Mitochondrial DNA mutations and polymorphisms in asthenospermic infertile men

In this study we performed a systematic sequence analysis of 6 mitochondrial genes (cytochrome oxidase I, cytochrome oxidase II, cytochrome oxidase III, adenosine triphosphate synthase6, ATP synthase8, and cytochrome b] in 66 infertile men suffering from asthenospermia (n = 34) in comparison to normospermic infertile men (n = 32) and fertile men (n = 100) from Tunisian population. A total of 72 nucleotide substitutions in blood cells mitochondrial DNA were found; 63 of them were previously identified and reported in the human mitochondrial DNA database (www.mitomap.org) and 9 were novel. We also detected in 3 asthenospermic patients a novel heteroplasmic missense mitochondrial mutation (m.9387 G>A) in COXIII gene (8.8 %) that was not found in any of normospermic infertile and fertile men. This mutation substituting the valine at position 61 to methionine in a conserved amino acid in the transmembrane functional domain of the polypeptide, induces a reduction of the hydropathy index (from +1.225 to +1.100) and a decrease of the protein 3D structures number (from 39 to 32) as shown by PolyPhen bioinformatic program.     

Composition of the essential oils in various organs at different developmental stages of Ammi visnaga (L.) Lam. from Tunisia

The composition of the essential oils isolated by hydrodistillation from various organs at different development stages of Ammi visnaga (L.) Lam. growing in Tunisia was determined by GC/MS analysis. In particular, the oil profiles of the leaves, stems, flower buds, roots, umbels, and fruits have been examined during the whole life cycle. The oil from the flowering aerial parts was characterized by a high content of isoamyl 2-methylbutanoate. After flowering and during desiccation and fructification, the umbels and fruits expressed a high content of linalool. The oils, extracted from the roots collected in the vegetatif, buds floral, and floral stages, were rich in monoterpene aldehydes, oxygenated monoterpenes, and monoterpene hydrocarbons. The highest level of non-terpene hydrocarbons was found at the flower-bud stage, represented by 61.3% of nonane. Among the monoterpenes, sabinene (12.5%) and β-pinene (8.5%) were identified in the flower buds.     

Expression patterns of Notch receptors and their ligands in human osteoarthritic and healthy articular cartilage

Notch pathway plays a pivotal role in cell fate determination. There is much interest surrounding its therapeutic potential, in osteoarthritis, but the expression profile of Notch-related molecules, as well as their relation with cartilage pathological parameters, remains unclear. The purpose of our study is to analyze the expression pattern of Notch family members, type II and type I collagen, in normal (healthy) and osteoarthritic human knee cartilage. Osteoarthritic cartilages were obtained from 3 patients undergoing a total knee replacement. Macroscopically normal cartilage was dissected from 3 human knees at the time of autopsy or surgery. Immunohistochemical staining was performed using Notch1,2,3 and 4, Delta, Jagged, type II collagen and type I collagen antibodies. In healthy cartilage, type II collagen was abundantly expressed while type I was absent. This latter increased proportionally to the osteoarthritic grade. Type II collagen expression remained intense in osteoarthritic cartilage. In healthy cartilage as well as in cartilage with minor lesions, Notch family member's proteins were not or just weakly expressed at the surface and in the cells. However, Notch molecules were over-expressed in osteoarthritic cartilage compared to healthy one. This expression pattern was different according to the cartilage zone and the severity of OA. Our data suggest that Notch signaling is activated in osteoarthritic cartilage, compared to healthy cartilage, with a much more abundant expression in the most damaged areas.     

Detection and frequency of Chlamydia trachomatis DNA in synovial samples from Tunisian patients with reactive arthritis and undifferentiated oligoarthritis

We aimed to determine the frequency of Chlamydia trachomatis DNA in the synovial compartment of 34 arthritic patients. Chlamydia trachomatis DNA was detected using a nested PCR targeting the cryptic plasmid, the 16S rRNA gene and the outer membrane protein 1 gene. The presence of serum immunoglobulin (Ig)G and IgA antibodies against C. trachomatis was studied by a microimmunofluorescence assay and by an enzyme-linked immunosorbent assay, respectively. Synovial samples from 20 of 34 (59%) patients [nine with reactive arthritis (ReA), seven with undifferentiated oligoarthritis (UOA), two with rheumatoid arthritis and two with osteoarthritis] were positive for at least one C. trachomatis DNA sequence by nested PCR. The high sensitivity results most likely from the combination of a standardized automated MagNA Pure extraction method, PCR targeting three different C. trachomatis genes and the screening for C. trachomatis in synovial tissue and fluid samples. There was no correlation between the presence of C. trachomatis DNA in the joint and a Chlamydia -specific serologic response. Our data support that PCR is the method of choice to establish the diagnosis of Chlamydia -induced arthritis in patients with ReA. We suggest that this diagnosis might also be considered in C. trachomatis -positive patients previously classified as UOA.     

Molecular Identification of <Emphasis Type="Italic">Malassezia</Emphasis> Species in Patients with <Emphasis Type="Italic">Malassezia</Emphasis> folliculitis in Sfax,Tunisia

Aim

Malassezia folliculitis is caused by the invasion of hair follicles by large numbers of Malassezia cells. Several Malassezia researches still use cultures, morphology and biochemical techniques. The aim of this study was to identify Malassezia species isolated from patients diagnosed with folliculitis, at the Parasitology and Mycology Laboratory of Sfax University Hospital, and to explore the genetic diversity of Malassezia by using PCR-RFLP and PCR-sequencing targeting the rDNA region of the Malassezia genome.

Patients and Methods

Specimens were taken from 27 patients with Malassezia folliculitis. For the molecular identification, PCR amplification of the 26S rDNAD1/D2 region was carried out using the Malup and Maldown primers and three restriction enzymes (BanI, MspI and HeaII) for RFLP analysis. The nucleotide sequences of each isolate were compared to those in the NCBI GenBank by using BLASTIN algorithm.

Results

Three species of Malassezia yeasts were identified among the 31 Malassezia strains isolated: M. globosa (83.9%), M. sympodialis (12. 9%) and M. furfur (3.2%). The sequence analysis of M. globosa showed six genotypes.

Conclusion

There is a high genotypic variability of M. globosa colonizing patients with folliculitis.

     

Olive mill wastewater stabilization in open-air ponds: impact on clay-sandy soil

The aim of this work was to study the natural biodegradation of the stored olive mill wastewater (OMW) in ponds and the infiltration as well as the impact on soil of the effluent in the evaporation pond used for the storage over the past eight years. For this, two approaches were considered. First, a laboratory-scale column was used for the infiltration of OMW through soil (clay and sand) to predict the effect of the clayey soil in reducing OMW pollution. Second, the ponds including the effluent annually stored and having this clayey structure were investigated. At the laboratory-scale, a modification of OMW contents was noticed, with the elimination of 95% of total suspended solids (TSS), 60% of chemical oxygen demand (COD), 40% of total organic carbon (TOC), 50% of total P, 50% of phenols and 40% of minerals (K+, Mg++ and Na+). The experimented soil was able to restrain the considerable effects of OMW pollution. In the ponds, the granulometric characteristics, the physico-chemical and the biological parameters of the soil profile from the contaminated pond were compared to those of a control soil, located near the contaminated pond. Property modifications of the contaminated soil were noted, especially pH, electrical conductivity, COD and microflora. These changes can be explained by the infiltration of OMW constituents, which were noticed in the soil layers, especially phenolic compounds that have a negative effect on the ground water.     

HPLC‐DAD Analysis,Antioxidant and Protective Effects of Tunisian Rhanterium suaveolens against Acetamiprid Induced Oxidative Stress on Mice Erythrocytes

The present study was performed to assess the HPLC‐DAD analysis as well as antioxidant and protective effects of Tunisian Rhanterium suaveolens (Rs) against acetamiprid (ACT) induced oxidative stress on mice erythrocytes. The in vitro assays showed that the methanolic extract of Rs has an impressive antioxidant effect proved by testing the total antioxidant and scavenging activities using BCB, DPPH and ABTS assays, respectively. Moreover, qualitative and quantitative analysis using HPLC‐DAD revealed the richness of Rs in polyphenols where p‐Coumaric, Apigenin‐7‐glucoside and Ferulic acid were detected as the most abundant polyphenols. In the in vivo experiment, ACT, used as a toxicity model, was given to mice at a dose of 20 mg/kg. The latter was the origin of hemolytic anemia characterized by a significant decrease in red blood cells, hemoglobin and hematocrit levels and an increase in bilirubin, LDH, osmotic fragility, reticulocytes and white blood cells number. Characteristic erythrocyte morphological alterations were also determined as spherocytosis, schistocytosis and dacryocystitis. The oxidative status of ACT‐treated mice was also altered manifested by a significant increase in MDA and GSH levels and a decrease in SOD, CAT and GPx activities. When receiving the Rs methanolic extract at a dose of 300 mg/kg, all the parameters cited above were restored in mice. These remarkable corrections could only confirm the important antioxidant effect and the noticeable protective properties that possess Rs owing to its broad range of secondary bioactive metabolites.     

P2X4 Assembles with P2X7 and Pannexin-1 in Gingival Epithelial Cells and Modulates ATP-induced Reactive Oxygen Species Production and Inflammasome Activation

We have previously reported that Porphyromonas gingivalis infection of gingival epithelial cells (GEC) requires an exogenous danger signal such as ATP to activate an inflammasome and caspase-1, thereby inducing secretion of interleukin (IL)-1β. Stimulation with extracellular ATP also stimulates production of reactive oxygen species (ROS) in GEC. However, the mechanism by which ROS is generated in response to ATP, and the role that different purinergic receptors may play in inflammasome activation, is still unclear. In this study, we revealed that the purinergic receptor P2X₄ is assembled with the receptor P2X₇ and its associated pore, pannexin-1. ATP induces ROS production through a complex consisting of the P2X₄, P2X_7, and pannexin-1. P2X₇−mediated ROS production can activate the NLRP3 inflammasome and caspase-1. Furthermore, separate depletion or inhibition of P2X₄, P2X₇, or pannexin-1 complex blocks IL-1β secretion in P. gingivalis-infected GEC following ATP treatment. However, activation via P2X₄ alone induces ROS generation but not inflammasome activation. These results suggest that ROS is generated through stimulation of a P2X₄/P2X₇/pannexin-1 complex, and reveal an unexpected role for P2X₄, which acts as a positive regulator of inflammasome activation during microbial infection.