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1.  Transcriptomic clues to understand the growth of Lactobacillus rhamnosus in cheese 
BMC Microbiology  2014;14:28.
Background
Lactobacillus rhamnosus is a non-starter lactic acid bacterium that plays a significant role during cheese ripening, leading to the formation of flavor. In long-ripened cheeses it persists throughout the whole time of ripening due to its capacity to adapt to changing environmental conditions. The versatile adaptability of L. rhamnosus to different ecosystems has been associated with the capacity to use non-conventional energy sources, regulating different metabolic pathways. However, the molecular mechanisms allowing the growth of L. rhamnosus in the cheese dairy environment are still poorly understood. The aim of the present study was to identify genes potentially contributing to the growth ability of L. rhamnosus PR1019 in cheese-like medium (CB) using a transcriptomic approach, based on cDNA-amplified fragment length polymorphism (cDNA-AFLP) and quantitative real-time reverse transcription-PCR (qPCR).
Results
Using three primer combinations, a total of 89 and 98 transcript-derived fragments were obtained for L. rhamnosus PR1019 grown in commercial MRS medium and CB, respectively. The cDNA-AFLP results were validated on selected regulated genes by qPCR. In order to investigate the main adaptations to growth in a cheese-mimicking system, we focused on 20 transcripts over-expressed in CB with respect to MRS. It is worth noting the presence of transcripts involved in the degradation of pyruvate and ribose. Pyruvate is a intracellular metabolite that can be produced through different metabolic routes starting from the carbon sources present in cheese, and can be released in the cheese matrix with the starter lysis. Similarly the ribonucleosides released with starter lysis could deliver ribose that represents a fermentable carbohydrate in environments, such as cheese, where free carbohydrates are lacking.
Both pyruvate degradation and ribose catabolism induce a metabolite flux toward acetate, coupled with ATP production via acetate kinase. Taking into account these considerations, we suggest that the energy produced through these pathways may concur to explain the great ability of L. rhamnosus PR1019 to grow on CB.
Conclusions
By a transcriptomic approach we identified a set of genes involved in alternative metabolic pathways in L. rhamnosus that could be responsible for L. rhamnosus growth in cheese during ripening.
doi:10.1186/1471-2180-14-28
PMCID: PMC3928093  PMID: 24506811
Lactobacillus rhamnosus; Cheese; cDNA-amplified fragment length polymorphism; Quantitative real-time reverse transcription-PCR
2.  Inflammation and colorectal cancer, when microbiota-host mutualism breaks 
Structural changes in the gut microbial community have been shown to accompany the progressive development of colorectal cancer. In this review we discuss recent hypotheses on the mechanisms involved in the bacteria-mediated carcinogenesis, as well as the triggering factors favoring the shift of the gut microbiota from a mutualistic to a pro-carcinogenic configuration. The possible role of inflammation, bacterial toxins and toxic microbiota metabolites in colorectal cancer onset is specifically discussed. On the other hand, the strategic role of inflammation as the keystone factor in driving microbiota to become carcinogenic is suggested. As a common outcome of different environmental and endogenous triggers, such as diet, aging, pathogen infection or genetic predisposition, inflammation can compromise the microbiota-host mutualism, forcing the increase of pathobionts at the expense of health-promoting groups, and allowing the microbiota to acquire an overall pro-inflammatory configuration. Consolidating inflammation in the gut, and favoring the bloom of toxigenic bacterial drivers, these changes in the gut microbial ecosystem have been suggested as pivotal in promoting carcinogenesis. In this context, it will become of primary importance to implement dietary or probiotics-based interventions aimed at preserving the microbiota-host mutualism along aging, counteracting deviations that favor a pro-carcinogenic microbiota asset.
doi:10.3748/wjg.v20.i4.908
PMCID: PMC3921544  PMID: 24574765
Colorectal cancer; Inflammation; Gut microbiome; Co-abundance groups
3.  Novel Targets of Sulforaphane in Primary Cardiomyocytes Identified by Proteomic Analysis 
PLoS ONE  2013;8(12):e83283.
Cardiovascular diseases represent the main cause of mortality in the industrialized world and the identification of effective preventive strategies is of fundamental importance. Sulforaphane, an isothiocyanate from cruciferous vegetables, has been shown to up-regulate phase II enzymes in cardiomyocytes and counteract oxidative stress-induced apoptosis. Aim of the present study was the identification and characterization of novel sulforaphane targets in cardiomyocytes applying a proteomic approach. Two-dimensional gel electrophoresis and mass spectrometry were used to generate protein profiles of primary neonatal rat cardiomyocytes treated and untreated with 5 µM sulforaphane for 1-48 h. According to image analysis, 64 protein spots were found as differentially expressed and their functional correlations were investigated using the MetaCore program. We mainly focused on 3 proteins: macrophage migration inhibitory factor (MIF), CLP36 or Elfin, and glyoxalase 1, due to their possible involvement in cardioprotection. Validation of the time-dependent differential expression of these proteins was performed by western blotting. In particular, to gain insight into the cardioprotective role of the modulation of glyoxalase 1 by sulforaphane, further experiments were performed using methylglyoxal to mimic glycative stress. Sulforaphane was able to counteract methylglyoxal-induced apoptosis, ROS production, and glycative stress, likely through glyoxalase 1 up-regulation. In this study, we reported for the first time new molecular targets of sulforaphane, such as MIF, CLP36 and glyoxalase 1. In particular, we gave new insights into the anti-glycative role of sulforaphane in cardiomyocytes, confirming its pleiotropic behavior in counteracting cardiovascular diseases.
doi:10.1371/journal.pone.0083283
PMCID: PMC3859650  PMID: 24349480
4.  Functional metagenomic profiling of intestinal microbiome in extreme ageing 
Aging (Albany NY)  2013;5(12):902-912.
Age-related alterations in human gut microbiota composition have been thoroughly described, but a detailed functional description of the intestinal bacterial coding capacity is still missing. In order to elucidate the contribution of the gut metagenome to the complex mosaic of human longevity, we applied shotgun sequencing to total fecal bacterial DNA in a selection of samples belonging to a well-characterized human ageing cohort. The age-related trajectory of the human gut microbiome was characterized by loss of genes for shortchain fatty acid production and an overall decrease in the saccharolytic potential, while proteolytic functions were more abundant than in the intestinal metagenome of younger adults. This altered functional profile was associated with a relevant enrichment in “pathobionts”, i.e. opportunistic pro-inflammatory bacteria generally present in the adult gut ecosystem in low numbers. Finally, as a signature for long life we identified 116 microbial genes that significantly correlated with ageing. Collectively, our data emphasize the relationship between intestinal bacteria and human metabolism, by detailing the modifications in the gut microbiota as a consequence of and/or promoter of the physiological changes occurring in the human host upon ageing.
PMCID: PMC3883706  PMID: 24334635
centenarians; extreme-aging; gut microbiome
5.  The Enterocyte-Associated Intestinal Microbiota of Breast-Fed Infants and Adults Responds Differently to a TNF-α-Mediated Pro-Inflammatory Stimulus 
PLoS ONE  2013;8(11):e81762.
Co-evolved as an integral component of our immune system, the gut microbiota provides specific immunological services at different ages, supporting the immune education during our infancy and sustaining a well-balanced immunological homeostasis during the course of our life. In order to figure out whether this involves differences in the microbial groups primarily interacting with the host immune system, we developed a non-invasive HT29 cell-based minimal model to fingerprint the enterocyte-associated microbiota fraction in infants and adults. After depicting the fecal microbial community of 12 breast-fed infants and 6 adults by 16S rDNA amplicon pools 454 pyrosequencing, their respective HT29 cell-associated gut microbiota fractions were characterized by the universal phylogenetic array platform HTF-Microbi.Array, both in the presence and absence of a tumor necrosis factor-alpha (TNF-α)-mediated pro-inflammatory stimulus. Our data revealed remarkable differences between the enterocyte-associated microbiota fractions in breast-fed infants and adults, being dominated by Bifidobacterium and Enterobacteriaceae the first and Bacteroides-Prevotella and Clostridium clusters IV and XIVa the second. While in adults TNF-α resulted in a profound impairment of the structure of the enterocyte-associated microbiota fraction, in infants it remained unaffected. Differently from the adult-type gut microbial community, the infant-type microbiota is structured to cope with inflammation, being co-evolved to prime the early immune response by means of transient inflammatory signals from gut microorganisms.
doi:10.1371/journal.pone.0081762
PMCID: PMC3841132  PMID: 24303069
6.  A Natural-Like Synthetic Small Molecule Impairs Bcr-Abl Signaling Cascades and Induces Megakaryocyte Differentiation in Erythroleukemia Cells 
PLoS ONE  2013;8(2):e57650.
Over the past years, we synthesized a series of new molecules that are hybrids of spirocyclic ketones as complexity-bearing cores with bi- and ter-phenyls as privileged fragments. Some of these newly-shaped small molecules showed antiproliferative, pro-apoptotic and differentiating activity in leukemia cell lines. In the present study, to investigate more in depth the mechanisms of action of these molecules, the protein expression profiles of K562 cells treated with or without the compounds IND_S1, MEL_T1, IND_S7 and MEL_S3 were analyzed using two-dimensional gel electrophoresis coupled with mass spectrometry. Proteome comparisons revealed several differentially expressed proteins, mainly related to cellular metabolism, chaperone activity, cytoskeletal organization and RNA biogenesis. The major results were validated by Western blot and qPCR. To attempt integrating findings into a cellular signaling context, proteomic data were explored using MetaCore. Network analysis highlighted relevant relationships between the identified proteins and additional potential effectors. Notably, qPCR validation of central hubs showed that the compound MEL_S3 induced high mRNA levels of the transcriptional factors EGR1 and HNF4-alpha; the latter to our knowledge is reported here for the first time to be present in K562 cells. Consistently with the known EGR1 involvement in the regulation of differentiation along megakaryocyte lineage, MEL_S3-treated leukemia cells showed a marked expression of glycoprotein IIb/IIIa (CD41) and glycoprotein Ib (CD42), two important cell markers in megakaryocytic differentiation, together with morphological aspects of megakaryoblasts and megakaryocytes.
doi:10.1371/journal.pone.0057650
PMCID: PMC3584047  PMID: 23460890
7.  Dietary supplementation with probiotics during late pregnancy: outcome on vaginal microbiota and cytokine secretion 
BMC Microbiology  2012;12:236.
Background
The vaginal microbiota of healthy women consists of a wide variety of anaerobic and aerobic bacterial genera and species dominated by the genus Lactobacillus. The activity of lactobacilli helps to maintain the natural healthy balance of the vaginal microbiota. This role is particularly important during pregnancy because vaginal dismicrobism is one of the most important mechanisms for preterm birth and perinatal complications. In the present study, we characterized the impact of a dietary supplementation with the probiotic VSL#3, a mixture of Lactobacillus, Bifidobacterium and Streptococcus strains, on the vaginal microbiota and immunological profiles of healthy women during late pregnancy.
Results
An association between the oral intake of the probiotic VSL#3 and changes in the composition of the vaginal microbiota of pregnant women was revealed by PCR-DGGE population profiling. Despite no significant changes were found in the amounts of the principal vaginal bacterial populations in women administered with VSL#3, qPCR results suggested a potential role of the probiotic product in counteracting the decrease of Bifidobacterium and the increase of Atopobium, that occurred in control women during late pregnancy. The modulation of the vaginal microbiota was associated with significant changes in some vaginal cytokines. In particular, the decrease of the anti-inflammatory cytokines IL-4 and IL-10 was observed only in control women but not in women supplemented with VSL#3. In addition, the probiotic consumption induced the decrease of the pro-inflammatory chemokine Eotaxin, suggesting a potential anti-inflammatory effect on the vaginal immunity.
Conclusion
Dietary supplementation with the probiotic VSL#3 during the last trimester of pregnancy was associated to a modulation of the vaginal microbiota and cytokine secretion, with potential implications in preventing preterm birth.
Trial registration
ClinicalTrials.gov NCT01367470
doi:10.1186/1471-2180-12-236
PMCID: PMC3493352  PMID: 23078375
8.  Tumor Necrosis Factor Alpha Modulates the Dynamics of the Plasminogen-Mediated Early Interaction between Bifidobacterium animalis subsp. lactis and Human Enterocytes 
The capacity to intervene with the host plasminogen system has recently been considered an important component in the interaction process between Bifidobacterium animalis subsp. lactis and the human host. However, its significance in the bifidobacterial microecology within the human gastrointestinal tract is still an open question. Here we demonstrate that human plasminogen favors the B. animalis subsp. lactis BI07 adhesion to HT29 cells. Prompting the HT29 cell capacity to activate plasminogen, tumor necrosis factor alpha (TNF-α) modulated the plasminogen-mediated bacterium-enterocyte interaction, reducing the bacterial adhesion to the enterocytes and enhancing migration to the luminal compartment.
doi:10.1128/AEM.07883-11
PMCID: PMC3302587  PMID: 22287006
9.  Unbalance of intestinal microbiota in atopic children 
BMC Microbiology  2012;12:95.
Background
Playing a strategic role in the host immune function, the intestinal microbiota has been recently hypothesized to be involved in the etiology of atopy. In order to investigate the gastrointestinal microbial ecology of atopic disease, here we performed a pilot comparative molecular analysis of the faecal microbiota in atopic children and healthy controls.
Results
Nineteen atopic children and 12 healthy controls aged 4–14 years were enrolled. Stools were collected and the faecal microbiota was characterized by means of the already developed phylogenetic microarray platform, HTF-Microbi.Array, and quantitative PCR. The intestinal microbiota of atopic children showed a significant depletion in members of the Clostridium cluster IV, Faecalibacterium prausnitzii, Akkermansia muciniphila and a corresponding increase of the relative abundance of Enterobacteriaceae.
Conclusion
Depleted in key immunomodulatory symbionts, the atopy-associated microbiota can represent an inflammogenic microbial consortium which can contribute to the severity of the disease. Our data open the way to the therapeutic manipulation of the intestinal microbiota in the treatment of atopy by means of pharmaceutical probiotics.
doi:10.1186/1471-2180-12-95
PMCID: PMC3404014  PMID: 22672413
10.  Relevance of Bifidobacterium animalis subsp. lactis Plasminogen Binding Activity in the Human Gastrointestinal Microenvironment ▿  
Applied and Environmental Microbiology  2011;77(19):7072-7076.
Human plasmin(ogen) is regarded as a component of the molecular cross talk between the probiotic species Bifidobacterium animalis subsp. lactis and the human host. However, up to now, only in vitro studies have been reported. Here, we demonstrate that the probiotic strain B. animalis subsp. lactis BI07 is capable of recruiting plasmin(ogen) present at physiological concentrations in crude extracts from human feces. Our results provide evidence that supports the significance of the B. lactis-plasmin(ogen) interaction in the human gastrointestinal tract.
doi:10.1128/AEM.00413-11
PMCID: PMC3187096  PMID: 21821753
11.  Oxalate-Degrading Activity in Bifidobacterium animalis subsp. lactis: Impact of Acidic Conditions on the Transcriptional Levels of the Oxalyl Coenzyme A (CoA) Decarboxylase and Formyl-CoA Transferase Genes ▿  
Applied and Environmental Microbiology  2010;76(16):5609-5620.
Oxalic acid occurs extensively in nature and plays diverse roles, especially in pathological processes. Due to its highly oxidizing effects, hyperabsorption or abnormal synthesis of oxalate can cause serious acute disorders in mammals and can be lethal in extreme cases. Intestinal oxalate-degrading bacteria could therefore be pivotal in maintaining oxalate homeostasis and reducing the risk of kidney stone development. In this study, the oxalate-degrading activities of 14 bifidobacterial strains were measured by a capillary electrophoresis technique. The oxc gene, encoding oxalyl-coenzyme A (CoA) decarboxylase, a key enzyme in oxalate catabolism, was isolated by probing a genomic library of Bifidobacterium animalis subsp. lactis BI07, which was one of the most active strains in the preliminary screening. The genetic and transcriptional organization of oxc flanking regions was determined, unraveling the presence of two other independently transcribed open reading frames, potentially responsible for the ability of B. animalis subsp. lactis to degrade oxalate. pH-controlled batch fermentations revealed that acidic conditions were a prerequisite for a significant oxalate degradation rate, which dramatically increased in cells first adapted to subinhibitory concentrations of oxalate and then exposed to pH 4.5. Oxalate-preadapted cells also showed a strong induction of the genes potentially involved in oxalate catabolism, as demonstrated by a transcriptional analysis using quantitative real-time reverse transcription-PCR. These findings provide new insights into the characterization of oxalate-degrading probiotic bacteria and may support the use of B. animalis subsp. lactis as a promising adjunct for the prophylaxis and management of oxalate-related kidney disease.
doi:10.1128/AEM.00844-10
PMCID: PMC2918965  PMID: 20601517
12.  Antibiotics and probiotics in chronic pouchitis: A comparative proteomic approach 
AIM: To profile protein expression in mucosal biopsies from patients with chronic refractory pouchitis following antibiotic or probiotic treatment, using a comparative proteomic approach.
METHODS: Two-dimensional polyacrylamide gel electrophoresis and matrix-assisted laser desorption/ionization-time of flight mass spectrometry were used to characterize the changes related to antibiotic therapy in the protein expression profiles of biopsy samples from patients with chronic refractory pouchitis. The same proteomic approach was applied to identify differentially expressed proteins in the non-inflamed pouch before and after probiotic administration.
RESULTS: In the first set of 2D gels, 26 different proteins with at least 2-fold changes in their expression levels between the pouchitis condition and antibiotic-induced remission were identified. In the second set of analysis, the comparison between mucosal biopsy proteomes in the normal and probiotic-treated pouch resulted in 17 significantly differently expressed proteins. Of these, 8 exhibited the same pattern of deregulation as in the pouchitis/pouch remission group.
CONCLUSION: For the first time, 2D protein maps of mucosal biopsies from patients with ileal pouch-anal anastomosis were provided, and differentially expressed proteins following antibiotic/probiotic treatment were identified.
doi:10.3748/wjg.v16.i1.30
PMCID: PMC2799914  PMID: 20039446
Chronic disease; Pouchitis; Antibiotics; Probiotics; Proteins; Gene expression
13.  Dynamics of Vaginal Bacterial Communities in Women Developing Bacterial Vaginosis, Candidiasis, or No Infection, Analyzed by PCR-Denaturing Gradient Gel Electrophoresis and Real-Time PCR▿  
Applied and Environmental Microbiology  2007;73(18):5731-5741.
The microbial flora of the vagina plays a major role in preventing genital infections, including bacterial vaginosis (BV) and candidiasis (CA). An integrated approach based on PCR-denaturing gradient gel electrophoresis (PCR-DGGE) and real-time PCR was used to study the structure and dynamics of bacterial communities in vaginal fluids of healthy women and patients developing BV and CA. Universal eubacterial primers and Lactobacillus genus-specific primers, both targeted at 16S rRNA genes, were used in DGGE and real-time PCR analysis, respectively. The DGGE profiles revealed that the vaginal flora was dominated by Lactobacillus species under healthy conditions, whereas several potentially pathogenic bacteria were present in the flora of women with BV. Lactobacilli were the predominant bacterial population in the vagina for patients affected by CA, but changes in the composition of Lactobacillus species were observed. Real-time PCR analysis allowed the quantitative estimation of variations in lactobacilli associated with BV and CA diseases. A statistically significant decrease in the relative abundance of lactobacilli was found in vaginal fluids of patients with BV compared to the relative abundance of lactobacilli in the vaginal fluids of healthy women and patients with CA.
doi:10.1128/AEM.01251-07
PMCID: PMC2074899  PMID: 17644631
14.  Binding of Human Plasminogen to Bifidobacterium▿  
Journal of Bacteriology  2007;189(16):5929-5936.
Bifidobacteria constitute up to 3% of the total microbiota and represent one of the most important health-promoting bacterial groups of the human intestinal microflora. The presence of Bifidobacterium in the human gastrointestinal tract has been directly related to several health-promoting activities; however, to date, no information about the specific mechanisms of interaction with the host is available. In order to provide some insight into the molecular mechanisms involved in the interaction with the host, we investigated whether Bifidobacterium was able to capture human plasminogen on the cell surface. By using flow cytometry, we demonstrated a dose-dependent human plasminogen-binding activity for four strains belonging to three bifidobacterial species: Bifidobacterium lactis, B. bifidum, and B. longum. The binding of human plasminogen to Bifidobacterium was dependent on lysine residues of surface protein receptors. By using a proteomic approach, we identified five putative plasminogen-binding proteins in the cell wall fraction of the model strain B. lactis BI07. The data suggest that plasminogen binding to B. lactis is due to the concerted action of a number of proteins located on the bacterial cell surface, some of which are highly conserved cytoplasmic proteins which have other essential cellular functions. Our findings represent a step forward in understanding the mechanisms involved in the Bifidobacterium-host interaction.
doi:10.1128/JB.00159-07
PMCID: PMC1952040  PMID: 17557824
15.  Gut microbiome of the Hadza hunter-gatherers 
Nature Communications  2014;5:3654.
Human gut microbiota directly influences health and provides an extra means of adaptive potential to different lifestyles. To explore variation in gut microbiota and to understand how these bacteria may have co-evolved with humans, here we investigate the phylogenetic diversity and metabolite production of the gut microbiota from a community of human hunter-gatherers, the Hadza of Tanzania. We show that the Hadza have higher levels of microbial richness and biodiversity than Italian urban controls. Further comparisons with two rural farming African groups illustrate other features unique to Hadza that can be linked to a foraging lifestyle. These include absence of Bifidobacterium and differences in microbial composition between the sexes that probably reflect sexual division of labour. Furthermore, enrichment in Prevotella, Treponema and unclassified Bacteroidetes, as well as a peculiar arrangement of Clostridiales taxa, may enhance the Hadza’s ability to digest and extract valuable nutrition from fibrous plant foods.
Gut microbes influence our health and may contribute to human adaptation to different lifestyles. Here, the authors describe the gut microbiome of a community of hunter-gatherers and identify unique features that could be linked to a foraging lifestyle.
doi:10.1038/ncomms4654
PMCID: PMC3996546  PMID: 24736369

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