Proteomic and metabolomic analysis of the carotenogenic yeast Xanthophyllomyces dendrorhous using different carbon sources

Background

Astaxanthin is a potent antioxidant with increasing biotechnological interest. In Xanthophyllomyces dendrorhous, a natural source of this pigment, carotenogenesis is a complex process regulated through several mechanisms, including the carbon source. X. dendrorhous produces more astaxanthin when grown on a non-fermentable carbon source, while decreased astaxanthin production is observed in the presence of high glucose concentrations. In the present study, we used a comparative proteomic and metabolomic analysis to characterize the yeast response when cultured in minimal medium supplemented with glucose (fermentable) or succinate (non-fermentable).

Results

A total of 329 proteins were identified from the proteomic profiles, and most of these proteins were associated with carotenogenesis, lipid and carbohydrate metabolism, and redox and stress responses. The metabolite profiles revealed 92 metabolites primarily associated with glycolysis, the tricarboxylic acid cycle, amino acids, organic acids, sugars and phosphates. We determined the abundance of proteins and metabolites of the central pathways of yeast metabolism and examined the influence of these molecules on carotenogenesis.Similar to previous proteomic-stress response studies, we observed modulation of abundance from several redox, stress response, carbohydrate and lipid enzymes. Additionally, the accumulation of trehalose, absence of key ROS response enzymes, an increased abundance of the metabolites of the pentose phosphate pathway and tricarboxylic acid cycle suggested an association between the accumulation of astaxanthin and oxidative stress in the yeast. Moreover, we observed the increased abundance of late carotenogenesis enzymes during astaxanthin accumulation under succinate growth conditions.

Conclusions

The use of succinate as a carbon source in X. dendrorhous cultures increases the availability of acetyl-CoA for the astaxanthin production compared with glucose, likely reflecting the positive regulation of metabolic enzymes of the tricarboxylic acid and glyoxylate cycles. The high metabolite level generated in this pathway could increase the cellular respiration rate, producing reactive oxygen species, which induces carotenogenesis.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1484-6) contains supplementary material, which is available to authorized users.     

Intradiscal application of rhBMP-7 does not induce regeneration in a canine model of spontaneous intervertebral disc degeneration

IntroductionStrategies for biological repair and regeneration of the intervertebral disc (IVD) by cell and tissue engineering are promising, but few have made it into a clinical setting. Recombinant human bone morphogenetic protein 7 (rhBMP-7) has been shown to stimulate matrix production by IVD cells in vitro and in vivo in animal models of induced IVD degeneration. The aim of this study was to determine the most effective dose of an intradiscal injection of rhBMP-7 in a spontaneous canine IVD degeneration model for translation into clinical application for patients with low back pain.MethodsCanine nucleus pulposus cells (NPCs) were cultured with rhBMP-7 to assess the anabolic effect of rhBMP-7 in vitro, and samples were evaluated for glycosaminoglycan (GAG) and DNA content, histology, and matrix-related gene expression. Three different dosages of rhBMP-7 (2.5 μg, 25 μg, and 250 μg) were injected in vivo into early degenerated IVDs of canines, which were followed up for six months by magnetic resonance imaging (T2-weighted images, T1rho and T2 maps). Post-mortem, the effects of rhBMP-7 were determined by radiography, computed tomography, and macroscopy, and by histological, biochemical (GAG, DNA, and collagen), and biomolecular analyses of IVD tissue.ResultsIn vitro, rhBMP-7 stimulated matrix production of canine NPCs as GAG deposition was enhanced, DNA content was maintained, and gene expression levels of ACAN and COL2A1 were significantly upregulated. Despite the wide dose range of rhBMP-7 (2.5 to 250 μg) administered in vivo, no regenerative effects were observed at the IVD level. Instead, extensive extradiscal bone formation was noticed after intradiscal injection of 25 μg and 250 μg of rhBMP-7.ConclusionsAn intradiscal bolus injection of 2.5 μg, 25 μg, and 250 μg rhBMP-7 showed no regenerative effects in a spontaneous canine IVD degeneration model. In contrast, intradiscal injection of 250 μg rhBMP-7, and to a lesser extent 25 μg rhBMP-7, resulted in extensive extradiscal bone formation, indicating that a bolus injection of rhBMP-7 alone cannot be used for treatment of IVD degeneration in human or canine patients.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-015-0625-2) contains supplementary material, which is available to authorized users.     

Antagonist-mediated down-regulation of toll-like receptors increases the prevalence of human papillomavirus infection in systemic lupus erythematosus

IntroductionPrevalence of an abnormal Papanicolaou smear was significantly increased in lupus patients in cross-sectional studies, associated with a higher prevalence of high-risk human papillomavirus (HPV) infection. The nucleic acid-specific Toll-like receptors (TLRs) locate at the endolysosomal compartments and trigger the induction of cytokines for the innate immune response. This study evaluated whether abnormal host innate immune response in lupus patients may enhance HPV persistence.MethodsProtein levels of TLRs 3, 7, 8 and 9 in cervical epithelial cells of lupus patients and controls with or without HPV infection were assessed using flow cytometry. Characteristics associated with the differential expression of TLRs in systemic lupus erythematosus (SLE) were elucidated. The effect and interferon-stimulated genes (ISGs) (ISG15 and Mx-1) gene expressions were then measured in oncogenic HeLa (HPV18), CaSki (HPV) and C33A (HPV negative) cell lines using flow cytometry and quantitative real-time PCR. Ex vivo productions of cytokines and interferon-gamma (IFN-γ) upon TLR ligands stimulations were subsequently measured using cytometric bead array and ELISA.ResultsFor subjects with HPV infection, levels of TLR3 and TLR7 were significantly lower in lupus patients compared with controls. Significantly decreased TLRs 7, 8 and 9 levels were observed in HPV-negative SLE compared to healthy controls. For SLE with and without HPV infection, TLR7 and 9 levels were significantly lower in infected SLE than those in HPV-negative patients. Independent explanatory variables associated with down-regulation of TLR7 level included HPV infection and a higher cumulative dose of prednisolone; while a higher cumulative dose of hydroxychloroquine and HPV infection were associated with down-regulation of TLR9 level. In cervical cell lines, TLRs 3, 7, 8, 9 protein levels and antiviral ISG15 and Mx-1 gene expressions were inhibited in two oncogenic HPV types. Functional data showed that the induction of pro-inflammatory cytokines by TLR ligands (R837, ssRNA and ODN2395) was greatly impaired in CaSki and HeLa than C33A cells.ConclusionsIn conclusion, prednisolone and TLR antagonist (hydroxychloroquine) may down-regulate protein levels of TLR7 and TLR9 in lupus patients, thereby decreasing the innate immune response against HPV infection. Upon infection, HPV further down-regulate TLR7 and 9 levels for viral persistence. Furthermore, reduction of nucleic acid-sensing TLRs 7, 8 and 9 in carcinogenic HPVs ensures that the expression of inducible pro-inflammatory cytokines is minimized to prevent the expression of antiviral ISGs (ISG15 and Mx-1) on a biologically relevant antiviral response.     

Structure of a murine norovirus NS6 protease-product complex revealed by adventitious crystallisation

Murine noroviruses have emerged as a valuable tool for investigating the molecular basis of infection and pathogenesis of the closely related human noroviruses, which are the major cause of non-bacterial gastroenteritis. The replication of noroviruses relies on the proteolytic processing of a large polyprotein precursor into six non-structural proteins (NS1-2, NS3, NS4, NS5, NS6(pro), NS7(pol)) by the virally-encoded NS6 protease. We report here the crystal structure of MNV NS6(pro), which has been determined to a resolution of 1.6 ?. Adventitiously, the crystal contacts are mediated in part by the binding of the C-terminus of NS6(pro) within the peptide-binding cleft of a neighbouring molecule. This insertion occurs for both molecules in the asymmetric unit of the crystal in a manner that is consistent with physiologically-relevant binding, thereby providing two independent views of a protease-peptide complex. Since the NS6(pro) C-terminus is formed in vivo by NS6(pro) processing, these crystal contacts replicate the protease-product complex that is formed immediately following cleavage of the peptide bond at the NS6-NS7 junction. The observed mode of binding of the C-terminal product peptide yields new insights into the structural basis of NS6(pro) specificity.     

LCA of a tomato crop in a multi-tunnel greenhouse in Almeria

Purpose

Protected crops have expanded significantly in the Mediterranean area over the last few decades as a successful means to provide abundant and high-quality produce. Although resources are generally used efficiently, greenhouse areas cause major environmental impacts. The aim of this work was to study, from an environmental point of view, the improvement capacity of greenhouse areas in the Mediterranean region and to assess several alternative agricultural practices to decrease their contribution to the environmental impacts in this system.

Materials and methods

The methodology used was life cycle assessment (LCA) based on a tomato crop grown in a multi-tunnel greenhouse in Almeria, on the southeast coast of Spain. The functional unit chosen was 1 ton of loose classic tomatoes. Five midpoint impact categories and one energy flow indicator were selected for their relevance. The agricultural practice alternatives evaluated were reduction of volume of substrate and fertilizers, extension of substrate and greenhouse life span and increase in renewable energy for electricity production.

Results and discussion

The results indicated that the main contributors to impact categories in the tomato production were structure, auxiliary equipment and fertilizers. Structure accounted for between 30 and 48?% of the contributions, depending on the impact category. The principal burdens in the auxiliary equipment stage were substrate and consumption of electricity. Fertilizers environmental impacts were due to emissions during their manufacture and application to the crop. In a best-case option, taking into account the best alternatives, contributions to the impact categories were reduced by between 17 and 30?%. The LCA methodology proved to be a useful tool to evaluate the environmental damage of this agricultural activity. The importance of including farm infrastructure in the assessment was demonstrated as it was a major contributor. The risk of eutrophication could be reduced by adjustment of the fertilizers?Cwater balance and implementation of a closed-loop irrigation system. Future technological improvements should be developed to increase yields and thereby directly reduce the environmental burdens per unit produce.

Conclusions

The present study served to assess the environmental impacts of a tomato crop in a multi-tunnel greenhouse on the coast of Almeria. The assessment was used to evaluate alternatives for improvement of cleaner production in greenhouse areas. Further research should focus on assuring the feasibility of the suggested options.     

Molecular and morphological phylogeny of hooded shrimps, genera Betaeus and Betaeopsis (Decapoda, Alpheidae): testing the center of origin biogeographic model and evolution of life history traits

The phylogenetic relationships of the alpheid shrimp genera Betaeus (Dana, 1852) (15 species) and Betaeopsis (Yaldwyn, 1971) (three species), collectively known as hooded shrimps, are analyzed with morphological, molecular (16S and H3) and combined "total evidence" (morphology+DNA) datasets. The tree topology resulting from morphological and combined analyses places Betaeus jucundus as sister to all the remaining species of Betaeus and Betaeopsis, rendering Betaeus paraphyletic. On the other hand, Betaeopsis is recovered as monophyletic. Betaeus australis is positioned as sister to the remaining species of Betaeus s. str. (excluding B. jucundus), which is composed of three well-supported and resolved clades. Mapping of biogeographic traits on the combined tree suggests at least two possible historic scenarios. In the first scenario, the North-East Pacific harboring the highest diversity of hooded shrimps (seven species of Betaeus), acted as the "center of origin", where species appeared, matured and eventually migrated toward peripheral regions. In the second scenario, Betaeus+Betaeopsis originated in the southern Indo-West Pacific and subsequently colonized the North-East Pacific, where a major radiation involving dispersal/vicariance events took place. The mapping of life history traits (symbiosis vs. free living and gregariousness vs. single/pair living) in the combined tree suggests (1) that different types of symbioses with dissimilar host organisms (sea urchins, abalones, other decapods, spoon worms) evolved independently more than once in the group (in B. jucundus and in various lineages of Betaeus s. str.), and (2) that gregariousness was ancestral in the Betaeus s. str. -Betaeopsis clade and later shifted toward single/pair living in several lineages.     

AFLP and breeding system studies indicate vicariance origin for scattered populations and enigmatic low fecundity in the Moroccan endemic Hypochaeris angustifolia (Asteraceae), sister taxon to all of the South American Hypochaeris species

We used Amplified Fragment Length Polymorphism markers (AFLP) and breeding system studies to investigate the population structure and reproductive biology of Hypochaeris angustifolia (Asteraceae: Cichorieae). This species is endemic to altiplanos of the Atlas Mountains (Morocco) where it occurs in scattered populations, and it is the sister species to c. 40 species of this genus in South America. PCoA, NJ, and Bayesian clustering, revealed that the populations are very isolated whilst AFLP parameters show that almost all populations have marked genetic divergence. We contend that these features are more in accord with a vicariance origin for the scattered populations of H. angustifolia, rather than establishment by long-distance dispersal. The breeding system studies revealed that H. angustifolia is a self-incompatible species, with low fecundity in natural and in experimental crosses, probably due to a low frequency of compatible phenotypes within and between the populations.     

Polyhydroxyalkanoate synthesis using different carbon sources by two enhanced biological phosphorus removal microbial communities

Polyhydroxyalkanoate (PHA) is a biodegradable plastic synthesised by bacteria as energy and carbon storage material. PHA production is mostly based on pure cultures operated under sterile conditions, which increase the costs of this biopolymer. The use of inexpensive mixed culture biomass, such as activated sludge, to produce biodegradable plastics from renewable waste streams has been proposed as an alternative.The effect of carbon sources (acetate, propionate, butyrate and glucose) on the type and quantity of PHA synthesis obtained with different enhanced biological phosphorus removal (EBPR) microbial communities enriched with acetate and propionate are reported in this work. Two sequencing batch reactors (SBRs) were seeded with biomass withdrawn from a non-EBPR wastewater treatment plant (WWTP). The same operational conditions were kept, but using acetate or propionate as the sole carbon source of each reactor. These conditions produced two microbial communities with different P-removal capacity. The results presented in this study show the effect of the carbon source on the PHA composition (amount of polyhydroxybutyrate (PHB), polyhydroxyvalerate (PHV) and polyhydroxy-2-methylvalerate (PH2MV)), which differed not just between substrates but also between the two EBPR communities used. In addition, some monomers not always analysed contribute significantly to the total amount of PHA, especially when using butyrate, showing that if they are not considered this can lead to erroneous calculated yields.     

Extent and consistency of linkage disequilibrium and identification of DNA markers for production and egg quality traits in commercial layer chicken populations

Background

The genome sequence and a high-density SNP map are now available for the chicken and can be used to identify genetic markers for use in marker-assisted selection (MAS). Effective MAS requires high linkage disequilibrium (LD) between markers and quantitative trait loci (QTL), and sustained marker-QTL LD over generations. This study used data from a 3,000 SNP panel to assess the level and consistency of LD between single nucleotide polymorphisms (SNPs) over consecutive years in two egg-layer chicken lines, and analyzed one line by two methods (SNP-wise association and genome-wise Bayesian analysis) to identify markers associated with egg-quality and egg-production phenotypes.

Results

The LD between markers pairs was high at short distances (r² > 0.2 at < 2 Mb) and remained high after one generation (correlations of 0.80 to 0.92 at < 5 Mb) in both lines. Single- and 3-SNP regression analyses using a mixed model with SNP as fixed effect resulted in 159 and 76 significant tests (P < 0.01), respectively, across 12 traits. A Bayesian analysis called BayesB, that fits all SNPs simultaneously as random effects and uses model averaging procedures, identified 33 SNPs that were included in the model >20% of the time (φ > 0.2) and an additional ten 3-SNP windows that had a sum of φ greater than 0.35. Generally, SNPs included in the Bayesian model also had a small P-value in the 1-SNP analyses.

Conclusion

High LD correlations between markers at short distances across two generations indicate that such markers will retain high LD with linked QTL and be effective for MAS. The different association analysis methods used provided consistent results. Multiple single SNPs and 3-SNP windows were significantly associated with egg-related traits, providing genomic positions of QTL that can be useful for both MAS and to identify causal mutations.