating migration, invasion, and proliferation of the relevant cutaneous resident cells. Three-dimensional chitosan nanofibrillar scaffolds produced by electrospinning were compared with evaporated films and freeze-dried sponges for their biological properties. The nanofibrillar structure strongly improved cell adhesion and proliferation in vitro. When implanted in mice, the nanofibrillar scaffold was colonized by mesenchymal cells and blood vessels. Accumulation of collagen fibrils was also observed. In contrast, sponges induced a foreign body granuloma. When used as a dressing covering full-thickness skin wounds in mice, chitosan nanofibrils induced a faster regeneration of both the epidermis and dermis compartments. Altogether our data illustrate the critical importance of the nanofibrillar structure of chitosan devices for their full biocompatibility and demonstrate the significant beneficial effect of chitosan as a wound-healing biomaterial.     

Effect of porcine respiratory coronavirus infection on lipopolysaccharide recognition proteins and haptoglobin levels in the lungs

Porcine respiratory coronavirus (PRCV) potentiates respiratory disease and proinflammatory cytokine production in the lungs upon intratracheal inoculation with lipopolysaccharide (LPS) at 1 day of infection. This study aimed to quantify LPS-binding protein (LBP), CD14 and haptoglobin in the lungs throughout a PRCV infection. LBP and CD14 recognize LPS and enhance its endotoxic activity, whereas haptoglobin dampens it. Gnotobiotic pigs were inoculated intratracheally with PRCV (n = 34) or saline (n = 5) and euthanized 1-15days post inoculation (DPI). Virus was detected in the lungs from 1 to 9DPI. Cell-associated CD14 in lung tissue increased up to 15 times throughout the infection, due to an increase in highly CD14+ monocyte-macrophages from 1 to 12DPI and CD14+ type 2 pneumocytes from 7 to 9DPI. LBP and soluble CD14 levels in bronchoalveolar lavage fluids were elevated from 1-12DPI, with up to 35- and 4-fold increases, respectively. Haptoglobin levels increased significantly (x4.5) at 7DPI. In addition, we found that PRCV could sensitize the lungs to LPS throughout the infection, but the response to LPS appeared less enhanced at the end of infection (7DPI). The marked increases in LBP, CD14 and haptoglobin were not correlated with the extent of the LPS response.     

Characterization of Erwinia amylovora strains from Bulgaria by pulsed-field gel electrophoresis

The aim of this study was to characterize genetically Bulgarian Erwinia amylovora strains using pulsed-field gel electrophoresis (PFGE) analysis. Fifty E. amylovora strains isolated from different hosts, locations, as well as in different years were analysed by PFGE after XbaI, SpeI, and XhoI digestion of the genomic DNA. The strains were distributed into four groups according to their XbaI-generated profile. About 82% of the strains displayed a PFGE profile identical to that of type Pt2. Three strains belonged to the Central Europe Pt1 type. Two new PFGE profiles, not reported so far, were established--one for a strain isolated from Malus domestica and another for all Fragaria spp. strains. The same grouping of the strains was obtained after analysis of the SpeI digestion patterns. On the basis of PFGE profiles, after XbaI and SpeI digestion, a genetic differentiation between the strains associated with subfamily Maloideae and subfamily Rosoideae was revealed. The presence of more than one PFGE profile in the population of E. amylovora in Bulgaria suggests a multiple source of inoculum.     

Functional incorporation of the pore forming segment of AChR M2 into tethered bilayer lipid membranes

Tethered bilayer lipid membranes (tBLMs) are robust and flexible model platforms for the investigation of various membrane related processes. They are especially suited to study the incorporation and function of ion channel proteins, where a high background resistance of the membrane is essential. Synthetic M2 peptides, analogues of the transmembrane fragment of the acetylcholine receptor, could be incorporated into two different membrane architectures. The functional reconstitution and the formation of a conducting pore are shown by electrochemical impedance spectroscopy (EIS). The pore is selective for small monovalent cations, while bulky ions cannot pass. This is a significant step towards a novel biosensing approach. We envision a device, where a stable and insulating membrane would be attached to an electronic read-out unit and embedded proteins would serve as actual sensing units.     

Rapid Species Diagnosis for Invasive Candidiasis Using Mass Spectrometry

Background

Matrix-assisted laser desorption ionisation time of flight mass spectrometry (MALDI TOF-MS) allows the identification of most bacteria and an increasing number of fungi. The potential for the highest clinical benefit of such methods would be in severe acute infections that require prompt treatment adapted to the infecting species. Our objective was to determine whether yeasts could be identified directly from a positive blood culture, avoiding the 1–3 days subculture step currently required before any therapeutic adjustments can be made.

Methodology/Principal Findings

Using human blood spiked with Candida albicans to simulate blood cultures, we optimized protocols to obtain MALDI TOF-MS fingerprints where signals from blood proteins are reduced. Simulated cultures elaborated using a set of 12 strains belonging to 6 different species were then tested. Quantifiable spectral differences in the 5000–7400 Da mass range allowed to discriminate between these species and to build a reference database. The validation of the method and the statistical approach to spectral analysis were conducted using individual simulated blood cultures of 36 additional strains (six for each species). Correct identification of the species of these strains was obtained.

Conclusions/Significance

Direct MALDI TOF-MS analysis of aliquots from positive blood cultures allowed rapid and accurate identification of the main Candida species, thus obviating the need for sub-culturing on specific media. Subsequent to this proof-of-principle demonstration, the method can be extended to other clinically relevant yeast species, and applied to an adequate number of clinical samples in order to establish its potential to improve antimicrobial management of patients with fungemia.