Molecular Cloning and Characterization of a Superoxide Dismutase (sod) Gene in Pneumocystis carinii

This work reports the isolation and characterization of a gene encoding a superoxide dismutase (SOD. EC.1.15.1.1.) from Pneumocystis carinii derived from rat. Sense and antisense oligonucleotides, deduced from SOD amino acid sequences from a wide variety of organisms, allowed amplification of a 669 bp genomic DNA fragment specific to this P. carinii. RACE-PCR was used to obtain the major pan of the complementary DNA; the 5- and 3'-genomic regions were obtained respectively from a Mbo I subgenomic library and from an amplified fragment using oligonucleotides designed from the cDNA sequence. Comparison of genomic and cDNA sequences showed an open reading frame of 660 bp interrupted by seven small introns. The deduced amino acid sequence contained 220 residues. Protein sequence alignment demonstrated the highest homology (50.5% identity. 70.3% similarity) with Saccharomyces cerevisiae manganese-SOD (MnSOD) suggesting that P. carinii SOD belongs to the mitochondrial MnSOD group. A putative targeting peptide found at the 5'-end of the P. carinii SOD sequence also suggested its mitochondrial localization.     

Lysozyme crystal growth, as observed by small angle X-ray scattering, proceeds without crystallization intermediates

A combination of small angle X-ray scattering and gel techniques was used to follow the kinetics of protein crystal growth as a function of time. Hen egg white lysozyme, at different protein concentrations, was used as a model system. A new sample holder was designed, in which supersaturation is induced in the presence of salt by decreasing the temperature. It had been shown previously that a decrease in temperature and/or an increase in crystallizing agent induces an increase in the attractive interactions present in the lysozyme solutions, the lysozyme remaining monomeric. In the present paper we show that similar behaviour is observed in NaCl when agarose gels are used. During crystal growth, special attention was paid to determine whether oligomers were formed as the protein in solution was incorporated in the newly formed crystals. From these first series of experiments, we did not find any indication of oligomer formation between monomer in solution and crystal. The results obtained are in agreement with the hypothesis that lysozyme crystals in NaCl grow by addition of monomeric particles. Received: 28 July 1997 / Revised version: 4 December 1997 / Accepted: 5 December 1997     

In vitro interactions between bone marrow stromal cells and hippocampal slice cultures

Bone marrow stromal cells (BMSCs) are capable of differentiating into various cell types including brain cells. Several groups have also demonstrated trophic effects of MSC grafts in experimental ischemia models. However, the underlying molecular mechanisms of these effects are not fully understood. We developed an “in vitro graft model” which consisted in a coculture of GFP-expressing BMSCs and hippocampal organotypic slice cultures. Total marrow cells (MCs) or BMSCs after one (BMSC^1P) or five passages (BMSC^5P) were transplanted on hippocampal slices. During the 10 days of our experiments, MCs and BMSC^1P migrated toward the tissue, but their total number remained constant. Conversely, the number of BMSC^5P decreased over the 10 days of the experiment, and no migration could be detected. Using immunohistochemistry, we observed that the hippocampal slices induced the expression of neural antigens in very few grafted cells, but MCs and BMSC^1P improved the conservation of the hippocampal slice culture. Similar experiments using BMSC^5P did not produce any significant change. We conclude that the number of passages greatly influence BMSCs survival rate, migration and neuroprotective capacities.     

Behavioral syndrome in a native and an invasive hymenoptera species

Recent studies have focused on the role of behavior in biological invasions. Individuals may differ consistently in time for several behavioral traits (personality) which covary (behavioral syndrome) resulting in different behavioral types, some of them favoring invasion. Social hymenopterans have a strong potential to be invaders and their success depends primarily on the foundresses’ ability to found viable colonies. They are expected to be active, explorative and bold for optimally establishing their nest. In Europe, 2 hornet species coexist: the native Vespa crabro and the invasive Vespa velutina. These 2 species may compete for nesting sites and we suggest that the initial success of V. velutina has been favored by its behavior in outperforming V. crabro for the traits involved in nest initiation. Here, we (i) defined the personality of V. crabro and V. velutina, (ii) tested for the existence of behavioral syndrome in these species, and (iii) compared their performances using an open‐field test. Our results show that V. crabro foundresses behave consistently but not V. velutina; this lack of consistency being mainly due to reduced variance among individuals. This result questions the possibility of detecting consistent behavioral differences in species having recently undergone a strong bottleneck. Both species exhibit the same correlations between activity, boldness and exploration and V. velutina clearly outperforms V. crabro for all traits. Our results suggest that activity, boldness, and exploration are implicated in both hornet nest initiation and invasion process which contributed to explain why social hymenopterans are so successful at colonization.     

Initial Data on the Molecular Epidemiology of Cryptosporidiosis in Lebanon

Cryptosporidium spp. represent a major public health problem worldwide and infect the gastrointestinal tract of both immunocompetent and immunocompromised persons. The prevalence of these parasites varies by geographic region, and no data are currently available in Lebanon. To promote an understanding of the epidemiology of cryptosporidiosisin this country, the main aim of this study was to determine the prevalence Cryptosporidium in symptomatic hospitalized patients, and to analyze the genetic diversity of the corresponding isolates. Fecal specimens were collected in four hospitals in North Lebanon from 163 patients (77 males and 86 females, ranging in age from 1 to 88 years, with a mean age of 22 years) presenting gastrointestinal disorders during the period July to December 2013. The overall prevalence of Cryptosporidium spp. infection obtained by modified Ziehl-Neelsen staining and/or nested PCR was 11%, and children <5 years old showed a higher rate of Cryptosporidium spp. The PCR products of the 15 positive samples were successfully sequenced. Among them, 10 isolates (66.7%) were identified as C. hominis, while the remaining 5 (33.3%) were identified as C. parvum. After analysis of the gp60 locus, C. hominis IdA19, a rare subtype, was found to be predominant. Two C. parvum subtypes were found: IIaA15G1R1 and IIaA15G2R1. The molecular characterization of Cryptosporidium isolates is an important step in improving our understanding of the epidemiology and transmission of the infection.     

Poor Transferability of Species Distribution Models for a Pelagic Predator,the Grey Petrel,Indicates Contrasting Habitat Preferences across Ocean Basins

Species distribution models (SDMs) are increasingly applied in conservation management to predict suitable habitat for poorly known populations. High predictive performance of SDMs is evident in validations performed within the model calibration area (interpolation), but few studies have assessed SDM transferability to novel areas (extrapolation), particularly across large spatial scales or pelagic ecosystems. We performed rigorous SDM validation tests on distribution data from three populations of a long-ranging marine predator, the grey petrel Procellaria cinerea, to assess model transferability across the Southern Hemisphere (25-65°S). Oceanographic data were combined with tracks of grey petrels from two remote sub-Antarctic islands (Antipodes and Kerguelen) using boosted regression trees to generate three SDMs: one for each island population, and a combined model. The predictive performance of these models was assessed using withheld tracking data from within the model calibration areas (interpolation), and from a third population, Marion Island (extrapolation). Predictive performance was assessed using k-fold cross validation and point biserial correlation. The two population-specific SDMs included the same predictor variables and suggested birds responded to the same broad-scale oceanographic influences. However, all model validation tests, including of the combined model, determined strong interpolation but weak extrapolation capabilities. These results indicate that habitat use reflects both its availability and bird preferences, such that the realized distribution patterns differ for each population. The spatial predictions by the three SDMs were compared with tracking data and fishing effort to demonstrate the conservation pitfalls of extrapolating SDMs outside calibration regions. This exercise revealed that SDM predictions would have led to an underestimate of overlap with fishing effort and potentially misinformed bycatch mitigation efforts. Although SDMs can elucidate potential distribution patterns relative to large-scale climatic and oceanographic conditions, knowledge of local habitat availability and preferences is necessary to understand and successfully predict region-specific realized distribution patterns.     

Diversification and Distribution of Ruminant Chlamydia abortus Clones Assessed by MLST and MLVA

Chlamydia abortus, an obligate intracellular bacterium, is the most common infectious cause of abortion in small ruminants worldwide and has zoonotic potential. We applied multilocus sequence typing (MLST) together with multiple-locus variable-number tandem repeat analysis (MLVA) to genotype 94 ruminant C. abortus strains, field isolates and samples collected from 1950 to 2011 in diverse geographic locations, with the aim of delineating C. abortus lineages and clones. MLST revealed the previously identified sequence types (STs) ST19, ST25, ST29 and ST30, plus ST86, a recently-assigned type on the Chlamydiales MLST website and ST87, a novel type harbouring the hemN_21 allele, whereas MLVA recognized seven types (MT1 to MT7). Minimum-spanning-tree analysis suggested that all STs but one (ST30) belonged to a single clonal complex, possibly reflecting the short evolutionary timescale over which the predicted ancestor (ST19) has diversified into three single-locus variants (ST86, ST87 and ST29) and further, through ST86 diversification, into one double-locus variant (ST25). ST descendants have probably arisen through a point mutation evolution mode. Interestingly, MLVA showed that in the ST19 population there was a greater genetic diversity than in other STs, most of which exhibited the same MT over time and geographical distribution. However, the evolutionary pathways of C. abortus STs seem to be diverse across geographic distances with individual STs restricted to particular geographic locations. The ST30 singleton clone displaying geographic specificity and represented by the Greek strains LLG and POS was effectively distinguished from the clonal complex lineage, supporting the notion that possibly two separate host adaptations and hence independent bottlenecks of C. abortus have occurred through time. The combination of MLST and MLVA assays provides an additional level of C. abortus discrimination and may prove useful for the investigation and surveillance of emergent C. abortus clonal populations.     

Individuals with higher metabolic rates have lower levels of reactive oxygen species in vivo

There is increasing interest in the effect of energy metabolism on oxidative stress, but much ambiguity over the relationship between the rate of oxygen consumption and the generation of reactive oxygen species (ROS). Production of ROS (such as hydrogen peroxide, H₂O₂) in the mitochondria is primarily inferred indirectly from measurements in vitro, which may not reflect actual ROS production in living animals. Here, we measured in vivo H₂O₂ content using the recently developed MitoB probe that becomes concentrated in the mitochondria of living organisms, where it is converted by H₂O₂ into an alternative form termed MitoP; the ratio of MitoP/MitoB indicates the level of mitochondrial H₂O₂ in vivo. Using the brown trout Salmo trutta, we tested whether this measurement of in vivo H₂O₂ content over a 24 h-period was related to interindividual variation in standard metabolic rate (SMR). We showed that the H₂O₂ content varied up to 26-fold among fish of the same age and under identical environmental conditions and nutritional states. Interindividual variation in H₂O₂ content was unrelated to mitochondrial density but was significantly associated with SMR: fish with a higher mass-independent SMR had a lower level of H₂O₂. The mechanism underlying this observed relationship between SMR and in vivo H₂O₂ content requires further investigation, but may implicate mitochondrial uncoupling which can simultaneously increase SMR but reduce ROS production. To our knowledge, this is the first study in living organisms to show that individuals with higher oxygen consumption rates can actually have lower levels of H₂O₂.