Variability in Aggressiveness of Rice Blast (Magnaporthe oryzae) Isolates Originating from Rice Leaves and Necks: A Case of Pathogen Specialization?

Rice blast, caused by Magnaporthe oryzae, causes yield losses associated with injuries on leaves and necks, the latter being in general far more important than the former. Many questions remain on the relationships between leaf and neck blast, including questions related to the population biology of the pathogen. Our objective was to test the hypothesis of adaptation of M. oryzae isolates to the type of organ they infect. To that aim, the components of aggressiveness of isolates originating from leaves and necks were measured. Infection efficiency, latent period, sporulation intensity, and lesion size were measured on both leaves and necks. Univariate and multivariate analyses indicated that isolates originating from leaves were less aggressive than isolates originating from necks, when aggressiveness components were measured on leaves as well as on necks, indicating that there is no specialization within the pathogen population with respect to the type of organ infected. This result suggests that the more aggressive isolates involved in epidemics on leaves during the vegetative stage of the crop cycle have a higher probability to infect necks, and that a population shift may occur during disease transmission from leaves to necks. Implications for disease management are discussed.     

Molecular characterization of deletion breakpoints in adults with 22q11 deletion syndrome

22q11 Deletion syndrome (22q11DS) is a common microdeletion syndrome with variable expression, including congenital and later onset conditions such as schizophrenia. Most studies indicate that expression does not appear to be related to length of the deletion but there is limited information on the endpoints of even the common deletion breakpoint regions in adults. We used a real-time quantitative PCR (qPCR) approach to fine map 22q11.2 deletions in 44 adults with 22q11DS, 22 with schizophrenia (SZ; 12 M, 10 F; mean age 35.7 SD 8.0 years) and 22 with no history of psychosis (NP; 8 M, 14 F; mean age 27.1 SD 8.6 years). QPCR data were consistent with clinical FISH results using the TUPLE1 or N25 probes. Two subjects (one SZ, one NP) negative for clinical FISH had atypical 22q11.2 deletions confirmed by FISH using the RP11-138C22 probe. Most (n = 34; 18 SZ, 16 NP) subjects shared a common 3 Mb hemizygous 22q11.2 deletion. However, eight subjects showed breakpoint variability: a more telomeric proximal breakpoint (n = 2), or more centromeric (n = 3) or more telomeric distal breakpoint (n = 3). One NP subject had a proximal nested 1.4 Mb deletion. COMT and TBX1 were deleted in all 44 subjects, and PRODH in 40 subjects (19 SZ, 21 NP). The results delineate proximal and distal breakpoint variants in 22q11DS. Neither deletion extent nor PRODH haploinsufficiency appeared to explain the clinical expression of schizophrenia in the present study. Further studies are needed to elucidate the molecular basis of schizophrenia and clinical heterogeneity in 22q11DS.     

Co-substrate composition is critical for enrichment of functional key species and for process efficiency during biogas production from cattle manure

Cattle manure has a low energy content and high fibre and water content, limiting its value for biogas production. Co-digestion with a more energy-dense material can improve the output, but the co-substrate composition that gives the best results in terms of degree of degradation, gas production and digestate quality has not yet been identified. This study examined the effects of carbohydrate, protein and fat as co-substrates for biogas production from cattle manure. Laboratory-scale semi-continuous mesophilic reactors were operated with manure in mono-digestion or in co-digestion with egg albumin, rapeseed oil, potato starch or a mixture of these, and chemical and microbiological parameters were analysed. The results showed increased gas yield for all co-digestion reactors, but only the reactor supplemented with rapeseed oil showed synergistic effects on methane yield. The reactor receiving potato starch indicated improved fibre degradation, suggesting a priming effect by the easily accessible carbon. Both these reactors showed increased species richness and enrichment of key microbial species, such as fat-degrading Syntrophomonadaceae and families known to include cellulolytic bacteria. The addition of albumin promoted enrichment of known ammonia-tolerant syntrophic acetate- and potential propionate-degrading bacteria, but still caused slight process inhibition and less efficient overall degradation of organic matter in general, and of cellulose in particular.     

Expression of chondroitin-4- O -sulfotransferase in Escherichia coli and Pichia pastoris

Chondroitin sulfates are linear sulfated polysaccharides called glycosaminoglycans. They are important nutraceutical and pharmaceutical products that are biosynthesized through the action of chondroitin sulfotransferases on either an unsulfated chondroitin or a dermatan polysaccharide precursor. While the enzymes involved in the biosynthesis of chondroitin sulfates are well known, the cloning end expression of these membrane-bound Golgi enzymes continue to pose challenges. The major chondroitin-4-sulfotransferase, Homo sapiens C4ST-1, had been previously cloned and expressed from mammalian CHO, COS-7, and HEK 293 cells, and its activity was shown to require glycosylation. In the current study, a C4ST-1 construct was designed and expressed in both Escherichia coli and Pichia pastoris in its non-glycosylated and glycosylated forms. Both constructs showed similar activity albeit different kinetic parameters when acting on a microbially prepared unsulfated chondroitin substrate. Moreover, the glycosylated form of C4ST-1 showed lower stability than the non-glycosylated form.     

Glycosyltransferases from Oat (Avena) Implicated in the Acylation of Avenacins

Plants produce a huge array of specialized metabolites that have important functions in defense against biotic and abiotic stresses. Many of these compounds are glycosylated by family 1 glycosyltransferases (GTs). Oats (Avena spp.) make root-derived antimicrobial triterpenes (avenacins) that provide protection against soil-borne diseases. The ability to synthesize avenacins has evolved since the divergence of oats from other cereals and grasses. The major avenacin, A-1, is acylated with N-methylanthranilic acid. Previously, we have cloned and characterized three genes for avenacin synthesis (for the triterpene synthase SAD1, a triterpene-modifying cytochrome P450 SAD2, and the serine carboxypeptidase-like acyl transferase SAD7), which form part of a biosynthetic gene cluster. Here, we identify a fourth member of this gene cluster encoding a GT belonging to clade L of family 1 (UGT74H5), and show that this enzyme is an N-methylanthranilic acid O-glucosyltransferase implicated in the synthesis of avenacin A-1. Two other closely related family 1 GTs (UGT74H6 and UGT74H7) are also expressed in oat roots. One of these (UGT74H6) is able to glucosylate both N-methylanthranilic acid and benzoic acid, whereas the function of the other (UGT74H7) remains unknown. Our investigations indicate that UGT74H5 is likely to be key for the generation of the activated acyl donor used by SAD7 in the synthesis of the major avenacin, A-1, whereas UGT74H6 may contribute to the synthesis of other forms of avenacin that are acylated with benzoic acid.     

Scale‐dependent strategies for coexistence of mesocarnivores in human‐dominated landscapes

Identifying factors influencing the distribution of and interactions within carnivore communities is important for understanding how they are affected by human activities. Species differ in their ability to adapt to humans depending on their degree of specialization in habitat use and feeding habits. This results in asymmetric changes in the ecology of co‐occurring species that can influence their interactions. We investigated whether human infrastructures and free‐ranging domestic dogs (a species typically associated with humans) influenced the co‐occurrence and habitat use of mesocarnivores in a landscape of high human population density in Maharashtra, India. We used 40 camera trap locations during 233 trapping nights and used Bayesian co‐occurrence occupancy models to investigate the habitat use and coexistence of species at different spatial scales. Additionally, we investigated their temporal overlap in space use. Indian foxes altered their habitat use both spatially and temporally in order to avoid free‐ranging domestic dogs and other larger competitors. The use of human infrastructure by jackals and jungle cats was limited by the presence of dogs. Our results illustrate how habitat use of smaller carnivore species changes both spatially and temporally in order to avoid larger competitors. We also show that the presence of species associated with humans mediates the influence of human infrastructures on the habitat use of mesocarnivores. We highlight the importance of acknowledging the potential impact of urbanization not only on single species, but also on the interactions within the community.     

Generation of site-distinct N-glycan variants for in vitro bioactivity testing

Glycosylation, a critical product quality attribute, may affect the efficacy and safety of therapeutic proteins in vivo. Chinese hamster ovary fed-batch cell culture batches yielded consistent glycoprofiles of a Fc-fusion antibody comprizing three different N-glycosylation sites. By adding media supplements at specific concentrations in cell culture and applying enzymatic glycoengineering, a diverse N-glycan variant population was generated, including high mannose, afucosylated, fucosylated, agalactosylated, galactosylated, asialylated, and sialylated forms. Site-specific glycosylation profiles were elucidated by glycopeptide mapping and the effect of the glycosylation variants on the FcγRIIIa receptor binding affinity and the biological activity (cell-based and surface plasmon resonance) was assessed. The two fusion body glycosylation sites were characterized by a high degree of sialic acid, more complex N-glycan structures, a higher degree of antennarity, and a site-specific behavior in the presence of a media supplement. On the other hand, the media supplements affected the Fc-site glycosylation heterogeneity similarly to the various studies described in the literature with classical monoclonal antibodies. Enzymatic glycoengineering solely managed to generate high levels of galactosylation at the fusion body sites. Variants with low core fucosylation, and to a lower extent, high mannose glycans exhibited increased FcγRIIIa receptor binding affinity. All N-glycan variants exhibited weak effects on the biological activity of the fusion body. Both media supplementation and enzymatic glycoengineering are suitable to generate sufficient diversity to assess the effect of glycostructures on the biological activity.     

Biosynthesis of Natural Flavanones in Saccharomyces cerevisiae

A four-step flavanone biosynthetic pathway was constructed and introduced into Saccharomyces cerevisiae. The recombinant yeast strain was fed with phenylpropanoid acids and produced the flavanones naringenin and pinocembrin 62 and 22 times more efficiently compared to previously reported recombinant prokaryotic strains. Microbial biosynthesis of the flavanone eriodictyol was also achieved.     

Development of an Ointment Formulation Using Hot-Melt Extrusion Technology

Ointments are generally prepared either by fusion or by levigation methods. The current study proposes the use of hot-melt extrusion (HME) processing for the preparation of a polyethylene glycol base ointment. Lidocaine was used as a model drug. A modified screw design was used in this process, and parameters such as feeding rate, barrel temperature, and screw speed were optimized to obtain a uniform product. The product characteristics were compared with an ointment of similar composition prepared by conventional fusion method. The rheological properties, drug release profile, and texture characteristics of the hot-melt extruded product were similar to the conventionally prepared product. This study demonstrates a novel application of the hot-melt extrusion process in the manufacturing of topical semi-solids.