Analysis of molecular interactions between yoghurt bacteria by an integrated genomics approach

The lactic acid bacteria (LAB) are a group of Gram-positive bacteria that ferment sugars such as lactose to produce mainly lactic acid. LAB are a group of industrially important microorganisms that are applied for the production of many fermented foods. These include foods produced with substrates from plant origin (e.g. sauerkraut and wine) and animal origin (e.g. fermented meats and dairy products such as yoghurt). The current market trends regarding sustainability and health-promoting foods demand more efficient and a more diverse range of fermentations. Most fermentations are carried out by multiple strains of different species. The interactions between consortium members are at the base of the performances of the individual microorganisms within a microbial ecosystem and therewith of the whole fermentation. These microbial interactions are often poorly understood. Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus are two LAB species that upon fermentation convert (bovine) milk into yoghurt. These two bacteria stimulate each other in growth and acid production. They produce exopolysaccharides (EPS), important for the texture of yoghurt, and characteristic flavor compounds such as acetaldehyde and diacetyl. However, the molecular basis of the mutualistic interactions between these two bacteria was poorly characterized. In this thesis research, a combination was used of screening, mixed culture transcription profiling, whole-genome metabolic modeling, experimental evolution and next-generation sequencing. This was done to unravel the molecular basis of the interactions between S. thermophilus and L. bulgaricus in milk. The results showed that interactions were primarily based on the exchange of metabolites (see Figure 1). Moreover, it was shown which genes or pathways were affected. Evidence was found that S. thermophilus provided L. bulgaricus with formic acid, folic acid (both involved in purine metabolism), long-chain fatty acids (by the action of lipolytic enzymes to break down milk fat) and CO2. The proteolysis by the exoprotease of L. bulgaricus, in turn, provided both species peptides, which are taken up by the cell and broken down into amino acids (AA) by intracellular peptidases. However, this probably did not yield a sufficient supply of branched-chain and sulfur AA, leading to a higher expression of the genes for biosynthesis of these AA in both species when grown in mixed culture. Moreover, EPS biosynthesis genes were induced in the mixed culture, leading to increased EPS production and a higher viscosity of the yoghurt. Figure 1. Schematic representation of the mutualistic interactions between S. thermophilus and L. bulgaricus in yoghurt. Solid arrows indicate interactions; dotted arrows indicate pathways that are affected by the interactions. Pathways that were for the first time shown to be regulated at the transcriptome level upon co-culture are indicated in bold. Pathways that were confirmed in our study to be regulated at the transcriptome level upon co-culture are underlined. EPS is hypothesized to promote the exchange of both bacteria. There was no evidence at the transcriptome level for the exchange of putrescine and ornithine. AA, amino acids; BCAA, branched-chain AA; EPS, exopolysaccharides; LCFA, long-chain fatty acids. A mixed culture genome-scale metabolic model confirmed that cross-feeding interactions between the yoghurt bacteria were based on purine and AA metabolism. Moreover, this model was used to show that the interactions provided a significant benefit to both bacteria, i.e. their biomass yield on lactose increased by around 50% in mixed culture. Experimental evolution revealed that it is possible to co-adapt a novel combination of strains of S. thermophilus and L. bulgaricus. It was shown that their mutual stimulation increased by optimizing their interactions by fine-tuning expression of pathways involved in the interactions. Furthermore, as little as ~1000 generations of co-culture was sufficient to transform the relatively slow growing mixed culture into one that showed similar performance as commercial starters with respect to key characteristics such as acidification rate and viscosity. Improved understanding of the described interactions that are at the base of the yoghurt fermentation provides us targets for the rational optimization of existing mixed culture fermentations and the rational development of industrially relevant mixed cultures, such as those containing probiotics. Moreover, the results are in particular interesting for the field of microbial ecology as they show how mutual nutritional dependencies evolve and structure the microbial composition of this ecosystem. <br/

Prognostic and predictive testing of molecular markers in breast cancer by real-time quantitative PCR

This chapter provides a general overview of breast cancer, including the relevance of measuring gene expression in the primary breast tumor in relation to the progression of the disease and the tumor response to treatment. To better understand the concept of breast cancer, extra emphasis will be put on breast cancer subtypes, staging and grading, and the tumor micro-environment that harbors the epithelial cancer cells. Currently available biomarkers to assess outcome of breast cancer patients in general (i.e. prognosis) as well as biomarkers currently available to assess patient outcome in response to therapy (i.e. prediction) will be discussed. After this it should become clear that there is still an urgent need for new biomarkers. How this search for additional biomarkers can be achieved by measuring mRNAgene expression in the primary tumor of patients diagnosed with breast cancer, will be explained in more detail

Diagnostic applications of cell-free and circulating tumor cell-associated miRNAs in cancer patients

Summary: Circulating tumor cells (CTCs) have rapidly developed as important cancer biomarkers after their enumeration proved to be prognostic in metastatic breast, colorectal and prostate cancer, and their rise or decline after the first cycle of therapy showed to predict therapy response. Besides mere counting, CTCs can be isolated and subsequently analyzed using various molecular applications, including miRNA expression analysis. Recently, miRNA expression profiling in primary tumors has yielded promising results. However, establishing miRNA expression in the circulation likely has advantages over determination in primary tumor tissue, further augmenting the potential applications of miRNA determination in oncology. Additionally to CTC-associated miRNAs, free circulating miRNAs have been identified in whole blood, plasma and serum. Since determination of miRNAs in peripheral blood, either cell-free or CTC-associated, is expected to become important in oncology, especially when linked to and interpreted together with epithelial CTCs, this review focuses on measuring miRNAs in the circulation of cancer patients

Overexpression of Colligin 2 in Glioma Vasculature is Associated with Overexpression of Heat Shock Factor 2

In previous studies we found expression of the protein colligin 2 (heat shock protein 47 (HSP47), SERPINH1) in glioma neovasculature while not in normal brain tissue. Generally, the regulation of heat shock gene expression in eukaryotes is mediated by heat shock factors (HSF). In mammals, three heat shock transcription factors, HSF-1, -2, and -4, have been isolated. Here we investigated the relation between the expression of colligin 2 and these heat shock factors at the mRNA level using real-time reverse transcriptase PCR (qRT-PCR) in different grades of astrocytic tumorigenesis, viz., low-grade glioma and glioblastoma. Endometrium samples, representing physiological angiogenesis, were included as controls. Since colligin 2 is a chaperon for collagens, the gene expression of collagen I (COL1A1) was also investigated. The blood vessel density of the samples was monitored by expression of the endothelial marker CD31 (PECAM1). Because NG2-immunopositive pericytic cells are involved in glioma neovascularization, the expression of NG2 (CSPG4) was also measured

Pleiotropic actions of suramin on the proliferation of human breast-cancer cells in vitro

Suramin, a non‐specific growth factor antagonist, is currently under investigation for treatment of cancer patients. We studied its action on 6 different human breast‐cancer cell lines in vitro. In complete growth medium, pleiotropic effects were observed with respect to cell proliferation, i.e. suramin is stimulatory at low concentrations and inhibitory at higher concentrations, for 4 of the 6 cell lines studied. The various cell lines showed marked differences with respect to the antiproliferative action of suramin, the Evsa‐T cells being by far the most sensitive ones. A suramin concentration of 100 μg/ml brought about a 100% stimulation of the proliferation of ZR/HERc cells, ZR 75.1 cells ectopically expressing a human epidermal growth factor receptor (EGF‐R) cDNA. Although less pronounced (10 to 60% stimulation), a similar response was observed for the parent ZR 75.1 cells, as well as for T‐47D and MDA‐MB‐231 cells. The non‐specificity of the action of suramin was established by the observation that suramin‐induced inhibition of cell proliferation could be abolished by insulin‐like growth factor‐1 (IGF‐I) or basic fibroblast growth factor (bFGF), and even by estradiol, both in complete growth medium and under defined serum‐free conditions. Our data indicate that suramin exerts pleiotropic effects on the proliferation of human breast cancer cells in vitro, and confirm the non‐specific nature of its action. The stimulatory effect of low concentrations of suramin on the proliferation of breast cancer cells may have important consequences for breast cancer patients treated with suramin. Copyrigh

Clinical significance of the nuclear receptor co-regulator DC-SCRIPT in breast cancer: an independent retrospective validation study

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High TWIST1 mRNA expression is associated with poor prognosis in lymph node-negative and estrogen receptor-positive human breast cancer and is co-expressed with stromal as well as ECM related genes

Introduction: The TWIST homolog 1 (TWIST1) is a transcription factor that induces epithelial to mesenchymal transition (EMT), a key process in metastasis. The purpose of this study was to investigate whether TWIST1 expression predicts disease progression in a large breast cancer cohort with long-term clinical follow-up, and to reveal the biology related to TWIST1 mediated disease progression.Methods: TWIST1 mRNA expression level was analyzed by quantitative real-time reverse polymerase chain reaction (RT-PCR) in 1,427 primary breast cancers. In uni- and multivariate analysis using Cox regression, TWIST1 mRNA expression level was associated with metastasis-free survival (MFS), disease-free survival (DFS) and overall survival (OS). Separate analyses in lymph node-negative patients (LNN, n = 778) who did not receive adjuvant systemic therapy, before and after stratification into estrogen receptor (ER)-positive (n = 552) and ER-negative (n = 226) disease, were also performed. The association of TWIST1 mRNA with survival endpoints was assessed using Kaplan-Meier analysis. Using gene expression arrays, genes showing a significant Spearman rank correlation with TWIST1 were used to identify overrepresented Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG)-annotated biological pathways.Results: Increased mRNA expression level of TWIST1 analyzed as a continuous variable in both uni- and multivariate analysis was associated with shorter MFS in all patients (hazard ratio (HR): 1.17, 95% confidence interval, (95% CI):1.09 to 1.26; and HR: 1.17, 95% CI: 1.08 to 1.26; respectively), in LNN patients (HR: 1.22, 95% CI: 1.09 to 1.36; and HR: 1.21, 95% CI: 1.07 to 1.36; respectively) and in the ER-positive subgroup of LNN patients (HR: 1.34, 95% CI: 1.17 to 1.53; and HR: 1.32, 95% CI: 1.14 to 1.53; respectively). Similarly, high TWIST1 expression was associated with shorter DFS and OS in all patients and in the LNN/ER-positive subgroup. In contrast, no association of TWIST1 mRNA expression with MFS, DFS or OS was observed in ER-negative patients. Genes h

BCAR4 induces antioestrogen resistance but sensitises breast cancer to lapatinib

BACKGROUND: High BCAR4 and ERBB2 mRNA levels in primary breast cancer associate with tamoxifen resistance and poor patient outcome. We determined whether BCAR4 expression sensitises breast cancer cells to lapatinib, and identifies a subgroup of patients who possibly may benefit from ERBB2-targeted therapies despite having tumours with low ERBB2 expression. METHODS: Proliferation assays were applied to determine the effect of BCAR4 expression on lapatinib treatment. Changes in cell signalling were quantified with reverse-phase protein microarrays. Quantitative reverse-transcriptase polymerase chain reaction (RT-PCR) of ERBB2 and BCAR4 was performed in 1418 primary breast cancers. Combined BCAR4 and ERBB2 mRNA levels were evaluated for association with progression-free survival (PFS) in 293 oestrogen receptor-alpha (ER)-positive patients receiving t RESULTS: BCAR4 expression strongly sensitised ZR-75-1 and MCF7 breast cancer cells to the combination of lapatinib and antioestrogens. Lapatinib interfered with phosphorylation of ERBB2 and its downstream mediators AKT, FAK, SHC, STAT5, and STAT6. Reverse transcriptase-PCR analysis showed that 27.6% of the breast cancers were positive for BCAR4 and 22% expressed also low levels of ERBB2. The clinical significance of combining BCAR4 and ERBB2 mRNA status was underscored by the finding that the gr CONCLUSION: This study shows that BCAR4 expression identifies a subgroup of ER-positive breast cancer patients without overexpression of ERBB2 who have a poor outcome and might benefit from combined ERBB2-targeted and antioestrogen therapy. British Journal of Cancer (2012) 107, 947-955. doi:10.1038/bjc.2012.351 www.bjcancer.com Published online 14 August 2012 (C) 2012 Cancer Research U