Colonization Phase Research Articles

Formulation of a 3D Printed Biopharmaceutical: The Development of an Alkaline Phosphatase Containing Tablet with Ileo-Colonic Release Profile to Treat Ulcerative Colitis

Powder bed printing is a 3D-printing process that creates freeform geometries from powders, with increasing traction for personalized medicine potential. Little is known about its applications for biopharmaceuticals. In this study, the production of tablets containing alkaline phosphatase using powder bed printing for the potential treatment of ulcerative colitis (UC) was investigated, as was the coating of these tablets to obtain ileo-colonic targeting. The printing process was studied, revealing line spacing as a critical factor affecting tablet physical properties when using hydroxypropyl cellulose as the binder. Increasing line spacing yielded tablets with higher porosity. The enzymatic activity of alkaline phosphatase (formulated in inulin glass) remained over 95% after 2 weeks of storage at 45 °C. The subsequent application of a colonic targeting coating required a PEG 1500 sub-coating. In vitro release experiments, using a gastrointestinal simulated system, indicated that the desired ileo-colonic release was achieved. Less than 8% of the methylene blue, a release marker, was released in the terminal ileum phase, followed by a fast release in the colon phase. No significant impact from the coating process on the enzymatic activity was found. These tablets are the first to achieve both biopharmaceutical incorporation in powder bed printed tablets and ileo-colonic targeting, thus might be suitable for on-demand patient-centric treatment of UC.

From Robert Keayne to Angola, Richard, and Grace: Bernard Bailyn and New England's Place in an Atlantic World

From Robert Keayne to Angola, Richard, and Grace: Bernard Bailyn and New England's Place in an Atlantic World

"Late" effectors from Leptosphaeria maculans as tools for identifying novel sources of resistance in Brassica napus.

The Dothideomycete Leptosphaeria maculans, causing stem canker (blackleg) of Brassica napus, secretes different cocktails of effectors at specific infection stages. Some effectors (“Late” effectors) are specifically produced during the long asymptomatic phase of stem colonization. By manipulating their expression so that they are overexpressed during cotyledon infection (OEC transformants of the fungus), we previously postulated that resistance genes operating in the stem may be involved in gene‐for‐gene relationship and thus contribute to quantitative disease resistance (QDR). Here, we selected 10 relevant new effector genes, and we generated OEC transformants to screen a collection of 130 B. napus genotypes, representative of the available diversity in the species. Five B. napus accessions showed a typical hypersensitive response when challenged with effectors LmSTEE98 or LmSTEE6826 at the cotyledon stage, and all belong to the semi‐winter type of the diversity panel. In addition, five winter‐type genotypes displayed an intermediate response to another late effector, LmSTEE7919. These new interactions now have to be genetically validated to check that they also correspond to gene‐for‐gene interactions. In all cases, they potentially provide novel resources, easy to breed for, and accounting for part of the quantitative resistance in a species for which we are currently facing limited resistance sources.

Should the colonisation of space be based on reproduction? Critical considerations on the choice of having a child in space

This paper aims to argue for the thesis that it is not a priori morally justified that the first phase of space colonisation is based on sexual reproduction. We ground this position on the argument that, at least in the first colonisation settlements, those born in space may not have a good chance of having a good life. This problem does not depend on the fact that life on another planet would have to deal with issues such as solar radiation or with the decrease or entire absence of the force of gravity. These issues could plausibly be addressed given that the planets or settlements we will feasibly colonise could be completely transformed through geoengineering processes. Likewise, the ability of humans to live in space could be enhanced through genetic modification interventions. Even if, however, the problems concerning survival in space were solved, we think that, at least in the first period of colonisation of space or other planets, giving birth to children in space could be a morally irresponsible choice since we argue, the life we ​​could give them might not be good enough. We contend that this is the case since when we decide to have a baby. We argue that it is not morally right to be content that our children have a minimally sufficient life worth living; before we give birth to children in space, we should make sure we can give them a reasonable chance of having a good life. This principle applies both on Earth - at least where you can choose - and for space travel.

Age structure dynamics in an eastern collared lizard population from founding to demographic stability

AbstractThe global decline of reptile species has raised much concern, and many translocation programs have been implemented in response to this decline. Most of these programs have not been successful, but one exception is the translocation program for eastern collared lizards (Crotophytus collaris collaris) that had experienced massive local extinction on glades in the Ozarks in central North America. Translocation coupled with glade restoration was initiated in the 1980s, including reintroductions starting in 1984 on Stegall Mountain in southern Missouri. The translocated populations underwent three distinct demographic phases: (1) an isolate phase with no net growth in population size, no colonization of nearby glades and almost no dispersal among populations, (2) a colonizing phase of high dispersal, colonization of new glades and population growth that started with the onset of prescribed woodland burning and (3) a stable metapopulation phase established by 2000 on Stegall Mountain in which the number of occupied glades, total population size and measures of genetic variation were roughly constant. This study uses distinct color phases to infer the age of 1162 marked individuals. Age probabilities were assigned to 391 individuals based on time‐of‐capture within the field season and snout‐vent length, as calibrated from following 529 individuals first captured as hatchlings. Age structure differed significantly between phases. The isolate phase was characterized by an old age structure. The colonization phase had a younger age structure and much more recruitment. The stable metapopulation phase had an intermediate age structure. These dynamic age structure attributes show that species cannot be regarded as static units, and ignoring this dynamism can result in poor conservation practice. These results also validate age‐structure as a monitoring device for a conservation program and a tool to identify significant shifts in the environment or management that have a conservation impact, in this case prescribed woodland burning.

Bioaccessibility of arsenic from contaminated soils and alteration of the gut microbiome in an in vitro gastrointestinal model

Arsenic exposure has been reported to alter the gut microbiome in mice. Activity of the gut microbiome derived from fecal microbiota has been found to affect arsenic bioaccessibility in an in vitro gastrointestinal (GI) model. Only a few studies have explored the relation between arsenic exposure and changes in the composition of the gut microbiome and in arsenic bioaccessibility. Here, we used simulated GI model system (GIMS) containing a stomach, small intestine, colon phases and microorganisms obtained from mouse feces (GIMS-F) and cecal contents (GIMS-C) to assess whether exposure to arsenic-contaminated soils affect the gut microbiome and whether composition of the gut microbiome affects arsenic bioaccessibility. Soils contaminated with arsenic did not alter gut microbiome composition in GIMS-F colon phase. In contrast, arsenic exposure resulted in the decline of bacteria in GIMS-C, including members of Clostridiaceae, Rikenellaceae, and Parabacteroides due to greater diversity and variability in microbial sensitivity to arsenic exposure. Arsenic bioaccessibility was greatest in the acidic stomach phase of GIMS (pH 1.5–1.7); except for GIMS-C colon phase exposed to mining-impacted soil in which greater levels of arsenic solubilized likely due to microbiome effects. Physicochemical properties of different test soils likely influenced variability in arsenic bioaccessibility (GIMS-F bioaccessibility range: 8–37%, GIMS-C bioaccessibility range: 2–18%) observed in this study.

The ScMo‐350 site, Haumi, Moʻorea (Society Islands): Preliminary analysis of coastal occupation spanning the colonization phase to classic phase

ABSTRACTIn the Society Islands, multi‐phase coastal sites with successive occupations spanning several centuries have been difficult to find and have been poorly studied, resulting in a vague understanding of this archipelago's founding cultural complex and its adaptations to specific island environments through time. Discovery and excavation of ScMo‐350, a well‐stratified coastal site in Haumi, Moʻorea, Society Islands, provides robust sample sizes for understanding the timing and function of early Māʻohi coastal settlements and their constellation of use through time. My discussion of the site focuses on four topics: methodological issues for identifying early colonization sites in Central Eastern Polynesia; site stratigraphy and dating; the timing of island colonization on Moʻorea and its comparison to the Faʻahia site on Huahine; and preliminary analysis of ScMo‐350 sub‐surface features and artifacts to understand site function and its change through time. Bayesian analysis of short‐lived samples from ScMo‐350's earliest deposits and those from the Faʻahia site are described in an effort to refine our understanding of the Society Islands’ Colonization Phase.

Spontaneous Riboflavin-Overproducing Limosilactobacillus reuteri for Biofortification of Fermented Foods.

Riboflavin-producing lactic acid bacteria represent a promising and cost-effective strategy for food biofortification, but production levels are typically insufficient to support daily human requirements. In this study, we describe the novel human isolate Limosilactobacillus reuteri AMBV339 as a strong food biofortification candidate. This strain shows a high natural riboflavin (vitamin B2) overproduction of 18.36 μg/ml, biomass production up to 6 × 1010 colony-forming units/ml (in the typical range of model lactobacilli), and pH-lowering capacities to a pH as low as 4.03 in common plant-based (coconut, soy, and oat) and cow milk beverages when cultured up to 72 h at 37°C. These properties were especially pronounced in coconut beverage and butter milk fermentations, and were sustained in co-culture with the model starter Streptococcus thermophilus. Furthermore, L. reuteri AMBV339 grown in laboratory media or in a coconut beverage survived in gastric juice and in a simulated gastrointestinal dialysis model with colon phase (GIDM-colon system) inoculated with fecal material from a healthy volunteer. Passive transport of L. reuteri AMBV339-produced riboflavin occurred in the small intestinal and colon stage of the GIDM system, and active transport via intestinal epithelial Caco-2 monolayers was also demonstrated. L. reuteri AMBV339 did not cause fecal microbiome perturbations in the GIDM-colon system and inhibited enteric bacterial pathogens in vitro. Taken together, our data suggests that L. reuteri AMBV339 represents a promising candidate to provide riboflavin fortification of plant-based and dairy foods, and has a high application potential in the human gastrointestinal tract.

Forest ecosystem development in European nemoreal-boreal forest (NE Poland) over the last 2200 years: Impact of human activity and climate change

Long-term ecological studies can provide useful information on forest ecosystem resilience against past climatic change and human caused disturbances. Here, we present a high-resolution 2200-year-long record of forest development in north-eastern Poland, Suwalki region, using paleobotanical proxies (pollen, plant macrofossils, and charcoal). We show that the pollen abundance of deciduous trees was higher than that of coniferous trees, indicating a near pristine state until 900 AD and a semi-natural forest state until 1500 AD. After 1500 AD, the proportion of coniferous tree taxa surpassed that of deciduous trees and have since remained the dominant forest component. The 17th century experienced massive deforestation coupled with a new phase of human colonization in the area that led to the continued and significant decline of deciduous tree cover, for example, Carpinus, Quercus, and Tilia. Cooling associated with the Little Ice Age may have played a role in Picea’s expansion in this area after 1450 AD. Despite significant climatic shifts associated with the warmer Roman Period or Medieval Climate Anomaly and colder Migration Period, as well as a more sustained human impact, Quercus remained a stable forest component until 1500 AD. The stability of Quercus is an important aspect for forest management strategies as future projections suggest warmer conditions and increased frequency of climate extremes will impact forest composition and structure. Our long-term data suggest that forests in the Suwałki region should contain more abundant deciduous tree species, that is, Quercus, whereas conifer cover should be reduced. We also show clear regional differences in the forest development in the Suwałki region, highlighting the importance of local hydrology, geomorphology, and degrees of human activity on the forest composition.

Liver macrophage subsets differentially regulate metastasis in pancreatic cancer

Abstract Metastasis is the main cause of morbidity and mortality in patients with cancer. In pancreatic ductal adenocarcinoma (PDA), the liver is the most common site of metastasis. During PDA progression, the liver microenvironment becomes altered to support metastatic seeding and colonization by disseminated tumor cells (DTC). Macrophages (MΦ), including liver-resident Clec4f+ Kupffer cells (KCs) and bone marrow derived MΦ (BMDMs), are the dominant immunological components of this niche, yet their precise roles in regulating metastasis remain poorly defined. Here, we sought to understand the interaction of DTCs with liver MΦ as they seed and colonize the liver. To model liver metastasis, mice received intraportal (iPo) injection of murine PDA cells with analysis 2 or 14 days later to examine metastatic seeding and outgrowth, respectively. Liver metastatic burden and immune cell populations were quantified by flow cytometry and immunohistochemistry. Liver MΦ were depleted using clodronate encapsulated liposomes (CEL) and KCs were depleted using Clec4fDTR mice administered diphtheria toxin. DTCs were found to interact with BMDMs more often than KCs during the seeding phase of metastasis. During colonization, KCs became excluded from metastatic colonies whereas BMDMs actively infiltrated. Although depletion of liver MΦ and KCs specifically did not affect metastatic seeding, MΦ depletion during the colonization phase inhibited metastatic colony outgrowth. Interestingly, BMDM infiltration into lesions was largely unaffected by CEL treatment, whereas KCs were uniformly depleted. This finding suggests that MΦ residing outside of metastatic lesions may be critical to supporting metastatic outgrowth. Supported by grants from NIH (T32 HL0439, F30 CA257287, R01 CA245323)

Availability and Metabolic Fate of Olive Phenolic Alcohols Hydroxytyrosol and Tyrosol in the Human GI Tract Simulated by the In Vitro GIDM-Colon Model.

Hydroxytyrosol (HTyr) and tyrosol (Tyr) are the most well studied phenolic alcohols of olive oil and olive products demonstrating numerous and significant beneficial health effects. However, their activity in the human organism as food bioactives is strongly associated with their bioavailability and metabolism, while manifested through their metabolites. Nevertheless, there are limited studies investigating their biotransformation and mainly catabolism by gut microflora under a holistic interpretation close to the human organism. Thus, in the present study, the GastroIntestinal Dialysis (GIDM)-colon model, a continuous flow in vitro dialysis system mimicking physiological conditions during human gastrointestinal digestion, was used to explore the metabolism of HTyr and Tyr as pure compounds. The GIDM–colon model simulates absorption from the lumen to the mucosa, followed by the colon phase using pooled human fecal suspensions. Samples were collected at different time points and analyzed via LC–Orbitrap MS. An integrated approach combining Multivariate Data Analysis (MVA) and thorough dereplication procedures led to the identification of HTyr and Tyr metabolites in different phases (gastric, small intestine, and colon), yielding also valuable information about metabolites kinetics. To our knowledge, this is the first study reporting full spectrometric data of HTyr and Tyr metabolites along with possible transformation mechanisms in the GI tract.

Transient Presence of Live Leptospira interrogans in Murine Testes.

ABSTRACTAnalysis of Leptospira dissemination and colonization of sex organs in rodents is of significant value as it queries the possibility of mammal-to-mammal venereal transmission. The aim of our study was to evaluate the presence and viability of Leptospira interrogans in testes of mice using models of infection that we previously developed. Using sublethal and lethal doses of bioluminescent strains of L. interrogans serovars Manilae and Copenhageni, we visualized the presence of leptospires in testes of C57BL/6 mice as early as 30 min and up to days 3–4 postinfection. This was confirmed by qPCR for the Copenhageni serovar after lethal infection of C3H/HeJ mice. In this model, no histopathological changes were noticed in testis. We further studied persistence of serovar Copenhageni in C3H/HeJ testes after lethal and sublethal infection, with different doses of leptospires. No viable leptospires were recovered from testes of lethally infected mice. However, we found live culturable Leptospira in testes of 19/19 (100%) sublethally infected mice at the acute phase but not at 15 days postinfection, which corresponds to the chronic phase of renal colonization. The data suggest that colonization of testes with live and potentially infectious leptospires is transient and limited to the spirochetemic phase of infection. Further studies are necessary to evaluate if presence of Leptospira in testes of mice leads to excretion in semen and to venereal transmission to female mice.IMPORTANCE Analysis of venereal transmission of Leptospira is important to determine if direct animal to animal transmission occurs, which could impact measures to prevent and treat leptospirosis. The goal of this study was to determine if live Leptospira colonize mouse testes. We found that colonization of mouse testes with live Leptospira was transient and limited to the acute spirochetemic phase of infection and that transient colonization of the testes was insufficient to cause histopathological changes.

Understanding photosynthetic biofilm productivity and structure through 2D simulation.

We present a spatial model describing the growth of a photosynthetic microalgae biofilm. In this 2D-model we consider photosynthesis, cell carbon accumulation, extracellular matrix excretion, and mortality. The rate of each of these mechanisms is given by kinetic laws regulated by light, nitrate, oxygen and inorganic carbon. The model is based on mixture theory and the behaviour of each component is defined on one hand by mass conservation, which takes into account biological features of the system, and on the other hand by conservation of momentum, which expresses the physical properties of the components. The model simulates the biofilm structural dynamics following an initial colonization phase. It shows that a 75 μm thick active region drives the biofilm development. We then determine the optimal harvesting period and biofilm height which maximize productivity. Finally, different harvesting patterns are tested and their effect on biofilm structure are discussed. The optimal strategy differs whether the objective is to recover the total biofilm or just the algal biomass.

Overexpression of the Potato Monosaccharide Transporter StSWEET7a Promotes Root Colonization by Symbiotic and Pathogenic Fungi by Increasing Root Sink Strength.

Root colonization by filamentous fungi modifies sugar partitioning in plants by increasing the sink strength. As a result, a transcriptional reprogramming of sugar transporters takes place. Here we have further advanced in the characterization of the potato SWEET sugar transporters and their regulation in response to the colonization by symbiotic and pathogenic fungi. We previously showed that root colonization by the AM fungus Rhizophagus irregularis induces a major transcriptional reprogramming of the 35 potato SWEETs, with 12 genes induced and 10 repressed. In contrast, here we show that during the early colonization phase, the necrotrophic fungus Fusarium solani only induces one SWEET transporter, StSWEET7a, while represses most of the others (25). StSWEET7a was also induced during root colonization by the hemi-biotrophic fungus Fusarium oxysporum f. sp. tuberosi. StSWEET7a which belongs to the clade II of SWEET transporters localized to the plasma membrane and transports glucose, fructose and mannose. Overexpression of StSWEET7a in potato roots increased the strength of this sink as evidenced by an increase in the expression of the cell wall-bound invertase. Concomitantly, plants expressing StSWEET7a were faster colonized by R. irregularis and by F. oxysporum f. sp. tuberosi. The increase in sink strength induced by ectopic expression of StSWEET7a in roots could be abolished by shoot excision which reverted also the increased colonization levels by the symbiotic fungus. Altogether, these results suggest that AM fungi and Fusarium spp. might induce StSWEET7a to increase the sink strength and thus this gene might represent a common susceptibility target for root colonizing fungi.

Structural and functional development of twelve newly established floodplain pond mesocosms.

Ecosystems are complex structures with interacting abiotic and biotic processes evolving with ongoing succession. However, limited knowledge exists on the very initial phase of ecosystem development and colonization. Here, we report results of a comprehensive ecosystem development monitoring for twelve floodplain pond mesocosms (FPM; 23.5 m × 7.5 m × 1.5 m each) located in south‐western Germany. In total, 20 abiotic and biotic parameters, including structural and functional variables, were monitored for 21 months after establishment of the FPMs. The results showed evolving ecosystem development and primary succession in all FPMs, with fluctuating abiotic conditions over time. Principal component analyses and redundancy analyses revealed season and succession time (i.e., time since ecosystem establishment) to be significant drivers of changes in environmental conditions. Initial colonization of both aquatic (i.e., water bodies) and terrestrial (i.e., riparian land areas) parts of the pond ecosystems occurred within the first month, with subsequent season‐specific increases in richness and abundance for aquatic and terrestrial taxa over the entire study period. Abiotic environmental conditions and aquatic and terrestrial communities showed increasing interpond variations over time, that is, increasing heterogeneity among the FPMs due to natural environmental divergence. However, both functional variables assessed (i.e., aquatic and terrestrial litter decomposition) showed opposite patterns as litter decomposition rates slightly decreased over time and interpond differences converged with successional ecosystem developments. Overall, our results provide rare insights into the abiotic and biotic conditions and processes during the initial stages of freshwater ecosystem formation, as well as into structural and functional developments of the aquatic and terrestrial environment of newly established pond ecosystems.

The complex response of continental silicate rock weathering to the colonization of the continents by vascular plants in the Devonian

The first forests appeared on the continents during the Givetian stage of the Devonian. The fossil record shows that, by the end of the Devonian, vascular plants and forests were common and widespread in the wet lowlands. Although the impact of this major event on chemical weathering of the continents is reasonably known, the coeval change in physical erosion has never been explored. Here, we build a mathematical description of the coupled response of the physical erosion and chemical weathering on the continents, to the colonization by vascular plants over the course of the Devonian. This spatially-resolved erosion model is coupled to the GEOCLIM model to simulate the response of the global carbon and alkalinity cycles, and of climate, to the colonization phase. A set of simulations is described, assuming an increased weatherability of the continental surface, and a change in physical erosion which can be either a decrease or an increase in response to the spreading of vascular plants. We explore first the initial pre-colonization and the final post-colonization steady states of the surficial Earth system. Then, we simulate the transient states of the Earth system in response to theoretical randomized scenarios for the colonization. We find that the pathways of the colonization have a major impact on the CO₂ history through the Devonian. Depending on the magnitude of the change in physical erodibility and chemical weatherability, and on the colonization scenario, atmospheric CO₂ evolution may display contrasting behaviors: from a uniform CO₂ decrease over the Devonian, to more complex patterns characterized first by a global warming from the end of the Givetian into the Frasnian, and then by a final cooling, in first order agreement with the proxy data for CO₂ and reconstructed climate evolution.

Arsenic in soils contaminated by arsenic-containing chemical weapons in a site of Jilin, China: fraction and bioaccessibility.

At the end of World War II, the Japanese abandoned arsenic (As)-containing chemical weapons (CWs) in China. During the long-term burial process, the As-containing agents leaked into the environment due to the corrosion of weapon shells. This study explored the surface distribution, fraction composition, and bioaccessibility of As in the soil contaminated by chemical weapons in a site of Jilin Province, China. Results showed that As was enriched in the soil of CWs buried and the maximum concentration of As in this area was 110mg/kg (dry weight). In terms of fraction, As primarily accumulated in amorphous Fe/Al-oxides bound and residual fractions. Moreover, from the perspective of fractions with potential environmental risks, As accounted for 45.6-82.0% and 61.0-80.7% of the fractions extracted by Wenzel and Shiowatana sequential extraction procedure (SEP), respectively. Bioaccessibility can also be used to assess environmental risks. The mean values of As bioaccessibility were as follows: gastric phase (15.0%) > colon phase (14.8%) > small intestinal phase (13.3%), and the As bioaccessibility was closely related to the Fe/Al oxide bound fraction. Compared with the surrounding farmland, the potential environmental risk of soil pollution was more significant in the CW burial areas. This study provided support for remediation of As-containing agent-contaminated soil in China.

Mycotoxin Interactions along the Gastrointestinal Tract: In Vitro Semi-Dynamic Digestion and Static Colonic Fermentation of a Contaminated Meal.

Aflatoxin B1 (AFB1) and ochratoxin A (OTA) naturally co-occur in several foods, but no studies have followed the fate of mycotoxins’ interactions along the gastrointestinal tract using in vitro digestion models. This study used a novel semi-dynamic model that mimics gradual acidification and gastric emptying, coupled with a static colonic fermentation phase, in order to monitor mycotoxins’ bioaccessibility by the oral route. AFB1 and OTA bioaccessibility patterns differed in single or co-exposed scenarios. When co-exposed (MIX meal), AFB1 bioaccessibility at the intestinal level increased by ~16%, while OTA bioaccessibility decreased by ~20%. Additionally, a significant increase was observed in both intestinal cell viability and NO production. With regard to mycotoxin–probiotic interactions, the MIX meal showed a null effect on Lactobacillus and Bifidobacterium strain growth, while isolated AFB1 reduced bacterial growth parameters. These results were confirmed at phylum and family levels using a gut microbiota approach. After colonic fermentation, the fecal supernatant did not trigger the NF-kB activation pathway, indicating reduced toxicity of mycotoxins. In conclusion, if single exposed, AFB1 will have a significant impact on intestinal viability and probiotic growth, while OTA will mostly trigger NO production; in a co-exposure situation, both intestinal viability and inflammation will be affected, but the impact on probiotic growth will be neglected.

Metabolomic changes in polyunsaturated fatty acids and eicosanoids as diagnostic biomarkers in Mycobacterium avium ssp. paratuberculosis (MAP)-inoculated Holstein–Friesian heifers

Mycobacterium avium subspecies paratuberculosis (MAP) is the causative organism of Johne’s disease, a chronic granulomatous enteritis of ruminants. We have previously used naturally MAP-infected heifer calves to document metabolomic changes occurring in MAP infections. Herein, we used experimentally MAP-inoculated heifer calves to identify biomarkers for MAP infections. At 2-weeks of age, 20 Holstein–Friesian (HF) calves were experimentally inoculated with MAP. These calves, along with 20 control calves, were sampled biweekly up to 13-months of age and then monthly up to 19-months of age. Sera were assessed using flow infusion electrospray high-resolution mass spectrometry (FIE-HRMS) on a Q Exactive hybrid quadrupole-Orbitrap mass spectrometer for high throughput, sensitive, non-targeted metabolite fingerprinting. Partial least squares-discriminate analysis (PLS-DA) and hierarchical cluster analysis (HCA) discriminated between MAP-inoculated and control heifer calves. Out of 34 identified metabolites, six fatty acyls were able to differentiate between experimental groups throughout the study, including 8, 11, 14-eicosatrienoic acid and cis-8, 11, 14, 17-eicosatetraenoic acid which were also detected in our previous study and so further suggested their value as biomarkers for MAP infection. Pathway analysis highlighted the role of the alpha-linoleic acid and linoleic acid metabolism. Within these pathways, two broad types of response, with a rapid increase in some saturated fatty acids and some n-3 polyunsaturated fatty acids (PUFAs) and later n-6 PUFAs, became predominant. This could indicate an initial anti-inflammatory colonisation phase, followed by an inflammatory phase. This study demonstrates the validity of the metabolomic approach in studying MAP infections. Nevertheless, further work is required to define further key events, particularly at a cell-specific level.

Cultivating Pleurotus ostreatus and Pleurotus eryngii mushroom strains on agro-industrial residues in solid-state fermentation. Part I: Screening for growth, endoglucanase, laccase and biomass production in the colonization phase

The influence of ten agro-residues, i.e., wheat straw (WS), poplar wood sawdust (PWS), grape pomace (GP), beech wood shavings (BWS), cotton cake (CK), corn cobs (CC), coffee residue (CR), olive pulp (OP), barley and oats straw (BOS) and rice bark (RB), used as alternative substrates for the cultivation of Pleurotus mushrooms, was examined during mycelial growth (colonization phase). Several native and commercial strains of P. ostreatus and P. eryngii were subjected to screening regarding their mycelial growth rates, biomass production and endoglucanase and laccase activities. The experimental results showed that the highest growth rates of P. ostreatus strains were noticed on BWS, BOS, CC and RB substrates, with the time of complete colonization varying between 16 and 36 days, whereas for P. eryngii a high linear growth rate was observed on CC, OP, CR and BOS with the colonization period ranging between 26 and 51 days. The maximum biomass production obtained on the various substrates for both Pleurotus species ranged from 115.32 to 454.42 mg/g d.w. for P. ostreatus and from 108.50 to 422.59 mg/g d.w. for P. eryngii. As for endoglucanase, the highest activities were observed in P. eryngii AMRL 163 cultures on PWS and BOS, i.e., 0.19 and 0.18 U/g d.w., respectively. P. eryngii AMRL 173–6 and P. ostreatus AMRL 150 cultivated on RB and BOS, synthesized significant laccase amounts, i.e., 2172.28 and 1987.25 U/g d.w., respectively. The effect of the substrate components on the growth parameters was considered and discussed. This study showed the industrial potential to convert important low-value agro-residues to fungal biomass and enzymes and eventually to valuable food products.