Improved pregnancy and birth rates with routine application of nonsurgical embryo transfer

Nonsurgical embryo transfer (NSET) of blastocysts to pseudopregnant female recipients provides many benefits over surgical implantation with less distress for the mice, no anesthesia or analgesia required and a considerable reduction in implantation time per mouse. Although a disposable device to perform NSET is on the market since 2009, it is not generally used in transgenic facilities, most likely because surgical implantation is efficient and inexpensive. Here, we report that with several refinements to the original protocol, the NSET method becomes very attractive and outperforms the traditional surgical transfer on basis of pregnancy rate, birth rate and implantation-related discomfort. Furthermore, repeated use of the same NSET device on several recipient females reduces the costs to a reasonable level. The data presented covers all embryo transfers over the last 5 years at the transgenic facility of the Netherlands Cancer Institute, of which the last 2 years were performed exclusively with NSET.     

Different iron sources to study the physiology and biochemistry of iron metabolism in marine micro-algae

We compared ferric EDTA, ferric citrate and ferrous ascorbate as iron sources to study iron metabolism in Ostreococcus tauri, Phaeodactlylum tricornutum and Emiliania huxleyi. Ferric EDTA was a better iron source than ferric citrate for growth and chlorophyll levels. Direct and indirect experiments showed that iron was much more available to the cells when provided as ferric citrate as compared to ferric EDTA. As a consequence, growth media with iron concentration in the range 1–100 nM were rapidly iron-depleted when ferric citrate—but not ferric EDTA was the iron source. When cultured together, P. tricornutum cells overgrew the two other species in iron-sufficient conditions, but E. huxleyi was able to compete other species in iron-deficient conditions, and when iron was provided as ferric citrate instead of ferric EDTA, which points out the critical influence of the chemical form of iron on the blooms of some phytoplankton species. The use of ferric citrate and ferrous ascorbate allowed us to unravel a kind of regulation of iron uptake that was dependent on the day/night cycles and to evidence independent uptake systems for ferrous and ferric iron, which can be regulated independently and be copper-dependent or independent. The same iron sources also allowed one to identify molecular components involved in iron uptake and storage in marine micro-algae. Characterizing the mechanisms of iron metabolism in the phytoplankton constitutes a big challenge; we show here that the use of iron sources more readily available to the cells than ferric EDTA is critical for this task.     

The lipidome and proteome of microsomes from the methylotrophic yeast Pichia pastoris

The methylotrophic yeast Pichia pastoris is a popular yeast expression system for the production of heterologous proteins in biotechnology. Interestingly, cell organelles which play an important role in this process have so far been insufficiently investigated. For this reason, we started a systematic approach to isolate and characterize organelles from P. pastoris. In this study, we present a procedure to isolate microsomal membranes at high purity. These samples represent endoplasmic reticulum (ER) fractions which were subjected to molecular analysis of lipids and proteins. Organelle lipidomics included a detailed analysis of glycerophospholipids, fatty acids, sterols and sphingolipids. The microsomal proteome analyzed by mass spectrometry identified typical proteins of the ER known from other cell types, especially Saccharomyces cerevisiae, but also a number of unassigned gene products. The lipidome and proteome analysis of P. pastoris microsomes are prerequisite for a better understanding of functions of this organelle and for modifying this compartment for biotechnological applications.     

Mechanisms of phosphate solubilization by fungal isolates when exposed to different P sources

The use of phosphate-solubilizing fungi is a promising biotechnological strategy in the management of phosphorus (P) fertilization, as it enables the utilization of rock phosphates (RP) or the recovery of P fixed in soil particles. The objective of our study was to evaluate fungal isolates for mechanisms of solubilization of P-bearing compounds, such as AlPO₄, FePO₄, Ca₃(PO₄)₂, Araxá RP, and Catalão RP. Four fungal isolates obtained from Brazilian soils were characterized in liquid media: Aspergillus niger FS1, Penicillium canescens FS23, Eupenicillium ludwigii FS27, and Penicillium islandicum FS30. A. niger FS1 was the only isolate able to solubilize all of the P sources, solubilizing 71, 36, 100, and 14 % of the P from AlPO₄, FePO₄, Ca₃(PO₄)₂, and RPs, respectively. Medium acidification was an effective solubilization mechanism, particularly for Ca₃(PO₄)₂. The other P sources were mainly solubilized through organic acids produced by the fungi. Oxalic acid, produced exclusively by A. niger FS1, and citric acid were decisive factors in the solubilization of AlPO₄ and FePO₄. Penicillium isolates produced more gluconic acid than A. niger FS1 in all treatments. However, this higher production did not result in higher solubilization for any of the P sources, showing that gluconic acid contributes little to the solubilization of the P sources evaluated. The higher capacity of medium acidification and the production of organic acids with stronger metal-complexation activity are characteristics that confer to A. niger FS1 a wider action on insoluble P sources. Consequently, this isolate qualifies as a promising candidate for application in the management of P fertilization.     

No major role for periconceptional folic acid use and its interaction with the MTHFR C677T polymorphism in the etiology of congenital anorectal malformations

Background: Both genetic and nongenetic factors are suggested to be involved in the etiology of congenital anorectal malformations (ARM). Maternal periconceptional use of folic acid supplements were inconsistently suggested to play a role in the prevention of ARM. Therefore, we investigated independent associations and interactions of maternal periconceptional folic acid supplement use and the infant and maternal MTHFR (methylenetetrahydrofolate reductase) C677T polymorphisms with the risk of ARM and subgroups of ARM. Methods: A case–control study was conducted among 371 nonsyndromic ARM cases and 714 population‐based controls born between 1990 and 2012 using maternal questionnaires and DNA samples from mother and child. Cases were treated for ARM at departments of Pediatric Surgery of the Radboud university medical center, Sophia Children's Hospital‐Erasmus MC Rotterdam, and the University Medical Center Groningen in The Netherlands and hospitals throughout Germany. Results: No association with folic acid use was present (odds ratio = 1.1; 95% confidence interval: 0.8–1.4) for ARM as a group. Infant and maternal MTHFR C677T polymorphisms were weakly associated with isolated ARM in particular. Lack of folic acid supplement use in combination with infants or mothers carrying the MTHFR C677T polymorphism did not seem to increase the risk of ARM or subgroups of ARM. The relative excess risks due to interaction did not clearly indicate interaction on an additive scale either. Conclusion: This first study investigating interactions between periconceptional folic acid supplement use and infant and maternal MTHFR C677T polymorphisms in the etiology of ARM did not provide evidence for a role of this gene–environment interaction. Birth Defects Research (Part A) 100:483–492, 2014. © 2014 Wiley Periodicals, Inc.     

Computer-Guided Trypanocidal Activity of Natural Lactones Produced by Endophytic Fungus of Euphorbia umbellata

Hundreds of millions of people worldwide are affected by Chagas’ disease caused by Trypanosoma cruzi. Since the current treatment lack efficacy, specificity, and suffers from several side-effects, novel therapeutics are mandatory. Natural products from endophytic fungi have been useful sources of lead compounds. In this study, three lactones isolated from an endophytic strain culture were in silico evaluated for rational guidance of their bioassay screening. All lactones displayed in vitro activity against T. cruzi epimastigote and trypomastigote forms. Notably, the IC₅₀ values of (+)-phomolactone were lower than benznidazole (0.86 vs. 30.78 μM against epimastigotes and 0.41 vs. 4.88 μM against trypomastigotes). Target-based studies suggested that lactones displayed their trypanocidal activities due to T. cruzi glyceraldehyde-3-phosphate dehydrogenase (TcGAPDH) inhibition, and the binding free energy for all three TcGAPDH-lactone complexes suggested that (+)-phomolactone has a lower score value (−3.38), corroborating with IC₅₀ assays. These results highlight the potential of these lactones for further anti-T. cruzi drug development.     

The fatty acyl-CoA reductase Waterproof mediates airway clearance in Drosophila

The transition from a liquid to a gas filled tubular network is the prerequisite for normal function of vertebrate lungs and invertebrate tracheal systems. However, the mechanisms underlying the process of gas filling remain obscure. Here we show that waterproof, encoding a fatty acyl-CoA reductase (FAR), is essential for the gas filling of the tracheal tubes during Drosophila embryogenesis, and does not affect branch network formation or key tracheal maturation processes. However, electron microscopic analysis reveals that in waterproof mutant embryos the formation of the outermost tracheal cuticle sublayer, the envelope, is disrupted and the hydrophobic tracheal coating is damaged. Genetic and gain-of-function experiments indicate a non-cell-autonomous waterproof function for the beginning of the tracheal gas filling process. Interestingly, Waterproof reduces very long chain fatty acids of 24 and 26 carbon atoms to fatty alcohols. Thus, we propose that Waterproof plays a key role in tracheal gas filling by providing very long chain fatty alcohols that serve as potential substrates for wax ester synthesis or related hydrophobic substances that ultimately coat the inner lining of the trachea. The hydrophobicity in turn reduces the tensile strength of the liquid inside the trachea, leading to the formation of a gas bubble, the focal point for subsequent gas filling. Waterproof represents the first enzyme described to date that is necessary for tracheal gas filling without affecting branch morphology. Considering its conservation throughout evolution, Waterproof orthologues may play a similar role in the vertebrate lung.     

Secreted Fungal Effector Lipase Releases Free Fatty Acids to Inhibit Innate Immunity-Related Callose Formation during Wheat Head Infection

The deposition of the (1,3)-β-glucan cell wall polymer callose at sites of attempted penetration is a common plant defense response to intruding pathogens and part of the plant’s innate immunity. Infection of the Fusarium graminearum disruption mutant Δfgl1, which lacks the effector lipase FGL1, is restricted to inoculated wheat (Triticum aestivum) spikelets, whereas the wild-type strain colonized the whole wheat spike. Our studies here were aimed at analyzing the role of FGL1 in establishing full F. graminearum virulence. Confocal laser-scanning microscopy revealed that the Δfgl1 mutant strongly induced the deposition of spot-like callose patches in vascular bundles of directly inoculated spikelets, while these callose deposits were not observed in infections by the wild type. Elevated concentrations of the polyunsaturated free fatty acids (FFAs) linoleic and α-linolenic acid, which we detected in F. graminearum wild type-infected wheat spike tissue compared with Δfgl1-infected tissue, provided clear evidence for a suggested function of FGL1 in suppressing callose biosynthesis. These FFAs not only inhibited plant callose biosynthesis in vitro and in planta but also partially restored virulence to the Δfgl1 mutant when applied during infection of wheat spikelets. Additional FFA analysis confirmed that the purified effector lipase FGL1 was sufficient to release linoleic and α-linolenic acids from wheat spike tissue. We concluded that these two FFAs have a major function in the suppression of the innate immunity-related callose biosynthesis and, hence, the progress of F. graminearum wheat infection.The molecular and physiological regulation of the biosynthesis of callose, which is a (1,3)-β-glucan polymer with some (1,6)-branches (Aspinall and Kessler, 1957), and its importance for plant development as well as plant defense are still under examination. Regarding the involvement of callose in plant defense responses, particular attention has been focused on the formation of cell wall thickenings in plants, so-called papillae, at sites of microbial attack. They were already described 150 years ago (deBary, 1863) and reported to commonly contain callose (Mangin, 1895). Since then, examinations have identified callose as the most abundant chemical constituent in papillae, which may also include proteins (e.g. peroxidases and antimicrobial thionins), phenolics, and other constituents (Aist and Williams, 1971; Sherwood and Vance, 1976; Mims et al., 2000). Papillae have been regarded as an early defense reaction that may not completely stop the pathogen; rather, they have been considered to act as a physical barrier to slow pathogen invasion (Stone and Clarke, 1992; Voigt and Somerville, 2009) and to contribute to the plant’s innate immunity (Jones and Dangl, 2006; Schwessinger and Ronald, 2012). The host plant can gain time to initiate defense reactions that require gene activation and expression, such as the hypersensitive reactions, phytoalexin production, and pathogenesis-related protein synthesis (Lamb and Dixon, 1997; Brown et al., 1998). However, our recent study revealed that callose can also act as a barrier that completely prevents fungal penetration. The overexpression of POWDERY MILDEW RESISTANT4 (PMR4), a gene encoding a stress-induced callose synthase, resulted in early elevated callose deposition at sites of attempted powdery mildew penetration in Arabidopsis (Arabidopsis thaliana; Ellinger et al., 2013). Interestingly, the pmr4 deletion mutant also showed an increased resistance to powdery mildew that, however, was induced at later stages of powdery mildew infection because an initial fungal penetration still occurred. In fact, the absence of the functional callose synthase PMR4 in the pmr4 mutant resulted in papillae that were free from callose but also induced a hyperactivation of the salicylic acid defense pathway, which was shown to be the basis of resistance in double mutant and microarray analyses (Jacobs et al., 2003; Nishimura et al., 2003). The callose synthase gene PMR4 from Arabidopsis belongs to the GLUCAN SYNTHASE-LIKE (GSL) family, genes that have been identified in higher plants including wheat (Triticum aestivum; Cui et al., 2001; Doblin et al., 2001; Hong et al., 2001; Østergaard et al., 2002; Voigt et al., 2006). The predicted function of these genes as callose synthases is generally supported by homology with the yeast FK506 SENSITIVITY (FKS) genes, which are believed to be subunits of (1,3)-β-glucan synthase complexes (Douglas et al., 1994; Dijkgraaf et al., 2002). Additionally, the predicted proteins encoded by the GSL genes correlate with the approximately 200-kD catalytic subunit of putative callose synthases. Li et al. (2003) showed that the amino acid sequence predicted from a GSL gene in barley (Hordeum vulgare; HvGSL1) correlates with the amino acid sequence of an active (1,3)-β-glucan synthase fraction.In this study, we aimed to examine the involvement of callose synthesis and callose deposition in plant defense against intruding fungal pathogens in the pathosystem wheat-Fusarium graminearum. We focused on the ability of wheat to inhibit a further spread of fungal pathogens after an initial, successful infection. This resistance to fungal spread within the host has been referred to as type II resistance and is part of a widely accepted two-component system of resistance, which includes type I resistance operating against initial infection (Schroeder and Christensen, 1963). For our analyses, we used the direct interaction between wheat as host and F. graminearum as a pathogen. On the one hand, Fusarium head blight (FHB) of wheat, caused by F. graminearum, is one of the most destructive crop diseases worldwide (McMullen et al., 1997; del Blanco et al., 2003; Madgwick et al., 2011) and classifies this fungus as a top 10 plant pathogen based on its importance in science and agriculture (Dean et al., 2012). On the other hand, only a limited number of wheat cultivars were identified that revealed FHB resistance. However, these cultivars did not qualify for commercial cultivation or breeding approaches due to inappropriate agronomic traits (Buerstmayr et al., 2009). Further elucidation of the mechanisms of spreading resistance could support the generation of FHB-resistant wheat cultivars.In this regard, we demonstrated that the secreted lipase FGL1 of F. graminearum is a virulence factor required for wheat infection (Voigt et al., 2005). A strong resistance to fungal spread was observed in a susceptible wheat cultivar after infection with the lipase-deficient F. graminearum strain Δfgl1. Light microscopy indicated barrier formation in the transition zone of rachilla and rachis of directly inoculated spikelets. In contrast, neither spreading resistance nor barrier formation was observed during F. graminearum wild type infection. An active role of lipases in establishing full virulence was also recently proposed for the plant pathogen Fusarium oxysporum f. sp. lycopersici, where reduced lipolytic activity due to the deletion of lipase regulatory genes resulted in reduced colonization of tomato (Solanum lycopersicum) plants (Bravo-Ruiz et al., 2013). Because the expression of the lipase-encoding gene LIP1 was induced in the biotrophic fungus Blumeria graminis during early stages of infection (Feng et al., 2009) and disruption of the putative secreted lipase gene lipA resulted in reduced virulence of the bacterial plant pathogen Xanthomonas campestris (Tamir-Ariel et al., 2012), a general importance of extracellular lipolytic activity during plant colonization is indicated.We evaluated a possible role of callose in plant defense by infecting wheat spikes with the virulent fungal pathogen F. graminearum wild type, the virulence-deficient F. graminearum deletion mutant Δfgl1, and the barley leaf pathogen Pyrenophora teres, the latter intended to induce strong plant defense responses as known from incompatible, nonhost interactions. The formation of callose plugs within the vascular bundles of inoculated spikelets and the callose synthase activity of infected spikelet tissue correlated directly with increased plant resistance. Subsequent analyses of free fatty acid (FFA) concentrations revealed that those polyunsaturated FFAs were enriched during wheat infection with the F. graminearum wild-type strain that could inhibit callose synthase activity in vitro as well as in planta and partially restored the virulence of the lipase-deficient F. graminearum strain Δfgl1. On the basis of these results, we propose a model for FHB where defense-related callose synthase is inhibited by specific FFAs whose accumulation is caused by the fungus during fungal infection; this inhibition is required for full infection of the wheat head.     

The art of isolating nitrogen-fixing bacteria from non-leguminous plants using N-free semi-solid media: a practical guide for microbiologists

Background and aim

Nitrogen-fixing bacteria or diazotrophs have been isolated for many years using different formulations of N-free semi-solid media. However, the strategies used to isolate them, and the recipes of these media, are scattered through the published literature and in other sources that are more difficult to access and which are not always retrievable. Therefore, the aim of this work was to collate the various methods and recipes, and to provide a comprehensive methodological guide and their use by the scientific community working in the field of biological nitrogen fixation (BNF), particularly with non-leguminous plants.

Methods

Procedures used for bacterial counting and identification either from rhizosphere soil or on the surface of, or within, plant tissues (to access “endophytic” bacteria) are presented in detail, including colony and cell morphologies. More importantly, appropriate recipes available for each N-free semi-solid culture medium that are used to count and isolate various diazotrophs are presented.

Results

It is recognized by those working in the field of BNF with non-legumes that the development of the N-free semi-solid medium has allowed a tremendous accumulation of knowledge on the ecology and physiology of their associated diazotrophs. At least 20 nitrogen-fixing species have been isolated and identified based on the enrichment method originally developed by Döbereiner, Day and collaborators in the 70’s. In spite of all the advances in molecular techniques used to detect bacteria, in most cases the initial isolation and identification of these diazotrophs still requires semi-solid media.

Conclusions

The introduction of the N-free semi-solid medium opened new opportunities for those working in the area of BNF with non-legumes not only for elucidating the important role played by their associated microorganisms, but also because some of these bacteria that were isolated using semi-solid media are now being recommended as plant growth-promoting inoculants for sugarcane (Saccharum sp.), maize (Zea mays) and wheat (Triticum aestivum) in Brazil and other countries. Further progress in the field could be made by using a combination of culture-independent molecular community analyses, in situ activity assessments with probe-directed enrichment, and isolation of target strains using modified or standard semi-solid media.