DuPont (Netherlands)

Objective— Inflammasomes are multiprotein complexes, and their activation has been associated with cardiovascular disease. Inflammasome activation leads to secretion of caspase-1 by innate immune cells, resulting in the activation of interleukin-1β. Recently, a potent and selective inhibitor of the NLRP3 inflammasome, MCC950, was described. In this study, we investigated the effect of MCC950 on atherosclerotic lesion development in apoE −/− mice. Approach and Results— First, we determined the efficacy of MCC950 in vitro. Bone marrow–derived macrophages and dendritic cells were stimulated with lipopolysaccharide and cholesterol crystals resulting in high levels of interleukin-1β release, which was inhibited by MCC950. In vivo MCC950 treatment reduced lipopolysaccharide–induced interleukin-1β secretion, without affecting the tumor necrosis factor-α response. Subsequently, atherosclerotic plaques were induced in Western-type diet fed apoE −/− mice by semiconstrictive perivascular collar placement at the carotid arteries, after which the mice received MCC950 (10 mg/kg) or vehicle control 3× per week intraperitoneally for 4 weeks. After euthanize, atherosclerotic plaque size and volume were quantified in hematoxylin-eosin–stained 10-µm cryosections throughout the artery. MCC950 treatment significantly reduced the development of atherosclerotic lesions as determined by maximal stenosis, average plaque size, and plaque volume. Although the amount of collagen and the necrotic core size were not affected, the number of macrophages in the plaque was significantly reduced on treatment. In addition, VCAM-1 (vascular cell adhesion molecule 1) and ICAM-1 (intercellular adhesion molecule 1) mRNA expression was significantly reduced in the carotids of MCC950-treated mice. Conclusions— These findings show that specific inhibition of the NLRP3 inflammasome using MCC950 can be a promising therapeutic approach to inhibit atherosclerotic lesion development.

Novel species of fungi described in this study include those from various countries as follows: Antarctica: Cadophora antarctica from soil. Australia : Alfaria dandenongensis on Cyperaceae , Amphosoma persooniae on Persoonia sp., Anungitea nullicana on Eucalyptus sp., Bagadiella eucalypti on Eucalyptus globulus , Castanediella eucalyptigena on Eucalyptus sp., Cercospora dianellicola on Dianella sp., Cladoriella kinglakensis on Eucalyptus regnans , Cladoriella xanthorrhoeae (incl. Cladoriellaceae fam. nov. and Cladoriellales ord. nov.) on Xanthorrhoea sp., Cochlearomyces eucalypti (incl. Cochlearomyces gen. nov. and Cochlearomycetaceae fam. nov.) on Eucalyptus obliqua , Codinaea lambertiae on Lambertia formosa , Diaporthe obtusifoliae on Acacia obtusifolia , Didymella acaciae on Acacia melanoxylon , Dothidea eucalypti on Eucalyptus dalrympleana , Fitzroyomyces cyperi (incl. Fitzroyomyces gen. nov.) on Cyperaceae , Murramarangomyces corymbiae (incl. Murramarangomyces gen. nov., Murramarangomycetaceae fam. nov. and Murramarangomycetales ord. nov.) on Corymbia maculata , Neoanungitea eucalypti (incl. Neoanungitea gen. nov.) on Eucalyptus obliqua , Neoconiothyrium persooniae (incl. Neoconiothyrium gen. nov.) on Persoonia laurina subsp. laurina , Neocrinula lambertiae (incl. Neocrinulaceae fam. nov.) on Lambertia sp., Ochroconis podocarpi on Podocarpus grayae , Paraphysalospora eucalypti (incl. Paraphysalospora gen. nov.) on Eucalyptus sieberi , Pararamichloridium livistonae (incl. Pararamichloridium gen. nov., Pararamichloridiaceae fam. nov. and Pararamichloridiales ord. nov.) on Livistona sp., Pestalotiopsis dianellae on Dianella sp., Phaeosphaeria gahniae on Gahnia aspera , Phlogicylindrium tereticornis on Eucalyptus tereticornis , Pleopassalora acaciae on Acacia obliquinervia , Pseudodactylaria xanthorrhoeae (incl. Pseudodactylaria gen. nov., Pseudodactylariaceae fam. nov. and Pseudodactylariales ord. nov.) on Xanthorrhoea sp., Pseudosporidesmium lambertiae (incl. Pseudosporidesmiaceae fam. nov.) on Lambertia formosa , Saccharata acaciae on Acacia sp., Saccharata epacridis on Epacris sp., Saccharata hakeigena on Hakea sericea , Seiridium persooniae on Persoonia sp., Semifissispora tooloomensis on Eucalyptus dunnii , Stagonospora lomandrae on Lomandra longifolia , Stagonospora victoriana on Poaceae , Subramaniomyces podocarpi on Podocarpus elatus , Sympoventuria melaleucae on Melaleuca sp., Sympoventuria regnans on Eucalyptus regnans , Trichomerium eucalypti on Eucalyptus tereticornis , Vermiculariopsiella eucalypticola on Eucalyptus dalrympleana , Verrucoconiothyrium acaciae on Acacia falciformis , Xenopassalora petrophiles (incl. Xenopassalora gen. nov.) on Petrophile sp., Zasmidium dasypogonis on Dasypogon sp., Zasmidium gahniicola on Gahnia sieberiana . Brazil : Achaetomium lippiae on Lippia gracilis , Cyathus isometricus on decaying wood , Geastrum caririense on soil, Lycoperdon demoulinii (incl. Lycoperdon subg. Arenicola ) on soil, Megatomentella cristata (incl. Megatomentella gen. nov.) on unidentified plant, Mutinus verrucosus on soil, Paraopeba schefflerae (incl. Paraopeba gen. nov.) on Schefflera morototoni , Phyllosticta catimbauensis on Mandevilla catimbauensis , Pseudocercospora angularis on Prunus persica , Pseudophialophora sorghi on Sorghum bicolor , Spumula piptadeniae on Piptadenia paniculata . Bulgaria : Yarrowia parophonii from gut of Parophonus hirsutulus . Croatia : Pyrenopeziza velebitica on Lonicera borbasiana . Cyprus : Peziza halophila on coastal dunes Czech Republic : Aspergillus contaminans from human fingernail. Ecuador : Cuphophyllus yacurensis on forest soil, Ganoderma podocarpense on fallen tree trunk. England : Pilidium anglicum (incl. Chaetomellales ord. nov.) on Eucalyptus sp. France : Planamyces parisiensis (incl. Planamyces gen. nov.) on wood inside a house. French Guiana : Lactifluus ceraceus on soil. Germany : Talaromyces musae on Musa sp. India : Hyalocladosporiella cannae on Canna indica , Nothophoma raii from soil. Italy : Setophaeosphaeria citri on Citrus reticulata , Yuccamyces citri on Citrus limon . Japan : Glutinomyces brunneus (incl. Glutinomyces gen. nov.) from roots of Quercus sp. Netherlands (all from soil): Collariella hilkhuijsenii , Fusarium petersiae , Gamsia kooimaniorum , Paracremonium binnewijzendii , Phaeoisaria annesophieae , Plectosphaerella niemeijerarum , Striaticonidium deklijnearum , Talaromyces annesophieae , Umbelopsis wiegerinckiae , Vandijckella johannae (incl. Vandijckella gen. nov. and Vandijckellaceae fam. nov.), Verhulstia trisororum (incl. Verhulstia gen. nov.). New Zealand : Lasiosphaeria similisorbina on decorticated wood. Papua New Guinea : Pseudosubramaniomyces gen. nov. (based on Pseudosubramaniomyces fusisaprophyticus comb. nov.). Slovakia : Hemileucoglossum pusillum on soil. South Africa : Tygervalleyomyces podocarpi</jats:ital

Natural carbohydrate polymers such as starch, cellulose, and chitin provide renewable alternatives to fossil fuels as a source for fuels and materials. As such, there is considerable interest in their conversion for industrial purposes, which is evidenced by the established and emerging markets for products derived from these natural polymers. In many cases, this is achieved via industrial processes that use enzymes to break down carbohydrates to monomer sugars. One of the major challenges facing large-scale industrial applications utilizing natural carbohydrate polymers is rooted in the fact that naturally occurring forms of starch, cellulose, and chitin can have tightly packed organizations of polymer chains with low hydration levels, giving rise to crystalline structures that are highly recalcitrant to enzymatic degradation. The topic of this review is oxidative cleavage of carbohydrate polymers by lytic polysaccharide mono-oxygenases (LPMOs). LPMOs are copper-dependent enzymes (EC 1.14.99.53-56) that, with glycoside hydrolases, participate in the degradation of recalcitrant carbohydrate polymers. Their activity and structural underpinnings provide insights into biological mechanisms of polysaccharide degradation.

BACKGROUND: Lytic polysaccharide monooxgygenases (LPMOs) are known to boost the hydrolytic breakdown of lignocellulosic biomass, especially cellulose, due to their oxidative mechanism. For their activity, LPMOs require an electron donor for reducing the divalent copper cofactor. LPMO activities are mainly investigated with ascorbic acid as a reducing agent, but little is known about the effect of plant-derived reducing agents on LPMOs activity. RESULTS: Here, we show that three LPMOs from the fungus Myceliophthora thermophila C1, MtLPMO9A, MtLPMO9B and MtLPMO9C, differ in their substrate preference, C1-/C4-regioselectivity and reducing agent specificity. MtLPMO9A generated C1- and C4-oxidized, MtLPMO9B C1-oxidized and MtLPMO9C C4-oxidized gluco-oligosaccharides from cellulose. The recently published MtLPMO9A oxidized, next to cellulose, xylan, β-(1 → 3, 1 → 4)-glucan and xyloglucan. In addition, MtLPMO9C oxidized, to a minor extent, xyloglucan and β-(1 → 3, 1 → 4)-glucan from oat spelt at the C4 position. In total, 34 reducing agents, mainly plant-derived flavonoids and lignin-building blocks, were studied for their ability to promote LPMO activity. Reducing agents with a 1,2-benzenediol or 1,2,3-benzenetriol moiety gave the highest release of oxidized and non-oxidized gluco-oligosaccharides from cellulose for all three MtLPMOs. Low activities toward cellulose were observed in the presence of monophenols and sulfur-containing compounds. CONCLUSIONS: Several of the most powerful LPMO reducing agents of this study serve as lignin building blocks or protective flavonoids in plant biomass. Our findings support the hypothesis that LPMOs do not only vary in their C1-/C4-regioselectivity and substrate specificity, but also in their reducing agent specificity. This work strongly supports the idea that the activity of LPMOs toward lignocellulosic biomass does not only depend on the ability to degrade plant polysaccharides like cellulose, but also on their specificity toward plant-derived reducing agents in situ.

The development of the caecal microbiota plays a role in the metabolism and immune competence of chickens. A detailed understanding of normal succession in the caecal microbiota can inform the use of probiotics and other interventions to optimise the caecal microbiota. The development of the microbiota in caecal mucus and lumen samples from three breeds of broiler chicken (Cobb 500, n = 36; Hubbard JA87, n = 38; and Ross 308, n = 36) was observed between 0 and 42 days post hatch. Chicks were housed in the same room of a climate-controlled, biosecure chicken housing unit. Between 0 and 14 days post hatch, chicks were kept in brooder pens ensuring a mixture of breeds in each brooder. From 22 days post hatch, chicks were removed from the brooders and kept in the same room. DNA was extracted from a pooled sample of caecal mucus and luminal contents from five birds of each breed at 0, 3, 7, 14, 21, 28 and 42 days post hatch. High-throughput Illumina sequencing was performed for the V4 hypervariable region of the 16S rRNA gene. The early caecal microbiota was characterised by poor diversity and dominance by one or two bacterial species. Early colonisers of the caecum included \textit{Bifidobacteriaceae}, \textit{Lachnospiraceae}, \textit{Bacteroidaceae} and \textit{Burkholderiaceae} with some amplicon sequence variants (ASVs) assigned to \textit{Ruminococcaceae}. Later colonisers of the caecal microbiota were most apparent from 14 d.p.h and included \textit{Ruminococcaceae}, \textit{Clostridiales} vadin BB60 group, \textit{Christensenellaceae} and \textit{Bacillaceae}. The caecal microbiota continued to change until 42 d.p.h when the microbiota was characterised by a high abundance of \textit{Bacteroidaceae}, \textit{Lachnospiraceae} and \textit{Ruminococcaceae}. The lumen microbiota was significantly different to the mucus with some ASVs assigned to \textit{Lachnospiraceae}, \textit{Ruminococcaceae}, \textit{Christensenellaceae} and \textit{Bacillaceae} showing increased abundance in the mucus. ASVs assigned to \textit{Bacteroidaceae}, \textit{Lactobacillaceae} and \textit{Burkholderiaceae} showed a preference for the lumen. Analysis of five caecal mucus samples from each breed at 42 days post hatch showed differences in microbiota composition between Ross and Cobb as well as between Ross and Hubbard. Since performance data was not collected no functional inferences as to the significance of this finding can be made.

Many fungi boost the deconstruction of lignocellulosic plant biomass via oxidation using lytic polysaccharide monooxygenases (LPMOs). The application of LPMOs is expected to contribute to ecologically friendly conversion of biomass into fuels and chemicals. Moreover, applications of LPMO-modified cellulose-based products may be envisaged within the food or material industry. Here, we show an up to 75-fold improvement in LPMO-driven cellulose degradation using polyphenol oxidase-activated lignin building blocks. This concerted enzymatic process involves the initial conversion of monophenols into diphenols by the polyphenol oxidase MtPPO7 from Myceliophthora thermophila C1 and the subsequent oxidation of cellulose by MtLPMO9B. Interestingly, MtPPO7 shows preference towards lignin-derived methoxylated monophenols. Sequence analysis of genomes of 336 Ascomycota and 208 Basidiomycota reveals a high correlation between MtPPO7 and AA9 LPMO genes. The activity towards methoxylated phenolic compounds distinguishes MtPPO7 from well-known PPOs, such as tyrosinases, and ensures that MtPPO7 is an excellent redox partner of LPMOs. The correlation between MtPPO7 and AA9 LPMO genes is indicative for the importance of the coupled action of different monooxygenases in the concerted degradation of lignocellulosic biomass. These results will contribute to a better understanding in both lignin deconstruction and enzymatic lignocellulose oxidation and potentially improve the exploration of eco-friendly routes for biomass utilization in a circular economy.

The development and succession of the microbiota in ileal mucus and lumen samples from three breeds of broiler chicken (Cobb 500, n = 36; Hubbard JA87, n = 38; and Ross 308, n = 36) was observed between 3 and 42 days post hatch (d.p.h). Chicks were housed in the same room of a climate-controlled, biosecure chicken housing unit. Between 0 and 14 d.p.h, chicks were kept in three circular brooder pens ensuring a mixture of breeds in each brooder. From 22 d.p.h, chicks were removed from the brooders and kept in the same room. DNA was extracted from a pooled sample of ileal mucus and luminal contents taken from five birds of each breed at 3, 7, 14, 21, 28 and 42 d.p.h. High-throughput Illumina sequencing was performed for the V4 hypervariable region of the 16S rRNA gene. The initial microbiota in the ileum varied between breeds. The common features were a low diversity and general dominance by one or two taxa such as Enterococcus or Escherichia with relatively low numbers of Lactobacillus. Escherichia became the most abundant genus in samples where Enterococcus was previously the dominant taxa. The next phase of development was marked by an increase in the abundance of Candidatus Arthromitus in the mucus and Lactobacillus in the lumen. The high abundance of Candidatus Arthromitus persisted between 7 and 14 d.p.h after which Lactobacillus became the most abundant genus in both the mucus and lumen. Dominance of the ileal microbiota by Lactobacillus was a transient feature. By 42 d.p.h, the relative abundance of Lactobacillus had fallen while a range of other taxa including Escherichia, Turicibacter and members of Clostridiales increased. This general pattern was followed by all breeds, however, the rate at which succession occured differed as Ross matured quicker than Cobb with Hubbard as an intermediate.

Abstract Background Trichoderma reesei is one of the best-known cellulolytic organisms, producing large quantities of a complete set of extracellular cellulases and hemicellulases for the degradation of lignocellulosic substances. Hence, T. reesei is a biotechnically important host and it is used commercially in enzyme production, of both native and foreign origin. Many strategies for producing enzymes in T. reesei rely on the cbh1 and other cellulase gene promoters for high-level expression and these promoters require induction by sophorose, lactose or other inducers for high productivity during manufacturing. Results We described an approach for producing high levels of secreted proteins by overexpression of a transcription factor ACE3 in T. reesei . We refined the ace3 gene structure and identified specific ACE3 variants that enable production of secreted cellulases and hemicellulases on glucose as a sole carbon source (i.e., in the absence of an inducer). These specific ACE3 variants contain a full-length Zn 2 Cys 6 binuclear cluster domain at the N-terminus and a defined length of truncations at the C-terminus. When expressed at a moderate level in the fungal cells, the ACE3 variants can induce high-level expression of cellulases and hemicellulases on glucose (i.e., in the absence of an inducer), and further improve expression on lactose or glucose/sophorose (i.e., in the presence of an inducer). Finally, we demonstrated that this method is applicable to industrial strains and fermentation conditions, improving protein production both in the absence and in the presence of an inducer. Conclusions This study demonstrates that overexpression of ACE3 variants enables a high level of protein production in the absence of an inducer, and boosts protein production in the presence of an inducer. It is an efficient approach to increase protein productivity and to reduce manufacturing costs.

Tomato leaf mold disease is caused by the biotrophic fungus Cladosporium fulvum. During infection, C. fulvum produces extracellular small secreted protein (SSP) effectors that function to promote colonization of the leaf apoplast. Resistance to the disease is governed by Cf immune receptor genes that encode receptor-like proteins (RLPs). These RLPs recognize specific SSP effectors to initiate a hypersensitive response (HR) that renders the pathogen avirulent. C. fulvum strains capable of overcoming one or more of all cloned Cf genes have now emerged. To combat these strains, new Cf genes are required. An effectoromics approach was employed to identify wild tomato accessions carrying new Cf genes. Proteomics and transcriptome sequencing were first used to identify 70 apoplastic in planta-induced C. fulvum SSPs. Based on sequence homology, 61 of these SSPs were novel or lacked known functional domains. Seven, however, had predicted structural homology to antimicrobial proteins, suggesting a possible role in mediating antagonistic microbe-microbe interactions in planta. Wild tomato accessions were then screened for HR-associated recognition of 41 SSPs, using the Potato virus X-based transient expression system. Nine SSPs were recognized by one or more accessions, suggesting that these plants carry new Cf genes available for incorporation into cultivated tomato.

. During hydrolysis, oligosaccharides bind at subsites -2 to +2 in the enzyme's active site. Chitinase Chi1 can be used for chitin valorisation and for production of chitin- and chito-oligosaccharides at industrial scale.

Lytic polysaccharide monooxygenases (LPMOs) have recently been shown to significantly enhance the degradation of recalcitrant polysaccharides and are of interest for the production of biochemicals and bioethanol from plant biomass. The copper-containing LPMOs utilize electrons, provided by reducing agents, to oxidatively cleave polysaccharides. Here, we report the development of a β-glucosidase-assisted method to quantify the release of C1-oxidized gluco-oligosaccharides from cellulose by two C1-oxidizing LPMOs from Myceliophthora thermophila C1. Based on this quantification method, we demonstrate that the catalytic performance of both MtLPMOs is strongly dependent on pH and temperature. The obtained results indicate that the catalytic performance of LPMOs depends on the interaction of multiple factors, which are affected by both pH and temperature.

The increased use of plant ingredients in aquafeeds over the last decades, as replacement for fish meal, has led to rising levels of undesired non-starch polysaccharides (NSP) and phytate. Both NSP and phytate degrading enzymes and probiotics have been widely studied. They can be used as a tool to deal with increasing levels of NSP and phytate in aquafeeds. However, studies combining both probiotics and enzymes are scarce in fish. The main objective of the present study was to assess the impact on Nile tilapia (Oreochromis niloticus) of enzymes and probiotics, as well as their synergistic effect. Parameters measured were: growth; nutrient digestibility; body composition; and the energy, nitrogen (N), P and Ca balance. Diets were supplemented with, and without, an enzyme mix (phytase at 1000 FTU/kg and xylanase at 6000 U/kg) and with, and without, probiotics (three strains of B. amyloliquefaciens at 60 mg/kg feed), according to a 2 × 2 factorial arrangement. This resulted in a control treatment (CON-CON) without enzymes and probiotics, an enzyme treatment (ENZ-CON), a probiotic treatment (CON-PRO) and a treatment with both enzymes and probiotics (ENZ-PRO). In total, 16 tanks (4 replicates/treatment) were used with 35 fish each (mean initial weight 39 g). Fish were restrictively fed equal amounts of dry matter for 42 days. Both enzymes (P < 0.001) and probiotics (P < 0.05) improved growth (g/d) and FCR when applied individually. The combination of enzymes and probiotics showed an interaction effect (P < 0.05) on growth and FCR. Enzymes improved growth to a greater extent than probiotics, whereas the combination of enzymes and probiotics did not further enhance growth. The CON-CON treatment had the highest FCR (1.33), the CON-PRO treatment a slightly lower FCR (1.27); the lowest FCR (1.11) was found for both treatments with enzymes (ENZ-CON and ENZ-PRO). Enzyme supplementation improved the digestibility of all nutrients (P < 0.01), whereas probiotics enhanced fat digestibility (P < 0.01). Additionally, enzyme supplementation increased retained P (mg/d), retained N (mg/d) and N efficiency (P < 0.001). Probiotic supplementation affected the energy requirements for maintenance (kJ/kg0.8/d; P < 0.05). Dietary supplementation of either enzymes or probiotics had positive effects on the measured parameters, but the combination of enzymes and probiotics did not have a synergistic effect.

Hypocrea jecorina cellulases are able to efficiently degrade cellulosic biomass to fermentable sugars at large, commercially relevant scales. H. jecorina Cel7A, cellobiohydrolase I, from glycoside hydrolase family 7, is the workhorse enzyme of the process. However, the thermal stability of Cel7A limits its use to processes where temperatures are no higher than 50 C. Enhanced thermal stability is desirable to enable the use of higher processing temperatures and to improve the economic feasibility of industrial biomass conversion. Here, we enhanced the thermal stability of Cel7A through directed evolution. Sites with increased thermal stability properties were combined, and a Cel7A variant (FCA398) was obtained, which exhibited a 10.4 C increase in T m and a 44-fold greater half-life compared with the wild-type enzyme. This Cel7A variant contains 18 mutated sites and is active under application conditions up to at least 75 C. The X-ray crystal structure of the catalytic domain was determined at 2.1 resolution and showed that the effects of the mutations are local and do not introduce major backbone conformational changes. Molecular dynamics simulations revealed that the catalytic domain of wild-type Cel7A and the FCA398 variant exhibit similar behavior at 300 K, whereas at elevated temperature (475 and 525 K), the FCA398 variant fluctuates less and maintains more native contacts over time. Combining the structural and dynamic investigations, rationales were developed for the stabilizing effect at many of the mutated sites.

Abstract The objective of this paper is to compare, under Dutch market conditions, the energy consumption and net costs of membrane-based advanced treatment processes for three water reuse types (i.e. potable, industrial, agricultural reuse). The water source is municipal wastewater treatment plant effluent. Results indicate that the application of reverse osmosis is needed to reclaim high quality water for industrial and potable reuse but not for irrigation water which offers significant energy savings but may not lead automatically to lower net costs. While a reclamation process for industrial reuse is economically most promising, irrigation water reclamation processes are not cost effective due to low water prices. Moreover, process operational expenditures may exceed capital expenditures which is important for tender procedures. A significant cost factor is waste management that may exceed energy costs. Water recovery rates could be significantly enhanced through the integration of a softener/biostabilizer unit prior to reverse osmosis. Moreover, the energy consumption of wastewater reclamation processes could be supplied on-site with solar energy. The possibility of designing a ‘fit for multi-purpose’ reclamation process is discussed briefly. This comparative analysis allows for better informed decision making about which reuse type is preferably targeted in a municipal wastewater reuse project from a process design perspective.

The variation in the length of gestation, the period from mating until parturition, was studied in 77 dogs of different breeds; the time for mating was determined by measuring peripheral blood progesterone levels. The mean length of gestation was 62.1 +/- 0.2 (S.E.M.) days, with a variation of 11 days. The number of pups appeared to influence the length of gestation. Length of gestation was negatively correlated (r = -0.96, P < 0.001, n = 44) with litter size in litters with 7 or fewer pups. The intra-breed variation in length of gestation in the five breeds represented by five or more bitches was 3-6 days. The mean gestation of Alsatians (60.1 +/- 0.5, n = 9) was shorter (P < 0.005) than that of the other breeds combined (62.3 +/- 0.3, n = 68). The primiparous/multiparous status of the bitch did not influence the length of gestation.

The sand dunes and inter-dune zones of the hyper-arid central Namib Desert represent heterogeneous soil habitats. As little is known about their indigenous edaphic bacterial communities, we aimed to evaluate their diversity and factors of assembly and hypothesized that soil physicochemistry gradients would strongly shape dune/interdune communities. We sampled a total of 125 samples from 5 parallel dune/interdune transects and characterized 21 physico-chemical edaphic parameters coupled with 16S rRNA gene bacterial community fingerprinting using T-RFLP and 454 pyrosequencing. Multivariate analyses of T-RFLP data showed significantly different bacterial communities, related to physico-chemical gradients, in four distinct dune habitats: the dune top, slope, base and interdune zones. Pyrosequencing of 16S rRNA gene amplicon sets showed that each dune zone presented a unique phylogenetic profile, suggesting a high degree of environmental selection. The combined results strongly infer that habitat filtering is an important factor shaping Namib Desert dune bacterial communities, with habitat stability, soil texture and mineral and nutrient contents being the main environmental drivers of bacterial community structures.

This study evaluated the effects of increasing the dose of a 6-phytase from Buttiauxella on phytate degradation, mineral, energy, and AA digestibility in weaned pigs fed complex diets based on wheat, corn, soybean meal, barley, and rapeseed meal. A negative control (NC) diet containing no added inorganic phosphorus (P) and a reduction of 0.1% calcium (Ca) and 36 kcal/kg ME was supplemented with Buttiauxella phytase at 0, 250, 500, 1,000, or 2,000 FTU/kg diet and tested against a nutritionally adequate, positive control (PC) diet. One phytase units (FTU) is the amount of enzyme that liberates 1 micromole of inorganic phosphate per minute from a sodium phytate substrate at pH 5.5 and 37 °C. Barrows (Topigs × Pietrian; initial mean body weight 19.3 kg) were housed individually in metabolic crates and fed the test diets in mash form via 2 equal meals per day for 9 d (fed at 2.5 times the maintenance energy requirement), with 8 replicate pigs per treatment, in 2 experimental runs (total n = 48). After a 3-d adaptation period, urine and feces were collected over 5 d for measurements of apparent total tract digestibility (ATTD) and retention of nutrients. On day 9, pigs were euthanized and ileal digesta collected for measurements of apparent ileal digestibility (AID) of nutrients. Phytase improved (P < 0.05) digestibility of all measured AA except Trp (P < 0.1), and AID P, nitrogen, phytate, ATTD P, Ca versus NC. Increasing phytase dose from 0 (NC) to 2,000 FTU/kg increased AID Lys, Cys, Thr, Val, Ile, Leu, mean AA, P, N, phytate, ATTD P, N, Na, energy, ME, P retention (g/d), and reduced P excretion (g/d) in a linear or exponential manner (P < 0.05). Phytase at 2,000 FTU/kg improved AA digestibility by between +3.1 percentage points (Trp) and +8.8 percentage points (Cys) versus NC (average +6.3 percentage points) (P < 0.05). Phytase inclusion at 2,000 FTU/kg reduced P excretion (g/d) by 57% versus PC (P < 0.05). In conclusion, increasing Buttiauxella phytase in the range of 0 to 2,000 FTU/kg increased phytate degradation, improved AA and P digestibility, and reduced P excretion in weaned pigs fed complex diets.

Abstract Background Myceliophthora thermophila is a thermophilic ascomycete fungus that is used as a producer of enzyme cocktails used in plant biomass saccharification. Further development of this species as an industrial enzyme factory requires a detailed understanding of its regulatory systems driving the production of plant biomass-degrading enzymes. In this study, we analyzed the function of MtXlr1, an ortholog of the (hemi-)cellulolytic regulator XlnR first identified in another industrially relevant fungus, Aspergillus niger . Results The Mtxlr1 gene was deleted and the resulting strain was compared to the wild type using growth profiling and transcriptomics. The deletion strain was unable to grow on xylan and d -xylose, but showed only a small growth reduction on l -arabinose, and grew similar to the wild type on Avicel and cellulose. These results were supported by the transcriptome analyses which revealed reduction of genes encoding xylan-degrading enzymes, enzymes of the pentose catabolic pathway and putative pentose transporters. In contrast, no or minimal effects were observed for the expression of cellulolytic genes. Conclusions Myceliophthora thermophila MtXlr1 controls the expression of xylanolytic genes and genes involved in pentose transport and catabolism, but has no significant effects on the production of cellulases. It therefore resembles more the role of its ortholog in Neurospora crassa , rather than the broader role described for this regulator in A. niger and Trichoderma reesei . By revealing the range of genes controlled by MtXlr1, our results provide the basic knowledge for targeted strain improvement by overproducing or constitutively activating this regulator, to further improve the biotechnological value of M. thermophila .

Immunocytochemical detection of bromodeoxyuridine (BrdU) labeling can be hampered by low BrdU incorporation levels. We describe here an amplification method for weak BrdU immunosignals. The tyramide signal amplification method based on catalyzed reporter deposition (CARD) uses fluorescein-labeled tyramide as a substrate for horseradish peroxidase. The enzyme catalyzes the formation of highly reactive tyramide radicals with a very short half-life, resulting in the binding of fluorescein-conjugated tyramide only at the site of the enzymatic reaction. MCF-7 cells were grown in vitro in medium containing charcoal-stripped fetal bovine serum supplemented by growth factors. Under these culture conditions, the BrdU immunosignal was hard to detect but could be enhanced specifically by the tyramide signal amplification system, resulting in clear-cut differences between BrdU-negative and BrdU-positive cells. This enabled rapid and objective quantification of the BrdU labeling index without the risk of underestimating the number of cells in S-phase. Therefore, this amplification of BrdU immunosignals might also prove valuable for in vivo cancer prognosis, cell kinetics studies, and computer-assisted image analyses.

Comparative effects of graded doses of two phytases on growth performance, nutrient and amino acid (AA) digestibility were investigated in corn-soybean meal-based broiler diets. A negative control diet (NC) with low phosphorus (P) content (1.8, 4.4 and 2.5 g/kg retainable, total and phytate P respectively) was supplemented with four doses of a Buttiauxella sp phytase (analyzed activity of 303 to 1046 FTU/kg) or an E. coli phytase (analyzed activity of 442 to 1811 FTU/kg), and tested against three positive control diets comprising the NC + 0.6, 1.2 or 1.8 g P from monocalcium phosphate (MCP)/kg feed and increased Ca content (+0.8 g/kg). A total of 1152 male Ross 308 broilers at 5 days of age were assigned to 72 cages with 16 birds/cage and 6 cages/treatment were given free access to pelleted diets until 21 days of age. Feed intake (FI), body weight and mortality were recorded and used to calculate body weight gain (BWG) and feed conversion ratio (FCR) during 5–20 days of age. Excreta was collected on day 18–20 to determine total tract retention of nutrients. Tibias (from 4 birds pooled per cage) and ileal digesta (from all birds and pooled per cage) were sampled at 21 days of age to determine tibia ash, ileal digestibility of nutrients and AA. Compared to NC, increasing phytase dose and MCP levels produced a stepwise increase in FI, BWG and reduction in FCR. Increasing phytase dose produced curvilinear increases in BWG, ileal P digestibility, retention and tibia ash (P < 0.01) but effects were greater for Buttiauxella vs. E. coli phytase. In contrast, only Buttiauxella phytase increased the ileal digestibility of protein and total AA (+3.7% on average vs. NC at 1046 FTU/kg, P < 0.05). Linear increases in ileal digestibility of all individual AA were seen with increasing dose of Buttiauxella (P < 0.05 in all cases) and were greatest for cysteine (+ 7.9% vs. NC at 1046 FTU/kg), whereas only 2 individual AA showed linear increases in digestibility in response to increasing dose of the E. coli phytase (cysteine and proline, P < 0.05). Ileal digestibility of Na (%) was markedly increased (less negative) by phytase, especially for the Buttiauxella phytase which produced a 96% increase in ileal Na digestibility at 1046 FTU/kg vs. NC. Across treatments, ileal digestibility of AA and Na were positively correlated (P < 0.001). These results provide insight into the ‘extra-phosphoric’ effects in broilers fed corn-soybean meal-based diets and suggest that the Buttiauxella and E. coli phytase had markedly different degrees of ‘extra-phosphoric’ effects on AA digestibility per standard FTU. The data showed that the AA digestibility response to increasing phytase dose do not follow the same response curve as for digestible P, and that the response curves are specific for different phytases.