Enzyme-Linked Lectinsorbent Assay Measures N-Acetyl-D-Glucosamine in Matrix of Biofilm Produced by Staphylococcus epidermidis

An enzyme-linked lectinsorbent assay (ELLA) was developed for quantification of in situ biofilm produced by Staphylococcus epidermidis in polystyrene 96-well tissue culture plates with phosphatase-labeled wheat germ agglutinin (WGA) as a specific probe for the GlcNAcβ-1,4 _n component of exocellular matrix material (EMM) that is responsible for intercellular adhesion and accumulation. The ELLA and the modified Christensen dye assay were used to test 13 laboratory strains of coagulase-negative staphylococci and 10 clinical isolates of S. epidermidis. Four biofilm-positive laboratory strains of S. epidermidis were positive by both tests, and six biofilm-negative strains were negative by both. One strain of S. hemolyticus was positive by the ELLA only. Two of the 10 clinical isolates of S. epidermidis were positive by both assays, two were negative by both, and the remaining were positive by the ELLA only. The ELLA was objective, reproducible, specific, sensitive, and useful for screening strains for their capacity to adhere to plastic, produce EMM, and form biofilm. Received: 15 February 1997 / Accepted: 16 April 1997     

Full Genome Sequence-Based Comparative Study of Wild-Type and Vaccine Strains of Infectious Laryngotracheitis Virus from Italy

Infectious laryngotracheitis (ILT) is an acute and highly contagious respiratory disease of chickens caused by an alphaherpesvirus, infectious laryngotracheitis virus (ILTV). Recently, full genome sequences of wild-type and vaccine strains have been determined worldwide, but none was from Europe. The aim of this study was to determine and analyse the complete genome sequences of five ILTV strains. Sequences were also compared to reveal the similarity of strains across time and to discriminate between wild-type and vaccine strains. Genomes of three ILTV field isolates from outbreaks occurred in Italy in 1980, 2007 and 2011, and two commercial chicken embryo origin (CEO) vaccines were sequenced using the 454 Life Sciences technology. The comparison with the Serva genome showed that 35 open reading frames (ORFs) differed across the five genomes. Overall, 54 single nucleotide polymorphisms (SNPs) and 27 amino acid differences in 19 ORFs and two insertions in the UL52 and ORFC genes were identified. Similarity among the field strains and between the field and the vaccine strains ranged from 99.96% to 99.99%. Phylogenetic analysis revealed a close relationship among them, as well. This study generated data on genomic variation among Italian ILTV strains revealing that, even though the genetic variability of the genome is well conserved across time and between wild-type and vaccine strains, some mutations may help in differentiating among them and may be involved in ILTV virulence/attenuation. The results of this study can contribute to the understanding of the molecular bases of ILTV pathogenicity and provide genetic markers to differentiate between wild-type and vaccine strains.     

El papel de la mitocondria en el dolor de la neuropatía diabética

Diabetes is a metabolic disease affecting approximately 300 million people worldwide. Neuropathy is one of its frequent complications, and may affect sensory, motor, and autonomic nerves. Its pathophysiology has not fully been elucidated. Several hypotheses have been proposed, and mitochondria have been suggested to play a significant role. This article reviews the mechanisms involved in mitochondrial dysfunction and development of diabetic neuropathy, consisting mainly of oxidative and inflammatory stress, changes in intracellular calcium regulation, apoptotic processes, and changes in mitochondrial structure and function that may lead to development of diabetic neuropathy.     

Vaccination with Recombinant Adenoviruses Expressing the Peste des Petits Ruminants Virus F or H Proteins Overcomes Viral Immunosuppression and Induces Protective Immunity against PPRV Challenge in Sheep

Peste des petits ruminants (PPR) is a highly contagious disease of small ruminants caused by the Morbillivirus peste des petits ruminants virus (PPRV). Two recombinant replication-defective human adenoviruses serotype 5 (Ad5) expressing either the highly immunogenic fusion protein (F) or hemagglutinin protein (H) from PPRV were used to vaccinate sheep by intramuscular inoculation. Both recombinant adenovirus vaccines elicited PPRV-specific B- and T-cell responses. Thus, neutralizing antibodies were detected in sera from immunized sheep. In addition, we detected a significant antigen specific T-cell response in vaccinated sheep against two different PPRV strains, indicating that the vaccine induced heterologous T cell responses. Importantly, no clinical signs and undetectable virus shedding were observed after virulent PPRV challenge in vaccinated sheep. These vaccines also overcame the T cell immunosuppression induced by PPRV in control animals. The results indicate that these adenovirus constructs could be a promising alternative to current vaccine strategies for the development of PPRV DIVA vaccines.     

Heart Rate and Systolic Blood Pressure Variability in the Time Domain in Patients with Recent and Long-Standing Diabetes Mellitus

Diabetes Mellitus (DM) affects the cardiovascular response of patients. To study this effect, interbeat intervals (IBI) and beat-to-beat systolic blood pressure (SBP) variability of patients during supine, standing and controlled breathing tests were analyzed in the time domain. Simultaneous noninvasive measurements of IBI and SBP for 30 recently diagnosed and 15 long-standing DM patients were compared with the results for 30 rigorously screened healthy subjects (control). A statistically significant distinction between control and diabetic subjects was provided by the standard deviation and the higher moments of the distributions (skewness, and kurtosis) with respect to the median. To compare IBI and SBP for different populations, we define a parameter, α, that combines the variability of the heart rate and the blood pressure, as the ratio of the radius of the moments for IBI and the same radius for SBP. As diabetes evolves, α decreases, standard deviation of the IBI detrended signal diminishes (heart rate signal becomes more “rigid”), skewness with respect to the median approaches zero (signal fluctuations gain symmetry), and kurtosis increases (fluctuations concentrate around the median). Diabetes produces not only a rigid heart rate, but also increases symmetry and has leptokurtic distributions. SBP time series exhibit the most variable behavior for recently diagnosed DM with platykurtic distributions. Under controlled breathing, SBP has symmetric distributions for DM patients, while control subjects have non-zero skewness. This may be due to a progressive decrease of parasympathetic and sympathetic activity to the heart and blood vessels as diabetes evolves.     

Secreted phospholipase A2-IIA modulates key regulators of proliferation on astrocytoma cells

Human group IIA secreted phospholipase A₂ (sPLA₂-IIA) has been characterized in numerous inflammatory and neoplastic conditions. sPLA₂-IIA can either promote or inhibit cell growth depending on the cellular type and the specific injury. We have previously demonstrated that exogenous sPLA₂-IIA, by engagement to a membrane structure, induces proliferation and activation of mitogen-activated protein kinases cascade in human astrocytoma cells. In this study, we used human astrocytoma 1321N1 cells to investigate the key molecules mediating sPLA₂-IIA-induced cell proliferation. We found that sPLA₂-IIA promoted reactive oxygen species (ROS) accumulation, which was abrogated in the presence of allopurinol and DPI, but not by rotenone, discarding mitochondria as a ROS source. In addition, sPLA₂-IIA triggered Ras and Raf-1 activation, with kinetics that paralleled ERK phosphorylation, and co-immunoprecipitation assays indicated an association between Ras, Raf-1 and ERK. Additionally, Akt, p70 ribosomal protein S6 kinase, and S6 ribosomal protein were also phosphorylated upon sPLA₂-IIA treatment, effect that was abrogated by N -acetylcysteine or LY294002 treatment indicating that ROS and phosphatidylinositol 3 kinase are upstream signaling regulators. As the inhibitors N -acetylcysteine, PD98059, LY294002 or rapamycin blocked sPLA₂-IIA-induced proliferation without activation of the apoptotic program, we suggest that inhibition of these intracellular signal transduction elements may represent a mechanism of growth arrest. Our results reveal new potential targets for therapeutic intervention in neuroinflammatory disorders and brain cancer in particular.     

The distribution of the ectoparasite fauna of Sebastes capensis from the southern hemisphere does not correspond with zoogeographical provinces of free-living marine animals

Aim We examined the ectoparasite fauna of Sebastes capensis over almost all its geographical distribution range (Chilean, Argentinean and South African coasts) to determine (1) whether the ectoparasites of this host show a zoogeographical pattern and, if so, (2) how this pattern is related to known zoogeographical patterns for free‐living organisms. Location Fish were captured from 20, 24, 30, 33, 36, 40, 45 and 52° S along the Chilean coast; 11° S on the Peruvian coast; 43° S on the Argentina coast; and 34° S on the South African coast. Methods From April to September 2003 and from April to August 2004, 626 fish were captured. The parasites were collected using standard parasitological techniques. At the component community level, zoogeographical distribution patterns were evaluated using cluster analysis. At the infra‐community level, patterns of similarity in parasite composition among localities were investigated with multivariate discriminant analyses. Results The ectoparasite fauna of S. capensis consists of six species distributed along the whole of the Chilean coast. Four other species are distributed only within the transitional zone between the northern warm temperate region (Peruvian faunistic province), extending from Peru to the northern Chilean coast up to c. 30° S, and the cold temperate region (Magellanic faunistic province). The component communities from latitudes 30 to 40° S showed higher ectoparasite species richness, while localities on the margins of the geographical range showed lower species richness. Cluster analysis indicated a grouping of localities consistent with the transitional zone. Argentina and South Africa always emerged as separate localities. Main conclusions The ectoparasite communities of S. capensis do not follow a distributional pattern concordant with the known biogeographical zones for invertebrates and/or fish along the south‐eastern Pacific. Therefore their ectoparasite fauna is not useful as a zoogeographical indicator, although it does allow us to distinguish the transitional zone of the south‐eastern Pacific. On a more extended geographical scale, it is possible to distinguish the ectoparasite communities of S. capensis in the south‐eastern Pacific (as a whole) from those of Argentina and South Africa.