Identification and characterization of a spore-like morphotype in chronically starved Mycobacterium avium subsp. paratuberculosis cultures

Mycobacteria are able to enter into a state of non-replication or dormancy, which may result in their chronic persistence in soil, aquatic environments, and permissive hosts. Stresses such as nutrient deprivation and hypoxia provide environmental cues to enter a persistent state; however, a clear definition of the mechanism that mycobacteria employ to achieve this remains elusive. While the concept of sporulation in mycobacteria is not novel, it continues to spark controversy and challenges our perceptions of a non-replication. We investigated the potential role of sporulation in one-year old broth cultures of Mycobacterium subsp. paratuberculosis (MAP). We show that dormant cultures of MAP contain a mix of vegetative cells and a previously unknown morphotype resembling a spore. These spore-like structures can be enriched for using sporulating media. Furthermore, purified MAP spore forms survive exposure to heat, lysozyme and proteinase K. Heat-treated spores are positive for MAP 16SrRNA and IS900. MAP spores display enhanced infectivity as well as maintain acid-fast characteristics upon germination in a well-established bovine macrophage model. This is the first study to demonstrate a new MAP morphotype possessing spore-like qualities. Data suggest that sporulation may be a viable mechanism by which MAP accomplishes persistence in the host and/or environment. Thus, our current understanding of mycobacterial persistence, pathogenesis, epidemiology and rational drug and vaccine design may need to be reevaluated.     

DNA fragmentation dynamics allows the assessment of cryptic sperm damage in human: Evaluation of exposure to ionizing radiation, hyperthermia, acidic pH and nitric oxide

Sperm DNA fragmentation (SDF) is not a static seminal parameter, since the longevity of sperm DNA decreases progressively with time following ejaculation or thawing. While the dynamics of SDF is a species-specific characteristic, in the case of humans, there is still significant variation within patients. To evaluate the suitability of the dynamic SDF assay to assess the adverse effects of agents that cause genetic damage, fresh semen samples from different donors were exposed in vitro to (1) increasing acute doses of ionizing radiation, (2) elevated temperature (41°C and 45°C), (3) acidic pH (pH 4) and (4) the nitric oxide (NO) donor sodium nitroprusside (SNP). Sperm DNA fragmentation was analyzed after an incubation period of chronic (24h), or acute (1h) exposure to each treatment followed by incubation at 37°C over a period of 24h. SDF was assessed using the sperm chromatin dispersion (SCD) test. Dynamic SDF for each treatment was analyzed using Kaplan-Meier survival curves. All agents, except for ionizing radiation, accelerated SDF kinetics following chronic exposure over a 24h period. Transient exposure to NO and heat but not acidic pH increased the basal (T0) level of SDF. Despite the removal of the three toxicants, the remaining sperm following acute exposure showed a decrease in their expected DNA longevity. It is concluded that the assessment of sperm DNA fragmentation dynamics is an effective methodological approach for revealing latent damage associated with toxicants that is not initially expressed following a single initial observation of SDF.     

Genome-wide association study of multiple sclerosis confirms a novel locus at 5p13.1

Multiple Sclerosis (MS) is the most common progressive and disabling neurological condition affecting young adults in the world today. From a genetic point of view, MS is a complex disorder resulting from the combination of genetic and non-genetic factors. We aimed to identify previously unidentified loci conducting a new GWAS of Multiple Sclerosis (MS) in a sample of 296 MS cases and 801 controls from the Spanish population. Meta-analysis of our data in combination with previous GWAS was done. A total of 17 GWAS-significant SNPs, corresponding to three different loci were identified:HLA, IL2RA, and 5p13.1. All three have been previously reported as GWAS-significant. We confirmed our observation in 5p13.1 for rs9292777 using two additional independent Spanish samples to make a total of 4912 MS cases and 7498 controls (ORpooled = 0.84; 95%CI: 0.80-0.89; p = 1.36 × 10-9). This SNP differs from the one reported within this locus in a recent GWAS. Although it is unclear whether both signals are tapping the same genetic association, it seems clear that this locus plays an important role in the pathogenesis of MS.     

Molecular magnetic materials from polyoxometalates

The significance of polyoxometalates in the field of molecular magnetism is discussed. We show that this kind of inorganic complexes provides remarkable examples for the study of the exchange interactions in clusters. On the other hand, we examine the possibility of using these metal oxide anions as magnetic components of molecular materials containing organic tetrathiafulvalenes as electron donor molecules.     

A new species of Hyphoderma (Meruliaceae, Polyporales) and its discrimination from closely related taxa

Thirty-five corticioid collections from the Canary Islands and Azores Archipelago were examined morphologically and subjected to molecular phylogenetic analysis. These specimens, almost all collected on endemic and/or xerophilic vegetation, were similar in morphological and ecological characteristics to Hypochnicium prosopidis from the Sonoran Desert (Arizona, USA) and Hyphoderma amoenum. Thirty-seven new ITS nrDNA sequences from these specimens, including the nomenclatural type of the above-mentioned species, were obtained and aligned with homologous sequences from GenBank. These collections were distributed in two strongly supported monophyletic clades. However, similar patterns of morphological variability shared by specimens included in both clades and their differences with related species suggest that they should be described as a single new species. Therefore Hyphoderma macaronesicum is proposed. Studies will be required to test, in a more robust multilocus genealogical framework, whether these populations constitute two cryptic species or whether they are the same taxon. The position of Hypochnicium prosopidis in the resolved tree and its morphological characters suggest that it should be included in Hyphoderma and the new combination Hyphoderma prosopidis is proposed.     

Using dot blot with immunochemical detection to evaluate global changes in SUMO-2/3 conjugation

Here we describe a non-invasive method for rapid and highly reproducible genotyping of transgenic mammals with ubiquitous expression of fluorophore reporters. Hair samples from transgenic mice and pigs with systemic expression of the fluorophore reporter Venus were analyzed with a fluorescence microscope in few minutes. The hair samples can be preserved for long-term storage at ambient temperature conditions. This non-invasive method is useful for genotyping of transgenic large animals and contributes to animal welfare by reducing stress and discomfort of the animals during sample collection.     

Determination of fatty acid and triacylglycerol composition of human very-low-density lipoproteins

The fatty acid composition of human very-low-density lipoproteins (VLDL) was studied in a population from western Andalusia with a diet in which the fat content came mainly from olive oil. The lipid composition of VLDL, including the fatty acid composition of the phospholipids and triacylglycerols, was examined by capillary gas chromatography. Twenty-five peaks were resolved, ranging in chain length from 14 to 24 carbon atoms, including geometric and positional isomers. The major fatty acids present in phospholipids were 16:0, 18:0, 18:1(n − 9) and 18:2(n − 6), and in triacylglycerols were 18:1(n − 9), 16:0 and 18:2(n − 6). The major triacylglycerol was POO, followed by PLO and OOO. MLP, PPS and LLL were absent. The presence of a large amount of OOO in this fraction demonstrates that the triacylglycerol composition of the VLDL depends on the type of diet consumed.     

An endogenous factor which interacts with synaptosomal membrane Na+, K+-ATPase activation by K+

In previous papers, the isolation of brain soluble fractions able to modify neuronal Na⁺, K⁺-ATPase activity has been described. One of those fractions-peak I-stimulates membrane Na⁺, K⁺-ATPase while another-peak II-inhibits this enzyme activity, and has other ouabain-like properties. In the present study, synaptosomal membrane Na⁺, K⁺-ATPase was analyzed under several experimental conditions, using ATP orp-nitrophenylphosphate (p-NPP) as substrate, in the absence and presence of cerebral cortex peak II. Peak II inhibited K⁺-p-NPPase activity in a concentration dependent manner. Double reciprocal plots indicated that peak II uncompetitively inhibits K⁺-p-NPPase activity regarding substrate, Mg²⁺ and K⁺ concentration. Peak II failed to block the known K⁺-p-NPPase stimulation caused by ATP plus Na⁺. At various K⁺ concentrations, percentage K⁺-p-NPPase inhibition by peak II was similar regardless of the ATP plus Na⁺ presence, indicating lack of correlation with enzyme phosphorylation. Na⁺, K⁺-ATPase activity was decreased by peak II depending on K⁺ concentration. It is postulated that the inhibitory factor(s) present in peak II interfere(s) with enzyme activation by K⁺.     

Littoral benthic macroinvertebrates of mountain lakes in the Tatra Mountains (Slovakia,Poland)

Littoral benthic macroinvertebrates of 45 mountain lakes in the Tatra Mountains were sampled using a semi-quantitative method in September 2000. A total of 32,852 specimens were identified to 93 taxa belonging to 14 higher taxonomic groups. Multivariate statistics (CCA, RDA) and nine biotic metrics (AQEM/STAR) were used to explain relationships between macroinvertebrate assemblages and environmental variables. Up to 57% of the ecological position of littoral macroinvertebrate assemblages were explained by variance of environmental variables divided into chemical, trophic, physical, catchment and location. Five types of Tatra lakes were recognized using CCA: A — strongly acidified lakes (small catchment, low pH, high concentration of TP, DOC, highest amount of POM in littoral); B — alpine acidified lakes (low amount of POM, low values of biotic metrics); C — alpine non-acidified lakes (high value of diversity index, predominance of Diptera); D — subalpine acidified lakes (high values of biotic metrics: number of families, proportion of crenal and rhithral taxa/total taxa); E — subalpine non-acidified lakes (high values of biotic metrics: number of families, number of genera, BMWP score, number of taxa and abundance of EPT taxa). RDA was used to design five levels of macroinvertebrate taxa acidification tolerance. The Tatra Acidification Index (TAI) was established to assess the acidification status of the lakes in the Tatra Mts.