Summary
The endospore‐forming rhizobacterium Bacillus subtilis– the model system for Gram‐positive organisms, is able to produce more than two dozen antibiotics with an amazing variety of structures. ...The produced anti‐microbial active compounds include predominantly peptides that are either ribosomally synthesized and post‐translationally modified (lantibiotics and lantibiotic‐like peptides) or non‐ribosomally generated, as well as a couple of non‐peptidic compounds such as polyketides, an aminosugar, and a phospholipid. Here I summarize the structures of all known B. subtilis antibiotics, their biochemistry and genetic analysis of their biosyntheses. An updated summary of well‐studied antibiotic regulation pathways is given. Furthermore, current findings are resumed that show roles for distinct B. subtilis antibiotics beyond the ‘pure’ anti‐microbial action: Non‐ribosomally produced lipopeptides are involved in biofilm and swarming development, lantibiotics function as pheromones in quorum‐sensing, and a ‘killing factor’ effectuates programmed cell death in sister cells. A discussion of how these antibiotics may contribute to the survival of B. subtilis in its natural environment is given.
The mouse mammary gland is widely used as a model for human breast cancer and has greatly added to our understanding of the molecular mechanisms involved in breast cancer development and progression. ...To fully appreciate the validity and limitations of the mouse model, it is essential to be aware of the similarities and also the differences that exist between the mouse mammary gland and the human breast. This introduction therefore describes the parallels and contrasts in mouse mammary gland and human breast morphogenesis from an early embryonic phase through to puberty, adulthood, pregnancy, parturition, and lactation, and finally the regressive stage of involution.
During pregnancy, the mouse mammary ductal epithelium branches and grows into the surrounding stroma, requiring extensive extracellular matrix (ECM) and tissue remodelling. It therefore shows ...parallels to cancer invasion. We hypothesised that similar molecular mechanisms may be utilised in both processes, and that assessment of the stromal changes during pregnancy-associated branching may depict the stromal involvement during human breast cancer progression.
Immunohistochemistry (IHC) was employed to assess the alterations within the mouse mammary gland extracellular matrix during early pregnancy when lateral branching of the primary ductal epithelium is initiated. Primary mouse mammary fibroblasts from three-day pregnant and age-matched non-pregnant control mice, respectively, were 3D co-cultured with mammary epithelial cells to assess differences in their abilities to induce branching morphogenesis in vitro. Transcriptome analysis was performed to identify the underlying molecular changes. A signature of the human orthologues of the differentially expressed matrisome RNAs was analysed by Kaplan-Meier and multi-variate analysis in two large breast cancer RNA datasets (Gene expression-based Outcome for Breast cancer Online (GOBO) und Kaplan-Meier Plotter), respectively, to test for similarities in expression between early-pregnancy mouse mammary gland development and breast cancer progression.
The ECM surrounding the primary ductal network showed significant differences in collagen and basement membrane protein distribution early during pregnancy. Pregnancy-associated fibroblasts (PAFs) significantly enhanced branching initiation compared to age-matched control fibroblast. A combined signature of 64 differentially expressed RNAs, encoding matrisome proteins, was a strong prognostic indicator of distant metastasis-free survival (DMFS) independent of other clinical parameters. The prognostic power could be significantly strengthened by using only a subset of 18 RNAs (LogRank P ≤ 1.00e-13; Hazard ratio (HR) = 2.42 (1.8-3.26); p = 5.61e-09). The prognostic power was confirmed in a second breast cancer dataset, as well as in datasets from ovarian and lung cancer patients.
Our results describe for the first time the early stromal changes that accompany pregnancy-associated branching morphogenesis in mice, specify the early pregnancy-associated molecular alterations in mouse mammary fibroblasts, and identify a matrisome signature as a strong prognostic indicator of human breast cancer progression, with particular strength in oestrogen receptor (ER)-negative breast cancers.
Fibulin-2 (FBLN2) is a secreted extracellular matrix glycoprotein which has been associated with tissue development and remodelling. In the mouse mammary gland, FBLN2 can be detected during ductal ...morphogenesis in cap cells and myoepithelial cells at puberty and early pregnancy, respectively. In an attempt to assign its function, we knocked down Fbln2 in the mouse mammary epithelial cell line EpH4. FBLN2 reduction led to an increase in the size of spheroidal structures when compared to scrambled control shRNA-transduced cells plated on Matrigel matrix. This phenotype was associated with a disruption of the collagen IV sheath around the epithelial spheroids and downregulation of integrin β1, suggesting a role for FBLN2 in stabilizing the basement membrane (BM). In contrast to mice, in normal adult human breast tissue, FBLN2 was detected in ductal stroma, and in the interlobular stroma, but was not detectable within the lobular regions. In tissue sections of 65 breast cancers FBLN2 staining was lost around malignant cells with retained staining in the neighbouring histologically normal tissue margins. These results are consistent with a role of FBLN2 in mammary epithelial BM stability, and that its down-regulation in breast cancer is associated with loss of the BM and early invasion.
Abstract
Background
Circulating microRNAs (miRNAs) are described as promising non-invasive biomarkers for diagnostics and therapeutics. Human studies have shown that haemolysis occurring during blood ...collection or due to improper sample processing/storage significantly alters the miRNA content in plasma and serum. Nevertheless, no similar research has been performed in dogs so far. We therefore investigated the effects of different degrees of haemolysis on the levels of selected miRNAs in serum and serum-derived extracellular vesicles (EVs) from dogs, by inducing a controlled in vitro haemolysis experiment.
Results
The abundance of miR-16, miR-92a, miR-191, miR-451 and miR-486 was significantly sensitive to haemolysis in serum and serum-derived EVs, while other selected miRNAs were not influenced by haemolysis. Furthermore, we found that the abundance of some canine miRNAs differs from data reported in the human system.
Conclusions
Our results describe for the first time the impact of haemolysis on circulating miRNAs not only in whole serum, but also in serum-derived EVs from dogs. Hence, we provide novel data for further analyses in the discovery of canine circulating biomarkers. Our findings suggest that haemolysis should be carefully assessed to assure accuracy when investigating circulating miRNA in serum or plasma-based tests.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
7.
Mammary Gland Involution as a Multi-step Process Stein, Torsten; Salomonis, Nathan; Gusterson, Barry A
Journal of mammary gland biology and neoplasia,
03/2007, Letnik:
12, Številka:
1
Journal Article
Recenzirano
Mammary gland involution is a highly complex multi-step process in which the lactating gland returns to a morphologically near pre-pregnant state. This developmental stage is characterized by a high ...degree of epithelial cell death, redevelopment of the mammary adipose tissue and tissue remodelling. Many factors involved have been described and these have been reviewed intensively in this journal (Furth, P. A., J. Mammary Gland Biol. Neoplasia, 4:123-127, 1999) and elsewhere. Microarray analysis technology has now not only allowed us to identify genes not previously associated with involution (Stein, T., Morris, J.S., Davis, C.R.,Weber-Hall, S.J., Duffy, M.A., Heath, V.J., et al., Breast Cancer Res., 6: R75-R91, 2004; Clarkson, R.W., Wayland, M.T., Lee, J., Freeman, T., Watson, C.J., Breast Cancer Res., 6: R92-R109, 2004; Clarkson, R.W., Watson, C.J., J. Mammary Gland Biol. Neoplasia, 8: 309-319, 2003), it has also enabled us to define multiple phases of the controlled regulatory response to forced weaning on the basis of their transcriptional profiles. This review provides a synthesis of published data, integrating the time course of transcriptional activity in the mouse mammary gland with a gene ontology approach to identify the pathways involved.
1 Department of Physiology and Biophysics, University of Colorado Health Sciences Center at Fitzsimmons, Aurora, Colorado
2 Department of Obstetrics and Gynecology, University of Colorado Health ...Sciences Center at Fitzsimmons, Aurora, Colorado
3 Department of Anesthesiology, University of Colorado Health Sciences Center at Fitzsimmons, Aurora, Colorado
4 Department of Pharmacology, University of Colorado Health Sciences Center at Fitzsimmons, Aurora, Colorado
5 Department of Pathology, University of Colorado Health Sciences Center at Fitzsimmons, Aurora, Colorado
6 Division of Cancer Sciences and Molecular Pathology, Western Infirmary, Glasgow, United Kingdom
The mammary gland of the lactating mouse synthesizes and secretes milk lipid equivalent to its entire body weight in a single 20-day lactation cycle, making it one of the most active lipid synthetic organs known. We test the hypothesis that multiple control points and potential regulatory mechanisms regulate milk lipid synthesis at the level of gene expression. The mammary transcriptome of 130 genes involved in glucose metabolism was examined at late pregnancy and early lactation, utilizing data obtained from microarray analysis of mammary glands from quadruplicate FVB mice at pregnancy day 17 and lactation day 2 . To correlate changes with physiological parameters, the metabolome obtained from magnetic resonance spectroscopy of flash-frozen glands at day 17 of pregnancy was compared with that at day 2 of lactation. A significant increase in carbohydrates (glucose, lactose, sialic acid) and amino acids (alanine, aspartate, arginine, glutamate) with a moderate increase in important osmolytes ( myo -inositol, betaine, choline derivatives) were observed in the lactating gland. In addition, diets containing 8% or 40% lipid were fed from lactation days 510 and mammary glands and livers of triplicate FVB mice prepared for microarray analysis. The results show that substantial regulation of lipid synthesis occurs at the level of mRNA expression and that some of the regulation points differ substantially from the liver. They also implicate the transcription factor SREBP-1c in regulation of part of the pathway.
lipid synthesis; microarray; metabolomics; dietary lipid; magnetic resonance spectroscopy
The tumour microenvironment comprises a diverse range of cells, including fibroblasts, immune cells and endothelial cells, along with extracellular matrix. In particular, fibroblasts are of ...significant interest as these cells are reprogrammed during tumorigenesis to become cancer‐associated fibroblasts (CAFs), which in turn support cancer cell growth. MicroRNAs (miRNAs) have been shown to be involved in this intercellular crosstalk in humans. To assess whether miRNAs are also involved in the activation of fibroblasts in dogs, we cocultured primary canine skin fibroblasts with the canine mast cell tumour cell line C2 directly or with C2‐derived exosomes, and measured differential abundance of selected miRNAs. Expression of the CAF markers alpha‐smooth muscle actin (ACTA2) and stanniocalcin 1 confirmed the activation of our fibroblasts after coculture. We show that fibroblasts displayed significant downregulation of miR‐27a and let‐7 family members. These changes correlated with significant upregulation of predicted target mRNAs. Furthermore, RNA interference knockdown of miR‐27a revealed that cyclin G1 (CCNG1) exhibited negative correlation at the mRNA and protein level, suggesting that CCNG1 is a target of miR‐27a in canine fibroblasts and involved in their activation. Importantly, miR‐27a knockdown itself resulted in fibroblast activation, as demonstrated by the formation of ACTA2 filaments. In addition, interleukin‐6 (IL‐6) was strongly induced in our fibroblasts when cocultured, indicating potential reciprocal signalling. Taken together, our findings are consistent with canine fibroblasts being reprogrammed into CAFs to further support cancer development and that downregulation of miR‐27a may play an important role in the tumour–microenvironment crosstalk.
Fibroblasts are reprogrammed during tumorigenesis to become cancer‐associated fibroblasts (CAFs), which support cancer cell growth. MicroRNAs are involved in this intercellular crosstalk. We show that after coculture of canine fibroblasts with the tumour cell line C2, miR‐27a is downregulated, while expression of CAF markers ACTA2 and stanniocalcin 1 confirmed the activation of fibroblasts. Additionally, RNA interference knockdown of miR‐27a by itself led to fibroblast activation.
It has been recently shown, that certain strains/isolates of
Bacillus subtilis
can be used as a probiotic for humans. The production of the macrocyclic sactibiotic subtilosin in
B. subtilis
ATCC 6633 ...is highly regulated. To improve the subtilosin productivity of
B. subtilis
, different growth conditions were compared for maximal expression of the
sbo
promoter that regulates the expression of the subtilosin biosynthetic gene cluster. Oxygen-limiting conditions led to a strong increase of
sbo
promoter activities compared to aerobic conditions, and accordingly, the subtilosin amount determined by reversed phase HPLC (7.8 mg/L) was 15-fold superior to the amount of aerobic grown cultures (0.5 mg/L). A further promising enhancement of the subtilosin yield was achieved using a deletion mutant that is avoiding the general transition state regulator protein AbrB. The subtilosin titer of 42 mg/L produced by Δ
abrB
cells grown under oxygen-limiting conditions corresponds to an 84-fold increase compared to the subtilosin titer obtained from
B. subtilis
wild type cells propagated in aerobic conditions. Furthermore, evidence is provided that oxygen-limiting conditions led to a strong decrease in the productivity of the lantipeptide subtilin suggesting contrary regulatory mechanisms for the
B. subtilis
antimicrobials subtilin and subtilosin.
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