Intérêt de l’étude de l’oxydation de l’ADN des spermatozoïdes par marquage de la 8-oxo-guanine en cytométrie en flux chez l’homme infertile

Oxidative stress in semen is essentially due to excessive production of oxygen-reactive species (ORS) essentially derived from leukocytes. DNA oxidation is due to the direct action of ORS which produce several adducts the most extensively studied of which is 8-oxo-guanine. Integrity of DNA is essential for the fertility of sperm and is an important subject of research for scientists and clinicians all over the world. Although evaluation of the global integrity of sperm DNA has considerably developed over recent years, few tests are available to document oxidative DNA damage. This study was designed to review the various tests of sperm DNA integrity commonly used in the literature and to present the results of our study on DNA oxidation with 8-oxo-guanine labelling by flow cytometry in infertile men. This study was based on 15 semen samples that were submitted to sperm analysis according to WHO guidelines, with determination of the leukocyte concentration by a cytochemical method revealing peroxidase in cytoplasmic granulations. The DNA oxidation study was performed with 8-oxo-guanine labelling by flow cytometry. Linear regression analysis showed a strong correlation between DNA oxidation and leukocyte count in the semen (p = 0.006, r = 0.7). A leukocyte cut-off of 250,000/ml of semen was associated with a significant increase of DNA oxidation (p = 0.03). 8-oxo-guanine can therefore be considered to be a biological marker of the direct action of oxidative stress on sperm DNA which appears to be susceptible to relatively low levels of ORS produced by leukocytes present at concentrations well below the limit of leukospermia defined by WHO.     

Genetic diversity of SSR and ISSR markers in wild populations of Brachypodium distachyon and its close relatives B. stacei and B. hybridum (Poaceae)

The genetic diversity of seven wild populations of the grass Brachypodium distachyon (2n = 10), 4 of B. stacei (2n = 20) and 13 of B. hybridum (2n = 30) from the Mediterranean and southern areas of the Iberian Peninsula was studied via the analysis of microsatellite (SSR) and inter-microsatellite (ISSR) markers. The 11 SSR markers analysed provided a total of 156 polymorphic fragments. The B. hybridum populations returned more fragments (98) than the B. stacei populations (87), and more than twice the number recorded for the B. distachyon populations (44). Some fragments were specific to the B. distachyon (3.85 %), B. stacei (27.56 %) or B. hybridum populations (18.58 %). The analysis of 16 ISSR markers returned similar results: the B. hybridum populations returned more polymorphic fragments than the B. distachyon or B. stacei populations. The analysis of molecular variance, with distances between individuals, populations and species estimated on the basis of the presence/absence of the SSR and ISSR fragments, showed most of the variation (67 %) to occur among populations. This was followed by differences among individuals within populations (24 %), and finally among species (9 %). Grouping based on UPGMA and principal coordinate analysis showed a clear separation of three groups corresponding to the populations of the same species. Principal component analysis, involving chromosome number, low-molecular weight-glutenin subunits and the most influential climatic and geographic factors, was also performed. This revealed an obvious separation among the populations of B. distachyon and B. hybridum. The index of Mediterraneity and altitude explained 70.56 % of the total variation associated with the first axis. A trend was seen towards a greater presence of B. distachyon forms in areas of the Peninsular interior and higher altitude, with the B. hybridum forms more common in regions of greater summer rainfall and a lower index of Mediterraneity.     

Proteomic analysis of the metabolic adaptation of the biocontrol agent <Emphasis Type="Italic">Pseudozyma flocculosa</Emphasis> leading to glycolipid production

The yeast-like epiphytic fungus Pseudozyma flocculosa is known to antagonize powdery mildew fungi through proliferation on colonies presumably preceded by the release of an antifungal glycolipid (flocculosin). In culture conditions, P. flocculosa can be induced to produce or not flocculosin through manipulation of the culture medium nutrients. In order to characterize and understand the metabolic changes in P. flocculosa linked to glycolipid production, we conducted a 2-DE proteomic analysis and compared the proteomic profile of P. flocculosa growing under conditions favoring the development of the fungus (control) or conducive to flocculosin synthesis (stress). A large number of protein spots (771) were detected in protein extracts of the control treatment compared to only 435 matched protein spots in extracts of the stress cultures, which clearly suggests an important metabolic reorganization in slow-growing cells producing flocculosin. From the latter treatment, we were able to identify 21 protein spots that were either specific to the treatment or up-regulated significantly (2-fold increase). All of them were identified based on similarity between predicted ORF of the newly sequenced genome of P. flocculosa with Ustilago maydis' available annotated sequences. These proteins were associated with the carbon and fatty acid metabolism, and also with the filamentous change of the fungus leading to flocculosin production. This first look into the proteome of P. flocculosa suggests that flocculosin synthesis is elicited in response to specific stress or limiting conditions.     

l-glutamine is a key parameter in the immunosuppression phenomenon

The present work describes the influence of both vitamin C (VC) and mechanical stimulation on development of the extracellular matrix (ECM) and improvement in mechanical properties of a chondrocyte-agarose construct in a regenerating tissue disease model of hyaline cartilage. We used primary bovine chondrocytes and two types of VC, ascorbic acid (AsA) as an acidic form and ascorbic acid 2-phosphate (A2P) as a non-acidic form, and applied uniaxial compressive strain to the tissue model using a purpose-built bioreactor. When added to the medium in free-swelling culture conditions, A2P downregulated development of ECM and suppressed improvement of the tangent modulus more than AsA. By contrast, application of mechanical stimulation to the construct both increased the tangent modulus more than the free-swelling group containing A2P and enhanced the ECM network of inner tissue to levels nearly as high as the free-swelling group containing AsA. Thus, mechanical stimulation and strain appears to enhance the supply of nutrients and improve the synthesis of ECM via mechanotransduction pathways of chondrocytes. Therefore, we suggest that mechanical stimulation is necessary for homogenous development of ECM in a cell-associated construct with a view to implantation of a large-sized articular cartilage defect.     

Preablative stimulated thyroglobulin in predicting dynamic risk stratification after 1 year in patients with differentiated thyroid cancer

The aimThe aim of the study is to establish the utility of stimulated preablative stimulated thyroglobulin (ps-Tg) as a predictor of response to therapy and to determine a possible cut-off for ps-Tg as prognostic tool.Patients and methodsA total of 73 consecutive patients who underwent total thyroidectomy and remnant ablation with radioactive iodine therapy (RIT) were reviewed retrospectively. Patients were classified according to the dynamic risk stratification 1 year after initial treatment. The ps-Tg values were compared among the groups. ROC curve analysis was performed.ResultsThe mean age at diagnosis was 43.85 (range: 17–75) with a female-to-male ratio of 4.6. Ps-Tg value after total thyroidectomy and before RIT ranged from 0,1 to 256 ng/mL. When patients were restaged, 74% had excellent response to treatment, 12.3% indeterminate and 13.7% incomplete response 1 year after initial therapy. ROC curve analysis showed that the optimal cut-off for ps-Tg was 15 ng/mL with a sensivity of 61%; a specificity of 87%; PPV of 61% and NPV of 87%. Among the group of patients showing an excellent response to treatment after 1 year, 87% had ps-Tg < 15 ng/mL.ConclusionPs-Tg before RIT is associated with dynamic risk stratification at 1 year after therapy in patients with DTC. Higher ps-Tg levels were found in patients that had indeterminate, and particularly incomplete, response.     

Teeth,fossil record and evolutionary history of the cowtail stingray Pastinachus Rüppell, 1829

Abstract

Hypolophin ‘dasyatids’ are a common group of large stingrays today frequenting the Indo-Pacific inshores. Being often harvested in their restricted area, few are known about their biology and their evolutionary history despite a very peculiar dental pattern making it easy to track their fossil record. An abundant material consisting of isolated teeth from Late Bartonian (38–40 Ma) lagoonal deposits of Djebel el Kébar, Tunisia, allows to describe a new stingray, Pastinachus kebarensis nov. sp. This taxon represents the oldest occurrence for this genus but also the oldest fossil record for hypolophins. A dental comparison of these fossils with 3D rendered models of fresh specimens testifies that early hypolophin representatives had already a strongly arcuate and bulbous upper jaw, interlocking with a broad and elongated tooth plate on the lower jaw. This new fossil and its fossil relatives (here updated), indicate a pre-Bartonian origination for hypolophins in western Neotethys, and reveal a rapid and widespread colonization of the proto-Mediterranean Sea, western Atlantic and Indo-Pacific coasts during the late Paleogene–early Neogene. Finally, it is worth noting that early hypolophin representatives seemingly entered freshwater habitats occasionally as modern cowtail stingrays do.     

Intérêt de la scintigraphie rénale au DMSA dans les pyélonéphrites aiguës de l’enfant

IntroductionAcute pyelonephritis is responsible for significant morbidity during the acute phase and distant irreversible kidney damage. However, diagnosis is often difficult in children. The aim of our study was to evaluate in children hospitalized for suspected acute pyelonephritis prevalence of acute lesions revealed by scintigraphy with ^99mTc labeled dimercaptosuccinic acid (DMSA) and the relationship between renal parenchymal lesions and different clinical factors and biological risks.MethodsThis is a prospective study collecting 40 children admitted with suspected infection of the high urinary tract. All have benefited from DMSA renal scintigraphy.ResultsThe average age of our patients was 3.25 years. The DMSA scan showed signs of acute pyelonephritis in 32.5% of cases. Taking this examination as a reference, fever was the most sensitive clinical signs (76.9%), but the specificity was only 48.1%. CRP (≥ 20 mg/mL) was the most sensitive (92.3%) but with a specificity of 40.7% organic signs. A high CRP was associated with acute pyelonephritis significantly (P = 0.0033). Sixty-three percent of refluxing kidneys were associated with acute pyelonephritis versus 20.8% of non-refluxing kidneys, with a significant difference (P = 0.007).ConclusionIn case of urinary tract infection in children, a complete clinical and laboratory table may be sufficient to retain the diagnosis of acute pyelonephritis. However, this possibility is rare. In the opposite case, a DMSA scan should be performed to confirm the diagnosis.     

Prepubertal goat oocytes from large follicles result in similar blastocyst production and embryo ploidy than those from adult goats

Developmental competence of oocytes from prepubertal females is lower than those from adult females. Oocyte development competence is positively related to follicular diameter. Most of the follicles of prepubertal goat ovaries are smaller than 3 mm. The aim of this study was to compare oocytes of two follicle sizes (< 3 mm and ≥ 3 mm) from prepubertal goats with oocytes from adult goats in relation to their in vitro production and quality of blastocysts. Oocytes from prepubertal goats were obtained from slaughterhouse ovaries and selected according to the follicle diameter whereas oocytes from adult goats were recovered in vivo by LOPU technique without prior selection of follicle size. COCs were IVM for 27 h, IVF at the conventional conditions with fresh semen and presumptive zygotes were cultured in SOF medium for 8 days. Blastocysts obtained were vitrified and after warming their blastocoele re-expansion and the ploidy by FISH technique were assessed. We found significant differences between blastocysts yield of oocytes recovered from follicles smaller than 3 mm of prepubertal goats compared to those from adult goats (5.45% vs 20. 83%, respectively) however, these differences disappear if oocytes were recovered form large follicles (18.07%). A total of 28 blastocysts were analysed and 96.43% showed mixoploidy. Age did not affect the number of embryos with abnormal ploidy or blastocyst re-expansion after warming. Furthermore, the percentage of diploid blastomeres per embryo was similar in the 3 groups studied, adult, prepubertal from follicles ≥ 3 mm and < 3 mm (68.6%, 80.8% and 73.6%, respectively). In conclusion, IVP of blastocysts coming from follicles larger than 3 mm of goats 45 days old were not different to the blastocysts produced from adult goats, both in terms of quantity and quality.