Purification and partial characterization of azoreductase from Enterobacter agglomerans

Azoreductase, an enzyme catalyzing the reductive cleavage of the azo bond of methyl red (MR) and related dyes, was purified to electrophoretic homogeneity from Enterobacter agglomerans. This bacterial strain, isolated from dye-contaminated sludge, has a higher ability to grow, under aerobic conditions, on culture medium containing 100mg/L of MR. The enzyme was purified approximately 90-fold with 20% yield by ammonium sulfate precipitation, followed by three steps of column chromatography (gel-filtration, anion-exchange, and dye-affinity). The purified enzyme is a monomer with a molecular weight of 28,000 Da. The maximal azoreductase activity was observed at pH 7.0 and at 35 degrees C. This activity was NADH dependent. The K(m) values for both NADH and MR were 58.9 and 29.4 microM, respectively. The maximal velocity (V(max)) was 9.2 micromol of NADH min(-1)mg(-1). The purified enzyme is inhibited by several metal ions including Fe(2+) and Cd(2+).     

Chemodiversity of Volatile Oils in Thapsia garganica L. (Apiaceae)

The chemical composition of the volatile oils obtained from the roots, leaves, flowers, and stems of Thapsia garganica of Tunisian origin was investigated by GC‐FID and GC/MS analyses. Sesquiterpene hydrocarbons and oxygenated monoterpenes were predominant in the oils of all plant parts. Bicyclogermacrene (21.59–35.09%) was the main component in the former compound class, whereas geranial (3.31–14.84%) and linalool (0.81–10.9%) were the most prominent ones in the latter compound class. Principal‐component (PCA) and hierarchical‐cluster (HCA) analyses revealed some common constituents, but also significant variability amongst the oils of the different plant parts. This organ‐specific oil composition was discussed in relation to their biological and ecological functions. For the evaluation of the intraspecific chemical variability in T. garganica, the composition of the flower volatile oils from four wild populations was investigated. Bicyclogermacrene, linalool, and geranial were predominant in the oils of three populations, whereas epicubenol, β‐sesquiphellandrene, and cadina‐1,4‐diene were the most prominent components of the oil of one population. PCA and HCA allowed the separation of the flower oils into three distinct groups, however, no relationship was found between the volatile‐oil composition and the geographical distribution and pedoclimatic conditions of the studied populations.     

Phylodynamics and human-mediated dispersal of a zoonotic virus

Understanding the role of humans in the dispersal of predominantly animal pathogens is essential for their control. We used newly developed Bayesian phylogeographic methods to unravel the dynamics and determinants of the spread of dog rabies virus (RABV) in North Africa. Each of the countries studied exhibited largely disconnected spatial dynamics with major geopolitical boundaries acting as barriers to gene flow. Road distances proved to be better predictors of the movement of dog RABV than accessibility or raw geographical distance, with occasional long distance and rapid spread within each of these countries. Using simulations that bridge phylodynamics and spatial epidemiology, we demonstrate that the contemporary viral distribution extends beyond that expected for RABV transmission in African dog populations. These results are strongly supportive of human-mediated dispersal, and demonstrate how an integrated phylogeographic approach will turn viral genetic data into a powerful asset for characterizing, predicting, and potentially controlling the spatial spread of pathogens.     

Molecular epidemiology of methicillin-resistant Staphylococcus hominis (MRSHo): low clonality and reservoirs of SCCmec structural elements

Background

Methicillin resistant Staphylococcus hominis (MRSHo) are important human pathogens in immunocompromised patients. However, little is known regarding its population structure and staphylococcal chromosomal cassette mec (SCCmec) content.

Methodology/Principal Findings

To assess the population structure and the SCCmec content of S. hominis, 34 MRSHo and 11 methicillin-susceptible S. hominis (MSSHo) from neutropenic patients collected over a 3-year period were studied. The genetic backgrounds of S. hominis isolates were analyzed by pulsed-field gel electrophoresis (PFGE) and SCCmec types were determined by PCR. Cassette chromosome recombinases (ccr) were characterized by PCR and ccrB sequencing. The 34 S. hominis isolates were classified into as many as 28 types and 32 subtypes (SID = 99.82%); clonal dissemination was occasionally observed. The main SCCmec structures identified were SCCmec type VI (4B) (20%), SCCmec VIII (4A) (15%), and a new SCCmec composed of mec complex A in association with ccrAB1 (38%); 27% of the isolates harbored non-typeable SCCmec. Overall, a high prevalence of mec complex A (73.5%), ccrAB1 (50%) and ccrAB4 (44%) were found. Importantly, ccrB1 and ccrB4 from both MRSHo and MSSHo showed a high nucleotide sequence homology with those found in S. aureus SCCmec I, VI and VIII respectively (>95%).

Conclusions/Significance

The S. hominis population showed a limited clonality and a low genetic diversity in the allotypes of ccr and classes of mec complex. Moreover, our data suggest that S. hominis might have been a privileged source of mec complex A, ccrB1 and ccrB4, for the assembly of primordial SCCmec types.     

Evidence of DNA rearrangements in the 128-kilobase pBtoxis plasmid of Bacillus thuringiensis israelensis

BUPM97 is a novel Tunisian isolate of Bacillus thuringiensis israelensis presenting insecticidal activity against Culex pipiens larvae. The δ-endotoxins pattern of this strain was different from that of the reference strain B. thuringiensis israelensis H14. Therefore, the study of its cry genes content was carried out by restriction-fragment-length-polymorphism (RFLP) using specific cry genes probes and by DNA sequencing. It was clearly demonstrated that in the strain BUPM97 the cry4A and cry10A genes were deleted from the B. thuringiensis israelensis 128-kb pBtoxis plasmid. In addition, a strong DNA sequence polymorphism was evidenced in the same plasmid downstream from the cry4B gene. This very particular DNA dynamic evidenced in this new strain of B. thuringiensis israelensis should be taken into consideration, regarding the strain stability during the industrial production of B. thuringiensis bioinsecticides.     

Effect of freezing–thawing process and quercetin on human sperm survival and DNA integrity

We aimed in the first part of our work to study the effect of cryopreservation on the human sperm DNA integrity and the activation of caspase 3, the main apoptosis indicator. In the second part, we were interested in testing the effect of quercetin, as an antioxidant, in preventing sperm damage during the freeze–thawing process. Seventeen semen samples were obtained from 17 men recruited for infertility investigations. Liquefied sperm was cryopreserved using spermfreeze®. Nine of the used samples were divided into two aliquots; the first one was cryopreserved with spermfreeze only (control) and the second one was cryopreserved with spermfreeze supplemented with quercetin to a final concentration of 50 μM. Sperm motility and viability were assessed according to WHO criteria. We used TUNEL assay and the Oxy DNA assay to assess sperm DNA integrity. Activated caspase 3 levels were measured in spermatozoa using fluorescein-labeled inhibitor of caspase (FLICA). Cryopreservation led to a significant increase in sperm DNA fragmentation, DNA oxidation and caspase 3 activation (p < 0.01). Supplementation of the cryopreservation medium with quercetrin induced a significant improvement in post thaw sperm parameters, compared to those of control, regarding sperm motility (p = 0.007), viability (p = 0.008) and DNA integrity (p = 0.02); however, it had no effect on caspase 3 activation (p = 0.3). We conclude that oxidative stress plays a major role in inducing sperm cryodamage but implication of apoptosis in this impairment requires further investigations. Quercetin could have protective effect during cryopreservation but further research is needed to confirm this effect.     

Structural basis of C-terminal β-amyloid peptide binding by the antibody ponezumab for the treatment of Alzheimer's disease

Alzheimer's disease, the most common cause of dementia in the elderly and characterized by the deposition and accumulation of plaques, is composed in part of β-amyloid (Aβ) peptides, loss of neurons, and the accumulation of neurofibrillary tangles. Here, we describe ponezumab, a humanized monoclonal antibody, and show how it binds specifically to the carboxyl (C)-terminus of Aβ40. Ponezumab can label Aβ that is deposited in brain parenchyma found in sections from Alzheimer's disease casualties and in transgenic mouse models that overexpress Aβ. Importantly, ponezumab does not label full-length, non-cleaved amyloid precursor protein on the cell surface. The C-terminal epitope of the soluble Aβ present in the circulation appears to be available for ponezumab binding because systemic administration of ponezumab greatly elevates plasma Aβ40 levels in a dose-dependent fashion after administration to a mouse model that overexpress human Aβ. Administration of ponezumab to transgenic mice also led to a dose-dependent reduction in hippocampal amyloid load. To further explore the nature of ponezumab binding to Aβ40, we determined the X-ray crystal structure of ponezumab in complex with Aβ40 and found that the Aβ40 carboxyl moiety makes extensive contacts with ponezumab. Furthermore, the structure-function analysis supported this critical requirement for carboxy group of AβV40 in the Aβ-ponezumab interaction. These findings provide novel structural insights into the in vivo conformation of the C-terminus of Aβ40 and the brain Aβ-lowering efficacy that we observed following administration of ponezumab in transgenic mouse models.     

Changes in growth and oxidative response of leaves and nodules in <Emphasis Type="Italic">Medicago ciliaris</Emphasis> during salt stress recovery

In the present study, we exposed Medicago ciliaris isolated from a saline sebhka and growing symbiotically with Sinorhizobium medicae to 100 mM of NaCl and followed this stress by a recovery period (complete NaCl withdrawal from the root medium for two or one weeks). This experiment was conducted in order i) to check whether a reduction in growth and nitrogen fixation could be reversed by alleviating salt stress and ii) to determine specific changes related to salt-induced growth and nitrogen fixation decline. Salt stress reduced growth of all organs (particularly nodules), leaf area, photosynthetic activity and nitrogen fixation. The depressive effect of salt was not linked to osmotic stress. The removal of saline conditions restored growth of all organs after 2 weeks of recovery, with aerial organs recovering quickly after 1 week. Both photosynthetic and nitrogen fixation recovered only after a 2 week period of recovery. Salt stress in these saline-tolerant plants was accompanied by oxidative damage (electrolyte leakage), which was more accentuated in nodules in comparison with leaves. The observed recovery in growth, nitrogen fixation and photosynthesis was mainly linked to a preferential preservation of shoots growth owing to the maintenance of SOD activity (namely the isoforms A₂ and A₃); such maintenance would allow higher photosynthetic activity permitting an adequate supply of nodules with photoassimilates and thus facilitating stress withstand.