Pangenome reconstruction of Lactobacillaceae metabolism predicts species-specific metabolic traits.

Strains across the Lactobacillaceae family form the basis for a trillion-dollar industry. Our understanding of the genomic basis for their key traits is fragmented, however, including the metabolism that is foundational to their industrial uses. Pangenome analysis of publicly available Lactobacillaceae genomes allowed us to generate genome-scale metabolic network reconstructions for 26 species of industrial importance. Their manual curation led to more than 75,000 gene-protein-reaction associations that were deployed to generate 2,446 genome-scale metabolic models. Cross-referencing genomes and known metabolic traits allowed for manual metabolic network curation and validation of the metabolic models. As a result, we provide the first pangenomic basis for metabolism in the Lactobacillaceae family and a collection of predictive computational metabolic models that enable a variety of practical uses.IMPORTANCELactobacillaceae, a bacterial family foundational to a trillion-dollar industry, is increasingly relevant to biosustainability initiatives. Our study, leveraging approximately 2,400 genome sequences, provides a pangenomic analysis of Lactobacillaceae metabolism, creating over 2,400 curated and validated genome-scale models (GEMs). These GEMs successfully predict (i) unique, species-specific metabolic reactions; (ii) niche-enriched reactions that increase organism fitness; (iii) essential media components, offering insights into the global amino acid essentiality of Lactobacillaceae; and (iv) fermentation capabilities across the family, shedding light on the metabolic basis of Lactobacillaceae-based commercial products. This quantitative understanding of Lactobacillaceae metabolic properties and their genomic basis will have profound implications for the food industry and biosustainability, offering new insights and tools for strain selection and manipulation.

A liquid chromatography-tandem mass spectrometry-based investigation of the lamellar interstitial metabolome in healthy horses and during experimental laminitis induction.

Lamellar bioenergetic failure is thought to contribute to laminitis pathogenesis but current knowledge of lamellar bioenergetic physiology is limited. Metabolomic analysis (MA) can systematically profile multiple metabolites. Applied to lamellar microdialysis samples (dialysate), lamellar bioenergetic changes during laminitis (the laminitis metabolome) can be characterised. The objectives of this study were to develop a technique for targeted MA of lamellar and skin dialysates in normal horses, and to compare the lamellar and plasma metabolomic profiles of normal horses with those from horses developing experimentally induced laminitis. Archived lamellar and skin dialysates (n = 7) and tissues (n = 6) from normal horses, and lamellar dialysate and plasma from horses given either 10 g/kg oligofructose (treatment group, OFT; n = 4) or sham (control group, CON; n = 4) were analysed. The concentrations of 44 intermediates of central carbon metabolism (CCM) were determined using liquid chromatography-tandem mass spectrometry. Data were analysed using multivariate (MVA) and univariate (UVA) analysis methods. The plasma metabolome appeared to be more variable than the lamellar metabolome by MVA, driven by malate, pyruvate, aconitate and glycolate. In lamellar dialysate, these metabolites decreased in OFT horses at the later time points. Plasma malate was markedly increased after 6 h in OFT horses. Plasma malate concentrations between OFT and CON at this time point were significantly different by UVA. MA of lamellar CCM was capable of differentiating horses developing experimental laminitis from controls. Lamellar malate, pyruvate, aconitate and glycolate, and plasma malate alone were identified as the source of differentiation between OFT and CON groups. These results highlighted clear discriminators between OFT and CON horses, suggesting that changes in energy metabolism occur locally in the lamellar tissue during laminitis development. The biological significance of these alterations requires further investigation.

Quorum-sensing linked RNA interference for dynamic metabolic pathway control in Saccharomyces cerevisiae.

Some of the most productive metabolic engineering strategies involve genetic modifications that cause severe metabolic burden on the host cell. Growth-limiting genetic modifications can be more effective if they are 'switched on' after a population growth phase has been completed. To address this problem we have engineered dynamic regulation using a previously developed synthetic quorum sensing circuit in Saccharomyces cerevisiae. The circuit autonomously triggers gene expression at a high population density, and was linked with an RNA interference module to enable target gene silencing. As a demonstration the circuit was used to control flux through the shikimate pathway for the production of para-hydroxybenzoic acid (PHBA). Dynamic RNA repression allowed gene knock-downs which were identified by elementary flux mode analysis as highly productive but with low biomass formation to be implemented after a population growth phase, resulting in the highest published PHBA titer in yeast (1.1mM).

The prevalence and impact of Fusarium head blight pathogens and mycotoxins on malting barley quality in UK.

Fusarium head blight (FHB) caused by Fusarium and Microdochium species can significantly affect the yield of barley grain as well as the quality and safety of malt and beer. The present study provides new knowledge on the impacts of the FHB pathogen complex on the malting and brewing quality parameters of naturally infected barley. Quantitative real-time PCR and liquid chromatography double mass spectrometry were used to quantify the predominant FHB pathogens and Fusarium mycotoxins, respectively, in commercially grown UK malting barley samples collected between 2007 and 2011. The predominant Fusarium species identified across the years were F. poae, F. tricinctum and F. avenaceum. Microdochium majus was the predominant Microdochium species in 2007, 2008, 2010 and 2011 whilst Microdochium nivale predominated in 2009. Deoxynivalenol and zearalenone quantified in samples collected between 2007 and 2009 were associated with F. graminearum and F. culmorum, whilst HT-2 and T-2, and nivalenol in samples collected between 2010 and 2011 correlated positively with F. langsethiae and F. poae, respectively. Analysis of the regional distribution and yearly variation in samples from 2010 to 2011 showed significant differences in the composition of the FHB species complex. In most regions (Scotland, the South and North of England) the harvest in 2010 had higher concentrations of Fusarium spp. than in 2011, although no significant difference was observed in the Midlands between the two years. Microdochium DNA was significantly higher in 2011 and in the North of England and Scotland compared to the South or Midlands regions. Pathogens of the FHB complex impacted negatively on grain yield and quality parameters. Thousand grain weight of malting barley was affected significantly by M. nivale and M. majus whilst specific weight correlated negatively with F. avenaceum and F. graminearum. To determine the impact of sub-acute infections of the identified Fusarium and Microdochium species on malting and brewing quality of naturally infected samples, selected malting barley cultivars (Optic, Quench and Tipple) were micromalted and subjected to malt and wort analysis of key quality parameters. F. poae and M. nivale decreased germinative energy and increased water sensitivity of barley. The fungal biomass of F. poae and F. langsethiae correlated with increased wort free amino nitrogen and with decreased extract of malt. DNA of M. nivale correlated with increased malt friability as well as decreased wort filtration volume. The findings of this study indicate that the impact of species such as the newly emerging F. langsethiae, as well as F. poae and the two non-toxigenic Microdochium species should be considered when evaluating the quality of malting barley.

TRI12 based quantitative real-time PCR assays reveal the distribution of trichothecene genotypes of F. graminearum and F. culmorum isolates in Danish small grain cereals.

Quantitative real-time PCR assays, based on polymorphisms in the TRI12 gene of the trichothecene pathway, were developed to identify and quantify the trichothecene genotypes producing 3-acetyl-deoxynivalenol (3ADON), 15-acetyl-deoxynivalenol (15ADON) or nivalenol (NIV) in the Fusarium graminearum species complex, Fusarium culmorum, Fusarium cerealis and Fusarium pseudograminearum. These assays were applied on a total of 378 field samples of cereal grain of wheat, barley, triticale, rye and oats collected from 2003 to 2007 to study the trichothecene genotype composition in Danish cereals. The three genotypes, 3ADON, 15ADON and NIV were found in all five cereal species, great annual variation in the occurrence of the trichothecene genotypes was evident with considerable variation between the samples. 3ADON was the dominant genotype in barley, triticale, rye and oats while 15ADON was most dominant in wheat. The NIV genotype was found at low levels in most samples. Study of genotype composition within the Danish F. graminearum and F. culmorum population was based on principal component analysis (PCA). PCA revealed that the dominating genotype of F. graminearum in wheat is 15ADON. For barley, the PCA analysis indicated that the F. graminearum population consisted of all three genotypes, and in triticale, the F. graminearum population consisted mainly of 15ADON genotype. F. culmorum/F. cerealis showed correlation to the NIV genotype in wheat and triticale but not in barley. F. culmorum/F. cerealis also showed some correlation to 3ADON especially in wheat and triticale. Selected wheat and barley samples from 1957 to 2000 showed low amounts of F. graminearum and F. culmorum in general but with a dominance of the 3ADON genotype. 15ADON was not detected in these samples, except for very low amounts in the sample representing the years from 1997 to 2000. Detection of low amounts of the 15ADON genotype in these historical samples and the relatively high amounts of 15ADON genotype in 2003 and following years correspond well with the occurrence of F. graminearum and indicates that the 15ADON genotype was introduced along with F. graminearum around 2000. The amounts of the 3ADON and 15ADON genotypes correlated well with the total amount of DON whereas the amounts of NIV genotype correlated well with the amount of NIV in wheat and triticale but not in barley where the results indicate that Fusarium poae may also contribute to the NIV content.

Manufactured RBC--rivers of blood, or an oasis in the desert?

Red blood cell (RBC) transfusion is an essential practice in modern medicine, one that is entirely dependent on the availability of donor blood. Constraints in donor supply have led to proposals that transfusible RBC could be manufactured from stem cells. While it is possible to generate small amounts of RBC in vitro, very large numbers of cells are required to be of clinical significance. We explore the challenges facing large scale manufacture of RBC and technological developments required for such a scenario to be realised.

Fusarium head blight of cereals in Denmark: species complex and related mycotoxins.

Quantitative real-time polymerase chain reaction differentiating 10 Fusarium spp. and Microdochium nivale or M. majus was applied to a total of 396 grain samples of wheat, barley, triticale, oat, and rye sampled across Denmark from 2003 to 2007, along with selected samples of wheat and barley from 1957 to 2000, to determine incidence and abundance of individual Fusarium spp. The mycotoxins deoxynivalenol (DON), nivalenol, zearalenone, T-2, and HT-2 were quantified using liquid chromatography-double mass spectrometry. Major differences in the Fusarium species complex among the five cereals as well as great yearly variation were seen. Fusarium graminearum, F. culmorum, and F. avenaceum were dominant in wheat, with DON as the dominant mycotoxin. F. langsethiae, F. culmorum, and F. avenaceum were dominant in barley and oat, leading to relatively high levels of the mycotoxins T-2 and HT-2. F. graminearum, F. culmorum, and F. avenaceum dominated in triticale and rye. The nontoxigenic M. nivale/majus were present in significant amounts in all cereal species. Wheat and barley samples from 1957 to 1996 exhibited no or very low amounts of F. graminearum, indicating a recent increase of this pathogen. Biomass and mycotoxin data exhibited good correlations between Fusarium spp. and their corresponding mycotoxins under field conditions.

Production of bacteriocins by Streptococcus bovis strains from Australian ruminants.

AIMS: To examine the prevalence of bacteriocin production in Streptococcus bovis isolates from Australian ruminants and the feasibility of industrial production of bacteriocin. METHODS AND RESULTS: Streptococcus bovis strains were tested for production of bacteriocin-like inhibitory substances (BLIS) by antagonism assay against Lactococcus lactis. BLIS production was associated with source animal location (i.e. proximity of other bacteriocin-positive source animals) rather than ruminant species/breed or diet. One bacteriocin showing strong inhibitory activity (Sb15) was isolated and examined. Protein sequence, stability and activity spectrum of this bovicin were very similar to bovicin HC5. Production could be increased through serial culturing, and increased productivity could be partially maintained during cold storage of cultures. CONCLUSIONS: BLIS production is geographically widely distributed in Eastern Australia, and it appears that the bacteriocin(+) trait is maintained in animals at the same location. The HC5-like bacteriocin, originally identified in North America, is also found in Australia. Production of bacteriocin can be increased through serial culturing. SIGNIFICANCE AND IMPACT OF THE STUDY: The HC5-like bacteriocins appear to have a broad global distribution. Serial culturing may provide a route towards commercial manufacturing for use in industrial applications, and purified bacteriocin from S. bovis Sb15 could potentially be used to prevent food spoilage or as a feed additive to promote growth in ruminant species.

Class switch recombination in selective IgA-deficient subjects.

Selective IgA deficiency is a common immunodeficiency in Caucasians, but the molecular basis of the disorder remains elusive. To address this issue we examined the molecular events leading to IgA production. Naive IgD positive B cells were purified from four donors with IgA deficiency and four control donors, all Caucasians. Stimulation of B cells from IgA-deficient donors with the cytokines transforming growth factor (TGF)-beta, interferon (IFN)-gamma or interleukin (IL)-10 in the presence of anti-CD40 antibodies showed reduced expression of both activation-induced cytidine deaminase (AID) and alpha germline transcripts (GLT) compared to controls. It was possible, however, to induce AID and alpha GLT when stimulating the cells with anti-CD40 antibody and TGF-beta in the combination with IL-10. Moreover, in anti-CD40 antibody-stimulated cultures, addition of IL-10 or IL-10 + TGF-beta in combination, induced IgA production, albeit lower than found in B cells from controls. The B cells from the IgA-deficient subjects were less effective in differentiating into CD138(+) X-box binding protein 1 (XBP-1)(+) plasma cells when stimulated with TGF-beta, IFN-gamma or IL-10. Interestingly, when adding IL-4 to TGF-beta alone or in combination with IL-10, the immunoglobulin production in B cells from IgA-deficient donors was comparable with those of normal controls. These data show that in healthy subjects in vitro IgA production can be up-regulated by addition of IL-10 to CD40-stimulated B cells, whereas a similar B cell differentiation does not occur in IgA-deficient subjects. Addition of IL-4, however, reverts this abnormality.

Method for the generation and cultivation of functional three-dimensional mammary constructs without exogenous extracellular matrix.

During puberty, pregnancy, lactation and post-lactation, breast tissue undergoes extensive remodelling and the disruption of these events can lead to cancer. In vitro studies of mammary tissue and its malignant transformation regularly employ mammary epithelial cells cultivated on matrigel or floating collagen rafts. In these cultures, mammary epithelial cells assemble into three-dimensional structures resembling in vivo acini. We present a novel technique for generating functional mammary constructs without the use of matrix substitutes.

Hemin reconstitutes proton extrusion in an H(+)-ATPase-negative mutant of Lactococcus lactis.

H(+)-ATPase is considered essential for growth of Lactococcus lactis. However, media containing hemin restored the aerobic growth of an H(+)-ATPase-negative mutant, suggesting that hemin complements proton extrusion. We show that inverted membrane vesicles prepared from hemin-grown L. lactis cells are capable of coupling NADH oxidation to proton translocation.

Osmolarity effects on observed insect cell size after baculovirus infection are avoided using growth medium for sample dilution.

Rates of cell size increase are an important measure of success during the baculovirus infection process. Batch and fed batch cultures sustain large fluctuations in osmolarity that can affect the measured cell volume if this parameter is not considered during the sizing protocol. Where osmolarity differences between the sizing diluent and the culture broth exist, biased measurements of size are obtained as a result of the cell osmometer response. Spodoptera frugiperda (Sf9) cells are highly sensitive to volume change when subjected to a change in osmolarity. Use of the modified protocol with culture supernatants for sample dilution prior to sizing removed the observed error during measurement.

Stoichiometric modeling of Clostridium acetobutylicum fermentations with non-linear constraints.

A stoichiometric model of Clostridium acetobutylicum and related strains has been previously derived. The stoichiometric matrix of the model contains a singularity which has prevented the calculation of a unique set of fluxes which describe the primary metabolic activity. To resolve the singularity, we have developed a non-linear constraint relating the acetate and butyrate uptake fluxes. Subsequently, we developed a software package utilizing a model independent heuristic global optimization approach to solve the resultant non-linear problem. We have validated the use of the non-linear constraint by correlating calculated butyrate production pathway flux profiles with measured intracellular pH profiles. Finally, we examined a controlled batch fermentation to determine that the acid formation pathways play critical roles throughout solventogenesis. The broader usefulness of reformulating the stoichiometric model as a constrained minimization problem is discussed.

Lag-burst kinetics in phospholipase A(2) hydrolysis of DPPC bilayers visualized by atomic force microscopy.

The lag-burst phenomenon in the phospholipase A(2) mediated hydrolysis of phospholipid bilayers is for the first time demonstrated in an atomic force microscopy (AFM) study. Simultaneous AFM measurements of the degree of bilayer degradation and the physical-chemical state of the membrane reveals growing nanoscale indentations in the membrane during the lag phase. It is argued that these indentations are domains of hydrolysis products (lysoPC/PC) which eventually trigger the burst. The rate of the rapid hydrolysis following the burst is found to be proportional to the length of the edge between membrane adsorbed and desorbed to the mica base. The observed maximal rate of membrane degradation is approx. 0.2 mmol lipid/min/mol lipase in solution.

Bioreactors for hematopoietic cell culture.

Hematopoietic cell culture, or ex vivo expansion of hematopoietic cells, is an enabling technology with many potential applications in bone-marrow transplantation, immunotherapy, gene therapy, and the production of blood products. Hematopoietic cultures are complex, with many different cell types of different stages of development present at any given point in time and never in steady state. Moreover, these cells interact strongly with each other and the environment through cytokines (growth factors) and adhesion molecules, as well as through their metabolism. Despite these significant challenges, cell products produced in bioreactors have shown promise in recent phase 1 clinical trials.

Characterization of hematopoietic cell expansion, oxygen uptake, and glycolysis in a controlled, stirred-tank bioreactor system.

Cultures of umbilical cord blood and mobilized peripheral blood mononuclear cells were carried out in a stirred bioreactor with pH and dissolved oxygen control. Expansion of total cells and colony-forming units granulocyte-macrophage was greatly enhanced by the use of a cell-dilution feeding protocol (as compared to a cell-retention feeding protocol). The specific oxygen consumption rate (qO2) for these cultures ranged from 1.7 x 10(-8) to 1.2 x 10(-7) micromol/(cell.h). The maximum in qO2 for each culture closely corresponded with the maximum percentage of progenitor or colony-forming cells (CFCs) present in the culture. The maximum qO2 values are slightly less than those reported for hybridomas, while the lowest qO2 values are somewhat greater than those reported for mature granulocytes. Examination of the ratio of lactate production to oxygen consumption in these cultures suggests that post-progenitor cells of the granulomonocytic lineage obtain a greater portion of their energy from glycolysis than do CFCs. The different metabolic profiles of CFCs and more mature cells suggest that monitoring the uptake or production of oxygen, lactate, and other metabolites will allow estimation of the content of several cell types in culture.

Effect of percutaneous ethanol injection therapy versus suppressive doses of L-thyroxine on benign solitary solid cold thyroid nodules: a randomized trial.

The results of studies using suppressive doses of L-T4 on benign solitary solid cold thyroid nodules have been conflicting. Recently, intranodular injection of absolute ethanol has been proposed as an effective treatment, but has been evaluated only in uncontrolled studies. Our objective was to evaluate the effect of two alternative medical treatment modalities, percutaneous ethanol injection therapy and L-T4, on the benign solitary solid cold thyroid nodule. In a prospective randomized clinical trial, 50 euthyroid patients with a single solid colloid thyroid nodule causing local discomfort were assigned to a single intranodular injection of sterile 98% ethanol (n = 25) or suppressive doses of L-T4 (n = 25). We aimed at an ethanol dose of 20-50% of the pretreatment nodular volume. The initial daily dose of L-T4 was 1.5 microg/kg BW and was adjusted monthly during the first 6 months to reduce serum TSH to subnormal levels (<0.40 mU/L). Thyroid nodule volume and total thyroid volume were assessed by ultrasound, and thyroid function was determined by routine assays before and during follow-up. Symptom scores before and at 12 months were evaluated by a questionnaire rating pressure symptoms and cosmetic symptoms. The median ethanol dose given was 21% [95% confidence interval (CI), 18;25] of the pretreatment nodule volume. In this group, the median reduction in nodule volume was 47% (CI, 33;57; P < 0.0001) compared to 9% (CI, -7;22; P = 0.09) in the L-T4 group. The difference between the two treatment regimens was statistically significant (P < 0.0001). The median reduction in perinodular thyroid volume was 20% (CI, 11;31; P = 0.03) in the L-T4 group, whereas no change was seen in the ethanol group (-2.5%; CI, -18;11; P = 0.9). Fourteen of 25 (56%) patients treated with ethanol injection and 8 of 25 (32%) treated with L-T4 had complete relief of symptoms at 12 months of follow-up (P = 0.09). No major side-effects were seen in either group. Percutaneous ethanol injection therapy administered as a single small dose results in a satisfactory clinical response in approximately 50% of patients by halving the nodule volume. The thyroid nodule-reducing effect of L-T4 suppressive therapy is insignificant, but a subjective satisfactory clinical response is seen in a subgroup of patients, probably explained by the concomitant reduction of perinodular thyroid volume.

Population balance model of in vivo neutrophil formation following bone marrow rescue therapy.

In this paper, we develop a simple four parameter population balance model of in vivo neutrophil formation following bone marrow rescue therapy. The model is used to predict the number and type of neutrophil progenitors required to abrogate the period of severe neutropenia that normally follows a bone marrow transplant. The estimated total number of 5 billion neutrophil progenitors is consistent with the value extrapolated from a human trial. The model provides a basis for designing ex vivo expansion protocols.

Real-time method for determining the colony-forming cell content of human hematopoietic cell cultures.

Glucose and lactate metabolic rates were evaluated for cultures of cord blood (CB) mononuclear cell (MNC), peripheral blood (PB) MNC, and PB CD34(+) cell cultures carried out in spinner flasks and in T-flasks in both serum-containing and serum-free media. Specific glucose uptake rates (q(gluc), in micromoles per cell per hour) and lactate generation rates (q(lac)) correlated with the percentage of colony-forming cells (CFC) present in the culture for a broad range of culture conditions. Specifically, the time of maximum CFC percentage in each culture coincided with the time of maximum q(gluc) and q(lac) in cultures with different seeding densities and cytokine combinations. A two-population model (Q(lac) = alpha[CFC] + beta([TC] - [CFC ]), where [TC] is total cell concentration; Q(lac) is volumetric lactate production rate in micromoles per milliliter per hour; alpha is q(lac) for an average CFC; and beta is q(lac) for an average non-CFC) was developed to describe lactate production. The model described lactate production well for cultures carried out in both T-flasks and spinner flasks and inoculated with either PB or CB MNC or PB CD34(+) cells. The values for alpha and beta that were derived from the model varied with both the inoculum density and the cytokine combination. However, preliminary results indicate that cultures carried out under the same conditions from different samples with similar initial CD34(+) cell content have similar values for beta and beta. These findings suggest that it should be possible to use lactate production data to predict the harvest time that corresponds to the maximum number of CFC in culture. The ability to harvest ex vivo hematopoietic cultures for transplantation when CFC are at a maximum has the potential to speed the rate at which immunocompromised patients recover. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 693-700, 1997.