Preservation at ultra-low temperature of in vitro cultured arbuscular mycorrhizal fungi via encapsulation–drying

At present, over 300 species of arbuscular mycorrhizal fungi (AMF) have been identified, most of which being stored in international collections. Their maintenance is mostly achieved in greenhouse via continuous culture on trap plants or in vitro in association with excised root organs. Both methods are work-intensive and for the former present the risk of unwanted contaminations. The in vitro root organ culture of AMF has become an alternative preventing contamination. Nevertheless, the risk for somaclonal variation during the sub-cultivation process cannot be excluded. A method for the long-term conservation that guarantees the stability of the biological material is thus highly demanded to preserve the microorganisms and their genetic stability. Here, 12 AMF isolates cultured in vitro in association with excised carrot roots were encapsulated in alginate beads and subsequently cryopreserved. Several protocols were tested taking into consideration culture age, alginate bead pre-drying, and rate of decrease in temperature. The viability of the AMF isolates was estimated by the percentage of potentially infective beads (%PIB) that measure the % of beads that contain at least one germinated propagule. Thermal behaviour of alginate beads was analysed by a differential thermal calorimeter before and after drying to estimate the frozen and unfrozen water during the cryopreservation process. It was shown that the spore damage was directly related to ice formation during cryopreservation. The encapsulation and culture age were also determinant parameters for the successful cryopreservation. Irrespective of the AMF isolate, the optimal procedure for cryopreservation comprised five steps: (1) the encapsulation of propagules (i.e. spores and mycorrhizal root pieces) isolated from 5 m old cultures, (2) the incubation overnight in trehalose (0.5 M), (3) the drying during 48 h at 27 °C, (4) the cryopreservation in the freezer at −130 °C following a two-step decrease in temperature: a fast decrease (∼12 °C min⁻¹) from room temperature (+20 °C) to −110 °C followed by a slow decrease in temperature (∼1 °C min⁻¹) from −110 °C to −130 °C, and (5) the direct thawing in a water bath (+35 °C). The % PIB was above 70 % for all the isolates and even above 95 % for 11 out of the 12 isolates after several months of storage at ultra-low temperature. All the isolates kept their capacity to associate to an excised carrot root in vitro and to reproduce the fungal life cycle with the production of several hundreds to thousands of spores after 2 m. This method opens the door for the long-term maintenance at ultra-low temperature of AMF isolates within international repositories.     

Effect of dexamethasone on development of in vitro–produced bovine embryos

Studies in somatic cells have shown that glucocorticoids such as dexamethasone (DEX) may trigger or prevent apoptosis depending on the cell type in culture. Because the dysregulation of apoptosis may lower in vitro embryo production efficiency, we sought to investigate the effects of supplementing IVC medium with DEX (0.1 μg/mL) on embryo morphology, development kinetics, and apoptosis rates of in vitro–produced bovine preimplantation embryos. Embryo morphology was graded on Day 7, and development rates were assessed on Days 4 and 7 of IVC. Apoptosis was evaluated via annexin/propidium iodide staining under fluorescence microscopy where a cell labeled with annexin, propidium iodide, or both would be considered apoptotic. An embryo was counted in the apoptosis rates, if it displayed at least one such labeled cell. Although DEX supplementation did not reduce apoptosis rates, it had a positive impact on developmental kinetics and cell number both on Days 4 and 7 of embryo culture. Presumably, such effect resulted from increased cell proliferation rather than a direct inhibition of apoptosis. Further studies may evaluate the mechanisms by which glucocorticoids may affect embryo development, as DEX supplementation could become a tool to improve in vitro embryo yield in mammalian species.     

Expression pattern of glucose metabolism genes in relation to development rate of buffalo (Bubalus bubalis) oocytes and in vitro–produced embryos

The expression pattern of glucose metabolism genes (hexokinase, phosphofructokinase, glucose-6-phosphate dehydrogenase [G6PDH], lactate dehydrogenase [LDH], and pyruvate dehydrogenase [PDH]) were studied in buffalo in vitro–matured oocytes and in vitro–produced embryos cultured under different glucose concentrations (0 mM, 1.5 mM, 5.6 mM, and 10 mM) during in vitro maturation of oocytes and culture of IVF produced embryos. The expression of the genes varied significantly over the cleavage stages under different glucose concentrations. Developmental rate of embryos was highest under a constant glucose level (5.6 mM) throughout during maturation of oocytes and embryo culture. Expression pattern of glucose metabolism genes under optimum glucose level (5.6 mM) indicated that glycolysis is the major pathway of glucose metabolism during oocyte maturation and early embryonic stages (pre-maternal to zygotic transition [MZT]) and shifts to oxidative phosphorylation during post-MZT stages in buffalo embryos. Higher glucose level (10 mM) caused abrupt changes in gene expression and resulted in shifting toward anaerobic metabolism of glucose during post-MZT stages. This resulted in decreased development rate of embryos during post-MZT stages. High expression of LDH and PDH in the control groups (0 mM glucose) indicated that in absence of glucose, embryos try to use available pyruvate and lactate sources, but succumb to handle the post-MZT energy requirement, resulting to poor development rate. Expression pattern of G6PDH during oocyte maturation as well early embryonic development was found predictive of quality and development competence of oocytes/ embryos.     

Candida species in tchapalo and bangui, two traditional alcoholic beverages from Côte d'Ivoire

The increase of infections due to non-Candida albicans species made it very necessary to conduct adequate characterization to be able to identify the species of Candida isolated from traditional fermented foods. In this study, based on their hue on Candida Chromogenic Agar medium, a total of 136 yeast strains were isolated from tchapalo and bangui. Molecular identification based on PCR-RFLP of internal transcribed spacers of rDNA (ITS) and sequencing of the ITS and the D1/D2 regions allowed us to assign these isolates to seven species: Candida tropicalis, Candida inconspicua, Candida rugosa, Saccharomyces cerevisiae, Kluyveromyces marxianus, Hanseniaspora guilliermondii, Trichosporon asahii. With the respect to each beverage, six species were found among with four species are regarded as opportunistic pathogens. From these, C. tropicalis, C. inconspicua and K. marxianus were the most commonly encountered. The enzyme activities of the potential pathogens assessed using API ZYM system showed that almost strains had esterase, esterase lipase, valine and cystine arylamidase, alpha chymotrypsin, alkaline phosphatase and naphthol phosphohydrolase activities. The activity of α-glucosidase was found only in C. tropicalis and C. inconspicua strains isolated from tchapalo while β-glucosidase activity was found in all strains from tchapalo and only in C. inconspicua isolated from bangui.     

The influence of reduced glutathione in fertilization medium on the fertility of in vitro–matured C57BL/6 mouse oocytes

It is well known that IVM oocytes show a decreased potential for fertility and development compared with in vivo–matured oocytes. In this study, we added reduced glutathione (GSH) to the fertilization medium during IVF to investigate its effect on the fertility and early embryo development of IVM oocytes. The fertilization rate for IVM oocytes and fresh sperm increased with the addition of GSH (0, 1.0, and 2.0 mM: 51%, 76%, and 70%). Moreover, the addition of GSH to the fertilization medium also improved the developmental potential compared with the control sample (0 mM). In addition, we performed IVF using IVM oocytes and frozen/thawed sperm that had been cryopreserved in a mouse bank. Results indicated a marked increase in the fertilization rate when 1.0 mM GSH was added to the fertilization medium compared with when no GSM was used (0.0 mM GSH: 2% (3/195); 1.0 mM GSH: 33% (156/468)). Furthermore, the fertilization rate improved dramatically via zona drilling using laser equipment (52%: 267/516), whereas normal offspring were obtainsed after transferring embryos created via IVF using IVM oocytes and frozen/thawed sperm. This is the first report in which offspring have been obtained via IVF using IVM oocytes and frozen/thawed sperm.     

Evaluation of sperm subpopulation structure in relation to in vitro sperm–oocyte interaction of frozen-thawed semen from Holstein bulls

The present study examined the relationship between the relative amount of high motile sperm and sperm–oocyte interactions obtained from Holstein bull ejaculates. Post-thaw sperm motility was analyzed using a computer-assisted sperm analyzer system and evaluated to determine the sperm motility subpopulations. Adhesion and penetration of zona pellucida (ZP) and pronucleus formation using post-thawed samples (15 ejaculates form 5 different bulls) with different percentages of sperm in the subpopulation with the fastest and most progressive subpopulation (subpopulation 4 [SP4]) were analyzed. The correlation between the proportion of sperm in SP4 and the number of spermatozoa bound to the zona pellucida (ZBA), the penetration rate, and the rate of pronucleus formation were calculated. A significant (P < 0.05) and positive correlation was found between the number of spermatozoa bound to the zona pellucida, the penetration rate, and the rate of pronucleus formation with the proportion of sperm in SP4 (r = 0.79, r = 0.66, and r = 0.63, respectively). Our results suggest that this specific high motile and progressive subpopulation is positively and significantly correlated with the ability of a thawed bull semen sample to interact properly with the oocyte and its extracellular vestments. These findings emphasize the relevance of analyzing semen subpopulation composition to predict bull sperm fertilizing ability and to select Holstein bulls for breeding purposes.     

Progress in antischistosomal N,N′-diaryl urea SAR

N,N′-Diaryl ureas have recently emerged as a new antischistosomal chemotype. We now describe physicochemical profiling, in vitro ADME, plasma exposure, and ex vivo and in vivo activities against Schistosoma mansoni for twenty new N,N′-diaryl ureas designed primarily to increase aqueous solubility, but also to maximize structural diversity. Replacement of one of the 4-fluoro-3-trifluoromethylphenyl substructures of lead N,N′-diaryl urea 1 with azaheterocycles and benzoic acids, benzamides, or benzonitriles decreased lipophilicity, and in most cases, increased aqueous solubility. There was no clear relationship between lipophilicity and metabolic stability, although all compounds with 3-trifluoromethyl-4-pyridyl substructures were metabolically stable. N,N′-diaryl ureas containing 4-fluoro-3-trifluoromethylphenyl, 3-trifluoromethyl-4-pyridyl, 2,2-difluorobenzodioxole, or 4-benzonitrile substructures had high activity against ex vivo S. mansoni and relatively low cytotoxicity. N,N-diaryl ureas with 3-trifluoromethyl-4-pyridyl and 2,2-difluorobenzodioxole substructures had the highest exposures whereas those with 4-fluoro-3-trifluoromethylphenyl substructures had the best in vivo antischistosomal activities. There was no direct correlation between compound exposure and in vivo activity.     

Ultrastructural observations on mehlis' gland in the monogeneans, Diplozoon paradoxum and Calicotyle kröyeri

Mehlis' gland of both Diplozoon paradoxum and Calicotyle kröyeri is composed of two cell types that taper to form ducts opening into the lumen of the ootype. The cells are invested with fibrous interstitial material and form a close structural relationship with surrounding parenchyma. The most prevalent cell type, the S₁ cell, is characterized by an extensive GER with narrow cisternae containing a finely granular material, and numerous Golgi stacks involved in the formation of multi-vesicular secretory bodies. In the S₂ cells the GER cisternae are greatly distended with a finely filamentous material and the Golgi give rise to dense secretory bodies with a packed fibrous appearance. There are species differences in the fine structure of the secretory bodies and these may reflect differences in the chemical composition of the glands. The ducts of the glands are lined with microtubules and are anchored to the ootype epithelium by septate desmosomes. They convey the secretory products to the ootype where they are released, apparently by exocytosis involving membrane fusion, into the lumen. The ootype is lined by a highly folded cellular epithelium which in D. paradoxum is ciliated. The cells contain profiles of GER and Golgi complexes and produce a third type of secretion which is also discharged into the ootype lumen.     

Ammonites diartianus d'Orbigny, 1850,Vascoceratidae du cénomanien supérieur de saint-calais (Sarthe)

Ammonites diartianus d'Orbigny is a Vascocerasoof late Cenomanian (Sciponoceras gracile Zone) age, occuring in both Sarthe (France) and southern England. It is the earliest vascoceratid known from France, and its recognition is of great significance, lending support to recent suggestions that the classic Vascoceras gamai - mundaeChoffat group are of Upper Cenomanian age.     

Alcaloïdes d'Alstonia vitiensis var. Novo ebudica monachino

Five alkaloids have been isolated from Alstonia vitiensis: pleïocarpamine, vincorine, cabucraline, alstovine (11-methoxycompactinervine) and quaternoxine; the latter two are new alkaloids.     

Visual learning and memory of Drosophila melanogaster wild type CS and the mutants dunce1, amnesiac, turnip and rutabaga

Dunce¹, amnesiac, turnip and rutabaga, mutants of Drosophila melanogaster deficient in olfactory learning and/or memory, were tested for visual learning ability and memory. All these mutants are able to learn conditioned visual information, but not as well as the corresponding wildtype CS. Memory of all four mutants is reduced during the first 30 min after training, but indistinguishable from that of the wildtype two hours after conditioning.     

Biogéographied'un groupe d'Échinides cénozoiques (Echinolampas et ses sous-genres Conolampas et Hypsoclypus)

Echinolampas is a subtropical genus living in rather shallow water; one may regards it as a climatic marker. The theory of continental drift affords a rather good explanation for its distribution in space during Cenozoic era. It appears in Old Wolrd during Paleocene and it occurs in Central America during Middle Eocene; that implies it had to cross the already broad Atlantic Ocean; but at that time this ocean is not as broad at it is now. Migration along the shelf area which rimmed North Atlantic might have been impossible, owing to disruption of land connection between Europe and North America. Probably the migration occured in low latitudes and pelagic larvae were transported by one of the two equatorial currents. Diversity of the genus has much decreased during Late Eocene. The cause may be chiefly due to climatic deterioration, resulting from marine communication between North Atlantic and Arctic Ocean. Echinolampas occurs for the first time in Australia during Oligocene. One may suggest the possibility of a link between this late evidence and the quite remote position till then of Australian continent. During Miocene, the relative decrease in Echinolampas diversity in the Mediterranean Basin occurs as a result of the welding between Asia and Africa.     

R-(-)-β-O-methylsynephrine, a natural product, inhibits VEGF-induced angiogenesis in vitro and in vivo

R-(-)-β-O-methylsynephrine (OMe-Syn) is an active compound isolated from a plant of the Rutaceae family. We conducted cell proliferation assays on various cell lines and found that OMe-Syn more strongly inhibited the growth of human umbilical vein endothelial cells (HUVECs) than that of other normal and cancer cell lines tested. In angiogenesis assays, it inhibited vascular endothelial growth factor (VEGF)-induced invasion and tube formation of HUVECs with no toxicity. The anti-angiogenic activity of OMe-Syn was also validated in vivo using the chorioallantonic membrane (CAM) assay in growing chick embryos. Expression of the growth factors VEGF, hepatocyte growth factor, and basic fibroblast growth factor was suppressed by OMe-Syn in a dose-dependent manner. Taken together, our results indicate that this compound could be a novel basis for a small molecule targeting angiogenesis.     

Nadaouiyeh – A Homo erectus in Acheulean context

The Middle East is apparently the most important passage for the dispersal of early hominins. Numerous archeological sites prove the existence of hominin populations in this region, but despite these rich cultural remains, hominin fossils are very rare. In 1996, a hominin left parietal was found in an Acheulean context. In addition, the faunal remains indicate a steppe environment. What does this single cranial fragment tell us? Based on new publications and in particular on recent finds, the value of isolated elements is discussed.     

The effect of interleukin-1β, interleukin-6, and tumor necrosis factor-α on estradiol-17β release in the myometrium: The in vitro study on the pig model

Estradiol-17β (E₂) is a potent regulator of early pregnancy and the estrous cycle in pigs. Production of E₂ occurs in the porcine myometrium, but the factors involved in its regulation are unknown. In this in vitro study, it was investigated whether interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α affect the release of E₂ from the porcine myometrium on Days 10 to 11, 12 to 13, and 15 to 16 of pregnancy and the estrous cycle. The expression of the cytochrome P450 family 19 (CYP19) gene and the presence of the aromatase cytochrome P450 protein in the myometrium confirmed the ability of the tissue to produce E₂. In gravid pigs, the expression of IL1RI mRNA and IL6R mRNA was markedly increased on Days 15 to 16 of gestation, whereas TNFRI mRNA was increased on Days 10 to 11 of gestation. In cyclic pigs, the expression of myometrial IL1RI mRNA did not differ among the studied days, although the expression of IL6R and TNFRI mRNAs was increased on Days 15 to 16. In gravid pigs, IL-1β, IL-6, and TNF-α increased myometrial E₂ secretion on Days 15 to 16 but did not affect E₂ release on Days 10 to 11 and 12 to 13 of pregnancy. In cyclic pigs, IL-1β, IL-6, and TNF-α did not increase myometrial E₂ release. In conclusion, IL-1β, IL-6, and TNF-α affected myometrial E₂ release in a manner that is dependent on the physiologic status of the female. The porcine myometrium expresses IL1RI, IL6R, and TNFRI genes and is the target tissue for IL-1β, IL-6, and TNF-α. In gravid pigs, IL-1β, IL-6, and TNF-α may increase myometrial release of E₂in vitro specifically on Days 15 to 16 of pregnancy. These findings may be of interest to researchers using pigs as an animal model for fetal programming.     

In vitro, ex vivo and in vivo models: A comparative analysis of Paracoccidioides spp. proteomic studies

Members of the Paracoccidioides complex are human pathogens that infect different anatomic sites in the host. The ability of Paracoccidioides spp. to infect host niches is putatively supported by a wide range of virulence factors, as well as fitness attributes that may comprise the transition from mycelia/conidia to yeast cells, response to deprivation of micronutrients in the host, expression of adhesins on the cell surface, response to oxidative and nitrosative stresses, as well as the secretion of hydrolytic enzymes in the host tissue. Our understanding of how those molecules can contribute to the infection establishment has been increasing significantly, through the utilization of several models, including in vitro, ex vivo and in vivo infection in animal models. In this review we present an update of our understanding on the strategies used by the pathogen to establish infection. Our results were obtained through a comparative proteomic analysis of Paracoccidioides spp. in models of infection.     

Acute toxicity,critical body residues,Michaelis–Menten analysis of bioaccumulation,and ionoregulatory disturbance in response to waterborne nickel in four invertebrates: Chironomus riparius, Lymnaea stagnalis, Lumbriculus variegatus and Daphnia pulex

We investigated the bioaccumulation and acute toxicity (48 h or 96 h) of Ni in four freshwater invertebrate species in two waters with hardness of 40 (soft water) and 140 mg L^− 1 as CaCO₃ (hard water). Sensitivity order (most to least) was Lymnaea stagnalis > Daphnia pulex > Lumbriculus variegatus > Chironomus riparius. In all cases water hardness was protective against acute Ni toxicity with LC50 values 3–3.5 × higher in the hard water vs. soft water. In addition, higher water hardness significantly reduced Ni bioaccumulation in these organisms suggesting that competition by Ca and Mg for uptake at the biotic ligand may contribute to higher metal resistance. CBR50 values (Critical Body Residues) were less dependent on water chemistry (i.e. more consistent) than LC50 values within and across species by ~ 2 fold. These data support one of the main advantages of the Tissue Residue Approach (TRA) where tissue concentrations are generally less variable than exposure concentrations with respect to toxicity. Whole body Ni bioaccumulation followed Michaelis–Menten kinetics in all organisms, with greater hardness tending to decrease B_max with no consistent effect on K_d. Across species, acute Ni LC50 values tended to increase with both K_d and B_max values — i.e. more sensitive species exhibited higher binding affinity and lower binding capacity for Ni, but there was no correlation with body size. With respect to biotic ligand modeling, log K_NiBL values derived from Ni bioaccumulation correlated well with log K_NiBL values derived from toxicity testing. Both whole body Na and Mg levels were disturbed, suggesting that disruption of ionoregulatory homeostasis is a mechanism of acute Ni toxicity. In L. stagnalis, Na depletion was a more sensitive endpoint than mortality, however, the opposite was true for the other organisms. This is the first study to show the relationship between Na and Ni.