Identification of genes required for glucan exopolysaccharide production in Lactobacillus johnsonii suggests a novel mechanism of biosynthesis

Lactobacillus johnsonii FI9785 makes two capsular exopolysaccharides-a heteropolysaccharide (EPS2) encoded by the eps operon and a branched glucan homopolysaccharide (EPS1). The homopolysaccharide is synthesized in the absence of sucrose, and there are no typical glucansucrase genes in the genome. Quantitative proteomics was used to compare the wild type to a mutant where EPS production was reduced to attempt to identify proteins associated with EPS1 biosynthesis. A putative bactoprenol glycosyltransferase, FI9785_242 (242), was less abundant in the Δ eps_cluster mutant strain than in the wild type. Nuclear magnetic resonance (NMR) analysis of isolated EPS showed that deletion of the FI9785_242 gene ( 242) prevented the accumulation of EPS1, without affecting EPS2 synthesis, while plasmid complementation restored EPS1 production. The deletion of 242 also produced a slow-growth phenotype, which could be rescued by complementation. 242 shows amino acid homology to bactoprenol glycosyltransferase GtrB, involved in O-antigen glycosylation, while in silico analysis of the neighboring gene 241 suggested that it encodes a putative flippase with homology to the GtrA superfamily. Deletion of 241 also prevented production of EPS1 and again caused a slow-growth phenotype, while plasmid complementation reinstated EPS1 synthesis. Both genes are highly conserved in L. johnsonii strains isolated from different environments. These results suggest that there may be a novel mechanism for homopolysaccharide synthesis in the Gram-positive L. johnsonii IMPORTANCE Exopolysaccharides are key components of the surfaces of their bacterial producers, contributing to protection, microbial and host interactions, and even virulence. They also have significant applications in industry, and understanding their biosynthetic mechanisms may allow improved production of novel and valuable polymers. Four categories of bacterial exopolysaccharide biosynthesis have been described in detail, but novel enzymes and glycosylation mechanisms are still being described. Our findings that a putative bactoprenol glycosyltransferase and flippase are essential to homopolysaccharide biosynthesis in Lactobacillus johnsonii FI9785 indicate that there may be an alternative mechanism of glucan biosynthesis to the glucansucrase pathway. Disturbance of this synthesis leads to a slow-growth phenotype. Further elucidation of this biosynthesis may give insight into exopolysaccharide production and its impact on the bacterial cell

The ?Dark Side? of Food Stuff Proteomics: The CPLL-Marshals Investigate

The present review deals with analysis of the proteome of animal and plant-derived food stuff, as well as of non-alcoholic and alcoholic beverages. The survey is limited to those system investigated with the help of combinatorial peptide ligand libraries, a most powerful technique allowing access to low- to very-low-abundance proteins, i.e. to those proteins that might characterize univocally a given biological system and, in the case of commercial food preparations, attest their genuineness or adulteration. Among animal foods the analysis of cow’s and donkey’s milk is reported, together with the proteomic composition of egg white and yolk, as well as of honey, considered as a hybrid between floral and animal origin. In terms of plant and fruits, a survey is offered of spinach, artichoke, banana, avocado, mango and lemon proteomics, considered as recalcitrant tissues in that small amounts of proteins are dispersed into a large body of plant polymers and metabolites. As examples of non-alcoholic beverages, ginger ale, coconut milk, a kola drink, almond milk and orgeat syrup are analyzed. Finally, the trace proteome of white and red wines, beer and aperitifs is reported, with the aim of tracing the industrial manipulations and herbal usage prior to their commercialization. The review ends with a comparison between mammalian and plant proteomics highlighting the difficulties besieging analysis of any vegetable proteome

Protein network analyses of pulmonary endothelial cells in chronic thromboembolic pulmonary hypertension

Chronic thromboembolic pulmonary hypertension (CTEPH) is a vascular disease characterized by the presence of organized thromboembolic material in pulmonary arteries leading to increased vascular resistance, heart failure and death. Dysfunction of endothelial cells is involved in CTEPH. The present study describes for the first time the molecular processes underlying endothelial dysfunction in the development of the CTEPH. The advanced analytical approach and the protein network analyses of patient derived CTEPH endothelial cells allowed the quantitation of 3258 proteins. The 673 differentially regulated proteins were associated with functional and disease protein network modules. The protein network analyses resulted in the characterization of dysregulated pathways associated with endothelial dysfunction, such as mitochondrial dysfunction, oxidative phosphorylation, sirtuin signaling, inflammatory response, oxidative stress and fatty acid metabolism related pathways. In addition, the quantification of advanced oxidation protein products, total protein carbonyl content, and intracellular reactive oxygen species resulted increased attesting the dysregulation of oxidative stress response. In conclusion this is the first quantitative study to highlight the involvement of endothelial dysfunction in CTEPH using patient samples and by network medicine approach

Anthocyanins promote learning through modulation of synaptic plasticity related proteins in an animal model of ageing

Anthocyanin-rich foods, such as berries, reportedly ameliorate age-related cognitive deficits in both animals and humans. Despite this, investigation into the mechanisms which underpin anthocyanin-mediated learning and memory benefits remains relatively limited. The present study investigates the effects of anthocyanin intake on a spatial working memory paradigm, assessed via the cross-maze apparatus, and relates behavioural test performance to underlying molecular mechanisms. Six-week supplementation with pure anthocyanins (2% w/w), administered throughout the learning phase of the task, improved both spatial and psychomotor performances in aged rats. Behavioural outputs were accompanied by changes in the expression profile of key proteins integral to synaptic function/maintenance, with upregulation of dystrophin, protein kinase B (PKB/Akt) and tyrosine hydroxylase, and downregulation of apoptotic proteins B-cell lymphoma-extra-large (Bcl-xL) and the phosphorylated rapidly accelerated fibrosarcoma (p-Raf). Separate immunoblot analysis supported these observations, indicating increased activation of extracellular signal-related kinase (ERK1), Akt Ser473, mammalian target of rapamycin (mTOR) Ser2448, activity-regulated cytoskeleton-associated protein (Arc/Arg 3.1) and brain-derived neurotrophic factor (BDNF) in response to anthocyanin treatment, whilst �-E-catenin, c-Jun N-terminal kinase (JNK1) and p38 protein levels decreased. Together, these findings suggest that purified anthocyanin consumption enhances spatial learning and motor coordination in aged animals and can be attributed to the modulation of key synaptic proteins, which support integrity and maintenance of synaptic function

A combined biomarker panel shows improved sensitivity for the early detection of ovarian cancer allowing the identification of the most aggressive Type II tumours.

Background: There is an urgent need for biomarkers for the early detection of ovarian cancer (OC). The purpose of this study was to assess whether changes in serum levels of lecithin-cholesterol acyltransferase (LCAT), sex hormone-binding globulin (SHBG), glucoseregulated protein, 78 kDa (GRP78), calprotectin and insulin-like growth factor-binding protein 2 (IGFBP2) are observed before clinical presentation and to assess the performance of these markers alone and in combination with CA125 for early detection. Methods: This nested case–control study used samples from the United Kingdom Collaborative Trial of Ovarian Cancer Screening trial. The sample set consisted of 482 serum samples from 49 OC subjects and 31 controls, with serial samples spanning up to 7 years pre-diagnosis. The set was divided into the following: (I) a discovery set, which included all women with only two samples from each woman, the first ato14 months and the second at 432 months to diagnosis; and (ii) a corroboration set, which included all the serial samples from the same women spanning the 7-year period. Lecithin-cholesterol acyltransferase, SHBG, GRP78, calprotectin and IGFBP2 were measured using ELISA. The performance of the markers to detect cancers pre-diagnosis was assessed. Results: A combined threshold model IGFBP2 478.5 ng ml 1 : LCAT o8.831 mg ml 1 : CA125 435 Uml 1 outperformed CA125 alone for the earlier detection of OC. The threshold model was able to identify the most aggressive Type II cancers. In addition, it increased the lead time by 5–6 months and identified 26% of Type I subjects and 13% of Type II subjects that were not identified by CA125 alone. Conclusions: Combined biomarker panels (IGFBP2, LCAT and CA125) outperformed CA125 up to 3 years pre-diagnosis, identifying cancers missed by CA125, providing increased diagnostic lead times for Type I and Type II OC. The model identified more aggressive Type II cancers, with women crossing the threshold dying earlier, indicating that these markers can improve on the sensitivity of CA125 alone for the early detection of OC

Harry Belafonte and the secret proteome of coconut milk

The proteome of coconut milk has been extensively mapped via capture at three pH values with combinatorial peptide ligand libraries (CPLL). A grand total of 307 unique gene products could be listed, 200 discovered via CPLL capture, 137 detected in the control, untreated material and 30 species in common between the two sets of data. This is by far the most extensive mapping of coconut milk, in which, up to the present, only a dozen proteins were known, those belonging to the high- to very-high abundance class. The database of coconut contains only 106 proteins: of those, only six are listed in our table. The vast majority of the classified proteins, thus, has been identified only by homologies with sequences deposited in the general viridiplantae database. This unique set of data could be the starting point for nutritionists and researchers involved in nutraceutics for enucleating some proteins responsible for some of the unique beneficial health effects attributed to coconut milk

In taberna quando sumus: a drunkard's cakewalk through wine proteomics

A review. Anal. of white and red wine trace proteomes via capture with combinatorial peptide ligand libraries (CPLL) is reported here. Most of the alc. beverages tested (all of Italian origin) were found to contain only traces of casein (on av. from 20 to 60 μg/L, with a detectability of as low as 1 μg/L) and not any grape protein any longer, as they had been fined with bovine casein (surprisingly also red wines for which the typical fining agent is egg albumin). However, anal. of untreated white wine (Recite, from Garganega grapes in the Veneto region) via CPLL capture indeed permitted to detect close to 100 unique gene products from the grapes, suggesting the possibility of proteotyping grand crus, i.e. those aged, high quality wines that should not be treated with fining agents. Thus the CPLL technique could become a formidable tool for traceability of beverages in particular and of foodstuff in general. For trace protein anal., a new, most powerful CPLL methodol. emerges: capture at pH=2.2 in 0.1 % trifluoroacetic acid (TFA) under the conditions mimicking reversed-phase mechanisms of adsorption

Horam nonam exclamavit: sitio. The trace proteome of your daily vinegar

The trace proteome of white-wine vinegar has been identified via capture with home-made combinatorial peptide ligand libraries under conditions mimicking reverse-phase capture, i.e. at pH 2.2 in presence of 0.1% trifluoroacetic acid. A total of 27 unique gene products have been identified, of which 10 specific of the database Vitis vinifera, 13 found in the general database Uniprot_viridiplantae and 4 in Swiss Prot_all entries. The most abundant species detected, on the basis of spectral counts, appears to be the whole genome shotgun sequence of line PN40024, scaffold_22 (a protein of the glycosyl hydrolase family). Curiously, up to the present, no information had been available on vinegar proteome

Assessment of the floral origin of honey via proteomic tools

The honey from chestnut, acacia, sunflower, eucalyptus and orange was analysed for its proteome content, in order to see if any plant proteins present would allow the proteo-typing of these different varieties. Since the total protein content turned out to be minute, 200. g of each honey type were diluted to 1. L and then added with ProteoMiner to enhance the visibility of the proteinaceous material. All bands visible in the SDS-PAGE profile of each type of honey were eluted, digested and identified by mass spectrometry in a LTQ-XL instrument. It turned out that all proteins identified (except one, the enzyme glyceraldehyde-3-phosphate dehydrogenase from Mesembryanthemum crystallinum) were not of plant origin but belonged to the Apis mellifera proteome. Among the total proteins identified (eight, but only seven as basic constituents of all types of honey) five belonged to the family of major royal jelly proteins 1-5, and were also the most abundant ones in any type of honey, together with \uce\ub1-glucosidase and defensin-1. It thus appears that honey has a proteome resembling the royal jelly proteome (but with considerably fewer species), except that its protein concentration is lower by three to four orders of magnitude as compared to royal jelly. Attempts at identifying additional plant (pollen, nectar) proteins via peptidome analysis were unsuccessful

In taberna quando sumus: A Drunkard’s Cakewalk Through Wine Proteomics

Analysis of white and red wine trace proteomes via capture with combinatorial peptide ligand libraries (CPLL) is reported here. Most of the alcoholic beverages tested (all of Italian origin) were found to contain only traces of casein (on average from 20 to 60 µg/L, with a detectability of as low as 1 µg/L) and not any grape protein any longer, as they had been fined with bovine casein (surprisingly also red wines for which the typical fining agent is egg albumin). However, analysis of untreated white wine (Recioto, from Garganega grapes in the Veneto region) via CPLL capture indeed permitted to detect close to 100 unique gene products from the grapes, suggesting the possibility of proteotyping grand crus, i.e. those aged, high quality wines that should not be treated with fining agents. Thus the CPLL technique could become a formidable tool for traceability of beverages in particular and of foodstuff in general. For trace protein analysis, a new, most powerful CPLL methodology emerges: capture at pH=2.2 in 0.1 % trifluoroacetic acid (TFA) under the conditions mimicking reversed-phase mechanisms of adsorption